The influence of the stimulus on normal sural nerve conduction velocity: a study of the latency of activation. Conduction along the sural nerve was studied in 64 normal subjects using near-nerve electrodes. Conduction velocities over the same nerve segments were calculated: (1) from the latency recorded from a site of stimulation to a site of recording (1R-method); and (2) from the difference in latency between 2 recording sites, the site of stimulation being situated elsewhere along the nerve (2R-method). Consistently faster velocities were seen with the 2R-method and could best be explained by a fixed delay of about 0.15 ms at the stimulus site (latency of activation, utilization time). This delay was markedly prolonged when a ramp rather than a rectangular stimulus was applied, though fast fibers were excited with both types of stimuli. The delay is thought to be dependent on the relationship between the density of current at the stimulus site and the threshold of responding fibers.


McArdle's disease and gout.

McArdle's disease and gout. We report the first case of McArdle's disease (muscle phosphorylase deficiency) and tophaceous gout. To examine the contribution of adenine nucleotide degradation to the disturbance of uric acid metabolism, we labeled the adenine nucleotide pool with [8-14C]adenine, and measured plasma and urine purines following vigorous exercise tests. Plasma and urinary hypoxanthine and xanthine concentrations and the specific radioactivity of urinary purines increased markedly, but plasma urate levels and uric acid excretion were not substantially modified. We suggest that, in this patient, the association of McArcle's disease with gout is coincidental.


Transcranial magnetic stimulation of the facial nerve: intraoperative study on the effect of stimulus parameters on the excitation site in man. Magnetic stimulation (magStim) of the intracranial facial nerve is performed in clinical and research settings, but the activation site is a matter of controversy. Latencies of nasalis muscle responses to magStim were, therefore, compared with those obtained by direct electrical stimulation of the facial nerve (a) at the root exit zone (REZ); (b) at the porus of the facial canal; and (c) in the stylomastoid fossa during microvascular decompression operations in the cerebellopontine angle (CPA). Measurements of latencies of the nasalis muscle response, obtained while the stimulating coil was placed over the parieto-occipital area of the scalp, indicated that it was the labryinthine segment of the facial canal, 5 to 16 mm distal to the CPA, that was activated. This would be in agreement with studies of physical models reported in the literature that showed (a) the strength of the electrical current generated by a magnetic field is particularly high close to a nerve foramen; and (b) excitation to magStim is most likely to occur where the induced electrical field changes rapidly over distance, i.e., at anatomical boundaries between media of high and low specific resistance. These characteristics are found at the end of the labyrinthine segment of the facial canal, where the facial nerve leaves the low-resistance cerebrospinal fluid and enters the high-resistance petrous bone. The site of neural excitation is robust and unaffected by stimulus intensity and current direction within a wide range, or by large changes in location of the coil.(ABSTRACT TRUNCATED AT 250 WORDS)


Twitch and tetanus in mdx mouse muscle.

Twitch and tetanus in mdx mouse muscle. We compared mdx and C57BL10 anterior tibial muscle force in situ (single pulse, multiple pulse, staircase, posttetanic potentiation, and fatiguing stimulation patterns) to define muscle strength, physiology, and fatigue resistance. The relatively hypertrophied mdx muscle showed: reduced strength (N/cm2), an increased twitch-tetanus ratio, and resistance to post-fatigue twitch slowing. These differences implicate altered mdx calcium regulation, and emphasize the importance of measuring both muscle function and morphology in mdx treatment trials.


Case-of-the-month: painful thigh mass in a young woman: diabetic muscle infarction. A 29-year-old female with type I diabetes mellitus developed pain, focal tenderness, and swelling in the posterior left thigh. Subsequent evaluation included a muscle biopsy, which revealed large confluent areas of necrosis and edema, compatible with a diagnosis of diabetic muscle infarction (DMI). Diabetic muscle infarction (DMI) is an unusual neuromuscular complication of diabetes mellitus. DMI begins with the acute onset of focal pain and swelling in the thigh. The anterior compartment (quadriceps muscle group) or posterior compartment (hamstring muscle group) are most frequently involved. The focal region of muscle damage can be noninvasively viewed by magnetic resonance imaging and radionuclide scans. Muscle biopsy demonstrates large confluent regions of muscle necrosis and edema. DMI needs to be differentiated from other processes that can cause leg pain in a diabetic patient.


Mimicry of elastin repetitive motifs by Theileria annulata sporozoite surface antigen. Theileria annulata is an important pathogen of cattle in the tropics. The gene sequence of a sporozoite surface antigen (SPAG-1) is reported. Data is also presented demonstrating that SPAG-1 is synthesised as a large precursor. This antigen, which is a candidate for inclusion in a subunit vaccine, shows a remarkable degree of molecular mimicry to the extracellular matrix protein elastin. It contains both repetitive motifs PGVGV and VGVAPG. Immunofluorescence using a monoclonal antibody against VGVAPG confirmed that this peptide is expressed on sporozoites as predicted. The presence of VGVAPG is particularly interesting since this is the ligand for elastin receptors on a range of cell types, including macrophages/monocytes which are a major class of host target cells. It is proposed that this antigen represents the ligand whereby T. annulata recognises its host cells.


Leishmania tarentolae taxonomic relatedness inferred from phylogenetic analysis of the small subunit ribosomal RNA gene. The sequence of the Leishmania tarentolae SSU rRNA (small subunit or 18S rRNA) gene was completely determined from 2 different strains and used to determine phylogenetic relationships between this organism and other trypanosomatids. Extensive structural similarities were observed between L. tarentolae and mammalian leishmanias the SSU rRNA. Phylogenetic reconstructions, using distance matrix or parsimony methods, showed large evolutionary distances between trypanosomes, either African and American, and L. tarentolae. Further analysis using intergenic rDNA spacer (IGS) sequences as probes in dot blot experiments confirmed the results obtained with the SSU rDNA comparisons. The data presented here clearly indicate that L. tarentolae is closely related to the mammalian parasite Leishmania donovani and highly divergent from trypanosomes.


Random mating of natural Plasmodium populations demonstrated from individual oocysts. DNA amplified from individual Plasmodium vivax oocysts, produced by feeding mosquitoes directly on naturally infected humans in Thailand, was used to study cross-mating of 2 polymorphs of the circumsporozoite (CS) gene, VK 210 and VK 247. Alleles were detected in matched blood parasites, sporozoites, and individual oocysts with oligoprobes specific to characteristic repeat units. Oocysts developing from 3 cases in which mixed alleles were present in the blood parasites had genotype frequencies, including hybrids, consistent with the Hardy-Weinberg equilibrium. There was apparently no barrier to hybridization of the 2 alleles nor a bias, as has been found in some laboratory experiments, favoring hybrid formation. These are the first measurements of cross-mating frequencies directly from natural Plasmodium infections and the first observations of genetic hybridization in P. vivax.


Mechanisms of microenvironmental pH regulation in the cuticle of Ascaris suum. The excretion kinetics of various organic acids by Ascaris suum were quantified to determine if the excretion of these metabolic end-products could generate and maintain a microclimate pH within the aqueous compartment of the cuticle. Ligated and nonligated A. suum were incubated in media buffered with 0.25 or 2.5 mM Hepes (initial pH 7.5) or 0.5 or 5 mM glycine (initial pH 3.25). The concentration of organic acids and the pH of the media were followed for 24 h. Several volatile fatty acids, including acetic, 2-methylbutyric, 2-methylvaleric, n-valeric, and n-butyric, were excreted at relatively high rates. Propionic, n-caproic, 2-methylcaproic, tiglic acid, and the non-volatile organic acids, lactic and succinic, were excreted more slowly. The organic acids were excreted at a constant rate and in apparently fixed molar concentration ratios. The accumulation of organic acids was associated with changes in pH of the medium until a limiting constant pH, in the vicinity of the pKa of the volatile fatty acids, was reached. The rate of organic acid excretion was not affected by initial medium pH, buffer capacity, or parasite ligation. The rate of pH change induced by the excretion of organic acids was also insensitive to whether ligated or nonligated A. suum were used, but was dependent on the initial buffer capacity of the medium. These results suggest that A. suum excrete the end-products of carbohydrate metabolism across the cuticle. The presence of organic acids in the aqueous pores of the cuticle creates and maintains a microclimate pH of about 5.0 +/- 0.3. This pH will influence the transport properties of weak acids and bases and should be considered in the design of delivery systems for anthelmintics.


Isolation, sequence and differential expression of the p58 gene family of Babesia bigemina. Four copies of the gene encoding the merozoite surface protein p58 from the protozoan hemoparasite Babesia bigemina were amplified from genomic DNA by polymerase chain reaction (PCR) techniques, molecularly cloned and subjected to DNA sequence analysis. The amplified DNA (Bbg7, Bbg9, Bbg13, Bbg14) could be placed into 2 classes with respect to its size and the length of the open reading frame (ORF). With the exception of a single base substitution, the sequence of Bbg13 is identical to the cDNA sequence published earlier [1]. The Bbg7 and Bbg14 copies of p58 diverged from Bbg13 sequence at regions towards the 3' and 5' ends, respectively. In contrast, Bbg9 has incorporated both regions of divergence within its sequence. Using a cloned strain of B. bigemina, RNA-PCR and Northern blot analyses demonstrate the in vivo transcription of 3 of the 4 copies, although one of the 3 expressed copies is present in very low abundance. The relative abundance and size of the two p58 mRNA species detected are consistent with the 58- and 55-kDa proteins detected by in vitro translation of B. bigemina poly(A)+ mRNA by immunoprecipitation with an anti-p58 monospecific antibodies. These results indicate that the gene encoding p58 exists as a multigene family that appears to be differentially expressed in the blood stage of the parasite's life cycle.


Cloning and expression in Escherichia coli of cDNAs encoding a 31-kilodalton surface antigen of Sarcocystis muris. Using polyadenylated RNA isolated from Sarcocystis muris cyst merozoites, we have constructed a cDNA library in the expression vector lambda ZAP. Immunoscreening with monoclonal and polyclonal antibodies directed against a 31-kDa surface antigen of S. muris [1] yielded a number of clones with insert sizes ranging between 1.1 kb and 1.3 kb. An additional clone with an insert length of 1.55 kb was isolated by screening with a labeled DNA probe derived from one of the cDNA clones. The cDNA sequence was found to contain an open reading frame specifying a polypeptide of 280 amino acids with a predicted size of 29.7 kDa. The deduced amino acid sequence is rich in serine and threonine (22%) and harbors a hypothetical N-terminal signal peptide sequence as well as a C-terminal glycosyl phosphatidylinositol anchor attachment site. The predicted amino acid sequence has been confirmed by peptide sequencing and an analysis of the overall amino acid composition of the 31-kDa protein. A recombinant protein was obtained which was recognized by the polyclonal antibodies directed against the 31-kDa antigen. Antiserum raised against the purified fusion protein specifically reacted with a 31-kDa protein from S. muris cystozoites. Southern blot analysis indicated that the corresponding gene exists as a single copy within the S. muris genome.


