Influence of repeated local stimulation on the splenic response to mucosal and parenteral immunization. It has been suggested that a regional immune response may be induced by local administration of small doses of antigen, whereas larger doses are capable of inducing not only a local but also a systemic immune response. Except with fluorescence, the study at the cellular level and in the mucosa of specific antibody-producing cells appears difficult. The circulation in the body of lymphocytes from the lamina propria of the gut allows the detection of antibody-producing cells in extra-intestinal lymphoid tissues. The administration of 4 times 10-9 sheep red blood cells (SRBC) by gastric intubation to BALB/c mice (repeated daily for 4 days) was found to cause the appearance in the spleen of plaque-forming cells which predominantly belonged to the IgA class and whose numbers reached a maximum at about day 8 or 9 after the onset of the immunization. This observation is relevant to splenic colonies from cells that had received their antigenic stimulation in the gut wall and thence had seeded out in the body. Booster administration of SRBC performed 3 months after the first antigenic contact induced in the spleen an immunological response similar to that observed in newly primed mice and in no way suggestive of immunologic memory. A secondary response is completely absent when the second intragastric immunization is initiated 10 days after the end of the first. The parenteral administration of 2 times 10-8 SRBC to mice primed by the oral route 15 days earlier is followed by a marked hyporeactivity of the spleen. In any case, the IgA system appears endowed with only a minimum, if any, immunological memory to non-living antigen. In some conditions local immunological stimulation may even cause tolerance.


Assessment of the efficacy of influenza vaccines against natural challenge. Assessment of influenza vaccines by serological conversion rates or by protection against artificial challenge in animals or man give only a preliminary and indirect guide to their expected efficiency in controlling the natural epidemic disease. Field trials are essential but demand careful advance planning and control before meaningful results can be obtained. This paper reviews critically the various attempts that have been made to establish an ideal trial procedure to overcome the many inherent difficulties in studying 'wild' influenza. Vaccines of appropriate antigenic content can prevent influenza in 75 percent or more of recipients for up to a year, and can decrease the severity and duration of the disease in those who do succumb. Attack rates can be reduced even in epidemics occurring within 8-14 days of vaccination. If a high proportion of a population is vaccinated, infection rates in the unvaccinated in the same environment may fall. The desirability of vaccinating (a) only high risk groups or (b) entire open population or (c) school children is discussed.


An attenuated influenza virus vaccine: reactogenicity, transmissibility, immunogenicity and protective efficacy. A more effective influenza virus vaccine must be capable of inducing a greater degree of protection against the disease while causing fewer unwanted side effects. If the vaccine is an attenuated one, it should not be transmissible unless it can incontrovertably be shown to be genetically stable and not mutate to a more virulent form. One approach for producing an attenuated strain has been to select for horse serum inhibitor resistance. Such a strain (ANN strain, Recherche et Industrie Thérapeutique) was tested for the above characteristics. The vaccine (containing 10-7 50 percent egg infectious doses) and placebo was administered in a double-blind fashion to 37 volunteers in 2 doses 2-3 weeks apart. Volunteers were housed so that a vaccinee and control shared a room for 4 days following immunization. The only side effects noted were mild rhinorrhea and sore throat in 3 vaccinees. There was no evidence of vaccine virus having been transmitted from a vaccinee to a control individual.A 4-fold or greater rise in serum antibody was noted in 13/19 vaccinees following either the 1st or 2nd dose. Only 1/18 controls showed such a rise. A similar nasal antibody rise was noted in 15/19 and 1/18 of the vaccine and placebo groups respectively. Following virulent challenge 7 of 18 in the control group, and 1 of 19 vaccinees, became ill.


The coronaviruses.

The coronaviruses. Coronaviruses of human origin have emerged as probable important etiologic agents of acute upper respiratory tract illnesses in adults; their role in the etiology of respiratory illnesses of childhood is less certain. The first coronavirus of human origin, B814, was described in 1965 and since that time only 31 additional strains have been recovered. Ten of these were originally recovered in human embryonic tracheal organ culturesonly, and the remainder in monolayer cell cultures. All of the latter group were antigenically related to a prototype strain designated 229e. As a result of the fastidious growth requirements of these agents, most of the knowledge concerning the clinical syndromes associated with them has come from chajlenge studies in volunteers and seroepidemiologic investigations. Coronaviruses have been shown to be associated with a substantial number of adult respiratory illnesses especially during certain periods when rhinovirus infections are infrequent. Progress in propagating coronaviruses and in detecting coronavirus infections has been achieved recently: viruses similar to 229E were successfully recovered in human embryonic intestine cell cultures from patients with upper respiratory tract illnesses; 30C strains (B814, LP and EVS) were recovered in L132 cell cultures from infectious nasal washings; OC43(and OC38) viruses which were originally recovered in OC and later adapted to grow in suckling mice were not only shown to directly hemagglutinate various erythrocytes but were also adapted to grow in MK cell cultures; OC43 virus was also found to induce hemadsorption of rat and mouse erythrocytes in certain cell cultures; and a coronavirus strain was detected by immune electron microscopy. Coronaviruses are also associated with certain diseases in animals.


Contagious bovine pleuropneumonia-protection following natural infection and vaccination. Cattle that recover from contagious bovine pleuropneumonia, the disease caused by Mycoplasma mycoides var. mycoides, are usually immune to reinfection. Prohylactic inoculation against the disease has been practiced for over a century and utilizes the fact that even virulent strains of M. mycoides inoculated subcutaneously into fully susceptible cattle only very rarely produce pneumonia, but instead produce subcutaneous lesions which may or may not kill the animals depending on various factors including the site of the inoculation and virulence of the strains. Animals that survive are immune to further local infections or to the natural disease. The most successful vaccines at present consist of live attenuated strains of M. mycoides which produce a slight local reaction at the site of inoculation. It appears that vaccines made from inactivated organisms are poorly immunogenic and may even sensitize the animals thereby causing them to react more severely to subsequent challenge.


The importance of mycoplasmas in respiratory infections. In contrast to the uncertain role of mycoplasmas in genital disease, there is no doubt that they produce respiratory disease. The first mycoplasma isolated, M. mycoidesis is the aetiological agent of bovine pleuropneumonia, a disease now confined to parts of Africa and Australia. M. capri causes a similar disease in goats which is geographically more widespread. Enzootic pneumonia of pigs, seen in various parts of the world, is due primarily to M. suipneumoniae. Although some strains of M. hyorhinis also seem capable of causing disease. Not all strains of a mycoplasma species are equally pathogenic; only some strains of M. pulmonis produce severe respiratory disease in mice and rats. Further, concomitant infection with other agents may result in more severe disease; this is particularly seen in M. gallisepticum infections of poultry. Of those mycoplasmas which infect man, only M. pneumoniae is known to cause respiratory disease, mainly involving the younger age groups. Mycoplasmas spread by close contact so that disease is seen in family groups or where there is crowding or herding. Factors in resistance to respiratory disease caused by mycoplasmas have been poorly understood but the importance of local immunity is becoming more clear.


Multiple metabolic functions of glucose in rat pancreatic islets. Metabolic interactions between glucose and amino acids were studied with isolated rat islets using glucose utilization and lactate formation as indicators. Certain amino acids (8-10 mM) are capable of greatly stimulating lactate formation from 5mM glucose. On a molar basis L-isoleucine is the most potent stimulator in a group of twenty-six amino acids. Aphysiological amino acid mixture (7.5-14 mM) or L-isoleucine (8 mM) profoundly altered the basic sigmoidal relation between glucose concentration in the medium and the rate of glucose utilization and lactate formation: with basal glucose (5 mM) both glucose utilization and lactate production were stimulated by the amino ACID MIXTURE and by L-isoleucine; at high glucose levels utilization was decreased by the amino acid mixture, but was unaffected by L-isoleucine, whereas lactate formation was decreased by both additions. The data indicate that amino acids may play a significant role in regulating the extent to which glucose serves as a fuel of pancreatic islet cells and in determining the pathways of glucose metabolism. In order to elucidate the mechanisms of the amino acid effect, studies with phloridzin, ouabain, iodoacetate, cytochalasin B, and Na+-deficiency were performed with the most effective amino acid, L-isoleucine. Each of these agents and Na+-deficiency substantially reduced or completely blocked the extra lactate formation induced by L-isoleucine (8-10 mM). The intracellular uptake of 14-CL-isoleucine by isolated islets was found to be Na+-independent, and uphill transport of this amino acid was not detectable, whether basal glucose was present in the medium or not. The action of iodoacetate in blocking glycolysis was reinvestigated. After forty-five minutes of exposure, 0.2mM iodoacetate completely blocks lactate formation as well as glucose utilization. Thisconfirms and extends earlier data for this laboratory and suggests that this SH-reagent indeed allows dissociation of the fuel and releasing functions of glucose.


Electrical and secretory manifestations of glucose and amino acid interactions in rat pancreatic islets. Interactions between glucose and amino acids in rat pancreatic islets were studied by recording the intracellular membrane potential and spike discharges from the isolated perfused pancreas.  It was found that L-isoleucine requires the presence of basal glucose (5 mM) in order to increase spike discharge from islet cells and depolarize the cell membrane.  Similarly basal glucose is needed for insulin release by L-isoleucine.  A physilolgical mixture of twenty amino acids also required the presence of basal glucose in order to increase spike activity and insulin release.  In contrast to L-isoleucine the amino acid mixture did not depolarize the beta-cells.  Iodoacetate, at concentrations previously shown to block glycolysis completely, did not interfere with any of these permissive actions of glucose, nor did iodoacetate alter the well known electrical manifestations of high levels of glucose itself (i.e. depolarization and increased spike discharge).  These data show that glucose plays a pre-eminent role as regulator of islet cell function, governing the efficacy of amino acids as beta-cells stimulants.  The results are most easily interpreted if one assumes that glycolysis is not required for glucose to exert its action.


Pregnancy in a juvenile diabetic after renal transplantation (class T diabetes mellitus). The successful outcome of a pregnancy in a juvenile diabetic after renal transplantation is reported.  It is proposed that class T be added to the classification of pregnancies complicated by diabetes mellitus.  Pregnancy prevention should be considered until significant longevity can be demonstrated in diabetics receiving renal transplants.


