[Experimental study of the regenerative potentialities of the cardiac and somatic musculatures]. In rats a portion of the cardiac wall was ablated, auto-, homo- and heterotransplantation of the myocardium into somatic muscles of the femur was made and somatic muscles were autotransplanted into the heart wall. The material was treated by general histological and histochemical methods. Spindle-shaped "cardiac myoblasts" were formed of muscle fibres of the wound sites remote from sutures, polygonal "intermediate" cells often disposed in the shape of epithelioid strands were formed closer to sutures as well as in auto- and homotransplants. In heterotransplants and wound portions near sutures degenerating cells were prevailing. Regenerative processes manifested themselves in the wound and myocardium transplants as mitoses of cardiac muscular nuclei, formation of "cardiac myoblasts" and "intermediate" cells, mitotically dividing. However, no secondary differentiation occurred. When autotransplanting somatic muscles in the cardiac wall, unlike myocardium transplants, there occurred seondary differentiation of myoblasts and myosymplasts with formation of muscular fibres. In later terms muscular elements of transplants died and at the site of wound a scar was formed.


[Functional state of the human adrenals according to morphological analysis data and some remarks on the method of studying them]. When studying the morphology of the adrenals sufficiently exact criteria for their evaluation are necessary. Some of them may be obtained from complex macro- and microscopical investigations with the use of biometry and mathematical statistics. The work analyzes macroscopical (weight, volume, specific gravity) and microscopical (qualitative -- plethora, content of lipids, foci of cytolysis, mosaity etc. and quantitative -- absolute and relative dimensions of zones, cyto- and karyometry, interrelationship of zones, their relationship with KNP as a whole) data. On their grounds the estimation of the functional state of the adrenal cortex is made depending on the mechanism and causes of death as well as on duration of the disease.


[Cerebral actinomycosis. Anatomo-clinical case report and review of the Brazilian literature]. A case of central nervous system actinomycosis is reported. A 33-year-old male complained of headache, vomiting and blurred vision lasting for eight days prior to admission. On examination, a right hemiparesis, as well an intracranial hypertension were detected. The cerebrospinal fluid showed mild lymphomononuclear hypercytosis. Necropsy disclosed three abscess in the cerebral hemispheres, in addition to moderate cerebral edema on the left side but without purulent leptomeningitis. Actinomyces filaments and granules were demonstrated in the cerebral and lung abscessess. The Brazilian literature on actinomycosis is reviewed and six published cases with nervous system involvement were found. Relevant clinical and anatomical aspects of the cases and of the present one are discussed.


[Prognosis of neurotic patients under ambulatory care]. The prognosis of neurotic patients followed on a outpatient service was determined by calculation of probabilities of "improvement", "worsening" or "lack of changes" on successive examinations. These criteria were established for evaluation of 157 patients, 20 to 59 years old, who entered the study during a 12 month observation period.


The diagnosis and management of keratomycoses. II. Medical and surgical management. Medical management of 61 cases of mycotic keratitis, including the use of natamycin (pimaricine) in 53 cases, resulted in successful healing in 46 cases. A final visual acuity of 20/40 or better was achieved in 25 cases by medical therapy alone. Thirteen cases were considered medical treatment failures, and 11 necessitated therapeutic surgery. A final visual acuity of 20/70 or better was achieved in six of these cases, including five of nine therapeutic penetrating keratoplasties. In four cases, ulceration had progressed despite natamycin treatment, but fungal cultures were negative at the time of therapeutic surgery.


Scleral strength impairment and recovery after diathermy. Compression force rupture experiments were conducted in dog eyes at varying time intervals after scleral diathermy to determine the time variable of scleral weakening and recovery. The sclera was weakened immediately after the diathermy, and there was progressive further weakening for three weeks and then gradual partial recovery to 60% of control eye strength at eight months, at which point the experiment was terminated.


[Bone tumours in the hip region (author's transl)]. 13 Patients with bone tumours in the hip region are presented. The frequent misinterpretation auf tumours in this locality and the faulty use of diagnostic possibilities are stressed. The use of homoioplastic transplantation after a large resection is presented as a therapeutic method for discussion.


[Therapy for ruptures of the rotator cuff of the shoulder (author's transl)]. The authors discuss the indication and various methods of repair of the rotator cuff tear. The results in 24 patients are evaluated with respect to postoperative pain and function.


[Ruptures of the extensor mechanism of the knee joint (author's transl)]. Ruptures of the quadriceps tendon and patellar ligament are rare among injuries of the extensor mechanism of the knee when compared to fractures of the patella. 39 cases of which 31 were re-examined are being discussed with regard to therapy and results. According to our findings, ruptures of the extensor mechanism are not exclusively an injury of higher age groups. Predisposing factors for ruptures of the quadriceps tendon and patellar ligament are preexisting disease or posttraumatic sequelae. Fresh or old injuries require different operative technics. Usually, fresh ruptures can be repaired by end-to-end sutures. Old injuries require plastic procedures. Different technics are being described. Postoperatively, cast fixation is mandatory. The post-operative period of remobilisation is largely depend upon preexisting degenerative joint changes as are the post-operative results. A significant increase in post-operative osteoarthritis did not occur. Early degenerative changes in the femoro-patellar joint as seen on tangential views by the Settegast technic lead to the conclusion that the operative reconstruction does not always take into consideration the biomechanics of the femoro-patellar articulation. Immediate operative reconstruction of the ruptured extensor mechanism of the knee joint seems mandatory.


Early jaundice after pig liver transplantation. Of a series of pigs surviving orthotopic liver allotransplantation with end-to-end anastomosis of the bile duct, 70% were noted to be jaundiced at the end of the first week after transplantation. Seven animals in a subsequent series were investigated biochemically, but operative cholangiography, and by liver biopsy seven days after transplantation, when the jaundice was maximal. There was definite cholangiographic obstruction in only one animal, and this finding was subsequently confirmed at autopsy. This was also the only animal in which bile culture was positive. Jaundiced animals appeared to have more marked histological evidence of rejection than non-jaundiced ones. The jaundice was probably a result of transient rejection and usually resolved spontaneously without immunosuppression.


Congenital defects of the nervous system of lambs. Sixty-seven defects of the nervous system of lambs were examined during a 3-year investigation. Thirteen (19.4 percent) were single defects, and 54 (80.6 percent) were associated with defects of other body systems. Twice as many male lambs as females were affected. The various defects were: hydrocephalus 15, spina bifida 9, meningocoele 8, perosomus elumbis 8, cyclopia 6, cerebellar agenesis 3, aprosopia 3, cranium bifidum 3, anencephaly 2, cerebellar fusion 2, meningo-encephalocoele 2, congenital ataxia 2, micrencephaly 2, cerebellar hypoplasia 1, and acrania 1. A number of lambs had more than 1 defect of the nervous system. Congenital defects of the nervous system were found in 1.5 percent of the 4,417 lambs autopsied.


Spina bifida with syringomyelia and meningocoele in a short-tailed cat. Spina bifida of the proximal coccygeal vertebral column with an associated meningocoele is described in a male, mixed breed, congenitally short-tailed cat. The meningocoele had opened onto the skin of the tail allowing periodic escape of cerebrospinal fluid. Other clinical signs exhibited by the animal were an abnormal hindquarter gait, paralysis of the tail and defective control of urination and defaecation.


The nature of the alkylation lesion in mammalian cells. Methylating agents may produce as many as nine alkylated purine and pyrimidine adducts in DNA, as well as forming phosphotriesters and inducing apurinic sites and strand breaks. Although some of these products are formed in proportionately small amounts, there are sufficient sites affected in the DNA of a mammalian cell to make even the most minor product of potential biological significance. It is not possible to specify the exact reaction sites resulting in biological damage, but it is possible to quantitate the excisiion-repair of such damage both in the bulk of the DNA and at DNA growing points. Excision-repair can be measured in the bulk of the DNA by determining the specific activity of the NaCl eluate of a benzoylated naphthoylated DEAE-cellulose column of extracts of cells after treatment and incubation in the presence of hydroxyurea and labeled thymidine. The average number of nucleotides inserted per methyl methanesulfonate-induced methyl group is 0.1, per apurinic site is 9. Repair in growing-point regions after methyl methanesulfonate treatment occurs to approximately the same extent as in the bulk of the DNA.


Enzymic mechanism of excision-repair in T4-infected cells. Excision of pyrimidine dimers from ultraviolet-irradiated DNA in a cell-free system of Escherichia coli infected with bacteriophage T4 consists of two different steps, one to induce a single-strand break at a point close to a pyrimidine dimer and the other to release dimer-containing nucleotide from the DNA. The enzymes responsible for these steps were isolated and the reactions were characterized; T4 endonuclease V introduces a break at the 5' side of a dimer and 5' in equilibrium 3' exonucleases, which are also induced by T4, act at the break to excise dimer-containing nucleotides. We isolated temperature-dependent v mutants, which exhibit increased sensitivity to UV at 42 degrees C but not at 30 degrees C, and found that the mutants induce temperature-sensitive T4 endonuclease V, indicating that the v gene of T4 is indeed the structural gene for T4 endonuclease V and that the enzyme is responsible for the first step of excision-repair. A possible mechanism of excision-repair in T4-infected cells is discussed.


Recovery of phage lambda from ultraviolet damage. Recovery of phage lambda from ultraviolet damage can occur, in the dark, through three types of repair processes as defined by microbiological tests: (1) host-cell reactivation, (2) prophage reactivation, and (3) UV reactivation. This paper reviews the properties of the three repair processes, analyzes their dependence on the functioning of bacterial and phase genes, and discusses their relationship. Progress in the understanding of the molecular mechanisms underlying the three repair processes has been relatively  slow, particularly for UV reactivation. It has been shown that host-cell reactivation is due to pyrimidine dimer excision and that prophage reactivation is due to genetic recombination (prereplicative). We provide evidence showing that neither of these mechanisms accounts for UV reactivation of phage lambda. Furthermore, UV reactivation differs from the other repair processes in that it is inducible and error-prone. Whether UV-damaged bacterial DNA is subject to a similar repair process is still an open question.


The Escherichia coli UV endonuclease (correndonuclease II). An endonuclease from Escherichia coli which acts specificially upon UV-irradiated DNA (correndonuclease II) and is absent from the uvrA and uvrB mutants has been isolated and partially chacterized. The enzyme is present in normal amounts in the urvC mutant. It elutes from phosphocellulose at about 0.25 M potassium phosphate (pH 7.5) and passes through dialysis tubing. The enzyme binds tightly to UV-irradiated DNA but does not bind to unirradiated DNA. The enzyme incises irradiated DNA to the 5' side of a pyrimidine dimer and leaves a 5'-phosphoryl terminus which can be resealed with polynucleotide ligase. The Km of the enzyme is about 1.5 X 10(-8) M dimers. Endonucleolytic activity of the enzyme is inhibited by caffeine with a KI of about 10mM.