Cloning of the gene encoding Leishmania donovani S-adenosylhomocysteine hydrolase, a potential target for antiparasitic chemotherapy. A full-length gene encoding the S-adenosylhomocysteine hydrolase (AdoHcyase) enzyme has been isolated from a genomic library of Leishmania donovani DNA in lambda GEM-11 by cross-hybridization to the full-length human AdoHcyase cDNA. The nucleotide sequence of the SalI fragment contained a single open reading frame that encoded a polypeptide of 438 amino acids (47,712 Da). After maximum gap alignment, the predicted amino acid sequence of the leishmanial AdoHcyase was 70-73% identical to AdoHCyases from higher eukaryotes. In addition, a data base search revealed that the primary structure of all AdoHcyase proteins was highly homologous to that of a protein encoded by a mRNA from Drosophila melanogaster that maps near the r element function of the Abd-b homeotic gene. In Northern blots, the SalI fragment hybridized to a 3.0-kb transcript that presumably encodes the parasite enzyme. Southern blot analysis of genomic DNA revealed that the AdoHcyase gene did not exist as a tandemly repeated array within the L. donovani genome. Moreover, monoclonal antibodies generated against human AdoHcyase recognized a leishmanial protein on immunoblots. Finally, the growth of L. donovani promastigotes could be arrested by micromolar concentrations of 3-deazaaristeromycin (C3Ari) and 9-(trans-2',trans-3'-dihydroxycyclopentanyl)adenine, 2 known inhibitors of mammalian AdoHcyase. C3Ari also induced a substantial expansion of the intracellular pools of both AdoHcy and S-adenosylmethionine (AdoMet), as well as a significant diminution of the AdoMet/AdoHcy ratio. Thus, AdoHcyase may have therapeutic potential for the selective treatment of diseases of parasitic origin.


Alternative splicing of the Schistosoma mansoni gene encoding a homologue of epidermal growth factor receptor. The complete coding DNA for a Schistosoma mansoni homologue of the epidermal growth factor receptor (SER) was characterized from cDNA clones obtained by homology to the tyrosine kinase domain of erbB. The DNA sequence predicts a 200-kDa translation product that contains a secretory leader, a cysteine-rich extracellular domain, a hydrophobic transmembrane sequence, and an intracellular tyrosine kinase domain. The SER transcript is present in cercariae and adult schistosomes. In addition to SER transcripts, schistosomes produce at least 3 variant transcripts encoding truncated SER products that include the secretory leader and a small portion of the extracellular domain followed by short sequences of unrelated, C-terminal amino acids. Based on these sequences, 2 of the variant mRNAs (class 2 and 5) appear to encode soluble, secreted proteins while one (class 4) encodes an SER variant protein with a hydrophobic C-terminus that may serve as a membrane anchor. Class 2 SER variant transcripts are present at levels comparable to SER transcripts in adult worms but are not detected in cercariae. Class 4 and 5 SER variant transcripts are also found within adult worms but at lower levels. Genomic cloning and characterization demonstrate that the variant SER transcripts arise through alternative splicing of the SER gene.


Brefeldin A inhibits protein secretion and parasite maturation in the ring stage of Plasmodium falciparum. The release of all newly synthesized soluble proteins from the ring stage of Plasmodium falciparum-infected erythrocytes was reversibly blocked by brefeldin A, indicating the presence of a conserved step of classical eukaryotic secretory export within the parasite. This implies that proteins exported to the erythrocyte cytosol undergo secretory release at the parasite plasma membrane and subsequent translocation across the vacuolar membrane. Along with inhibiting protein export brefeldin arrested parasite maturation, but the cells remained viable even after 24 h in the presence of the drug. The results suggest that secretory export may be important for development, but not for immediate survival, at the ring stage.


Utilization of a carbohydrate reserve comprised primarily of mannose by Leishmania donovani. Promastigotes from late log phase and 3-day stationary phase cultures of Leishmania donovani were collected, washed in buffer, and the cell pellet was treated with boiling KOH. A putative carbohydrate storage material was then precipitated and washed in ethanol/LiBr. This material did not liberate glucose when treated with amyloglucosidase, indicating that it was not glycogen. Acid hydrolysis released a hexose which was identified as mannose by several criteria. Considerably more of this mannan-like carbohydrate is present in cells from 3-day stationary phase than from late log phase cultures, consistent with the ability of 3-day stationary phase cells to survive in non-nutrient buffer and maintain oxygen consumption for longer than log phase cells. The amount of this mannan-like compound decreased by over 50% during a 3-h incubation in buffer of cells from 3-day stationary phase cultures. The presence of glucose during the incubation prevented the utilization of this carbohydrate, consistent with the possibility that it serves as an energy reserve.


Regulation of hsp70 expression in Trypanosoma cruzi by temperature and growth phase. The steady-state level of the hsp70 mRNAs of Trypanosoma cruzi cultured at different temperatures and growth conditions has been analyzed by Northern blotting. We show that only one size class of hsp70 mRNA, of about 2.2 kb, is transcribed from the hsp70 cluster and that its transcription is constitutive at 28 degrees C. However, after a heat shock treatment at 37 degrees C for 2 h of logarithmically growing parasites, the abundance of the hsp70 mRNA increased about 4-fold. A similar increase was observed at 28 degrees C when the parasite culture reached the stationary phase of growth. On the other hand, a heat shock at 42 degrees C did not change the steady state level of the 2.2-kb size class of hsp70 mRNA. However, accumulation of transcripts of high molecular weight was detected when stationary growing parasites were cultured at 42 degrees C for 2 h. Also at 37 degrees C the steady state level of the alpha- and beta-tubulin mRNAs of logarithmically growing parasites exhibited a slight increase but only after a period of 24 h. Analysis by one-dimensional immunoblots of the Hsp70 levels showed that at 37 degrees C the abundance of the protein was 4-fold higher than at 28 degrees C. Immunoblots of high-resolution two-dimensional gel electrophoresis showed, moreover, that various isoforms of this protein are constitutively expressed at 28 degrees C and that some of them have a specific pattern of induction at 37 degrees C. We observed, moreover, that the heat shock induces the expression of a series of proteins while it causes repression of others.


Characterisation of melarsen-resistant Trypanosoma brucei brucei with respect to cross-resistance to other drugs and trypanothione metabolism. An arsenical resistant cloned line of Trypanosoma brucei brucei was derived from a parent sensitive clone by repeated selection in vivo with the pentavalent melaminophenyl arsenical, sodium melarsen. The melarsen-resistant line was tested in vivo in mice against a range of trypanocidal compounds and found to be cross-resistant to the trivalent arsenicals, melarsen oxide, melarsoprol and trimelarsen (33, 67 and 122-fold, respectively). A similar pattern of cross-resistance was found in vitro using a spectrophotometric lysis assay (greater than 200-fold resistance to melarsen oxide and greater than 20-fold resistance to both trimelarsen and melarsoprol). Both lines were equally sensitive to lysis by the lipophilic analogue phenylarsine oxide in vitro, suggesting that the melamine moiety is involved in the resistance mechanism. Although trypanothione has been reported to be the primary target for trivalent arsenical drugs [1], levels of trypanothione and glutathione were not significantly different between the resistant and sensitive lines. Statistically significant differences were found in the levels of trypanothione reductase (50% lower in the resistant clone) and dihydrolipoamide dehydrogenase (38% higher in the resistant clone). However, the Km for trypanothione disulphide, the Ki for the competitive inhibitor Mel T (the melarsen oxide adduct with trypanothione) and the pseudo-first order inactivation rates with melarsen oxide were the same for trypanothione reductase purified from both clones. The melarsen-resistant line also showed varying degrees of cross-resistance to the diamidines: stilbamidine (38-fold), berenil (31.5-fold), propamidine (5.7-fold) and pentamidine (1.5-fold). Cross-resistance correlates with the maximum interatomic distance between the amidine groups of these drugs and suggests that the diamidines and melaminophenyl arsenicals are recognised by the same transport system.


The interaction of arsenical drugs with dihydrolipoamide and dihydrolipoamide dehydrogenase from arsenical resistant and sensitive strains of Trypanosoma brucei brucei. D,L-dihydrolipoamide and D,L-dihydrolipoic acid react to form stable complexes with melarsen oxide with association constants of 5.47 x 10(9) and 4.51 x 10(9) M-1, respectively. These complexes possess 6-membered cyclic dithioarsenite rings which are 10-fold less stable than the 5-membered rings found in the trypanocidal drugs melarsoprol and trimelarsen, but 500-fold more stable than the 25-membered macrocyclic ring formed between melarsen oxide and dihydrotrypanothione. L-Lipoic acid concentrations in arsenical sensitive and resistant cloned lines of Trypanosoma brucei brucei have been determined by bioassay using a mutant of Escherichia coli auxotrophic for lipoate. The arsenical resistant strain was found to contain significantly less lipoic acid than the sensitive strain (19.2 +/- 4.3 and 9.7 +/- 2.9 pmol (10(8) cells)-1, respectively). The activity of the plasma membrane-associated dihydrolipoamide dehydrogenase was found to be slightly, but significantly increased in the arsenical resistant strain (34.7 +/- 1.4 and 47.8 +/- 3.7 mU mg-1, respectively). However, the Km for dihydrolipoamide and the inactivation kinetics with melarsen oxide were not significantly different between these strains. Estimates of the ratio of substrate to enzyme are of the order of 12:1 and 6:1 for arsenical sensitive and resistant strains, respectively, suggesting that these components are likely to be intimately associated with each other in the plasma membrane. These findings implicate lipoic acid, but not dihydrolipoamide dehydrogenase, in resistance to arsenical drugs, either through the mechanism of uptake or as the final target of these drugs.


Characterization of the gene encoding sporozoite surface protein 2, a protective Plasmodium yoelii sporozoite antigen. Sporozoite surface protein 2 (SSP2) is a 140-kDa, protective sporozoite surface protein from Plasmodium yoelii distinct from the circumsporozoite protein (CSP). A genomic clone containing the SSP2 gene was isolated and sequenced to determine its size, structural organization and deduced primary amino acid sequence. The coding sequence consists of a single, long open reading frame encoding 826 amino acids. The overall structure of SSP2 is similar to that of the CSP, consisting of a central region of immunogenic amino acid repeats flanked by non-repetitive sequence. SSP2 has one copy of a thrombospondin repeat motif in common with several cell adhesion molecules as well as with the CSP and the thrombospondin related anonymous protein (TRAP) of P. falciparum. Additionally, SSP2 shares substantial sequence similarity to TRAP, suggesting that TRAP is the analogue of SSP2 in P. falciparum.


