The isolation and characterization of the pyruvate kinase-encoding gene from the yeast Yarrowia lipolytica. The dimorphic yeast, Yarrowia lipolytica, has been developed as a useful expression/secretion system for heterologous proteins such as chymosin and tissue plasminogen activator. To further develop this expression system, we have cloned the gene (PYK) encoding the highly expressed glycolytic enzyme, pyruvate kinase (PYK). Genomic clones were selected by their specific hybridization to synthetic oligodeoxyribonucleotide probes based on regions of the enzyme that were conserved through evolution. The clones identified by hybridization contained overlapping DNA inserts. We have confirmed the identity of the cloned gene based on two criteria: (1) the nucleotide sequence of the proposed PYK gene predicts a protein that is highly homologous to the corresponding Saccharomyces cerevisiae enzyme, and (2) PYK-specific activity was increased twofold when wild-type Y. lipolytica strains were transformed with the isolated DNA. Interestingly, we found that the open reading frame of the Y. lipolytica PYK gene was interrupted by an intron. This represents the first report of an intron in a Y. lipolytica gene.


Characterization of a highly expressed lignin peroxidase-encoding gene from the basidiomycete Phanerochaete chrysosporium. The genomic clone, LG2, encoding LiP2, the major lignin peroxidase (LiP) isozyme from Phanerochaete chrysosporium strain OGC101, was isolated and characterized. The 5'-untranslated region of LG2 contains sequences similar to CRE and XRE promoter elements. Comparison with its transcript indicates that eight introns, each less than 59 bp, interrupt the coding sequence. Comparison with genes encoding other LiP isozymes shows five related patterns of intron location, whose incidence coincides with described LiP structural subfamilies. Codon bias indices calculated for all known P. chrysosporium genes, including trpC and genes encoding LiP, MnP, and exo-cellobiohydrolase I, demonstrate that LG2 has the most biased codon usage. We conclude that subdivisions of the LiP family may be based on intron location in the encoding genes, and that ranking of isozyme production levels can be estimated by the extent of bias in codon usage in the cognate gene.


Development of a prokaryotic expression vector that exploits dicistronic gene organization. For unknown reasons, levels of expression of foreign genes inserted into expression vectors in Escherichia coli have frequently been undetectable. The most critical step in the successful production of foreign proteins seems to be the initiation of translation. Since most prokaryotic genes are transcribed in a polycistronic form, we have devised a new prokaryotic expression system utilizing dicistronic gene organization. Downstream from a strong promoter and the gene encoding glutathione S-transferase from Schistosoma japonicum, various foreign genes were connected via a ribosome-binding site, a stop codon and a start codon. The VH domain of an immunoglobulin fused to the alpha subunit of tryptophan synthase, FK506-binding protein, cyclophilin, and a domain of a major histocompatibility complex antigen were successfully produced in E. coli as discrete polypeptides by this method.


The yptV1 gene encodes a small G-protein in the green alga Volvox carteri: gene structure and properties of the gene product. Small G-proteins encoded by ras-like genes are ubiquitous in eukaryotic cells. These G-proteins are believed to play a role in central processes, such as signal transduction, cell differentiation and membrane vesicle transport. By screening genomic and cDNA libraries of the colonial alga, Volvox carteri f. nagariensis, with ypt DNA probes from Zea mays, we have identified the first member of a ypt gene family, yptV1, within a green alga. The 1538-bp yptV1 gene of V. carteri consists of nine exons and eight introns and has three potential polyadenylation sites 210, 420 and 500 bp downstream from the UGA stop codon. The derived 203-amino-acid polypeptide, YptV1, exhibits 81% similarity with Ypt1 from mouse, with the corresponding genes sharing four identical intron positions. Recombinant YptV1 (reYptV1) produced in Escherichia coli retains the ability to bind GTP after SDS-PAGE and immobilization on nitrocellulose. Immunological studies using polyclonal antibodies against reYptV1 indicate that the protein is present in the membrane fraction of a V. carteri extract and is expressed throughout the whole life-cycle of the alga. Similar to other Ras-like proteins, YptV1 contains two conserved C-terminal cysteine residues suggesting post-translational modification(s), such as isoprenylation or palmitoylation, required for membrane anchoring. The presumptive role of YptV1 in cytoplasmic vesicle transport is briefly discussed.


Sequence analysis of the indirect flight muscle actin-encoding gene of Drosophila simulans. A genomic clone of the Drosophila simulans indirect flight muscle actin-encoding gene (Act88F) has been isolated, and the sequence of a 3358-nucleotide segment corresponding to the Drosophila melanogaster Act88F transcription unit is presented. The ACt88F homologs in these two sibling species encode identical proteins and the general genomic organization of the Act88F locus is highly conserved, including the location of the transcription start point, and the size and position of intron/exon junctions. Substitutions within the 5' flanking region, however, are clearly nonuniform and the regions of lowest divergence coincide with regions that have been implicated in transcript accumulation and the regulation of tissue-specific expression. Silent substitutions within the coding regions have been compared to five other gene homologs in these sibling species. The rate of silent substitution at these loci varies more than threefold, suggesting selection at the codon level.


Identification of a genomic DNA fragment containing the Drosophila melanogaster ovarian tumor gene (otu) and localization of regions governing its expression. We have identified a genomic DNA fragment which restores fertility to mutants of the ovarian tumor locus (otu) of Drosophila melanogaster. Germ-line transformants bearing this fragment express otu mRNA with the same tissue specificity as, and at levels comparable to, the wild-type otu gene. Transcription from the otu promoter, P(otu), which lacks a TATA element, appears to be initiated at multiple transcription start points (tsp) within an 80-bp region. Deletion of sequences upstream of the tsp indicates that a region between nucleotides -190 and -310 is required for proper expression from the otu gene. A DNA fragment containing 452 bp upstream and 126 bp downstream from the tsp is able to direct expression of the Escherichia coli lacZ gene in the germ cells of the ovary and testis, indicating that cis-acting regulatory elements governing these expression patterns are located in a 578-bp region surrounding the multiple tsp.


Ependymins from the cerebrospinal fluid of salmonid fish: gene structure and molecular characterization. So far, ependymins (Epds) have been sequenced only from cypriniform fish, and in the past all attempts have failed to characterize, on a molecular level, homologous Epd proteins in higher vertebrates. Therefore, rainbow trout (Oncorhynchus mykiss) Epds, which represent the predominant proteins of the cerebrospinal fluid, have been N-terminally sequenced and the encoding cDNA subsequently cloned using the polymerase chain reaction. Surprisingly, only 40-42% of the amino acids are identical with the corresponding sequences from goldfish (Carassius auratus), and no convincing immunological cross-reactivity is observed with an antiserum raised against purified Epds from C. auratus. O. mykiss possesses two highly homologous genes encoding Epds (Om-I, Om-II), a feature typical of a quasi-tetraploid species. Western analysis, using two specific antibodies against Epds from O. mykiss, revealed a variety of different glycosylation variants. In contrast to C. auratus, Epds from O. mykiss probably do not form disulfide-linked dimers. The structure of one Epd gene and its flanking regions have been determined for the Atlantic salmon (Salmo salar). Six exons were deduced by comparison with the corresponding cDNA sequence from O. mykiss (almost 98% homology with Om-II).


Rainbow trout mitochondrial DNA: sequence and structural characteristics of the non-coding control region and flanking tRNA genes. We have determined the sequence of the 1003-bp control region (also referred to as the 'displacement-loop region') and flanking tRNA genes in the mitochondrial DNA (mtDNA) of the rainbow trout, Oncorhynchus mykiss. This region has the same overall structure (i.e., trnT-trnP-control region-trnF) as in mammalian and amphibian (Xenopus laevis) mitochondrial (mt)DNAs. The trout control region contains apparent homologues of the conserved sequence blocks (CSB) and termination-associated sequences identified in all other vertebrate mtDNA control regions; however, it is distinguished by having an imperfect direct repeat (68/73 bp; 77% positional identity) in the right domain (proximal to the phenylalanine tRNA gene), downstream from CSB-3 in the direction of heavy-strand transcription. Within the control region, rainbow trout mtDNA shares considerable sequence similarity with the mtDNAs of Atlantic cod, Gadus morhua (Johansen et al., Nucleic Acids Res. 18 (1990) 411-419] and white sturgeon, Acipenser transmontanus (Buroker et al., Genetics 124 (1990) 157-163). The highest level of identity in pairwise comparisons is 60-70% over about 80 bp in the right domain (encompassing CSB-2 and CSB-3). About 270 bp comprising the central domain of the control region (encompassing a polypyrimidine tract) are more moderately conserved (55-60% identity in pairwise comparisons), while the left domain is highly divergent. Comparison of five trout mitochondrial tRNA sequences with their human and X. laevis homologues emphasizes the strong A+T substitution bias shown by the human sequences.


Characterization of the murine thymidine kinase-encoding gene and analysis of transcription start point heterogeneity. We have determined the molecular organization and transcription start points (tsp) for the murine gene (TK) encoding thymidine kinase. The exon/intron structure and sequences present at the splice junctions of the mammalian TK genes have been highly conserved; however, the promoter sequences of these genes have diverged widely. Both the human and Chinese hamster TK promoter regions contain CCAAT and TATA consensus motifs, whereas the mouse promoter has neither element. This difference between species is reflected in that, unlike the hamster and human TK genes, transcription initiates from numerous specific tsp within a 100-bp region in the mouse TK gene. The complex pattern of tsp seen in the endogenous gene was not maintained in transfected cell lines containing TK promoter::beta-globin (HBB) fusions. Transcription from the murine TK:HBB fusion genes initiated from a small number of tsp that were clustered downstream from the ATG in hybrids containing TK coding sequences, and in the HBB 5' UTR in hybrids that did not. Few or no specific tsp were detected from the upstream sites used in the endogenous mouse TK gene.


Genomic organization and tissue expression of the murine gene encoding the protein beta-aspartate methyltransferase. Two overlapping clones containing the entire 684-nucleotide (nt) sequence encoding murine protein beta-aspartate methyltransferase (EC 2.1.1.77) were isolated from a genomic library. Partial nt sequence analysis of the two clones revealed that the protein carboxyl methyltransferase (PCMT)-encoding sequence is distributed among seven exons, ranging from 32 to 339 bp in length, within 25 kb of genomic DNA. Three exons correspond to regions of primary structure which are strongly conserved among a number of eukaryotic and prokaryotic enzymes which utilize S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy). The 5'-flanking region of the PCMT-encoding gene (PCMT) contains an 800-bp G+C-rich region with potential binding sites for transcription factor ETF, but lacks a TATA box and binding sites for other known transcription factors. Multiple PCMT mRNAs were detected on Northern blots of RNA extracted from murine brain, testis, liver and kidney. The overall abundance of PCMT mRNAs in each tissue paralleled the measured specific activity of the PCMT. Comparison of the genomic sequence information with the 3'-untranslated regions (UTRs) of two cDNA clones from a murine testis library indicated that PCMT mRNA precursors undergo alternative splicing. The structure and widespread expression of PCMT are characteristics of vertebrate housekeeping genes.


