Inhibition of glucagon and insulin secretion by somatostatin in the rat pancreas perfused in situ. Perfusion of growth hormone inhibitory factor (somatostatin) into rat pancreas inhibited secretion of glucagon and insulin into medium containing 5.5 mM glucose. A 15-min infusion of arginine (20 mM) greatly increased glucagon and insulin secretion. When perfused simultaneously with arginine, somatostatin (55 nM) abolished the increase in glucagon secretion. The acute phase of insulin secretion in response to arginine was attenuated by somatostatin, and subsequent secretion was decreased to control levels. Pretreatment for 5 min with somatostatin blocked even acute-phase insulin secretion in response to arginine. Somatostatin did not affect basal or glucose-stimulated secretion of insulin from rat pancreatic islets isolated by the collagenase technique. Arginine-stimulated secretion of insulin was enhanced by somatostatin in isolated islets. These results demonstrate a direct effect of somatostatin on the pancreas to inhibit secretion of glucagon and insulin. The failure of somatostatin to inhibit insulin secretion in pancreatic islets may be due to alterations in the beta cells produced by the isolation procedure. It is also possible that the effect of somatostatin on insulin secretion may be mediated indirectly.


Subcellular localization of LH releasing activity in the rat hypothalamus. Separation of particulate fractions associated with LH releasing activity has been effected by differential centrifugation followed by sucrose density gradient centrifugation. The fractions were monitored for LH releasing activity by an in vitro incubation with rat pituitaries and for subcellular organelles by electron microscopy. LH released into the incubation medium and contained in the pituitaries was measured by radioimmunoassay (RIA). After differential centrifugation only the "crude mitochondrial fraction" caused an increase of LH release accompanied by a depletion of pituitary LH and a rise in total LH. Further fractionation of this pellet on a discontinuous sucrose gradient resulted in three opaque bands and three clear areas which were removed as sic fractions. Only one fraction, not associated with myelin, synaptosomes, or mitochondria, caused an increase in LH release and total LH while pituitary LH remained unchanged. This fraction appears to be predominantly composed of electron dense vesicles of varying sizes. Also of interest is another fraction which decreased LH release and was again not associated with myelin, synaptosomes, or mitochondria. The decreased release was associated with an increased pituitary LH while total LH remained unchanged. Of the six subcellular fractions obtained from the "crude mitochondrial pellet" only these two caused significant changes in LH release. Two other neural tissues, the cerebellum and cerebral cortex, were similarly processed. None of these subcellular fractions significantly altered LH release and/or synthesis.


Effects of cervical stimulation and anti-LH releasing hormone serum on LH releasing hormone content in the hypothalamus. Hypothalamic content of LHRH was radioimmunoassayed after cervical stimulation on the morning of day of estrus' LHRH content rose 3-fold by 120 min after the stimulation, reaching a level equivalent to that of day 5 of pseudopregnancy. The increase was not steady as there were two early periods when the content did not increase. Measurement of pituitary LH content revealed a marked increase followed by a sudden decrease which corresponded respectively to the two periods when LHRH levels failed ro tise. These changes in pituitary LH level suggest that both the synthesis and release of LH in the pituitary are promoted by the action of LHRH released from the hypothalamus. LH changes in the pituitary were abolished iv injection of 2 ml LHRH antiserum at the time of cervical stimulation, but pseudopregnancy was still induced and diestrus prolonged 4 days longer than in the control group. Progestin concentrations in ovarian vein blood were comparable between anti-LHRH-treated and control groups. These results suggest that the LH released by cervical stimulation is dispensable for the induction of pseudopregnancy. The marked elevation of LHRH in the hypothalamus observed immediately after cervical stimulation must be related to the regulatory mechanism of the characteristic secretory pattern of LH during the period of pseudopregnancy.


Hypothalamic control of the pituitary-testicular relation in fetal rats: measurement of collective volume of Leydig cells. On the 20th day of gestation in pregnant rats, the male fetuses were subjected to surgical hypophysectomy by decapitation or to intracranial paraffin injection which compressed the fetal brain. Autopsy was done on the 22nd day of gestation. The collective volume of Leydig cells in the left fetal testis was estimated by Chalkley's method. Decapitation of a fetus caused a significant retardation in increase of the collective volume of Leydig cells 2 days later. In fetuses given an intracranial paraffin injection, the Leydig cell volume was increased significantly compared with normal fetuses on the 20th day of gestation but was far smaller than that in their intact littermates. This effect of paraffin compression was completely prevented by injections of LRH. The Leydig cell volume remained extremely small in decapitated fetuses given LRH. The observations suggest that in the male rat the hypothalamus begins to govern a pituitary gonadotropic function before birth.


Absence of luteinizing hormone-releasing hormone (LH-RH) from catecholaminergic neurons. 6-Hydroxy-dopamine was administered into the third ventricle of adult male rats. After 2 successive 250 mug doses of 6-OH-dopamine, the levels of norepinephrine and dopamine in the median eminence and arcuate nucleus fell to 15-20% of the control value. The amount of luteinizing hormone releasing hormone (LH-RH) in these same hypothalamic areas, however, was unchanged. It is concluded that LH-RH containing cells are distinct from catecholaminergic neurons.


Purification and characterization of 30-S ribosomal proteins from Bacillus stearothermophilus. Twenty-three proteins were identified by two-dimensional eletrophoresis on polyacrylamide-gel slabs in the 30-S ribosomal subunit of Bacillus stearothermophilus strain 799. They were designated as B-S1 through B-S21, B-Sa and B-Sb and purified on carboxymethyl-cellulose and Sephadex G100 in the presence of 6 M urea. Their molecular weight was estimated by dodecyl-sulfate-gel electrophoresis and their amino acid composition was determined after acid hydrolysis. Results obtained for the individual proteins were essentially similar to those for Escherichia coli 30-S proteins with some characteristic differences.


Somatic antigen of Shigella dysenteriae type 3. Structural features of specific polysaccharide chain. On mild acid hydrolysis of lipolysaccharide from Shigella dysenteriae type 3 the O-specific polysaccharide (hapten) was obtained which appeared to be acidic branched hexosaminoglycan. The repeating unit of this polysaccharide represents a pentasaccharide composed of two D-galactose residues, N-acetyl-D-galactosamine, D-glucose and unidentified acidic component. On the basis of methylation analysis, periodate oxidation, partial acid hydrolysis and chromic anhydride oxidation it is concluded that the structure of the chemical repeating unit of polysaccharide is (see article) where Glcp is glucopyranose, Galp is galactopyranose, Galf is galactofuranose, GalNAcp is 2-acetamido-2-deoxygalactopyranose and where the configuration of galactofuranoside glycosidic linkage and the structure of the acidic monosaccharide A are not known.


Childhood mortality, family size and birth order in pre-industrial Europe. Based on parish registers, demographic histories of Crulai (France), Tourouvre-au-Perche (France), and Geneva (Swizertland) established the childhood mortality experienced by complete sibships during periods of at least half a century before the Fench revolution. These observations may be presented as frequenceis in incomplete five-dimensional contingency tables. The five dimensions are: survival (living or dead), completed sibship size, birth order, type of family (according to completeness of information about family), and epoch (period in which the family lived). This paper reanalyzes these published data, using hierarchical log-linear models to discern which interations among the five variables can justifiably be inferred from the data. The neonatal and infant mortality rates of firstborn are probably higher than those of later sibs (in Crulai and Tourouvre). But mortality by age 20 (in Geneva) is associated strongly with the epoch, type of family, and family size, and not significantly with birth order. The increase in mortality with completed family size is insufficient to select, in an evolutionary sense, for limited family size.


Structure of the gastric mucosa in acute infectious bacterial gastroenteritis. It is now well documented that a characteristic mucosal lesion of the proximal small intestine is present in acute nonbacterial gastroenteritis. To determine whether a gastric mucosal lesion also accompanies this illness, stool filtrate containing Norwalk agent was given orally to 15 volunteers after base line biopsies of gastric fundal and/or antral mucosa had been obtained. Gastric fundal and/or antral biopsies were then obtained serially between 24 and 168 hr after administration of the inoculum. Nine volunteers developed symptoms of gastroenteritis. gastric biopsies from those with normal base line fundal and/or antral biopsies remained normal during and after clinical illness. Those volunteers who had mild to moderate gastritis in their base line biopsies showed persistence but no progression of the lesion during illness. In 4 of the volunteers who became ill, intestinal biopsies were available and showed the typical gastroenteritis lesion. These results indicate that acute infectious nonbacterial gastroenteritis induced by Norwalk agent is not associated with histologically detectable gastric mucosal lesion.


A rapid pull-through technique for measuring lower esophageal sphincter pressure. Because disadvantages are inherent in the conventional station pull-through technique (SPT) currently used for recording lower esophageal sphincter pressure (LESP), a more suitable recording method is needed. In study, we evaluated a rapid pull-through technique (RPT) for recording LESP and compared the results with those obtained by SPT. The RPT features rapid withdrawal of recording sensors across the LES during a 10- to 20-sec interval of suspended respiration. This method avoids recording "artifact" caused by respiratory LES motion and provides precise end points for scoring and measurement. In 12 normal subjects, LESP measured by RPT (24.3 plus or minus 0.5 mm Hg) was comparable, but significantly greater, than LESP measured by SPT (21.1 plus or minus 9.1 mm Hg). Analysis of LESP scores for the two recording methods revealed that both intraobserver and interobserver error were substantially less for RPT than SPT (P less than or equal to 0.01). Further correlation of LESP between studies was greater for the RPT than the SPT recording method. We conclude that RPT represents a convenient method for recording LESP which is easier to perform and yields more reproducible values than LESP recording by SPT.


Effect of glucagon on meal-induced gastric secretion in man. The effect of glucagon on gastric acid and pepsin secretion, basal or stimulated by a meal, pentagastrin and histamine, was studied in duodenal ulcer patients. Intravenous glucagon infused in graded doses ranging from 6.2 to 50 mug per kg-hr produced a dose-related inhibition of pentagastrin-induced acid secretion reaching about 40% of the control level at the dose of 50 mug per kg-hr. Acid inhibition was paralleled by a decrease in the pepsin output and serum calcium level and was accompanied by a rise in the blood glucose concentration. Glucagon used in a standard dose of 25 mug per kg-hr produced about 50% inhibition of acid secretion induced by a meal (measured by intragastric titration) accompanied by a significant decrease in the serum gastrin level measured by radioimmunoassay. Histamine-induced secretion was only slightly inhibited by glucagon, and the degree of inhibition for acid (25%) and pepsin (20%) secretion was statistically insignificant.