Studies of alloxan toxicity on the beta cell. The ability of sugars to protect the beta cell  from alloxan diabetes is highly stereospecific.  The alpha anomer, which is present in equilibrium in both glucose and 3-O-methyl glucose (3-OMG) at approximately 34 per cent, provides greater protection than the beta anomer.  The greater protection of the alpha anomer of glucose is present fifteen seconds between its administration and alloxan, but there is no difference in protection following a thirty-second interval.  The nonmetabolized analogue, 3-OMG, provides even greater protection than glucose, and this higher affinity is expressed both by the lower dose necessary to provide protection, as well as by the higher dose of mannoheptulose needed to remove thr protection.  Mannoheptulose not only removes the protection provided by exogenous glucose but sensitizes the beta cell to the toxic effects of alloxan in the fasting state, probably by inhibiting the protection provided by endogenous glucose.  Mannoheptulose is able to remove glucose protection before, with, or after the administration of glucose prior to alloxan injection.  Finally, the protective effect of both glucose and 3-OMG is time-related, and the protection not only is due to absolute concentration but also appears to be affected by a changing concentration.


Comparative effects of metoclopramide and bethanechol on lower esophageal sphincter pressure in reflux patients. The effects of oral metoclopramide, 10 and 20 mg, bethanechol, 25 mg, and placebo on lower esophageal sphincter pressure (LESP) were studied in 15 men with symptoms of gastroesophageal reflux and basal LESP less than 11 mm Hg. Each drug produced a significant increase in LESP when compared to placebo. Metoclopramide, 20 mg, produced a greater increase than either metoclopramide, 10 mg, or bethanechol, 25 mg. Serum gastrin concentrations were not altered by any of the drugs. Side effects were unremarkable. The LESP increasing effect of metoclopramide might be useful in treatment of gastro-esophageal reflux.


Fluorescence histochemistry of the gastrin cell infetal and adult man. Immunoreactive gastrin cells were demonstrated in the human antral and duodenal mucosa. No gastrin immunoreactivity was seen in pancreatic islets. Formaldehyde- or Bouin-fixed material was used throughout for immunohistochemistry. Antral and duodenal gastrin cells, as well as pancreatic A2-cells, displayed strong formaldehyde- ozone- and formaldehyde-HCl-induced fluorescence, whereas the pancreatic A1-cells (claimed to store gastrin) were nonfluorescent. In the fetus, gastrin cells appeared first in the duodenal mucosa (earliest stage examined, 11 weeks) and much later in the antrum. The fluorescence histochemical properties of the fetal gastrin cells were not different from the adult.


Hepatitis B surface antigen (HBS Ag) subtypes and indices of clinical disease. Serial serum samples of prospectively studied patients exposed to plasma containing both adw and ayw subtypes of hepatitis B surface antigen (HBS Ag) were studied for the relationship of HBS Ag subtype to several indices of clinical type B hepatitis. Seventy-four patients circulated HBS Ag that could be subtyped; 50 subtype adw, 22 ayw, and 2 adyw. Although the average incubation period among adw cases was shorter than among ayw cases (P less than 0.05), no additional significant differences were detected between these two groups of patients when analyzed for severity and chronicity of disease. The chronic HBS Ag carrier state developed in 16% (8 of 50) adw cases and in 9% (2 of 22) ayw cases, but this difference was not statistically significant. Judging from these data, it was concluded that the severity and outcome of type B hepatitis are probably independent of HBS Ag subtypes d and y.


Colonoscopy. A review.

Colonoscopy. A review. The technique of colonoscopy has introduced an exciting new modality for the diagnosis and treatment of diseases of the colon. It is not a panacrea, for, as with any technique, limitations are always present; but it offers the single most far reaching advance for the diagnosis and treatment of diseases of the colon that has been offered in the last decade. The next 10 years should indeed prove exciting.


Action of morphine sulfphate on stimulated gastric secretion in dogs. In dogs morphine sulphate suppresses both 2-deoxy-D-glucose and pentagastrin-stimulated acid and pepsin secretion, but not histimaine- and pilocarpine-stimulated acid. This is consistent with the view that morphine both depresses acetylcholine release from cholinergic endings in the stomach and that pentagastrin acts by liberation of acetylcholine from these endings.


[Single dose treatment of trichomoniasis]. The present regiman for the treatment of trichomoniasis with Tinidazol is 150 mg. twice daily for 7 days with a total dose of 2,100 mg. The success rate of this regimen is 85-90%. With a single dosage treatment of 2,000 mg. (Four 500 mg. tablets for the patient and her sexual partner) the success rate improved to 100%. Toxic side effects were not observed. The treatment was well tolerated and well accepted.


[Present situation of prenatal diagnosis of neural tube defects]. Anencephaly and spina bifida both are malformations of unknown etiology which are caused by an absent or incomplete closure of the neural tube. The risk of recurrence for the malformations is 5% in families with one child with a neural tube defect and 10%, if two such children have already been born. Neural tube defects can be diagnosed already before 20th week of gestation by ultrasonics and alpha-fetoprotein measurement in amniotic fluid, if an underdeveloped neurocranium or a free communication between amniotic and cerebrospinal fluid is present. By these diagnostic techniques and possibly following interruption the probability in risk families can be significantly lowered.


Reciprocal translocations and translocation disomics of aspergillus and their use for genetic mapping. Two new techniques are described for genetic mapping of reciprocal translocations in A. nidulans, which can be used to locate centromeres and meiotically unlinked markers. They both make use of unbalanced disomics from heterozygous translocation crosses. These are mainly hyperhaploids of two classes: either typical-looking n + 1 with a normal chromosome in addition to a haploid set containing the translocation, or translocation disomics. When large chromosome segments are involved, such disomics, as well as stable aneuploids and duplication types, show characteristic phenotypes and can be classified visually. The first method maps translocation breaks qualitatively, since translocated markers can be identified when translocation disomics are analyzed for heterozygous markers. The second method measures meiotic linkage of any marker to the translocation breaks when allele ratios in the balanced haploid sectors of either or both classes of disomics are determined: linked markers show reciprocal deviations from 1:1--In addition, it can be shown that frequencies of nondisjunction and recovery of specific translocation disomics both depend on the relative position of the break within a chromosome arm. Such information can provide a rough estimate of the positions of breaks for a new translocation.--Using these techniques, as well as mitotic mapping in homo- and heterozygous translocation diploids, four reciprocal translocations were mapped. from these results, information on the sequence and orientation of most of the "meiotic fragments" of the current maps (groups III, VI, VII and VIII) was obtained, and the position of the centromeres of groups VI and VII were identified. Translocation disomics are also used to map meiotically unlinked single genes, e.g. oliA of group VII, to specify chromosome segments.


Mitotic chromosome loss in a disomic haploid of Saccharomyces cerevisiae. Experiments designed to characterize the incidence of mitotic chromosome loss in a yeast disomic haploid were performed; The selective methods employed utulize the non-mating property of strains disomic for linkage group III and heterozygous at the mating type locus. The principal findings are: (1) The grequency of spontaneous chromosome loss in the disome is of the order 10- minus 4 per cell; this value approximates the frequency in the same population of spontaneous mitotic exchange resulting in homozygosity at the mating type locus. (2) The recovered diploids are pure clones, and thus represent unique events in the disomic haploid. (3) Of the euploid chromosomes recovered after events leading to chromosome loss, approximately 90% retain the parental marker configuration expected from segregation alone; however, the remainder are recombinant for marker genes, and are the result of mitotic exchanges in the disome, especially in regions near the centromere. The recombinant proportion significantly exceeds that expected if chromosome loss and mitotic exchange in the disome were independent events. The data are consistent with a model proposing mitotic nondisjunction as the event responsible for chromosome loss in the disomic haploid.


The distribution of mucosal antibodies in the bowel of patients with Crohn's disease. Specimens of intestine from 24 patients with histologically proven Crohn's disease have been studies by an immunofluorescent technique for the distribution of immunoglobulins A, G, M, E, and D. A marked reduction of IgA in lymphoid and epithelial cells was noted in those areas of the bowel which, although histologically affected, were not ulcerated; in the histologically unaffected areas of the bowel the distribution of IgA was similar to that found in control sections of normal bowel. There was an increase in IG7 in the affected areas but the numbers of lymphoid cells staining for IgD did not differ from those found in normal controls; in 12 cases of Crohn's disease, however, IgG appeared to be present in bound form on the surface of, and in, the epithelial cells. It is postulated that the local deficiency in IgA in the affected areas may be secondary to a local cell-mediated immune reaction, to a focal metaplasia of the surface epithelium, or to patchy bindings of anticolon antibodies to the surface epithelium. The effect of this local deficiency of IgA will be to allow for an increased focal entry of antigenic material into the bowel wall, and it is suggested that many of the pathological features of Crohn's disease are explicable on the basis of deep penetration of a variety of antigens.


Influence of exocrine and endocrine pancreatic function on intestinal brush border enaymatic activities. Digestive enzymatic activities (disaccharidases, alkaline phosphatase, peptide hydrolases) have been determined in the mucosa of 14 patients with chronic pancreatitis. All had an abnormal secretin-pancreozymin test. Four patients had insulin-dependent diabetes mellitus, four a pathological glucose tolerance test. Nine patients had steatorrhoea. Maltase, sucrase, and alkaline phosphatase activity was significantly elevated in patients with exocrine pancreatic insufficiency, whereas those of lactase, trehalase, and peptide hydrolase were normal. Patients with steatorrhoea had higher maltase and sucrase activity than those without steatorrhoea, whereas decreased glucose tolerance had no effect on brush border enzymatic activity. It is suggested thatdecreased exocrine rather than decreased endocrine pancreatic function is responsible for the increase in intestinal disaccharidase and alkaline phosphatase activity, possible by the influence of pacreatic enzymes on the turnover of brush border enzymes from the luminal side of the mucosal membranes or by direct hormonal stimulation though cholecystokinin.


Release of 14C label and complement killing of Escherichia coli. When Escherichia coli labelled with 14C were killed by complement, characteristic 14C compounds were released even when complete cell disintegration was prevented or delayed by removal of lysozyme. Treatment with heated serum only resulted in the loss of small molecular weight compounds. Separation of the products was made easier if whole serum was replaced by a salt-precipitated fraction which contained no albumin or lysozyme but retained antibody and complement. Fractionation of the bacterial products on Sephadex G-200 showed two radioactive peaks containing lipids and proteins of which a preliminary examination was made. The release of these compounds was related to complement action, since they were not found when bacteria were killed by streptomycin or ultra-violet light in the absence of complement. Nor were they found when resistant bacteria were treated with complement and survived. The possible modes of action of complement on bacterial cell walls are discussed.