Insulin increases transcription of rat gene 33 through cis-acting elements in 5'-flanking DNA. Gene 33 is a multihormonally-regulated rat gene whose transcription is rapidly and markedly enhanced by insulin in liver and cultured hepatoma cells. To examine the mechanism by which insulin regulates transcription, we have constructed chimeric plasmids in which expression of the bacterial cat gene, encoding chloramphenicol acetyltransferase (CAT), is governed by gene 33 promoter elements and contiguous sequences in DNA flanking the transcription start point (tsp). When transfected into H4IIE hepatoma cells, these constructs gave rise to stably transformed cell lines producing the bacterial CAT enzyme. This expression was increased by insulin treatment in a fashion resembling the effect of this hormone on transcription of the native gene. In vitro transcription assays in nuclear extracts also revealed increased transcription of the chimeric plasmids when the extracts were prepared from insulin-treated rat hepatoma cells. The results demonstrate that induction by insulin is mediated by cis-acting nucleotide sequences located between bp -480 to +27 relative to the tsp.


The carbamyl phosphate synthetase promoter contains multiple binding sites for C/EBP-related proteins. The promoter of the gene (CPS) encoding rat carbamyl phosphate synthetase I has been mapped 5' to a segment of about 525 nucleotides upstream from the transcription start point and, when analyzed in liver nuclear extracts, contained six well-defined protein-recognition elements, designated CPS sites I-VI. All six elements were recognized, with varying affinities, by CAAT and enhancer-binding protein (C/EBP alpha) produced in bacteria. Oligodeoxyribonucleotides corresponding to CPS site II or to the C/EBP alpha-recognition element of the ALB promoter, site D, competed with the six CPS-promoter elements in footprinting assays. However, mutagenesis of the C/EBP alpha-recognition element, 5'-GTTGCAAC, at the core of site II was sufficient to abolish transactivation of the CPS promoter by C/EBP alpha in co-transfected HepG2 cells. These findings indicate that the CPS promoter contains multiple recognition elements for factors with DNA-binding specificities similar to C/EBP proteins. Activation by C/EBP alpha, however, requires promoter site II.


Production of mature bovine pancreatic ribonuclease in Escherichia coli. The coding sequence for the bovine pancreatic ribonuclease (RNase) precursor has been cloned and produced in Escherichia coli using the polymerase chain reaction (PCR) technique. A PCR amplification has been carried out utilizing as template the recombinant plasmid, pQR138, which contains the coding sequence for the RNase precursor, and primers that allow for the addition of new sequences at the 5' and 3' ends of the coding sequence. The resultant fragment contains two coding sequences, one for a hexapeptide and the other for pre-RNase. This fragment has been cloned into the expression vector, pKK223.3, under the control of the tac promoter, to form a two-cistron vector. Upon induction with IPTG, E. coli cells harboring this construct generate a bicistronic mRNA which upon translation produces a hexapeptide and pre-RNase. The RNase precursor is efficiently translocated into the periplasmic space of E. coli. Upon translocation, the signal sequence is removed generating mature RNase. Formation of the disulfide bridges in RNase is facilitated by the oxidative environment of the periplasm and a fully active protein is obtained. RNase produced in E. coli has been purified to homogeneity by cation-exchange chromatography, and the removal of the signal sequence has been verified by N-terminal sequencing. The total process from inoculation of media to obtaining pure and fully active recombinant RNase is achieved in 48 h.


Transcription of the chicken anemia virus (CAV) genome and synthesis of its 52-kDa protein. This paper describes the expression of the chicken anemia virus (CAV) genome, a recently characterized single-stranded circular-DNA virus of a new type [Noteborn et al., J. Virol. 65 (1991) 3131-3139]. The major transcript from the CAV genome is an unspliced mRNA of about 2100 nucleotides (nt). Its transcription start point and poly(A)-addition site are located at nt 354 and 2317 of the CAV sequence, respectively. In vitro translation experiments provide evidence that the major CAV open reading frame encodes a 52-kDa protein by using the fifth AUG as a start codon of the unspliced CAV mRNA.


The Drosophila melanogaster ribosomal protein L17A-encoding gene. The structure and sequence of the gene encoding the Drosophila melanogaster homolog of the human and yeast large-subunit ribosomal protein L17A (rpL17A) is presented. The deduced amino acid (aa) sequence of 140 residues exhibits 87% and 77% identity to that of the human (140 aa) and yeast (137 aa) rpL17As, respectively. The D. melanogaster rpL17A gene is single copy and maps at 58F6-59A3, a chromosome region encompassing a previously characterized Minute locus, M(2)I. Despite this extensive homology in their protein products, the D. melanogaster and yeast rpL17A genes display different exon-intron structures, with the first D. melanogaster intron mapping within the 5'-untranslated mRNA leader. The rpL17A gene gives rise to a single 600-nucleotide transcript present throughout development, and is located close to another similarly expressed gene. The 5' end of the D. melanogaster rpL17A mRNA contains a polypyrimidine tract displayed by several mammalian rp genes and involved in translational control of their expression.


Cloning and sequence of a functionally active cDNA encoding the mouse ubiquitin-activating enzyme E1. A cDNA encoding the ubiquitin-activating enzyme, E1, was isolated from the mouse mammary carcinoma cell line, FM3A, and shown to complement mutant mouse cells deficient in the enzyme. The 3495-bp cDNA encodes 1058 amino acids (aa), and shares extensive homology with the human E1 enzyme at both the nucleotide and aa sequence levels.


Factors interacting with the rat carbamyl phosphate synthetase promoter in expressing and nonexpressing tissues. The proximal promoter of the rat carbamyl phosphate synthetase-encoding gene (CPS) contains at least six potential cis-acting regulatory elements (sites I-VI), as judged by DNase I footprint analysis using rat liver nuclear extracts; all six regions bind proteins with DNA recognition properties similar to those of CAAT and enhancer-binding protein alpha (C/EBP alpha) [Lagacé et al., Gene 118 (1992) 231-238]. In contrast, nuclear extracts from kidney, brain and spleen contain proteins that recognize CPS promoter sites II, V and VI, but not sites I, III and IV. Mutation of the octameric sequence (5'-GTTGCAAC) within site II, which is a recognition element for C/EBP alpha, abolished binding of nuclear proteins to site II oligodeoxyribonucleotides (oligos) in all tissues. As well, the site II mutation reduced the level of in vitro transcription from the CPS promoter by about 50% in liver and spleen nuclear extracts, but had a negligible effect in brain and kidney extracts. The fact that promoter activity was observed in extracts of tissues that do not express the endogenous CPS gene (i.e., brain, kidney and spleen) indicates that these tissues, nevertheless, contain factors with the potential to activate transcription through a limited number of CPS promoter elements. Tissue-specific regulation, therefore, must involve steps to prevent these factors from acting on the endogenous CPS promoter in situ.


Sequence of a rat TIS11 cDNA, an immediate early gene induced by growth factors and phorbol esters. We report here the nucleotide sequence of a rat TIS11 cDNA, an immediate early gene, induced by nerve growth factor and epidermal growth factor, by 12-O-tetradecanoyl phorbol-13-acetate, and other stimuli in PC12 pheochromocytoma cells. The deduced protein consists of 320 amino acid residues with two tandem repeats of a putative Zn(2+)-finger motif.


Plasmid pLTRpoly: a versatile high-efficiency mammalian expression vector. Plasmid pLTRpoly is an expression vector enabling high-level expression of introduced genes in a variety of cell types. A large multiple cloning site (MCS) and the availability of the full-length nucleotide sequence facilitate the generation of constructs using this vector. Here, constructs made with pLTRpoly have been tested in NIH3T3 mouse fibroblasts.


Sequence of a cDNA clone encoding a rat Reg-2 protein. We report here the nucleotide sequence of a rat cDNA clone encoding a protein homologous to the Reg (regenerating) protein. The encoded protein, designated Reg-2, shows 60%, 78% and 61% similarities with the reported amino acid sequences of the rat, bovine and human proteins, respectively.


Repeat sequences in the gene encoding the human D4 dopamine receptor. The gene encoding the human D4 dopamine receptor has evolved by gaining at least five internal repeats which are located within exons 3 and 4, and in the intervening intron 3 sequence. The amino acid sequence in the cytoplasmic loop of the receptor, involved in G protein coupling, has been altered by these gene changes.


Cloning of a cDNA encoding a receptor related to the formyl peptide receptor of human neutrophils. We cloned a cDNA (RFP) encoding a receptor (RFP) related (70% overall nucleotide homology) to the formyl peptide receptor of human neutrophils (hFPR). RFP is a seven-transmembrane-domain receptor and its distribution is limited to myeloid cells. Domain sequence comparison with hFPR reveals highly conserved regions and provides clues to putative domains involved in ligand binding and receptor desensitization.


Health care for all: a crisis of cost and access. A roundtable discussion: Part 1. Primary care physicians are feeling the impact of a healthcare system in crisis. Many Americans have no health insurance, paperwork is consuming more and more of physicians' time, and concerns about controlling costs seem to overshadow the value of medical decision-making. Medicare protects the elderly, but low reimbursement fees act as a disincentive to accepting them as patients. Although there is a general consensus that reforms are needed, there are many different opinions on how this should be achieved. In this first part of a three-part roundtable discussion, seven experts in the fields of aging, medicine, and government discuss flaws in our current system and factors that are interfering with reform.


Detection of polymorphisms of human DNA after polymerase chain reaction by miniaturized SDS-PAGE. PCR followed by SDS-PAGE in miniaturized non-denaturing gels permits in some cases the identification of single base pair substitutions in small DNA fragments and therefore, the study of human DNA polymorphisms. The usefulness of the system in forensic science is investigated by typing the HLA-DQA1 locus and the VNTR recognized with the probe pMCT118 (locus D1S80) and it shows to be advantageous over previously published methods for typing the MCT118 system, whereas in HLA-DQA1 typing for forensic casework, both dot-blot with ASO probes and this method could be complementary.


Complement component C3a or C3a desArg as a new marker for estimation of local vital reactions in incised skin wounds. The anaphylatoxin C3a or its desArg form (C3a/desArg) generated during complement activation could be detected in the vicinity of incised skin wounds of guinea pigs using immunoblotting methods. The C3a/desArg peptides were detectable immediately after injury in local sites up to 3 mm from the wound edge. In subsequent determinations of up to at least 3-day-old antemortem wounds, the maximum concentration of these peptides was largely localized up to 6 mm from the wound edge at 2 h after injury. In postmortem wounds, however, these peptides were undetectable. When they were released in antemortem wounded tissues they could be detected up to 1 day at 22 degrees C after death. These results suggest that the detection of C3a/desArg in wounds using immunoblotting methods can be useful for distinguishing ante- from postmortem wounds.


