Possible roles of the pancreatic D-cell in the normal and diabetic states. The A-, D-, and B-cells--the islet cells that contain, respectively, immunoreactive glucagon, somatostatin, and insulin--are distributed within a specialized heterocellular region of the islets of Langerhans as if to permit heterologous contacts between all three cell types. Inasmuch as each one of the three secretory products of these three cell types influences the secretion of at least one of its neighboring cells, "paracrine" influence on islet hormone secretion becomes a reasonable hypothesis. Glucagon stimulates both insulin and somatostatin release, while insulin and somatostatin both inhibit glucagon release, providing the basis for a feedback relationship through which A-cell secretion may be restrained. In addition, glucagon-mediated insulin secretion may be estrained by glucagon-stimulated somatostatin release. Such intercellular relationships could help determine the composition of the insulin and glucagon mistures released within a given metabolic setting.


Contractile proteins and pancreatic beta-cell secretion. Much indirect evidence suggests, but does not prove, that insulin secretion depends on contractile proteins similar to those of skeletal muscle and cilia. Such proteins constitute a molecular basis for the emiocytotic extrusion of insulin granules. It is likely that the secretory machinery is complex, requiring over eight proteins. The available evidence is consistent with a model of saltatory granule movement oriented by microtubules and powered by actomyosin contraction in response to elevations in cytosol calcium. Because most diabetics secrete some insulin and because relatively little of the stored B-cell insulin is released in response to hyperglycemia, further research into the molecular mechanism of insulin granule release is needed.


Hyperplasia of "pancreatic polypeptide"-cells in the pancreas of juvenile diabetics. The cellular composition of the pancreatic islets of juvenile diabetics was studied, using recently developed immunocytochemical methods. B-cells were identified only in juvenile diabetics with a disease of short duration. In chronic juvenile diabetics, the islets which are classically viewed as "atrophic", were shown to be composed of glucagon- and of somatostatin-cells. Another type of islets which commonly occurs in the pancreas of juvenile diabetics, i.e. the ribbon-like type first described by Cecil in 1911, appeared to be composed almost exclusively of "pancreatic polypeptide" (HPP)-cells. It is suggested that hyperplasia of the HPP-cells in the pancreas of juvenile diabetics results from an atypical type of islet regeneration induced by a severe and prolonged injury to the pancreatic endocrine tissue.


Effects of L-leucine on the insulin production, oxidative metabolism and mitochondrial ultrastructure of isolated mouse pancreatic islets in tissue culture. This study was performed to evaluate whether L-leucine is able to relieve the structural and functional alterations previously described in pancreatic islets exposed in vitro for a prolonged time to a subnormal glucose concentration (3.3 mM). It was found that both the impairment of secretion and the decreased rate of biosynthesis of insulin characteristic of islets cultured for one week in 3.3 mM glucose were prevented by adding 15 mM L-leucine to the culture medium. Further more, the rates of tritiated water production and glucose or leucine oxidation were significantly enhanced after culture in the presence of L-leucine. The rate of DNA synthesis as estimated by the incorporation of tritiated thymidine was, however, unchanged by the presence of L-leucine in the culture medium. Leucine cultured islet cells displayed ultrastructural signs of high functional activity. A detailed morphometric examination revealed fewer but hypertrophic mitochondria. The present results suggest that L-leucine can replace glucose in several respects as a long-term stimulus of the pancreatic B-cells, possibly by acting as a metabolic substrate.


An apparent inhibition of insulin biosynthesis resulting from inhibition of transport of neutral amino acids by arginine. At concentrations higher than 10 mM, the cationic amino acid, arginine, inhibited the incorporation of neutral amino acids such as alanine, threonine, valine and leucine into insulin in the presence of glucose. This inhibitory effect probably did not result from the stimulatory effect of argnine on insulin release because, in the absence of glucose, arginine failed to stimulate insulin release but nevertheless inhibited the incorporation of leucine into insulin. This inhibitory effect of arginine was shared by another basic amino acid, histidine, but not by lysine. Arginine inhibited the incorporation of leucine not only into insulin but also into other islet proteins. This inhibition was not accompanied by any disturbance of glucose metabolism in the islet cells. Further studies indicated that the inhibition of incorporation resulted primarily from the interference of uptake of the neutral amino acids by arginine.


Developmental studies of vitellogenesis in a dipteran insect, Chironomus thummi. Vitellogenesis of developing oocytes of a Dipteran insect Chironomus thummi has been investigated. The onset of yolk deposition is marked by the differentiation of the oolemma including the formation of microvilli and endocytosis. These changes are accompanied by the appearance of small electron dense granules, similar in density to the yolk platelets, arising through the sequential accumulation of material into the matrices of the multivesicular bodies (MVBs). These latter structures are produced in the previtellogenic oocytes of the pharate pupae and early pharate adults. Often the limiting membrane of the MVBs bears bristle coats resembling those of the coated vesicles of pinocytotic origin, suggesting that it is through the fusion with the pinocytotic vesicles that the accumulation of dense material in the MVBs results. That the Mvbs transform into structures resembling yolk granules is supported by statistical analysis which indicates that the decrease in the number of electron-dense MVBs coincides with the increase in the occurrence of small dense yolk granules. In the late pharate adult stage the yolk granules are considerably larger than those of earlier stages. It is during this period that at least one type of electron-dense granule occurs at the oocyte follicle cell border, and that these apparently contribute to the formation of the vitelline envelope. The results of the present study indicate that preformed oocytic elements, the MVBs, play a strategic role in the formation and arrangement of the yolk granules in Chironomus. Since these structures account for the bulk of the ooplasm, it appears that the MVBs are at least partly responsible for the correct ordering of the cytoplasmic constituents of the oocytes, which is critical for the proper development and differentiation of the embryo.


Effects of diet on glucaric acid concentration in bile and the formation of calcium bilirubinate gallstones. The authors reported previously that beta-glucuronidase in bile, especially during biliary infection with Escherichia coli, plays a substantial role in producing cium bilirubinate gallstones. In the present study, bile levels of glucaro-1:4-lactone (measured as glucaric acid) the leading inhibitor of beta-glucuronidase, were measured in both man and in rats fed high, medium, and low protein-fat diets. Glucaric acid and total bilirubin in bile correlated well in human controls but not in gallstone patients. In animal experiments, the levels of these substances in bile were high in rats on high protein-high fat and low in those on low protein-low diets. These data suggest that when bile is infected with E. coli, calcium bilirubinate gallstones seemed to form more easily in patients on low protein-low fat diet than in those consuming food rich in protein and fat. On the other hand, the ratio of lecithin to cholesterol was higher in low protein-low fat rats than in high protein-high fat rats, suggesting that cholesterol gallstones were more likely to form on the latter diet. The animal, clinical, epidemiological, and dietary data are consistent with the known trend to a decreased incidence of calcium bilirubinate and an increased incidence of cholesterol gallstones in Japan.


Unusual complication of the Sengstaken-Blakemore tube. Reported here is the first description of innominate vein obstruction by esophageal tamponade. The syndrome is characterized by local edema of the area drained by the vein. The diagnosis can be made by phlebography. Deflation of the balloon corrects the problem.


[Investigations on the contraceptive action of the Graefenberg ring. Historical review (author's transl)]. The biological activity of 2 Graefenberg rings was tested in vitro by electrone microscopy and X-ray microanalysis. The investigation showed that Graefenberg rings made of silver contain a varying amount of copper. The Graefenberg rings showed dissociation of copper in vitro which probably also occurred in the uterine cavity. The effect of the copper on the endometrium was probably a factor in the good contraceptive effectiveness of the Graefenberg silver ring compared for the silk thread intrauterine pessaries.


[The treatment of threatened abortion (author's transl)]. In a prospective study 265 patients with threatened abortions were divided into 2 groups. One group of 134 patients received treatment with Oestradiol benzoat and hydroxyprogesterone capronat. The other group of 131 patients received no hormonal treatment. Of the group treated with hormones 40.7% of the women had an abortion. Of the group treated with placebos 39.7% of the patients had an abortion. There was therefore no difference in the success rate of the treatment of threatened abortion irrespective of the administration of hormones or placebos. The value of hormonal therapy in the treatment of threatened abortion is therefore questionable.


Variation in the number of genes coding for salivary amylase in the bank vole, clethrionomys glareola. A Danish population of bank voles is polymorphic for three electrophoretically different salivary amylases; A, H and S, of which A is the most common. Both single-, double- and triple banded phenotypes were observed, and in several crosses two electrophoretic forms cosegregated. In addition to the qualitative variation, some individuals show consistent quantitative variation in the relative activities of their amylase bands. This variation has been qualified by spectrophotometrical measurements of the relative amounts of amylase protein in the various bands. --Seventy wild chromosomes were analyzed by determining the amounts of amylase they produced when heterozygous with a laboratory stock chromosome known to carry two closely linked amylase genes, both coding for a fourth electrophoretic variant, B. The amount of A-protein divided by half the amount of B-protein was used as an estimate of the number of A-genes on the tested chromosomes. The wild chromosomes fell into three clearly distinguishable classes: 9 clustered around a gene number estimate of one, 45 chromosomes yielded estimates around two genes, and the gene number estimate of the remaining 16 was close to three. The integer values of the gene number estimates and the cosegregation of electrophoretically different salivary amylases are consistent with the model that the population is polymorphic for chromosomes with either one, two, or three closely linked amylase genes. It is suggested that such gene number variation may be more common than generally recognized, and some other reported cases of quantitative enzyme variation, for instance that of human red cell acid phosphatase, are interpreted in terms of variation in the number of genes involved.


Proteinase mutants of Saccharomyces cerevisiae. Fifty-nine mutants with reduced ability to cleave the chymotrypsin substrate N-acetyl-DL-phenylalanine beta-naphthyl ester have been isolated in S. cerevisiae. All have reduced levels of one or more of the three well-characterized proteinases in yeast. All have reduced levels of proteinase C (carboxy-peptidase Y). These mutations define 16 complementation groups.


The effect of ochre suppression on meiosis and ascospore formation in Saccharomyces. The effect of altered tyrosyl-tRNAs on the developmental process of sporulation was examined. Mutations in eight independent loci resulting in tyrosine-inserting nonsense suppressor were tested for their effects on sporulation. Different levels of inhibition were found ranging from SUP3-omicron, which caused the greatest reduction of sporulation (7-17% of wild type), to SUP11-omicron which caused no reduction in sporulation. Since the SUP3-omicron mutation exhibited the greatest effect, it was studied in detail. Although SUP3-omicron is a dominant nonsense suppressor, its effect on sporulation is recessive. Expression of the sporulation deficiency is dependent upon the stage of transfer from glucose growth medium (i.e., log, early stationary, etc.) to sporulation medium. SUP3-omicron/SUP3-omicron diploid cells transferred from log or early stationary phase are capable of sporulation, whereas cells transferred after early stationary phase (i.e., after adaptation to respiration) exhibit poor sporulative ability. Sporulation events were examined under restrictive conditions to observe those events completed by SUP3-omicron/SUP3-omicron diploids. The early events of sporulation occur in these cells. Later events are completed by progressively fewer cells. Premeiotic DNA synthesis occurred in approximately 40% of the cells, nuclear segregation occurred in 20%, and finally, only 2% formed asci. The fact that fewer late-sporulation events occur under restrictive conditions can be explained by increased efficiency of suppression.