Comparison of methods for the detection of Mycoplasmal contamination of cell cultures: a review. Several reviews in recent years have emphasized the problems created by mycoplasmal contamination of cultured cells (1-5). Because of the hazards of interpreting data derived from mycoplasma contaminated cells, most cell biologists routinely screen their cultures for the presence of these organisms. In recent years, the limitations of standard microbiological testing for mycoplasmas have become increasingly apparent and have led to the development of several new biochemical techniques for detection of these organisms. The aim of this review is to describe and compare available detection techniques and to evaluate their relative efficacy. Those properties of mycoplasmas that are relevant to their role as cell culture contaminants will be briefly discussed.


Effect of Corynebacterium parvum on the class and subclass of antibody produced in the response of different strains of mice to sheep erythrocytes. Several strains of mice were injected with sheep erythrocytes (SRBC) using C. parvum as adjuvant. The adjuvant effects on the amounts of class and subclass of antibody produced were ranked in the order IgG2b greater than IgG2a and IgM greater than IgG1. In addition, these effects were shown to vary depending on the time of administration of C. parvum relative to antigen. C parvum was shown to have no adjuvant effect on the response of congenitally athymic mice when given at the same time as the antigen, SRBC. On the basis of the reported observations it is suggested that certain of the adjuvant effects of C. parvum require T-cell function.


Hydrolysis of oligopeptides by sera used in cell and tissue culture. Sera commonly used in cell and tissue culture as medium supplements possess high peptidase activity. Pligopeptides incubated with 1% serum are rapidly hydrolyzed to intermediate length peptides and the constituent amino acids. Hydrolysis of lysine peptides is difficult to verify by a quantitative ninhydrin procedure because of reaction of the lysine epsi-lon-amino group and serum components with ninhydrin. Attempts to evaluate oligopeptides as sole sources of indispensable amino acids for cultured mammalian cells are of doubtful value when serum is used as a medium supplement.


Naturally occurring double-stranded RNA and immune responses. Effects on plaque-forming cells and antibody formation. A highly purified preparation of double-stranded RNA, obtained from virus-like particles in Penicillium cultures, was found to affert humoral immune responses in mice differentially depending on its time of administration in realtion to antigen. Double-stranded RNA administered with antigen, or a few hours after antigen, produced a variable degree of enhancement of plaque-forming cell numbers or agglutinating antibody levels depending on the antigen involved. Administration of double-stranded RNA 24 hours before antigen invariably produced a suppressed response. In mice which were either specifically hyporesponsive (tolerant) or non-specifically hyporesponsive (due to age or immunosuppressive drugs) double-stranded RNA administered with antigen resulted in a nearly normal immune response.


T cell-dependent helper and suppressive influences in an adoptive IgG antibody response. When immune spleen cells of mice immunized to a hapten carrier preparation 4 days previously were transferred to normal syngeneic hosts, they began to produce 7S antibody (presumably of the IgG class), provided that relatively small numbers of cells (about 1/10 spleen equivalent) were transferred. Increasing the number of transferred cells resulted in less IgG antibody formed. Depletion of the immune spleen cells of T cells by treatment with anti-theta serum and complement prevented IgG antibody formation. IgG antibody production by untreated and anti-therta serum-treated immune spleen cells could be enhanced (reinduced) by addition of small numbers of cells enriched for carrier-activated T cells. These suggest that T cells are necessary to stimulate antigen-activated B cells into IgG antibody production. Larger numbers of 'carrier-activated T cells' depressed IgG antibody production. Both enhancement and depression could be demonstrated to be antigen-specific. IgG antibody production high numbers of transferred immune spleen cells could be induced by treating the cells prior to transfer with suboptimal amounts of anti-theta serum and complement. It is argued that this results from the elimination of a T cell-dependent suppressor influence arising during a normal immune response.


T cell-dependent mediator in the immune response. III. The role of non-specific factor (NSF) in the in vitro immune response. Evidence is presented which suggests that T cell-dependent nonspecific factor (NSF) cannot be the sole mediator of 'co-operation' for all types of thymus-dependent antibody response. NSF can replace the requirement for T cells in responses to hapten conjugated to particulate carriers, but cannot do so in responses to hapten conjugates of soluble thymus-dependent carriers.


The inter-relationship of antigenic structure, thymus-independence and adjuvanticity. IV. A general model for B-cell induction. Polymerized flagellin and E. coli lipopolysaccharide both express adjuvanticity in vivo and in vitro for responses to hapten conjugated to sheep erythrocytes, and hapten conjugated to soluble thymus-dependent antigens. In the case of erythrocyte-bound hapten, adjuvanticity is expressed in the absence of thymus-derived cells (T cells). However, in the case of responses to soluble thymus-dependent conjugates, carrier-specific T cells would appear to be necessary for adjuvanticity to be expressed. On the basis of these observations an hypothesis for the mechanism of B-cell induction and tolerance is proposed.


C3 receptors on direct plaque-forming cells. Using a rosette technique it is shown that only a small proportion of direct plaque-forming cells posses detectable C3 receptors 5 and 7 days after antigenic stimulation. The significance of this result is discussed.


A series of tibial fractures treated conservatively. Two hundred and two fractures of the tibia have been reviewed. They were treated by a standard, conservative method; with a Phemister graft when union was delayed and a pseudarthrosis threatened. The classifications of Charnley (1968) and Nicoll (1964) have proved to be useful in assessing results and these complement each other. The factors that influence bony union include damage to the interosseous membrane, the degree of the displacement and comminution of the fracture, and injury to soft tissues. Infection has a very unfavourable influence on bone union and leads to the majority of bad sequelae. The fundamental object in reduction is to obtain as perfect apposition and alignment of the fragments as possible. Imperfect apposition, however, with normal alignment does not affect bone union severely. The problem arising from the use of an above-knee walking plaster that affects the patient physically, psychologically and socially, may be solved by the early use of a below-knee plaster. The functional sequelae are the result of soft-tissue damage rather than of immobilization. The Chutro-Phemister type of graft has proved to be a safe method in preventing pseudarthrosis. The analysis of the results obtained with this conservative method is sufficient reason against choosing surgical treatment for these fractures.


The place and efficacy of operative management of fractured clavicle. A study of 22 cases of operative fixation of the fractured clavicle has been made. This has shown that with internal fixation and onlay bone grafting, highly successful results can be attained. There were no cases of non-union and there have been no significant complications. The indications for such surgery are presented from the literature. It is clear that the operation is not commonly indicated, but when performed is safe and highly successful.


Prophylaxis with dextran 70 against thrombosis in patients with fractures of the upper end of the femur. A trial was undertaken to assess the effect of dextran 70 (Macrodex) in prophylaxis against thrombosis in womem over 65 years with fractures of the upper end of the femur. The dosage used was 500 ml at operation and 500 ml on the first postoperative day. At this dosage given at this time there was no difference in the overall incidence of clinically diagnosed deep venous thrombosis in the group of patients given dextran 70 compared with the control group. There was a tendency for thrombosis to occur earlier in the control group. The mortality rates at 3 weeks and even at 6 months were less in the treated group than in the control series.


Oncogenesis by interspecific interaction of malignant murine and non-malignant hamster cells in vitro. A clone of Cloudman S91 murine melanoma was fused in vitro with non-malignant hamster cheek pouch cells by means of lysolecithin, and the putative hybrid progeny cells, HCP-MM, were found to be highly malignant in hamster, but not in appropriate mice. A malignant clone of HCP-MM cells was shown to have hamster species-specific surface antigens (as demonstrated by immunofluorescence and the cytotoxic antibody) and hamster-like lactate dehydrogenase and NAD-dependent malate dehydrogenase isoenzyme profiles. Nevertheless, chromosomes similar to those of both murine and hamster parental cells could be distinguished in cells of this malignant clone and in hamster tumor grafts by the method of trypsin-Giemsa banding. A majority of the murine chromosomes, however, appeared to be lost. This study indicates that a murine melanoma previously found untransplantable in hamsters could produce a highly malignant and lethal tumor for hamsters after being mixed in vitro with non-malignant hamster cells, in the presence of a fusing chemical. It is not as yet certain whether the production of transformed cells in vitro and of highly malignant tumors in the hamster (both with predominantly hamster properties) required heterosynkarion formation between the murine melanoma and hamster cheek pouch cells. Nevertheless, our results suggest that the presence of the murine melanoma, and possibly the interaction of its genome with non-malignant hamster cells, was implicated in this process.


Mycetoma in Iran.

Mycetoma in Iran. The subject of mycetoma is reviewed and 7 cases are reported for the first time from southern Iran.  Four were causes by Actinomyces israelii and one each by Nocardia asteroides, Actinomadura madurae and Allescheria boydii.


Silver sulfadiazine for control of burn wound infections. Silver sulfadiazine (Silvadene, U.S.; Flamazine, U.K.; Flammazine, N.; Sulplata, S.A.) is the newest topical antimicrobial agent available following worldwide clinical trials. Good control of infection is achieved without pain or other demonstrable side effects, using either dressings or the exposure technic. Many burned areas kept free of infection heal without grafting. Where necessary, early preparation for and good take of grafts has been attained by utilizing this new therapeutic agent. Markedly reduced mortality from burn wound sepsis has generally been observed.


Bifunctional reagents as vapour- and liquid-phase fixatives for immunohistochemistry. Out of a total of eleven bifunctional reagents tested as fixatives for light microscopic immunohistochemistry, four were found satisfactory when applied in the vapour phase to freeze-dried blocks. These were diethylpyrocarbonate, as observed in earlier studies, dimethyladipimidate, p-benzoquinone, and diacetyl. Adequate but less satisfactory liquid-phase fixation was provided by three reagents (dimethyladipimidate, dimethylsuberimidate, p-benzoquinone). None of the eleven reagents gave optimal preservation of antigencity and structure when tested at the ultrastructural level. Encouraging results were obtained with p-benzoquinone, however.


Immunofluorescent localization of the female sex steroids in the porcine ovary. Antibodies against progesterone 20-(o-carboxymethyl) oxime and oestrone 17-(o-carboxymethyl) oxime have been applied to porcine ovaries as the first layer in the indirect immunofluorescent technique. In the Graafian follicles, both steroids have been demonstrated within single cells of the theca interna. In the corpora lutea, the same steroids were found in the cells assembled at the periphery of the organ, as well as around the large vessels. Degenerating cells present in the corpora albicans revealed positive immunofluorescence together with distinct autofluorescence. Similarly, the atretic follicles and fragments of the interstitial gland showed both immunofluorescence and autofluorescence. The immunofluorescent localization of steroids was confirmed by the similar distribution of sudanophilic substances. However, the differentiation between the two hormones investigated was not certain.