Biostatistical basis of individualization and segregation analysis using the multilocus DNA probe MZ 1.3: results of a collaborative study. A collaborative study using the multilocus minisatellite DNA probe MZ 1.3 was carried out to investigate segregation information, mutation rate, DNA fragment frequencies as well as band sharing characteristics. The fingerprint patterns of 393 children as well as 694 unrelated individuals were analysed after digestion of DNA with the restriction enzyme HinfI. A mutation rate of 1% per meiosis or 0.04% per band was found with a mean number of 26 bands/individual. It was shown that maternal and paternal fragments are inherited in equal proportions. Population frequencies of restriction fragments demonstrated a distribution with increasing frequencies in the small fragment size range below 10 kb as well as the absence of very common or very rare fragments. Our data can be used to calculate simple exclusion probabilities based on the number of non-maternal bands in the child.


HLA-DQA1 allele and genotype frequencies in a northern Italian population. HLA-DQA1 typing of 227 randomly selected Northern Italian people by the use of polymerase chain reaction are reported. The combined use of commercial Amplitype HLA-DQalpha system and four sequence-specific oligonucleotide probes allows the definition of 8 alleles and 36 genotypes, arranged according to World Health Organisation nomenclature. Seven of these genotypes are not observed among the analyzed samples. Allele frequencies range from 1.5 to 35.7% and genotype observations do not deviate significantly from Hardy-Weinberg equilibrium; observed heterozygosity is 0.8238 with an allelic diversity value of 0.79 and the power of discrimination is 0.925. Our Italian population sample shows differences from other Caucasian samples both for allele and genotype frequencies. This locus typing for the 8 defined alleles provides a rapid and sensitive method in individual identification and paternity investigation.


Drug abuse in western Denmark during the eighties. I. Drugs of abuse. A description is given of the availability and quality of drugs seized in the Danish illicit drug market during the eighties. Cannabis was the most prevalent illegal drug throughout the decade. Seizures of heroin predominated among the 'hard' illegal drugs until 1987 but during the late eighties a significant increase in amphetamine seizures was observed. Most of the cocaine was seized in transit, apparently not meant for the Danish market. Medical drugs, particularly benzodiazepines and the synthetic opiates methadone and ketobemidone, comprised an increasing part of the illegal drug market during the period. No seizures of designer drugs were seen. The street level purities of both heroin and amphetamine were, on average, 30-40%.


Drug abuse in western Denmark during the eighties. II. Fatal poisonings among drug abusers. Cases of fatal poisoning among drug addicts (n = 246) and abusers of medical drugs (n = 138) are discussed on the basis of the availability of illicit and medical drugs during the eighties. Propoxyphene was frequently the cause of death among both drug addicts and abusers of medical drugs. Deaths caused by analgesics and psychoactive drugs, mainly barbiturates and antidepressants, predominated among abusers of medical drugs. During the late eighties the number of deaths caused by barbiturates and propoxyphene declined as a result of changes in the distribution regulations for both drugs. Heroin/morphine accounted for only one-third of the poisonings in drug addicts. No trends concerning the prevalence of deaths caused by these drugs were observed throughout the decade. The number of deaths caused by the synthetic opiates methadone and ketobemidone, however, increased during the late eighties, probably due to an increased availability of these drugs in the illicit drug market. Alcohol was detected in half of all the poisoning cases and benzodiazepines in one-third.


Prevalence of HIV-1 antibodies in drug addicts autopsied at the University Institute of Forensic Pathology in Copenhagen 1988-1991. In the period 1988-1991 the HIV-1 antibody test was performed in a total of 389 autopsied drug addicts. The HIV-1 incidence rose from 6% in 1988 to 14% in 1990 and 14% in 1991. There was no significant difference between the cumulated incidence in males and females. The HIV-1-positive addicts were compared to the HIV-1 negative addicts as regards mean age, duration of abuse, residence and cause and manner of death. Except for residence, where significantly more HIV-1-positive than negative addicts lived in Copenhagen, there were no significant differences between the two groups. In about one-third of the HIV-1-positive cases, equal both for males and females, the HIV-1-positivity was not known beforehand. The results are discussed in relation to the findings in other countries and to the findings in selected groups of living addicts in Copenhagen. It is concluded, that the diagnosis of HIV-1 infection in autopsied drug addicts is important from epidemiologic and hygienic points of view.


Women, homicide and alcohol in Cape Town, South Africa. Little is known about the complex circumstances culminating in the homicidal death of a woman. The records of 248 female homicides and suicides admitted to the Salt River State Mortuary between January 1990 and July 1991 were reviewed with specific attention to mode of death and blood alcohol concentration (BAC). Female homicide levels reflected the intensity of violence in specific residential areas. White females were far more likely to die a suicidal, as opposed to homicidal, death (Odds Ratio = 31.94; 95% Confidence Interval, 11.63-90.24). Blunt homicide predominated amongst White females, who were substantially older than the Coloured and African subjects. A BAC greater than 0.1 g per 100 ml was found in 56% of all female homicides studied. An association was found between increasing BAC and sharp homicide amongst women. A high BAC may represent a self-destructive element in a risk-taking situation amongst female homicide victims.


[Psychiatry and psychoanalysis--Eugen Bleuler's "dementia praecox or group of schizophrenias" (1911)]. Unlike many psychiatrists around 1900, Eugen Bleuler had a friendly, even partly affirmative attitude towards psychoanalysis. In his famous book "Dementia Praecox or Group of Schizophrenias" (1911) he emphasized the influence of psychoanalysis on his theory about schizophrenia. In this article we describe Bleuler's fundamental thoughts on psychology and psychotherapy in order to look for a special disposition to accept psychoanalysis. Psychoanalysis seemed to him to be one of the most promising attempts to develop a systematic psychotherapy founded on a scientific psychology. In particular, Freud's hypothesis about the wish-fulfilling character of dreams influenced Bleuler's understanding of delusion and other symptoms of schizophrenia.


[The Leonhard classification of endogenous psychoses--initial biological findings and differential therapeutic considerations]. Current systems of classification of endogenous psychoses, the APA's Diagnostic and Statistical Manual and the International Classification of Diseases in particular, aim at a synthesis of Kraepelin's prognosis-orientated diagnostic scheme with the symptomatological approach as established by Eugen Bleuler and Kurt Schneider. The restriction to few diagnostic entities has remained unchanged to the present day and appeared at first to be confirmed and validated by modern pharmaco-psychiatry. Kraepelin's dichotomy has failed to make a major contribution to the etiologic clarification of endogenous psychoses. New diagnostic concepts beyond those currently applied in clinical psychiatry and biological psychiatric research must therefore be considered and should lead towards a more differentiated approach to psycho-pathological phenomena. Karl Leonhard's classification of endogenous psychoses which rests upon the pioneer work of Carl Wernicke and Karl Kleist is part of a tradition in psychopathology emphasizing the interface of biology and psychopathology. It might provide a basis for future empirical research in biological psychiatry and pharmacopsychiatry.


[Xanthogranuloma of the choroid plexus of the lateral ventricle (case report)]. We report on a case of a 59-year old man. During work he died suddenly and unexpectedly. Autopsy revealed myocardial infarction as cause of death. We also found symmetrical granulomatous proliferations proceeding from choroid plexuses of the lateral ventricles of the brain. Results of histological and chemical investigation proved the tumours to be xanthogranulomas. Despite their unusual size they did not cause any neurological or psychiatric symptoms. The patient had been known to have hypercholesterinemia for years.


[What are the psychiatrist's expectations from nuclear magnetic resonance tomography and spectroscopy?]. Magnetic resonance imaging (MRI) of the brain has several advantages over CAT-scans (CT). MRI will gradually replace CAT-scans for most of the diagnostic examinations in psychiatry. MRI has an advantage in detecting discrete lesions of the brain. This is demonstrated by a review of MRI studies with schizophrenic and alcohol dependent patients. Magnetic resonance spectroscopy (MRS) is suitable for the in-vivo investigation of brain functions. MRS might provide the greatest advantage for future research in psychiatry. This is demonstrated by different studies, where the concentration and the kinetics of psychopharmacological agents in the brain were measured in-vivo.


[Secretion of insulin and pancreatic polypeptide from the isolated perfused ventral and dorsal portions of the rat pancreas]. The stimulation of the secretion of insulin and pancreatic polypeptide (PP) by carbachol, three different neuropeptides (gastrin-releasing peptide (GRP), vasoactive intestinal polypeptide (VIP) and neuromedin C (NC)), and glucose was investigated using the isolated perfused ventral part of the rat pancreas, with or without atropine. We used a specific radioimmunoassay for the secretion of rat PP. In addition, we studied the secretion of insulin and PP from the dorsal part of the rat pancreas in response to GRP and carbachol. The secretion of insulin and PP in response to carbachol and GRP were dose-dependent in range of 10(-9) M to 10(-6) M. The responses of insulin and PP to carbachol (10(-7) M) were biphasic, and were abolished by atropine (10(-5) M). Those in response to GRP (10(-7) M) were transient, monophasic and not inhibited by atropine (10(-5) M). VIP (10(-7) M) stimulated PP slightly and insulin obviously. Such secretion was not influenced by atropine (10(-5) M). Neuromedin C showed a bioactivity similar to that of GRP with respect to the releases of insulin and PP. Glucose stimulated a biphasic response of insulin, but no response of PP. The relative secretion of PP from the dorsal vs. the ventral portions of the rat pancreas following stimulation by carbachol and GRP (10(-7) M) was 19% and 22%, respectively, while that of insulin was 289% and 382%, respectively. These data show that peptidergic nerves may play a role in the secretion of PP as with that of insulin.(ABSTRACT TRUNCATED AT 250 WORDS)


Exposure to hepatitis B virus in the general population of Hisayama, Japan: significance of isolated antibody to hepatitis B surface antigen in general population. Cross-sectional survey on the prevalence of hepatitis B serological markers was performed in 2,411 residents who accounted for 74.4% of the population aged 40 and over and living in Hisayama Town, Japan, in 1983. Overall prevalences were 40.7% for both anti-HBs and anti-HBc, 6.1% for isolated anti-HBs and 5.4% for isolated anti-HBc. The condition with isolated anti-HBs was different from those with isolated anti-HBc and both anti-HBc and anti-HBs as follows. The titer of anti-HBs in isolated anti-HBs positive samples was significantly lower than that in both anti-HBs and anti-HBc positive ones (46.2 +/- 5.4 vs. 83.2 +/- 2.8, mean +/- SE, p less than 0.001). The presence of isolated anti-HBs was neither significantly more frequent in males nor related to the risk of liver damages in contrast with that of anti-HBc with or without anti-HBs. These findings suggest that isolated anti-HBs pattern with the absence of anti-HBc in general population was not due to prior HBV infection, but due to natural immunization with HBsAg.