Pathogenesis of Salmonella-associated arithritis in the rat. The distribution of joint lesions in rats with Salmonella-associated arthritis (SSA), as determined in a detailed survey, resembles to a great extent the pattern of small joint involvement in human rheumatoid arthritis. Such lesions, though regularly induced in the rat by the intravenous injection of live S.enteritidis, could not be evoked by the heat-killed organisms injected by various routes with and without extrinsic adjuvants. Efforts to transfer SAA from sensitized donors to either normal or primed recipients, employing lymphoid ce-ls from several sources, also failed repeatedly. Two observations, however, virtually exclude the possibility that joint damage in SAA can be the direct result of sustained intra-articular sepsis. First, the inoculation of as many as 10-3 viable inflammation. Second, the incidence of SAA was significantly lower in weanling rats in the adult controls although the growth and distribution of intravenously injected S. enteritidis was virtually identical in the two groups. Together these observations indicate that the joint damage occurring in SAA is determined by the host and not by the infecting organism. From this, it seems fair to conclude that the destructive arthritis characteristic of this syndrome is immunologically mediated.


Isolation of mitogenic and adjuvant active fractions from various species of Nocardiae. Delipidated lysozyme digests of Nocardia opaca, N. corallina, and N. rubra have been fractionated by Sephadex filtration. The mitogenic and adjuvant activities of the fractions thus obtained have been investigated. All fractions are mitogenic except the last fraction of N.rubra, but the N. opaca products induce a stronger stimulation of mouse spleen lymphocytes than the corresponding fractions of the two other species. The activity of the first Sephadex fractions of each strain has been compared to other mitogens (concanavalin A, lipopolysaccharide). All fractions are adjuvant, although one of them, the last Sephadex fraction of N. rubra, does not contain peptidoglycan; its activity must thus be attributed to another kind of molecule. Fractionation of the first Sephadex fraction of N. opaca by centrifugation in glacial acetic acid led to a separation of adjuvant and mitogenic activities.


Deregulation of mouse antibody-forming cells in vivo in cell culture by streptococcal pyrogenic exotoxin. An unregulated, elevated rebound of antibody levels in rabbits was shown to follow late (10 to 15 days) after steptococcal pyrogenic exotoxin (SPE)-induced immunosuppression. Because of that result we have suggested that SPE acts by preferentially inhibiting a regulatory cell which normally limits the extent of full expression of antibody formation by B-cells. We are currently testing this hypothesis in mice. NIH (Swiss Webster) mice (+/+) or NIH (Swiss Webster) mice heterozygous (+/nu) for the mutant athymic nude gene and phenotypically normal showed an elevated plaque-forming cell (PFC) response to sheep erythrocytes (SE) late (10 to 15 days) after immunosuppressive SPE treatment similar to that described in rabbits. Homozygous nude mice (nu/nu) that are phenotypically athymic normally show a reduced early (4 day) PFC response to SE (a T-cell-dependent antigen) as compared with +/nu littermates or +/+ parent strain mice. This cryptic early 4-day response was improved by injection of purified endotoxin (a B-cell mitogen), but these relatively elevated nude PFC responses had decreased to normal control (SE only)nude PFC levels before 10 days. In similar SE-injected nude mice treated instead with SPE, no elevation at 4 days was observed and, more pertinently, the late (10 to 15 day) elevated rebound of PFC levels observed in normal response controls (+/nu or +/+) was not observed. Similar experiments were subsequently conducted in Marbrook-type spleen PFC cultures during periods of 12 days. The results of these experiments paralleled the in vivo results above, and in addition showed that SPE induced a large proliferation of either +/+ or +/nu cells (T-and B-cells) in culture but had no such effect on nu/nu cells (B-cells) in culture. Purified endotoxin, the Bcell mitogen, had a better sparing effect on nu/nu cells in this respect. These results are consistent with our premise that SPE inhibits preferentially the function of a regulator of the antibody response. The regulator appears to be a T-cell and is likely a suppressor T-cell.


Immune responses of specific pathogen-free and gnotobiotic mice to antigens of indigenous and nonindigenous microorganisms. Strains of indigenous Escherichia coli, Bacteroides, and Lactobacillus were isolated from the gastrointestinal tracts of specific pathogen-free (SPF) mice. Nonvaccinated SPF mice exhibited in their spleens low numbers of plaque-forming cells (PFC) and rosette-forming cells reacting with antigens of these andigenous bacteria. PFC reacting with these bacterial antigens were not detected in infant SPF mice until 7 days after birth. Compared with nonvaccinated controls, SPF mice vaccinated parenterally with indigenous E. coli or Bacteroides produced a moderate increase in the numbers of specific PFC. Thus, the SPF mouse is capable of responding immunologically after vaccination with microbes indigenous to its intestinal tract. However, more PFC reacting with homologous vaccine antigens were detected after parenteral vaccination of SPF mice with nonindigenous E. coli O127:B8, E.coli O14, or B. fragilis than after parenteral vaccination with indigenous E. coli or Bacteroides. Gnotobiotic mice orally monoassociated with these nonindigenous bacteria exhibited greater immune responses to antigens of the bacteria used to monoassociation than did gnotobiotes monoassociated with the indigenous microbes. The results are consistent with the hypothesis that mice are more responsive immunologically to antigens of nonindigenous bacteria than they are to antigens of certain microbes indigenous to their gastrointestinal tracts.


Test for enterotoxigenic Escherichia coli using Y-1 adrenal cells in miniculture. A rapid, potentially clinically useful test for detection of enterotoxigenic Escherichia coli is described. Whole bacterial cultures of enterotoxigenic E. coli, when briefly exposed to Y1 adrenal cells in tissue miniculture, effect a rounding response in the tissue culture that can be discerned at 18 to 24 h. The tissue culture technique agreed with the rabbit ileal loop in all 58 enterotoxigenic and 52 non-enterotoxigenic E. coli strains tested.


In vitro interaction of Bacteroides fragilis with polymorphonuclear leukocytes and serum factors. The sera and polymorphonuclear leukocytes (PMNs) of healthy human subjects were tested against several isolates of Bacteroides fragilis. All sera killed most of the fecal isolates, but were active aganist only one of the clinical isolates. The degree of killing was directly related to the serum concentration but was independent of the bacterial inoculum within the range of bacterial concentrations studied. The serum bactericidal activity was heat-labile. Engulfment and killing of B. fragilis by PMNs were demonstrated consistently.  The opsonim involved in phagocytosis was also heat liable. Calculations involving log-transformed data permitted the quantitative study of the separate and combined effects of serum and PMNs on bacteria which were killed by serum alone. Whereas serum alone usually killed 0.5 log of serum-sensitive bacteria, the addition of PMNs was usually associated with one log further killing. The studies reported here demonstrate the presence of heat-labile serum factors in normal human sera which killed B. fragilis directly and which promote its phagocytosis and killing by PMNs. These observations provide a foundation for investigations into host defense mechanisms against anaerobes.


Association of Salmonella typhimurium with, and its invasion of, the ileal mucosa in mice. A wild-type strain of Salmonella typhimurium and three mutant rough colonial variants of the wild type were compared for their ability to become associated with and invade the ileal mucosa of germfree and specific-pathogen-free mice. The rough-mutant strains differed from the wild type in having incomplete lipopolysaccharides lacking one or more sugars in the polysaccharide moiety. The wild-type and mutant strains also differed one from the other in the types of appendages (flagella, pili) on their surfaces. Depending upon the dosage of bacteria given, all mutant strains as well as the wild type could associate with and invade the intestinal mucosa of infected gnotobiotic mice. If the infecting dosage was high enough, at least two of the mutant strains and the wild type invade the intestinal mucosa of the specific-pathogen-free animals. O antigen, flagella, or pili do not appear to be essential for the association of S. typhimurium with the mucosal surface of the mouse ileum. O antigen on the bacterial cell surface may be important, but not essential, for invasion of the ileal mucosa.


Phagocytosis of Mycoplasma salivarium by human polymorphonuclear leukocytes and monocytes. Mycoplasmacial activity was exhibited by human peripheal blood leukocytes in the absence of detectable specific antiserum. After incubation of varying concentrations of Mycoplasma salivarium with leukocytes, changes in colony-forming units (CFU) of this species per millilter occurred. The most noticeable decrease in CFU per milliliter was then the incubation mixtures contained five mycoplasmas per leukocyte. At this ratio, the mycoplasmacial min of incubation. Continued incubation demonstrated a tenfold decrease in CFU per milliliter by 4 h. Electron micrographs of incubated mixtures of human leukocytes and M. salivarium showed this mycoplasma to be phagocytized by monocytes and neutrophils whenever mutual contact or pseudopodial formation occurred. The process was continuous. Numerous phagocytic vacuoles developed which contained multiple ingested microorganisms. After the cytoplasmic granules of the leukocytes fused with the phagocytic vacuole, the phagocytized mycoplasmas became disrupted and unrecognizable.


Effect of local corticosteroids on antibody-forming cells in the eye and draining lymph nodes. Significant numbers of antibody-forming cells (AFC) have been found in the cornea, uveal tract, and draining lymph nodes after the intracorneal injection of bovine gamma-globulin (BGG). To study the effect of locally administered corticosteroids on these antibody-forming tissues, we made unilateral intracorneal injections of rabbit eyes with BGG. These we followed immediately with subconjunctival injections of 10 mg. of triamcinolone suspension, and then with a second round of 10 mg. injections seven days later. A control group of animals received the BGG injections followed by two subconjunctival saline injections. We killed the animals on postinjection days 6, 9, 12, 15, and 21, and tested the draining lymph nodes, homolateral uveal tissue, and homolateral cornea for AFC by a modification of the Jerne placque technique. The local steroids had no effect on the number of AFC produced in the draining lymph nodes or on the circulating antibody response, but they reduced the number of AFC in the homolateral uveal tracts and corneas. Clinically there was less inflammatory response in the steroid-treated eyes than in the control eyes. The possible mechanisms by which corticosteroids achieve their anti-immunologic and anti-inflammatory benefits are discussed.


Fracture faces of fenestrations and junctions of endothelial cells in human choroidal vessels. Replicas of freeze-fractured endothelial cells of human choroidal capillaries show fenestrations with a central thickening, as described in thin-section studies. Between the endothelial cells there are discontinuous zonulae occludentes (i.e., maculae or fasciae occludentes), composed of ridges on face A and grooves on face B of the split-cell membrane. They are also present along the sutures of the endothelial cells of venules. Close to the intercellular suture gap junctions, probably connecting capillary endothelial cells and pericytes, are found.


Sutures and bladder wound healing in the experimental animal. An incision was made in the bladder of each of 81 female rats, then closed with various types and sizes of sutures and by different methods in order to determine the optimum suture and method to facilitate watertight closure and wound healing. Chromic catgut, collagen, and polyglycolic acid sutures, of different sizes, were used. Pressure studies and microscopic examinations of the wound were done at different intervals. All sutures lasted long enough to allow healing of the wound and none broke during pressure studies. A two-layer continuous closure with fine suture was the most watertight. Tissue reaction was almost absent when polyglycolic acid suture was used and was minimal with fine chromic catgut.