The gentamicin antibiotics. 6. Gentamicin C2b, an aminoglycoside antibiotic produced by Micromonospora purpurea mutant JI-33. A mutant strain of Micromonospora purpurea, designated var. JI-33, produced an antibiotic complex consisting primarily of gentamicin C1a. A further product of this fermentation was identical to a very minor component isolated from the fermentation of the parent organism and named gentamicin C2b. Physical measurements indicated its structure to be 6'-N-methylgentamicin C1a and this was confirmed by synthesis from gentamicin C1a. The in vitro antibacterial activity of gentamicin C2b was very similar to that of the gentamicin C complex. Antibiotic XK-62-2, produced by Micromonospora sagamiensis, appears to be identical to gentamicin C1b.


A new prodiginne (prodigiosin-like) pigment from Streptomyces. Antimalarial activity of several prodiginnes. Two prodigiosin-like pigments from Streptomyces sp. were shown to be undecylprodiginine (i) and butylcycloheptylprodiginine (v). The antimalarial activity of five prodiginine pigments is given.


Insertion of a minor protein into the outer membrane of Escherichia coli during inhibition of lipid synthesis. The antibiotic cerulenin, a specific inhibitor of fatty acid synthetase systems, was used to demonstrate that a minor protein component of the outer membrane of Escherichia coli, which serves as the receptor for the phage lambda, can be synthesized and inserted into the outer membrane during inhibition of lipid synthesis.


Thermosensitive mutants of Escherichia coli K-12 altered in the catalytic Subunit and in a Regulatory factor of the glutamy-transfer ribonucleic acid synthetase. The glutamyl-transfer ribonucleic acid synthetase (GluRS) of a partial revertants (ts plus or minus) of the thermosensitive (ts) mutant strain JP1449 (LOcus gltx) and of a ts mutant strain EM111-ts1 with a lesion in or near the locus gltx have been studied to find the relation between these two genetic loci known to influence the GluRS activity in vitro and the presence of a catalytic subunit and of a regulatory subunit in the GluRS purified from Escherichia coli K-12. The ts character of strain JP1449-18ts plus or minus is co-transduced with the marker dsdA at the same frequency as is the ts character of strain JP1449. Its purified GluRS is very thermolabile and its Km for glutamate is higher than that of a wild-type GluRS. These results indicate that the locus gltX is in the structural gene for the catalytic subunit of this enzyme. The location of the mutation causing the partial ts reversion in strain JP1449-18ts plus or minus is discussed. The GluRS purified from the ts mutant strain EM111-ts1 has the same stability as the wild-type enzyme, but its Km forglutamate increases with the temperature, suggesting that the locus gltE codes for a regulatory factor, possibly for the polypeptide chain that is co-purified with the catalytic subunit.


Reduced plasma membrane permeability in a multiple cross-resistant strain of Saccharomyces cerevisiae. Single nuclear gene inheritance was shown to be responsible for increased resistance to: eight diverse inhibitors of mitochondrial function (antimycin, carbonylcyanide-m-chlorophenylhydrazone, chloramphenicol, oligomycin, tetracycline, triethyltin bromide, triphenylmethylphosphonium bromide and triton-X-165); and an inhibitor of cytoplasmic protein synthesis (cycloheximide). Continuous monitoring of oxygen uptake during respiratory adaptation showed that anerobic pretreatment of resistant cells sensitized respiratory adaptation to chloramphenicol and antimycin. However, since a depression of mitochondrial function by catabolite repression did not result in sensitization to antimycin, alteration of the mitochondrial membrane does not appear to be responsible for resistance to mitochondrial inhibition. Alteration of cellular binding sites was not responsible for resistance since in vitro mitochondrial protein synthesis was sensitive to chloramphenicol and in vitro mitochondrial respiration was sensitive to oligomycin, carbonylcyanide-m-chlorophenylhydrazone, and antimycin. Autoradiography of an ethylacetate-ethanol extract of [14C]chloramphenicol-treated resistant cells indicated that resistance was not due to enzymatic modification of inhibitors. The maintenance of an antimycin-resistant respiration by protoplasts of resistant cells ruled out the involvement of the cell wall in cellular resistance. The reduced transport of [14C]chloramphenicol by resistant cells (1% of normal cells) indicated that a single nuclear gene mutation can alter the permeability of the plasma membrane to many diverse inhibitors.


Existence of two levels of repression in the biosynthesis of methionine in Saccharomyces cerevisiae: effect of lomofungin on enzyme synthesis. Derepression of a methionine biosynthetic enzyme (homocysteine synthase) has been studied after repression either by exogenous methionine or by exogenous S-adenosylmethionine (SAM). Lomofungin, which inhibits the synthesis of ribosomal precursor and messenger ribonucleic acid but not of protein in Saccharomyces cerevisiae, has been used in this system. It has been shown that the addition of this antibiotic prevents the derepression of homocysteine synthase after repression by exogenous methionine but not after repression by exogenous SAM. These experiments with lomofungin and the kinetics of repression after addition of methionine or SAM to the growth medium provide evidence that the repression induced by exogenous methionine acts at the transcriptional level whereas the repression induced by exogenous SAM acts at the translational level.


Biochemical and regulatory effects of methionine analogues in Saccharomyces cerevisiae. The effect of three methionine analogues, ethionine, selenomethionine, and trifluoromethionine, on the biosynthesis of methionine in Saccharomyces cerevisiae has been investigated. We have found the following to be true. (i) A sharp decrease in the endogenous methionine concentration occurs after the addition of any one of these analogues to growing cells. (ii) All of them can be transferred to methionine transfer ribonucleic acid in vitro as well as in vivo with, as a consequence, their incorporation into proteins. In the absence of radioactive trifluoromethionine, this conclusion results from experiments of an indirect nature and must be taken as an indication rather than a direct demonstration. (iii) Ethionine and selenomethionine can be activated as homologues of S-adenosylmethionine, whereas trifluoromethionine cannot. (iv) All of them can act as repressors of the methionine biosynthetic pathway. This has been shown by measuring the de novo rate of synthesis of methionine in a culture grown in the presence of any one of the three analogues.


Characterization of transmissible genetic elements from sucrose-fermenting Salmonella strains. Two of seven sucrose-fermenting Salmonella strains obtained from clinical sources were found capable of conjugal transfer of the sucrose fermentation (Scr+) property to the Escherichia coli K-12 strain WR3026. The genetic elements conferring this Scr+ property, designated scr-53 and scr-94, were then conjugally transmissible from Escherichia coli WR3026 Scr+ exconjugants to other strains of Escherichia coli at frequences of 5 times 10- minus 6 to 5 times 10- minus 3 for the scr-53 element and 10- minus 6 to 10- minus 5 for the scr-94 element. In Escherichia coli hosts, both of these elements were compatible with F-lac and with each of six previously characterized transmissible lac elements. No antibiotic resistance characteristics or colicin production were discovered to be associated with either scr-53 or scr-94. Neither scr element generated a male host response to the female-specific phage phiII, but the scr-53 element rendered its Escherichia coli host sensitive to the male-specific phage R-17. Escherichia coli hosts containing scr-53 were susceptible to lysis by P1vir, and transduction of the scr-53 element was accomplished with this phage. The scr-53 element was isolated from Escherichia coli WR3026, Scr+ transductants, and Escherichia coli WR2036 Scr+ exconjugants as a covalently closed circular deoxyribonucleic acid molecule with a molecular weight (determined by electron microscopy) of approximately 52 times 10-6. Receipt of the scr-94 element rendered Escherichia coli hosts of this element unsusceptible to lysis by P1vir, although adsorption of the phage by an Escherichia coli WR3026 exconjugant containing scr-94 occurred as efficiently as it did on WR3026 itself. Repeated examination of Escherichia coli strains harboring scr-94, as well as of the Salmonella strain which initially contained it, did not reveal the presence of circular deoxyribonucleic acid. The synthesis of the sucrose cleaving enzyme was inducible in Escherichia coli exconjugants containing either scr-53 or scr-94.


Events following prophage Mu induction.

Events following prophage Mu induction. Escherichia coli strains lysogenic for a thermoinducible Mu prophage (Mu cts62) undergo rapid lysis about 50 min after heat induction. Induction of Mu cts62 apparently causes damage to the host sequences in which Mu is inserted. The normal expression of A, BU, and X genes of Mu is needed for this specific deleterious effect on the prophage-containing host sequences. Mu deoxyribonucleic acid can be shown to reintegrate extensively at different sites on the host genome during the lytic cycle after prophage induction or after infection of sensitive cells by clear-plaque mutants of Mu. We estimate that approximately 10 copies of Mu deoxyribonucleic acid are inserted per chromosome during vegetative growth. The episome rescue method for detecting vegetative Mu deoxyribonucleic acid insertion, in which an episome is transferred from the lytically infected cells to F- receipient cells, can be applied to study Mu integration without requiring the host cells to survive. It also provides an easy system to isolate Mu insertions in transmissible episomes and plasmids.


Homology between the deoxyribonucleic acid of fertility factor P and Vibrio cholerae chromosomal deoxyribonucleic acid. The deoxyribonucleic acid (DNA) of the Vibrio cholerae fertility factor P was isolated by the dye-buoyant density method and hybridized to V. cholerae chromosomal DNA. The DNA of this fertility plasmid had between 35 to 40% homology with the V. cholerae chromosomal DNA. Little or no homology was detected between the P factor DNA and DNA of the Escherichia coli sex factor F.


Isolation and characterization of mutator strains of Escherichia coli K-12. A selection procedure was devised to select for mutants of Escherichia coli K-12 with enhanced rates of spontaneous frameshift mutation. Three types of mutants were isolated. Two of the mutations apparently represent alleles of previously isolated mutL13 and mutS3. The third type of mutation, represented by two alleles, lies between lysA and thyA, and has been designated mutR. mutR increases the rate of spontaneous frameshift mutation and also the rate of base substitution mutations. The mutator phenotype is recessive. Reversion of a lac amber mutation located on an episome is increased in the presence of the mutator, indicating that mutR can act in trans. No change in sensitivity to ultraviolet irradiation or mitomycin C could be found when mutR34 was compared to the isogenic mutR+ strain. The mutator's activity was little affected by the type of medium in which the strain was grown. Deoxyribonucleoside triphosphate pools were normal in mutR34. Intergenic recombination frequencies were the same in mutR and mutR and mutR+ strains, but a two- to threefold increase in intragenic recombination was observed in Hfr times Fminus crosses when the recipeint was mutR34 as compared with mutR+. This increase appeared independent of the distance between the two markers within the gene in which the crossover took place.