Prevalences of hepatitis B surface antigen carriers and liver damages in the general population of Hisayama, Japan. The prevalences of hepatitis B surface antigen (HBsAg) carriers and liver damages were studied in 2,411 residents aged 40 and over and living in Hisayama, Japan in 1983. Hepatitis B virus (HBV) associated markers were all measured by radioimmunoassay. HBsAg carriers were found in 2.3 per cent of the residents. Hepatitis B e antigen and antibody to hepatitis B e antigen were positive in 8.9 per cent and 80.4 per cent, respectively, of HBsAg carriers. The prevalences of liver damages in HBsAg carriers were compared with 1095 who had none of HBV markers (neither anti-HBc nor anti-HBs). The prevalences of abnormal aminotransferase level in sera were not different between HBsAg carriers and those who had none of HBV markers. A history of jaundice and/or hepatitis was evident in 32.3 per cent of male carriers and 24.0 per cent of female ones, being significantly more than those without HBV markers (13.1 per cent and 5.8 per cent, p less than 0.05 and p less than 0.005, respectively). These results indicate that, among HBsAg carriers aged 40 and over, few have active clinical signs of hepatitis, although about 20 per cent of them have histories of symptomatic hepatitis due to hepatitis B.


[Human immunoglobulin heavy-chain gene intronic enhancer segment HE2 (microB) functions as a major B-cell specific cis-element at cellular level as well as in transgenic mice]. Immunoglobulin heavy-chain (IgH) gene expression is regulated in a large part of the IgH gene intronic enhancer which is composed of certain protein-binding motifs. These motifs act as a DNA element which is important for the regulation of the IgH gene transcription. Two of these motifs, HE2 and E6 in the human IgH gene intronic enhancer, are considered to affect the B cell-specific gene expression. To examine the true function of the HE2 and E6 motifs in vivo, we established transgenic mouse lines. A 3'deletion mutant of the IgH gene intronic enhancer which includes an active HE2 motif showed almost the same degree of transcriptional activity and tissue-specificity as the whole IgH gene intronic enhancer. Still more, using the luciferase-assay, it was confirmed at cellular level that the HE2 motif plays an important role in the tissue-specific activity of the IgH gene intronic enhancer. On the other hand, the E6 motif was unable to sufficiently produce the B cell-specific expression in vivo and showed enhancer activity in some non-B cells. In transgenic mice, the IgH gene intronic enhancer functioned not only in B-lymphocytes but also in chorid plexus cells which are similar to glial cells. This suggests the existence of common gene expression control mechanisms between the immune system and the nervous system.


Removal of intrahepatic cholesterol stones by the combination of percutaneous pneumatic balloon dilatation and cholangioscopic lithotomy. A 53-year-old man presented with a common hepatic duct stone and was also found to have an incarcerated stone, which was thought to consist of cholesterol, in the intrahepatic bile duct of the right lobe. After operative choledocholithotomy, removal of the intrahepatic cholesterol stone was accomplished with percutaneous cholangioscopic lithotomy (PCL) via a choledochal drainage route after pneumatic balloon dilation of a stricture, followed by internal stenting. Although case reports of primary cholesterol hepatolithiasis with some characteristic clinical features have increased recently in Japan, the natural history and clinical course after non-surgical intervention remain uncertain because of the rarity of this disease entity and lack of long-term follow-up studies. We concluded that intrahepatic cholesterol stones can be removed with percutaneous cholangioscopic lithotomy (PCL) without hepatectomy because ductal inflammatory changes and liver parenchymal atrophy, which are indications for surgical intervention, are seldom encountered in primary cholesterol hepatolithiasis.


Primary papillary adenocarcinoma in the third portion of the duodenum. We herein report a rare case of papillary adenocarcinoma which occurred in the third portion of the duodenum. The patient was a 62-year-old Japanese male who was admitted due to vomiting and right lower abdominal pain. No abnormal findings were found in the laboratory examinations. After a diagnosis of primary duodenal carcinoma was made by radiologic, endoscopic and ultrasonographic studies, a pancreatoduodenectomy was performed. The histology of the resected specimen revealed papillary adenocarcinoma, with invasion reaching to the pancreatic body. Some characteristic features of the disease are also reviewed.


[A study on clonal elimination of auto-reactive thymocytes in bone marrow chimera mice]. Bone marrow (BM) chimera mice were established by injecting BM cells from B10 H-2 congenic or recombinant mice (Mls-1b) into lethally irradiated AKR (Mls-1a) mice in order to elucidate what type of cells were responsible for intrathymic clonal elimination of self-reactive V beta 6+T cells that are reactive to Mls-1a plus I-E products. When I-E+ mice were donors, V beta 6+ SP thymocytes were not eliminated. However, in chimeras where B10 (I-Ab, I-E-) or B10.A(4R)(I-Ak, I-E-) mice were donors, variable proportions of V beta 6+ SP cells were observed. These differences appeared to be attributable to the difference in affinity between class II antigens expressed on BM derived cells and Mls-1a on recipient cells. When AKR mice were reconstituted with BM cells from both B10 and AKR mice, V beta 6+ SP thymocytes were eliminated according to frequencies of the AKR derived cells. These findings collectively indicate that the BM derived thymic stromal cells are essential for the clonal elimination of V beta 6+ cells. However, in GVHR chimeras prepared by injecting of both BM cells and splenic T cells from the same donor mice, V beta 6+ cells were not eliminated at all any periods after BMT. Significantly high number of V beta 6+ SP thymocytes seen 1 week after BMT were shown to be the splenic T cellsinjected with BM cells or their descendants. By contrast, the proportions of the V beta 6+ SP cells in GVHR chimeras 5 weeks after BMT fell within the same range as those of normal donor mice. These V beta 6+ cells were derived from BM precursors. These results reveal that acute GVHR in the thymus results in abrogation of clonal elimination of self reactive T cells in the thymus.


[Quantitative analysis of DNA synthesis and cell condensation in the oligodactylic rat hand plate]. In order to clarify the teratogenic mechanism of oligodactyly which occurs during the hand plate development, pre-axial oligodactyly was induced in rat by myleran treatment on day 12.0 of gestation. This animal model was consecutively analyzed in the process of the hand plate development. DNA synthesis was quantified by a BrdU/ant-BrdU immuno histochemical study, and cell damage was assessed on the number of reactive macrophages. From these experiments, the following results were obtained. 1) In the oligodactylic rat embryos, the number of DNA-synthesizing cells was uniformly reduced in mesenchyme compared to that of the normal embryos on day 13.0. 2) In the pre-axial areas of the diseased hand plate, the number of DNA-synthesizing cells in ectoderm decreased significantly on day 13.5, and the reduction of DNA-synthesis was observed in all areas on day 14.0. 3) The damaged cells in the diseased hand plate were distributed uniformly, regardless of the lack of finger. Thus, it could be speculated that in this myleran induced oligodactylic model both the deficit of mesenchymal cells and the lack of ectoderm derived induction in the fingers were closely implicated in the development of oligodactyly.


[Effects of nonthermal stresses on brown adipose tissue thermogenesis]. Repetitive intermittent immobilization stress has been shown to induce an improved cold tolerance through an enhanced capacity of nonshivering thermogenesis (NST). In the present study, effects of immobilization (3 hrs daily for 4-5 weeks), exercise training (running with treadmill 30 min daily, 30 m/min under 8 degrees inclination for 4-5 weeks) and chronic corticosterone treatment (subcutaneous injection at a dose of 0.3 mg/100 g for 4-5 weeks) were investigated on in vitro and/or in vivo thermogenesis of rat interscapular brown adipose tissue (BAT). BAT thermogenesis in vitro was measured in the minced tissue blocks in Krebs-Ringer phosphate buffer using a Clark type oxygen electrode. DNA content per whole BAT pad was greater in the stressed rats, while it was not affected by exercise training and corticosterone. Noradrenaline-and glucagon-stimulated oxygen consumptions were significantly greater in the stressed rats, while significantly smaller in the trained rats as compared with respective controls. Corticosterone treatment failed to affect those values in terms with both per mg tissue and per whole tissue pad, except the less noradrenaline-stimulated oxygen consumption in terms with per mg tissue and DNA. In vivo thermogenesis was assessed by the changes of temperatures in colon (Tcol), BAT (TBAT) and tail skin (Tsk) induced by noradrenaline or glucagon infusion under anesthesia Noradrenaline and glucagon increased the TBAT and the extent of increase was greater in the stressed rats. These results indicated: 1. Repetitive immobilization stress induces the tissue hyperplasia and enhances thermogenic activity of BAT. 2. Exercise training suppresses BAT thermogenesis. 3. Chronic corticosterone administration does not affect BAT thermogenesis. It may be concluded that the enhancing or suppressing effect of nonthermal stress on BAT thermogenesis is due to other factor(s) than corticosterone.


[Spinal neurons activated by ventral root afferents in rats: their physiological properties, distribution in the spinal cord and rostral projection]. The present experiment was designed to locate spinal neurons which are activated by afferent fibers in the ventral roots, and once found, to study their receptive field and rostral projection. After dorsal roots L1 approximately S4 on the left side were cut under a binocular microscope, stimulus was applied to ventral roots and extracellular responses were recorded using a glass microelectrode filled with fast green FCF. Sixty-four neurons were activated by stimulating ventral roots at 1.2-27.8 x T of the lowest threshold fibers of the ventral root. Six neurons were classified as wide dynamic neurons and 29 as high threshold neurons according to their response patterns to the periphery. Twelve neurons out of 29 were unresponsive to cutaneous stimulation, and located lamina V, suggesting that they innervate visceral organs. Thirty-five neurons (54.6%) were nociceptive nerons, most of which were located in laminae V and VI. In 6 neurons out of 35 nociceptive neurons (17.1%), rostral projection was confirmed by the collision block method and the ability to respond to high frequency stimuli with constant latency. Our results suggest that some spinal neurons activated primary affernt fibers in ventral roots stimulation are nociceptive and some of them are rostral projecting neurons which are antidromically activated ventral quadrants of the spinal cord.


[Expression of granulocyte-macrophage colony-stimulating factor mRNA by inflammatory cells in the sarcoid lung and its clinical significance]. T lymphocytes and alveolar macrophages accumulating in the lower respiratory tract of patients with pulmonary sarcoidosis are known to be activated to produce several cytokines, presumably leading to granuloma formation within the lung. I hypothesized that these cells produce colony-stimulating factors (CSF), which have been shown to affect the proliferation and function of monocyte/macrophage-lineage cells. To test this hypothesis, I tried to detect mRNA encoding CSFs in cells obtained by bronchoalveolar lavage using a reverse transcription-polymerase chain reaction. Macrophage-CSF mRNA was detected in all subjects examined and interleukin 3 mRNA in none. Granulocyte-macrophage CSF (GM-CSF) mRNA was detected in 15 of 20 patients with pulmonary sarcoidosis, whereas it was detected in none of the farmer's lung disease patients and normal controls. The sarcoid patients whose BAL cells expressed GM-CSF mRNA had more active disease than those patients whose BAL cells did not, as judged from clinical and laboratory findings. These results indicate that GM-CSF produced by the inflammatory cells plays a substantial role in the formation or maintenance of the sarcoid lesion.