A genetic fine structure analysis of the suppressor 3 locus in Saccharomyces. A meiotic fine structure map of a yeast tyrosine-inserting ochre suppressor, SUP3-omicron, was constructed. This was accomplished by examining ten intragenic suppressor-inactive revertants for their relationship to each other and to the original SUP3-omicron mutation. The second-site revertants map on both sides of the SUP3-omicron mutation. The meiotic map length based on the summation of short intervals is 45+/10(5) asci.


Controlled trial of synthetic D-penicillamine and prednisone in maintenance therapy for active chronic hepatitis. In view of promising, but uncontrolled, reports of the use of D-penicillamine in active chronic hepatitis, a randomised, prospective, controlled trial of this drug against prednisone was carried out. Of the 35 patients entered, 18 received D-penicillamine (increasing to 1-2 g daily) and 17 prednisone (15 mg daily). In all patients the disease had already been brought under biochemical control with corticosteroids. During the first year of the trial, the treatment of nine patients in the D-penicillamine group was discontinued (two because of lack of disease control and seven because of side-effects) compared with six patients in the prednisone group (four because of lack of disease control, one because of side-effects, and one because of the development of carcinomatosis. Detailed statistical analysis of the liver function tests in the patients remaining in the trial at the end of the year showed no significant differences. D-penicillamine is associated with a higher frequency of side-effects than is prednisone. However, in some patients it is as satisfactory as prednisone in keeping the disease under control.


Correlation between hepatic morphology and immunoglobulins and antibodies to Escherichia coli in cirrhosis. Increased antibody production and hypergammaglobulinaemia in cirrhosis are probably to a large extent due to decreased hepatic extraction of antigens. The deceased extraction is presumably related to changed microcirculation caused by damaged anatomical structure of the liver. It is therefore to be expected that immunoglobulin and antibody levels in serum in cirrhotic patients are related to the degree of certain morphological changes of the liver. This hypothesis has been tested. In 50 patients with cirrhosis, 28 alcoholics and 22 non-alcoholics, the degree of architectural destruction, the degree of fibrosis, the degree of fatty infiltration, and the degree of "activity" were compared with immunoglobulins G, A, and M and E. coli O antibody levels. The comparison was carried out within each of the aetiological groups. Identical relationships were found in both groups. Patients with completely destroyed lobular architecture had higher levels of E. coli O antibodies than patients with partly destroyed architecture. Patients with severe fibrosis had higher IgA and E. coli O antibody levels than patients with moderate or slight fibrosis. Patients with moderate and severe steatosis and patients with no or slight steatosis had the same immunoglobulin and E. coli O antibody levels. Patients with active cirrhosis had higher IgG levels than patients with inactive cirrhosis. When architectural destruction and fibrosis were combined significantly higher IgG, IgA, IgM, and E. coli antibodies were found in the group with the most severe changes. These findings support the hypothesis that immunoglobulin and antibody levels are related to the degree of morphological changes in the liver--namely, destruction of lobular architecture, fibrosis, and "activity".


Zinc in ulcerative colitis: a therapeutic trial and report on plasma levels. A double-blind controlled trial of oral zinc sulphate as adjuvant treatment in idiopathic ulcerative colitis or proctitis in relapse is reported. Fifty-one patients were treated, and the clinical and sigmoidoscopic improvement in the zinc treated patients was similar to that in patients receiving placebo. No difference was found between plasma zinc levels in a further 46 patients with idiopathic ulcerative colitis or proctitis and those obtained in a group of healthy controls.


Controlled trial of Duogastrone in duodenal ulcer. A double-blind placebo-controlled trial of carbenoxolone, as 50 mg Duogastrone capsules, and in a dose of 200 mg daily for 12 weeks, was carried out in 40 ambulant subjects with endoscopically diganosed duodenal ulceration, of whom 34 were available for final analysis. Each patient was seen every two weeks and endoscoped at four, eight and 12 weeks. Serum carbenoxolone was measured at each visit. Complete ulcer healing occurred in a significantly greater number of patients receiving Duogastrone than placebo, the significance being greater after four and eight weeks treatment (P less than 0-01) than at 12 weeks (P less than 0-02). While significant symptomatic improvement also was achieved (P less than 0-05), but only after 12 weeks on Duogastrone, there was much closer correlation between ulcer healing and symptom relief, 69% on Duogastrone returning to normal, compared with 22% of controls (P less than 0-02). Rise of systolic blood pressure and reduction in serum potassium levels, especially during the last four treatment weeks, were the most common effects noted in patients taking Duogastrone, and five patients required thiazide diuretics and potassium supplements. Higher serum carbenoxolone levels were found in patients with healed ulcers as well as in those with more marked side-effects.


Correlation between survival, ability to rejoin DNA and stability of DNA after preirradiation inhibition of protein synthesis in a rec mutant of Escherichia coli K12. A 90 min inhibition of protein synthesis induced by starvation for amino acids (AA-) or by treatment with chloramphenicol (CAP) prior to UV irradiation (2.5 Jm-2) increased the resistance of the strain Escherichia coli K12 SR19 to UV radiation more than ten-fold. Under these conditions, cultures in which protein synthesis was inhibited before the UV irradiation rejoin short regions of DNA synthesized after the irradiation to a normal-size molecule, whereas an exponentially growing culture does not rejoin DNA synthesized after UV irradiation to a molecule of a normal size. In the exponentially growing culture both the parental and the newly synthesized DNA are unstable after the irradiation. In cultures with inhibited protein synthesis only the parental DNA is somewhat unstable. In Escherichia coli K12 SR19 where protein synthesis was inhibited before the irradiation, a correlation between the survival of cells, the ability to rejoin short regions of DNA synthesized after UV irradiation and a higher stability of both parental and newly synthesized DNAs could be demonstrated.


[Problems and tasks of psychology in terminal kidney failure in childhood]. After a review on aspects of the role of the chronically ill the social competence of psychological prossess of adaptation and effects of the disease on the family of the child with chronic renal failure are reported. Futhermore, psychological aspects of renal transplantation as well as tasks and structures of psychological management are discussed.


Inhibition of protein kinase activity from Trypanosoma cruzi and Trypanosoma gambiense by 3'-deoxyadenosine. 3'-Deoxydadenosine was found to be a potent inhibitor of nucleoside-stimulated protein kinase activity from culture forms of Trypanosoma cruzi and bloodstream forms of Trypanosoma gambiense. The type of inhibition by 3'-deoxyadenosine was competitive with respect to ATP. The inhibition constants for 3'-deoxyadenosine were determined to be 0.11mM and 0.085mM for the enzyme from T. cruzi and T. gambiense, respectively. The apparent Km value for ATP was 0.2mM for both enzymes. 2'-Deoxyadenosine was less effective as inhibitor of the protein kinase activity. The inhibition constants were calculated to be 0.8mM and 0.67mM, respectively.


Synthesis and biological activity of position 1 analogs of LH-RH. (Formyl-sarcosine)1-LH-RH (I), (acetyl-sarcosine)1-LH-RH (II), (2-pyrrolidone-4-carboxylic acid)1-LH-RH (III), (N-methyl-2-pyrrolidone-4-carboxylic acid)1-LH-RH (IV, hydroxyproline1-LH-RH (V) and (cylcopentane-carboxylic acid)1-LH-RH (VI) were synthesized by solid phase methods on a benzhydrylamine resin support. Peptides I-IV were assayed for LH- and FSH-releasing activity over a 4-h period after subcutaneous infection into immature male rats in order to detect any prolongation ofactivity. The peptides were found to have the following integrated LH-releasing activities compared with LH-RH: I, 64%; II, 72%; III, 19%; IV, 58%. None of the peptides were found to be longer acting than LH-RH. Peptides V and VI were far less active, 0.001% and 1.4%, respectively.


Morphology and physiology of salivary myoepithelial cells. Myoepithelial cells, which occur in many but not all exocrine glands, are usually quite conspicuous in salivary glands. Their contractile nature seems established beyond doubt, and the morphological similarities to smooth muscle cells are close enough to suggest that they contract by a similar, if not the same, mechanism. Morphologically speaking, they seem to possess a dual autonomic innervation, but, functionally speaking, the sympathetic control seems usually to be more important that the parasympathetic. While their contraction undoubtedly causes a transitory increase in the rate of delivery of saliva to the oral cavity, the principal function of the cells seems to be to prevent distension of the endpieces during secretion. This is achieved partly by providing support for the endpiece and partly by a widening and shortening of the intercalated ducts, which has the effect of lowering the outflow resistance.


Microvascular changes in canine renal allograft rejection: a correlative microangiographic and histologic study. The progressive microvascular changes occurring during unmodified renal allograft rejection were studied in 22 dogs utilizing post-mortem barium sulfate perfusion microangiography. Sixteen dogs (7 autografts, 8 ischemia controls and 1 hydronephrotic animal) made up a control group. The microradiographic findings were correlated with intravenous urography, coagulation profiles and histopathologic studies. Vascular obstruction and obliteration occur from the outer cortex inward and involve sequentially, capillaries, efferent arterioles, glomeruli, afferent arterioles and interlobular arteries. A-V communications develop late and are due to a loss in vascular integrity. Medullary vascular obliteration occurs subsequent to cortical changes. The microangiographic changes mirror the hstologically evident cellular infiltrate, vascular damage and eventual hemorrhagic necrosis. The appearance of fibrin split products in the plasma late in rejection and the dramatic vascular occlusive changes seen microangiographically suggest that localized intravascular coagulation occurs during rejection.


Microangiography of rejected human kidney transplants. A microangiographic study of 17 rejected and surgically removed human kidney transplants, three transplants removed for other reasons and two adequately perfused and preserved but not transplanted cadaver kidneys was performed. Transplants rejected with acute clinical course showed poor or variable glomerular filling and tapering cortical arteries with no impairment of medullary perfusion, more extensively in those transplants showing poor initial function. Chronic rejection, independent of other parameters showed lack of cortical perfusion, and glomerular filling; impaired perfusion of whole segments, arterial wall narrowing and changes secondary to kidney shrinkage. The radiographic patterns of chronic and acute rejection were distinguishable from each other and from nonrejected specimens.