Multiple gene loci for a single species of glycine transfer ribonucleic acid. The study of suppressors of tryptophan synthase A protein missense mutations in Escherichia coli has led to the establishment of two nonadjacent genetic loci (gly V and gly W) specifying identical nucleotide sequences for a single isoaccepting species of glycine transfer ribonucleic acid (tRNA GLY 3 GGU/C). In one case, suppression of the missense mutation trpA78 was due to a mutation in a structural gene (gly W) for tRNA Gly 3 GGU/C. This mutation resulted in a base change in the anticodon and modification of an A residue adjacent to the 3' side of the anticodon, leading to the production of a tRNA Gly 3 UGU/C species. The resulting glyW51 (SU UGU/C) allele was mapped by interrupted mating and was located at approximately 37 min on the Escherichia coli genetic map. Other suppressor mutations affecting the primary sequence of tRNA Gly GGU/C and giving rise to the Ins and SU+A58 phenotypes were positioned at 86 min (glyV). Several independently arising missense suppressor mutations resulting in the SU+A78 phenotypes were isolated and mapped at these two genetic loci (glyV and glyW). The ratio of appearance of suppressor mutations at glyV and glyW suggests that there are three of four tRNAGly3 GGU/C structural gene copies at the glyV locus to one copy at the glyW locus. Structural genes for tRNA ly isoacceptors are now known at four distinct locations on the Escherichia coli chromosome: glyT (77 MIN), TRNA Gly 2 GGA/G; gly U (55 min), tRNAGly-1 minus; and gly V (86 MIN) AND GLYW (37 min), tRNAGly 3 GGU/C.


Five control systems preventing transfer of Escherichia coli K-12 sex factor F. The transfer inhibition systems of 28 Fin+ plasmids have been characterized, using Flac mutants insensitive to inhibition by R100 or R62. All F-like plasmids (except R455) and one N group plasmid determined systems analogous to that of R100; this is designated the FinOP system. None of these plasmids could supply a FinP component of the transfer inhibitor able to replace that of F itself. In addition to the FinOP and R62 transfer inhibition systems described previously, new systems were encoded by the F-like plasmid R455, the I-like plasmid JR66a, and the group X plasmid R485. Besides inhibiting F transfer, JR66a also inhibited F pilus formation and surface exclusion, whereas R485 inhibited only pilus formation and R455 inhibited neither. All three R factors inhibited transfer of J-independent Flac elements, indicating that they act directly on one or more genes (or products) of the transfer operon, rather than directly via traJ. The tral products and transfer origin sequences of two Fin+ F-like plasmids, ColB2 and R124, appear to have similar specificities to those of F itself.


Formation of chromatographically unique species of transfer ribonucleic acid during amino acid starvation of relaxed-control Escherichia coli. Examination of the transfer ribonucleic acid (tRNA) produced by starving, relaxed-control (rel minus) strains of Escherichia coli for required amino acids revealed the occurrence of a number of chromatographically unique subspecies. Leucine starvation results in the formation of new isoacceptor species of leucine-, histidine-, arginine-, valine-, and phenylalanine-specific tRNA and quantitative changes in the column profiles of serine, glycine, and isoleucine tRNA. Evidence that the unique tRNA species are synthesized de novo during amino acid starvation comes from the findings that the major unique leucine isoacceptor species is not formed in stringent control cells or in rel minus cells starved for uracil or treated with rifampin. Furthermore, heat treatment of the unique leucine tRNA does not alter its chromatographic behavior, indicating that the species is not an aggregate or nuclease-damaged form of a normal isoacceptor tRNA. The methyl acceptor activities of tRNA from leucine-starved and nonstarved rel+ or rel minus cells were found to be essentially the same. This result and the finding that the chromatographic behavior of the unique leucine-specific tRNA was not altered after treatment with tRNA methylase suggests that gross methyl deficiency is probably not the biochemical basis for the occurrence of the unique species.


Hycanthone and its congeners as bacterial mutagens. Hycanthone methanesulfonate (HCT) was shown to induce "forward" and "reverse" mutations in Salmonella typhimurium and Escherichia coli. Mutational effects of HCT on S. tyhhimurium TA1532 were concentration and time dependent. A comparison of mutagenic potency for TA1532 was made between HCT and the frame-shift mutagens quinacrine and ICR-191. An investigation of structure-activity relationships revealed the substituent in the 4-position of ring A to be critical for mutagenicity. Activity was found when this group was a hydroxymethyl (i.e., HCT) or an aldehyde (Win 25,315), but the analogues having a carboxyl group (Win 25,850) or methyl group (lucanthone) in this positionwere inactive. Removal of a single ethyl group from the side chain did not affect mutagenic activity inasmuch as the potency of desethyl HCT (Win 27,262) equaled that of HCT on a molar basis. A marginal activity was found with a sample of HCT sulfoxide (win 27,266), but this sample was found to contain traces of HCT. The HCT analogue with a terminal N-oxide in the side chain (Win 29,329) was inactive at the concentration tested.


Rapid cessation of phospholipid synthesis in fructose-1,6-diphosphate aldolase mutants of Escherichia coli. Escherichia coli GH352, which was originally described as a temperature-sensitive strain containing a thermolabile acyl coenzyme A:monoacylglycerol 3-phosphate acyltransferase, does not now contain a thermolabile form of this enzyme. It has a defect in fructose-1,6-diphosphate aldolase and at least one additional temperature-sensitive lesion. Both strains GH352 and NP315, a temperature-sensitive aldolase mutant, show rapid cessation of 32-P1 incorporation into nucleic acids and phospholipids at 42 C. These characteristics of strain GH352 are therefore no longer attributed to thermolabile phospholipid synthesis, but can be attributed to the fructose-1,6-diphophate aldolase lesion.


Mapping of sul, the suppressor of lon in Escherichia coli. The suppressor sul, which is allele specific for the ultraviolet sensitivity gene lon, has been mapped by conjugation and transductional crosses in Escherichia coli K-12 and B/r. Previously, sul was reported to lie in the azi region of the Escherichia coli chromosome. Evidence is presented which positions sul close to and clockwise of fabA on the Escherichia coli map. Cotransductional frequencies of 31.3% were obtained between sul and pyrD, and frequencies of 82% were obtained between sul and fabA. Also, the mucoid phenotype of K-12 lon strains grown on minimal glucose agar plates at 37 C was not significantly effected in sul derivatives. No differences between the sul of Escherichia coli B/r and that of K-12 derivatives with regard to map location or effect on mucoid production were observed.


Simple downshift and resulting lack of correlation between ppGpp pool size and ribonucleic acid accumulation. The growth rate of Escherichia coli can be limited by the availability of carbon and energy. To impose such a limitation, alpha-methylglucoside (alpha MG), a non-metabolizable analogue, can be used to decrease uptake of glucose by competition for the transport of this sugar. Varying the ratio of glucose to alphaMG allowed shifts in growth rate without simultaneous qualitative changes in the growth medium and permitted examination of the immediate changes accompanying such shifts. Stringent (rel+) as well as relaxed (rel minus) strains were able to rapidly curtail their accumulation of ribonculeic acid (RNA) after a downshift imposed by decreasing glucose transport into the cell. Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) accumulated in both rel+ and rel minus strains after a degrease in growth rate. However, the accumulation of ppGpp in relaxed derivatives was very slow, and there was no direct or obligatory correlation between the level of ppGpp and the rate of RNA accumulation. This latter conclusion is supported by measurements of ppGpp levels and rates of RNA accumulation after restoration of maximal growth rates by addition of excess glucose.


Effect of ribonuclease on the association of deoxyribonucleic acid with the membrane in Escherichia coli. The Mg-2+-Sarkosyl crystals (M band) procedure was used to study the effect of ribonuclease (RNase) A on the association of Escherichia coli deoxyribonucleic acid (DNA) with membrane. Incubation of gently prepared cell extracts with RNase results in the release of DNA from membrane. This effect appears to result from the activation, by RNase, of endonuclease I and subsequent limited activity of this deoxyribonuclease. In support of this explanation, it is demonstrated (i) that the extent of the RNase-induced loss of DNA from membrane is directly correlated with the endogenous level of endonuclease I, and (ii) that endonucleolytic activity occurs when gently lysed cell preparations are incubated in the presence of RNase.


Potassium translocation in yeast mitochondria and its relationship to ergostrol biosynthesis. The energy-dependent transport and accumulation of K+ in respiring mitochondria has been found to inhibit the S-adenosylmethionine: delta-24-sterol methyltransferase enzyme of Saccharomyces cerevisiae. Potassium cation translocation is discussed as a possible regulatory mechanism over the biosynthesis of ergosterol.


Reinitiation of chromosome replication in the presence of chloramphenicol under an integratively suppressed state by R6K. The autonomous replication of an R plasmid, R6K (amp, str) was shown not to be affected by chloramphenicol. It provoked integrative suppression and gave rise to Hfr strains when integrated into the chromosome of a strain of Escherichia coli K-12 with a temperature-sensitive mutation in the gene, dnaA. An Hfr strain designated as Hfr(R6K) no. 1 was thus obtained and characterized. It was not completely stable as shown by a plating efficiency of 0.6 at 42 C relative to that at 30 C. The density labeling and the ultracentrifugation analysis suggested that the deoxyribonucleic acid replication in this Hfr strain did not stop immediately after completion of the round already started before temperature shift-up and the addition of chloramphenicol. These observations are discussed in relation to a possibility that the chromosome replication of this Hfr strain is under the control of the integrated plasmid at a nonpermissive temperature.


Lipophilic chelator inhibition of electron transport in Escherichia coli. The lipophilic chelator bathophenanthroline inhibits electron transport in membranes from Escherichia coli. The less lipophilic 1,10-phenanthroline, bathophenanthroline sulfonate, and alpha,alpha-dipyridyl have little effect. Reduced nicotinamide adenine dinucleotide oxidase is more sensitive to bathophenanthroline inhibition than lactate oxidase activity. Evidence for two sites of inhibition comes from the fact that both reduced nicotinamide adenine dinucleotide menadione reductase and duroquinol oxidase activities are inhibited. Addition of uncouplers of phosphorylation before bathophenanthroline protects against inhibition.


Morphological change in the early stages of the mating process of Rhodosporidium toruloides. The events which occur in the early stages of the mating process of the yeast Rhodosporidium toruloides between strains M-919 (mating type A) and M-1057 (mating type a) were investigated. In preliminary experiments we determined the frequency of mating by two newly designed methods: the liquid culture method and the membrane-filter microculture method. The mating frequencies of strains M-919 and M-1057 were 89% in the liquid culture method and 62% in the membrane-filter microculture method. The early stages in the mating process included the following events: (i) M-919 cells produce constitutively the extracellular inducing substance (A factor), (ii) M-1057 cells receive A factor, and in response to it they form mating tubes and secrete another inducing substance (a factor), (iii) M-919 cells receive a factor, and in response to it they form mating tubes, (iv) mating tubes elongate to the cells or the tubes of mating partner, (v) tips of the growing tubes recognize the opposite mating type cells or their tubes, followed by cell-to-cell fusion.