DNA fingerprints: a tool for identification and determination of the relationships between species and strains of Leishmania. Using nonradioactive hybridization, the multilocal probes 33.15, F10 and (CAC)5 were shown to recognize multiple minisatellite regions in nuclear DNA of Leishmania, producing on Southern blots complex banding patterns typical of DNA fingerprints. We used the 33.15 probe to study 14 different strains belonging to 6 different species from both the sub-genus Viannia and the Leishmania mexicana complex of the sub-genus Leishmania. Distinct DNA fingerprints were obtained for each strain, permitting their identification. On the other hand, each strain showed little or no clonal variation. The information from the fingerprinting maps could be used for constructing phenograms and cladograms of the species and strains of Leishmania.


Sulfate metabolism in Entamoeba histolytica. Sulfate fluxes and sulfate metabolites in Entamoeba histolytica were characterized employing [35S]sulfate as a marker. Sulfate was taken up both across the plasma membrane and by pinocytosis; in growth medium (sulfate concentration, 1.1 mM) total uptake was 1.5 mumol h-1 (5 x 10(7) cells)-1. The fate of sulfate within the cells was investigated by thin-layer chromatography. Major metabolites (together greater than 3 mumol (5 x 10(7) cells)-1) were monoethyl sulfate and 3-cholesteryl sulfate; both these products were released into the growth medium. As minor components we identified the activated sulfate derivatives, adenosine-5'-phosphosulfate and 3'-phosphoadenosine-5'-phosphosulfate. In addition, up to 10% of the sulfate taken up was incorporated into high-molecular weight material (possibly proteoglycans). We propose that sulfurylation of cholesterol may play a role in controlling membrane sterol content.


Characterisation of the secretory acetylcholinesterases from adult Nippostrongylus brasiliensis. The biochemical nature and relationship between the different isoforms of acetylcholinesterase (AChEs) secreted by adult Nippostrongylus brasiliensis was investigated, primarily via staining for enzyme activity and active-site labelling with [3H]-diisopropylfluorophosphate (DFP). Analysis by 1-dimensional SDS-PAGE under non-reducing conditions revealed the existence of 2 proteins of 74-kDa and 39-kDa, and each protein resolved as 2 species by isoelectric focusing. Both AChEs were co-purified via affinity chromatography on 9-[N beta-(epsilon-aminocaproyl)-beta-aminopropylamino]-acridine-coupled Sepharose 6B, and utilised to raise a polyclonal rabbit antiserum. Examination of the expression of secretory AChEs by adult worms during their residence in the gastrointestinal tract showed that the initial secretion of both forms on day 4 post-infection switched to predominant secretion of the 39-kDa protein by day 8. Immunoprecipitation of 35S-labelled products of in vitro translation via RNA from day 4 and day 8 worms predicted a single primary translation product of 59 kDa. These data suggested that the 'switching' event seen in vivo most likely corresponded to processing of the 74-kDa molecule. This interpretation was supported by limited digestion with V8 protease and chymotrypsin, which showed that the 74-kDa and the 39-kDa proteins possessed structural similarities.


Intramolecular mapping of Plasmodium falciparum P126 proteolytic fragments by N-terminal amino acid sequencing. Protein P126 (also called P140, P113, SERA, SERP1) is a major parasitophorous vacuole antigen of Plasmodium falciparum. This protein is processed upon merozoite release into 2 fragments of 73 kDa (P73) and 50 kDa (P50), which are found in the culture medium. P73 is composed of 2 polypeptides of 47 and 18 kDa linked by disulfide bridges. In the presence of leupeptin, an inhibitor of serine and cysteine proteases which inhibits merozoite release, a 56-kDa intermediate product (P56) is recovered in the culture medium instead of P50. In order to map these proteolytic fragments on the 126-kDa precursor, we purified them from Plasmodium falciparum culture medium by immunoadsorption, SDS-electrophoresis and Western blotting on PVDF membrane and determined the N termini of P126, P73 (P47 and P18), P50 and P56. Comparison of these sequences with the amino acid sequence deduced from the P126 gene allowed the mapping of the different fragments on the precursor. P47 was at the N-terminal and P18 at the C-terminal end of P126. P56 and P50 had the same N-termini and were located in the middle of P126. This latter result indicates that the proteolysis of P56-P50 occurs at the C-terminus of P56. The peptide bonds cleaved by leupeptin-insensitive activities are Glu-Thr and Gln-Asp; C-terminal sequencing of P50 will be needed to identify the leupeptin-sensitive cleavage site.


Arginine metabolism during culture of Giardia intestinalis. The effect of arginine on the growth and metabolism of Giardia intestinalis trophozoites was determined. Supplementation of the normal growth medium (Diamond's TYI-S-33) with 5 or 10 mM arginine accelerated trophozoite growth over the first 2 days. There was a corresponding rapid utilisation of arginine, with none being detectable after this time. The decrease was associated with the appearance in the growth medium of 1 mol of ornithine and 2 mol of ammonia per mol of arginine utilised, the stoichiometry being consistent with the operation of the arginine dihydrolase pathway. Subsequently, there was a decrease in the ammonia concentration in the medium. Removal of arginine from the medium by pretreatment with arginase substantially decreased cell growth. In TYI-S-33 medium containing no added glucose, instead of the normal 50 mM glucose concentration, arginine supplementation also increased cell growth over the first 2 days, with concurrent stoichiometric production of ornithine and ammonia. However, in these conditions, the ammonia concentration remained elevated. This suggests that under normal conditions there is re-uptake of ammonia, which is glucose dependent. The observations confirm the operation of a functional arginine dihydrolase pathway in G. intestinalis. The concordance of cessation of rapid growth with the depletion of arginine, and the beneficial effect on growth of arginine supplementation suggests that arginine availability is a limiting factor during the initial stages of rapid growth. It would appear that arginine is a major potential energy source during the initial stages of giardial growth, and that supplementation of Diamond's TYI-S-33 medium with additional arginine may provide an improved in vitro culture medium.


Gastrointestinal damage associated with the use of nonsteroidal antiinflammatory drugs. Long-term use of nonsteroidal antiinflammatory drugs (NSAIDs) may lead to inflammation of the small intestine associated with occult blood and protein loss. The aim of this study was to investigate the prevalence and structural correlates of this enteropathy. We examined the stomach, duodenum, and small intestine of 713 patients post mortem. Of these patients, 249 had had NSAIDs prescribed during the six months before death and 464 patients had not. All visible small intestinal lesions were removed for histologic examination, and specific etiologic factors were sought. The prevalence of nonspecific small-intestinal ulcers and ulcers of the stomach and duodenum was compared in the two groups of patients. Nonspecific small-intestinal ulceration was found in 21 (8.4 percent) of the users of NSAIDs and 3 (0.6 percent) of the nonusers (difference, 7.8 percent; 95 percent confidence interval, 5.0 to 10.6 percent; P less than 0.001). Three patients who were long-term users of NSAIDs were found to have died of perforated nonspecific small-intestinal ulcers. Ulcers of the stomach or duodenum were found in 54 (21.7 percent) of the patients who used these drugs and 57 (12.3 percent) of those who had not (difference, 9.4 percent; 95 percent confidence interval, 3.9 to 15.1 percent; P less than 0.001). Patients who take NSAIDs have an increased risk of nonspecific ulceration of the small-intestinal mucosa. These ulcers are less common than ulcers of the stomach or duodenum, but can lead to life-threatening complications.


Role of plasminogen-activator inhibitor type 1 in the pathogenesis and outcome of the hemolytic uremic syndrome. Deposition of fibrin in glomeruli and renal failure are characteristic features of the hemolytic uremic syndrome. An inhibitor of glomerular fibrinolysis has been detected in plasma from children with this disorder. In this study, we define the inhibitor and show that its plasma level is correlated with the outcome of the disease. Plasminogen-activator inhibitor type 1 (PAI-1) in plasma was measured with an assay employing a specific monoclonal antibody in 40 consecutive children hospitalized with the hemolytic uremic syndrome: 12 who recovered adequate renal function (serum creatinine, less than or equal to 2.0 mg per deciliter [177 mumol per liter]) without dialysis, 23 who recovered adequate renal function after peritoneal dialysis, and 5 who did not recover adequate renal function after undergoing dialysis. At presentation, plasma PAI-1 levels were higher in the patients with the hemolytic uremic syndrome than in nine children with other forms of acute renal failure. That the inhibitor was PAI-1 was indicated by the fact that it was a potent inhibitor of tissue plasminogen activator, was acid-resistant, and was not inhibited by denaturation (all unique traits of PAI-1) and that it was neutralized by an antibody specific for PAI-1. Multivariate discriminant-function analysis revealed that the duration of elevated PAI-1 activity was strongly correlated with the outcome of the disease (P less than 0.001). Peritoneal dialysis reduced plasma PAI-1 levels dramatically. Our studies suggest that PAI-1 is the circulating inhibitor of fibrinolysis in the hemolytic uremic syndrome. Normalization of plasma PAI-1 levels (e.g., by peritoneal dialysis) is correlated with improvement in renal function. However, the possibility that increased plasma levels of PAI-1 are either causes or effects of the hemolytic uremic syndrome is not unequivocally established by these studies.


Relation of skin capillary pressure in patients with insulin-dependent diabetes mellitus to complications and metabolic control. Microvascular disease is a major problem in patients with diabetes mellitus. It has been suggested that diabetic microangiopathy may result from an increase in capillary blood flow and capillary hypertension, but direct evidence of capillary hypertension in such patients is lacking. We measured capillary pressure at the summit of the capillary loop by direct microcannulation of skin nail-fold capillaries and a dynamic method of pressure measurement in 29 patients with insulin-dependent (Type I) diabetes and 29 normal subjects matched for age and sex. Among the diabetic patients, 7 had had diabetes for less than one year, 12 had incipient nephropathy (albumin excretion, 20 to 200 micrograms per minute), and 10 had overt nephropathy (albumin excretion, greater than 200 micrograms per minute). In addition, seven patients with no evidence of nephropathy were studied before and after three months of improved glycemic control. The median capillary pressure in the diabetic patients was 20.4 mm Hg (range, 13.6 to 25.3), as compared with 16.7 mm Hg (range, 12.8 to 22.8; P less than 0.001) in the normal subjects. The values were higher in each subgroup of diabetic patients than in the corresponding group of normal subjects, but the values did not differ among the three subgroups of diabetic patients. In the seven patients who were studied before and after three months of improved glycemic control, the median capillary pressure fell from 20.0 mm Hg (range, 18.5 to 21.7) to 17.8 mm Hg (range, 14.1 to 20.3; P = 0.02). Nail-fold capillary hypertension may develop early in the course of diabetes, before the emergence of microvascular disease, and may be influenced by changes in metabolic control.