The effect of intravenous sodium diatrizoate on the function of the transplanted canine kidney. The response of the canine renal autograft and allograft was measured following intravenous injection of 1 cc/kg 50% sodium diatrizoate (50% Hypaque). There was no significant effect on glomerular filtration rate or renal plasma flow immediately after injection; likewise there was no significant change in GFR 24 hours following injection compared to control animals.


Probability of survival in hypertensive and nonhypertensive patients on maintenance hemodialysis. The actuarial survival rate for 58 unselected patients who entered a program of maintenance hemodialysis and transplantation was found to be 43.0 +/- 8.3 (SE)% for the six-year period of observation. The survival rate was considerably lower in hypertensive patients as well as in patients with familial Mediterranean fever with amyloidosis, all of whom were nonhypertensive. When the patients with familial Mediterranean fever were excluded from the non-hypertensive group, the expected survival rate of this group became greater than that of the hypertensive group, the difference being about 25% in five years and about 50% in six years. This difference in the survival rate approaches that between normotensive subjects and untreated severely hypertensive patients in the general population. It is concluded that hypertension is a serious limiting factor in the survival of patients on chronic hemodialysis, and that the difference in survival between the hypertensive and the non-hypertensive patients is attributable to hypertension.


Rhinoscleroma.

Rhinoscleroma. Rhinoscleroma may present a diagnostic problem when encountered outside endemic region. Two young men from Gaza with tumor-like masses of scleroma in the nasal cavities and the nasopharynx are described. Histological examination of biopsy material from the nasal lesions showed pathognomonic Mikulicz cells, and cultures grew Klebsiella rhinoscleromatis. Both patients responded well to antibiotic treatment.


[Description of human hair and skin by Martin Frobenius Ledermüller (1719-1769)]. As a result of his research extended over several years the lawyer and naturalist Martin Frobenius Ledermüller published in 1761 the "Mikroskopische Gemüths- und Augen-Ergötzung". His book contains 100 coloured copper etchings dealing with all aspects of nature and a detailed accompanying text. Two copper etchings which depict human hair and skin will be reproduced here along with excerpts of the original descriptions.


[The present status of vestibular testing (author's transl)]. Proceeding from the standard aims of every vestibular test, modern fine-quantitative investigatory methods with or without electronystagmographic recording in opposition to the systematic search for spontaneous and provoked nystagmus are presented with a view to their value for medical practice. Examination of spontaneous nystagmus and provoked nystagmus including the fistula symptom and coarse-quantitative excitability tests still represents the most important and most profitable part of the vestibular investigation. For modern research methods standardization of nomenclature and restriction of their plurality are as necessary as a method, which takes in not only single parameters but the whole data contents of vestibular reactions. The practicability of the method is documented by the author's own research work in this field.


The pig synovium, II. Some properties of isolated intimal cells. I. The effect of trypsinization. Certain physiological and immunological properties of isolated intimal synoviocytes were studied in various in vitro systems. Suspensions of the synoviocytes were obtained by incubating sheets of joint capsule in a solution of trypsin and then scraping off the intimal cells with a small knife. Synovium was examined immediately after trypsinization, but before scraping, by light and electron microscopy. The intimal cells had withdrawn many of their long processes, acquired lamelliform pseudopodia, and somewhat resembled cells of the A-type (Barland et al. 1962). To test the viability of the trypsinized synovium, fragments of trypsinized joint capsule were explanted in organ culture with the intima in contact with a Millipore substrate (Fell et al. 1976). After two days the intimal synoviocytes had become branched and, as in untreated control explants, now resembled B-cells. II. Phagocytosis and opsonic adherence. In a freshly prepared suspension of synoviocytes scraped from trypsinized synovial tissue many cells were still branched, but during 1 1/4 hours' incubation in serum-containing medium the majority had withdrawn their processes and become rounded. Cells in the branched form had little capacity for phagocytosis, but most of those in the rounded form were actively phagocytic. After suspensions of intimal cells had been incubated with opsonized sheep erythrocytes, most of the rounded, but none of the few remaining branched cells, had formed rosettes. Intimal cells from scraped synovial tissue were maintained in Sykes-Moore chambers for periods of up to 48 hours. Although the young pig synovium contains only a small proportion of macrophage-like (A-type) cells, in the cultures the cell population consisted of cells indistinguishable from macrophages, with a few small colonies of typical fibroblasts. In marked contrast to the fibroblasts, the macrophage-like cells in the Sykes-Moore cultures were highly phagocytic and formed conspicuous opsonic rosettes. Excessive phagocytosis inhibited subsequent rosetting by the macrophage-like cells. III. The effect of antiserum, with and without complement, on synovial cells. In view of earlier work by Fell & Barratt (1973) the effect of rabbit antiserum to pig erythrocytes (AS) with and without serum complement (C') on intimal synoviocytes in a Sykes-Moore chamber was investigated. AS+C' either lysed the macrophage-like cells or caused them to fuse into multinucleate giant cells. In the presence of AS without C', the macrophage-like cells formed large lakes of multinucleated cytoplasm. Fibroblasts in the same cultures were much more resistant to lysis by AS+C' and did not form multinucleate giant cells when exposed to AS either with or without C'.


Norvaline accumulation by regulatory mutants of Serratia marcescens. A gene coding for desensitized L-threonine dehydratase was transduced with phage PS20 into a leucine accumulator of Serratia marcescens Sr41. The transductant converted L-threonine to alpha-ketobutyrate, a precursor of both norvaline and isoleucine. An isoleucine-valine auxotroph of the transductant accumulated large amount of norvaline from L-threonine as well as from D-threonine.


Mutational biosynthesis by idiotrophs of Micromonospora purpurea. I. Conversion of aminocyclitols to new aminoglycoside antibiotics. By mutation and strain improvement techniques idiotrophs of Micromonospora purpurea, the gentamicin-producing organism, were obtained which require an exogenous source of 2-deoxystreptamine in order to produce gentamicin. Streptamine incorporation afforded a mixture of 2-hydroxygentamicin C as a complex of essentially the C1 and C2 components whereas 2-deoxystreptamine when incorporated by the same idiotroph afforded the same mixture of C1, C2 and C1a gentamicins as the parent (m1) organism. The 2-hydroxygentamicin C complex exhibited broad-spectrum antibiotic activity with an in vitro potency less than that for the gentamicin C complex, but with greater activity against selected gentamicin C resistant organisms. The LD 50 (i.v.) in mice of the 2-hydroxygentamicin C complex indicated that it had approximately half the toxicity of the gentamicin C complex. 2, 5-Dideoxystreptamine affordeda C1, C2, and C1a mixture of 5-deoxygentamicins, which also had broad spectrum activity, and exhibited improved activity against several gentamicin-acetylating strains of resistant bacteria. The LD50 (i.v.) in mice of the 5-deoxygentamicin C complex indicated that it was about 2.5 times more toxic than the gentamicin C complex. Two derivatives of 2,5-dideoxystreptamine afforded the same mixture of 5-deoxygentamicins. 2-Epistreptamine upon supplementation to a broth containing growing cultures of these idiotrophs also produced antibiotic.


Comparison of endotoxin and leukocytic pyrogen pyrogenicity in newborn guinea pigs. Guinea pigs under 8 days of age generally are unable to develop fever (viz., deltaTre greater than 0.5 degrees C) in response to a standardized dose of endotoxin (2 mug/kg iv of Salmonella enteritidis [SE]). This study was undertaken to determine whether this lack of responsiveness might be due to an incapacity of leukocytes from young neonates to produce sufficient leukocytic pyrogen (LP). Three series of experiments were performed at Ta = 27 degrees C: guinea pigs aged 0-2, 4, and 8 days were injected iv with: a) 2, 4, 8, or 16 mug/kg of SE, b) 0.1, 0.5, or 1.0 ml of LP generated by 8 mug of SE/25 X 10(6) leukocytes from adult guinea pigs (LPa), or c) 0.1 or 1.0 ml of LP generated by 8 mug of SE/25 X 10(6) leukocytes from 0-5-, 6-12-, and 13-16-day-old guinea pigs (LPn). Adult guinea pigs received iv 1.0 ml of LPa or LPn. The results revealed that fever could be induced in these animals from birth, but the required doses of SE, LPa and LPn were greater the younger the guinea pigs. Under these conditions, LPn, regardless of the donors' ages, produced fever in all the recipients. It is concluded that the pyrogenic unresponsiveness of newborn guinea pigs to endotoxin may be related not to an inability of leukocytes from these neonates to elaborate LP, but rather to an insensitivity of, presumably, their hypothalamic febrogenic mechanisms to low levels of LP.


Air embolism with positive-pressure ventilation of rats. Although air embolism is known to occur in humans and animals when the lung is overdistended, very few cases have been reported to be associated with positive-pressure ventilation. We have observed that air embolism occurs in rats ventilated with high inspiratory pressures (70 cmH2O) associated with high end-expiratory pressures (10 cmH2O). However, it does not occur in normal rats if the end-expiratory pressure is less than 5 cmH2O or the peak inspiratory pressure is below 60 cmH2O when the frequency of ventilation is 30. Hemorrhagic shock predisposes to air embolism, whereas conditions with pulmonary edema (fluid overloading, lung injury from ventilation with high inspiratory and low expiratory pressures, or oxygen toxicity) decrease the probability of its occurrence.


Use of three optical systems to obtain a microscopic profile of asbestos and other nonopaque particulate matter larger than 5 mum in parenteral drugs. A comparative microscopic study of chrysotile fibers was conducted, using 3 sequential optical systems: plane polarized light (PL), phase contrast (PC), and Nomarski differential interference contrast (NDIC) for complementary image analyses. The introduction of NDIC for qualitative and quantitative analyses of particulate matter greater than 5 mum resolves the problem of diffraction halos encountered in PC microscopy. High optical contrast, the instrumental characteristic of NDIC, is particularly useful for the detection of chrysotile fibers at a magnification of 500 X.


Establishment of exponential growth after a nutritional shift-up in Escherichia coli B/r: accumulation of deoxyribonucleic acid, ribonucleic acid, and protein. The accumulation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein was followed in cultures of Escherichia coli B/r during exponential growth in different media and for 2 h after a nutritional shift-up from succinate minimal medium (growth rate [mu1] = 0.67 doublings per h) to glucose plus amino acids medium (mu2 = 3.14 doublings per h). During postshift growth of the culture, the amounts of RNA (R), DNA (D), and protein (P) increased such that the ratios of the increments (delta R/delta P; delta D/delta P) were constants (k1, k2). This implies that the rates of accumulation of nuclei1:k2:1. These constants change from their preshift value to their final postshift value (i.e., k1 and k2) within a few minutes after the shift. k1 is a function of the activity of ribosomes, whereas k2 is related to the initiation of rounds of DNA replication. These parameters and the observed change in the doubling time of RNA (= mu2/mu1) were used to derive kinetic equations that describe the accumulation of DNA, RNA, protein, and cell mass during the 2- to 3-h transition period after a shift-up. The calculated kinetics agree closely with the observed kinetics.