Cutaneous everted nipple ureterostomy.

Cutaneous everted nipple ureterostomy. Normal dog ureter transplanted to the skin by the everted nipple ureterostomy technique was found to be far superior to the skin flap ureterostomy in nevovascularization and healing of the stoma. The results suggest a possibility of successful application in the human; recent clinical experience supports animal data.


Experimental lipid A-induced nephritis in the dog. The effect of radioactive lipid A, obtained from Escherichia coli, on the kidney of adult dogs and puppies was investigated. Injection of lipid A into the temporarily occluded renal pelvis of adult dogs caused abacterial interstitial nephritis in all animals tested. The intensity and duration of the kidney response coorelated well with the lipid A dose administered. Lipid A was demonstrated autoradiographically in the renal cortex, extra- and intracellularly. Radioactivity was still present after 10 weeks in 16 of 20 examined dogs. In the remaining four dogs the interstitial nephritis had subsided. Thirteen of 14 puppies in which the immunologic defense mechanisms had not yet developed showed no histologic reaction in the kidney after the same procedure as in the adult dogs. Lipid A appeared in the renal parenchyma through the blood stream rather than through the retrograde route. Lipid A antibody titers could be detected only in adult dogs, never in puppies. Lipid A apparently provoked an immunologic process in the kidney damaged by the operative manipulations, thus resulting in an abacterial interstitial nephritis. The possibility that chronic abacterial pyelonephritis can be induced clinically through lipid A and thus may have a pathogenesis similar to abacterial interstitial nephritis is discussed.


Experimental pyelonephritis in the monkey. II. The prognostic value of radionuclide evaluation of the urinary tract. The radionuclide scintiphoto study, a noninvasive technique, is abnormal in acute pyelonephritis. A delay in excretion of the isotope has also been observed in dehydrated studies in chronic pyelonephritis. To further elucidate this abnormal pattern in pyelonephritis, experimental renal infection was produced in monkeys. Dehydrated 131-I-hippuran scintiphoto studies were conducted weekly for 8 weeks after the initial infection. The scintiphoto studies showed significant delay in excretion of radionuclide in all bacteriuric monkeys at 1 week. After this time, delayed excretion was seen in only those monkeys later classified as having chronic pyelonephritis. This was statistically significant (P smaller than 0.01).


Benurestat, a urease inhibitor for the therapy of infected ureolysis. A single oral administration of the urease inhibitor benurestat (2-(p-chlorobenz-amido)acetohydroxamic acid) to the human at 15 or 25 mg per kg produced, for 4 hr, mean urinary levels of inhibitory activity that were 700 to 1900 times that equivalent concentration of benurestat required to inhibit Proteus mirabilis urease by 90 per cent. In the rat these same dosage levels produced urinary inhibitory activity equivalent to 16 to 140 fold that required for 90 per cent urease inhibition. Benurestat administration, 25, 50, or 100 mg per kg, caused a decrease in the urinary excretion of ammonia from rats with experimental P. mirabilis genitourinary tract infection. The formation of struvite calculi was inhibited under these conditions. Nitrofurantoin, sulfamethoxazole, and ampicillin also slowed the formation of struvite calculi in infected rats and together with benurestat a potentiation of the inhibition of calculi formation was secured. Some combination therapies composed of benurestat plus an antibacterial agent, sulfamethoxazole or ampicillin, were effective in promoting the net dissolution of formed calculi. The number of viable bacteria present in the bladders of infected rats was significantly less after the administration of benurestat plus nitrofurantoin, sulfamethoxazole, or ampicillin than the respective numbers that were obtained from control infected rats or from rats administered either component of the combination separately.


Succinic dehydrogenase as a measure of tissue viability in the cadaver kidney. Influence of perfusing solutions. Succinic dehydrogenase (SDHG) has been studied in the rabbit cadaver kidney. Increasing warm ischemia time decreases the ability of the organ to utilize oxygen and therefore lowers its SDHG activity. The addition of perfusing solutions further decreases the SDHG activity; saline has a more adverse affect than either Perfudex or albumin.


Canine granulocytopathy syndrome: neutrophil dysfunction in a dog with recurrent infections. A male Irish Setter dog had a clinical history of recurrent life-threatening bacterial infections, with associated periods of pyrexia and severe neutrophilia. Examination of a mandibular lymph node biopsy made when the patient was 10 weeks old revealed subacute diffuse suppurative lymphadenitis with reticuloendothelial hyperplasia. Circulating leukocytes isolated from the dog when it was 5 months old had a marked bactericidal defect when compared with cells from clinically normal dogs of the same age. The clinical syndrome in the affected patient resembled that observed in the granulocytopathies described in man and other animals.


Stanford cardiac transplantation. A review. The cardiac transplantation experience at Stanford University School of Medicine commenced in 1959 with the development of a comprehensive surgical method for successful orthotopic cardiac transplantation in experimental animals. The steps involved included atrial level transection and anastomosis, local myocardial hypothermia and cardiopulmonary bypass. From the first Stanford clinical case in January 1968 until May 1974, 73 transplants have been carried out in 71 patients. Cardiac transplantation is indicated in myopathic disease of an advanced nature with the prospect of imminent death. Contraindications are high pulmonary vascular resistance and intercurrent infection. Relative contraindications are advanced age and insulin-dependent diabetes. Previous cardiac surgery procedures are not contraindications to transplantation. Maintenance immunosuppression requires azathioprine and prednisone indefinitely. Antithymocyte globulin is used for a short time in the postoperative period. Anticoagulants and antiplatelet agents are also used. Therapy for rejection crises includes methylprednisolone, in gram amounts, and actinomycin D. The diagnosis of graft rejection is based on a decrease in electrocardiographic voltage, transvenous endomyocardial biopsy and clinical examination of the patient. The overall survival rate is 43% at one year and 39% at two years. The longest-living survivor in the series is doing a regular day's work five years after transplantation.


The modified bovine carotid artery as a vascular substitute in man. Personal experience and review of the literature. The modified bovine heterograft is one of the vascular prostheses currently available for clinical use. This report reviews the mode of preparation of the heterograft and its physical and biological qualities. The experience of the authors with this heterograft for vascular reconstruction in 32 patients and for the construction of s.c. arteriovenous (A-V) fistulas in 80 patients is reported. The modified bovine heterograft was found to be a satisfactory prosthesis when used in vascular reconstruction above the inguinal ligament or for the construction of A-V fistulas for hemodialysis. In reconstructions in the femoropopliteal segment of the circulation, the use of the heterograft by the authors was associated with an unacceptable rate of morbidity and graft failures. Experience reported in the literature has so far been incomplete and inconclusive, with one-year patency rates ranging between 20 and 74%.


Dimensions of the pet population problem. In 1973, an estimated $4.5 billion was spent on the combined care and feeding of pets, whereas about 18 million neglected pets were impounded, costing our society nearly $125 million. About 72,000 dogs and cats must die or be killed each day to maintain zero growth in the population. Humane shelters and pounds have become slaughterhouses. Neglected pets constitute a hazard to the health and quality of life of man and other animals. Sizable segments of society are becomming hostile toward dogs and cats.


New azasteroidal antifungal antibiotics from Geotrichum flavo-brunneum. III. Biological activity. The A25822 antibiotic complex consists of seven biologically active factors. A comparative study of these factors determined that factor B possessed the greatest antifungal activity. The minimal inhibitory concentration of A25822B against isolates of Candida albicans was less than 0.3 similar to 5.0 mug/ml, Trichophyton mentagrophytes was inhibited at less than 0.0312 mug/ml. Other pathogenic fungi such as Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatitidis, Sporotrichum schenckii, and Microsporium gypseum were very susceptible to A25822B. Only limited antibacterial activity of A25822B was found. Parenteral or oral administration of 50 mg/kg of A25822B significantly extended the average survival time of mice infected with C. albicans. Doses of 20 mg/kg of A25822B caused a greater than ten-fold reduction in the number of Candida cells recovered from kidneys of infected mice. A solution of 0.5% or 0.25% A25822B applied topically was effective against an experimental dermatophyte infection on guinea pigs. A peak blood level of 3 mug/ml was achieved in mice following a 100 mg/kg dose of A25822B. Combination of A25822B with a polyene antibiotic in vitro showed antagonism.


New azasteroidal antifungal antibotics from Geotrichum flavo-brunneum. I. Discovery and fermentation studies. Although Geotrichum species occur ubiquitously, antibiotic production by members of this genus has not previously been reported. The antibiotic complex designated A25822, consisting of one major and six minor structurally-related components active primarily against Candida and Trichophyton, represents a new family of naturally-occurring compounds. Approximately 90% of the antibiotic activity synthesized remained associated with the fungal cell mass, from which it was recovered by multiple methanolic extractions for quantitation. Antibiotic production was enhanced by tryptophan, iron, zinc, and high levels of dextrin.


Uptake of adenosine 5'-monophosphate by Escherichia coli. Adenosine 5'-monophosphate is dephosphorylated before its uptake by cells of Escherichia coli. This is demonstrated by using a radioactive double-labeled culture, and with a 5'-nucleotidase-deficient, mutant strain. The adenosine formed is further phosphorolyzed to adenine as a prerequisite for its uptake and incorporation. The cellular localization of the enzymes involved in the catabolism of adenosine 5'-monophosphate is discussed.


Cellular content of ribonucleic acid and protein in Saccharomyces cerevisiae as a function of exponential growth rate: calculation of the apparent peptide chain elongation rate. The average cellular content of ribonucleic acid and protein was determined in cultures of Saccharomyces cerevisiae growing exponentially at different rates in a variety of media. Estimations of the proportion of total cellular ribonucleic acid that is made up of ribosomal ribonucleic acid were used to calculate the average number of ribosomes per cell at the different growth rates. The fraction of ribosomes actively engaged in translation was estimated by sucrose gradient centrifugation of ribosomes and polysomes. These data were used in a calculation of the apparent time taken for the addition of an amino acid to the growing polypeptide chain; this value was found to vary linearly with growth rate over a fivefold range of doubling times.


Characterization of lipopolysaccharides from Escherichia coli K-12 mutants. Chemical analyses of the carbohydrate composition of lipopolysaccharides (LPS) from a number of LPS mutants were used to propose a schematic composition for the LPS from Escherichia coli K-12. The formula contains four regions: the first consists of lipid A, ketodeoxyoctonoic acid, and a phosphorous component; the second contains only heptose; the third only glucose; and the fourth additional glucose, galactose, and rhamnose. LPS from E. coli B may have a similar composition but lacks the galactose and rhamnose units. A set of LPS-specific bacteriophages were used for comparing three mutants of Salmonella with a number of LPS mutants of E. coli K-12. The results confirm that there are basic similarities in the first and second regions of the LPS structure; they also support the four region divisions of the LPS formula. Paper chromatography was used for characterization of 32-P-labeled LPS from different strains of E. coli and Salmonella. The Rf values for LPS varied from 0.27 to 0.75 depending on the amounts of carbohydrates in the molecule. LPS from all strains studied was homogenous except for strain D31 which produced two types of LPS. Mild acid hydrolysis of labeled LPS liberated lipid A and two other components with phosphate, one of which was assigned to the first region. It is suggested that paper chromatography can be used in biosynthetic studies concerning regions 2 to 4.