Predisposing factors for severe, uncontrolled hypertension in an inner-city minority population. Hypertensive emergency and urgent hypertension are the most severe forms of uncontrolled hypertension and are now seen predominantly in poor, minority populations. We studied the characteristics of the medical care received by patients with these conditions in order to identify risk factors for severe, uncontrolled hypertension. Using a case-control study design, we interviewed 93 patients with severe, uncontrolled hypertension who presented in the hospital emergency room and 114 control patients with hypertension; both groups were seen at two New York City hospitals from 1989 through 1991. All the patients were black or Hispanic. Multiple logistic-regression models were used to adjust for age, sex, race or ethnic background, education, smoking status, alcohol-related problems, and use of illicit drugs during the previous year. After additional adjustment for lack of health insurance, severe, uncontrolled hypertension was found to be more common among patients who had no primary care physician (adjusted odds ratio, 3.5; 95 percent confidence interval, 1.6 to 7.7) and among those who did not comply with treatment for their hypertension (adjusted odds ratio, 1.9; 95 percent confidence interval, 1.4 to 2.5). Lack of health insurance was marginally associated with severe, uncontrolled hypertension (adjusted odds ratio, 1.9; 95 percent confidence interval, 0.8 to 4.6) after adjustment for lack of a primary care physician and noncompliance with antihypertensive treatment. Patients without a primary care physician and without health insurance were more likely to have their blood pressure checked and receive prescriptions for blood-pressure medications in emergency rooms than in physicians' offices or clinics. Characteristics of both the health care system and patients' behavior are associated with severe, uncontrolled hypertension. Improving access to primary care physicians, through health insurance or other means, may be an effective strategy for improving control of hypertension in disadvantaged minority populations.


[Kinetics of thermal killing of microorganisms in egg yolk]. Egg yolk is relating to its germ content and enzyme activity only restricted storable. Unpasteurized egg yolk get spoiled between a few hours and days. The rapid spoilage can be reduced by a pasteurization and following cold storage. The storage stability of egg yolk can be increased by additives, e.g. salt or saccharose. Kinetic results of the thermal microorganisms destruction in the egg yolk with and without stabilizing additives are presented in this article. It will be shown that the evaluation method from REICHART gives representative results for mixed cultures, too. A further evaluation method by a differential equation will be demonstrated and discussed.


Toxicological evaluation of rapeseed products in a subacute feeding study in rats. Groups of 15 male rats were fed ad libitum for 4 weeks standard diet containing 0, 2.5, 5 or 10% rapeseed protein isolate (RPI), 2.5, 5 or 10% rapeseed extraction residue (RER) or 10% casein. Body weight gain and food intake were recorded weekly. Clinical chemistry analyses, haematology, urinalysis, organ weights and histology were performed terminally. Serum alanine aminotransferase was decreased by RPI, RER and casein, while serum albumin only increased in rats fed RER. Microscopic examinations revealed abnormalities in liver and kidneys of animals at the 10% RPI, RER and casein levels. The absolute liver weights showed increases in the 5 and 10% RER groups and in the 10% RPI group. The relative kidney weights were reduced at all RER levels and in the 2.5% RPI group. Antithyroid activity of the rapeseed products were not noted.


Study on some trace elements content of certain internal organs and muscles of lambs. The aim of the investigations was the determination of the lead, cadmium, copper, zinc and iron content. It was found that lead and cadmium content in the kidneys and livers of lambs from the Sedziszów region was by several times higher than in the case of lambs from an agricultural region (control). The average lead concentration in 31% of kidneys and livers coming from the lambs of the Sedziszów region exceeded the permissible norm.


[The effect of thyroid- and sex hormones on triglyceride secretion in rat liver]. The influence of thyroxine, estradiol and testosterone on the triacylglycerol (TG) secretion rate out of the liver into the blood as well as on concentrations of FFA, triglycerides, and glucose in serum was determined. All of these experiments were carried out under different nutritional conditions (non-fasting, and 10 or 16 h fasting, respectively). Accordingly the results after hormone application were compared with three groups of controls. Within these control groups increasing time of fasting caused exclusively an enhancement of FFA concentrations, while the TG secretion rate and other parameters were not influenced. Concerning all hormones effects on several lipid parameters have been observed. In particular, a decrease of FFA concentrations in comparison with controls after 16 h fasting was evident. In these cases, the TG secretion rate and TG concentration in serum were simultaneously lowered. Both alterations may be a consequence of a diminished mobilization of FFA in adipose tissue. Increases of FFA concentrations in serum after hormone application, compared with the corresponding controls, only occurred after administration of thyroxine and testosterone, while changes of the TG secretion rate and the TG concentration in serum were scarcely observed. Final remarks about the actual influence of hormones on the investigated parameters, especially TG secretion rate were drawn including results of a previous paper, where catecholamines, insulin, and prednisolone had been tested under the same conditions.


Some relationships between addiction and drug delivery to the brain. Hemodynamic radioisotope studies of brain blood flow in humans have been correlated with the delivery of some common addictive drugs. Both lipophilic and hydrophilic tracers were used in the hemodynamic studies. Iodoantipyrine is lipophilic and is completely cleared by brain during a single brain circulatory passage, as are cocaine and nicotine. Iodohippurate is hydrophilic, so its brain clearance after IV injection resembles that of morphine. The earlier studies performed in humans have been related here with recent studies of blood brain penetration of drugs of abuse. As presented, these separate studies are consistent with the proposed hypothesis that the interval between drug intake and perceived effect is a significant consideration when explaining severity of addiction. The shorter the time interval between drug intake and its perceived effect, the more severe the addiction appears to be. This relationship may explain differences in severity of addiction to the same drug taken by various routes of administration.


Opioid peptide drug development: transport of opioid chimeric peptides through the blood-brain barrier. The use of chimeric peptides and physiologic-based strategies for drug delivery through the BBB may be applied to opioid peptides (dynorphins, endorphin analogs, and enkephalin analogs), and these agents have considerable advantages in drug development with respect to drug addiction. Recent research has allowed for the development of efficacious BBB drug transport vectors, as well as the demonstration that these vectors allow for shuttling of opioid peptides through the BBB in vivo. The major challenge to future research is the development of coupling strategies that allow for the release of biologically active opioid peptide from the drug transport vector following its cleavage by disulfide reductase enzymes, which are abundant in brain. These coupling strategies must be developed in advance since the amino groups necessary for coupling are incorporated into the opioid peptide at the level of solid-state synthesis.


Pathways into, through, and around the fluid-brain barriers. The potential intracellular and extracellular pathways that blood-borne substances may follow for circumventing the fluid-brain barriers and entry to the CNS are numerous. The extracellular avenues, patent to blood-borne protein the size of IgM, and movement of blood-borne macromolecules through perivascular clefts deep into the CNS complicate the interpretation and identification of bona fide transcytosis through the BBB. The often-stated belief in literature reviews of the BBB that nonfenestrated cerebral endothelia fail to engage in endocytosis and possess few vesicles under normal conditions is invalid. Endocytic vesicle formation and vesicular traffic among constituents of the endomembrane system are no different in BBB endothelia than in other cell types. Available biochemical and morphological data advocate the transcytosis of blood-borne protein and peptides through nonfenestrated cerebral endothelia. However, absence of demonstrable endocytic activity at the abluminal front compared with a very prominent endocytic activity at the luminal surface of BBB endothelia argues against bidirectional membrane trafficking through the BBB and supports the concept of a brain-blood barrier. The latter is no less significant functionally than the BBB and may be more so in deterring transendothelial transfer of peptides and proteins bidirectionally through the nonfenestrated cerebral endothelium. The difficulty in interpreting transcytosis through BBB endothelia is not encountered for epithelia of the blood-CSF barrier at the level of the choroid plexus. Choroid epithelia engage in endocytosis circumferentially; hence, the potential for transcytosis and circumvention of the blood-CSF barrier through an intraepithelial route exists bidirectionally in the choroid plexus.


The basic neurobiology of addiction.

The basic neurobiology of addiction. This overview on cocaine's addiction liability is presented in this monograph on the blood-brain barrier (BBB) because the National Institute on Drug Abuse has been given the task of finding pharmacotherapies to treat addiction. Knowledge about the BBB might help researchers design better drugs or approaches to keep the "good" drugs inside and/or the "bad" drugs outside. It is to be hoped that the BBB community can be convinced that drug abuse is an exciting area and that work on biological barriers has something to offer.


An overview of the multiple functions of the blood-brain barrier. The BBB formed by brain capillary endothelial cells is now recognized as more than an impermeable wall. It has specific transport systems that facilitate the uptake of important nutrients and hormones and active pumps that help to regulate the concentrations of ions and metabolites in the brain's ISF. Enzymes present in the endothelial cell metabolize neurotransmitters, drugs, and toxins before they can enter the brain and disrupt its function. Many of these properties are likely to be under regulation either by neurotransmitters and hormones released in the brain or by those present in the systemic circulation. As a result of this constellation of diverse functions, the brain capillary endothelial cell is able to efficiently supply the brain with the metabolites that it requires while contributing to the maintenance of the brain's ionic homeostasis and protecting it from circulating toxins.


A small metalloribozyme with a two-step mechanism. An RNA molecule consisting of an asymmetric internal loop of six nucleotides can be rapidly and specifically cleaved by Pb2+ in the presence of Mg2+. The 5' cleavage product terminates with a 3' phosphomonoester generated from a 2',3'-cyclic phosphodiester reaction intermediate. This two-step reaction mechanism resembles that of many protein ribonucleases but has not previously been observed for reactions catalysed by RNA.


Genetic divergence, speciation and morphological stasis in a lineage of African cichlid fishes. Since their discovery at the turn of the century, the species assemblages of cichlid fishes in the East African Lakes Victoria, Malawi and Tanganyika have fascinated evolutionary biologists. Many models have attempted to account for the 'explosive' evolution of several hundred species within these lakes. Here we report a case of surprisingly large genetic divergence among populations of the endemic Tropheus lineage of Lake Tanganyika. This lineage of six species contains twice as much genetic variation as the entire morphologically highly diverse cichlid assemblage of Lake Malawi and six times more variation than the Lake Victoria species flock. Although it is highly variable in coloration, this group of species and its closest relatives have not undergone appreciable morphological change. The observed geographic pattern of genetic variation suggests that major lake level fluctuations affected the distribution and speciation of this lineage of cichlid fishes.


The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Tuberculosis is responsible for one in four of all avoidable adult deaths in developing countries. Increased frequency and accelerated fatality of the disease among individuals infected with human immunodeficiency virus has raised worldwide concern that control programmes may be inadequate, and the emergence of multidrug-resistant strains of Mycobacterium tuberculosis has resulted in several recent fatal outbreaks in the United States. Isonicotinic acid hydrazide (isoniazid, INH) forms the core of antituberculosis regimens; however, clinical isolates that are resistant to INH show reduced catalase activity and a relative lack of virulence in guinea-pigs. Here we use mycobacterial genetics to study the molecular basis of INH resistance. A single M. tuberculosis gene, katG, encoding both catalase and peroxidase, restored sensitivity to INH in a resistant mutant of Mycobacterium smegmatis, and conferred INH susceptibility in some strains of Escherichia coli. Deletion of katG from the chromosome was associated with INH resistance in two patient isolates of M. tuberculosis.