Mutants of Escherichia coli "cryptic" for certain periplasmic enzymes: evidence for an alteration of the outer membrane. Mutants in which the expression of periplasmic enzymes by whole cells is reduced (termed "cryptic") are also found to show greatly reduced uptake of labeled adenosine 5'-monophosphate (5'-AMP), providing a rapid assay for crypticity. The crypticity of 3'- and 5'-nucleotidase has been examined as a function of substrate concentration. The Km for 3'- or 5'-AMP increases in the cryptic mutants when whole cells are used as the enzyme source. The Vmax is not altered. Electrophoretic analysis of protein prepared from cell envelopes showed that three cryptic mutants have a polypeptide absent from the outer membrane and a relatively high proportion of a polypeptide in the inner membrane. Analysis of the molar ratios of constituent sugars of the lipopolysaccharides showed no differences between three cryptic mutants and the parent strain. One cryptic mutant (3--41), however, has altered sensitivity to phage T4. By selection for phage resistance, derivatives of the cryptic mutants that are deoxycholate sensitive have been obtained. These mutants are no longer cryptic. We suggest that cryptic mutants have an altered outer membrane, with decreased permeability to 3'- and 5'-AMP, as a result of an altered polypeptide.


Volume-related mitochondrial deoxyribonucleic acid synthesis in zygotes and vegetative cells of Saccharomyces cerevisiae. The synthesis of mitochondrial deoxyribonucleic acid (DNA) in Saccharomyces cerevisiae cells has been examined during conjugation, in preconjugal conditions, and in control cultures that were not exposed to obverse diffusible sex factors. The ratios of mitochondrial to nuclear DNA varied from about 0.1 in control cells, to about 0.3 in alpha cells exposed for 180 min to cell-free culture medium from a cells, and to about 0.4 in conjugating cells 150 min after mixing. The enhanced levels of mitochondrial DNA during preconjugal and conjugal conditions seem correlated with enhanced cell volumes. Likewise, amounts of mitochondrial DNA in vegetative cells were found to be correlated with cytoplasmic volumes.


EcoRI cleavage sites in the argECBH region of the Escherichia coli chromosome. The EcoRI cleavage of deoxyribonucleic acids (DNAs) from lambdadarg phages, carrying argECBH, has been examined. The phages are derived from the heat-inducible, lysis-defective strain lambda y199,  and their bacterial DNA, including argECBH, is derived from Escherichia coli K-12. Such cleavage of the phage DNAs, in each case, produces the D, E, and F segments of lambda. Additionally, these DNAs yield segments, ordered from left to right, of length (in kilobases [kb]) determined by electron microscopy and 0.7% agarose slab gel electrophoresis as follows: lambdadarg13 (ppc argECBH bfe), 13.9, 2.8, 1.5, and 5.6; lambdadarg14 (ppc argECBH), 3.0, 2.0, 17.3, and 6.2; and lambdadarg23 (argECBH), 18.4 and 6.2. For lambdadarg13 sup102 DNA, the segment analogous to the 13.9-kb segment measures 12.2 kb. The direction from left to right corresponds to the clockwise orientation of the E. coli genetic map. The EcoRI segments define five cleavage sites near the arg region of the E. coli chromosome. For each of the DNAs, the arg genes occur on the largest segment produced. The 17.3-kb segment, being entirely bacterial, represents the argECBH-bearing EcoRI segment of the E. coli chromosome. The location of the arg genes was demonstrated electron microscopically in heteroduplex experiments.


Isolation and characterization of lambda pleu bacteriophages. In the Escherichia coli lysogen HfrH73 described by Shimada et al. (1973), none of the enzymes coded for by the leucine operon is synthesized due to an insertion of phage lambda into cistron leuA. The orientation of lambda in the chromosome is ara leuDCB lambda JAN leuA. After heat induction of the lysogen, plaque-forming transducing phages of two types are formed at low frequency. One type (e.g., lambda pleu9) transduces leuD, leuC, and leuB strains to prototrophy. The other type (e.g., lambda pleu 13) transduces leuA strains to prototrophy. lambda pleu 13 forms lysogens at low frequency (about 0.2%) by integration into the leucine operon. These lysogens are unstable, segregating phage-sensitive clones at high frequency (about 1%). Phages carrying different portions of the leucine operon were formed by aberrant excision after heat induction of strain CV437 (leuA371 lambda pleu13). A phage carrying the entire leucine operon (lambda K2) was constructed by a cross between lambda pleu9 and lambda pleu13. An analysis of leucine-forming enzyme levels in strains lysogenized with lambdaK2 indicated that leuO and leuP are present and functional in lambda K2. leu-specific messenger ribonucleic acid from E. coli hybridizes to the heavy (r) strand of lambdaK2. The leucine operon of lambda G4 pleuABCD (an S7 derivative of lambda K2) exists intact on a 7.3 x 10(6)-dalton fragment (lambdaG4EcoRI-B) generated by cleavage with endonuclease EcoRI. Heteroduplexes formed between lambda G4 and lambda show a 5.4 x 10(6)-dalton piece of bacterial deoxyribonucleic acid (DNA) replacing a 4.5 x 10(6)-dalton piece of lambda DNA starting at 0.46 fractional unit on the map of lambda. Fragment lambda G4EcoRI-B has about 0.6 x 10(6) daltons of lambda DNA from the b2 region at one end and about 1.4 x 10(6) daltons of lambda DNA from the int region at the other end.


Further mapping of IS2 and IS3 in the lac-purE region of the Escherichia coli K-12 genome: structure of the F-prime ORF203. The sequence organization of the F-prime ORF203 was determined by heteroduplex analysis. This large, type II F-prime (Scaife, 1967) contains lac, proC, and purE genes derived from the W1485 subline of Escherichia coli K-12. The IS3 and IS2 elements previously found in the lac-proC-purE region derived from the 58-161 subline (Hu et al., 1975) are also present in the same locations in the bacterial deoxyribonucleic acid (DNA) from the W1485 subline. Recombination between the IS2 region of F and an IS2 element located between lac and proC on the bacterial DNA apparently led to the formation of the perental Hfr, OR21. IS2 is thus directly repeated, with one copy of each element appearing at each of the two junctions between F and the bacterial sequences on ORF203. The F plasmid is found together with ORF203 in the plasmid DNA, and this probably forms from ORF203 by recombination between the directly repeated IS2 elements. ORF203 appears to have been excised from the Hfr chromosome by recombination between the IS3 sequence alpha3beta3 located counterclockwise of lac and the directly repeated IS3 sequence alpha4beta4 located clockwise of purE.


Genetic characterization of the temperature-sensitive and suppression phenotypes of Escherichia coli mutant N4316. Escherichia coli mutant N4316 is temperature sensitive and exhibits temperature-dependent suppression. These phenotypes are due to separate genes, as shown by reversion and mapping studies. The suppressor mutation was mapped and lies near argF.


Stability of the plasma membrane in Saccharomyces cerevisiae enriched with phosphatidylcholine or phosphatidylethanolamine. Spheroplasts from Saccharomyces cerevisiae NCYC 366, enriched in phosphatidylethanolamine after growth in medium supplemented with 1 mM ethanolamine, were more resistant to osmotic lysis than were spheroplasts from cells grown in the presence of 1 mM choline and enriched in phosphatidylcholine.


Carnitine acetyltransferase: candidate for the transfer of acetyl groups through the mitochondrial membrane of yeast. On the basis of its specific activity and its affinity for acetyl-coenzyme A, carnitine acetyltransferase appears to be the most likely candidate for acetyl group transfer out of yeast mitochondria.


Genetics of expression of asparaginase II activity in Saccharomyces cerevisiae. Expression of asparaginase II activity in Saccharomyces cerevisiae requires the participation of the products of at least two cistrons, asp2 and asp3, which are unlinked on the yeast genetic map.


Control of cell division in Escherichia coli: effect of amino acid starvation. The effect of amino acid starvation on cell division was studied in cells of Escherichia coli B. In this bacterial strain, deprivation of a required amino acid resulted in synchronous cell division upon restoration of the amino acid. This synchronization was apparently due to a shift forward in the cell cycle during the starvation. As a consequence, the cells divided at a size that was smaller than normal.


Influence of the stringent control system on the transcription of ribosomal ribonucleic acid and ribosomal protein genes in Escherichia coli. The fraction of the total ribonucleic acid (RNA) synthesis rate that is messenger RNA (mRNA) for ribosomal protein (r-protein) and ribosomal RNA (rRNA) has been estimated in valS(Ts) rel+ stringent and valS(Ts) relA1 relaxed strains of Escherichia coli during a partial inhibition of valyl-transfer RNA aminoacylation. The partial inhibition was accomplished by shifting the strains from the permissive growth temperature of 29.5 degrees C to the semipermissive temperature of 35.5 degrees C. The RNA synthesized at the elevated temperature was pulse labeled with [3H]uracil. The fraction of the total incorpoarted 3H radioactivity in r-protein mRNA or in rRNA was estimated by specific hybridization to the transducing phages gammaspc1, which carries about 15 r-protein genes and lambdailv5, which carries an rRNA transcription unit. The results clearly demonstrate that the rel gene influences the fraction of the total RNA synthesis rate that is r protein mRNA and rRNA; in the rel+ strain they are significantly increased relative to control cultures. This indicates that the expression of the genes coding for the RNA and protein component of the ribosome are most likely regulated at the level of transcription. Furthermore, it appears that the distribution of functioning RNA polymerase between rRNA genes, r-protein genes, and other types of genes is influenced by the rel gene control system; presumably this influence is mediated through the unusual nucleotide guanosine tetraphosphate.


Repression and inhibition of transport systems for branched-chain amino acids in Salmonella typhimurium. Kinetics of the transport systems common for entry of L-isoleucine, L-leucine, and L-valine in Salmonella typhimurium LT2 have been analyzed as a function of substrateconcentration in the range of 0.5 to 45 muM. The systems of transport mutants, KA203 (ilvT3) and KA204 (ilvT4), are composed of two components; apparent Km values for uptake of isoleucine, leucine, and valine by the low Km component are 2 muM, 2 to 3 muM, and 1 muM, respectively, and by the high Km component 30 muM, 20 to 40 muM, and 0.1 mM, respectively. The transport system(s) of the wild type has not been separated into components but rather displays single Km values of 9 muM for isoleucine, 10 muM for leucine, and 30 muM for valine. The transport activity of the wild type was repressed by L-leucine, alpha ketoisocaproate, glycyl-L-isoleucine, glycyl-L-leucine, and glycyl-L-methionine. That for the transport mutants was repressed by L-alanine, L-isoleucine, L-methionine, L-valine, alpha-ketoisovalerate, alpha-keto-beta-methylvalerate, glycyl-L-alanine, glycyl-L-threonine, and glycyl-L-valine, in addition to the compounds described above. Repression of the mutant transport systems resulted in disappearance of the low Km component for valine uptake, together with a decrease in Vmax of the high Km component; the kinetic analysis with isoleucine and leucine as substrates was not possible because of poor uptake. The maximum reduction of the transport activity for isoleucine was obtained after growing cells for two to three generations in a medium supplemented with repressor, and for the depression, protein synthesis was essential after removal of the repressor. The transport activity for labeled isoleucine in the transport mutant and wild-type strains was inhibited by unlabeled L-alanine, L-cysteine, L-isoleucine, L-leucine, L-methionine, L-threonine, and L-valine. D-Amino acids neither repressed nor inhibited the transport activity of cells for entry of isoleucine.