Endogenous messenger ribonucleic acid-directed polypeptide chain elongation in a cell-free system from the yeast Saccharomyces cerevisiae. An in vitro protein-synthesizing system from the yeast Saccharomyces cerevisiae has been made by a modification of the procedure for preparation of the Krebs ascites system. The protein synthetic activity is directed by endogenous messenger. Amino acid incorporation occurs over a broad range of magnesium and potassium concentration, being maximal at 6 and 85 mM, respcetively. The activity of this in vitro system is due to the elongation of polypeptides whose synthesis was initiated in vivo. The cell extract does not initiate synthesis with endogenous messenger ribonucleic acid (RNA), since 1 muM pactamycin, which blocks initiation on prokaryotic or eukaryotic ribosomes invitro, fails to decrease amino acid incorporation. Ten micromolar cycloheximide, however, inhibits incorporation by 87%. Moreover, this system is not stimulated by rabbit reticulocyte polysomal RNA, which directs the synthesis of hemoglobin in extracts of Krebs ascites cells. The translation of this messenger is not masked by high endogenous incorporation, because autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing [35-S]methionine-labeled products shows that no hemoglobin is made. Preincubation of this system, which reduces the high endogenous incorporation by 80%, does not increase its capacity to be stimulated by either rabbit reticulocyte RNA or yeast polyriboadenylic acid-containing RNA. Polyuridylic acid, however, does stimulate polyphenylalanine incorporation. The failure of the yeast lysate to be stimulated by or to translate added natural messenger RNA, its insensitivity to low levels of pactamycin but inhibition by cycloheximide, and its relatively high magnesium optimum (the same as that for polyuridylic acid) suggest that it elongates but does not initiate polypeptide chains.


Isolation and characterization of lambda transducing bacteriophages for argF, argI and adjacent genes. Two genes for ornithinetranscarbamylase exist in strain Escherichia coli K-12, argI, at 85 min, and argF, at 7 min. In an attempt to compare the deoxyribonucleic acid material of these two genes, the lambda transducing phages carrying a portion of the argI region, lambda dvalS argI, lambda pvalS, and lambda dvalS pyrB, and of the argF region, lambda dargF, have been isolated. Their structure, including that of phi 80dargF previously isolated, was studied by the method of heteroduplex mapping. In this paper, the results of this mapping are reported.


Electron microscopic heteroduplex studies of sequence relations among plasmids of Escherichia coli: structure of F13 and related F-primes. The structure of F13, a plasmid containing lac, purE, and proC, has been determined by heteroduplex analysis. As expected for an F-prime formed by a type II excision event, it contains all the sequences of F plus a large segment of Escherichia coli chromosomal deoxyribonucleic acid. There is a sequence of F with coordinates 16.3-17.6F which has been shown in other studies to be the insertion sequence IS2. This IS2 occurs twice on F13, once at each of the two junctions of F deoxyribonucleic acid with chromosomal deoxyribonucleic acid. The sequence alpha beta which occurs twice on F with coordinates 93.2-94.5/OF and 13.7-15.0F occurs an additional three times, twice in an inverted order relative to the alpha beta sequences of F, on the chromosomal sequences of F13. The structures of the plasmids F13-4 and F210 have been determined. The common sequences of F13 with F152-1 (a derivative of F152, the classical F2gal) and with F13-4 and F210 have been mapped. These results partially map lac, proC, tsx, and purE on F13. On the basis of all of these results, it is proposed that Hfr 13 (the parent of F13) was formed by recirpocal recombination between IS2 on F and an IS2 resident at a point between lac and proC on the chromosome of the F+ parent of Hfr 13. It is proposed that this IS2 and the several alpha beta sequences on the chromosomal part of F13 are hot spots for recombination with F, i.e., for Hfr formation. The point of origin and direction of transfer of many Hfr's can be explained by this hypothesis. In particular, the sequence relations of F42-1 (Flac) and of F152-1 (F 2gal) with F13 are completely consistent with this model.


Electron microscope heteroduplex studies of sequence relations among bacterial plasmids: identification and mapping of the insertion sequences IS1 and IS2 in F and R plasmids. Heteroduplex experiments between the plasmid R6 and one strand of the deoxyribonucleic acid (DNA) of a lambda phage carrying the insertion sequence IS1 show that IS1 occurs on R6 at the two previously mapped junctions of resistance transfer factor (RTF) DNA with R-determinant DNA. From previous heteroduplex experiments, it then follows that IS1 occurs at the same junctions in R6-5, R100-1, and R1 plasmids. Heteroduplex experiments with the DNA from a lambda phage carrying the insertion sequence IS2 show that one copy of IS2 occurs in R6, R6-5, and R100-1 (but not R1) at a point within the RTF with coordinates 67.5 TO 68.9 kilobase units (kb). In an accompanying paper, Ptashne and Cohen (1975) show that the insertion sequence IS3 occurs on R6 and R6-5. R100-25, a traC mutant, differs from its parent R100-1 only in that it contains an additional copy of IS1 inserted within the tra gene region of 82.1 kb. R100-31, atraX, TC-s mutant of R100-1, is deleted in R100-1 sequences starting at one of the IS3 termini (46.9 kb) and extending with RTF to 61.0 kb. Heteroduplex studies of F plasmids with the DNA of a lambda phage bearing insertion sequence IS2 show that the sequence of F with coordinates 16.3-17.6F is IS2. The occurrence of IS1 at the two junctions of R-determinant DNA and RTF DNA in R plasmids provides a structural basis to explain the mechanism of the previously observed formation of molecules containing one RTF unit and several tandem copies of the R-determinant unit, when R plasmids in Proteus mirabilis are grown in the presence of antibiotics, and the segregation of an R plasmid into an RTF unit and an R-determinant unit. In general, correlation of our results with previous studies shows that insertion sequences play a role in a variety of F- and R-related intra- and intermolecular recombination phenomena.


Occurrence of insertion sequence (IS) regions on plasmid deoxyribonucleic acid as direct and inverted nucleotide sequence duplications. Insertion sequence (IS) regions have been identified previously as a cause of strongly polar mutations in Escherichia coli and several bacteriophages. The present experiments indicate that genetically characterized IS regions occur on bacterial plasmid deoxyribonucleic acid (DNA) as both direct and inverted DNA sequence duplications. The DNA insertion which has been shown previously (Sharp et al., 1973) to control expression of tetracycline resistance in the R6-5 plasmid, and which occurs as directly and inversely repeated DNA sequences adjacent to the region believed to contain the tetracycline resistance gene, has been identified as IS3. A second genetically characterized insertion sequence (IS1) has been identified as a direct DNA duplication occurring at both junctions of the resistance transfer factor and R-determinant components of R6-5 and related plasmids. A model is presented for the reversible dissociation of resistance transfer factor and R-determinant components of co-integrate R plasmids at the sites of DNA sequence homology provided by the repeated IS regions.


Photosynthetic sulfide oxidation by Chloroflexus aurantiacus, a filamentous, photosynthetic, gliding bacterium. Chloroflexus, a newly described genus of filamentous, photosynthetic, gliding bacteria, oxidizes sulfide anaerobically under photoautotrophic or photoheterotrophic growh conditions and deposits elemental sulfur outside the cell. The formation of sulfur granules outside the cell supports the idea that this organism is related to the green sulfur bacteria (Chlorobiaceae).


Location of the Escherichia coli K-12 ruv gene affecting septum formation after inhibition of deoxyribonucleic acid synthesis. Escherichia coli ruv gene was located at 36.1 min on the chromosome by P1 transduction experiments and the gene order his - supD - uvrC, dar4 - ruv - eda - fadD - pps was proposed. Complementation analysis by an F' factor carrying genes in the his region indicated that ultraviolet light sensitivity genes, ruv and uvrC, consist of different cistrons and wild-type alleles of these genes are dominant over the mutant alleles.


A third kasugamycin resistance locus, ksgC, affecting ribosomal protein S2 in Escherichia coli K-12. A third kasugamycin-resistant mutant affecting ribosomal protein S2 has been isolated from Escherichia coli K-12. Mating and transduction revealed that this newly recognized kasugamycin resistance locus, designated as ksgC, is located at 0.1 to 0.2 min from purE.


Biochemical studies on radiation-sensitive mutations in bacteriophage T4-1. Mutants of bacteriophage T4 which exhibit increased sensitivity to ultraviolet radiation were isolated and their abilities to induce an enzyme system to excise pyrimidine dimers were examined.  Among 16 mutants isolated, 12 mutants were found to be defective in inducing T4 endonuclease V, which catalyzes the formation of dimer-specific breaks in ultraviolet-irradiated DNA.  A leaky v mutant, which exhibits intermediate ultraviolet sensitivity, was also isolated; the mutant induces a low level of endonuclease V and excises only a small amount of kimers in vitro.  Three other mutants, as well as x and y mutants, were able to induce both T4 endonuclease V and dimer-excision enzyme (5'yields 3' exonuclease).


Mode of replication of plasmid lambda-dv-1. About 20 copies of plasmid lambda-dv are perpetuated per host chromosome in Escherichia coli K12 cells.  The mode of DNA replication of this plasmid lambda-dv was studied, using the density-labelling technique followed by banding in DsCl.  It was shown that all the copies of plasmid lambda-dv are potentially capable of replicating roughly once a cell generation.  Their replication occurs by random, that is a copy of the plasmid is taken out at random for replication from a pool, to which the two replicas resulting from replication are returned.  Chloramphenicol did not inhibit the initiation of a new round of replication of the plasmid molecules, indicating that selection from the replication pool does not require concomitant protein synthesis.