Functional and phenotypic analyses of thymic low density adherent cells from murine bone marrow chimeras--influence on thymocyte differentiation. Previous studies from this laboratory demonstrated a malfunction of the thymus of C3H mice to induce normal level of thymocyte differentiation. Thymocytes developed in the C3H thymus showed depressed proliferative responses to stimulation with anti-CD3 antibody compared with those developed in the other strains. This study was conducted to analyze immunological functions of the thymic stromal cell population in the C3H mice. Using allogeneic bone marrow (BM) chimeras established by reciprocal combination of AKR and C3H mice as donor or recipient, antigen presenting cell (APC) function of low density adherent cells (LDAC) in the thymus was analyzed. The thymic LDAC from C3H mice or [AKR----C3H] BM chimeras where AKR were BM donors and C3H were recipients contained high proportion of Mac-1+ cells as compared to AKR mice or [C3H----AKR] chimeras. The proportion of Mac-1+ cells paralleled the IL-1 secreting ability of the LDAC. Thus, the higher proportion of Mac-1+ cell in the thymus may be responsible for low accessory function observed in C3H thymuses. However, when APC function was analyzed using various T cell hybridomas or a T cell line, the APC functions did not necessarily correlate to the proportions of Mac-1+ cells and amounts of IL-1 produced by the LDAC. When proliferative responses of thymocytes to anti-CD3 stimulation were analyzed in the presence of prostaglandins, PGE-2 inhibited more profoundly the responses of [AKR----C3H] and normal C3H mice than those of [C3H----AKR] and normal AKR mice. Furthermore, a prostaglandin inhibitor, indomethacin, reversed the depressed responses of the former thymocytes which had developed in the C3H thymus. These findings suggest that the hyporesponsiveness of thymocytes from [AKR----C3H] chimeras to anti-CD3 stimulation may be attributable to their increased sensitivity to prostaglandin produced by LDAC.


[A structure of ordering and display system by adopting computer for receipt in a medical clinic]. Online and display system for the result of laboratory examination were constructed by adopting a computer for receipt. The computer for receipt was expanded to a new schedule file and a management schedule file. Consequently, reservation system of medical clinic was made and check seats of order decreased in number. It was considered that patients can understand conditions of their illness and results of laboratory examination very easily with this system.


Annual survey: executive compensation under fire. Compensation of health care executives has continued to grow, largely pushed along by such factors as continuing turnover in top management positions in hospitals, according to the 1992 Hay hospital management/professional survey of compensation and benefits data from over 1,200 U.S. hospitals. And although pay increases are moving up faster among top hospital executives than in industry, overall hospital executive pay still lags. In addition, survey results look at such issues as linking executive pay to various measures of performance.


Survey: many CEOs overlook PR staff's role in strategic planning. Many CEOs say public relations is vital to their hospital's success, but don't give their PR officers a strategic role, a recent survey shows. CEOs with narrow views are "losing a great opportunity," says Richard Norling, president and CEO of Fairview Hospital and Healthcare Services, Minneapolis.


Molecular screening and fetal diagnosis of beta-thalassemia in the Italian population. This paper reports our experience of molecular screening and fetal diagnosis of beta-thalassemia in 457 at risk couples of Italian descent. Molecular screening was carried out by dot blot analysis on amplified DNA with oligonucleotide probes complementary to the eight most common mutations in Italians [beta zero 39 (C----T); beta zero 6 (-A); beta+ -87 (C----G); beta+ IVSI nt 110 (G----A); beta zero IVSI nt 1 (G----A); beta+ IVSI nt 6 (T----C); beta zero IVSII nt 1 (G----A); beta+ IVSII nt 745 (C----G)]. By using this approach, we have been able to define the mutation in 92.8% of cases. The rest (all but four) were defined by direct sequencing and this led to the detection of nine rare mutations [beta zero 76 (-C); beta+ IVSI nt 5 (G----A); beta+ IVSI nt 5 (G----C); beta+ IVSI -1 (cod 30) (G----C); beta+ -87 (C----T), beta zero -290 bp del.; beta+ -101 (C----T)], and to the characterization of a novel mutation consisting of the deletion of the G at the invariant AG of the IVSII splice acceptor site of the beta-globin gene (beta IVSII nt 850 -1 bp). In the remaining four cases, the beta-globin gene showed entirely normal sequences and the beta-globin gene cluster was intact, as indicated by Southern blot analysis. Fetal diagnosis was carried out by dot blot analysis with the oligonucleotide probes defined in the parents. The procedure is simple and reliable, and the results can be obtained within 1 week of sampling. No misdiagnosis has so far occurred. The results indicate that fetal diagnosis of beta-thalassemia by DNA analysis may be obtained in practically all cases (even in a population showing marked heterogeneity of beta-thalassemia) by the combination of dot blot analysis for detecting common mutations, and direct sequencing for defining those that are uncommon.


The majority of the marker chromosomes in Japanese patients with stigmata of Turner syndrome are derived from Y chromosomes. DNA analyses of 41 individuals with stigmata of Turner syndrome and a 45,X/46,X+mar or 46,X+mar karyotype were carried out. Southern-blot analysis employing 17 Y-specific probes were used to determine whether the marker chromosome was Y-chromosomal in origin. Of the 41 DNA samples from these patients, 23 contained detectable Y-chromosomal DNA. Points of chromosome breakage were distributed over the entire length of the Y long arm. Three individuals, who carry different portions of the Y chromosome, had developed gonadoblastoma. GBY (the gonadoblastoma locus on the Y chromosome) is mapped proximal to DYS132, midway between the 13 Yq loci that we have studied. We also used a polymerase chain reaction technique that could detect 7 loci over the length of the Y chromosome. This technique may be useful for the rapid assessment of marker chromosomes, especially for evaluating the risk of gonadoblastoma.


No evidence for linkage of familial hypertrophic cardiomyopathy and chromosome 14q1 locus D14S26 in a Chinese family: evidence for genetic heterogeneity. To understand the molecular basis of familial hypertrophic cardiomyopathy (FHC) in the Chinese population, a family with FHC was investigated. Nineteen family members who were 16 years of age or older were examined by M-mode or two-dimensional echocardiography. Eight members were diagnosed to be affected echocardiographically or clinically. Lymphocytes isolated from 20 family members were successfully transformed into permanent lymphoblastoid cell lines by Epstein-Barr virus. Three genomic DNA probes (CRI-L436, CRI-L329, and pSC14) that were derived from chromosome 14q1 loci and demonstrated to be linked closely to FHC were used to probe this family. Using the techniques of restriction fragment length polymorphism (RFLP) and linkage analysis, the probe CRI-L436, which recognized locus D14S26, was found informative in this family. The lod scores were -2.0 at theta = 0.025 and -1.49 at theta = 0.05. Thus, there was no evidence of linkage between the locus D14S26 and the gene for FHC in the pedigree studied. In addition, polymerase chain reaction (PCR) amplification did not indicate a mutation on exon 13 of the beta cardiac myosin heavy chain gene as previously reported. Our data suggest that FHC is a genetically heterogeneous disease.


Molecular cytogenetic analysis of a familial 8p23.1 deletion associated with minimal dysmorphic features, seizures, and mild mental retardation. We report a family in which three members presented with minimal phenotypic abnormalities, normal intelligence to mild mental retardation, and a cytogenetically terminal chromosome deletion at band 8p23.1 Whole chromosomal painting with a chromosome 8-specific DNA library confirmed this familial chromosome abnormality as a deletion, while fluorescence in situ hybridization with telomeric probes demonstrated the presence of telomeres at the deletion site. Coagulation studies were additionally performed to evaluate the purported location of the coagulation factor VII regulator gene at 8p23.1. A review of the clinical findings of seven cases of del(8)(p23.1) is presented.


Identification of crossovers in Wilson disease families as reference points for a genetic localization of the gene. Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. A minimum recombinant analysis using D13S22, ESD, RB1, D13S31, D13S55, D13S26, D13S39, and D13S12, all localized at 13q14-q22, has been carried out in 20 WD families of Northwest-European origin. No inconsistencies have been observed with respect to locus order or location of the WD locus (WND) compared with previous linkage studies. D13S31 was mapped as the closest marker proximal to WND, whereas D13S55 and D13S26 were mapped as the closest markers distal to WND. We have identified a crossover between WND and D13S31 in one family and a crossover between WND and D13S55 in another. These crossover sites can be used as reference points for new chromosome 13q14-q21 markers, and are therefore important for a more accurate mapping of the WD locus.


Fragile site (16) (q22). III. Segregation analysis. The rare autosomal fragile site, fra (16) (q22), is the most common of all rare autosomal fragile sites and has a heterozygote frequency of about 5%. Evidence for it was found following the segregation expected from a simple codominant trait with complete penetrance; this is in contrast to a variety of other rare autosomal fragile sites. Based on the analysis of 12 families in which fra (16) (q22) is segregating, we found that, whereas complete penetrance could be confirmed, the transmitting parent was significantly more likely to be of the female sex. On the other hand, there was no evidence for preferential transmission to offspring of either sex.


Physical fine mapping of genes underlying X-linked deafness and non fra (X)-X-linked mental retardation at Xq21. Linkage studies and cytogenetically visible deletions associated with nonspecific X-linked mental retardation (XLMR) and a specific form of deafness (DFN3) have indicated that the genes responsible for these disorders are located at Xq21. Using DNA probes from this region, we have studied several overlapping deletions spanning different parts of Xq21. This has enabled us to assign the DFN3 gene and a gene for nonspecific XLMR to an interval that encompasses the locus DXS232 and that is flanked by DXS26 and DXS121.


Beta-globin gene cluster haplotypes in Yanomama Indians from the Amazon region of Brazil. Six polymorphic restriction enzyme sites in the beta-globin gene cluster were investigated in Yanomama Indians from the Amazon region of Brazil, using the polymerase chain reaction (PCR) technique. Four haplotypes were identified; the haplotype frequency distribution is similar to those reported for Polynesians, Micronesians and most Asian populations.


Ornithine transcarbamylase (OTC) deficiency in a female patient with a de nova deletion of the paternal X chromosome. A girl with ornithine transcarbamylase (OTC) deficiency was investigated for molecular and cytogenetic abnormalities that might explain this phenotype. Analysis with polymorphic DNA markers indicated that the patient did not inherit paternal alleles of the OTC locus, but that she did inherit the proximal locus DXS7 and the long arm of chromosome X. High-resolution cytogenetic analysis of the patient indicated a deletion of Xp11.4-p21, whereas both parents had normal karyotypes. Since the mother might be heterozygous according to biochemical tests, a second mutation within the maternal OTC gene cannot be excluded.