The role of DNA polymerase I in excision-repair. The ability of three different DNA polymerase I mutants of Escherichia coli to carry out excision-repair was examined. Strains having the same genetic origin but carrying either the polAl, polA107, resAl, or pol+ alleles were compared. The rate of ultraviolet-induced dimer excision was slightly reduced, relative to that found in Pol+ strains, in the PolAl strains; greatly reduced in the PolA107 strains; and found not to occur in the resAl strain. Ultraviolet-light-induced repair synthesis as determined by the ultraviolet-stimulated incorporation of 3H-labeled 5-bromo-2'-deoxyuridine into DNA of the parental density showed that the polAl mutation resulted in an increase in repair replication, while the presence of the polA107 allele caused a reduction in the amount of repair synthesis relative to that of the Pol+ strain. The ResAl strain, however, showed no ultraviolet stimulation of the incorporation of the density label. These observations indicate that DNA polymerase I plays a key role in the excision-repair process in E. coli.


Involvement of Escherichia coli DNA polymerase-I-associated 5' in equilibrium 3' exonuclease in excision-repair of UV-damaged DNA. From comparative studies between Escherichia coli PolA107 cells (lacking 5' in equilibrium 3' exonucleoytic activity associated with DNA polymerase I) and the isogenic wild-type strain, and between the purified DNA polymerase I preparations isolated from these strains, it can be concluded that the 5' in equilibrium 5' exonuclease is involved in excision of pyrimidine dimers in E. coli. Evidence is presented that the polA107 mutation is located on that part of the DNA polymerase I gene coding for the small fragment on which 5' in equilibrium 3' exonucleolytic activity is found.


Exonuclease VII of Escherichia coli.

Exonuclease VII of Escherichia coli. A new exonuclease of Escherichia coli K12, exonuclease VII, has been purified 1700-fold and characterized. The enzyme is specific for single-stranded DNA and can  initiated hydrolysis at both 5' and 3' termini. It is also capable of thymine-dimer excision in vitro. The limit products of the reaction are oligonucleotides, predominantly in the range of tetramers to dodecamers. DNA is hydrolyzed by the enzyme in a processive fashion. Mutants of E. coli have been isolated having reduced levels of exonuclease VII activity in crude extracts. Mapping studies place the exonuclease VII locus between 45 and 56 minutes on the E. coli K12 linkage map.


Repair replication in permeabilized Escherichia coli. We have examined the modes of DNA synthesis in Escherichia coli strains made permeable to nucleoside triphosphates by treatment with toluene. In this quasi in vitro system, polymerase-I-deficient mutants exhibit a nonconservative mode of synthesis with properties expected for the resynthesis step of excision-repair. This UV-stimulated DNA synthesis can be performed by either DNA polymerase II or III and it also requires the uvrA gene product. It requires the four deoxynucleoside triphosphates; but, in contrast to the semiconservative mode, the ATP requirement can be partially satisfied by other nucleoside triphosphates. The ATP-dependent recBC nuclease is not involved. The observed UV-stimulated mode of DNA synthesis may be part of an alternate excision-repair mechanism which supplements or complements DNA-polymerase-I-dependent repair in vivo.


Repair by genetic recombination in bacteria: overview. DNA molecules that have been damaged in both strands at the same level are not subject to repair by excision but instead can be repaired through recombination with homologous molecules. Examples of two-strand damage include postreplication gaps opposite pyrimidine dimers, two-strand breaks produced by X-rays, and chemically induced interstrand cross-links. In ultraviolet-irradiated bacteria, the newly synthesized DNA is of length equal to the interdimer spacing. With continued incubation, this low-molecular-weight DNA is joined into high-molecular-weight chains (postreplication repair), a process associated with sister exchanges in bacteria. Recombination is initiated by pyrimidine dimers opposite postreplication gaps and by interstrand cross-links that have been cut by excision enzymes. The free ends at the resulting gaps presumably initiate the exchanges. Postreplication repair in Escherichia coli occurs in recB- AND RECC but is greatly slowed in recF- mutants. RecB and recC are the structural genes for exonuclease V, which digests two-stranded DNA by releasing oligonucleotides first from one strand and then from the other. The postreplication sister exchanges in ultra-violet-irradiated bacteria result in the distribution of pyrimidine dimers between parental and daughter strands, indicating that long exchanges involving both strands of each duplex occur. The R1 restriction endonuclease from E. COli has been used to cut the DNA of a bacterial drug-resistance transfer factor with one nuclease-sensitive site, and also DNA from the frog Xenopus enriched for ribosomal 18S and 28S genes. The fragments were annealed with the cut plasmid DNA and ligated, producing a new larger plasmid carrying the eukaryotic rDNA and able to infect and replicate in E. coli.


Analysis of temperature-sensitive recB and recC mutations. The in vivo pleiotropic effects associated with the temperature-sensitive recB270 and recC271 mutations have been correlated with the in vitro behavior of the recBC nucleases coded for by these alleles. The ATP-dependent breakdown of double-stranded DNA is essential for cell viability, radiation repair, and genetic recombination. Temperature sensitivity can be suppressed in vitro and in vivo.


Recombination and postreplication repair. The available data concerning postreplication repair are summarized. In Escherichia coli, recombination is implicated in this repair because the recA+ gene is necessary and because strand exchanges occur that extend over long regions. Other experiments involving phage-induced resistance also point to an interrelation between recombination and repair. In this phenomenon, gene products of lambda bacteriophage are introduced into bacteria, resulting in an increased resistance of the cells when they are subsequently exposed to X rays.


Ultraviolet-light-induced incorporation of bromodeoxyuridine into parental DNA of an excision-defective mutant of Escherichia coli. Bromodeoxyuridine-containing regions approximately 1.5 X 10(4) Nucleotides in length, and at intervals equivalent to the pyrimidine dimer content of the DNA, have been observed in the parental DNA of an excision-defective strain of Escherichia coli exposed to 10 ergs mm-2 at 254 nm followed by prolonged incubation in the presence of bromodeoxyuridine.


Distribution of pyrimidine dimers during postreplication repair in UV-irradiated excision-deficient cells of Escherichia coli K12. During postreplication repair in excision-deficient mutants of Escherichia coli K-12, pyrimidine dimers are gradually lost from UV-irradiated DNA. Our data indicate that dimers are transferred, by a process which may involve genetic exchange, into daughter strands made after irradiation. Dimer transfer appears to continue through several rounds of replication, resulting in the gradual dilution of dimers into successive generations of DNA molecules.


Postreplication repair gap filling in an Escherichia coli strain deficient in dnaB gene product. Gaps in daughter-strand DNA synthesized after exposure of Escherichia coli E279 to ultraviolet light are filled during reincubation at 30 degrees C for 20 min. Escherichia coli E279 is phenotypically DnaB- when incubated at 43 degrees C. Cells incubated at 43 degrees C were tested for their ability to complete postreplication repair gap filling. It is concluded that the dnaB gene product is essential for postreplication repair gap filling and that the inhibition seen is not initially the result of degradation.


Replication and expression of constructed plasmid chimeras in transformed Escherichia coli--a review. EcoRI restriction-endonuclease-generated fragments of bacterial plasmids isolated from Staphylococcus aureus or Escherichia coli, or of amplified DNA coding for the 18S and 28S ribosomal RNA of Xenopus laevis, have been linked to the pSC101 plasmid replicon and introduced into E. coli by transformation. The constructed plasmid chimeras can be cloned as stable replicons in E. coli, where they synthesize RNA and/or protein products specified by their component genes.


SOS repair hypothesis: phenomenology of an inducible DNA repair which is accompanied by mutagenesis. A hypothesis was proposed several years ago that Escherichia coli posses an inducible DNA repair system ("SOS repair") which is also responsible for induced mutagenesis. Some characteristics of the SOS repair are (1) it is induced or activated following damage to DNA, (2) it requires do novo protein synthesis, (3) It requires several genetic functions of which the best-studied are recA+ and lex+ of E. coli, and (4) the physiological and genetic requirements for the expression of SOS repair are suspiciously similar to those necessary for the prophage induction. The SOS repair hypothesis has already served as the working hypothesis for many experiments, some of which are briefly reviewed. Also, some speculations are presented to stimulate further discussions and experimental tests.


Thermal enhancement of ultraviolet mutability in a dnaB uvrA derivative of Escherichia coli B/r: evidence for inducible error-prone repair. DNA damage triggers coordinate expression of a cluster of diverse functions in  Escherichia coli, including prophage induction, filamentous growth, and "aberrant" reintiation of DNA replication at the chromosomal origin. The "SOS repair" hypothesis proposes that one of these coordinately inducible functions is an error-prone system of DNA repair ("SOS repair") which is responsible for ultraviolet mutagenesis. In dnaB strains, incubation of 42 degrees C stops DNA synthesis and induces lambda prophage and should, therefore, also induce the postulated error-prone repair activity. Thermal posttreatment of a dnaB urvA derivative of E. coli B/r is found to enhance the yield of ultraviolet-light-induced mutations as much as 50-fold, while having no such effect in the dnaB+ parent strain. The results support the SOS repair hypothesis. The possibility is discussed that the inducible repair system is a mutagenic DNA polymerase.


Indirect suppression of radiation sensitivity of a recA- strain of Escherichia coli K12. It has been shown previously that the radiation sensitivity of LexA strains of Escherichia coli K-12 can be suppressed by thermosensitive mutations (designated tsl) that are closely linked to the lexA locus. These are thought to be intragenic suppressors that reduce the activity of the diffusible product that gives rise to the LexA- phenotype (Mount et al., 1973). When a recA mutation is crossed into a suppressed tsl- strain, the extreme radiation sensitivity usually conferred by a recA mutation is considerably reduced without any detectable change in genetic recombination deficiency. Suppression of UV sensitivity depends upon the activity of the uvrA+ product. We propose that at least part of the radiation sensitivity of a recA- strain is due to a DNA repair defect that is different from inability to perform genetic exchanges and depends upon the presence of the lexA+ product. We hypothesize that the lexA+ product is a repressor of the synthesis of repair enzymes. In recA+ cells with DNA lesions, repressor is inactivated leading to enzyme induction but this does not occur in recA- cells. tsl mutations inactivate repressor leading to constitute enzyme synthesis and bypassing the need for recA+ product to inactivate the lexA+ product.