Isolation and properties of a species produced by the partial dissociation of aspartate transcarbamylase from Escherichia coli. A species produced by the reaction of aspartate transcarbamylase (C6R6) with 6 to 12 eq of p-hydroxymercuribenzoate was isolated by DEAE-Sephadex chromatography. Purified material was completely dissociated with mercurials and the relative amounts of catalytic (C) and regulatory (R) subunits were determined by three methods: (a) quantitative cellulose acetate electrophoresis; (b) Lowry analysis after separating the catalytic and regulatory subunits by sucrose gradient centrifugation; (c) dissociation of the species with sodium dodecyl sulfate and determination of the relative amounts of catalytic and regulatory chain by sodium dodecyl sulfate gel electrophoresis. All three methods gave consistent results, indicating that the molecule consists of 75% (by weight) catalytic chain and 25% regulatory chain. The molecular weight determined by gel filtration, sedimentation velocity, and sedimentation equilibrium experiments was found to be approximately 270,000. These observations establish that this species has the structure C6R4, and is produced by the release of a single regulatory dimer R2 from the intact aspartate transcarbamylase complex. This protein (C6R4) contains 20 cysteines and four zinc ions, consistent with the proposed subunit structure. The purified intermediate C6R4 contains no mercury. The parent molecule C6R6 can be reconstituted from C6R4 by incubation with isolated regulatory subunit (R2) in the presence of zinc and beta-mercaptoethanol. Titration of C6R4 yields an end point which corresponds to the addition of 1 mol of regulatory subunit (R2) per mol of C6R4. The intermediate is quite stable at neutral pH but tends to disproportionate into aspartate transcarbamylase and catalytic subunit after prolonged storage or at elevated pH. The kinetic properties of this species have been investigated. The specific activity of C6R4 is virtually identical with that of the native enzyme but the regulatory properties are substantially reduced. Both homotropic and heterotropic interactions are reduced but not abolished, indicating that the intact structure C6R6 is not required for the allosteric transitions involved in regulation.


Interactions of plasma retinol-binding protein with its receptor. Specific binding of bovine and human retinol-binding protein to pigment epithelium cells from bovine eyes. Bovine and human retinol-retinol-binding protein (RBP) were iodinated to high specific activity. At least 34% of the iodinated material was native and retained its retinol chromophore as judged by its characteristic ability to bind to plasma prealbumin. Bovine and human retinol-125I-RBP were found to bind specifically to intact isolated pigment epithelium cells from bovine eyes. The specific binding was complete in about 1 min with an estimated second order rate constant of about 10-8 M-minus 1 S-minus 1 (at 22 degrees). The specific binding was temperature dependent with the binding at 0 degrees being some 7-fold slower than at 22 degrees. The specific binding of both human and bovine retinol-125I-RBP was a linear function of the number of binding sites (number of cells) and was saturable with respect to retinol-125I-RBP. Bound iodinated retinol-RBP was rapidly displaced by the addition of unlabeled retinol-RBP, indicating that the specific binding process was a surface phenomenon and was not due to endocytosis. At saturation about 3.7 to 5.2 times 10-4 molecules of either bovine or human retinol-125I-RBP bound to one bovine pigment epithelium cell. The dissociation constant for the binding between retinol-125I-RBP and pigment epithelium receptor was estimated to be about 5 times 10-minus 12 M. Addition of human prealbumin (thyroxine-binding prealbumin) did not affect the binding of either human or bovine retinol-125I-RBP to pigment epithelium cells. Retinol-125I-RBP did not bind specifically to isolated bovine rod photoreceptor outer segments. Human apo-RBP was less effective in displacing bound retinol-125I-RBP than either native or reconstituted human retinol-RBP. These results suggest a mechanism whereby, after delivering its retinol to the cell, apo-RBP is displaced from the specific receptor on pigment epithelium cell by another retinol-RBP molecule. This postulated mechanism makes it possible to control the delivery of retinol to the target cell by the relative plasma concentrations of apo- and retinol-RBP and their relative affinities for the specific receptor binding site.


Deoxycytidine transport in the presence of a cytidine deaminase inhibitor and the transport of uracil in Escherichia coli B. Tetrahydrouridine, a cytidine deaminase inhibitor, prevents periplasmic degradation of deoxycytidine by Escherichia coli B. It does not inhibit deoxycytidine transport and therefore allows an accurate determination of deoxycytidine transport. Data obtained using tetrahydrouridine show that deoxycytidine is transported in E. coli B as the intact nucleoside by an active transport process, with a K-m of 6 times 10-minus 6 M. Cytidine and deoxyadenosine inhibit transport competitively, whereas guanosine has no effect on transport. Arsenate or KCN greatly reduces transport. In a mutant resistant to the nucleoside antibiotic, showdomycin, the active transport of deoxycytidine is lost, and residual slow uptake occurs by passive diffusion. Uracil is accumulated in E. coli B by an active transport process with a K-m of 5 times 10-minus 7 M.


The sequence of amino acid residues around the oxidation-reduction active disulfide in yeast glutathione reductase. A 14-residue peptide containing the oxidation-reduction active cystine residue from yeast glutathione reductase has been isolated from proteolytic digests of the enzyme in which the free sulfhydryl groups had been reacted with N-ethylmaleimide. The sequence of this disulfide-containing peptide was found to be:(see article). The sequence was highly homologous with the active cystine regions in Escherichia coli and pig heart lipoamide dehydrogenase. The sequences of three of the postulated four thiol-containing regions of the enzyme are also presented, as well as evidence supporting the view that the enzyme is composed of two identical subunits.


The mechanism of aminoacylation of transfer ribonucleic acid. Reactivity of enzyme-bound isoleucyl adenylate. Isoleucyl adenylate bound to isoleucine:tRNA ligase of Escherichia coli (EC 6.1.1.5; isoleucyl-tRNA synthetase) transfers the isoleucine moiety to tRNA-Ile-E. coli with a half-time of about 35 s at 0 degrees and pH 7.6 in the presence of spermine or Mg2+. If a limited amount of tRNA-Ile is supplied to a mixture of free enzyme and enzyme-bound [14c]isoleucyl adenylate in a medium containing spermine, ATP, and [3H]isoleucine, almost none of the resultant isoleucyl tRNA is derived from preformed enzyme-bound [14C]isoleucyl adenylate. Almost all of the isoleucyl tRNA formed results directly from reaction of free enzyme, ATP, and isoleucine with tRNA. Similar but less clearcut results are obtained when Mg2+ is substituted for spermine. We conclude that isoleucyl adenylate bound to isoleucine:tRNA ligase is not a significant intermediate in the synthesis of isoleucyl tRNA under these conditions.


Cross-linking of Escherichia coli succinic thiokinase. I. Reaction with diiminoesters and dimaleimides. Treatment of the tetrameric alpha2 beta2 protein succinic thiokinase of Escherichia coli with dimethylsuberimidate (DMS) yielded five protein species detectable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These five protein species had estimated molecular weight values of 29,500, 41,000, 73,000, 117,000, and 132,000, and corresponded to alpha monomer, beta monomer, alpha beta dimer, alpha2 beta trimer and alpha2 beta2 tetramer, respectively. In all cases, the cross-linking produced predominantly the 73,000 molecular weight dimer with respectively lesser amounts of the tetramer and trimer. Succinic thiokinase was also cross-linked by reaction with N, N'-o-phynylenedimaleimide or with N, N'-P-phenylenedimaleimide. In these instances, treatment produced the alpha beta dimer as the only oligomeric species. Ammonolysis of isolated tetramer, trimer, and dimer produced by DMS treatment gave the 29,500 molecular weight monomer (alpha monomer) and the 38,500 molecular weight monomer (beta monomer). The absence of dimers of like subunits and the predominance of the dimer of unlike subunits are consistent with a quaternary structure of the native enzyme in which unlike subunits are closely associated but like subunits are not. Under certain conditions, an additional dimer of approximately 60,000 molecular weight was produced. This appeared to result from cleavage of the beta chain of an alpha beta dimer. Phosphorylation of native succinic thiokinase with [gamma-32P]ATP and [gamma-32P]GTP showed radioactivity only in the alpha monomer. Phosphorylation of enzyme before or after cross-linking showed radioactivity in all cross-linked bands except the beta monomer. Experiments in which the enzyme was titrated with [14C]DMS and trinitrobenzenesulfonate revealed that approximately half of the available amino groups reacted with the diiminoester, but that a small fraction of these (smaller than 20%) had reacted bifunctionally


Purification and properties of 7, 8-diaminopelargonic acid aminotransferase. The enzyme 7, 8-diaminopelargonic acid aminotransferase utilizes S-adenosyl-L-methionine to transaminate the biotin precurson 7-keto-8-aminopelargonic acid and form the next intermediate in the pathway, 7, 8-diaminopelargonic acid. The enzyme has been purified nearly 1000-fold from an extract of a regulatory mutant of Escherichia coli which is derepressed for the enzymes of the biotin operon. The extract was treated with protamine sulfate, ammonium sulfate, and subjected to acid and heat treatments. Subsequently, the enzyme was chromatographed on columns of DEAE-cellulose, phosphocellulose, hydroxylapatite, and two Sephadex G-100. The resulting purified preparation was judged 86% homogeneous by the scanning of of a stained disc gel. The enzymatic activity was associated with the major band in gels run at two different gel concentrations and two different pH values. The cofactor, pyridoxal phosphate, can be resolved from the enzyme in the presence of phosphate buffer after incubation with the amino donor, S-adenosyl-L-methionine. A molecular weight estimation of 94,000 plus or minus 10, 000 has been obtained by gel filtration and sucrose gradient sedimentation studies. Gel electrophoresis in the presence of sodium dodecyl sulfate, shows a single subunit with a molecular weight of 47, 000 plus or minus 3, 000 indicating a dimeric enzyme. A neutral compound was detected in the acidified reaction mixture which was derived from the methionine moiety of S-adenosyl-L-methionine and was present in amounts equivalent to the 7, 8-diaminopelargonic acid produced in the reaction mixture. It is suggested that the keto product of the reaction, i.e. S-adenosyl-2-oxo-4-methylthiobutyric acid, may decompose nonenzymatically under the conditions of the reaction to form 5'-methylthioadenosine and the neutral compound, 2-oxo-3-butenoic acid.


Biosynthesis of 7, 8-diaminopelargonic acid from  7-keto-8-aminopelargonic acid and S-adenosyl-L-methionine. The kinetics of the reaction. The transamination of 7-keto-8-aminopelargonic acid by 7, 8-diaminopelargonic acid aminotransferase of Escherichia coli requires S-adenosyl-L-methionine as the amino donor. Initial velocity studies of this reaction revealed a parallel pattern of reciprocal plots characteristic of a ping-pong mechanism. m-Keto-8-aminopelargonic acid showed strong substrate inhibition which was competitive with S-adenosyl-L-methionine. The Michaelis constants determined for S-adenosyl-L-methionine and 7-keto-8-aminopelargonic acid were 0.20 mM and 1.2 muM, respectively. The Vmax of 0.16 mumol/mg/min corresponds to a turnover number for the enzyme of only 17 molecules/molecule enzyme/min. The Km values for the interaction of pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate with the apoenzyme were determined to be 32 muM and 21 muM, respectively. Two classes of inhibitors were observed: (a) those which showed competitive inhibition with respect to S-adenosynd (b) those which showed noncompetitive inhibition with respect to both substrates. In the former group were S-adenosyl-L-(2-hydroxy-4-methylthio)butyric acid and adenosine. In the latter were S-adenosyl-L-ethionine, adenine, and 8-keto-7-aminopelargonic acid. L-Methionine, S-methyl-L-methionine, inosine, and hypoxanthine were not significantly inhibitory. Certain conformations of the substrates in the active site of the enzyme have been proposed which explain: (a) the requirement for the sulfonium ion of S-adenosyl-L-methionine for activity but not for binding to the enzyme, and (b) the ability of 7-keto-8-aminopelargonic acid to bind to the pyridoxal form of the enzyme as a potent substrate inhibitor.