A dominant mutation in the COL1A1 gene that substitutes glycine for valine causes recurrent lethal osteogenesis imperfecta. Type I collagen chains of a proband from a family with recurrent lethal osteogenesis imperfecta (OI) migrated as a doublet when submitted to gel electrophoresis. Cyanogen bromide (CNBr) peptide mapping demonstrated that the post-translational over-modifications were initiated in alpha 1ICB7. Chemical cleavage of cDNA-RNA heteroduplexes identified a mismatch in the alpha 1I cDNA; this mismatch was subsequently confirmed by sequencing a 249-bp fragment amplified by the polymerase chain reaction. A G to T transition in the second base of the first codon of exon 41 resulted in the substitution of glycine 802 by valine. This mutation impaired collagen secretion by dermal fibroblasts. The over-modified chains were retained intracellularly and melted at a lower temperature than normal chains. Collagen molecules synthesized by parental fibroblasts had a normal electrophoretic mobility, but hybridization of genomic DNA with allele-specific oligonucleotides revealed the presence of the mutant allele in the mother's leukocytes. The mutation was not detected in her fibroblasts consistent with the protein data. These results support the hypothesis that somatic and germ-line mosaicism in the phenotypically normal mother explain the recurrence of OI.


Molecular studies of parental origin and mosaicism in 45,X conceptuses. The present report summarizes molecular studies of parental origin and sex chromosome mosaicism in forty-one 45,X conceptuses, consisting of 29 spontaneous abortions and 12 liveborn individuals with Turner syndrome. Our studies indicate that most 45,X conceptuses have a single, maternally derived X chromosome, regardless of whether the conceptus is liveborn or spontaneously aborted. In studies of mosaicism, our identification of X- and Y-chromosome mosaics among 45,X spontaneous abortions indicates that mosaicism does not ensure survival to term of 45,X fetuses. However, the incidence of sex chromosome mosaicism is substantially higher in liveborn than in aborted 45,X conceptuses, indicating that the presence of a second cell line increases the likelihood of survival to term.


Crossed renal ectopia with pelvic lipomatosis: a new syndrome involving chromosome 1. An 18-year-old male patient is described who possesses both kidneys on one side (crossed renal ectopia), together with pelvic lipomatosis. In general, lipomatosis is benign, but here the tissue shows the rare feature of malignancy. Chromosomally, the patient is typically characterised by somatic translocations involving chromosome 1 (37% metaphases); these almost always exhibit a whole chromosome translocation with chromosome 6 (35%), although involvement of chromosome 1 with chromosome 8 is present in 2% of metaphases. Other chromosomal features encountered in Giemsa-stained and G-banded preparations from lymphocyte cultures include the prevalence of a small Y chromosome in 25% of metaphases, the presence of marker dots in 20%, and acrocentric associations in 8%-10% of metaphases. However, more than 50% of metaphases have a normal 46XY karyotype with a normal-sized Y-chromosome. Crossed renal ectopia with pelvic lipomatosis can therefore be assigned to a new syndrome characterised by a whole-chromosome translocation involving chromosomes 1 and 6.


Geographic distribution and genealogy of mutation 207 of the lipoprotein lipase gene in the French Canadian population of Québec. Mutations in the lipoprotein lipase (LPL) gene, leading to partial or total inactivation of the enzyme, result in a hereditary clinical syndrome called familial LPL deficiency. The French Canadian population, which is primarily and historically located in the province of Québec, has the highest worldwide frequency of LPL-deficient patients. We have analyzed the prevalence, spatial distribution, and genealogy in the Québec population of a LPL gene mutation, M-207 (P207L in conventional notation), which changes the amino acid proline to leucine in position 207 of the LPL protein and inactivates the enzyme. Our results show that M-207 is the most prevalent LPL gene mutation among French Canadians and accounts for the largest proportion of LPL-deficient patients in this population. Genealogical reconstruction of French Canadian LPL-deficient patients point to 16 founders of M-207, all of whom migrated to Québec in the early seventeenth century from the north-western part of France, especially from the region of Perche. Most of the carriers of M-207 are, at present, found in Charlevoix, Saguenay-Lac-St-Jean regions of eastern Québec. On the basis of the number of homozygote M-207 LPL-deficient patients so far identified, we estimate that there are at least 31,000 carriers of this mutation in the province of Québec. This constitutes a large pool of individuals at risk for atherosclerosis and other lipid-related diseases, since LPL deficiency is considered to be a significant contributing factor in the etiology and development of these diseases.


Two new human hemoglobin variants caused by unusual mutational events: Hb Zaïre contains a five residue repetition within the alpha-chain and Hb Duino has two residues substituted in the beta-chain. With rare exceptions, the more than 600 human hemoglobin variants described are caused by a single point mutation. Other abnormal features, such as unequal crossing-over, frameshift mutagenesis or double mutations in the same polypeptide chain, have seldom been encountered. We report two new variants caused by such rare mutational events. Hb Zaïre [alpha 116(GH4)-His-Leu-Pro-Ala-Glu-117 (GH5)] is the second example in which a short amino acid sequence is inserted within the alpha-chain. This abnormal hemoglobin results from a tandem repetition of 5 amino-acid residues, from sequence 112 through 116, at the end of the GH corner. Hb Duino is an unstable hemoglobin. It presents within the same beta-chain, the association of two rare point mutations; these substitutions are those found in Hb Newcastle [beta 92(F8)His----Pro] and in Hb Camperdown [beta 104(G6)Arg----Ser]. Family studies demonstrated that the Hb Newcastle abnormality was a de novo mutation of a gene already carrying the Hb Camperdown substitution.


Assignment of the gene for human spasmolytic protein (hSP/SML1) to chromosome 21. A human cDNA corresponding to the porcine pancreatic spasmolytic protein (PSP) was isolated, and the recombinant clone was originally termed hSP for human spasmolytic protein. Later, the term SML1 for spasmolysin was suggested for the human gene. This protein shows a remarkable sequence homology to pS2, a protein coded by an estrogen-induced gene isolated from the breast carcinoma cell line MCF-7. Although, at the DNA level, the gene sequences pS2 and hSP/SML1 display insufficient homology for cross-hybridization, their expression in tumor cells occurs with remarkable coordination. The human pS2 gene sequence has been assigned to chromosome 21, and we have therefore attempted to map the hSP/SML1 gene by using cDNA and Southern blotting of genomic DNAs from a panel of human-rodent somatic cell hybrids carrying different complements of human chromosomes. Interestingly, the hSP/SML1 gene is also localized on chromosome 21.


A novel homozygous missense mutation in the protein C (PROC) gene causing recurrent venous thrombosis. A novel homozygous CCC----CTC (Pro 247----Leu) substitution was detected in the protein C genes of a patient, born to consanguineous parents, with inherited type 1 protein C deficiency and recurrent venous thrombosis. Since one of four heterozygous relatives was also clinically affected, the condition appears to be inherited as an incompletely recessive trait in this family.


Two different missense mutations at Arg 178 of the protein C (PROC) gene causing recurrent venous thrombosis. Non-identical missense mutations were identified at Arg 178 in the protein C genes of two patients with heterozygous type 1 protein C deficiency and recurrent venous thrombosis.


A polymorphic complex dinucleotide repeat at the telomeric D8S7 locus. Two cosmids isolated from a flow-sorted chromosome 8 library by hybridization with pSW50 (D8S7) were screened for GT microsatellite sequences. Both contained a positive 900-bp Hind III-Xba I fragment. Sequencing revealed a complex dinucleotide repeat. Flanking oligonucleotide primers were synthesized, and the polymerase chain reaction products produced by these primers were typed within the CEPH panel of families. A two-allele polymorphism was identified that is in linkage disequilibrium with a previously described insertion-deletion polymorphism at this locus.


BF and C3 genetic polymorphisms in Kaingang Indians from southern Brazil. The distribution of C3 and BF variants was determined in a sample of 239 Kaingang Indians. The corresponding gene frequencies were as follows: BF*S = 0.9393, BF*F = 0.0356, BF*S05 = 0.0251, C3*S = 0.9769, C3*F = 0.0231. The presence of the BF*S05 allele, which has previously been found only in a Brazilian population, suggests that this allele originated in Amerindians. The comparatively low degree of polymorphism with high frequencies of BF*S and C3*S is in accordance with the relatedness of the Kaingang with other Amerindians, Eskimos and Asian populations.


Limited genetic diversity in polynesians reflected in the highly polymorphic 3'HVR alpha-globin gene marker. Polynesians from five distinct island groups were studied with DNA probe alpha 3'HVR, a highly polymorphic VNTR (variable number of tandem repeats) minisatellite region associated with the alpha-globin gene cluster. Results showed a paucity of genetic heterozygosity together with clustering of alpha 3'HVR alleles with alpha-globin DNA haplotypes and alpha-globin gene rearrangements. This restricted diversity is consistent with population bottlenecks in the colonization of Polynesia.


Complement genetic markers in schizophrenia: C3, BF and C6 polymorphisms. Polymorphic variants of C3, BF and C6 complement factors have been investigated in schizophrenic patients subdivided according to the existence or not of a family history of both schizophrenia and other psychiatric disorders. To analyze the contingency tables, besides the usual methods, log-linear models have been fitted. Significant associations have been found in the C3 system, with a decrease of C3*F in patients (contradicting previous findings), and in the BF system, with a decrease of FS phenotype among patients (confirming some previous results). No association has been found for the C6 polymorphism (in accordance to previous results). Therefore, the present findings only partially confirm previous results and do not clarify the relationship between complement genetic markers and schizophrenia, stressing some statistical difficulties.


Serum protein groups (Hp, GC, C3) in patients with gastric carcinoma. The phenotypes and gene frequencies of 3 serum protein systems (Hp, GC and C3) were studied in 114 consecutive patients from all over Greece with gastric carcinoma. Healthy Greeks studied previously in our Department served as controls. No significant differences were found between patients and controls concerning Hp. Significant differences were found in the GC and C3 systems; GC 2-1 and C3F phenotype as well as C3*F gene frequencies were significantly higher in patients than in controls.


Complementation in Zellweger syndrome: biochemical analysis of newly generated peroxisomes. The Zellweger syndrome is characterized by a defect which results in the abnormal biogenesis of peroxisomes. As a consequence, metabolic activities associated with peroxisomes such as the oxidation of very long chain fatty acids, the synthesis of plasmalogens, and the catabolism of phytanic and pipecolic acids are impaired. Since this disorder is genetically heterogeneous and several complementation groups are known, we were able to study the normalization of peroxisomal activity during the process of complementation. The restoration of catalase and dihydroxyacetone phosphate acyltransferase activities peaked within 3-4 days postfusion while the oxidation of lignoceric acid was much delayed (7-8 days). Electron microscopy indicated that by 6 days following hybridization, peroxisome structure and density in heterokaryons was comparable to normal control cells. The heterogenous biochemical response during peroxisome normalization could be due to several factors including a possible requirement for restoration of peroxisomal structural integrity for maximum activation of certain metabolic pathways.


Hereditary nonspherocytic hemolytic anemia due to pyruvate kinase deficiency: a prevalence study in Quebec (Canada). Fifty-eight cases of hereditary nonspherocytic hemolytic anemia due to pyruvate kinase deficiency were collected from both laboratories that have performed the assay since 1972 and from attending physicians in the province of Quebec. Using the postal addresses of the probands, a prevalence map was constructed according to the administrative regions of the province of Quebec. The prevalence was found to be higher in eastern Quebec (1/81,838) than in western Quebec (1/139,086). Fifty probands were of French Canadian origin whereas the remaining 6 were recent immigrants.