The role of DNA polymerase I in genetic recombination and viability of Escherichia coli. The rate of formation of high-molecular-weight daughter DNA in the conditionally lethal double mutant polA12 uvrE502, incubated at nonpermissive temperature, was slower than that in the single polA12 mutant. There exist at least two pathways determining viability of Escherichia coli cells: one of them is dependent on polA+ and recB+ genes, while another is polA+ and recB+ genes, while another is polA recB independent but requires the uvrE+ gene and can be blocked by exonuclease I. The RecF but not the RecBC pathway of genetic recombination was found to be absolutely dependent on the polymerizing activity of DNA polymerase I. The involvement of DNA polymerase I in genetic recombination in the recB- C- sbsB strain and viability in the uvrE- or recB- strains suggest the existence of the common steps required for the accomplishing of the RecF pathway of recombination and for viability of E. coli.


The role of the rec genes in the viability of Escherichia coli K12. Mutations in the recA, recB, or recC genes significantly reduce the growth rate and viability of Escherichia coli. Cultures of rec- strains are compose of three populations of cells: viable cells, nonviable but residually dividing cells, and nonviable and nondividing cells. Nondividing cells can be separated from dividing cells by penicillin treatment and velocity sedimentation. Nondividing cells of all rec- strains are greatly reduced in their ability to synthesize DNA. recB- recC- and recA- and recB- recC- nondividing cells contain DNA. This DNA synthesized in dividing cells and segregated into the nondividing cells. recA- nondividing cells contain little or no DNA. recA- recB- recC- nondividing cell DNA accumulates single-strand breaks.


DNA turnover and strand breaks in Escherichia coli. The extent of DNA turnover has been measured in a dnaB mutant of Escherichia coli, temperature sensitive for semiconservative DNA replication. At the nonpermissive temperature about 0.02% of the deoxynucleotides in DNA are exchanged per generation period. This turnover rate is markedly depressed in the presence of rifampicin. During thymine starvation strand breaks accumulate in the DNA of E. coli strains that are susceptible to thymineless death. Rifampicin suppresses the appearance of these breaks, consistent with our hypothesis that transcription may be accompanied by repairable single-strand breaks in DNA. DNA turnover is enhanced severalfold in strands containing 5-bromodeoxy-uridine in place of thymidine, possible because the analog (or the deoxyuridine, following debromination) is sometimes recognized and excised.


Excision-repair of gamma-ray-damaged thymine in bacterial and and mammalian systems. The selective excision of products of the 5,6-dihydroxy-dihydrothymine type (t') from gamma-irradiated or OSO4-oxidized DNA or synthetic poly[d(A-T)] was observed with crude extracts of Escherichia coli and isolated nuclei from human carcinoma HeLa S-3 and Chinese hamster ovary cells. The results with E. coli extracts allow the following conclusion: (1) The uvrA-gene product is not required for t' excision. (2) Radiation-induced strand breakage is not required for product excision. (3) Experiments with extracts of E. coli polAexl showed that the 5' in equilibrium 3' exonuclease associated with polymerase I is responsible for the removal of t'. (4) Experiments with extracts of E. coli endo I lig 4 and the ligase inhibitor nicotinamide mononucleotide showed that polynucleotide ligase accomplishes the last strand resealing step in the excision-repair of t'. Isolated nuclei from HeLa and Chinese hamster ovary cells possess the necessary enzymes for the selective excision of t' from gamma-irradiated or osmium tetroxide oxidized DNA. Approximately 25 to 35% of the products were removed from DNA within 60 min. Unspecific DNA degradation was very low. Radiation-induced strand breakage is not required for product removal.


Kinetics of photoreactivation.

Kinetics of photoreactivation. This paper summerizes experimental work (most of which is published) in which light flashes were used for an analysis of photoenzymatic repair in vivo and in vitro. The method permits determination of the reaction rate constants for the formation, dark dissociation, and repair photolysis of enzyme-substrate complexes under various conditions, and estimation of the number of photoreactivating enzyme molecules present. Investigation of these characteristics is basic for understanding of the overall photoreactivation kinetics observed in biological systems, its dependence on experimental parameters, and possibly its biological significance.


Dependence upon growth medium and the polA, polC, recA, recB, recC, and exrA genes of separate branches of the uvr Gene-dependent excision-repair process in Escherichia coli K12 cells. The repair of single-strand breaks which arise in DNA during the uvr gene-dependent excision-repair process was examined in certain radiation-sensitive strains of Escherichia coli K12. The results suggest that the excision-repair process may be divided into two branches, one which is dependent on the presence of growth medium and also the rec+ exr+ genotype, and a second which can occur in buffer (growth medium independent) and is largely dependent on DNA polymerase I. DNA polymerase III appears to be involved in the growth-medium-dependent branch of excision repair, and also in the residual growth-medium-independent repair which occurs in polA1 cells. Chloramphenicol, dinitrophenol, and impurities present in some brands of agar all appear to inhibit the growth-medium-dependent branch of excision-repair. The similarities of the two branches of excision-repair to two known pathways for the repair of X-ray-induced DNA chain breaks are discussed.


Near-UV photoproduct(s) of L-typtophan: an inhibitor of medium-dependent repair of X-ray-induced single-strand breaks in DNA which also inhibits replication-gap closure in Escherichia coli DNA. Near-UV photoproducts of L-tryptophan (TP), which are especially toxic for recombination-deficient (rec) mutants, were found to inhibit medium-dependent repair of X-ray-induced single-strand breaks. This inhibitor also slows the rate of closure of replication gaps, suggesting that these two processes may have a common pathway (or share a required step which TP can inhibit).


DNA repair in DNA-polymerase-deficient mutants of Escherichia coli. Escherichia coli mutants deficient in DNA polymerase I, in DNA polymerases I and II, or in DNA polymerase III can efficiently and completely execute excision-repair and postreplication repair of the UV-damaged DNA at 30 degrees C and 43 degrees C when assayed by alkaline sucrose gradients. Repair by Pol I- and Pol I-, Pol II- cells is inhibited by 1-beta-D-arabinofuranosylcytosine (araC) at 43 degrees C but not at 30 degrees C, whereas that by Pol III- cells is insensitive to araC at any temperature. Thus, either Pol I or Pol III is required for complete and efficient repair, and in their absence Pol II mediates a limited, incomplete dark repair of UV-damaged DNA.


Repair of cross-linked DNA in Escherichia coli. The repair of DNA containing interstrand cross-links in Escherichia coli was studied by following the temporal sequence of DNA-related metabolic events in cells exposed to psoralen plus light. Mutations in some genes controlling replication, recombination, and repair strongly influence these specific events. Results reported here are consistent with a cross-link repair mechanism involving sequential excision and recombination.


Recovery of the priming activity of DNA in x-irradiated Escherichia coli. The template activity of DNA in RNA synthesis in vitro has been studied in Escherichia coli B/r and Bs-1 after exposure to X-rays and postirradiation incubation in growth medium for 60 min at 37 degrees C. The incubation of E. coli B/r after irradiation with 9.3 krad results in the increase of the priming activity of DNA practically to that of unirradiated cells, while after exposure to 18.6 krad the incubation leads to a partial increase in its priming activity. As for E. coli Bs-1, the incubation of the bacteria irradiated with 9.3 krad causes a slight recovery in the priming activity of DNA.


Repair of double-strand breaks in Micrococcus radiodurans. Micrococcus radiodurans has been shown to sustain double-strand breaks in its DNA after exposure to X-radiation. Following sublethal doses of X-rays (200 krad in oxygen or less), the cells were able to repair these breaks, and an intermediate fast-sedimenting DNA component seemed to be involved in the repair process.


The repair of double-strand breaks in chromosomal DNA of yeast. Ionizing radiation induces double-strand breaks in the nuclear DNA of the yeast Saccharomyces cerevisiae with an efficiency of approximately 0.6 X 10(-10) breaks being repaired. Based on the efficiency of break production, the sensitivity of a rad52 mutant, and the absence of radiation-induced recombination in such a mutant, it is proposed that the corresponding gene product may be involved in double-strand break repair. A model involving recombination and DNA synthesis is described for this type of repair.


The fate of UV-induced pyrimidine dimers in the nuclear and mitochondrial DNAs of Saccharomyces cerevisiae on various postirradiation treatments and its influence on survival and cytoplasmic "petite" induction. The photoreactivability of UV-induced pyrimidine dimers in the nuclear and mitochondrial DNAs of Saccharomyces cerevisiae has been investigated in conjunction with the fate of these photoproducts following postirradiation dark incubation in saline and nutrient media. In all instances, survival and "petite" induction were measured. An attempt has been made to relate these results to present ideas on the repair of UV damages in DNA.


Genetic control of radiation sensitivity and DNA repair in Neurospora. Radiation sensitivity in the fungus Neurospora crassa is under the control of at least eight distinct loci and is also affected by cytoplasmic factors. Although radiation-sensitive mutants which affect inter- or intragenic meiotic recombination have not been isolated, mutants which are defective in the repair of pyrimidine dimers have been found. Evidence from both mutational and biochemical studies shows that Neurospora has an excision-repair system for pyrimidine dimers which is very similar to the one found in Escherichia coli. Wild-type strains excise dimers, but two mutants, uvs2 and upr1, are UV sensitive and excision defective. Like the E. coli excision-defective mutants, the Neurospora mutants show a greatly increased frequency of UV-induced mutation at low UV doses, and they do not affect recombination. However, they differ from the E. coli mutants in being significantly more sensitive to ionizing radiation than wild-type strains. A third mutant, uvs6, resembles the DNA polymerase-I-negative mutants of E. coli. It is sensitive to both UV and X-irradiation, has a wild-type pattern of UV-induced mutation, and increases spontaneous deletion frequencies. Its polymerases have not been examined. The high frequency of UV-induced mutation in excision-defective strains suggests that a "mutation prone" system of DNA repair exists in Neurospora. This is supported by the ppoperties of the uvs3 strain, which shows no UV-induced mutation. Like postreplication-repair-defective E. coli mutants, it is UV and ionizing radiation sensitive and sensitive to both monofunctional and bifunctional alkylating agents. This mutant is sterile. As expected, the double mutant uvs3 upr1 strain is much more sensitive to UV than either single-mutant strain. Two other loci, muc2 and gs6, may affect DNA repair. Mutations at the five remaining loci, uvs1, uvs4, uvs5, gs3, and gs20, lead to a constellation of properties unlike those of any DNA-repair-deficient E. coli mutant. The occurrence of these mutations could mean that other DNA repair systems exist in Neurospora, or, like the lon mutants of E. coli, they might indicate that cell sensitivity to radiation damage can be increases in other ways.