The steady state kinetic parameters and non-processivity of Escherichia coli deoxyribonucleic acid polymerase I. A steady state kinetic study of Escherichia coli DNA polymerase I has been carried out using poly[d(A-T)] as the template-primer substrate. The results of substrate saturation and product inhibition kinetic studies suggest an altered Ordered Bi Bi mechanism for the enzyme. The Michaelis constants for polymer, d-atp, and dTTP are 5 nM (3'-OH ends), 1 muM, and 2 muM, respectively. The apparent equilibrium constant for the reaction, Keq equals [PPi]/[dNTP], was estimated as greater than or equal to 500. No quaternary complex of enzyme, template, and both deoxynucleoside triphosphates was detected. Single turnover experiments at 4 degrees indicated that the enzyme functions non-processively under the specified conditions, that is, dissociates after each catalytic step. The results at higher temperature were consistent with dissociation within 30 steps. Furthermore, at 4 degrees a burst of incorporation stoichiometric with the amount of enzyme was observed upon initiation of the reaction, indicating that the rate-limiting step in the steady state occurs after phosphodiester bond formation. There is a linear Arrhenius dependence of the initial reaction on temperature in the range 4-40 degrees, with an apparent Ea equals 17 kcal/mol. The rate equations appropriate for template-dependent polymerases which dissociate after each catalytic step have been derived.


Characterization of fragments of 16 S ribonucleic acid protected against pancreatic ribonuclease digestion by ribosomal protein S4. Digestion of reconstituted complexes of 16 S RNA and ribosomal protein S4 with pancreatic ribonuclease, followed by resolution of the products on polyacrylamide gels of low ionic strength, gives rise to a protected ribonucleoprotein fragment with a mobility of 7 S. Fingerprinting of the RNA moiety indicates the presence of sequences covering the 5' one-third of the 16 S RNA. After its extraction from the acrylamide gels, the protected RNA from the complex can be separated into two components by sedimentation in sucrose gradients. One of these retains the ability to reassociate specifically with S4, as judged from its stoichiometry of binding in the presence of excess S4 and from its selection of S4 alone from a mixture of unfractionated 30S ribosomal proteins. It spans the same regions of the 16 S RNA as the total protected RNA, but lacks an internal sequence of about 120 nucleotides. This component contains a number of nicks which give rise to discrete bands upon electrophoresis in urea. The principal subfragments include Sections L through F (230 nucleotides), B through I'' (160 nucleotides), and part of section C'' (35 to 40 nucleotides). It is proposed that the binding site for S4 lies within these noncontiguous sequences, in support of the hypothesis that S4 makes multiple contacts with the 16 S RNA.


The role of S-adenosylmethionine in the cleavage of deoxyribonucleic acid by the restriction endonuclease from Escherichia coli K. The restriction endonuclease from Escherichia coli K specifically cleaves foreign DNA in the presence of S-adenosylmethionine, ATP, and Mg2+. The role of S-adenosylmethionine in this reaction has been studied by following the specific binding of the enzyme to unmodified DNA. The results indicate that S-adenosylmethionine acts as an allosteric effector. However, the rate-limiting step in the activation of the enzyme is not the binding of the effector itself, but an event subsequent to it. The interaction of the S-adenosylmethionine with two mutant K restriction endonucleases isolated previously has also been investigated. One of them, which is defective in restriction, can be activated in a manner similar to the wild type enzyme, while the other one, which lacks both restriction and modification activities (due to a mutation in the subunit responsible for DNA recognition), shows no such effect.


Nucleotide sequence determination of bacteriophage T4 species I ribonucleic acid. The nucleotide sequence of T4 species I RNA, one of several stable RNA's specifically coded for by bacteriophage T4, has been determined using 32-P-labeled material from T4-infected cultures of Escherichia coli. The purified RNA species which has been sequenced has been shown to hybridize well to T4 DNA (Wilson J.H., Kim, J.S., and Abelson, J.N. (1972) J. Mol. Biol. 71, 547-556). The sequence is: pCGAUUCGAGGAAAUAUCUUUGCCGUAAGCCGAGUAGCGUUUUUGACGGAACGUUCGGAUAUGGUUGAGAUAUGGCCUUUUAAAAUAUUGAGUAGCGUCAACUACUUAAUAACCGGGUUCGAAUCCCGGCGUUUCGU-CAA-OHACA-OH. Species I RNA which is 140 nucleotides long is also found to occur in shorter versions with 135 to 136 nucleotides which terminate with a 3'-phosphate. The molecule can be arranged in a secondary structure which shows some striking similarities to the classic cloverleaf pattern of a tRNA. The molecule is specifically cleaved by an E. coli nuclease into three segments by cleavage at a double-stranded region in the molecule. The function of species I RNA is unknown, but evidence presented elsewhere (Paddock, G.V., and Abelson, J. (1975) J. Biol. Chem. 250, 4207-4219) indicates that the gene for this RNA molecule has been preserved in evolution. The position of a mutation within species I RNA has been determined. This mutation results in incorrect processing of the RNA and lower relative yields of the RNA are present.


Nucleotide sequence determination of bacteriophage T2 and T6 species I ribonucleic acids. The nucleotide sequences of species I RNA coded for by bacteriophages T2 and T6 have been analyzed using 32-P-labeled material from T2 and T6-infected cultures of Escherichia coli. The T1 and pancreatic ribonuclease digestion products were partially analyzed and the results were compared with nucleotide sequences from T4 species I RNA to obtain a minimum estimate of the number of nucleotide sequence differences among the three species I RNAs. Analysis of fragments obtained by digestion with epsilon-carboxymethyl-lysine-41-pancreatic ribonuclease and with E. coli Q13 S30 crude extract was also performed to provide some additional confirmation for the nucleotide sequences that were derived for the T2 and T6 species I RNAs. T2 species I RNA was found to be different at three positions in the nucleotide sequence, and unlike T4 species I RNA, contained in addition the modified nucleotide, psi, in a region where the proposed secondary structure is identical to the TpsiC-loop of a tRNA. T6 species I RNA was found to contain nucleotide differences from the T4 species I RNA sequence at four positions. The U at position 119 in the sequence appears to be modified to psi only to a small extent. While a biological function for species I RNA is unknown, the fact that there is over 97% homology in the sequences suggests strong evolutionary pressures to retain the nucleotide sequence in the T-even genomes.


Localization of D-lactate dehydrogenase in native and reconstituted Escherichia coli membrane vesicles. In the preceding paper the preparation and characterization of antiserum to purified D-lactate are described. In this paper the effects of the antibody on D-lactate dehydrogenase activity and D-lactate-dependent active transport in native Escherichia coli ML 308-225 membrane vesicles and ML 308-225dld-3 vesicles reconstituted with D-lactate dehydrogenase are described. The results demonstrate that D-lactate dehydrogenase is inaccessible to antibody in native ML 308-225 vesicles, but readily accessible to antibody in reconstituted dld-3 vesicles. The findings indicate that D-lactate dehydrogenase is located on the inner surface of native ML 308-225 vesicles and on the outer surface of reconstituted dld-3 vesicles. The results with the native vesicle preparations also provide further evidence that virtually none of the vesicles is inverted or sufficiently damaged to allow access of antibody to D-lactate dehydrogenase. In addition, experiments are presented which demonstrate that an impermeable electron carrier, reduced 5-N-methylphenazonium-3-sulfonate, drives active transport in native ML 308-225 vesicles as well as its permeable analogue reduced phenazine methosulfate. Thus, reduction of the respiratory chain from either side of the vesicle membrane is able to drive active transport. Ca2+, Mg2+-stimulated ATPase is also inaccessible to antibody in ML 308-225 vesicles unless the preparation is subjected to ultrasonic sound, incubated in Tris buffer at pH 9.0, or homogenized vigorously. Moreover, as opposed to D-lactate dehydrogenase and cytochrome b1, ATPase is readily lost from the membrane during the preparation of vesicles.


Interaction of deoxycholate and of detergents with the coat protein of bacteriophage f1. The major coat protein of bacteriophage f1, which is localized in the host membrane during phage maturation, has a hydrophobic binding site capable of binding deoxycholate and a variety of detergents to form a soluble particle, and in that respect, resembles many membrane proteins. The soluble particle has properties that suggest it is formed by simple insertion of protein into a deoxycholate or detergent micelle, but molecular weight measurements show that the protein is present as a dimer, even in sodium dodecyl sulfate, indicating the existence of unusually strong forces for self-association. A by-product of the investigation has been to show that detergents can be very helpful in the fractionation of the constituent molecules of the virus: deoxycholate-solubilized virus is readily fractionated by gel chromatography into DNA, A protein, and B protein, with virtually no cross-contamination.


A historical review of the histology of patent autogenous vein grafts and vein patches. The literature on the histological changes occurring in autogenous vein grafts and patches was reviewed. Over 307 grafts (235 animal and 72 human) and 30 patches (28 animal and 2 human) have been described. Grafts were investigated from patients who died on the operating table up to 22 years after operation. Intimal thickening and various changes in the media (including necrosis and replacement fibrosis) occurred. Dilatation of vein grafts has occurred, and anerysm formation has been reported. Atheroma has been described in the grafts from 5 patients.


Operations for lymphoedema of the lower limbs. A study of the results in 108 operations utilizing vascularized dermal flaps. The results of operations for lymphoedema of the lower limbs have been reviewed. In experienced hands the results are very satisfactory. Modifications of the buried dermis flap operation of Thompson have been found to give the best results in patients where the local skin has been in good condition. The chief complication has been the occurrence of localized areas of necrosis, chiefly at the edges of the flaps. It has not been a serious complication but has sometimes prolonged the stay in hospital. It has been associated with haematoma formation. Methods of avoiding this and other complications are discussed in order to improve results still further. There have been no serious complications and no deaths in this series.