Blocking of bacteriophages phi W and phi 5 with lipopolysaccharides from Escherichia coli K-12 mutants. In the preceding paper we presented a formula for the composition of lipopolysaccharides (LPS) from Escherichia coli K-12. This formula contains four regions defined from analyses of LPS from four key strains, the parent and mutants which had lost one, two, or three regions of their carbohydrates. Support for the formula was derived from the susceptibility of the key mutants to several bacteriophages. One of these, phage phi W, was found specific for strains which had lost region 4. In this paper we described inactivation in vitro of phage phi W and its host-range mutant phi 5, using LPS devoid of regions 2 to 4. The blocking of phi W was found to require about 0.15 M concentrations of monovalent cations and to be inhibited by low concentrations of calcium and magnesium ions. One particle of phage phi W required 2 times 10-16 g of LPS devoid of region 4 for stoichiometric inactivation. Phage phi 5 was blocked by both heptose-less LPS (devoid of regions 2 to 4) and glucose-less LPS (devoid of regions 3 to 4) but was unaffected by LPS devoid of region 4. LPS from a heptose-less mutant of Salmonella minnesota showed the same inactivation ability as did LPS from heptose-less strains of E. coli K-12. Lipid A was prepared from LPS containing all four regions. Such lipid A was found to inactivate phi 5, whereas both the polysaccharide moiety as well as the intact LPS were without effect. It is suggested that lipid A is part of the receptor site for phage phi 5.


Genetic analysis of thymidine-resistant and low-thymine-requiring mutants of Escherichia coli K-12 induced by bacteriophage Mu-1. Four genes, dra, tpp, drm, and pup, that specify enzymes involved in the catabolism of nucleosides and deoxynucleosides in Escherichia coli are known to be very closely linked in the order dra-tpp-drm-pup. By infecting cells with the phage Mu-1 and isolating low-thymine-requiring derivatives of a strain lacking thymidylate synthetase and also thymidine-resistant mutants of a dra-strain, it has been possible to select for strains in which Mu-1 is inserted in this gene cluster. Making use of the polar effect of Mu-induced mutations on more distal genes in the same transcriptional unit, evidence is presented that dra and tpp are co-transcribed from a promoter to the left of dra, and drm and pup are co-transcribed from a promotor located between tpp and drm. Residual levels of purine nucleoside phosphorylase in drm- mutants induced by phage Mu seem to indicate that a weak promotor lies between drm and pup. From a strain in which Mu-1 is inserted in drm, a mutant has been isolated that has a deletion extending into tpp. Since this strain lacks thymidylate synthetase, it is unable to grow on minimal medium containing thymine. Mutants isolated from this strain that can grow on minimal medium containing thymine have been shown to have increased levels of the enzyme uridine phosphorylase.


Effect of a leu-linked mutation on the valine sensitivity of acetohydroxy acid synthase activity in Escherichia coli. A spontaneous leu-linked mutation (ilvH2015) in Escherichia coli K-12 made the strain resistant to 1 mM valine and l mM glycylvaline (Val-r) and caused the isoleucine and valine biosynthetic enzyme, acetohydroxy acid synthase, to be less sensitive to feedback inhibition by valine than the wild-type enzyme. Transfer of the ilvDAC deletion into a strain carrying ilvH2015 abolished the effect of the marker on the acetohydroxy acid synthase and rendered it as sensitive to valine as the enzyme in the isogenic control strain without the Val-r marker under both repressing and limiting conditions. In contrast, auxotrophy caused by transfer of an ilvC lesion into the Val-r strain did not interfere with the effect of ilvH2015 on valine sensitivity of acetohydroxy acid synthase. In addition, the presence of the Val-r marker produced minor but significant pleiotropic effects on several other isoleucine and valine biosynthetic enzymes but did not cause derepression of the ilv gene cluster. These studies suggest some type of interaction between a product produced by a gene close to leu and the isoleucine and valine biosynthetic enzymes.


Mutation affecting regulation of synthesis of acetohydroxy acid synthetase in Escherichia coli K-12. Altered regulation of synthesis of acetohydroxy acid synthetase (AHAS) was previously reported in a mutant of Escherichia coli strain K-12. The mutant strain, growing in minimal medium, exhibits a partial growth limiatation and derepression of AHAS, owing to deficient synthesis of isoleucine. The genetic lesion (ilvE503) causing the isoleucine limitation was shown to cause derepression of a valine-sensitive AHAS activity. The derepression effect of the ilvE503 mutation upon synthesis of AHAS was conclusively demonstrated by introducing both the ilvE503 allele and an altered AHAS (ilv-521) into the same cell. Evidence is presented that suggests the presence of multiple genetic regions for synthesis and control of the valine-sensitive AHAS activity.


Enzymatic production of deoxyribonucleic acid double-strand breaks after ultraviolet irradiation of Escherichia coli K-12. We have observed the enzymatic production of deoxyribonucleic acid (DNA) doublestrand breaks in Escherichia coli K12 after ultraviolet irradiation. Doublestrand breaks appeared in wild-type, polA1, recB21, recA, and exrA strains after incubation in minimal medium. THE UVRA6 strain showed no evidence of double-strand breakage under the same conditions. Our data suggest that uvr+ cells, which are proficient in the incision step of excision repair, accumulate double-strand breaks in their DNA as a result of the excision repair process, i.e., arising from closely matched incisions, excision gaps, or incisions and gaps on opposite strands of the DNA twin helix. Furthermore, strains deficient in excision repair subsequent to the incision step (i.e., polA, rec, exrA) showed more double-strand breaks than the wild type strain. The results raise the possibility that a significant fraction of the lethal events in ultraviolet-irradiated, repair-proficient (uvr+) cell may be enzymatically-induced DNA double-strand breaks.


mut-25, a mutation to mutator linked to purA in Escherichia coli. The mutation mut-25 that results in a mutator phenotype is closely linked to purA on the chromosome of Escherichia coli. The gene order in this region is ampA mut-25 purA. purA mut-25 double mutants retained mutator activity indicating that mut-25 is not a mutation in the purA gene. The repair mutations uvrA6, recA56, and exrA1 had no effect on mutation frequencies in mut-25 strains, and mut-25 strains were normally resistant to ultraviolet irradiation. Frequencies of host range mutations were not increased in phages T1, T2, and T7 grown on mut-25 strains. mut-25 could act trans, reverting the trpA46 mutation either on the chromosome or on an F episome. The transitions AT yields GC (adenine-thymine yields guanine-cytosine) and GC yields AT were induced by mut-25.


Suppression of initiation-negative strains of Escherichia coli by integration of the sex factor F. Data are presented suggesting that the most critical factor determining whether an Hfr dnaAts strain can synthesize deoxyribonucleic acid and form colonies at temperatures that are nonpermissive for corresponding F- strains is neither the site of insertion of F nor the presence of additional mutations in the F particle or the chromosome; it is whether the particle is capable of autonomous replication at the temperature used. Consequently, suppression of the DnaA phenotype in Hfr strains occurs at 40 C but not, in most of them, at 42 C without the occurrence of additional mutations. The site of insertion of F may also be important since it is shown that in one Hfr dnaA strain partial suppression does occur at 42 C. In addition, it is shown that strains exhibiting suppression by integration of F at 40 C on minimal agar plates do not do so at this temperature on nutrient agar plates.


Positive selection of general amino acid permease mutants in Saccharomyces cerevisiae. It was found that D-stereoisomers of natural amino acids inhibit the growth of Saccharomyces cerevisiae cells. Kinetic and genetic evidence showed that d-amino acids enter the cell by the general amino acid permease. Two classes of S. cerevisiae mutants resistant to d-amino acids were isolated. One class of mutants appeared to be defective in the general amino acid permease specified by the gene gap. In the second class, the activity of general amino acid permease was affected by ammonium ions. Mutants of the second class were isolated in a yeast strain with the general amino acid permease insensitive to the presence of ammonium ions in culture media. The mutation affecting the permease, amc, occurred in a locus unlinked to gap.


Urea transport in Saccharomyces cerevisiae. Urea transport in Saccharomyces cerevisiae occurs by two pathways. The first mode of uptake is via an active transport system which: (i) has an apparent Km value of 14 muM, (ii) is absolutely dependent upon energy metabolism, (iii) requires pre-growth of the cultures in the presence of oxaluric acid, gratuitous inducer of the allantoin degradative enzymes, and (iv) is sensitive to nitrogen repression. The second mode of uptake which occurs at external urea concentrations in excess of 0.5 mM is via either passive or facilitated diffusion.


Genetic and phenotypic characterization of dnaC mutations. The dna-1, dna-2, dna-7, and dna-28 mutations, all of which are located near min 89.5 on the E. coli linkage map, have been characterized further. As previously demonstrated for dna-2 and dna-28, neither the dna-1 nor dna-7 mutation affects the ability of a strain to produce bacteriophage lambda at temperatures non-permissive for the continued replication of the bacterial chromosome. The reported temperature-sensitive inhibition of lambda production in a strain carrying dna-7 is shown to be a consequence of a thermosensitive host specificity mutation in the hsm gene and not of the dna-7 mutation. The four dna mutations are recessive to the wild type and define a single dnaC cistron according to standard complementation criteria. Unlike other characterized dnaC mutants, however, strains carrying the dnaC1 or dnaC7 alleles exhibit an abrupt cessation of deoxyribonucleic acid synthesis at 42 C that appears to be more compatible with a defect in deoxyribonucleic acid chain elongation rather than in initiation. The possibility that the apparent elongation defect is actually a composite effect of residual synthesis and deoxyribonucleic acid degradation is raised by the net deoxyribonucleic acid degradation observed in the dnaC1 strain at 42 C. Several alternative possibilities for the function of the dnaC gene product are suggested.


Growth of bacteriophages MS2 and T7 on streptomycin-resistant mutants of Escherichia coli. Streptomycin-resistant mutants of an Hfr strain of Escherichia coli K were examined for their ability to support the growth of male-specific ribonucleic acid phage MS2 and female-specific deoxyribonucleic acid phage T7. Normally, the Hfr strain allows propagation of MS2 and is lysed by it (efficiency of plating equal to 1), whereas the same strain restricts propagation of T7 and is not lysed by it (efficiency of plating smaller than 10-7). Twenty-four isolates out of 26 independently obtained streptomycin-resistant mutants are partially or completely derestricted for propagation of T7; efficiency of plating of T7 in such strains ranges from 10-3-1. Depending on their response to plating of MS2 and T7, the streptomycin-resistant mutants can be divided into four classes. The mutants in all four classes continue to be "male" in conjugation with F- strains. Genetic analysis is presented to show that restriction of MS2, derestriction of T7, and resistance to streptomycin are the pleiotropic effects of a single mutation at the strA locus.