DNA electrophoresis in microlithographic arrays. We have used optical microlithography to fabricate capped quasi-two-dimensional obstacle courses in SiO2. We report here observations using epifluorescence microscopy of the electrophoresis and length fractionation of large DNA molecules confined in arrays. Simple reptation theory, based on the work of deGennes, predicts that at low electric fields the electrophoretic mobility of a polymer of length L much greater than the persistence length p scales inversely with L (ref. 2). But elongation of the coil in the matrix at sufficiently strong electric fields results in a length-independent electrophoretic mobility. The application of suitably timed pulsed electric fields restores the fractionating power of gels for long molecules but the protocols of pulsed-field electrophoresis are semi-empirical because the complex and ill-understood gel matrix plays a critical role in fractionation. Microlithographically constructed obstacle arrays, with their low dimensionality, small volume and extremely reproducible topography, will make it possible to understand the motion and fractionation of large polymer molecules in complex but well characterized topologies.


Candidate proto-oncogene bcl-3 encodes a subunit-specific inhibitor of transcription factor NF-kappa B. The NF-kappa B subunits p50 and p65 and the product of the rel proto-oncogene are members of a growing class of transcription factors with a unique DNA-binding and dimerization domain. Nuclear transfer of each of these factors is controlled by cytoplasmic inhibitors, and regulated by specific stimuli. The inhibitors I kappa B-alpha and -beta and pp40 recognize either p65 or the c-rel protein. We show here that the proto-oncogene bcl-3, believed to be involved in certain human B-cell leukaemias, encodes a protein that functions as an I kappa B-like molecule for native NF-kappa B but is specific for the p50 subunit. The ankyrin repeat domain of the bcl-3 product is shown to mediate complex formation with NF-kappa B dimers by contracting the conserved dimerization domain of NF-kappa B.


Automated real-time immunoassay of biomolecules. ImmunoDetection is a novel technique combining perfusion chromatography technology with antibodies to perform the steps of an immunoassay in a flow-through column format. Sensitive and precise measurements are performed in seconds to minutes using automated liquid chromatography instrumentation.


[Word association and schizophrenia--an empirical study]. Since Bleuler's (1911) work on dementia praecox, numerous studies on associative disturbances in schizophrenics have been published. Methodological flaws and difficulties in interpreting the disturbances consistently have brought about a certain dilemma: they are unanimously considered a basic disorder in schizophrenia, but the problem of their reliable evaluation has remained unanswered. 43 schizophrenics, 15 affective psychotics, and 46 normals were investigated in order to compare different forms of the word association test (WAT). The schizophrenics gave less common responses than the normals in the free association task. However, the two groups differed more widely in their performance when asked to respond "individually", "commonly", or using a multiple-choice associative test. The affective psychotics were similar in their response behaviour, partly to the schizophrenics (free and common response conditions; multiple-choice test), partly to the normals (individual response condition). The relationship of the WAT to personality characteristics (Eysenck's psychoticism), the influence of psychopathology and the course of the disease, the patients' well-being and self-report of speech and thought disorders are discussed.


[Artificial diseases (self-induced diseases)--a review]. Factitious disorders have gained greater significance in all medical specialties during recent years, and continue to pose difficult problems. At present factitious disorders can be divided into four sub-types, the pretence and/or production of somatic and/or psychic medical symptoms being central to all. Apart from disturbance of ego structure the self and the body image (self) a specific disturbance of the doctor-patient relationship is of pathognomonic value. An integrated therapeutic approach (combination of palliative measures, somatic therapy and psychotherapy) seems to be most successful. In the differential diagnosis, factitious diseases have to be delineated from many other diseases in which self-destructive behaviour may occur.


[Coping with depressive disorders].

[Coping with depressive disorders]. Coping behavior was assessed in 40 depressed patients during their hospitalization and 2 months later. Patients completed an antidepressive activity questionnaire and were interviewed. External assessment and patient self-assessment showed significant differences between patients with monopolar or bipolar depressive disorders and those with neurotic/reactive disorders. Patients treated with antidepressants reported more frequent and effective coping behaviors. There is a fundamental difficulty in distinguishing differentiate between symptoms of depression and certain coping behaviors (eg withdrawal). Only a longitudinal analysis of individual cases could help us to differentiate.


[Results of electroconvulsive therapy in restrictive indications. A retrospective study of 15 years]. In Tübingen ECT is restricted to severely ill patients who do not respond to other somatic therapies; especially to patients with endogenous depression and pernicious catatonia. Between 1976 and 1990, 45 patients were treated with ECT, of whom 22 suffered from endogenous depression and 10 from pernicious catatonia. Thirteen patients with other diagnoses (schizophrenic and schizoaffective psychoses, borderline schizophrenia and obsessive-compulsive disorder) were treated with ECT for severe depressive states after failure of psychopharmacological therapy. A positive therapeutic response to ECT was observed in 46% of patients with endogenous depression and in all 10 with pernicious catatonia. In the patients with schizophrenia and schizoaffective psychosis, borderline schizophrenia and obsessive-compulsive disorder, an amelioration of the depressive or anxiety syndrome was observed only in individual cases. Side effects of ECT were transit syndromes (20%), reversible amnestic syndromes (20%) and cardiac arrhythmias (6%). According to our results, ECT is highly effective in therapy-resistant endogenous depression and pernicious catatonia, and therefore remains a necessary part of psychiatric therapy.


[Prognosis, long-term sequelae and occupational reintegration 2-4 years after severe craniocerebral trauma]. 122 patients with severe head injuries were assessed in terms of their global outcome and social and vocational reintegration 2 to 4 years after trauma. Some patients were studied with regard to physical, psychological, psychosocial, and cognitive long-term consequences. Less than half of the patients (43%) were found to be without employment handicap, while the remainder showed various degrees of occupational difficulty. Most of the patients had significant long-term problems related to cognitive and psychosocial dysfunction. The cognitive disorder, physical handicap, age, and duration of unconsciousness are all important prognostic factors for vocational reintegration, neuropyschological impairment being the single most important factor.


[Differential neuroleptic treatment of schizophrenic psychoses in puerperium: advantages of the atypical neuroleptic clozapine]. Clozapine is an atypical neuroleptic drug characterised by specific pharmacological properties and clinical side effects, which are different from those observed with conventional neuroleptics such as drugs of the phenothiazine or butyrophenone classes. This report suggests a new clinical indication for clozapine, based on its negligible influence on plasma prolactin levels: the pharmacotherapy of schizophrenic psychoses in the post-partum period and in patients suffering from acute mastitis.


Nosography and immunopathogenesis of viral hepatitis. Five viruses are responsible for the vast majority of cases of viral related hepatitis. They have been named hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV). The more recent literature concerning the viral structure, the epidemiology, the serological identification, the clinical course and the prevention of each type of hepatitis is reviewed. HBV is not directly cytopathic. Hepatitis is a consequence of the destruction of the virus-infected cells. The efficient elimination of the virus relies on the viral antigenic determinants (HBs, pre-S1, pre-S2, HBc, HBe) and on the immune system of the host. The viral persistence may be caused by defect of the host immunity (interferon production, T and B lymphocyte function) or by factors related to the virus such as a genome mutation (lack of HBe formation). Some evidence suggesting an immunopathogenetic mechanism also for HCV, HDV and HAV is reported.


Prevention of viral hepatitis in dialysis units: isolation and technical management of dialysis. The contamination of dialysis machines is an important risk factor for the spread of viral hepatitis in hemodialysis units. The use of reserved machines in separate rooms is a safe prophylactic measure.


Renal transplant recipients and chronic liver disease: statistical evaluation of predisposing factors. 278 azathioprine and methylprednisolone (AZA)-treated and 406 ciclosporin (CS) treated patients with a kidney graft functioning for more than 1 year were investigated for the presence of chronic liver disease (CLD), defined as an increase in transaminases of 1.5 times the upper normal limits for a period of at least 12 months. The prevalence of CLD was 36 and 27% in the two groups, respectively. The univariate analysis showed that male sex, alcohol abuse and HBsAg positivity correlated with CLD onset in the AZA group while blood transfusions, length of dialysis treatment, pretransplantation CLD, HBsAg positivity and ferritin levels over 800 ng/ml correlated with CLD onset in CS. The multivariate analysis identified male sex and HBsAg positivity in the AZA group and age over 18 years, high ferritin levels and HBsAg positivity in the CS group as risk factors predictive of CLD onset. Liver failure represented the 4th cause of death in the AZA group but 1 of the 2 most important causes of death in CS in the long term. However, these drawbacks were overcome by the overall low mortality rate in CS. Therefore, renal transplantation should not be refused to patients positive for HBsAg and/or with preexisting liver disease.


Prevalence of hepatitides in our hemodialyzed population. We evaluated the prevalence of hepatitis in our hemodialysed population (65 patients, 37 M and 28 F). Screening for A and B hepatitis was tested with the RIA method and research of the anti-HCV with the immunoenzymatic method (Ortho HCV ELISA test of 2nd generation). 15 patients (23.07%) were anti-HCV positive (anti-HCV+); 23 (35.38%) showed positivity for 1 or more markers of B hepatitis (HBV+). A meaningful greater prevalence of B virus infections in anti-HCV+ patients (86.66%), compared to negatives, (20.00%) resulted. All non-A, non-B hepatitides are anti-HCV+. The dialytic treatment of the anti-HCV+ patients was meaningfully longer than in the negatives (p less than 0.05). The prevalence of the seropositive patients to B and C virus is not correlated to the number of transfusions, while it is to the number of surgical operations carried out in the predialytic period. This information suggests common pathogenetic mechanisms between the 2 forms of hepatitis and increased probability to find anti-HCV+ with a longer dialytic treatment.


Retrospective study on the prevalence of B and non-A, non-B hepatitis in a dialysis unit: 17-year follow-up. To evaluate the prevalence of HBV, non-A, non-B hepatitis (NANBH) and HCV, their evolution and the route of transmission, we performed a retrospective study (17 years) on 65 hemodialyzed patients (mean dialytical age 5 years, range 1-16) who developed acute viral hepatitis. Twenty-six percent of them were affected by HBV; 5 of them (29%) became chronic. Fourty-eight of 65 patients were affected by NANBH; in 1990, 42 of them were tested for antibody to HCV with the C-100 Elisa assay: 27 patients (64%) were positive. Seven of 27 patients demonstrated normal values of ALT. The evaluation of intrafamiliar transmission of HBV showed 8 sexual partners infected. In the family, no one was anti-HCV positive. In agreement with the current literature, we have observed: (1) a decrement in HBV infection; (2) that HCV is the major cause of NANBH; its rate of chronicity is over 50%; (3) that the prevalent mode of HCV transmission is the parenteral route while the sexual route seems to be negligible.