Concerning pyrimidine dimers as "blocks" to DNA replication in bacteria and mammalian cells. Irradiated UV-sensitive bacteria which are mutant in the uvrA6 locus, and cannot excise pyrimidine dimers, are capable of continued DNA replication with the same orientation as that in unirradiated cells. Replication of dimer-containing DNA is also accomplished by irradiated mammalian cells, suggesting that pyrimidine dimers do not act as absolute blocks to DNA synthesis. Thus, the role that dimers play in reducing the amount and size of DNA synthesized after exposure must be reassessed.


Response of homozygous and heterozygous xeroderma pigmentosum cells to several chemical and viral carcinogens. The topics discussed include the relationship between the carcinogenic property of various compounds and their capacity to elicit DNA damage that results in a DNA repair synthesis; the metabolic activation of precarcinogens; the in vivo use of DNA repair synthesis in the identification of tissues that will give rise to neoplasms; the possible involvement of DNA repair in the sensitivity of human cells to the chromosome-damaging and lethal effect of chemical carcinogens; the different responses of homozygous xeroderma pigmentosum (XP) cells, heterozygous XP cells, and controls to some but not all chemical carcinogens and oncogenic viruses; and the possible link between DNA damage, its repair, and viral-induced neoplastic transformation.


Improved rapid method for electron microscopy of macro- and micro-tissue cultures. A rapid method of embedding macro- and micro-tissue cultures is described. The procedure involves use of a preshaped BEEM capsule for pelleting, fixation and embedding of tissue culture cells for electron microscopy. To obtain good contrast specimens prepared by this rapid method need a rather longer staining time with uranyl acetate than specimens prepared by standard methods. THis procedure takes only about 200 min from fixation and embedding to double staining with uranyl acetate and lead citrate. It should be useful for both monolayer tissue cultures and organ cultures in diagnostic and research studies on viral agents.


Epimastigotes and trypomastigotes of Trypanosoma cruzi revealed by scanning electron microscopy. Epimastigotes and trypomastigotes of Trypanosoma cruzi were observed by scanning electron microscopy. These two forms could easily be differentiated by examination of the undulating membrane and kinetoplast. Subpellicular microtubules form parallel helices in epimastigotes while they are arranged parallel to the logitudinal axis in trypomastigotes. Some epimastigotes and trypomastigotes showed a "knob-like" swelling at the end of their bodies.


Synthesis of thiol-containing analogues of puromycin and a study of their interaction with N-acetylphenylalanyl-transfer ribonucleic acid on ribosomes to form thioesters. 1. The thiol-containing analogue of puromycin, 6-dimethylamino-9-{1'-[3'-(2''-mercapto-3''-phenylpropionamido)-3'-deoxy-beta-d-ribofuranosyl]}purine (XVII) in which the primary amino group of the antibiotic is replaced with a thiol grouping, was synthesized chemically (compound XVII is abbreviated to thiopuromycin). 2. Thiopuromycin (XVII) was found to be active in releasing N-[(3)H]acetylphenylalanine from its tRNA carrier as the thioester, N-acetylphenylalanylthiopuromycin (XIX) in the Escherichia coli ribosomal system. The reaction product (XIX) was synthesized chemically from thiopuromycin and N-acetylphenylalanine and found to be stable to hydrolysis in the standard incubation medium at pH7.6. dl-Phenyl-lactylpuromycin (XXI), the hydroxy analogue of puromycin, was also synthesized chemically and shown to release N-acetylphenylalanine from its tRNA carrier in the E. coli ribosomal system, thus confirming the previous results of Fahnestock et al. [Biochemistry (1970) 9, 2477-2483]. 3. In marked contrast with the results obtained in the E. coli system, both thiopuromycin (XVII) and hydroxypuromycin (XXI) were found to be inactive in releasing N-acetylphenylalanine from its tRNA carrier in the rat liver ribosomal system.


Genetics of patent ductus arteriosus.

Genetics of patent ductus arteriosus. A genetic study of 119 correlative cases of patent ductus arteriosus has been performed. The isolated cases showed female predominance, but there was a three times higher risk for the sibs of male cases, suggesting a multifactorial causative mechanism. Dermatoglyphs presented an excess of arches in male, and a tendency to distal axial triradius in female. There were 15 cases belonging to polymalformative syndromes, such as mongolism, Turner, Noonan, Holt-Oram, Lewis and rubella embryopathy.


Relationship between the chemotherapeutic effectiveness of ciclacillin and ampicillin and the time of their administration in experimental infections. In an attempt to explain the discrepancy between the weak in vitro activity and good clinical efficacy of ciclacillin, a time-dosage-efficacy study was made in order to investigate the relationship of the effectiveness of this antibiotic to the interval between experimental infection and administration in comparison to ampicillin, which because of its similar antimicrobial spectrum and completely different pharmacokinetic properties was particularly suitable for use in the study. Various single oral doses of both antibiotics were administered once to NMRI (SPF) mice at various intervals (0, 1, 2 or 3 h) following experimental infection with E. coli WT 102, E. coli 3033 or E. coli 026:B6 and the CD50's determined and compared statistically. It was demonstrated that the chemotherapeutic effectiveness of both antibiotics was markedly dependent on the interval between experimental infection and administration. Whereas ampicillin was superior to ciclacillin when drug and infective organism were administered simultaneously (0 h), ciclacillin was superior to ampicillin when it was administered 3 h after experimental infection. Both antibiotics were about equally effective when administered 1 or 2 h after infection. The difference in the serum concentrations and rates of absorption and excretion of the two drugs is assumed to be the reason for this phenomenon, and the pharmacokinetic characteristics of ciclacillin, in particular its rapid and almost complete absorption and rapid attainment of high peak serum levels, are discussed as at least a partial explanation of the difference in its in vitro and in vivo activities.


The pharmacodynamics of orally taken verapamil and verapamil retard as judged by their negative dromotropic effects. The widely recognized, negative dromotropic effects of alpha-isopropyl-alpha-[N-methyl-N-homaveratryl)-gamma-aminopropyl]-3-4-dimethoxyphenylacetonitrile (verapamil, Isoptin) on arterio-venous (A-V) nodal conductions were studied using long-term atrial stimulation (AS) in 7 healthy volunteers whose mean age was 30 years. A control group consisting of three of the volunteers who underwent long-term atrial stimulation without medication was established. Since an isolated, well-known drug effect was studied over a long period of time, it was felt that this was an acceptable method for reaching valid pharmacodynamic conclusions. Atrial stimulation was carried out at hourly intervals up to 14-16 h and continued to the highest possible rate without the occurrence of 2nd degree A-V block before and following oral application of verapamil, 240 mg. The stimulus-Q (S-Q) prolongation at increased pace rates was compared to the control. Verapamil retard was tested in a similar course of investigation the following day. The results clearly demonstrated that both forms of verapamil are effective when taken orally. Verapamil acts 2 h after oral application. The peak effectiveness of the drug is found at 5 h and at this point A-V block can occur at low atrial stimulated rates. The effect of verapamil retard is obvious only after 6 h but is still apparent after 14 h and it remains constant with no peak in effectiveness. Characteristics of the resulting regressions allow to distinguish the effect of verapamil on A-V conduction from influences that might be mediated by the diurnal changes in tone of the autonomic nervous system only. The effect of verapamil after oral application is more pronounced with higher heart rates. The pharmacodnaymics of verapamil evaluated with regard to a single well-known pharmacological effect, have not necessarily to be identical with the pharmacokinetics and the bioavailability of the drug.


[Human pharmacokinetics and metabolism of 14C-labeled gliquidone (AR-DF 26)]. Investigations of pharmacokinetics and metabolism of 14C-labelled 1-cyclohexyl-3 (p-[2-(3,4-dihydro-7-methoxy-4,4-dimethyl-1,3-dioxo-2-(1H)-isoquinolyl)-ethyl]-phenyl)-sulfonyl)-urea (AR-DF 26; gliquidone; Glurenorm) after i.v. and oral applications were carried out on 10 healthy volunteers. After oral application of 15 mg AR-DF 26, maximum blood levels of 370 ng/ml blood or 700 ng/ml plasma, respectively, were reached. After i.v. as well as oral application of AR-DF 26 in men the bulk of the substance applied was excreted with the faeces and only 5 percent were found in urine. After both modes of application the metabolites in the faeces were found to be quantitatively and qualitatively similar. The structure of 4 metabilites was demonstrated by isotope dilution analysis.


Clinical experience with use of ultrasound sphygmomanometer. The performance of an ultrasound sphygmomanometer (Arteriosonde 1217) has been compared with that of the London School of Hygiene and Tropical Medicine and Hawksley sphygmomanometers. The Arteriosonde gave closely similar values of systolic blood pressure of the other instruments but diastolic blood pressure lay midway between phase 4 and phase 5 of the Korotkoff sounds. Observers using the Arteriosonde showed a significant preference for even terminal digits.


Maximal exercise testing in patients with spontaneous angina pectoris associated with transiet ST segment elevation. Risks and electrocardiographic findings. Six patients with spontaneous angina associated with transient ST segment elevation had a multistate maximal exercise (bicycle) test. In 5 patients, typical electrocardiographic changes were recorded during exercise, namely ST segment elevation often accompanied by an increase in the voltage of the R wave and a widening of the QRS complex. Four of these patients developed severe rhythm disturbances: ventricular tachycardia (2 cases) and ventricular flutter (1 case) were the reason for early interruption of the test in 3 patients, while 1 patient had a short run of ventricular tachycardia after exercise. These rhythm disturbances which spontaneously regressed in all cases were consistently preceded by obvious ST elevation and in 2 patients were attended by slight chest discomfort. Maximal exercise testing of patients suspected of variant angina provides important diagnostic information in many patients, but the risks of potentially lethal arrhythmias should be considered and resuscitation facilities should always be immediately available.


Non-invasive left ventricular volume determination by two-dimensional echocardiography. Twenty patients undergoing routine left ventricular single-plane angiography have been investigated by an ultrasonic triggered B-scan technique to provide a two-dimensional cross-sectional image of the left ventricle in end-systole end-diastole. An area-length method has been used to establish the correlation between the angiographic and the echocardiographic assessments of left ventricular chamber volume (r equals 0.88) and ejection fraction (r equals 0.81). Differences between the two techniques are discussed, and it is concluded that in approximately 80 per cent of patients triggered B-scanning may provide a safe, non-invasive, and convenient technique for the determination of volumes and certain functional parameters, especially in patients with dilated hearts and irregular left ventricular shape, where M-scanning is known to be less reliable.