The first case of familial amyloidotic polyneuropathy (FAP Met30) in the Finnish population. Finnish hereditary amyloidosis-Meretoja (FAP type 4) is the predominating type of hereditary amyloidosis in the Finnish population, found in more than 200 individuals. We present a Finnish family with familial amyloidotic polyneuropathy (FAP Met30), a type of amyloidosis hitherto not described in the Finnish population. Genealogical tracing back to the 18th century revealed no connections with Swedish FAP families, but introduction from Sweden is the most probable origin of the FAP Met30 gene.


A rapid method for diagnosis of the Lebanese allele in the low-density lipoprotein receptor gene. The Lebanese allele in the low-density lipoprotein receptor gene is one of the alleles which results in the disease familial hypercholesterolemia. We describe a rapid method for detection of the Lebanese allele, using the polymerase chain reaction to amplify part of exon 13, intron 14 and all of exon 14. The amplified DNA is then digested with HinfI which distinguishes between the normal and Lebanese alleles. A previously unidentified HinfI site is described in the intron. HinfI fragments are separated using polyacrylamide gel electrophoresis, and visualized by ethidium bromide staining.


Uniparental origin of sex chromosome polysomies. The parental origin of the additional sex chromosomes in 8 cases with high-order sex chromosome polysomies was determined using DNA polymorphisms. The additional sex chromosomes were paternally derived in 3 48,XXYY cases, and maternal in origin in 1 48,XXXY case and 4 49,XXXXY cases. Thus, all extra chromosomes, within a particular patient, were always derived from only one parent. Their most likely origin was successive nondisjunction at the first and second meiotic division in one germ cell. The mechanism involved remains unclear, but appears to be independent of parental ages.


GC and C3 serum groups in peptic ulcer.

GC and C3 serum groups in peptic ulcer. The phenotypes and gene frequencies of two serum protein systems (GC, C3) were studied in 238 consecutive patients with peptic ulcer from all over Greece. 173 patients had duodenal ulcer and 65 had gastric ulcer. Healthy Greeks studied previously in our department served as controls. No significant differences were found between the studied groups and the controls as far as GC was concerned. However, significant differences emerged in C3; the C3*F gene was almost twice more common and the C3F phenotype almost three times more frequent in patients than in controls.


Interaction between ABO and haptoglobin systems in a Bengalee population. Blood samples from 2,232 individuals of a Bengalee Caste Hindu population were investigated in an attempt to confirm the association between the ABO and haptoglobin (HP) systems previously found in populations of European origin. Indians differ from Europeans in having lower HP*1 and higher ABO*B frequencies. In spite of this, as in previous studies, a weak HP/ABO association was found with a significantly lower HP*1 allele frequency in blood group O versus other ABO groups.


Haplotype analysis and polymorphic frequency data at two factor IX loci (F9-192 and F9-Hha1) in a Chinese population. Gene frequencies and haplotypes for two factor IX markers, F9-192 and F9-Hha1, were determined in a Chinese population. The frequencies for F9-192 are 0.86 for A and 0.14 for G. For F9-Hha1, the gene frequencies are 0.69 for - and 0.31 for +. Haplotype analysis revealed that the two polymorphic sites are in linkage equilibrium and, thus, are useful markers for genetic counseling of Asian families in which hemophilia B exists.


PI*E Tripoli: a new allele in the alpha-1-antitrypsin system. Genetic variants of the human serum alpha 1-antitrypsin (PI system) were analyzed in a population sample of 110 unrelated Libyans. Four common PI M variants and 3 rare ones, including a new anodal variant designated PI E Tripoli (PI ET) were identified. The estimated allele frequencies were: PI*M1 = 0.623; PI*M2 = 0.205; PI*M3 = 0.132; PI*M4 = 0.018; PI*ET = 0.005; PI*S = 0.005, and PI*T = 0.014.


Genetic markers in alcoholic liver cirrhosis. 11 genetic markers were typed in 157 individuals suffering from alcoholic cirrhosis, and compared with a random sample of healthy individuals. No significant differences were found for transferrin, specific group component, orosomucoid, esterase D, phosphogluconate dehydrogenase and adenylate kinase. Strong associations between alcoholic cirrhosis and alpha-1-antitrypsin PI*Z allele, haptoglobin HP*1 allele and acid phosphatase ACP AC phenotype were observed. The biological significance of these associations and their relationships with the development of alcoholic cirrhosis are also discussed.


The structure of the Polar Eskimo genealogy. The structure of the genealogy of the Polar Eskimos from the Thule District of North Greenland is studied by a variety of computational and graphical means, some of them novel. It is shown that although the level of inbreeding in the population is low, the genealogy is intricately connected, as if conforming to the requirement that spouses are as unrelated as is possible in a small population.


Distribution of F13A phenotypes in Spain: a particularly high frequency of the F13A*2 allele. The distribution of the phenotypes for coagulation factor XIIIA subunit (F13A) of autochthonous individuals from the following five Spanish populations was studied: Galicia, Castilla-León, Castilla-La Mancha, Extremadura and Western Andalusia. The frequency values obtained for F13A*2 ranged from 0.248 to 0.311. To date, these values are the highest recorded in the world.


In vitro characterization of repair synthesis initiated by T4 endonuclease V on a synthetic DNA substrate. The size of the repair patch produced by E. coli DNA polymerase (Pol I) following the removal of a pyrimidine dimer from DNA in response to the nicking activity of T4 endonuclease (T4 endo V) was determined. A 48-bp DNA containing a pyrimidine dimer at a defined location was labelled in the damaged strand and incubated with T4 endo V and E. coli endonuclease IV. Subsequently, DNA synthesis by DNA Pol I was carried out in the presence of four dNTPs, ATP and DNA ligase. Analysis of the reaction products on a sequencing gel revealed a ladder of only 4-oligonucleotides, 1-4 nucleotides greater in length than the fragment generated by the combined nicking activities of T4 endo V and E. coli endonuclease IV. Thus we conclude that the in vitro repair patch size of T4 endo V is 4 nucleotides and that in some cases the repaired DNA is not ligated.


Stereochemical aspects of interaction of DNA binding domain of human progesterone receptor with d(AGGTCATGCT)2. Structures of (i) 66 amino-acid fragment (residues 567-633) from DNA binding domain of human progesterone receptor (hPR), (ii) a ten base pair DNA sequence d(AGGTCATGCT)2 from hormone responsive element (HRE) and (iii) a complex of these two are optimised by computer modelling and molecular mechanics technique using extensive steric constraints from secondary structure predictions, comparison with the structures of known metalloproteins, geometric constraints imposed by tetrahedral coordination with the zinc ion and comparison with structures of DNA binding domains of human glucocorticoid and estrogen receptors (hGR and hER). Structure of the complex was obtained using genetic modification data on steroid receptors and general consensus about protein-DNA interaction. DNA is in distorted B conformation. Sequence dependent as well as protein-induced conformation changes are noticed. There is change in propeller twist, buckle and angle between glycosyl bonds. However, H-bonding network is preserved. The complex is stabilized with eighteen hydrogen-bonds, mainly between peptide side-chains and backbone phosphate. There are five specific H-bonds between basic amino acid side chains, Lys 22, Lys 26 and Arg 27, and DNA bases, A1, G3, G16 and A17. Gly 19, Ser 20 and Val 23 are in close proximity of DNA.


Characterization of concanavalin A-binding neuronal and glial surface glycoproteins from human foetal brain. Neuronal and glial surface glycoproteins have been isolated from human foetal brains by affinity chromatography on 8 M urea or 6 M guanidine-treated Con A-Sepharose 4B at 4 degrees C and three groups of glycoproteins of molecular mass 65-73 kDa, 52-63 kDa and 43-48 kDa have been identified on SDS/PAGE. These glycoproteins exhibited anomalous behaviour on SDS/PAGE, indicating the existence of a gradation of mutually interconvertible protein-SDS aggregates in dynamic equilibrium with one another. Deglycosylation and deacylation did not alter the SDS/PAGE multiple band pattern. Purified glycoproteins contained 160 +/- 90 micrograms carbohydrate/mg protein, and a sialic acid content of 25 +/- 5 nmole/mg protein. The N-terminals were blocked. The glycoproteins moved preferentially on acid/urea/PAGE. Sepharose 6B gel filtration in the absence of lipid and detergents resolved the glycoproteins into an excluded peak I and a low molecular mass peak II. Peaks I and II were non-interconvertible on Sepharose 6B gel filtration or on reversed phase HPLC in an isopropanol/water/TFA gradient system. Both peaks rendered a single fast moving band of identical mobility on acid/urea/PAGE, suggesting that peak I was possibly a micellar aggregate of the monomeric peak II. The glycoproteins were refractory to digestion by trypsin or pronase and reacted identically towards various lectins.(ABSTRACT TRUNCATED AT 250 WORDS)


Lectin binding to complex carbohydrate at the interface: a study by resonance energy transfer. The binding affinity of the oligosaccharide moiety of a neutral glycosphingolipid, asialoGM1, towards Ricinus communis agglutinin (RCAI) was determined for the first time by fluorescence resonance energy transfer (RET). The asialoGM1 was incorporated into a phospholipid (DMPC) vesicle doped with dansylated DPPE and then titrated with an increasing amount of the galactose specific RCAI. The efficiency of RET was determined by a saturable increase in the quenching of 'donor' fluorescence, i.e. the 'trp' residue of RCAI, due to the energy transfer from the 'acceptor' dansyl group on the surface of the vesicle. The apparent binding constant was found to be in the range of 10(5)-10(6) M-1 at 27 degrees C.


Rapid isolation of human plasma anti-alpha-galactoside antibody using sugar-specific binding to guar galactomannan or agarose. A method of purifying the naturally occurring antibody to alpha-galactoside moiety (anti-alpha-Gal) in human plasma by a single-step affinity chromatography on cross-linked guar galactomannan (CLGG) or agarose (Sepharose 4B) is described. IgG nature of the two preparations, as revealed by agar gel diffusion, as well as their preference for alpha-anomer of galactose, as revealed in inhibition of their agglutination of trypsinized rabbit erythrocytes by sugars, identified them with anti-alpha-Gal. The antibody binding capacity of Sepharose 4B was only a third of that of CLGG. Both gels showed similar dependence on ionic strength for binding. The pH optimum for binding of anti-alpha-Gal to CLGG was 8.0. Significantly anti-alpha-Gal binding to Sepharose was unaffected by CNBr activation and ligand coupling to the gel, thus warning that contaminating plasma could introduce artifacts in agarose-based chromatography of human tissue biomolecules.