Non-A, non-B hepatitis in patients on dialysis: retrospective evaluation of measures taken to limit the diffusion of the virus. 329 patients were evaluated from 1978 to 1990. 40 new cases of non-A non-B hepatitis were observed, all regarding patients on hemodialysis. Since the beginning of 1989, we introduced measures to limit the diffusion of the virus: reduction to a minimum of blood transfusions and a new disinfection protocol for monitors, instruments and surroundings. Of the 40 new cases, 30 took place between January 1985 and March 1989, only 2 later. If we consider the annual incidence before and after the introduction of preventive measures, we can observe a significant reduction in NANB hepatitis incidence in the last 2 years.


Anti-hepatitis C virus epidemiological study in two dialysis centers in Florence. We studied the prevalence and incidence of anti-hepatitis C virus (HCV) antibodies in 350 patients during 15 months and looked for some risk factors. We found a significant correlation between anti-HCV positivity and length of dialysis treatment and treatment in more than one center. We propose some prophylactic rules.


Pharmacokinetics of recombinant human erythropoietin in dialysis patients after single and multiple subcutaneous administrations. The pharmacokinetics of recombinant human erythropoietin (rhEPO) were evaluated after single intravenous and single subcutaneous administration of 40 U/kg to 8 patients with dialysis treatment. All patients suffered from renal anemia with a hematocrit less than or equal to 24% and were treated with 40 U/kg rhEPO subcutaneously, three times a week for 6 weeks. At the end of the treatment period, kinetics of rhEPO were repeated. After the initial subcutaneous rhEPO dose, the following results were obtained: maximum plasma concentration 39.5 (26.7-56.9) U/l, area under the curve (AUC) 1,122 (582-3,220) U.h.1-1 and terminal half-life 13.2 (2.6-53.1) h. The corresponding data after multiple rhEPO doses were: maximum rhEPO plasma concentration 26.3 (9.4-49.1) U/l, AUC 724 (407-1,464) U.h.1-1 and terminal half-life 14.2 (3.5-24.4) h. There were no statistical significant differences between the two investigations. From the present study, it can be concluded that after a treatment period of 6 weeks with multiple subcutaneous rhEPO doses, rhEPO absorption as well as rhEPO elimination are unchanged.


Effectiveness and safety of recombinant human erythropoietin in predialysis patients. Austrian Multicenter Study Group of r-HuEPO in Predialysis Patients. Experience with erythropoietin in the treatment of anemia in predialysis patients is limited. A practical treatment regimen which minimized the number of outpatient visits was investigated. The Austrian multicenter study included 123 patients. At baseline, the treatment protocol mandated once weekly the administration of 10,000 U recombinant human erythropoietin (r-HuEPO) subcutaneously. The follow-up period was 3 months, and dose adjustments were made at montly intervals. At baseline, the mean values for creatinine were 6.2 +/- 0.2 mg/dl, and for hemoglobin (Hb) 9.0 g/dl. During 3 months of therapy, mean Hb increased to 10.8 g/dl and creatinine to 6.6 mg/dl. The initial r-HuEPO weekly dose was 10,000 U. The mean dose after 3 months was 9,000 +/- 4,000 U. There was no significant alteration of the slope of the reciprocal creatinine curve or of blood pressure values. No side effects occurred during the 3-month treatment period. In conclusion, the results of this multicenter trial demonstrate that using a simple once-weekly subcutaneous treatment regime, r-HuEPO can be administered safely and effectively in predialysis patients.


Anemia and chronic renal failure: a therapeutical approach by reduced glutathione parenteral administration. Authors report on the effect of reduced glutathione parenterally administered on the anemic status in patients suffering from chronic renal failure and undergoing hemodialysis. Twenty patients were studied for 180 days and were divided into two age- and sex-matched groups. The first group (10 patients) received placebo, the second group (10 patients) received the treatment (1,200 mg of reduced glutathione). Reduced glutathione and placebo were given for 120 days in a randomized double-blind fashion and the following measurements were performed: red blood cells reduced and oxidized glutathione, plasma reduced and oxidized glutathione, hematocrit, hemoglobin, reticulocytes, serum iron, transferrin, indirect bilirubin, urea, creatinine, calcium, phosphate, parathyroid hormone and alkaline phosphatase. In the treated group, during the supplementation period, there was an increase in the levels of red blood cells and plasma reduced glutathione, hematocrit and hemoglobin and a concomitant decrease in plasma oxidized glutathione and reticulocytes with a maximum effect on the 120th day of therapy. In the placebo-treated group there were no significant variations of the parameters considered during the study period. When the therapy, on patients undergoing treatment, was terminated there was a drop in the analyzed parameters, which fell to pretreatment values at the subsequent controls. These findings seem to indicate that reduced glutathione could represent a useful drug in the treatment and management of anemia in patients affected by chronic renal failure.


Comparison of lipids, apoproteins and associated enzyme activities between diabetic and nondiabetic end-stage renal disease. Lipids, apoproteins and associated enzyme activities in type 2 diabetic end-stage renal disease (ESRD) were compared with that in nondiabetic ESRD and normal controls. Of the 40 uremic patients with non-insulin-dependent diabetes mellitus, 20 patients were receiving stable continuous hemodialysis treatment (CHT). Of the 39 patients with nondiabetic ESRD, 21 were undergoing CHT. Patients with nondiabetic ESRD exhibited elevated levels of serum triglyceride and a marked reduction in high-density-lipoprotein (HDL) cholesterol. Concentrations of serum apolipoprotein (Apo) C-3 were higher than in controls, whereas mean levels of serum Apo E were lower. The concentrations of serum Apo A-1 and Apo A-2 decreased with diminished lecithin: cholesterol acyltransferase activity. Lipoprotein lipase activity decreased in undialysed patients, and hepatic triglyceride lipase activity decreased significantly throughout the observation. Patients with diabetic ESRD exhibited elevated serum Apo B and normal serum Apo E levels, besides the lipid and Apo abnormalities observed in nondiabetic ESRD. Moreover, a prominent reduction in serum Apo A-1 was found in dialysed diabetic patients. The Apo B/Apo A-1 ratio was significantly higher in diabetic ESRD than in nondiabetic patients undergoing CHT. These results indicate that lipid abnormalities are accelerated in diabetic ESRD and may constitute a serious risk for the development of atherosclerosis.


Heparan sulfate proteoglycans are lost in patients with diabetic nephropathy. The pathogenesis of diabetic nephropathy relative to the changes in the glomerular extracellular matrices was investigated. Renal tissues from 10 diabetic patients were immunostained with antibodies directed against heparan sulfate proteoglycans (HS-PGs), laminin, type IV collagen and fibronectin. Seven patients were nephrotic and had advanced glomerulosclerosis with nodular lesion, while the other 3 had no renal manifestations or minor glomerular tissue alterations. Controls included kidneys removed from patients with renal tumors and specimens obtained by renal biopsy from patients with IgA nephropathy. Relationships among proteinuria, intensity of fluorescence and glomerular changes were studied. In diabetes 3 patients with minor glomerular lesions were found to have no changes in various components of extracellular matrices. A marked reduction in the intensity of staining with anti-HS-PG antibodies was observed in renal specimens from patients with nodular glomerulosclerosis and proteinuria, while a mild decrease in the intensity of fluorescence was observed in tissues stained with antilaminin antibodies. An increase compared to normal control sample findings in type IV collagen and fibronectin was observed in the mesangium of sclerosing glomeruli. No loss of HS-PG was observed in patients with IgA nephropathy. These results indicate that glomerular extracellular matrix HS-PG is lost in association with diabetic nephropathy; this loss results in alteration of the charge-selective properties of glomerular capillaries. This alteration may, in part, be the cause of the proteinuria associated with diabetic nephropathy.


Determinants of intact parathyroid hormone and free 1,25-dihydroxyvitamin D levels in mild and moderate renal failure. Parameters of calcium and phosphate metabolism were measured in 27 patients with mild renal failure [glomerular filtration rate (GFR) 40-90 ml/min], 12 patients with moderate renal failure (GFR 20-39 ml/min) and in 12 healthy subjects. GFR was determined by technetium-99m diethylenetriamine penta-acetic acid clearance. Intact parathyroid hormone (PTH) was measured by a sensitive immunochemiluminometric assay and somatomedin-C was determined by radioimmunoassay. Both 1,25-dihydroxyvitamin D [1,25(OH)2D] and vitamin-D-binding protein were measured allowing calculation of the free 1,25(OH)2D index. By linear regression and multivariate analysis, PTH was negatively and independently correlated with GFR, plasma bicarbonate and 25-hydroxyvitamin D [25(OH)D] while free 1,25(OH)2D was positively correlated with GFR. Increased PTH secretion and reductions in 1,25(OH)2D were present in mild renal failure patients before any changes in plasma calcium, phosphate and bicarbonate were noted. Plasma alkaline phosphatase was significantly higher in mild chronic renal failure patients compared to normal subjects, possibly indicating early effects of the secondary hyperparathyroidism on the skeleton. Somatomedin-C did not correlate with the free 1,25(OH)2D index or a measure of 1,25(OH)2D production. It is concluded that the secondary hyperparathyroidism which occurs very early in the onset of chronic renal failure may be due to a reduction in the circulating concentration of 1,25(OH)2D consequent upon the renal failure. Low plasma bicarbonate and 25(OH)D also appear to be determinants of a raised PTH concentration. The compensatory increase in PTH presumably maintains extracellular calcium and phosphate levels constant but with possible deleterious effects on the skeleton.


Decreased renal clearance of xanthine and hypoxanthine in a patient with renal hypouricemia: a new defect in renal handling of purines. Renal handling of urate, xanthine and hypoxanthine was studied in a hypouricemic patient who had increased plasma concentrations of xanthine and hypoxanthine. The patient, a 50-year-old man, had been suffering from Parkinson's disease, while neither systemic disorders nor particular renal diseases known to affect plasma purine levels were found. His serum urate level was 58 +/- 6 mumol/l (healthy controls for males, 310 +/- 48 mumol/l, mean +/- SD) and the renal uric acid clearance was 3 times higher than that of the controls, establishing a diagnosis of renal hypouricemia. Xanthine and hypoxanthine concentrations in the plasma were elevated to 1.3 +/- 0.1 mumol/l (controls, 0.5 +/- 0.3) and 5.9 +/- 3.5 mumol/l (controls, 1.6 +/- 0.4), respectively. Both renal xanthine and hypoxanthine clearance was only half the value of the controls, indicating reduced urinary excretion of xanthine, and hypoxanthine appears to be responsible for their elevation in plasma. A probenecid loading test revealed no response of urinary urate excretion but normal responses of xanthine and hypoxanthine excretion. However, urinary excretion of urate, xanthine or hypoxanthine did not respond at all to pyrazinamide administration. These findings indicate that the patient had a defective renal handling of xanthine and hypoxanthine as well as urate.