Hypertrophic obstructive cardiomyopathy. Assessment by echocardiographic and Doppler ultrasound techniques. Hypertrophic obstructive cardiomyopathy is a disease of the myocardium that can be assessed echocardiographic and transcutaneous Doppler ultrasound techniques. Four patients are presented with various patterns of the disease, and the frequently familial incidence is illustrated. The importance of ultrasonic evidence for asymmetric septal hypertrophy in all stages is emphasized and evidence of reduced septal contractility demonstrated. Abnormalities of mitral valve motion, slow diastolic closure rate and systolic anterior movement of the anterior leaflet, are shown in the obstructive form of the disease. Also partial mid-systolic aortic valve closure and aortic cusp flutter are shown with outflow obstruction. The outflow tract gradient can be calculated from mitral valve to septum systolic distances. Transcutaneous Doppler ultrasound shows a normal aortic velocity pattern in nonobstructive disease while consistent abnormalities are present with severe resting obstruction. Isoprenaline can be used to alter the normal velocity pattern associated with a minimal resting gradient to an abnormal pattern indicating the development of significant obstruction.


Localization of left ventricular ischaemia in angina pectoris by cineangiography during exercise. Cineangiography of the left ventricle during exercise has been used in an attempt to define the area of ischaemic myocardium in patients suffering from angina pectoris in whom coronary artery surgery was contemplated. A correlation was established between the site of coronary artery obstruction and the area of abnormal myocardial contraction. This method of localization of regional left ventricular ischaemia may furnish useful diagnostic information when coronary reconstructive surgery is contemplated in patients with exercise-induced angina pectoris.


Positive end-expiratory pressure in anaesthetized spontaneously breathing horses. Horses breathing spontaneously under halothane anaesthesia were subjected to expiratory resistance by the introduction of a water-trap into the expiratory limb of a circle absorber. Resistances of 10 and 20 cm H2O produced no significant increase in PaO2 (P greater than 0.05) during halothane/air and halothane/oxygen anaesthesia. The imposition of resistance was associated with an increase in PaCO2 and a significant increase in mixed venous PCO2. In three animals subjected to 20 cm H2O resistance under halothane/air anaesthesia, the cardiac output was reduced (P less than 0.01). It was concluded that the indiscriminate application of end-expiratory pressure has no place in routine equine anaesthesia.


The effect of anaesthesia and intermittent positive pressure ventilation with different frequencies on the anatomical and alveolar deadspace. Deadspace was measured in nine healthy subjects in the supine position, premedicated but awake and breathing spontaneously at a rate of 12 b.p.m. and subsequently under anaesthesia with artificial ventilation with frequencies of 12 and 24 b.p.m. The minute volume was kept at a relatively constant value. The physiological deadspace was calculated using the Bohr equation and the division into anatomical and alveolar deadspace was made with the aid of capnography. Physiological deadspace was increased by anaesthesia and IPPV, mainly as a consequence of increased rebreathing in the apparatus deadspace. There was no significant change in the anatomical deadspace. Thus, the expected reduction in deadspace brought about by endotracheal intubation was nullified by an increase in the anatomical deadspace distal to the carina. The VDanat/VT ratio remained constant on changing the respiratory frequency. A significant alveolar deadspace was measured during spontaneous breathing. This was unchanged by the induction of anaesthesia and the institution of artifical ventilation. On changing the frequency, the VDalv/VT ratio remained constant. It is concluded that both the anatomical and the alveolar deadspaces increasing with increasing tidal volume, but are unaffected by the breathing rate.


Absorption and sedative effects of diazepam after oral administration and intramuscular administration into the vastus lateralis muscle and the deltoid muscle. The absorption of diazepam 10 mg after oral administration and intramuscular administration into the vastus lateralis muscle or the deltoid muscle was compared in a double-blind cross-over study in eight healthy subjects. Serum diazepam concentrations were measured, and the presence of tiredness was noted 20, 40, 60, 90 and 150 min after the drug administration. Peak concentrations in serum were 209+/-49, 152+/-60 and 143+/-62 ng/ml ((means+/-SD) at 90, 60 and 60 min after oral, shoulder and thigh administration respectively. Absorption was more rapid after intramuscular than after oral administration, serum mean diazepam concentrations at 20 min after oral administration being only 26% of those after shoulder administration. The rapid rate of absorption from the shoulder was associated with a more rapid feeling of tiredness and a greater sedative effect than after oral or thigh administration. There was no evidence that diazepam induced its own metabolism after one or two administrations. The results suggest that, if rapid preanaesthetic medication with diazepam is needed, shoulder administration might be superior to oral or thigh administration.


Current practice of hypothermia in British cardiac surgery. A questionnaire was sent to all centres of cardiac surgery in the United Kingdom, enquiring into their current use of hypothermia. Moderate hypothermia without cardiopulmonary bypass and the Drew technique of profound hypothermia are becoming less popular, each technique being used in only two of the 30 centres which replied. Moderate hypothermia as an integral part of the cardiopulmonary bypass is used on occasions in 24 centres. Although some centres use moderate hypothermia out of habit, the main benefits from its use are considered to be the protection afforded to the myocardium and a greater safety margin in the event of technical difficulties. Profound hypothermia, usually induced by means of the pump oxygenator, followed by circulatory arrest is becoming increasingly popular for the correction of complex congenital anomalies in infants.


Benzoctamine-a study of the respiratory effects of oral doses in human volunteers and interactions with morphine in mice. A double-blind trial has been performed to investigate the respiratory effects of low oral doses of benzoctamine, and to compare them with diazepam and a placebo. The displacement of the carbon dioxide response curves indicated that whilst diazepam caused respiratory depression, benzoctamine had a variable effect. Some volunteers showed depression, but most showed stimulation. Peak respiratory effects were seen 1 hr after oral administration, returning to normal 2-3 hr after administration. It is suggested that oral drugs given for premedication need to be administered at least 2 hr before operation to obtain maximum sedative effects at a time when respiratory effects are returning to normal. In animal experiments it has been shown that the analgesic actions of morphine are diminished by concurrent administration of benzoctamine, and that the depression of respiratory rate caused by morphine is enhanced.


Pulmonary eosinophilia after substitution of aerosol for oral corticosteroid therapy. Three patients with chronic asthma developed pulmonary eosinophilia while oral prednisolone was being withdrawn after the introduction of treatment with beclomethasone dipropionate aerosol. These observations suggest that the development of pulmonary eosinophilia in patients with chronic asthma can be prevented by the systemic administration of corticosteroids, but not by a corticosteroid aerosol given by inhalation. It is considered that radiological examination of the chest should be carried out at frequent intervals during and for a few months after the substitution of corticosteroid aerosol therapy for oral corticosteroid therapy in order to ensure that those patients who are liable to develop pulmonary eosinophilia will be identified at an early stage. These precautions are even more essential when there is a previous history of pulmonary eosinophilia.


The effect of a tubercle lipid adjuvant on the distribution of injected foreign red blood cells. Increased localization occured of injected foreign red cells in the spleen and lungs of animals treated with a tubercle bacillary lipid adjuvant given intravenously. The distribution changes varied depending upon the time interval between injections of the adjuvant and the foreign red cells. These changes offer an explanation of the augmentation of haemolysins and haemagglutinins previously shown for the lipid.


Cellular and humoral aspects of host resistance in murine salmonellosis. Mice were challenged with a highly virulent strain of Salmonella typhimurium by intraperitoneal injections. At relatively low infecting doses, immunizations with either viable attenuated or heat killed Salm. typhimurium were found to be equally protective against otherwise fatal infections. Pre-opsonization of virulent salmonellae significantly increased the survival rate of mice infected with small numbers of the pathogen. By a cell culture method, peritoneal macrophages of mice were shown to be innately capable of destroying the ingested virulent Salm. typhimurium. Macrophages from previously infected mice did not appear to have any significant increase in their bactericidal activity against salmonellae, but they possessed cytophilic antibodies specific against the H and the O antigens of Salm. typhimurium. It is believed that humoral elements play an important role in acquired immunity in murine salmonellosis by opsonization of the pathogen.


Repeated tissue sampling with a dental broach. A trial in cutaneous leishmaniasis. A simple and almost painless technique for repeated sampling of dermal infiltrates with a dendritic broach was used in the diagnosis of cutaneous leishmaniasis. This technique can be used to advantage to obtain organisms and cell underneath crusted lesions and to evaluate not only the number of organisms but also the cell pattern at different depths of the lesion. It can be used repeatedly on the same lesions without appreciably disturbing the natural progress of the disease and is therefore well suited to monitor the in vivo effects of therapeutic agents on organisms. Its use in twenty-one cases of cutaneous leishmaniasis and one case of cutaneous leprosy is described.


Efficacy of miconazole in the topical treatment of tinea pedis in sportsmen. A double-blind trial of 2% miconazole in a cream and in a powder base and of the respective vehicles was done in a group of forty-five young sportsmen regularly training and using the showers in a gymnasium. After 4 weeks treatment, the mycological cure rate using the active preparation was 60% compared to 24% for the placebo (P less than 0-05).


Coexistence of lichen planus and bullous pemphigoid. A immunopathological study. A 43-year-old white man presented with a generalized eruption of lichen planus and tense blisters within the lichenoid lesions and also on clinically normal skin. Direct immunofluorescence (IF) studies revealed immunological and histopathological characteristics of lichen planus in the lichenoid lesions and of bullous pemphigoid in the bullous lesions, and indirect IF studies showed that the patient had circulating antibasement membrane antibodies. The coexistence of both disorders may indicate a possible link between the pathology in the junctional zone in lichen planus and the appearance of antibasement membrane zone antibodies and bullous lesions, respectively.


A prospective study of the effects of weekly oral methotrexate on liver biopsy. The results are reported of a prospective study of liver histological findings in twenty-five patients with severe psoriasis subjected to liver biopsy before and at intervals after the onset of treatment with methotrexate by an intermittent dosage schedule. The pre-methotrexate liver biopsy findings in forty-one other severe psoriatics are also summarized. Of the twenty-five treated patients, four showed a mild inflammatory cell infiltrate and two mild fibrosis before treatment. During treatment only one developed a grade 2 infiltrate and one other grade 2 fibrosis. None has developed cirrhosis. Where minor abnormalities developed, their fluctuating and reversible nature is demonstrated.