Ultraviolet light sensitivity of Escherichia coli K-12 strains carrying ruv mutations in combination with uvrA or lon mutant alleles. Strains of Escherichia coli K12 have been prepared that carry the ruv mutation in combination with lon or uvrA mutant alleles. The ruv minus uvrA minus double mutant is more sensitive to ultraviolet light than the urvA minus single mutant, whereas the strain with ruv and ion mutations shows the same sensitivity to ultraviolet light as the ruv minus single mutant.


Nucleotide pools and regulation of ribonucleic acid synthesis in yeast. Nucleotide pools were measured in growing and amino acid-starved Saccharomyces cerevisiae. During amino acid starvation there are neither significant changes in the endogeneous nucleoside triphosphate pool levels nor measurable synthesis of guanosine 5'-diphosphate, 3'-diphosphate. Stable ribonucleic acid synthesis does not appear to be regulated by changes in the triphosphate pools or by the unusual nucleotide guanosine 5'-diphosphate, 3'-diphosphate.


Sensitive methods for the detection and characterization of double helical ribonucleic acid. We have evaluated three methods which respond specifically to stable RNA-RNA duplexes and have compared their utility for examining several sorts of nucleic acids. We find that these methods, stepwise chromatography on Whatman CF11-cellulose; digestion with Escherichia coli RNase III; and specific inhibition of globin synthesis in vitro in rabbit reticulocyte lysates, are able to distinguish between stable double-stranded RNA and single-stranded RNA in the expected manner. The most sensitive method, inhibition of globin synthesis, responds to double-stranded RNA concentrations below 0.1 ng per ml. We have used the predominantly single-stranded RNA from several RNA bacteriophages of E. coli to test both the sensitivity and selectivity of these methods. The three viral RNAs tested contain low levels of double-stranded RNA which can be readily removed, leaving RNA which is not recognized as double-stranded RNA, despite indications from physical and sequencing studies that secondary structure is present. In particular, a potential hairpir loop of known sequence has been isolated from phage f2 RNA. Its properties were found to depart significantly from those of RNA-RNA duplexes by those two of our three methods capable of testing RNA of this size. Analysis of two eukaryotic mRNA populations by these methods was complicated by the presence of poly(A). Synthetic poly(A) chromatographs like double-stranded RNA on cellulose CF11 columns, and we could distinguish it from reovirus double-stranded RNA only at elevated temperatures.


Deoxyribonucleic acid polymerase III of Escherichia coli. Purification and properties. DNA polymerase III has been purified 4,500-fold from the Escherichis coli mutant, HMS83, which lacks DNA polymerases I and II. When subjected to disc gel electrophoresis, the most purified fraction exhibits a single major protein band from which enzymatic activity may be recovered. Polyacrylamide gel electrophoresis under denaturing conditions produces two protein bands with molecular weights of 140,000 and 40,000. The sedimentation coefficient of the enzyme is 7.0 S, and the Stokes radius is 62 A. Taken together these tow parameters indicate a native molecular weight of 180,000. Purified DNA polymerase III catalyzes the polymerization of nucleotides into DNA when provided with both a DNA template and a complementary primer strand. The newly synthesized DNA is covalently attached to the 3' terminus of the primer strand. Because the extent of polymerization is only 10 to 100 nucleotides, the best substrates are native DNA molecules with small single-stranded regions. The most purified enzyme preparation is devoid of endonuclease activities. In addition to the two exonuclease activities described in the accompanying paper, purified polymerase III also catalyzes pyrophosphorolysis and the exchange of pyrophosphate into deoxynucleoside triphosphates. DNA polymerase III has also been isolated from wild type E. coli containing the other two known DNA polymerases. Futhermore, the enzyme purified from three different polC mutants exhibits altered polymerase III activity, confirming that polC is the structural gene for DNA polymerase III (Gefter, M., Hirota, Y., Kornberb, T., Wechsler, J., and Barnoux, C. (1971) Proc. Natl. Acad. Sci. U. S. A. 68, 3150-3153).


Fluorescence studies on the active sites of porcine pepsin and Rhizopus-pepsin. Fluorescence studies on the interaction, with porcine pepsin, of oligopeptides bearing a mansyl (Mns, 6-(N-methylanilino)-2-naphthalenesulfonyl) or dansyl (Dns, 5-dimethylaminonaphthalene-1-sulfonyl) group at the NH2 or COOH terminus have provided further evidence showing that the probe group is drawn into the extended active site largely as a consequence of the specific binding of the peptide portion of the substrate. The active site does not appear to have appreciable intrinsic affinity for the mansyl or dansyl group, and the principal contribution to the specific peptide-protein interaction is provided by the sensitive L-phenylalanyl-L-phenylalanyl (Phe-Phe) unit of the substrates tested. The pepsin inhibitor pepstatin can displace substrates such as Mns-(Gly)n-Phe-Phe-OR or Gly-Gly-Phe-Phe-NHNH-Mns from the active site of porcine pepsin; in these circumstances the mansyl group is bound weakly at a separate, nonspecific locus, distinct from the active site, which can accept the mansyl group of Mns-Gly-Gly-OR or mansylamide. In the interaction with substrates such as Mns-(Gly)n-Phe-Phe-OR or Dns-(Gly)n-Phe-Phe-OR, the above conclusions for porcine pepsin also apply to Rhizopus-pepsin. With substrates such as Gly-Gly-Phe-Phe-NHNH-Mns, however, the active site of Rhizopus-pepsin shows less affinity for the fluorescent probe group than does that of porcine pepsin, suggesting structural differences between the two acid proteinases in the region of their extended active sites which bind the COOH-terminal portion of small oligopeptide substrates.


The structural basis for the resistance of Escherichia coli formylmethionyl transfer ribonucleic acid to cleavage by Escherichia coli peptidyl transfer ribonucleic acid hydrolase. Escherichia coli formylmethionly-tRNA-tMet is unique among N-acylaminoacyl-tRNAs in its resistance to cleavage by peptidyl-tRNA hydrolase. Chemical modification of tRNA-fMet with sodium bisulfite converts fMet-tRNA-fMet into a good substrate for the hydrolase. The products of the enzymatic cleavage are free tRNA-fMet and formylmethionine. Bisulfite treatment produces cytidine to uridine base changes at several sites in the tRNA structure. One of these modifications results in formation of a new hydrogen-bonded base pair at the end of the acceptor stem of tRNA-fMet. We have shown that this modification is responsible for the observed change in biological activity. Enzymatic cleavage appears to be facilitated by the presence of a 5-terminal phosphate at the end of a fully base-paired acceptor stem, because removal of the 5-phosphate group from N-acetylphenylalanyl-tRNA-Phe or bisulfite-modified fMet-tRNA-FMet reduced the rate of hydrolysis of these substrates. The unpaired base at the 5 terminus of unmodified fMet-tRNA-fMet appears to reduce susceptibility of the tRNA to hydrolytic attack both by positioning the 5-phosphate in an unfavorable orientation and by directly interfering with enzymatic binding. The unusual structure of the acceptor stem of this E. coli tRNA thus plays a critical role in maintaining the viability of the organism by preventing enzymatic cleavage of the fMet group from the bacterial initiator tRNA.


Subunit interactions in aspartate transcarbamylase. The interaction between catalytic and regulatory subunits and the effect of ligands. The interaction between the catalytic subunit (c3) and the regulatory subunit (r2) of aspartate transcarbamylase from Escherichia coli was studied by measuring the reversible formation of the c3r6 complex as a function of r2 concentration. Conversion to the native enzyme was prevented by using a very low concentration of c2 (40 ng per ml) in the presence of bovine serum albumin. A simple hyperbolic r2 saturation curve was obtained suggesting the presence of only one kind of c:r domain. From the association constant for the formation of c3r6, the free energy of c:r interaction can be estimated to be about -10 Cal per mole. Neither CTP nor ATP appears to affect the strength of c:r interaction in this complex. Succinate in the presence of carbamyl phosphate promotes tighter binding. At higher concentration of c3 and nonsaturating levels of r2, conversion to the native enzyme (c3r6) takes place. This renaturation process is second order with respect to the concentration of c3 and is virtually irreversible. Renaturation is inhibited by saturating levels of r2 and to some extent by both CTP and ATP. The effect of ligands on c:r interactions reported here may have significance in the allosteric mechanism of the native enzyme.


Subunit interactions in aspartate transcarbamylase. A model for the allosteric mechanism. The conformational changes in aspartate transcarbamylase upon binding of substrates or regulatory ligands and the effects of alterations in the subunit structure on the allosteric interactions are reviewed. The available information including recent results from studies of the c3r6 complex (c denotes the catalytic polypeptide and r, the regulatory polypeptide) is considered in terms of the existing models for the discrepancies between experimental observations and the present models could be resolved by postulating an important role for r:r interactions in the allosteric mechanism. A new model is presented in which an obligatory conformational change upon binding of substrates results in an alteration in the relative orientation of c versus r. As a consequence of symmetry conservation, the r:r domain is shifted to a position of higher potential energy. By favoring one or the other alternative r:r domains, CTP and ATP can respectively enhance and reduce the sigmoidal character of substrate saturation. The model is shown to be consistent with all of the important known properties of the enzyme. Because the heterotropic effects of CTP or ATP are postulated to operate via a mechanism separate from that for the homotropic effects of the substrates, this model accounts satisfactorily for the observation by Kerbiriou and Herve (Kerbiriou, D., and Herve, G. (1973) J. Mol. Biol. 78, 687-702) that homotropic effects can be abolished whereas heterotropic effects are retained in the altered enzyme from Escherichia coli grown in the presence of 2-thiouracil.


Ethidium binding affinity of circular lambda deoxyribonucleic acid determined fluorometrically. Ethidium-binding isotherms for purified circular lambda DNA, isolated from a superinfected lysogen, and for linear lambda DNA, isolated from the purified phage, were constructed from fluorescence measurements of ethidium-DNA MIXTURES. The measurements were made in 0.01 M Tris-HC1-0.001 M EDTA,pH 7.1, buffer at 20 degrees and in the same buffer containing 0.1, 0.4, or 1.0 M NaC1. When NaC1 was present, differences in the binding affinity for supercoiled and linear DNA could be quantitated. As the ethidium concentration was increased, supercoiled lambda DNA molecules bound the intercalating dye first more and then less avidly than nonsupercoiled ones. The number of potential supercoils in a circular lambda DNA molecular in the absence of dye was calculated from the amount of dye bound when it exhibited the same affinity for dye as its linear counterpart. The point of equivalent affinity shifted from 0.053 mol of dye bound per mol of nucleotide in 0.1 M NaC1 to 0.067 mol in 1.0 M NaC1. This corresponds to the removal of 164 and 206 supercoiling turns per molecule and superhelix densities in the absence of dye equal to 0.036 and 0.045 superhelical turns per 10 base pairs. If this difference in the number of supercoils reflects a salt-dependent change in the average rotation angle between base pairs of the Watson-Crick helix the angle differs by 0.32% in the two ionic environments.