Two cases in whom pyrazinamide does not inhibit the uricosuric action of benzbromarone. The 2 subjects were a diabetic male with renal hypouricemia and a healthy male with normouricemia. In these subjects, 200 mg of benzbromarone increased fractional uric acid clearance (FUa) and 3.0 g of pyrazinamide decreased FUa. However, pyrazinamide did not inhibit the uricosuric action of benzbromarone at all on the administration of 3.0 g of pyrazinamide together with 200 mg of benzbromarone. These results indicated that in these cases, the relative role of each component could not be determined on the basis of the hypothetical four-component model.


Role of platelet cytosolic calcium in regulation of blood pressure in patients on chronic hemodialysis. We measured blood pressure, platelet cytosolic calcium concentration ([Ca2+]i) and vasoactive hormones in 20 anuric normotensive patients on chronic hemodialysis (HD) to clarify the role of cytosolic calcium in regulation of blood pressure. All measurements were performed at the end of HD (post-HD) and at the beginning of the next HD (pre-HD). During the HD-HD interval, mean blood pressure increased from 85 +/- 4 to 94 +/- 4 mm Hg (mean +/- SEM, p less than 0.05), but the median [Ca2+]i in platelets did not show any significant change (133 +/- 5 vs. 136 +/- 5 nmol/l). Although all patients were within their ideal body weight at post-HD, platelet [Ca2+]i did not correlate with mean blood pressure. By contrast, platelet [Ca2+]i correlated with mean blood pressure (r = 0.489, p less than 0.05) at pre-HD. Moreover, platelet [Ca2+]i at post-HD also correlated with changes in mean blood pressure during the HD-HD interval (r = 0.532, p less than 0.05). In addition, there were negative correlations between platelet [Ca2+]i and plasma renin activity both post- and pre-HD (r = -0.652, p less than 0.005; r = -0.673, p less than 0.005; respectively). These results suggest that cytosolic calcium plays important roles in regulation of renin secretion and in the elevation of blood pressure induced by volume expansion in patients on chronic HD.


Trace elements in dialysis fluids and assessment of the exposure of patients on regular hemodialysis, hemofiltration and continuous ambulatory peritoneal dialysis. Forty-four elements (Al, Sb, As, Ba, Be, B, Br, Cd, Ce, Co, Cr, Cs, Cu, Eu, Ga, Au, Hf, In, Ir, Fe, La, Lu, Mn, Hg, Mo, Nd, Ni, Pb, Rb, Sm, Sc, Se, Ag, Sr, Ta, Tb, Tl, Th, Sn, W, U, V, Zn, Zr) have been determined in the dialysate for hemodialysis (HD) and fluids for hemofiltration (HF) and continuous ambulatory peritoneal dialysis (CAPD). Multiple determinations have been performed for each dialysis fluid. Several trace elements (TE) showed remarkably elevated average levels; moreover, different bathes of the same commercial product may present a wide variability in TE concentration. The data point out the pivotal role of dialysis fluids in contributing to TE imbalance in dialysis patients and allow the assessment of the potential element exposure of patients on regular dialytic treatment. Patients on HD treatment would be exposed on a weekly basis to milligrams of Al, B, Ba, Br, Cu, Fe, Ni, Pb, Rb, Sr and Zn; on HF, the highest exposures are due to Al, B, Br, Fe, Pb and Zn; on CAPD to B, Br, Fe and Zn. The weekly exposure for several TE appears to be 50- to 12,000-fold higher than the corresponding values on the amount absorbed via the diet (HD: Au, Ba, Be, Ce, Ga, La, Sc, Ta, Th, V, Zr; HF: Be, Ce, Ta, Th, V, Zr; CAPD: Au, Be, Ce, Ga, V, Zr).(ABSTRACT TRUNCATED AT 250 WORDS)


Glutathione monoethyl ester moderates mercuric chloride-induced acute renal failure. Glutathione (GSH)-dependent reactions are an important cellular defense against ischemic or oxidative injury, although their role in toxin-induced renal cellular injury is less clear. Because of the known sulfhydryl reactivity of mercury (M), we hypothesized that GSH could modify mercuric chloride (MC)-induced acute renal failure (ARF). Therefore, we evaluated the effects of glutathione monoethyl ester (GE), which produces high intrarenal levels of GSH, on the nephrotoxicity of MC. GE treatment in normal rats did not alter their creatinine clearance (CCr), fractional sodium (CNa/CCr) or lysozyme (CLy/CCr) excretion, but histologically resulted in prominent proximal tubular vacuolization. GE pretreatment in rats with MC-induced ARF resulted in partial preservation of their CCr, CNa/CCr and CLy/CCr. Renal histology also demonstrated a reduction in tubular necrosis. M content in the renal cortex 3 following MC was lower in the MC + GE group, but levels were higher in the liver and inner stripe/inner medulla as compared to animals receiving MC alone. No differences were seen in the outer stripe at 3 h or in any of the tissues 24 h following MC injection. Thus, GE moderated MC-induced ARF, likely by providing a large intracellular sulfhydryl pool and thereby reducing M reactivity with endogenous cellular proteins and enzymes.


Synthesis of fibronectin by isolated glomeruli from nephrectomized hypertensive rats. The accumulation of extracellular matrix (ECM) is an important feature of most forms of progressive glomerular diseases. In order to examine the relationship between ECM synthesis and glomerulosclerosis, we evaluated fibronectin synthesis by glomeruli with the immunoprecipitation of conditioned media from isolated glomeruli in 5/6 nephrectomized spontaneously hypertensive rats (5/6N-SHR). There was no difference in blood pressure between 5/6N-SHR and control SHR throughout the experiment. Two weeks after the nephrectomy, most of the glomeruli were intact and no difference in the synthesis of fibronectin was observed between either groups. Twenty weeks after the nephrectomy, marked glomerulosclerosis associated with an increase in urinary protein was revealed in 5/6N-SHR but no glomerular lesions in control SHR. The synthesis of fibronectin by isolated glomeruli increased in 5/6N-SHR compared to control SHR. The administration of enalapril or hydralazine + reserpine + hydrochlorothiazide markedly attenuated the glomerular sclerosis and urinary protein excretion to a comparable degree, although the later therapy reduced blood pressure more effectively. These antihypertensive therapies also suppressed fibronectin synthesis in the 5/6N-SHR group at week 20. In conclusion, increased synthesis of glomerular fibronectin appeared to contribute to the glomerulosclerosis caused by subtotal nephrectomy and hypertension.


[Research and surgical treatment of epilepsy]. The currently available surgical procedures for the treatment of epilepsy, from fundamental data to therapeutic results, including various means of investigation are reported. The work is based on a review of the literature and on the cases studied by the two teams from the Universities of Montreal and Bordeaux who share the same concept of epilepsy surgery. The patient groups of the two teams include 316 S.E.E.G., 214 cortectomies, 39 callosotomies and 2 multiple sub-pial transsections. In the first part, the authors attempt to demonstrate that the epileptic focus corresponds to the region where the seizures arise, that this focus is not directly comparable to the region where inter-ictal spikes are recorded and sometimes becomes autonomous from the causal lesion. The epileptic phenomenon has a definite harmful effect on cerebral functions and a probable self-aggravating potential. The second chapter summarizes the clinical data on which the indications and contraindications are based. These obviously depend on whether the intervention is intended to be curative or palliative. Various non-invasive and invasive investigations are then reviewed, according to their relative importance and the experience of each team. The main points developed are: the electroclinical correlations during seizures, the symptomatological data for differentiating between temporal and frontal lobe seizures, the contribution of M.R.I. in demonstrating the epileptogenic and epileptic lesions, the electrophysiological information suggesting that S.E.E.G. remains the most informative mean of investigation. The various methods of investigation of assessing electrical, functional (cerebral blood flow, metabolism) and morphological aspects of epilepsy, supply non-redondant findings about the localisation of the epileptic focus. The chapter on surgical techniques mainly discusses the various modes of implantation of subdural and intracerebral electrodes and reports the same rate of morbidity in both cases. Orthogonal teleradiography is still perfectly suited to the implantation of intracerebral electrodes. S.E.E.G. is still the most anatomically precise technique. However, in certain conditions, extraoperative E.Co.G. is more adequate. New surgical modalities have recently appeared such as the multiple subpial transsections which allow treatment of epileptic foci unapproachable by cortectomy and such as modified techniques of hemispherectomy, which by decreasing morbidity, renew interest in them. In the chapter on surgical results, the authors emphasize the methodological problems of evaluation that partly account for their wide variability. The results obtained with the various surgical modalities are reviewed. The outcome in cortectomies is discussed at length in terms of the data from the literature as well as the results reported by both teams.(ABSTRACT TRUNCATED AT 400 WORDS)


Modulation of the in vitro electrophysiological effect of corticosterone by extracellular calcium in the hippocampus. The electrophysiological effects of various concentrations of corticosterone (CORT) on the hippocampus were investigated using extracellular recordings from CA1 hippocampal slice preparations subjected to four different extracellular concentrations of calcium. In all cases, the CORT effect was manifested by a PS amplitude decrease without affecting the slope of either the input volley or the EPSP. Our results showed that, for the lowest extracellular calcium concentrations (1.3 and 2.5 mM), the inhibitory effect of CORT on the PS amplitude appeared only with the supra-physiological 10,000 nM CORT concentration, whereas for the highest ones (3.13 and 5.32 mM) this effect was observed, respectively, with 5 and 0.05 nM CORT concentrations. The antiglucocorticoid RU 486, used in combined application with CORT, blocked the electrophysiological effect of CORT. The possible involvement of calcium-dependent mechanisms at the CORT receptor level or in the final cellular response are discussed.