Deafness complicating mucous membrane pemphigoid: a case report. A patient with moderately severe mucous membrane pemphigoid is described. The onset of deafness was associated with the appearance of postero-superior retraction prockets in both eardrums. It is postulated that this was due to mucous membrane pemphigoid involving the mucosa of the middle ear. We are not aware of this complication having been described previously.


The deoxyribonucleic acid (DNA) skin test in systemic lupus erythematosus. I. Clinical evaluation. A clinical evaluation of the intradermal DNA-test was carried out on a series of patients with untreated or with treated definite systemic lupus erythematosus (SLE), or with suspected SLE with or without circulating antinuclear factors. A saline solution of a commercially available DNA-preparation was used. The course of the DNA reaction was followed for 24-48 h after the injection. All nine cases of untreated definite SLE had a positive DNA test 6 h after the injection, and eight cases a positive result at 24 h. All seven patients with definite SLE who were on low-dosage systemic steroid treatment had a clinically positive. DNA test at 6 h. In all except one of these cases the test was still positive at 24 h. All five patients with definite SLE on antimalarial treatment had a positive test at 6 h which had become negative at 24 h after the injection. Eleven of the twelve patients with suspected SLE and circulating antinuclear factors had a positive DNA test at 6 h, which in nine cases persisted for 24 h. On the other hand, of the eleven ANF negative patients with various connective tissue diseases five had a positive test at 6 h. In only one of these cases it persisted for 24 h. Two of the eighteen control patients with various dermatoses exhibited a positive test at 6 h, and in one of these the positive reaction persisted for 24 h. It is concluded that the intradermal skin test using native DNA is a useful diagnostic tool for the detection of SLE when the reaction is followed up for 24 h. Antimalarial treatment seems to decrease skin reactivity to native DNA.


Folate binding proteins.

Folate binding proteins. The physical and physiologic properties of serum, milk and cellular FABP have been presented. Several investigators have shown that FABP with similar characteristics is present throughout the body fluids and tissue. Thus, FABP appears to be established as a protein which can significantly influence folate metabolism.


The growth of human bone marrow in diffusion chambers. Diffusion chambers containing normal human bone marrow were implanted intraperitoneally into normal and irradiated mice and cultured for various periods. Granulocytes, macrophages and lymphocytes comprised the majority of the cells harvested in all cultures. The most impressive cell growth occurred when the chambers were implanted into primary hosts irradiated with 840 R and then retransplanted on Day 8 of the culture into secondary hosts irradiated with the same dose. Linear relationships were found between the number of nucleated cells inoculated into the chambers and the number of cells harvested after 8 or 10 days of culture in heavily irradiated hosts.


The localization of factor V within normal human platelets and the demonstration of a platelet-factor V antigen in congenital factor V deficiency. Separation of human platelets from plasma by a modified gel-filtration technique reveals very low levels of factor-V activity of the platelet suspension. Repeated freezing and thawing increases the factor-V activity in various factor-V assays. This activity neutralized the inactivating effect of a rabbit-antihuman factor V antibody to plasma factor V, while intact platelets had almost no such capacity. Washed and normal platelets and gel filtered platelets showed marked positive fluorescence after treatment with antifactor V serum and FITC labelled sheep antirabbit immunoglobulin. Fluorescence was inhibited by previous incubation of the antifactor V serum and platelet lysates. Platelets of a factor V deficient patient showed the same fluorescence pattern as normal platelets indicating that they contained a factor V antigen. These platelets showed after lysis no effect in various factor V assays. From these studies it is concluded that the localization of factor V is within the platelets.


Skin window studies in polycythaemia rubra vera. The inflammatory response was studied in patients with primary polycythaemia by means of a modified skin window technique. In untreated patients, the overall cellularity was a prominent feature and, as compared with the controls, the 48 h preparations showed a significantly greater percentage of granulocytes with a corresponding decreased percentage of macrophages. In the peripheral blood of these patients, both total white cells and granulocyte counts were significantly higher than in the control subjects. A highly significant relationship between the blood and skin window neutrophils was demonstrated. In polycythaemia patients in remission, the results were generally similar to the controls, except that there was still a highly significant increase in the percentage of skin window basophils. The results provided no evidence of a defect in cell migration or in the phagocytic activity of macrophages in polycythaemia vera. Possible mechanisms of the cutaneous basophilia and its relevance to the manifestation of this disorder are discussed.


Advanced non-Hodgkins' lymphomas: response to treatment with combination chemotherapy and factors influencing prognosis. Response to combination chemotherapy with cyclophosphamide, vincristine and prednisone (COP) and specific factors influenceing prognosis were evaluated in 70 patients with stage III or IV non-Hodgkin's lymphomas. Complete remissions (CR) were observed in 40% of patients, with a total response rate of 88%. The median duration of CR was 8 months, but 50% of patients were in continuing CR. High rates of CR were associated with: (a) well-differentiated lymphocytic cytology, (b) tumour nodularity, (c) normal blood lymphocyte counts at diagnosis, and (d) disease stage IIIA. These same factors also showed a positive correlation with 5-year-survival rates from diagnosis. It is suggested that patients with advanced non-Hodgkin's lymphomas do not form a homogeneous treatment group, but warrant a differential therapeutic approach based on the prognostic determinants stated above. The study provides further support for the prognostic usefulness of Rappaport's classification of these tumours.


Detection of carriers of haemophilia: a 'blind' study. A 'blind' study has been made to try to find out if it is possible to diagnose carriers of haemophilia. A group of 34 obligatory carriers of haemophilia were compared with 34 normal women. Levels of factor VIII activity, factor VIII-related antigen, factor V and ratio of factor VIII activity to factor VIII-related antigen were measured. In the carrier group the mean level of factor VIII activity and the mean level of the ratio of activity to antigen were each approximately half of those found in the normal women. The mean level of factor V was the same in both groups of women. By setting the lower limit of normal at the lowest level of the different factors found in the normal women, 12 out of 34 (35%) carriers could be distinguished on the basis of their factor VIII level alone; 24 out of 34 (71%) could be detected on the basis of the ratio of factor VIII activity to factor VIII related antigen and 25 out of 34 (73%) could be detected if both factor VIII activity and the ratio were taken into account. It is concluded that consideration of both the level of factor VIII activity and the ratio of factor VIII activity to factor VIII-related antigen is of some value in detecting carriers of haemophilia. The number of carriers detected (73%) in the present study is not as high as that found by other workers.


The detection of intracellular antigens in human leucocytes by immunoperoxidase staining. An immunoperoxidase procedure is described for staining intracellular leucocyte antigens in peripheral blood and bone marrow smears. Brief exposure of cell smears to a buffered formol acetone mixture was found to give optimal fixation, combining good cellular morphology with preservation of antigenic reactivity. The immunoperoxidase method is superior to immunofluorescence in that it provides a permanent preparation which can be counterstained with orthodox reagents and viewed by conventional light microscopy. In addition the technique is considerably more sensitive than immunofluorescence procedures. Immunoglobulin was demonstrated in plasma cells, Türk cells and a minority of peripheral blood lymphocytes. Lysozyme was found in cells of the neutrophil series from promyelocytes to mature granulocytes. Monocytes stained for lysozyme but the reaction was less intense than in neutrophils and some monocytes were devoid of activity. Lactoferrin stained strongly in mature neutrophil polymorphs and metamyelocytes, but was weak or absent in earlier myeloid cells. These reaction patterns are in keeping with previous reports on the distribution of these antigens in human leucocytes. In the case of immunoglobulin and lysozyme it was possible to abolish leucocyte staining by incubation of the specific antisera with the appropriate purified antigen, providing additional proof of the specificity of the reactions. Anti-ferritin antisera stained granulocytes and myeloid precursors strongly, and reached weakly with a minority of monocytes. These latter observations are not entirely in accordance with published data on the leucocyte distribution of ferritin and may be attributed to antibody activity of unknown specificity in the anti-ferritin antiserum.


Reading chest radiographs for pneumoconiosis by computer. Computer programs for measuring simple pneumoconiosis in radiographs are described and assessed. The 36 films studied had been read by 11 skilled human observers and a 'radiological score' of pneumoconiotic severity was therefore available for each film. The computer assigns to each square grid of side 3-6 mm a measure which reflects the unevenness of the density distribution in that grid. The 'computed score' is defined as the mean diversity over all relevant grids in both lung fields. On the set of 36 films the correlation between radiological score and computed score was 0-88. By contrast, the correlation between the score assigned by a single observer and the average of the scores assigned by the other 10 was in the range 0-95 to 0-98. The program can use the computed score to classify a film into one of the four major International Labour Office (ILO) U/C categories, the success rate of this process being 80% compared with those quoted by other workers in the range 45%-65%. If the films used in this study be typical, then the program described may form the basis of an automatic method for measuring pneumoconiosis in epidemiological work.


Lung function in textile workers.

Lung function in textile workers. Acute changes in ventilatory function during a workshift with exposure to hemp, flax, and cotton dust were measured on Mondays in a group of 61 textile workers, all working on carding machines. In addition, single-breath diffusing capacity (DLCOSB) was measured before dust exposure on Monday in 30 of the 61 workers. Large acute reductions during dust exposure were recorded in maximum expiratory flow rate at 50% VC (MEF50%), ranging from 38 to 22%. Acute reductions of FEV1-0 were considerably smaller, ranging from 17 to 9%. There was a statistically significant increase in residual volume (RV) with very small and insignificant changes in total lung capacity (TLC). Although preshift FEV1-0 and FVC were decreased, DLCOSB was within normal limits. Plethysmographic measurements in six healthy volunteers exposed to hemp-dust extract confirmed the results obtained in textile workers, that is, that TLC does not change significantly during dust-induced airway constriction and that maximum expiratory flow rate at 50% VC (MEF50%) is a more sensitive test than FEV1-0 in detecting acute ventilatory changes caused by the dust extract.


Kinetic studies on sex difference in susceptibility to chronic benzene intoxication--with special reference to body fat content. The sex difference in the susceptibility to haematopoietic disorders induced by benzene was studied kinetically with a special reference to its relation with the body fat content. In rats of both sexes with a large body fat content, benzene was eliminated more slowly and remained in the body for a longer time than in rats with a small body fat content. In accord with this finding, the decrease in white blood cell numbers during a chronic benzene exposure was observed only in the groups of rats which had a large volume of fat tissue. In an experimental human exposure, the elimination of benzene was slower in the females than in the males. The kinetic study revealed that the slower elimination in the females is due primarily to the bulky distribution of body fat tissue in that sex. From these results obtained from the experimental exposure of men and rats to benzene, it was concluded that the human female, with her massive body fat tissue, shows an inherent disposition to be susceptible to a chemical such as benzene which has a high affinity with fat tissue.