Purification of closed circular lambda deoxyribonucleic acid and its sedimentation properties as a function of Sodium chloride concentration and ethidium binding. The sedimentation of circular lambda DNA suggests that the molecular undergoes significant changes in shape and super-coiling as the NaC1 concentration increases. Closed circular lambda DNA, species I, isolated and purified from superinfected immune bacteria, sediments in sucrose gradients of low ionic strength at a rate 2.0 times faster than linear lambda DNA, species III. The addition of ethidium causes the sedimentation rate of species I DNA to decrease until enough dye is bound to remove 121 supercoils per molecule. At this point, species I co-sediments with nicked and nonsupercoiled species II. Futher additions of ethidium cause the sedimentation rate to increase until the relative rate of species I is again at least twice that of species III. This classical behavior is altered when NaC1 is present in the buffer. In 1.0 M NaC1 the changes in S are complex. Initially, species I sediments 1.55 times faster than species III. Titration with ethidium caused a decrease in S to an early minimum value, than an increase to a first maximum, followed by a decrease to the S of species II. At this point enough dye has intercalated to remove 208 superhelical turns. Further additions of dye introduce supercoils and cause S to increase again. In 0.1 to 0.4 M NaC1 the relative S of species I is 1.69 and 1.59, respectively. If titrated with ethidium, S first increases to a maximum value then decreases to the minimum rate when enough dye is bound to remove 158 and 183 supercoils, respectively. The results indicate an increase in the superhelix density from 0.026 turns per 10 base pairs in buffer alone to 0.045 in the same buffer with 1.0 M NaC1. If this change in superhelix density results from a concomitant change in the average rotation angle between base pairs in the Watson-Crick helix, the addition of 1.0 M NaC1 alters the rotation angle by 0.68 degrees per base pair.


The nucleotide sequence in the promoter region of the fene for an Escherichia coli tyrosine transfer ribonucleic acid. The sequence of 29 nucleotides in the region preceding the initiation of the transcription of the Escherichia coli tyrosine tRNA gene has been determined. This is: (5')  ---GGGGCGCATCATATCAAATGACGCGCCGC--- (3') (3') ---CCCCGCGTAGTATAGTTTACTGCGGCGGCG--- (5'). The general approach used for the sequence determination involved the DNA polymerase I-catalyzed elongation of suitable deoxyribopolynucleotide primers when hybridized to the 1-strand of phi80psuIII plus DNA at the appropriate site. Sequences of the newly grown oligonucleotide chains were determined by a combination of two-dimensional fingerprinting following partial exonucleolytic degradation, nearst neighbor analyses, and determination of pyrimidine tracts. Primer elongations were carried out in a controlled and stepwise manner and the newly grown oligonucleotide chains were kept short by incorporating the following features into the method: (a) the insertion of a ribonucleotide unit at or near the 3' terminus of the primers; (b) the use of a maximum of three nucleoside 5'-triphosphates in the first stage of the elongation reaction isolation of the elongated primer, and its reuse in a second step together with different sets of deoxy-nucleoside triphosphates; and (c) elongation of the primer using all of the four nucleoside triphosphates with one of the triphosphates being supplied in a limiting concentration.


Degradation of abnormal proteins in Escherichia coli. Formation of protein inclusions in cells exposed to amino acid analogs. Cells of Escherichia coli selectively degrade proteins that have incorporated amino acid analogs. Within 1 hour after exposure of cells to canavanine, 50% of the analog-containing proteins were degraded to acid-soluble form. At the same time, no net loss of canavanine-containing protein occurred from the 100,000 X g supernatant. Instead, most of the proteins containing the analog, unlike normal ones, accumulated in particulate fractions sedimenting at 10,000 X g or 100,000 X g. They were then lost from these fractions concomitant with the degradation of the abnormal proteins. The loss of such proteins from particulate fractions accounted for all of the protein degraded to acid-soluble form. Similar observations were obtained after incorporation of other analogs or puromycin. The 10,000 X g pellets correspond to amorphous dense intracellular granules visible in electron micrographs of cells exposed to canavanine. Upon removal of the analog, these granules disappeared, simultaneously with the degradation of the analog-containing proteins. These pellets do not resemble a degradative organelle, like the lysosome; they are not osmotically sensitive, do not exclude inulin, are not enclosed by a membrane, and do not show autolytic activity. The proteins in the granules could be solubilized by sodium dodecyl sulfate but not by Triton, NaC1, dithiothreitol, RNase, DNase, or phospholipase. The proteins extracted from the pellet with sodium dodecyl sulfate tend to become particulate again upon removal of this detergent. Incorporation of canavanine caused a normally soluble polypeptide, the monomer of beta-galactosidase, to be inactive and found in the sedimentable fraction. These findings suggest that (a) the presence of amino acid analogs in proteins can make them less soluble, and (b) the inclusions are formed by the spontaneous precipitation of abnormal proteins rather than by an active granule-forming process.


Identification of a ribosomal protein necessary for thiostrepton binding to Escherichia coli ribosomes. A ribosomal protein necessary for thiostrepton binding to Escherichia coli ribosomes has been identified using the following criteria: 1. A loss in the thiostrepton binding ability of the ribosome was correlated with the selective removal of ribosomal protein L11. This was achieved by a comparison of the thiostrepton binding ability of 50 S ribosomal subunits treated with 1 M NH4C1 and 50% ethanol at 37 degrees which still contained protein L11, and subunits treated successively at 0 degrees and 37 degrees in the same medium, from which protein L11 had been removed. 2. The thiostrepton binding ability of a ribosomal core containing only seven proteins, produced by treatment of 50 S subunits with 4 M LiC1, was fully restored by the rebinding of protein L11, obtained by Sephadex G-100 fractionation of the 1 M LiC1 split protein fraction from 50 S subunits. In addition, treatment of the 1 M LiC1 split protein fraction with an IgG specific for protein L11, uniquely inhibited the restoration of activity. 3. Thiostrepton binding to the 4 M LiC1 core, reconstituted with the 1 M LiC1 split protein fraction, was blocked by treatment with a monovalent antibody fragment (Fab) prepared against protein L11, but not by treatment with antibodies specific for the proteins of the 4 M LiC1 core. We conclude, therefore, that protein L11 is required for the ribosomal binding of thiostrepton.


Assembly of ribosomal proteins L7, L10, L11, and L12, on the 50 S subunit of Escherichia coli. We have determined the in vitro assembly sequence of ribosomal proteins L7, L10, L11, and L12 on Escherichia coli 50 S subunits by reconstitution experiments with the use of various ribosomal core particles and split protein fractions produced by treatment of 50 S subunits with 1 m NH4Cl and 50% ethanol. Proteins L7, L10, L11, and L12 were removed by a two-step treatment, first at 0 degrees, then at 37 degrees. Small amounts of proteins L1, L5, and L6 were also removed under these conditions. A one-step extraction of 50 S subunits at 0 degrees removed only proteins L7 and L12, while a similar one-step extraction of intact 50 S subunits at 37 degrees removed proteins L7, L12, and L10. Two-dimensional gel electrophoresis of the protein components and measurement of the ribosome-elongation factor G-guanosine diphosphate complex formed with the various reconstituted particles showed that the binding of proteins L7 and L12 is dependent on the binding of protein L10 and in turn, that the binding of protein L10 is dependent on the binding of protein L11.


Initiation and transcription of a set of transfer ribonucleic acid genes in vitro. Using RNA polymerase purified from Escherichia coli, DNA isolated from the bacteriophage T4, and a bacterial supernatant fraction containing the necessary processing enzymes, a set of transfer RNAs can be formed in vitro. To characterize the site or sites of initiation of this tRNA transcription, rifampicin-resistant complexes of RNA polymerase, DNA, and either ATP (UTP and CTP) or GTP (UTP and CTP) were formed, and tRNA was transcribed from these stabilized sites. It is concluded that transcription of the entire set is initiated by ATP. To study the transcription of the tRNAs, the time sequence of the appearance of individual species was determined during synchronous transcription of a preformed RNA polymerase-DNA complex. The appearance of three RNA species is found to be consistent with the sequential transcription of a large polycistronic cluster; the order and distances, inferred from the times of transcription, are as required by the existing gene map. It is concluded that the initiation of tRNA transcription can occur, without accessory factors, with the insertion of ATP at a single or a few closely spaced sites, and that the tRNAs encoded by the bacteriophage T4 are present in a single operon.


Chemical modification of yeast 3-phosphoglycerate kinase. Sulfhydryl reagents, as well as mild hydrogen peroxide oxidation, do not inhibit the activity of yeast phosphoglycerate kinase, indicating that the single thiol group and 3 methionine residues present in the enzyme are not essential for activity. Nitration of phosphoglycerate kinase by tetranitromethane inhibits the enzyme by reaction with a single tyrosine residue. Substrates provide partial protection against inactivation by nitration. Circular dichroism spectra indicate that no conformational changes occur upon nitration. However, perturbation of the microenvironment surrounding the aromatic amino acid residues, particularly tyrosine, was observed. The same perturbation was observed on addition of the substrate 3-phosphoglycerate kinase to native phosphoglycerate kinase. The role of lysine in the action of yeast phosphoglycerate kinase has been studied by modification with O-methylisourea, 2-methoxy-5-nitrotropone, and pyridoxal phosphate. Guanidination shows that there are lysines essential for phosphoglycerate kinase; extrapolation to zero activity indicates that there are three essential lysines as judged by nitrotroponylation and three essential lysines when the enzyme is reacted with pyridoxal phosphate. Substrates afford partial protection and extrapolation to total protection indicates that up to three lysines are protected by MgITP and one lysine by 3-phosphoglycerate. Spectrofluorescence and optical rotatory dispersion measurements show that there is no detectable conformational change for the guanidinated phosphoglycerate kinase and that there are slight changes in the spectra suggesting that there may be slight conformational changes for the nitrotroponylated and the pyridoxal phosphate-modified enzymes.