Effects of angiotensin II on LHRH release, as measured by in vivo microdialysis of the anterior pituitary gland of conscious female rats. The present experiments assessed the effects of central administration of angiotensin II (Ang II) on mean levels of luteinizing hormone-releasing hormone (LHRH) in the extracellular fluid of the anterior pituitary gland, monitored by in vivo microdialysis. Ovariectomized rats were tested under two conditions: (1) nonhormone-treated where Ang II infusion inhibits luteinizing hormone (LH) release, and (2) ovarian hormone-treated where Ang II stimulates LH secretion. Animals were ovariectomized and chronic guide cannulae were implanted, one into the lateral cerebral ventricle for infusion of Ang II and one directed toward the anterior pituitary gland for the insertion of the microdialysis probe. Approximately 1 week later, the dialysis probe was inserted and cemented into place. The length of the dialysis probe transected the pituitary gland from its dorsal to ventral aspects. Dialysis samples were collected at 15-min intervals. Levels of LHRH were continuously monitored in nonhormone-treated animals, prior to and during intracerebroventricular (i.c.v.) infusion of Ang II. The dialysis probe was removed at the end of the experiment. One week later, the same animals were treated with estrogen and progesterone and dialysis of the anterior pituitary gland was performed 3 days later using a protocol identical to the first dialysis sampling session. A separate group of animals was tested to confirm the effects of lateral ventricle infusion of this dose of Ang II on LH release. There were no detectable values of LHRH in dialysis samples from non-hormone-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)


Angiotensinogen secretion by single rat pituitary cells: detection by a reverse haemolytic plaque assay and cell identification by immunocytochemistry. A reverse haemolytic plaque assay (RHPA) for angiotensinogen was developed in rat hepatoma H4 cells and applied to investigate the possible secretion of angiotensinogen from rat pituitary cells in primary culture. Over a 24-hour incubation period in Cunningham chambers plaques with a mean area of 2,800 +/- 430 and 590 +/- 220 microns2/plaque (SD, n = 6) formed around all viable H4 cells and 2.8 +/- 0.59% of viable pituitary cells respectively. As a positive control PRL secretion from lactotrophs was routinely checked by the RHPA and shown to form plaques with a mean area of 4,050 +/- 1,850 microns2/plaque after a 4-hour incubation. By comparing plaque size in H4 cells with angiotensinogen release in cell culture, as quantified by radioimmunoassay, the secretion rate of angiotensinogen from pituitary cells was calculated as 22 +/- 8 ng/10(6) cells/24 h. Plaque-forming cells consisted of two morphologically distinct populations; 78% being small cells (less than 6 microns diameter) containing little cytoplasm and 22% were large (greater than 9 microns diameter) cells with an abundant cytoplasm. Immunocytochemical staining of pituitary cells after formation of plaques with anti-angiotensinogen, anti-LH and anti-PRL antiserum showed that the large plaque-forming cells were gonadotrophs and none were lactotrophs. All plaque-forming cells stained for angiotensinogen but only 44% of the viable cells which stained for angiotensinogen actually formed plaques. The possibility that cellular angiotensinogen was imported from extracellular sources was investigated by incubation of pituitary cells with pure 125I-angiotensinogen for periods up to 24 h. No uptake of the radiolabelled protein was found.(ABSTRACT TRUNCATED AT 250 WORDS)


Influences of hypothyroidism on TRH concentrations and preproTRH mRNA levels in rat hypothalamus: a simple and reliable method to detect preproTRH mRNA level. To gain further insight into the regulation of hypothalamic TRH by thyroid hormones, we measured TRH concentration in specific hypothalamic nuclei and preproTRH mRNA levels in the anterior hypothalamus. Adult male rats were decapitated 1, 7, 14 days after thyroidectomy. Micropunches by the method of Palkovitz from seven hypothalamic nuclei and median eminence were used for measurement of TRH by radioimmunoassay. As compared with normal levels, TRH concentration significantly decreased in the median eminence and five hypothalamic nuclei including paraventricular nucleus (PVN), posterior nucleus, anterior nucleus, arcuate nucleus, and ventromedial nucleus pars lateralis, by 7 days after thyroidectomy. No significant changes were observed in dorsomedial nucleus or ventromedial nucleus pars medialis until 14 days after thyroidectomy. A rapid and simple method to detect specific mRNA for preproTRH was developed using the polymerase chain reaction and a single anterior hypothalamic section. PreproTRH mRNA levels in the anterior hypothalamus increased approximately twice 7 days after thyroidectomy. These data indicate that thyroidectomy caused a marked increase in preproTRH mRNA levels of the anterior hypothalamus, while it significantly reduced TRH concentrations not only in PVN and median eminence but also in other specific hypothalamic nuclei, suggesting that these nuclei might be involved in the thyrotropin regulation in the hypothalamus.


Evidence that circadian variations of circulating melatonin levels in fetal and suckling rats are dependent on maternal melatonin transfer. Although the circadian variation of melatonin content in the pineal gland appears during the 3rd week of extrauterine life, recent studies suggest that the fetus perceives the day length through maternal melatonin transfer. Accordingly, we determined serum melatonin concentrations in pregnant and lactating rats and in their offsprings during the day (D) and at night (N). As compared with nonpregnant adult female rats (D: 6.0 +/- 0.5 and N: 112.0 +/- 5.0 pg/ml), significant increases of serum melatonin concentrations, both during the day and at night, were observed in 21-day pregnant (D: 21.0 +/- 2.6 and N: 222.0 +/- 7.2 pg/ml) and in 10-day (D: 20.0 +/- 2.6 and N: 145.0 +/- 12.3 pg/ml) and 20-day (D: 19.0 +/- 1.2 and N: 140.0 +/- 8.0 pg/ml) lactating animals, while a profound decrease was found in 5-day (D: 13.0 +/- 1.0 and N: 50.0 +/- 5.6 pg/ml) lactating rats. In addition, melatonin levels were significantly higher during the night than during the day in all the experimental groups. High levels and circadian variation of serum melatonin were detected also in 21-day-old fetuses (D: 13.0 +/- 0.8 and N: 108.0 +/- 4.8 pg/ml) and in 5- and 10-day-old suckling (in 5-day-old, D: 13.0 +/- 0.6 and N: 71.0 +/- 3.1 pg/ml; in 10-day-old, D: 17.0 +/- 1.4 and N: 63.0 +/- 3.8 pg/ml) rats.(ABSTRACT TRUNCATED AT 250 WORDS)


Hypothalamic regulation of microtubule-associated protein phosphorylation in lactotrophs. Endogenous phosphorylation of microtubule-associated proteins was examined in cultures of anterior pituitary cells from estradiol-treated rats. The cells were incubated for 1 h with 32P-orthophosphate and challenged for different times with removal of dopamine (DA), the addition of TRH in the presence of DA, or the transient removal of DA followed by addition of TRH. Microtubules were bundled by taxol followed by electrophoretic separation of the phosphorylated proteins and autoradiography. Within 10 s to 1 min of any of the treatments increased labeling of eight phosphoproteins (64, 80, 95, 110, 125, 155, 205 and 300 kDa) appeared in autoradiograms. The pattern of labeling in response to DA withdrawal was longer-lasting than that induced by TRH, whose effect disappeared by 10 min. The administration of TRH after a transient 10-min withdrawal of DA increased the magnitude and prolonged the duration of the effect of TRH. The 80-kDa microtubule-associated protein comigrated with the well characterized heat-stable, acid-soluble protein substrate for protein kinase C (PKC). The migration of the proteins following two-dimensional polyacrylamide gel electrophoresis and autoradiography was identical. Furthermore, the sequential extraction of microtubule-associated proteins followed by extraction of heat-stable, acid-soluble proteins showed a phosphoprotein of Mr 80 kDa. These observations suggest that the ubiquitous, heat-stable, acid-soluble 80-kDa phosphoprotein that is a specific substrate for PKC is associated with microtubules in lactotrophs. Furthermore, the levels of microtubule-associated phosphoprotein are increased following hormonal activation of PKC, although it is unclear whether this increase represents translocation of the phosphoprotein or phosphorylation of a previously associated protein.


Slowing of electroencephalogram and choline acetyltransferase activity in post mortem frontal cortex in definite Alzheimer's disease. Twenty-five (96%) of 26 patients with histologically verified moderate to severe Alzheimer's disease had abnormal electroencephalograms. The patients with the slowest (5-6 Hz) dominant occipital rhythms had significantly lower choline acetyltransferase activity in the post mortem frontal cortex than the patients with highest rhythm (8-9 Hz) (analysis of covariance adjusted for the neuropsychological test score). Concentrations of dopamine, noradrenaline or serotonin in the frontal cortex did not differ in the patient groups with the slowest and highest rhythms. Neither did scores of senile plaques or neurofibrillary tangles differ between these groups. In Alzheimer patients, the frequency of the dominant occipital rhythm correlated with the total score of the neuropsychological test (r = 0.58, P less than 0.01) and with the subscales of praxic functions and expressive speech, memory and general reasoning. The results suggest that the cholinergic deficit may contribute to the slowing of the electroencephalogram found in patients with Alzheimer's disease.


The barbiturate thiopental reduces ATP levels during anoxia but improves electrophysiological recovery and ionic homeostasis in the rat hippocampal slice. The barbiturate anesthetic thiopental enhances recovery of the evoked population spike recorded from rat hippocampal slices after short periods of anoxia. Thiopental reduces changes in sodium, potassium and calcium but enhances the fall in ATP levels during anoxia. The postsynaptic population spike recorded from the CA1 pyramidal cell region of the slices treated with thiopental (600 microM) recovered to 67% of the preanoxic amplitude after 3.5 min of anoxia. There was less recovery (24%) when a lower concentration of thiopental (250 microM) was used. Untreated slices recovered to only 10% of their preanoxic amplitude after 3.5 min of anoxia. Other studies have demonstrated that maintaining ATP levels during anoxia may be an important mechanism of protection. In contrast to those studies, thiopental was protective although it enhanced the fall of ATP levels after 3.5 min of anoxia in the CA1 region and after 3.5 and 5 min in the dentate region. Thus enhanced recovery of the population spike with thiopental is not due to its preservation of ATP levels. This result allows a clear separation of improved ATP levels during anoxia from other mechanisms of protection. We therefore looked for other mechanisms of protection. Sodium and potassium levels were measured after 10 min of anoxia. In untreated tissue, sodium levels in the slice rose and potassium levels fell significantly. In thiopental-treated tissue, changes in sodium and potassium caused by anoxia and by veratridine under normoxic conditions were significantly reduced. During anoxia calcium-45 uptake increases; thiopental significantly reduces this uptake.(ABSTRACT TRUNCATED AT 250 WORDS)


Desensitization of GABA-induced currents in cultured rat hippocampal neurons. The basic characteristics of desensitization of the GABAA receptor were investigated in cultured rat hippocampal neurons (three days to four weeks in vitro) using whole cell patch clamp techniques. GABA at 10-500 microM was perfused on to neurons for 30 or 60 s, with 60 s intervals of wash with control bath solution between perfusions. Desensitization, evaluated by peak-to-plateau ratio and time constants of current decay (tau), was dose-dependent and culture age-dependent. Desensitization was observed as early as three days in culture, the earliest time tested. At all ages, higher concentrations of GABA induced both larger and faster desensitization. Desensitization was markedly voltage-dependent and decreased with depolarization; peak-to-plateau ratio went from 6.3 to 1.4 and tau went from 4.6 to 26.8 s when holding potentials were changed from -80 mV to +30 mV. Low concentrations of GABA (1-2 microM) perfused for 2-60 s, which did not induce any current, had no effect on the maximal response nor desensitization produced by a subsequent application of 100 microM GABA. This finding suggests that GABA receptors were not desensitized without first being activated.