A technique for measuring outcome in group psychotherapy. A technique for measuring a patient's progress and outcome in group psychotherapy is presented. The various measured parameters are derived from the Symptom Check List plus individualized repertory grid rating scales. This measurement model was tested on an out-patient group over 18 months and shows potential as a device for measuring outcome in diverse types of psychotherapy groups.


Pituitary responsiveness to synthetic luteinizing hormone releasing hormone (LH-RH) during the menstrual cycle and in female hypogonadism. The pituitary responsiveness to synthetic luteinizing hormone releasing hormone (LH-RH) in female patients with primary and secondary hypogonadism was tested and compared with that in normal women with regular menstrual cycles. A 200 mug dose of synthetic LH-RH was injected intravenously into 15 normal menstruating women, 8 patients with primary hypogonadism and 5 patients with secondary hypogonadism and the serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) response to LH-RH was investigated using double-antibody radioimmunoassay. In normal women, the LH response to LH-RH in the preovulatory phase was greater than that seen in the follicular (p less than 0-05) and luteal phases (p less than 0-05) of the menstrual cycle. In contrast, no significant difference in the FSH response was demonstrated among the three different phases of the cycle. The serum LH response to LH-RH in primary hypogonadism was greater than that in the follicular (p less than 0-01) and luteal phases (p less than 0-01) of the cycle. The serum FSH response in primary hypogonadism was markedly greater than that in the follicular (p less than 0-01), preovulatory (p less than 0-01) and luteal phases (p less than 0-01) of the menstrual cycle. Conversely, the LH levels after LH-RH administration in secondary hypogonadism were significantly lower than in the follicular (p less than 0-01), preovulatory (p less than 0-01) and luteal phases (p less than 0-01) of the cycle. The FSH response in secondary hypogonadism was slightly below that in the follicular phase (p less than 0-05). Thus, the FSH response to LH-RH in primary hypogonadism and the LH response in secondary hypogonadism seemed to be sufficiently characteristic to have diagnostic value.


Therapeutic surgery in failures of medical treatment for fungal keratitis. Medical treatment failure necessitated surgery in nine cases of fungal keratitis. Therapeutic surgery eliminated fungal infection in seven cases, and useful vision was retained in five out of six penetrating keratoplasties. In three cases Natamycin (Pimaricin) therapy rendered fungi non-viable, but two were demonstrable by histopathology. These results suggest that antifungal treatment should be applied for as long as possible before therapeutic surgery in order to improve the final visual outcome.


Corneal dellen in the limbal approach to rectus muscle surgery. A retrospective study of 170 cases of horizontal rectus muscle surgery performed during the years 1969 and 1970 at the University of Illinois Eye and Ear Infirmary revealed a 6-5 per cent incidence of dellen with the limbal approach to the muscle and a 2-2 per cent of incidence of dellen with the nonlimbal or direct approach to the muscle. This was significant to the 5 per cent level of confidence by the chi2 test. We feel that this finding of an increased incidence of dellen with the limbal approach complements the current theory of desiccation as the cause of corneal dellen. We do not recommend abandonment of the limbal approach to rectus muscle surgery because of this finding, but suggest that surgeons scrutinize more carefully their postoperative patients for dellen. If dellen do not occur careful trimming of the conjunctiva to avoid bunching and thickening may perhaps eliminate them.


Synthesis of deoxyguanosine polyphosphates and their interactions with the guanosine 5'-triphosphate requiring protein synthetic enzymes of Escherichia coli. A chemical synthesis of deoxyguanosine analogs of the guanosine polyphosphates accumulated by bacteria during the stringent response is described. Both deoxyguanosine 3'-diphosphate 5'-triphosphate (d-pppGpp) and deoxyguanosine 3'-diphosphate 5'-diphosphate (d-ppGpp) were prepared, as well as the by-products deoxyguanosine 3'-monophosphate 5'-triphosphate (d-pppGp) and deoxyguanosine 3'-monophosphate 5'-diphosphate. A significant difference between d-(p)ppGpp and guanosine 3'-diphosphate 5'-tri- or 5'-diphosphate (p)ppGpp) is that the 3'-pyrophosphate moiety is alkali stable in the deoxyguanosine and alkali labile in the guanosine polyphosphates. The new GTP analogs d-pppGp and d-pppGpp were compared to GTP, dGTP, and pppGpp in their ability to support reactions catalyzed by the Escherichia coli protein synthetic enzymes initiation factor 2, elongation factor Tu, and elongation factor G (EF-G). Like pppGpp, both d-pppGp and d-pppGpp showed substantial deficiency only in reactions requiring EF-G. While d-pppGpp closely resembled pppGpp in its very low activity with EF-G, d-pppGp was somewhat more active. Nevertheless, d-pppGp was a poor substrate in EF-G-dependent translocation. Qualitatively and quantitatively its support of translocation was very similar to the reaction driven by periodate-oxidized and borohydride-reduced GTP, a derivative of GTP in which the ribose ring has been cleaved between the 2'- and 3'-hydroxyl groups.


Characterization of an inhibitor causing potassium chloride sensitivity of an RNA polymerase from T4 phage-infected Escherichia coli. The nature of the inhibition by salt (KCl) of DNA-dependent RNA polymerase from T4 phage-infected Escherichia coli (T4 enzyme) was studied using holoenzyme preparations, core enzyme and sigma fractions obtained by phosphocellulose column chromatography, and sigma fractions further purified by gradient centrifugation in the presence and absence of 6 M urea. We showed with holoenzyme preparations that salt inhibits the formation of rifampicin-resistant preinitiation complexes. The inhibition was considerably reduced when a nonionic detergent (particularly of the Triton series) was included in the reaction mixtures. With T4 core enzyme and T4 sigma fractions together with the same fractions from uninfected cells (host enzyme fractions) and different DNA templates, we showed that the T4 sigma fraction plays a role in the salt-sensitive activity with T4 DNA. The salt sensitivity of the T4 sigma fraction was antagonized by Triton; it was not a function of sigma fractions isolated from phage cultures infected in the presence of chloramphenicol. As reported previously (Stevens, A. (1973), Biochem. Biophys. Res. Commun. 54, 488), the T4 sigma fraction inhibited the activity of host sigma when they were present together in reaction mixtures, particularly in the presence of salt. T4 sigma further purified by centrifugation in glycerol gradients had the same properties as the cruder fraction, and the T4-specific polypeptide of mol wt 10000 (Stevens, A. (1972), Proc. Natl. Acad. Sci. U.S.A. 69, 603) was found in the same fractions. If the glycerol gradients contained 6 M urea, the mol wt 10000 polypeptide was separated from the salt-stimulated sigma. Fractions containing the small polypeptide could be added back to produce the salt-inhibitory effects. The inhibitory activity of both the crude sigma fraction and the fractions containing the small polypeptide was inactivated at 65 degrees C. The results suggest that the mol wt 10000 protein is a salt-promoted inhibitor, but the small amounts of it which are present in purified fractions of the T4 enzyme have not yet allowed its isolation in large enough quantities to permit a detailed study of its properties.


Synthesis of guanosine 5'-di- and -triphosphate derivatives with modified terminal phosphates: effect on ribosome-elongation factor G-dependent reactions. A series of GTP and GDP analogues modified in the terminal phosphate has been synthesized and their activities were investigated in elongation factor G dependent reactions. All of the analogues, with the exception of guanosine 5'-O-(3-thiotriphosphate), were not hydrolyzed by EF-G and ribosomes, but were competitive inhibitors of the ribosome-dependent EF-G GTPase. The most active inhibitors were P3-fluoro P1-5'-guanosine triphosphate and P3-methyl P1-5'-guanosine triphosphate with a Ki of 1.0 X 10(-6) and 2.5 X 10(-6) M, respectively. The activity of the GTP alkyl ester derivatives decreased with increasing number of carbon atoms in the side chain. GTP analogues were much more effective inhibitors than the corresponding GDP derivatives. This points out the necessity of the presence of at least three negative charges in the phosphate chain of the nucleotide for an effective interaction with the active site of the ribosomal EF-G GTPase. Guanosine 5'-O-(3-thiotriphosphate), which was hydrolyzed at one-third the rate of GTP, was able to support poly(U)-directed poly(phenylalanine) polymerization. Possible mechanisms of ribosome-EF-G GTP hydrolysis that arise from our results are discussed. Activity of the nucleotide analogues in EF-G-ribosome complex formation compared well with their ability to inhibit ribosome-dependent EF-G GTPase, P3-fluoro P1-5'-guanosine triphosphate and P3-methyl P1-5'-guanosine triphosphate being again the most effective ones. The stabilizing action of fusidic acid on the EF-G-ribosome complex formation induced by the various nucleotides could not be correlated to any of the structural modifications of the substrate. Guanylyl methylene diphosphonate was displaced more readily than GDP from the EF-G-ribosome complex by GTP analogues insensitive to fusidic acid.


Yeast DNA photolyase: molecular weight, subunit structure, and reconstruction of active enzyme from its subunits. Yeast DNA photolyase, purified by affinity chromatography, ran as a single component when analyzed by either electrophoresis on polyacrylamide gradient gels or by sedimentation velocity through 5-20% sucrose gradients containing 0.4 M KCl, and, therefore, was considered homogeneous. The molecular weights of photolyase, determined by these methods, were 130000 and 136000, respectively. When the enzyme was examined by electrophoresis on sodium dodecyl sulfate polyacrylamide gradient gels, it dissociated into two bands whole molecular weights were 60000 and 85000. After the enzyme was sedimented through sucrose gradients in the presence of 1.0 M KCl, two absorbance maxima, which corresponded to polypeptides of 54000 and 82500, were found in the fractions collected. Thus, the enzyme consists of two dissimilar subunits. When the two fractions that exhibited maximal absorbance were mixed together, a time-dependent increase in activity occurred, demonstrating that active enzyme could be reconstituted from these subunits. Analysis of sucrose gradients containing 1.0 M salt for photolyase activity showed that it was present exclusively in the region of the gradient corresponding to 68200 in agreement with a previous report (J. Cook and T. Worthy (1972), Biochemistry 11, 388). These active fractions were found in the overlap region between the two subunits, and their activity was attributed to reconstitution of the enzyme during the assay.