Energy-dependent binding of dansylgalactosides to the beta-galactoside carrier protein. Fluorescent beta-galactosides (1-(N-dansyl)amino-beta-D-galactopyranoside (DG0), 2'-(N-dansyl)aminoethyl-beta-D-thiogalactopyranoside (DG2), 2'-(N-dansyl)aminoethyl-beta-D-galactopyranoside (oxy-DG2), and 6'-(N-dansyl)aminohexyl-beta-D-thiogalactopyranoside (DG6)) competitively inhibit lactose transport by membrane vesicles from Escherichia coli ML 309-225, but are not actively transported. An increase in the fluorescence of these dansylgalactosides is observed upon addition of D-lactate, imposition of a membrane diffusion potential (positive outside), or dilution-induced, carrier-mediated lactose efflux. The increase is not observed with 2'-(N-dansyl)aminoethyl-beta-D-thioglucopyranoside nor with membrane vesicles lacking the beta-galactoside transport system. Moreover, the D-lactate-induced fluorescence increase is blocked or rapidly reversed by addition of beta-galactosides, sulfhydryl reagents, inhibitors of D-lactate oxidation, or uncoupling agents. The fluorescence increase exhibits an emission maximum at 500 nm and excitation maxima at 345 nm and at 292 nm. The latter excitation maximum is absent unless D-lactate is added, indicating that the bound dansylgalactoside molecules are excited by energy transfer from the membrane proteins. Titration of vesicles with dansylgalactosides in the presence of D-lactate demonstrates that the lac carrier protein constitutes 3 to 4% oof the total membrane protein, and that the affinity of the carrier for substrate is directly related to the length of the alkyl chain between the galactosidic and the dansyl moieties of the dansylgalactosides. In addition, there is excellent agreement between the affinity constants of the various dansylgalactosides as determined by fluorimetric titration and their apparent Kis for lactose transport (KDs and/or apparent Kis are approximately 550, 3o, 40, and 5 muM FOR DG0, DG2, oxy-DG2, and DG6, respectively). Polarization of fluorescence measurements with DG2 and DG6 demonstrate a dramatic increase in polarization on addition of D-lactate which is reversed by addition of lactose or anaerobiosis. These findings provide strong evidence for the contention that the fluorescence changes observed on "energization" of the membrane are due to binding of the dansylgalactosides per se, rather than binding followed by transfer into the hydrophobic interior of the membrane


Photoinactivation of the beta-galactoside transport system in Escherichia coli membrane vesicles with 2-nitro-4-azidophenyl-1-thio-beta-D-galactopyranoside. 2-Nitro-4-azidophenyl-1-thio-beta-D-galactopyranoside (azidophenylgalactoside) is a competitive inhibitor of lactose transport in membrane vesicles isolated from Escherichia coli ML 308-225, exhibiting an apparent Ki of 75 muM. The initial rate and steady state level of [3H]azidophenylgalactoside accumulation are markedly stimulated by the addition of D-lactate to vesicles containing the lac transport system, and kinetic studies reveal an apparent Km of 75 muM. Membrane vesicles devoid of the lac transport system do not take up significant amounts of azidophenylgalactoside in the presence or absence of D-lactate. When exposed to visible light in the presence of D-lactate, azidophenylgalactoside irreversibly inactivates the lac transport system. Strikingly, photolytic inactivation is not observed in the absence of D-lactate. Kinetic studies of the inactivation process yield a KD of 77 muM. Since lactose protects against inactivation and azidophenylgalactoside does not inactivate amino acid transport, it is apparent that these effects are specific for the lac transport system. The results are consistent with the proposal that the lac carrier protein is inaccessible to substrate in the absence of energy coupling.


Accumulation of lipid-soluble ions and of rubidium as indicators of the electrical potential in membrane vesicles of Escherichia coli. We have studied the accumulation of dibenzyldimethyl-ammonium ion (DDA+) by respiring membrane vesicles of Escherichia coli, as an index of the generation of an electrical gradient during respiration. Nonrespiring vesicles accumulated DDA+ when K+ efflux was induced by valinomycin or monactin. By various criteria this was shown to be the exchange of one cation for another, independent of metabolism and coupled entirely by electrical forces. Uptake of DDA+ by respiring vesicles was inhibited by ionophores that translocate electrical charge and by reagents that block the respiratory chain. Oxamate and p-chloromercuribenzoate inhibited accumulation of DDA+ but did not dissipate a preformed pool; the reason appears to be that these reagents are less inhibitory to transport after lactate oxidation has begun than they are in resting vesicles. Uptake does not appear to involve a biological carrier, but requires trace amounts of a lipid-soluble anion such as tetraphenylboron, which has a catalytic role in DDA+ translocation. Respiring K+ vesicles accumulated substantially less DDA+ than did Na+ vesicles. Na+ was expelled from the vesicles concurrently with DDA+ uptake, whereas Rb+ and K+ were not. Thus, DDA+ uptake, whereas Rb+ and K+ were not. Thus, DDA+ uptake may be limited in the latter case by the availability of anionic groups. This explanation was supported by the finding that the addition of nigericin doubled the capacity of K+ vesicles to take up DDA+, presumably by providing a route for K+ to exit in exchange for H+. Parallel experiments on the valinomycin-dependent accumulation of Rb+ by respiring vesicles indicate that this process is analogous to the uptake of DDA+. Ionophores that elicit electrogenic K+ movement also induced respiration-linked transport. Proton-conducting ionophores and several inhibitors of respiration block Rb+ uptake and dissipated a preformed gradient. Preincubation of the vesicles with oxamate or p-chloromercuribenzoate inhibited Rb+ uptake, but their addition to respiring vesicles again did not cause efflux. Rb+ and DDA+ complete for uptake when present simultaneously. We conclude that the accumulation of both DDA+ and Rb+ occurs in response to an electrical gradient, vesicle interior negative, produced by respiration.


Partial purification and properties of an endoribonuclease isolated from human KB cells. With the use of a precursor to Escherichia coli tRNA-Tyr as a substrate, we have detected and partially purified a novel endoribonuclease from the cytoplasm of human KB tissue culture cells. This activity, which we have called RNase NU, cleaves the tRNA precursor at two sites in that part of the molecule which is not included in the mature tRNA sequence and which is normally degraded in vivo. In keeping with this observation, we have found that, of a variety of substrates tested, only those which are unstable in vivo are attacked by RNase NU. RNase NU can be purified from the 0.2 M NH4Cl wash of ribosomes followed by ammonium sulfate fractionation and DEAE-Sephadex chromatography. RNase NU cleaves RNA to create 3'-phosphate-terminated oligonucleotides. It has a pH optimum near 8.0, requires either a monovalent cation (NH4+ is most efficient) or Ca-2+ for optimal activity, and is inhibited by 0.1 M PO4-3-. In the course of purifying RNase NU we have detected and studied the intracellular distribution of other ribonuclease activities in human KB cells.


Characterization of ribonuclease NU cleavage sites in a bacteriophage phi80-induced ribonucleic acid. Ribonuclease NU, an endoribonuclease isolated from human KB tissue culture cells, can cleave a bacteriophage phi80-induced RNA at four distinct sites. Nucleotide sequence analysis of the eight cleavage products has shown that the enzyme produces oligonucleotides terminating in 3'-phosphate groups, and that the four cleavage sites are in the only nonhydrogen-bonded region of the substrate. Various aspects of the cleavage reaction with this RNA and with other substrates are discussed.


Purification and properties of a soluble factor required for the deoxyribonucleic acid-directed in vitro synthesis of beta-galactosidase. The DNA-directed in vitro synthesis of beta-galactosidase has been investigated in a system dependent on Escherichia coli ribosomes, a salt wash of the ribosomes, and a supernatant fraction. Fractionation of the supernatant has made it possible to obtain dependencies on RNA polymerase and another protein factor for beta-galactosidase synthesis. The other factor (called L factor) cannot be replaced by a variety of proteins known to be required for transcription and translation. It has been purified to homogeneity and has a molecular weight of approximately 65,000. Although it is required for the in vitro synthesis of beta-galactosidase, it has no effect on total DNA-dependent amino acid incorporation under the conditions of the incubation. However, total RNA synthesis is depressed by the addition of L factor in a manner similar to what is observed with rho factor could not replace L factor in beta-galactosidase synthesis.


Threonine deaminase from Salmonella typhimurium. Relationship between regulatory sites. Kinetic analysis of the biosynthetic threonine deaminase, EC 4.2.1.16, from Samonella typhimurium yields hyperbolic substrate saturation curves in the absence of, and higher order substrate saturation curves in the presence of, L-isoleucine. L-Valine reverses this effect of L-isoleucine by restoring the hyperbolic substrate saturation curves. The inhibition of enzyme activity and the reversal of valine stimulation is a function of a second order concentration of L-isoleucine, whereas antagonism of inhibition is a function of first order concentration of valine. The antagonistic effects on enzyme activity of L-isoleucine and of L-valine appear as competitive in diagnostic plots. Threonine deaminase possesses two L-isoleucine binding sites (Kd equals 3.6 muM) and one L-valine binding site (Kd equals 26 muM); the binding of these ligands appear competitive. Exclusion of L-valine requires the binding of 2 molecules of L-isoleucine whereas binding of a single L-valine molecule prevents the binding of 2 L-isoleucine molecules. Cooperative binding of L-isoleucine is not observed under any of the conditions tested. Two cases, expressed in terms of modified Adair equations and based upon the assumption that L-threonine also serves as an activator ligand which binds to the L-valine site, are presented. Case I states that liganding of the activator sites must percede substrate-binding at the active site, and Case II states that the activator site liganding is required solely for reactivation of the L-isoleucine-inhibited enzyme. Analysis of kinetic data by a curve-fitting process suggests that Case II described the relationship between the activator site and the L-isoleucine sites. An enzymatically inactive derivative of threonine deaminase, prepared by reduction with borohydride, binds isoleucine and valine in a manner similar to native holoenzyme. Binding of L-threonine and L-valine to the derivatized enzyme is competitive. The Kd for threonine binding is 3 mM, which is in excellent agreement with the Kd determined by the curve fitting process. It is concluded that the modulation of threonine deaminase activity is wrought by interaction between inhibitor sites and an activator site rather than inhibitor and active sites and that induced transitions rather than concerted transitions more adequately describe the underlying regulatory principle.