Escherichia coli K-12 structural kdgT mutants exhibiting thermosensitive 2-keto-3-deoxy-D-gluconate uptake. A specific method is described for selecting thermosensitive mutants of Escherichia coli K-12 able to grow on 2-keto-3-deoxy-D-gluconate (KDG) and D-glucuronate at 2, but not at 42 degrees C. The extensive analysis of one such mutant is consistent with the conclusion that the carrier molecule responsible for KDG and glucuronate uptake becomes thermolabile. (i) Growth on a variety of carbon sources is perfectly normal at 28 and 42 degrees C, whereas in the same temperature range it gradually diminishes on KDG and glucuronate. (ii) The apparent Km value for KDG is about twofold in the range 25 to 40 degrees C. In the same temperature range, the Vmax values for KDG influx are higher for the mutant compared with those of the wild-type strain, but the optimum temperature is 34 degrees C instead of 38 degrees C. On the contrary, the Vmax values for glucuronate influx are lower for the mutant than for the parental strain, and the optimum temperature for both strains is shifted beyond 40 degrees C. (iii) The activation energies for KDG and glucuronate uptake are about twofold higher in the mutant than in the wild-type strain. (iv) Kinetics of counterflow under deenergized conditions (overshoot) at different temperatures indicate that the defect is located in the translocation step rather than in the processes involved in energy coupling. (v) The first-order rate constants for thermal denaturation are, respectively, 2.5- and 5-fold higher at 40 and 30 degrees C in the mutant than in the wild-type strain, and the activation energy for thermal denaturation is lower. (vi) The carrier molecule in the mutant is also much more sensitive to denaturation by N-ethylmaleimide. (vii) Four independent thermosensitive mutations and one revertatn were located by transduction in or near the kdgT locus, defined previously as the site of nonconditional KDG transport-negative mutations. These results support the conclusion that kdgT represents the structural gene coding for the KDG transport system.


Accumulation of nucleotides by starved Escherichia coli cells as a probe for the involvement of ribonucleases in ribonucleic acid degradation. The acid-soluble ribonucleic acid degradation products formed by Escherichia coli cells starved for a carbon source have been identified. They comprise oligonucleotides, nucleoside diphosphates, 5'- and 3'-nucleoside monophosphates, nucleosides, and free bases. The majority of these products are excreted phates, nucleosides, and free bases. The majority of these products are excreted into the medium, and only small and constant amounts are kept in the pool. During carbon starvation at elevated temperatures, mutants deficient in ribonuclease I do not form oligonucleotides and 3'-nucleoside monophosphates, and mutants that contain a modified form of polynucleotide phosphorylase do not accumulate nucleoside diphosphates. 5'-Nucleoside monophosphates do accumulate, however, in a mutant containing thermoabile ribonuclease II, under conditions where more than 95% of all enzyme activity had been destroyed. The data presented confirm the participation of ribonuclease I and polynucleotide phosphorylase in the final steps of ribonucleic acid degradation and indicate that an exonuclease forming 5'-nucleoside monophosphates is also involved.


Escherichia coli membrane proteins with an affinity for deoxyribonucleic acid. From the membrane fraction of Escherichia coli K-12 strain, four protein fractions (peaks I, IIa, IIb, and III) which have affinity for deoxyribonucleic acid (DNA) have been isolated. The molecular weights of these proteins are between 12,000 and 8,000. Only the peak III fraction contains a protein that binds preferentially to single-stranded DNA, whereas the others contain proteins that bind also to double-stranded DNA. The binding activity of the peak IIb protein is inhibited in the presence of polyuridylic acid. Peak I and peak IIa protein fractions behave like hydrophobic proteins.


Mode of action of yeast toxins: energy requirement for Saccharomyces cerevisiae killer toxin. The role of the energy status of the yeast cell in the sensitivity of cultures to two yeast toxins was examined by using 12K release from cells as a measure of toxin action. The Saccharomyces cerevisiae killer toxin bound to sensitive cells in the presence of drugs that interfered with the generation or use of energy, but it was unable to efflux 12K from the cells under these conditions. In direct contrast, the Torulopsis glabrata pool efflux-stimulating toxin induced efflux of the yeast 42K pool was insensitive to the presence of energy poisons in cultures. The results indicate that an energized state, maintained at the expense of adenosine 5'-triphosphate from either glycolytic or mitochondrial reactions, is required for the action of the killer toxin on the yeast cell.


Ultraviolet induction of prophage lambda during inhibition of deoxyribonucleic acid synthesis by hydroxyurea. Hydroxyurea inhibited synthesis of certain deoxyribonucleic acid (DNA) precursors and causes the cessation of DNA synthesis. It did not cause induction of lambda. Superinfection of an irradiated lysogen with lambdaind- could prevent induction, but the percentage of cells protected decreased as the time between irradiation and superinfection increased. The presence of hydroxyurea did not increase the time during which cells could be rescued by superinfection. The accumulation of DNA precursors after ultraviolet or ionizing radiation was not necessary for the induction of lambda prophage to occur.


Transient rates of synthesis of five amionacyl-transfer ribonucleic acid synthetases during a shift-up of Escherichia coli. The steady-state levels of a number of aminoacyl-transfer ribonucleic acid synthetases are known to be positively correlated with growth rate in Escherichia coli. To describe the regulation of these enzymes during a nutritional shift-up, use was made of the recent identification of polypeptide chains of several synthetases in whole cell lysates resolved by the O'Farrell two-dimensional gel system. A culture growing in acetate minimal medium was shifted to glucose-rich medium and pulse labeled with [3H]leucine and [3H]isoleucine for 30- or 6-s intervals during the 20 min after the shift. After mixing with a uniformly [35S]sulfate-labeled reference culture, the samples were subjected to two-dimensional gel electrophoresis. The 3H/35S ratio in the resolved synthetase polypeptides provided an accurate estimation of their transient rates of synthesis. Five aminoacyl-transfer ribonucleic acid synthetases (those for argnine, glycine, isoleucine, phenylalanine, and valine) exhibited an increase in formation within 30 to 90 s after the shift-up. The magnitude of the increases corresponded to the final steady-state values and were reached within 2 to 3 min. The addition to rifampin revealed that the increase in the differential rate of valyl-transfer ribonucleic acid synthetase formation was the result of increased messenger ribonucleic acid transcription and not of a relaxation of some translation restriction.


Delayed ultraviolet light-induced cessation of respiration by inadequate aeration of Escherichia coli. Inadequately aerated Escherichia coli B/r cultures did not shut their respiration off 60 min after ultraviolet light (52 M/m2 at 254 nm) as they did when well supplied with oxygen. Since cessation of respiaration is associated with cell death, the result suggested that oxygen toxicity by superoxide radicals generated by cell metabolism might be responsible for cell death. The specific activity of superoxide dismutase, which scavenges O2- radicals, increased twofold after 90 min of adequate aeration, but the specific activity of catalase remained constant. Respiration and viability of irradiated cells were affected not at all by the presence of superoxide dismutase and only slightly by the presence of catalase. Metal ions such as Mn2+ and Fe2+ inducers of superoxide dismutase, had no effect on respiration and viability. When irradiated cells were incubated under N2 for 90 min, the respiration, growth, and viability time-course responses were the same as for the cells not exposed to anareobiosis. We conclude that superoxide anions generated at the time of irradiation play no part in cessation delays the ultraviolet light-induced synthesis of proteins responsible for the irreversible cessation of respiration.


Influence of methionine biosynthesis on serine transhydroxymethylase regulation in Salmonella typhimurium LT2. The enzyme serine transhydroxymethylase (EC 2.1.2.1; L-serine:tetrahydrofolate-5,10-hydroxymethyltransferase) is responsible both for the synthesis of glycine from serine and production of the 5,10-methylenetetrahydrofolate necessary as a methyl donor for methionine synthesis. Two mutants selected for alteration in serine transhydroxymethylase regulation also have phenotypes characteristic of metK (methionine regulatory) mutants, including ethionine, norleucine, and alpha-methylmethionine resistance and reduced levels of S-adenosylmethionine synthetase (EC 2.5.1.6; adenosine 5'-triphosphate:L-methionine S-adenosyltransferase) activity. Because this suggested the existence of a common regulatory component, the regulation of serine transhydroxymethylase was examined in other methionine regulatory mutants (metK and metJ mutants). Normally, serine transhydroxymethylase levels are repressed three- to sixfold in cells grown in the presence of serine, glycine, methionine, adenine, guanine, and thymine. This does not occur in metK and metJ mutants; thus, these mutations do affect the regulation of both serine transhydroxymethylase and the methionine biosynthetic enzymes. Lesions in the metK gene have been reported to reduce S-adenosylmethionine synthetase levels. To determine whether the metK gene actually encodes for S-adenosylmethionine synthetase, a mutant was characterized in which this enzyme has a 26-fold increased apparent Km for methionine. This mutation causes a phenotype associated with metK mutants and is cotransducible with the serA locus at the same frequency as metK lesions. Thus, the affect of metK mutations on the regulation of glycine and methionine synthesis in Salmonella typhimurium appears to be due to either an altered S-adenosylmethionine synthetase or altered S-adenosylmethionine pools.


Effects of galU mutation on flagellar formation in Escherichia coli. Two mutants of Escherichia coli strictly deficient in uridine-diphosphoglucose pyrophosphorylase activity (galU) were found to have very small numbers of flagellar filaments and hooks. In these mutants, both the rate of flagellin (flagellar protein) synthesis and the amount of messenger ribonucleic acid specific for flagellin were considerably lower than in the parental strains. Motile revertants from the galU mutants were isolated and were found to carry a suppressor mutation, which was mapped in the flaH cistron. These strains formed swarms under conditions of catabolite repression; their intracellular concentration of cyclic adenosine 5'-monophosphate was the same as that in the parental strains. These results suggest that the outer membrane affects flagellar formation through the flaH gene product.