Physiochemical properties of DNA binding proteins: gene 32 protein of T4 and Escherichia coli unwinding protein. The single-stranded DNA binding protein coded for by gene 32 of bacteriophage T4 and a similar protein isolated from uninfected Escherichia coli both induce characteristic changes in the circular dichroism (CD) of single-stranded nucleic acids. These CD changes have been adapted as an assay of protein-DNA complex formation. Far-ultraviolet CD spectra show the secondary structure of the two proteins to be similar with approximately 20% alpha helix, approximately 20% beta structure, and 60% random coil. Both proteins show prominent Cotton effects arising from their aromatic chromophores. Nitration of five of the nine tyrosyl residues of gene 32 protein prevents DNA binding, while prior formation of the DNA complex protects all tyrosyl residues from nitration. The tyrosyl residues may participate in gene 32 protein-DNA binding by intercalation between bases of the single strand. In contrast, no tyrosyl residues can be nitrated in the E. coli protein suggesting that surface tyrosyls do not play a part in binding of E. coli protein to DNA. Approximately 50 amino acids can be cleaved from the gene 32 protein with trypsin. This cleavage also occurs spontaneously in infected cell extracts. The remaining protein of mol wt 30000 has the same CD spectra and DNA binding properties as the native protein. The physicochemical properties can be correlated with previous work on the structures and functions of the group of DNA "unwinding proteins".


Studies on the cooperative binding of the Escherichia coli DNA unwinding protein to single-stranded DNA. The cooperative binding of the Escherichia coli DNA unwinding protein to single-stranded DNA has been studied by electron microscopy. Analysis of the electron microscopic data by means of a simple statistical mechanical model has yielded a value of 3.8-7.6 X 10(10) l./mol for the cooperative binding constant in 0.15 M NaCl. Studied under elevated salt conditions have shown that the average DNA protein complex length is 50% of the length found at 0.04 or 0.15 M NaCl.


Phosphatidylethanolamine molecular species of fatty acid auxotroph of Escherichia coli grown with elaidate. Monoacetyldiglycerides derived from the phosphatidylethanolamine molecular species of the fatty acid auxotroph of Escherichia coli grown with elaidate at 37 degrees C were fractionated on thin-layer plates of silica impregnated with silver nitrate and were identified by gas chromatography-mass spectrometry with an OV-17 column and gas chromatography with a Silar-10C column. Phosphatidylethanolamine was made up of the following molecular species: 1-16 : 0-2-16 : 0 (1.2%), 1-14 : 0-2-trans-16 : 1 (1%), 1-16 : 0-2 trans-16 : 1 (3.5%), 1-16 : 0-2-trans-18 : 1 (26.4%), 1-16 : 0-2-cis-16 : 1 (3.8%), 1-trans-18 : 1-2-trans-16 : 1 (13.2%), 1-trans-18 : 1-2-trans-18 : 1 (44.9%), 1-trans-18 : 1-2-cis-16 : 1 (4.5%) and trans-18 : 1-cis-18 : 1 (1.5%).


Binding of alpha-ketoisovalerate to alpha-isopropylmalate synthase. Half-of-the-sites and all-of-the-sites availability. Binding of alpha-ketoisovalerate to alpha-isopropylmalate synthase (3-hydroxy-4-methyl-3-carboxyvalerate 2-oxo-3-methylbutyrate-lyase (CoA-acetylating), EC 4.1.3.12) from Salmonella thyphimurium has been studied by equilibrium dialysis. When alpha-ketoisovalerate is the only ligand present, no more than two sites per enzyme tetramer can be saturated under the conditions chosen. The binding is non-cooperative with a dissociation constant of 6.6+/- 0.4 muM. Binding of alpha-ketoisovalerate has also been studied in the presence of propionyl-CoA. This compound was selected because of its close similarity to the natural substrate acetyl-CoA. It is a competitive inhibitor with respect to acetyl-CoA while reacting only extremely sluggishly as as substrate itself. The presence of propionyl-CoA has a profound effect on alpha-ketoisovalerate binding. The number of sites available to alpha-ketoisovalerate increases to about four per tetramer. At the same time, the dissociation constant for alpha-ketoisovalerate increases approx. 4-fold. These results suggest that the active conformation of alpha-isopropylmalate synthase is not obtained unless both substrates are present. They also support the notion, based on previous studies with the feedback inhibitor L-leucine, that alpha-isopropylmalate synthase has a tendency to form "functional dimers".


Enzymatic epimerization of D-erythro-dihydroneopterin triphosphate to L-threo-dihydroneopterin triphosphate. An enzyme has been discovered in Escherichia coli that catalyzes the conversion of the triphosphate ester of 2-amino-4-hydroxy-6-(D-erythro-1',2',3'-trihydroxypropyl)-7,8-dihydropteridine, (i.e. D-erythro-dihydroneopterin triphosphate) to an epimer of this compound, L-threo-dihydroneopterin triphosphate. The enzyme, which is here named "D-erythro-dihydroneopterin triphosphate 2'-epimerase," needs a divalent cation (Mg2+ or Mn2+ is most effective) for maximal activity. Its molecular weight is estimated at 87 000-89 000. Little or no activity can be detected if either the monophosphate or the phosphate-free form of the substrate is incubated with the enzyme. Evidence is presented to establish that all three phosphate residues of the substrate are retained in the product and that the product is of the L-threo configuration.


Low molecular weight urinary proteins. I. Partial amino acid sequences of the retinol-binding proteins of man and dog. Human and dog retinol-binding proteins were isolated and their physico-chemical characteristics compared. Partial amino acid sequences of the first 50 residues were determined for both proteins and found to be remarkably similar. Only five residues were shown to be different; all could be due to single base pair mutations. However, immunological cross-reactivity was not detected between the two proteins with specific antisera prepared in rabbits against the human and dog retinol-binding proteins.


Isolation of outer membrane proteins of Escherchia coli and their characterization on polyacrylamide gel. Proteins from the outer membrane of Escherichia coli were studied on a ureadodecyl sulfate polyacrylamide gel by electrophoresis. A polyacrylamide gel containing sodium dodecyl sulfate and urea gave an excellent resolution of outer membrane proteins. Seventeen protein bands were reproducibly observed on a gel. By use of Sephadex G-200, DEAE-cellulose and polyacrylamide gel, eight proteins were purified to near homogeneity. Five of them were found to be heat-modifiable proteins. The behavior of these purified proteins was studied on a polyacrylamide gel under three different electrophoretic conditions, which had been used for the analysis of cell envelope proteins. Thus correspondence was made between these purified proteins and envelope proteins reported by other investigators.


Characterization of a low density cytoplasmic membrane subfraction isolated from Escherichia coli. We have used freeze fracture electron microscopy to study the distribution of membrane proteins in the cytoplasmic membrane of Escherichia coli W3110. While these proteins were distributed randomly at the growth temperature (37 degrees C), there was extensive protein lipid segregation when the temperature was lowered, resulting in bare patches containing no visible particles (protein), and areas of tightly packed or aggregated particles. To understand the segregation process, we have separated the bare patches from the particle rich membrane areas. Lysis of spheroplasts at 0 degrees C leads to cytoplasmic membrane fragments with different amounts of membrane particles per unit area; such fragments have been separated on isopycnic sucrose gradients. The bare patches occurred as low density membranes which were completely devoid of particles. They were compared to normal density cytoplasmic membranes with respect to fatty acid composition, protein distribution as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their content of several cytoplasmic membrane marker enzymes. The phospholipid to protein ratio of low density membranes was five times greater than that of normal membranes; unsaturated fatty acids were more abundant in the low density membranes. Most proteins had disappeared from the low density membranes. One protein, which had an apparent molecular weight of 26000 on sodium dodecyl sulfate gels appeared to be concentrated in the low density membranes; it accounted for about 50% of the total protein found in this membrane fraction. Of the cytoplasmic membrane markers tested, NADH oxidase and succinate dehydrogenase were excluded, while D-lactate dehydrogenase remained, and even appeared to be concentrated in the low density membranes. These results indicate that while most membrane proteins are associated with the fluid portion of the bilayer, some proteins evidently associate preferentially with phospholipids in the gel or frozen state.


Effect of tetraphenylboron upon the uptake of the lipophilic cation dibenzyldimethylammonium by yeast cells. The rate of uptake of the lipophilic cation dibenzyldimethylammonium by yeast cells is increased by tetraphenylboron. However, tetraphenylboron increases also the equilibrium partition of dibenzyldimethylammonium between cells and medium, probably because a complex between tetraphenylboron and dibenzyldimethylammonium is trapped inside the cells. Accumulation of dibenzyldimethylammonium in the presence of tetraphenylboron is not reversed by dinitrophenol, whereas accumulation of the lipophilic cation in the absence of tetraphenylboron appears to be almost completely reversible.


In vitro reassembly of the membranous vesicle from Escherichia coli outer membrane components. Role of individual components and magnesium ions in reassembly. A method was developed for the reassembly of membranous vesicle from the sodium deoxycholate-dissociated outer membrane components of Escherichia coli. The removal of the detergent by dialysis and the presence of Mg2+ were essential for the reassembly. Membrane protein alone did not form any membranous structure. Closed membranous vesicles similar to the native outer membrane were reassembled only when protein was mixed with both lipopolysaccharide and phospholipid in deoxycholate solution and subsequently dialized. The membrane showed a distinct trilaminar structure with a center-to-center distance between two dark lines of 53 A, which is a characteristic of the native outer membrane. This characteristic trilaminar  structure was shown to be due to the presence of lipopolysaccharide. Phospholipd was required for the vesicularization of membrane. Lipopolysaccharide and/or phospholipid formed a membranous structure in the absence of protein, while the morphology of their negatively stained sample was quite different from that of the native outer membrane unless the outer membrane protein was added to the reassembly mixture. The protein from the cytoplasmic membrane was unable to reform membranous vesicle with lipopolysaccharide and phospholipid, indicating that the reassembly system discriminated outer membrane proteins from cytoplasmic proteins.


Induction of radioresistance in Escherichia coli. The effect of prior treatment by inducing agents on the radioresistance of cells of Escherichia coli has been studied. In order to separate the induction process from the radiation-damage process, cells were first treated with inducing agents such as ultraviolet light, ionizing radiation, or nalidixic acid, allowed to become induced by incubation for 50 min and then given rifampin to prevent further induction. They were then tested for radiation sensitivity. It was found that all strains tested except recA-, lex-, and recB showed very apparent protection. Induction by UV had the most effect and by nalidixic acid the least. The time course of development of protection was observed in one case: it is 50% established in 15 min. The absence of effect in recA- and lex- is explainable by the fact that these cells cannot be induced, for example, for prophage or the inducible inhibitor of post-irradiation DNA degradation. We suggest that the inducible inhibitor of postirradiation DNA degradation is one factor in a recovery system possessed by E. coli cells.