Investigations concerning the mode of action of 3,4-dihydroxybutyl-1-phosphonate on Escherichia coli. Experiments were performed to evaluate the ability of the enzymes of Escherichia coli involved in glycerol 3-phosphate metabolism to recognize phosphonic acid analogues of the natural substrate. Neither the catabolic membrane-bound glycerol-3-phosphate dehydrogenase nor the acyl coenzyme A: glycerol-3-phosphate acyltransferase can use 3,4-dihydroxybutyl-1-phosphnate or 2,3-dihydroxypropyl-1-phosphonate are inhibitors of the reduction of dihydroxyactone phosphate as substrates. The 4-carbon phosphonic acid analogue does not exhibit inhibitory activity for either of these enzymes. While the 3-carbon phosphonic acid analogue has no inhibitory effect upon the catabolic dehydrogenase, it does appear to have a slight but reproducible inhibitory effect on the acyltransferase. Glycerol 3-phosphate and 3,4-dihydroxybutyl-1-phosphonate by glycerol 3-phosphate:NAD (P) oxidoreductase. rac-2,3-Dihydroxypropyl-1-phosphonate does not appear to be recognized by this enzyme. The apparent K-i for snglycerol 3-phosphate is 19 muM and for D-3,4-dihydroxybutyl-1-phosphonate it is 42 muM. In addition the glycerol 3-phosphate:NAD(P) oxidoreductase catalyzes the reduction of 4-hydroxy-3-oxobutyl-1-phosphonate (apparent K-m of 182 muM), a phosphonic acid analogue of dihydroxyacetone phosphate. 3,4-Dihydroxybutyl-1-phosphonate is both a competitive inhibitor (apparent Ki of 740 muM) and a substrate (apparent K-m of 450 muM) for the CDP-diglyceride: glycerol 3 phosphate phosphatidyltransferase but it has no effect upon CDP-diglyceride:L-serine phosphatidyltransferase. The relationship ofthese in vitro studies to in vivo investigations is discussed.


Studies on tyrosine phenol lyase. Modification of essential histidyl residues by diethylpyrocarbonate. Tyrosine phenol-lyase of Escherichia intermedia is inactivated by treatment with diethylpyrocarbonate at pH 6.0 AND 4 degrees. Spectrophotometric studies show that the inactivation is stoichiometric, with a modification of 2 histidyl residues per molecule of the enzyme. Finding that this inactivation is largely reversed by treatment with hydroxylamine indicates that the inactivation is mainly due to modification of the histidyl residues. No changes in the sulfhydryl content or in the aromatic amino acids are observed as a result of this modification. The modified tyrosine phenol-lyase retains most of its ability to form a nearly normal complex with its coenzyme, pyridoxal phosphate. This has been shown by studies of its absorption, by the determination of pyridoxal phosphate, and by reduction of the holoenzyme with tritiated sodium borohydride. The modified enzyme also appears to form a Schiff base intermediate with L-alanine. The modified holoenzyme fails to catalyze the exchange of the alpha-hydrogen of L-alanine with tritium from tritiated water. This is consistent with a catalytic role for modified histidyl residues at the active site of the enzyme; this role is the removal of the alpha-hydrogen of substrates.


N-Acetylglutamate synthase of Escherichia coli regulation of synthesis and activity by arginine. N-Acetylglutamate synthase, the first enzyme of arginine biosynthesis, was stabilized in crude extracts from Escherichia coli. At 4 degrees the enzyme lost less than 5% of activity per day. L-Arginine repressed the formation of N-acetylglutamate synthase. Under conditions of genetic or physiological derepression, a specific activity of approximately 50 nmol per min per mg of protein was measured. No activity (i.e. less than 0.2 nmol per min per mg of protein) could be detected in extracts from cells grown under conditions of repression, whereas an intermediate level was found in cell cultivated on minimal medium. In a 6-fold purified preparation L-arginine inhibited the enzyme. Of 11 precursors and analogues of arginine tested only O-[L-norvalyl-5]-isourea inhibited N-acetylglutamate synthase as strongly as L-agrinine.


The fate of ribosomes in Escherichia coli cells starved for a carbon source. The disappearance of ribosomes in Escherichia coli cells starved for a carbon source was studied. We used a series of mutants, some of them lacking in ribonuclease I(RNase I, EC 2.7.7.17), and other containing various combinations of modified polynucleotide phosphorylase (PNPase, EC 2.7.7.8) and modified ribonuclease II (RNase II, EC 3.1.4.1). RNA was prepared from the starved mutant cells and separated on polyacrylamide gels. The results obtained indicate that 23 S RNA degradation is similar in all strains that lack RNase I, and is slightly increased in the strain that contains this enzyme. The extent of 16 S RNA degradation is identical in all strains tested. RNA species in the size of 4 S and smaller accumulate in mutants containing modified forms of PNPase and RNase II. The appearance of an RNA species 10% smaller than 16 S RNA (d16 S RNA) was observed in all strains that contain unmodified RNase II. Analysis of ribosomes and polysomes and their RNA content indicated that polysomes are converted to monosomes and these, in turn, to ribosomal subunits. No RNA degradation products were found in polysomes, 70 S, OR 50 C particle; 30 S subunits contained 16 S RNA as well as the d16 S RNA species. Subunits are degraded to a similar extent in all strains lacking RNase I, and at a slightly faster rate in the strain that contains RNase I. The RNA to protein ratio in subunits prepared from starved cells is similar to that of unstarved cultures. Very little degradation of ribosomal proteins occurs in these mutants during carbon starvation. The proteins released from degraded ribosomes are found in the fast sedimenting (20,000 times g) pellet. Cell viability studies indicated a direct correlation between the capacity of the mutants to recovery from starvation and their capacity to degrade RNA. Thus a biological necessity for degradation of ribosomes during starvation is implied. Based on these data we propose that the endonucleolytic degradation of ribosomal RNA is the primary event in starvation degradation. It takes place in ribosomal subunits, which fall apart after the endonucleoltic attack. The RNA pieces produced by this cleavage are degraded to nucleotide by RNase II and PNPase. The ribosomal proteins attach to the cell membrane.


A kinetic study of dihydrolipoyl transacetylase from bovine kidney. The mammalian pyruvate dehydrogenase complex contains a core, consisting of dihydrolipoyl transacetylase, to which pyruvate dehydrogenase and dihydrolipoyl dehydrogenase are joined. This report describes studies on the kinetic mechanism of the transacetylase-catalyzed reaction between [1-14C]acetyl-CoA and dihydrolipoamide. This reaction appears to be a model of the physiological reaction, in which the acetyl group is transferred from the S-acetyldihydrolipoyl moiety, bound covalently to the transacetylase, to CoA. The model reaction is not affected by pyruvate dehydrogenase or dihydrolipoyl dehydrogenase, their substrates and products, or by removal of the covalently bound lipoyl moiety. These findings, together with the results of initial velocity, product inhibition, and dead-end inhibition studies, indicate that the model reaction and, apparently, the physiological reaction as well, proceeds via the Random Bi Bi (rapid equilibrium) mechanism. It appears that at the catalytic center of the transacetylase there are two adjacent sites, one that binds CoA and acetyl-CoA and another that binds dihydrolipoamide and S-acetyldihydrolipoamide (or the corresponding forms of the covalently bound lipoyl moiety.


Angiography in the diagnosis of osteoid-osteoma of the hand. Angiographic findings characteristic of osteoid-osteoma are described in two cases of osteoid-osteoma of the bones of the wrist. While the etiology of the disease is not understood, a developmental relationship between the osteoid-osteoma nidus and increased vascularity is suggested.


Pelvic displacement osteotomy for chronic hip dislocation in myelodysplasia. Twelve children with lumbar-level myelodysplasia (average age, eight and three-quarter years) underwent twenty-one pelvic displacement osteotomies for subluxated or dislocated hips. Nineteen of the twenty-one hips remained reduced on three-year follow-up. Gains in gait pattern, ease of bracing, and reduced pelvic obliquity were noted. Active function about the hips was not improved, nor was there a decrease in the amount of bracing needed following the osteotomy. Pelvic displacement osteotomy can be utilized in selected cases as part of the over-all management of chronic hip dislocation in myelodysplasia.


The immunogenicity of fresh and frozen allogeneic bone. Both fresh and frozen allogeneic bone elicit both acellular and humoral immune response. This response includes the development of enhancing factors which block detectable immunity and probably protect the graft from rejection. There seems to be no evidence of an alteration in immunogenicity by freezing of the graft. The importance of these observations lies in the potential technique of employing fresh viable allografts; prior freezing and tissue matching for HL-A transplantation antigens should not be necessary.


Physical and biological aspects of repair in dog cortical-bone transplants. The amount of repair and the time required to accomplish repair of four-centimeter segmental fibular transplants in twenty-one male adult dogs were determined at from two to forty-eight weeks after transplantation by torsional stress testing, microradiography, and tetracycline labeling. The transplanted cortical bone was greatly weakened at from six weeks to six months but was nearly normal at one year. The strength of the transplant appeared to be related to the amount of porosity of the matrix rather than to the quality or completeness of biological repair. Spatially, the repair was ordered rather than random. The initial resorption caused increased porosity which was slowly offset by apposition of new bone, a process which was dependent on general skeletal metabolic activity. Although physical strength was near normal at forty-eight weeks, only 60 per cent of the transplants had been remodeled.


Neglected rupture of the Achilles tendon. Treatment by V-Y tendinous flap. A method is described that achieves end-to-end anastomosis of ruptures of the Achilles tendon. A sliding tendinous flap is developed over the proximal portion of the tendon by making an inverted V incision which is then repaired in a Y fashion. The procedure is recommended for patients with neglected ruptures of the Achilles tendon in which end-to-end anastomosis is otherwise impossible.


The torn shoulder capsule.

The torn shoulder capsule. Tears of the shoulder capsule attended by retraction of the edges of the tear, inability to close the gap, and pathological changes in the biceps tendon may be very effectively managed by: (1) a transacromial exposure with a V-shaped osteotomy and subsequent replacement of the acromion with bone screws, and (2) standard repairs of the shoulder capsule after the biceps tendon has been reseated in a newly constructed, more laterally located groove in the head of the humerus. The follow-up results document the value of these procedures done on a group of fourteen patients.


Observations on the male nucleus during fertilization in the fern Pteridium aquilinum. Fertilization of eggs of Pteridium under controlled conditions has revealed that archegonia containing viable eggs require more than 30 min flooding before opening. Syngamy appears to take place by an engulfing action, and no evidence was found of a 'membrane-fusion' kind of conjugation. Although the gametic nucleus lacks a recognizable envelope, as soon as it comes into contact with the female cytoplasm an envelope is reconstituted from an opaque layer at the surface of its chromatin. This membrane also undergoes growth, the outer layer extending into the cytoplasm and surrounding spermatozoid mitochondria. The male envelope remains distinct from the female, and lacks evaginations and well defined pores. The male nucleus lies in irregular loops within a funnel, filled with egg cytoplasm, formed by the microtubular ribbon of the gamete. The mouth of the funnel is bordered by the multi-layered structure. Karyogamy is initiated at the anterior end of the male nucleus, the orifice connecting the 2 nuclei widening from about 0-1 to 2 mum in diameter during fusion. The contents of the male nucleus flow through the mouth of the funnel into the female, the chromatin relaxing locally, but retaining a rod-like form. The envelope of the male nucleus is probably resorbed at the site of junction. There is no evidence that it contributes significantly to the envelope of the zygotic nucleus.