Spermidine-Deoxyribonucleic acid interaction in vitro and in Escherichia coli. The binding of spermidine to deoxyribonucleic acid (DNA) was studied by equilibrium dialysis in a wide range of salt concentrations. The association constants ranged from 6 x 10(5) M-1 in 1 mM sodium cacodylate, pH 7.5, to 3 x 10(2) M-1 in 0.3 M NaCl. MgCl2 reduced spermidine-DNA interaction even more than NaCl so that in moderate-ionic-strength solutions (0.3 M NaCl, 0.002 M MgCl2) there was little detectable binding. Low-ionic-strength media were used to isolate DNA from Escherichia coli by a method shown to minimize loss of spermidine from the DNA. Considerable spermidine was associated with E. coli DNA, but control experiments indicated that complex formation had taken place during or after lysis of the cells. Exogenous DNA or ribonucleic acid added to spheroplasts at the time of their lysis caused most of the cellular spermidine to be scavenged by the extra nucleic acid. The data suggest that spermidine is relatively free in the cell and thereby capable of strong (high-affinity) associations with nucleic acids only after the ionic strength of the cell environment is lowered.


"Active" one-carbon generation in Saccharomyces cerevisiae. A new mutation introducing a one-carbon requirement (e.g., formate) for the glycine-supplemented growth of a serine-glycine auxotroph (ser1) was correlated with a lack of glycine decarboxylase activity. The presence of oxalate decarboxylase activity or glyoxylate decarboxylase activity did not overcome the one-carbon requirement. Another mutation characterized by the absence of oxalate decarboxylase activity did not introduce a one-carbon requirement. The presence and physiological significance of glycine decarboxylase activity in Saccharomyces are thus inferred.


Escherichia coli mutants deficient in exonuclease VII. Mutants of Escherichia coli having reduced levels of exonuclease VII activity have been isolated by a mass screening procedure. Nine mutants, five of which are known to be of independent origin, were obtained and designated xse. The defects in these strains lie at two or more loci. One of these loci, xseA, lies in the interval between purG and purC; it is 93 to 97% co-transducible with guaA. The order of the genes in this region is purG-xseA guaA,B-purC. The available data do not allow xseA to be ordered with respect to guaA,B. Exonuclease VII purified from E. coli KLC3 xseA3 is more heat labile than exonuclease VII purified from the parent, E. coli PA610 xse+. Therefore, xseA is the structural gene for exonuclease VII. Mutants with defects in the xseA gene show increased sensitivity to nalidixic acid and have an abnormally high frequency of recombination (hyper-Rec phenotype) as measured by the procedure of Konrad and Lehlman (1974). The hyper-Rec character of xseA strains is approximately one-half that of the polAex1 mutant defective in the 5' leads to 3' hydrolytic activity of deoxyribonucleic acid polymerase I. The double mutant, polAex1 xseA7, is twice as hyper-Rec as the polAex1 mutant alone. The xseA- strains are slightly more sensitive to ultraviolet irradiation than the parent strain. Bacteriophages T7, fd, and lambdared grow normally in xseA- strains.


Aminoacyl adenylate, a normal intermediate or a dead end in aminoacylation of transfer ribonucleic acid. The shape of the time curve for the aminoacylation of tRNA has been investigated using five different amino acid:tRNA ligases. Four of these enzymes showed a lag in the time curve during the early phase of the first catalytic turnover of the enzyme. In each case, the lag period could be abolished by preincubating the ligase with amino acid, ATP, and Mg2+ under conditions known to give an aminoacyl adenylate-enzyme complex. With all five ligases the steady state rate of transfer from the preformed aminoacyl-adenylate complex to tRNA was approximately the same as that of the overall reaction.


Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. A rapid and convenient method for peptide mapping of proteins has been developed. The technique, which is especially suitable for analysis of proteins that have been isolated from gels containg sodium dodecyl sulfate, involves partial enzymatic proteolysis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by polyacrylamide gel electrophoresis. The pattern of peptide fragments produced is characteristic of the protein substrate and the proteolytic enzyme and is highly reproducible. Several common proteases have been used including chymotrypsin, Staphylococcus aureus protease, and papain.


Isolation of wheat germ agglutinin-resistant clones of Chinese hamster ovary cells deficient in membrane sialic acid and galactose. Several clones of Chinese hamster ovary cells have been selected for their resistance to the toxic effects of wheat germ agglutinin. The clones do not bind wheat germ agglutinin as well as parent cells and are 5- to 250-fold more resistant to the toxic effects of the lectin. Of three clones studied in detail, all exhibit a decrease in wheat germ agglutinin binding affinity. Two have normal numbers of wheat germ agglutinin binding sites, while one (Clone 13) has a 65% decrease in binding sites. Crude membrane preparations of the clones have a decrease in sialic acid content relative to parent cells, and Clone 13 membranes are also deficient in galactose, while the mannose and hexosamine contents of all three clones are normal. The membrane sugar deficiencies affect both glycoproteins and glycolipids. Sialyl-lactosylceramide is the major glycolipid in parent cells, while Clones 1 and 1021 have lactosylceramide and Clone 13 has glucosylceramide as the predominant glycolipid. Labeling experiments with N-[G-3H]acetylmannosamine suggest that Clone 1021 cells have a block in the transfer of sialic acid from CMP-sialic acid to glycoprotein and glycolipid acceptors. Yet CMP-sialic acid:glycoprotein sialyl-transferase activity in cell lysates of Clone 1021 cells is 80% of normal. While CMP-sialic acid:lactosylceramide sialyl-transferase activity is only 25% of normal, it can be restored to normal or elevated levels by sodium butyrate induction without an associated increase in cellular sialyl-lactosylceramide content. Similarly, the galactose-deficient Clone 13 can synthesize UDP-galactose and has normal levels of UDP-galactose:glycoprotein galactosyltransferase and UDP-galactose:glucosylceramide galactosyltransferase when assayed in vitro. The glycosyltransferases of both these clones can utilize their own glycoproteins as sugar acceptors in in vitro assays. These data suggest that the variant cells fail to carry out specific glycosyltransferase reactions in vivo despite the fact that they possess the appropriate nucleotide sugars, glycoprotein and glycolipid acceptors, and glycosyltransferases.


Metabolic regulation of aminoacyl-tRNA synthetase biosynthesis in bakers' yeast. The specific activities of 15 aminoacyl-tRNA synthetases in Saccharomyces cerevisiae were measured after growth under a variety of conditions that produced a range of cell-doubling times. The specific activity of each synthetase increased as cell-doubling time decreased. Control experiments eliminate the possibility that these results are due to preferential recovery of synthetases, or to the presence of activators in the faster growing cultures or inhibitors in the slower growing ones. These observations run counter to the expectation that synthetases in bacteria and yeast are negatively regulated by free amino acids, or, more likely, by aminoacyl-tRNA. In fact, as the growth medium was enriched, generation times decreased, and synthetase and aminoacyl-tRNA levels increased. It is suggested that cytoplasmic aminoacyl-tRNA synthetases may be more or less coordinately controlled such that their response to growth follows the pattern observed for ribosome production and RNA synthesis. This suggests the possibility of coordinated response of genes for components of the protein synthetic apparatus.


Studies on the mechanism of the adenosylcobalamin-dependent diol dehydrase reaction by the use of analogs of the coenzyme. A series of 16 analogs of 5'-deoxy-5'-adenosylcobalamin (adenosylcobalamin) were examined for their effects on the diol dehydrase system of Klebsiella pneumoniae (Aerobacter Aerogenes). Four analogs, ara-adenosyl-, aristeromycyl-, 3-isoadenosyl-, and nebularylcobalamin, were able to function as coenzymes in the diol dehydrase reaction, coenzyme activity decreasing in that order. Like the native holoenzyme, complexes of the enzyme with these four analogs show a cob(II)alamin-like absorption peak or shoulder in the presence of 1,2-propanediol. Analogs containing hypoxanthine, cytosine, or benzimidazole do not function as coenzymes, but are weak competitive inhibitors in the presence of adenosylcobalamin. Analogs in which the D-ribosyl moiety is replaced by L-ribose or by an alkyl chain of 2 to 6 carbons are inactive as coenzymes, but act as competitive inhibitors with extremely high affinity for the apoenzyme. Complexes with the inactive analogs showed visible spectra similar to those of the corresponding free cobalamins. Upon anaerobic photolysis and subsequent aeration, complexes with the first group of inactive analogs produced unusually stabilized cob(II)alamin, while complexes with the second group of inactive analogs were readily photolyzed to a hydroxocobalamin-enzyme complex. Complexes with adeninylpentyl- and L-adenosylcobalamin were stable to light under the same conditions. These findings suggest that both the ribose and the adenine moiety of the nucleoside participate in enzyme-coenzyme interaction, involving not only the binding to the apoenzyme but also the activation of the carbon-cobalt bond.


Primary structure of rabbit skeletal muscle troponin-T. Sequence determination of the NH2-terminal fragment CB3 and the complete sequence of troponin-T. The amino acid sequence of CB3, the NH2-terminal fragment of troponin-T, and the alignment of all six cyanogen bromide (CB) fragments are reported. Fragment CB3, comprised of 70 residues, has eight of the nine prolines of troponin-T. As observed in other proteins of the myofibrillar system, its NH2 terminus is blocked by an acetyl group. Methionine-containing "overlap" peptides isolated from a peptic digest of troponin-T as well as 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine cleavage of the protein were used to order the fragments as CB3-CB2-CB5-CB4-CB7-CB6. The complete sequence of troponin-T, a single polypeptide chain of 259 amino acids having a molecular weight of 30,500, is presented.


Inhibition of DNA replication in Escherichia coli by dibromophenol and other uncouplers. DNA replication in Escherichia coli is inhibited by uncouplers such as 2,4-dibromophenol and 3,3'4',5-tetrachlorosalicylanilide. Inhibition occurs in either aerobically or anaerobically growing cells or in cells made permeable by toluene. With anaerobically growing cells, inhibition by dibromophenol is reversible and occurs under conditions in which there is no change in pools of ATP or deoxynucleoside triphosphates. With toluenized cells, inhibition is not due to breakdown of deoxynucleoside triphosphates. The rates of protein and RNA synthesis are not inhibited either in vivo or in toluenized cells by concentrations of dibromophenol or tetrachlorosalicylanilide which inhibit replication. It is generally believed that uncouplers inhibit many other cellular processes by collapsing a proton gradient across a membrane. However, the relative effectiveness of eight uncouplers and related compounds to inhibit replication did not parallel their ability to transport protons into E. coli cells. Therefore, the inhibition by uncouplers does not suggest that replication depends on a chemiosmotic process. A possible explanation for the uncoupler sensitivity is provided by the finding that many of the purified enzymes tested, including DNA polymerases II and III, are inhibited by dibromophenol and tetrachlorosalicylanilide.


