Subcellular fractions of vascular smooth muscle exhibiting calcium transport properties.

Vascular smooth muscle relies on two general sources of activator calcium, one the extracellular fluid and the other intracellular sites in the coupling between stimulation at the external membrane and tension development. Electron microscopic studies have demonstrated at least two subcellular elements, mitochondria and sarcoplasmic reticulum, that may participate in the release and/or sequestration of this activator calcium. In order to obtain quantitative answers to questions concerning the rates of calcium transport by these subcellular elements attempts to use differential centrifugation to obtain subcellular fractions from whole muscle homogenate have been reported. Mitochondrial enriched preparations have received little quantitative study as yet, although they possess active calcium uptake mechanisms. Nonmitochondrial preparations having active calcium uptake mechanisms have been found in a variety of smooth muscles. The method of preparation and properties of these nonmitochondrial preparations show a great deal of variation, indicating they are probably a heterogeneous mixture of sarcoplasmic reticulum and plasma membrane. Some preliminary experiments on the effects of vasoactive agents and cyclic AMP on the calcium transport by these nonmitochondrial preparations have yielded conflicting results. Resolution of these problems awaits clear identification of the calcium transport mechanisms with their appropriate subcellular structure.


Nutrition and cancer.

There is increasing epidemiological evidence that nutrition plays a dominant role in the pathogenesis of several types of human cancers. There is considerable epidemiological evidence showing that alcoholism in part because of associated nutritional deficiencies, significantly increases the risk of smokers for cancer of the alimentary tract. There is also some suggestion that nutritional deficiencies may relate to cancers of the stomach, cervix, and thyroid. Of particular importance, and based on relatively new concepts, are data indicating that overnutrition significantly affects the development of certain cancers, including cancers of the colon and pancreas, kidney, breast, ovary endometrium, and prostate. Except for cancer of the endometrium, and kidney cancer in women, there is no significant relationship to obesity. Rather, the evidence suggests both epidemiologically and experimentally that the etiological factors relate to a high intake of fats and possibly other variables associated with high fat intake. While we are investigating the mechanistic nature of the epidemiological and experimental observations, the question that needs to be asked is whether it is not prudent for us to associate ourselves with the recommendation of our colleagues in the cardiovascular disease field who call on both individuals and the food industry to practice a "Prudent Diet," i.e., one that is lower in total calories, total fat, saturated fats and cholesterol than is the present American diet.


Carcinogens occurring naturally in foods.

Humans are susceptible to the carcinogenic action of a small group of organic and inorganic chemicals in certain industrial, medical, and social habit exposures. A larger number and wider variety of chemical carcinogens, primarily organic compounds, are known for experimental animals. Chemical carcinogens are also found among the metabolites of living cells. No common structure is evident among chemical carcinogens, and a majority of these agents are precarcinogens that require metabolic activation into reactive electrophilic ultimate carcinogens. These strong electrophiles combine covalently with nucleophilic sites in DNAs, RNAs, and proteins in target tissues. One or more of these adducts appear to initiate carcinogenesis. About 20 naturally occurring organic chemical carcinogens, primarily metabolites of green plants and fungi, are known; some occur in some human foods. Many other naturally chemical carcingens doubtless exist among the vast number of uncharacterized nonnutritive minor components of living systems, some of which are sources of human foods. The electrophilic forms of chemical carcinogens are mutagenic, and mammalian tissue-mediated mutagenicity assays appear promising in the detection of potential chemical carcinogens. These assays should serve at least as a prescreen for conventional lifetime tests in rodents for the carcinogenic activity of food components and contaminants. Epidemiological approaches appear necessary to evaluate the importance of the naturally occurring chemical carcinogens in the occurrence of human cancer.


Prevention of chemical carcinogenesis by vitamin A and its synthetic analogs (retinoids).