Modulation of T cell functions by sperm-specific lactate dehydrogenase: fluorescence analysis of immune competent cells and local graft versus host reaction. Immune responses to a well-defined sperm-specific isogenic lactate dehydrogenase-C4 (LDH-C4) have been studied in C57Bl/Ks (H-2d) mice after immunization through intra-rectal route. Presence of anti-LDH-C4-antibodies in the sera of females immunized in presence or absence of adjuvant suggested that the immune system of mice becomes exposed to sperm antigens following intrarectal insemination. LDH-C4 primed lymphocytes from both males and females, when transferred in F1 hybrids, suppressed stimulation index of local graft versus host reaction. However, contrary to females, male counterparts which did not elicit measurable anti-LDH-C4-antibody titer, showed the presence of a higher proportion of Ly2+ and Ia+ fluorescence labelled cells in the spleen of LDH-C4 administered mice. Results suggest that males are more susceptible for immune suppression of T cell functions through generation of T suppressor cells. Sex differences in relation to immune deviation by intra-rectal administration of sperm-specific LDH-C4 in mice and their consequences in AIDS and AIDS-related complex diseases are described.


Conjugated dienes in lipids of apolipoprotein B containing lipoproteins of normal and type 2 (non-insulin-dependent) diabetic patients. Conjugated dienes present in the fatty acyl chains of cholesterol esters and triglycerides associated with plasma apolipoprotein B containing lipoproteins of normal and Type 2 (non-insulin-dependent) diabetic patients (n = 17) have been analysed using second derivative electronic absorption spectroscopy. Characteristic spectral patterns for both normal subjects and Type 2 diabetic patients were observed. Cis, trans and trans, trans conjugated dienes in cholesterol esters of lipoprotein B of Type 2 patients and normal subjects were found to be 41.74 +/- 0.51 mg/litre, 8.20 +/- 0.20 mg/litre (p less than 0.01) and 24.70 +/- 0.33 mg/litre, 9.22 +/- 0.06 mg/litre (p less than 0.01), respectively. Levels of these dienes in triglyceride fraction were 21.21 +/- 0.52 mg/litre, 7.72 +/- 0.02 mg/litre (p greater than 0.05) and 15.49 +/- 0.36 mg/litre, 7.91 +/- 0.11 mg/litre (p greater than 0.05), respectively.


Impact of chromium on lipoperoxidative processes and subsequent operation of the glutathione cycle in rat renal system. Oral administration of K2Cr2O7 to male albino rats at an acute dose of 1500 mg/kg body wt/day for 3 days brought about sharp decrease in the activities of glucose-6-phosphate dehydrogenase and glutathione reductase of kidney epithelial cells. The scavenging system of kidney epithelium is also affected as evident by the highly significant fall in the activities of glutathione peroxidase, superoxide dismutase and catalase which ultimately leads to the increase in lipid peroxidation value in kidney cortical homogenate. However, glutathione-s-transferase activity in cytosol and glutathione and total thiol content in cortical homogenate were not altered. Chronic oral administration of K2Cr2O7 (300 mg/kg body wt/day) for 30 days to rats lead to elevation in the activities of glutathione peroxidase, glutathione reductase, glutathione-s-transferase, superoxide dismutase and catalase with no change in glucose-6-phosphate dehydrogenase activity in epithelial cells. This might lead to the increase in glutathione and total thiol status and decrease in lipid peroxidation value in whole homogenate system.


Absorption and fluorescence studies on interaction between cationic dyes and Klebsiella K7 capsular polysaccharide. Interaction of cationic dyes, pinacyanol chloride, acridine orange and phenosafranin, with Klebsiella K7 capsular polysaccharide has been investigated by spectrophotometric and spectrofluorometric measurements. The acidic polysaccharide induce a metachromatic blue shift of the absorption band of pinacyanol chloride from 600 nm to 495 nm, indicating strong metachromasy. Stoichiometry of polyanion and dye cation (1:1.5) in the polymer-dye compound formed by the interaction between pinacyanol chloride dye and K7 polymer indicate that both glucuronic acid and pyruvic acid act as the potential anionic sites for interaction. Both spectrophotometric titration of pinacyanol chloride and spectrofluorometric titration of acridine orange and phenosafranin dyes by the polymer gave quite comparable equivalent weights for the polymer. Dye-polymer interaction studies indicated induction of metachromasy in the cationic dye by the anionic biopolymer, establishing its chromotropic character.


Isolation and characterization of the haemolymph lipoprotein of desert locust, Schistocerca gregaria Forskal. A yellow lipoprotein was isolated and purified from haemolymph of desert locust, Schistocerca gregaria Forskal, by precipitation at low ionic concentration and by gel filtration. Sephadex G-200 resolved haemolymph into five protein peaks. First peak was of lipoprotein with molecular weight of 501,000 +/- 7000 (n = 3) and on electrophoresis, it separated into three bands. Maximum lipids from lipoprotein could be extracted with chloroform: methanol solvent system and were 52% by weight.


Location of chloroplast membrane resonances in high resolution 13C-NMR spectrum of spinach leaves. Spinach leaves were studied by high resolution 13C-NMR spectroscopy. The spectra obtained, though highly complex, showed some distinct resonances assignable to carbons of lipids of the chloroplast membranes, thereby offering a novel way for studying the chloroplast membranes in situ.


Effect of neonatal pineal ablation on estradiol receptors in mammary glands of rats housed under varying photoperiods. Estradiol receptors (ER) in mammary glands of female Holtzman rats, either intact or neonatally pinealectomised and housed in 10:14 or 24:0 L:D (light:dark) schedule were studied at 30, 40, 50 +/- 5 and 65 +/- 5 days of age. Whereas ER were detectable only at the age of 60-65 days in intact rats housed in 10:14 L:D, they were present as early as 30 days onwards in the pineal ablated group. In the 24:0 L:D pinealectomised group, though mammary gland ER were maximum around 40 days of age and temporarily undetectable around day 50, they had stabilized around the age of 60-65 days. The data demonstrates for the first time, the modulation of ER in rat mammary glands in response to varying photoperiods as well as pineal ablation. Earlier reports on incidence of chemically induced mammary tumours have been compared to the receptor modulation.


Aflatoxin B1 induced hepatocarcinogenesis in neonatal rats. Role of cell replication on aflatoxin B1 (AFB1) induced hepatocarcinogenesis was investigated in neonatal rats showing persistence of cell replication in the liver for 21 days of post natal life. Adult (8-10 weeks old) rats displaying no hepatocytic proliferation served as controls. Three doses of AFB1 were administered to both the groups at intervals of 48 hr with the doses starting on 10th day of age in the neonatal group. Appearance of phenotypically altered preneoplastic hepatocytes was quantitated in both the groups. A significantly higher incidence of preneoplastic foci was recorded in neonatal rats as compared to adult animals. The results suggest that presence of cell replication in neonatal rats at the time of AFB1 administration enhances the process of hepatocarcinogenesis.


In vivo growth inhibitory effect of Withania somnifera (Ashwagandha) on a transplantable mouse tumor, Sarcoma 180. Withania somnifera is a medicinal plant used in the treatment of a variety of ailments in the Ayurvedic system. Alcoholic extract of the root of the plant was injected(ip) at daily doses of 200 to 1000 mg/kg body wt for 15 days starting from 24 hr after intradermal inoculation of 5 x 10(5) cells of S-180 in BALB/c mice. Solid tumor growth was monitored for 100 days. Doses of 400 mg/kg and above produced complete regression of tumor after an initial growth, the percentage of complete response (CR) increasing with increasing drug dose. A 55% CR was obtained at 1000 mg/kg drug administration, but this dose also produced some mortality among the animals. A significant increase in the volume doubling time and growth delay was seen when the drug dose was increased from 500 to 750 mg/kg body wt, but further increase in drug dose to 1000 mg/kg did not produce any significant increase in these responses. Cumulative doses of 7.5 to 10 g at daily doses of 500 or 750 mg/kg seems to produce a good response in this tumor.


Late afternoon administration of melatonin is prosomatotrophic and exerts androgen independent effects on erythropoiesis in male house sparrow Passer domesticus. In the third week of September 1989, birds were purchased locally and acclimated to their housing conditions in a room fully exposed to natural day length (average: 11.96 hr) and temperature (26 degrees +/- 2 degrees C) for 2 weeks. Birds were in the regressive phase of their annual gonadal cycle. In the first experiment 24 birds were selected randomly and were divided into 3 groups of 8 birds each. Initial body weight and bill color score were recorded. The birds of group-I and group-II were injected daily with 5 and 10 micrograms of melatonin in 0.1 ml of vehicle, respectively. The birds of group-III were injected with vehicle only and treated as control. Injections were given daily between 1700 and 1730 hrs over a period of 10 days. At the termination of the experiment, the birds were weighed, sacrificed, bill color scored, blood collected and immediately processed to determine the number of erythrocytes and hemoglobin concentration. The mean body weight loss amounted to 9.6% in vehicle-treated house sparrow. Birds receiving low and high doses of melatonin maintained their initial body weight. Melatonin significantly accelerated the rate of bleaching of bill color. Results clearly indicate that in house sparrow, melatonin produces prosomatotrophic and antigonadotrophic effects. The low dose of melatonin stimulated erythropoiesis significantly. In the second experiment, melatonin nullified the castration-induced decline in the number of circulating red cells. This clearly suggests that the influence of melatonin on erythropoietic machinery appears to be independent of testicular hormone(s).


DNA polymorphism in Cab locus of tomato induced by tissue culture. Plants were regenerated from callus induced from leaf disc explants of a tomato F1 hybrid heterozygous for three marker loci anthocyaninless (a), without anthocyanin (aw), and hairless (hl). Regenerants were studied for somaclonal variation at the phenotypic level by scoring for variation in the marker loci, and at the DNA level by probing geomic DNA blots with a chlorophyll a/b binding protein (Cab-3C) cDNA sequence. While no variation was observed at the phenotypic level in over 950 somaclones studied, DNA polymorphism for the Cab locus could be detected in two out of 17 somaclones tested. Tissue culture induced variation at the phenotypic level for specific loci is very low (less than 0.001 for a, aw or hl) but DNA sequence changes are induced at much greater frequency (approximately 0.1 for a multicopy gene family such as Cab).


Effects of acute or chronic opioid agonists and their modulation by diurnal rhythmicity and satiety states on food intake in rats. The effects of acute or chronic treatment with mu and k opioid agonists were investigated on food intake during light (0-6 hr) and dark (6-24 hr) phases in free fed and fasted rats. In free fed rats, morphine (MOR, 5 mg/kg, ip), a mu-agonist, induced a hyperphagic response during both light and dark phases, whereas ketocyclazocine (KCZ, 1 mg/kg, ip), a k-agonist, enhanced food intake only during the light phase. Chronic MOR (x 7 days) produced a further enhancement of hyperphagia in the light phase and attenuated the dark phase response. Chronic KCZ, however, had opposite effects, i.e. tolerance to light phase hyperphagia and an enhancement in the dark phase response. In fasted rats, neither MOR nor KCZ appreciably enhanced food intake after acute administration but chronic treatment potentiated the acute opioid effects. These results are discussed in light of the role of diurnal rhythmicity, satiety states and receptor (mu and k) specificity/interactions in the opioidergic regulation of food intake.