Noncoordinated transcription in the absence of protein synthesis in yeast. In the yeast Saccharomyces cerevisiae we have carried out a detailed study of the response of transcription to the inhibition of translation. Measurement of the incorporation of labeled bases and nucleosides into the nucleoside triphosphate pools revealed that amino acid deprivation brings about a 10- to 50-fold inhibition of such labeling. Therefore accurate comparisons of RNA synthesis using such precursors are difficult to obtain. To overcome this problem we have turned to the use of L-[methyl-3H] methionine as a precursor, because the labeling of the S-adenosylmethionine pool is relatively unaffected by the rate of protein synthesis. Using this precursor, we have observed that in the absence of protein synthesis the transcription of ribosomal RNA is reduced by 80%, the transcription of messenger RNA is reduced by about 25%, and the transcription of transfer RNA is reduced by less than 20%. These results are obtained when protein synthesis is inhibited either by deprivation of an amino acid or by the addition of cycloheximide. Ribosomal precursor RNA synthesized in the absence of protein synthesis is fully methylated. We conclude that the transcription of rRNA is the primary target of stringent control. Furthermore the inhibition of protein synthesis, itself, may be the trigger for this response.


Energy coupling to net K+ transport in Escherichia coli K-12. Energy coupling for three K+ transport systems of Escherichia coli K-12 was studied by examining effects of selected energy sources and inhibitors in strains with either a wild type or a defective (Ca2+, Mg2+)-stimulated ATPase. This approach allows discrimination between transport systems coupled to the proton motive force from those coupled to the hydrolysis of a high energy phosphate compound (ATP-driven). The three K+ transport systems here studied are: (a) the Kdp system, a repressible high affinity (Km=2 muM) system probably coded for by four linked Kdp genes; (b) the Trka system, a constitutive system with high rate and modest affinity (Km=1.5 mM) defined by mutations in the single trkA gene; and (c) the TrkF system, a nonsaturable system with a low rate of uptake (Rhoads, D.B., Waters, F.B., and Epstein, W. (1976) J. Gen. Physiol. 67, 325-341). Each of these systems has a different mode of energy coupling: (a) the Kdp system is ATP-driven and has a periplasmic protein component; (b) the TrkF system is proton motive force-driven; and (c) the TrkA system is unique among bacterial transport systems described to date in requiring both the proton motive force and ATP for activity. We suggest that this dual requirement represents energy fueling by ATP and regulation by the proton motive force. Absence of ATP-driven systems in membrane vesicles is usually attributed to the requirement of such systems for a periplasmic protein. This cannot explain the failure to demonstrate the TrkA system in vesicles, since this system does not require a periplasmic protein. Our findings indicate that membrane vesicles cannot couple energy to ATP-driven transport systems. Since vesicles can generate a proton motive force, the inability of vesicles to generate ATP or couple ATP to transport (or both) must be invoked to explain the absence of TrkA in vesicles. The TrkF system should function in vesicles, but its very low rate may make it difficult to identify.


Dissimilarity in protein chain elongation factor requirements between yeast and rat liver ribosomes. Factor requirements for yeast and rat liver ribosomes were determined in several different reactions using either yeast or liver factors. In polymerization assays yeast ribosomes required a factor in addition to elongation factor 1 (EF-1) and elongation factor 2 (EP-2). The third factor (EF-3) requirement was observed with EFs from either yeast or liver for both poly(U)-directed polyphenylalanine synthesis and elongation of endogenous peptidyl-tRNA. No significant effect of EF-3 was observed with liver risomes in either assay. In contrast to results with polypeptide synthesis EF-3 was not required for EF-1 dependent binding of [3H]Phe-tRNA or the translocation-dependent formation of N-acetylphenylalanylpuromycin. Up to 2-fold stimulation of the binding reaction was observed with saturating levels of either yeast or liver EF-1. No effect of EF-3 was observed on ribosome-EF-2-GDP-fusidic acid complex formation. The data suggest that the yeast EF-3 may be a loosely bound ribosomal protein which is not required for a specific step in the elongation cycle but is involved in the coordination of the partial reactions required for polymerization.


Transient kinetic studies on the allosteric transition of phosphoglycerate dehydrogenase. Stopped flow spectrophotometry was used to investigate the kinetics of the transition of the phosphoglycerate dehydrogenase (3-phosphoglycerate: NAD oxidoreductase, EC 1.1.1.95) reaction from the active to the inhibited rate upon the addition of the physiological inhibitor serine. The transition was characterized by a single first order rate constant (kobs,i) which was independent of enzyme concentration. At pH 8.5, kobs,i increased in a hyperbolic manner with serine concentration from 2 to 8 s-1. The increase in kobs,i occurred at serine concentrations where the steady state inhibition was virtually complete. These results indicate that serine inhibition is an allosteric process involving a conformational change in the enzyme. A model is presented in which serine at low concentrations binds exclusively to the inhibited state of the enzyme and shifts the equilibrium toward that state; at high serine concentrations, serine binds to the active state, facilitating its conversion to the inhibited state. An alternative model, which we favor, proposes two classes of inhibitor binding sites. The kinetics of the fluorescence quenching of enzyme-bound NADH by serine (Sugimoto, E., and Pizer, L.I. (1968) J. Biol. Chem. 243, 2090-2098), measured by stopped flow fluorimetry, was also characterized by a single first order rate constant (kobs,f.q.) which was independent of enzyme concentration. At pH 8.5, kobs,f.q. ranged from 0.4 s-1 at low serine concentrations to 1.1 s-1 at high serine concentrations. These results indicate that the fluorescence quenching induced by serine is a manifestation of a structural change in the enzyme. Enzyme and excess NADH were mixed with substrate and serine in the stopped flow instrument, and enzyme-bound NADH fluorescence was monitored by exciting through the protein at 285 nm. A rapid fluorescence quenching process, which occurred within the mixing time, was followed by a slower fluorescence enhancement process which terminated in a steady state level corresponding to the quenched fluorescence of the enzyme NADH serine complex. The rapid quenching was the result of substrate binding (Dubrow, R., and Pizer, L.I. (1977) J. Biol. Chem. 252, 1539-1551). The fluorescence enhancement was characterized by a single first order rate constant whose value for a given serine concentration corresponded with Kobs,j. This data shows that the quenched state of the enzyme-NADH-complex is the state which is directly responsible for the inhibition of enzyme activity. During catalysis the quenched state is achieved from a different initial conformation, and consequently at a different rate, than in the absence of substrate. kobs,j and kobs,f.q. were also measured using glycine, another inhibitor. The ultraviolet difference spectrum between enzyme and enzyme plus serine was determined and proposed to be the result of the same structural change which is responsible for the fluorescence quenching by serine.


Effects of prostaglandins and oxytocin on calcium release from a uterine microsomal fraction. A microsomal fraction resembling striated muscle sarcoplasmic reticulum was isolated from uterine smooth muscle. ATP induces calcium accumulation in this fraction. Increased temperature enhances calcium accumulation and calcium-activated ATPase. In the absence of ATP, approximately 35% of the intrinsic calcium exchanges with the 45Ca in the incubation medium. In the presence of ATP, exchange of intrinsic calcium with 45Ca increases by an amount which equals the ATP-dependent calcium binding. In preparations partially preloaded with calcium, a steady state of bound calcium is reached when the ATP is exhausted. Calcium is released under these conditions by prostaglandins E2 and F2alpha, but not by PGF1beta. The antibiotic ionophores X537A and A23187, as well as oxytocin, also release calcium previously accumulated under ATP stimulation. None of these agents, with the exception of oxytocin, release intrinsic calcium. Thus, the effect of prostaglandins resembles that of the ionophores, suggesting an ionophoretic action of these prostaglandins. The release of calcium conforms with the in vivo smooth muscle contracting action of these agents.


Limited proteolysis of nitrate reductase purified from membranes of Escherichia coli. The heterogeneous form of nitrate reductase released from the membrane fraction of Escherichia coli by heat treatment was converted to a new electrophoretic form by incubation with trypsin. As a result of the trypsin treatment, the heat-released enzyme was converted from an associating-dissociating system to a nonassociating monomer (Mr approximately 200,000) which retained full enzymatic activity. Several distinct subunits in the 47,000- to 59,000-dalton range were converted to a single 43,000-dalton subunit during the trypsin treatment, while the other major subunit (155,000 daltons) was unaffected. Nitrate reductase extracted from the membrane fraction with deoxycholate and ammonium sulfate was composed of two apparently homogeneous subunits (155,000 and 59,000 daltons). The detergent-extracted enzyme preparation was converted by trypsin to an electrophoretic form very similar to the product of trypsin treatment of the heat-released enzyme with an identical subunit composition (155,000 and 43,000 daltons). These results demonstrate that the heterogeneous subunits present in the heat-released enzyme are produced during heat treatment by proteolytic cleavage of a single 59,000-dalton subunit. The fragments removed by trypsin treatment are implicated in the self-associating properties of the heat-released enzyme.


Bacteriophage T4 RNA ligase. Reaction intermediates and interaction of substrates. Bacteriophage T4 RNA ligase catalyzes the ATP-dependent ligation of a 5'-phosphoryl-terminated nucleic acid donor to a 3'-hydroxyl-terminated nucleic acid acceptor. We have identified adenylylated DNA and RNA reaction intermediates in which the AMP moiety is attached by a pyrophosphate bond to the 5'-phosphoryl group of the donor. A large amount of DNA-adenylate accumulates during the reaction and the dependence of joining and adenylylation on chain length are similar. The adenylylated donor is joined by ligase to an acceptor in the absence of ATP, and AMP is released stoichiometrically in this reaction. The acceptor is not only a substrate in the reaction but also a cofactor for adenylylation of the donor; in the absence of a 3'-hydroxyl group the activated intermediate does not form. The activated DNA need not join to the acceptor that initially stimulated activation but can also join to another acceptor. This process of acceptor exchanges has proven useful for promoting the cyclization of small DNA substrates and the synthesis of DNA co-polymers.


Function of initiation factor 1 in the binding and release of initiation factor 2 from ribosomal initiation complexes in Escherichia coli. 1. Studies on the function of initiation factor 1 (IF-1) in the formation of 30 S initiation complexes have been carried out. IF-1 appears to prevent the dissociation of initiation factor 2 (IF-2) from the 30 S initiation complex. The factor has no effect on either the initial binding of IF-2 nor does it increase the amount of IF-2 dependent fMet-tRNA and GTP bound to the 30 S subunit. Bound fMet-tRNA remains stable to sucrose gradient centrifugation even in the absence of IF-1. 2. It is postulated that the presence of IF-2 on the 30 S complex is necessary so that at the time of junction with the 50 S subunit to form a 70 S complex, the 70 S-dependent GTPase activity of IF-2 can hydrolyze GTP. This hydrolysis provides a means by which GTP can be removed to facilitate formation of a 70 S initiation complex active in peptidyl transfer. In support of this postulate, it was observed that 30 S initiation complexes formed in the absence of IF-1 could be depleted of their complexes were still able to accept 50 S subunits to form 70 S complexes which could still donate fMet-tRNA into peptide linkages. These results indicate that 30 S complexes lacking GTP do not require IF-2 for formation of active 70 S complexes. 3. IF-1, which is required to prevent dissociation of IF-2 from the 30 S initiation complex, is also required for release of IF-2 from ribosomes following 70 S initiation complex formation. The mechanisms of the release of IF-2 has been studied in greater detail. Evidence is presented which rules out the presence of a stable IF-2 GDP complex on the surface of the 70 S ribosome following GTP hydrolysis and of any exchange reactions between IF-1 and guanine nucleotides necessary for effecting the release of IF-2. IF-2 remains on the 70 S initiation complexes after release of guanine nucleotides and can be liberated solely by addition of IF-1.


Early acute rejection in renal homotransplantation. Angiographic-pathologic correlation. Early acute rejection occurs between two and ten days after renal homotransplantation and has been recognized in eight of 156 renal transplants. Four of the eight patients developed spontaneous renal rupture unrelated to biopsies. Pathologically, there was acute passive congestion with areas of tubular necrosis, venous thrombi and perivenous lymphoplasia. Glomerular and arterial immunogenic injury was slight. Radiologically, the kidneys were enlarged with stretching of the interlobar and segmental arteries, decreased peripheral filling and a homogeneous nephrogram. Renal vein filling defects were a frequent feature.


Potential hazards of diagnostic radiation. There are no precise data for determining the extent of somatic damage from small doses of radiation used in diagnostic radiology. Diagnostic radiation given to pregnant women, knowingly or unknowingly, should rarely reach teratogenic levels causing brain and eye abnormalities. Evidence suggests that it does increase the risk of childhood malignancy, especially leukemia. Although rapidly growing tissues seem most susceptible, all radiation probably carries a very small risk of carcinogenesis. Genetic damage is equally difficult to estimate. Diagnostic radiation of females, even in childhood, may be related to an increased incidence of Down's syndrome in older mothers. Radiation also causes point mutations, which may explain the increase of some genetic abnormalities in progeny of older fathers. Whenever an abdominal or pelvic radiograph is ordered before the end of the reproductive period, there must be a potential benefit to balance the small risk involved.


A comparison of the distribution of actin and tubulin in the mammalian mitotic spindle as seen by indirect immunofluorescence. Rabbit antibodies against actin and tubulin were used in an indirect immunofluorescence study of the structure of the mitotic spindle of PtK1 cells after lysis under conditions that preserve anaphase chromosome movement. During early prophase there is no antiactin staining associated with the mitotic centers, but by late prophase, as the spindle is beginning to form, a small ball of actin antigenicity is found beside the nucleus; After nuclear envelope breakdown, the actiactin stains the region around each mitotic center, and becomes organized into fibers that run between the chromosomes and the poles. Colchicine blocks this organization, but does not disrupt the staining at the poles. At metaphase the antiactin reveals a halo of ill-defined radius around each spindle pole and fibers that run from the poles to the metaphase plate. Antitubulin shows astral rays, fibers running from chromosomes to poles, and some fibers that run across the metaphase plate. At anaphase, there is a shortening of the antiactin-stained fibers, leaving a zone which is essentially free of actin-staining fluorescence between the separating chromosomes. Antitubulin stains the region between chromosomes and poles, but also reveals substantial fibers running through the zone between separating chromosomes. Cells fixed during cytokinesis show actin in the region of the cleavage furrow, while antitubulin reveals the fibrous spindle remnant that runs between daughter cells. These results suggest that actin is  a component of the mammalian mitotic spindle, that the distribution of actin differs from that of tubulin and that the distributions of these two fibrous proteins change in different ways during anaphase.


Ultrastructural study of spontaneous bone marrow rosette-forming cells. Mouse bone marrow contains spontaneous rosette-forming cells (RFC) which include more than 70% T-cell precursors, as assessed by their transformation into theta-positive cells after incubation with thymic hormone. Such spontaneous RFC, examined in C57B1/6 mouse bone marrow by electron and scanning electron microscopy, have consistently been shown to be small, inactive mouse lymphocytes when macrophages have been eliminated by cell preincubation. These data suggest that thymic hormone target cells include small quiescent lymphocytes.


The two pools of pituitary gonadotropin: regulation during the menstrual cycle. Information on the relative activity and on the functional relationships between the acutely releasable (1st) and reserve (2nd) pools of pituitary LH during the course of the normal menstrual cycle was obtained via a 4 h LRF infusion (0.2 mug/min X 4 h). This was immediately followed by 3 pulses of LRF (10 mug at 2 h intervals) to assess further the size of the acutely releasable pool after activation of the reserve pool by the infusion. Our observations indicate that two functional pools of LH are present in all phases of the menstrual cycle and that comparative pool size or activity is influenced profoundly by ovarian steroid feedback as well as by the pattern of input of hypothalamic LRF. From the early to the late follicular phase, in synchrony with the rising levels of E2, the size of the 2nd pool is preferentially augmented. A small increase in the 1st pool activity is not apparent until the late follicular phase when a 5-fold increase in the size of the 2nd pool is also attained. During the mid-luteal phase and in association with relatively high progesterone (P) and E2, th large 2nd pool is maintained as in the late follicular phase but the 1st pool is strikingly smaller. Activation of the 2nd pool of LH by the LRF infusion (priming) increases the acutely releasable LH (1st pool) in all three phases of the cycle, as evidenced by an enhanced response to the 1st but not subsequent pulses of LRF at the end of the infusion as compared with non-infused controls. This priming effect is likely a reflection of activation or "shifting" of LH from the larger 2nd pool to the smaller 1st pool. It is found that this priming effect is greatest during the mid-luteal phase as compared to other phases of the cycle. During the days of mid-cycle LH surge, a dramatic reversal of the relative activity of the two pools in observed and this is manifested by an enormous increase in the activity of the 1st relative to the 2nd pool. In contrast to other phases of the cycle, the release of LH from the 2nd pool is not sustained and this premature decline in LH release despite continuous LRF infusion appears to be due to pituitary depletion of LH as evidenced by the failure of the pituitary response to pulses of LRF immediately following the infusion...


Usefulness of urinary gonadotropin measurements to assess luteinizing hormone releasing factor (LRF) responsiveness in hypogonadotropic states. Multiple blood sampling techniques and short-term, timed urine collections were employed before and after luteinizing hormone releasing factor (LRF) administration to 51 individuals on 58 occasions. Correlation of blood and urine per cent responses to LRF were significant for LH (P less than .05) and FSH (P less than .001), indicating that urine measurements provide an adequate means of assessing response to LRF. In 7 patients with basal blood gonadotropin levels below assay sensitivity, urine measurements provided the only means of accurately determining a response to LRF. Per cent response to LRF was negatively correlated with basal LH levels in the urine (P less than .02) but not in the blood. A significant negative correlation between basal levels and per cent response was demonstrated for FSH in blood and urine (P less than .01). Accurate measurement of basal gonadotropins and the expression of LRF responses as per cent increments aided in distinguishing between patients with hypothalamic and pituitary diseases. A marked response to LRF in the presence of very low basal LH levels was found in patients with hypothalamic disorders, a finding revealed only by using urine determinations. Low per cent responses to LRF were seen primarily in patients with pituitary disease, a situation most clearly delineated by blood FSH measurements.


Endocrine and cytogenetic studies in a patient with Turner's phenotype and a ring chromosome. A 27-year-old woman with secondary amenorrhea and some of the somatic stigmata of Turner's syndrome was found to have a ring chromosome. Laparoscopy and ovarian biopsy showed hypoplastic ovaries and an absence of primordial follicles. Endocrine evaluation showed a normal 24-h mean LH level (12.5 mIU/ml), an elevated FSH level (28 mIU/ml) and a normal plasma estradiol level (64 pg/ml). The augmented FSH and normal  LH response to LH-RH is similar to what is found in men with germinal cell aplasia (Sertoli-cell only). The synchronous initiations of normal LH and abnormally augmented FSH secretory episodes in this patient suggests the absence or decrease of some factor normally produced by the ovarian follicle which modulates the release of FSH in response to LH-RH.


Metabolic clearance and plasma half disappearance time of exogenous gonadotropin releasing hormone in normal subjects and in patients with liver disease and chronic renal failure. The metabolic clearance rate (MCR) and half disappearance time (t 1/2) of gonadotropin releasing hormone (GnRH) has been measured during and after cessation of constant infusion of exogenous GnRH. Studies were performed on normal subjects and patients with severe renal and liver disease. GnRH was quantified by a sensitive and specific radioimmunoassay which does not measure GnRH fragments. The MCR of GnRH in normal subjects was 1640+/-59.7 ml/min (23.7+/-1.8 ml/min/kg), similar to values found in 4 patients with liver disease. However in chronic renal failure an MCR of only 631+/-62 ml/min (9.1+/-0.7 ml/min/kg) was obtained. The t1/2 of GnRH after infusion was linear for 8-10 min, after which a much slower component was observed. The t1/2 of the first component ranged from 5.5 to 8 min in normal subjects, 6.5-8 min in patients with liver disease but prolonged (12-16.5 min) in patients with renal failure. It would appear that GnRH is cleared rapidly in normal subjects, that moderate liver dysfunction does not alter this, but that impaired renal function significantly prolongs the t1/2 and lowers the MCR. The kidney might be an important catabolic organ for infused GnRH; alternatively, uremia might impair catabolism non-specifically.


Carbohydrate metabolism and insulin resistance in myotonia dystrophica. Carbohydrate metabolism was studied in fourteen patients with myotonia dystrophica (MD) using oral glucose, fructose and galactose tolerance tests. Insulin responses to tolbutamide, glucagon, arginine and leucine were determined and insulin resistance was measured with exogenous iv insulin. Glucose tolerance was impaired in twelve of the four teen subjects while hyperinsulinism was found in all patients studied. Insulin response to the various substances was excessive. The insulin tolerance test revealed insulin resistance in all patients and this generally correlated well with the degree of hyperinsulinism to provocative tests. Serum galactose levels after an oral load were much lower in MD compared to normal subjects and were associated with a correspondingly greater rise in glucose, indicating an increased conversion of galactose to glucose. A similar response to oral galactose was found in diabetics. The hyperinsulinism seen with the fructose and galactose tests corresponded well to the rise in glucose during the test. Urinary sorbitol excretion was normal. It is concluded that the impaired carbohydrate metabolism seen in MD is due to peripheral insulin resistance affecting various organs including the liver and it is suggested that the excessive beta-cell response is secondayr to the peripheral resistance.