An approach to chemoprevention of common forms of epithelial cancer, during the period of preneoplasia, is described. Vitamin A and its synthetic analogs (retinoids) are potent agents for control of cell differentiation in many epithelial tissues. Direct effects of retinoids on normal and preneoplastic cell differentiation can be measured in organ culture. In experimental animals, deficiency of dietary retinoids enhances susceptibility to chemical carcinogenesis. Natural retinoids, fed at high dietary levels, have some ability to prevent chemical carcinogenesis in epithelial tissues of bronchi, trachea, stomach, uterus, and skin of experimental animals. However, natural retinoids have limited usefulness for chemoprevention of cancer because of inadequate tissue distribution and excessive toxicity. Synthetic retinoids have been made and shown to be more potent and less toxic for prevention of cancer in animals. Several structural modifications of the ring and terminal portions of the retinoid molecule have significant biological activity; modification of the side chain has been more difficult. The potential future usefulness of this approach to cancer prevention in man will depend on further synthetic modification of the retinoid molecule.


[Mitogenic activity of Brucella fractions on cultured lymphocytes].

LPS from smooth B. melitensis extracted by the phenol-water method has no mitogenic activity for lymphocytes of men or mice uninfected by brucellosis, in amounts that are not toxic in vitro for these cells. Mitogenic activities of other B. melitensis fractions are evidenced only in amounts 100-fold greater than an effective dose of LPS from E. coli. We interpret these findings as reflecting a virgin immune status against brucella antigens.


[Postoperative bacterial endocarditis after cardiac prosthesis (author's transl)].

The infection of an endocardial prosthesis, either valvular or septal patch, is a frequent and dangerous post-operative complication. Eleven patients having postoperative endocarditis (9 on valve prosthesis and 2 on ventricular septal patch) are the subject of this paper. Six of the 7 infected "early" had Gram-negative bacteria in the blood cultures, while staphylococus aureus, streptococcus viridans and klebsiella pneumoniae were responsible for 3 of the 4 "late" infections. In 2 patients, one "early" and one "late" the causative bacteria were not identified. The definite prevalence of Gram-negative flora in post-operative endocarditis may have been facilitated by the routine use of antibiotics after open-heart surgery. Three of the 11 patients survived, one after replacement of the infected prosthesis and two after prolonged specific antibiotic treatment. On the basic of this experience, compared with other written reports, the authors propose to treat postoperative endocarditis medically for approximately 5 weeks, with full doses of specific antibiotics, reserving for surgical treatment only the cases of prosthetic malfunction, left atrial thrombosis, peripheral embolization and in those patients where the medical treatment fails.


A prospective controlled trial of azathioprine in primary biliary cirrhosis.

Between 1968 and 1974, azathioprine has been used in a controlled prospective trial to treat patients with symptomatic but precirrhotic primary cirrhosis. Forty-five patients were admitted, of whom 22 were given azathioprine in a dose of 2 mg per kg of body weight. During the 1st year, serum aspartate transaminase levels showed a significant change in favor of the treated group, but improvement did not continue. Throughout the trial, serum alkaline phosphatase, bilirubin, cholesterol, albumin and immunoglobulin M values showed no significant change. Titers of serum mitochondrial antibodies tended to become negative more often in the treated than the untreated. Pruritus cannot be assessed objectively, but seemed less in the treated than in controls. Serial hepatic biopsy specimens showed the development of cirrhosis equally in the two groups. Survival, as judged by the life table method, was similar for the first 5 years of the trial. There was, however, a significant difference in favor of the treated group in the 6th year, although the number of patients available for assessment at that time was extremely small.


Pepsin secretion, pepsinogen, and gastrin in "Barrett's esophagus." Clinical and morphological characteristics.

Four cases of Barrett's esophagus are presented. Three cases presented with significant esophageal bleeding and one case presented with high esophageal stricture. Gastrointestinal panendoscopy was done in each case and multiple biopsies were taken. The biopsies were utilized for histomorphology, pepsinogen agar gel electrophoresis, and tissue gastrin assays. Tissue gastrin levels in esophageal mucosa were elevated in 2 cases when compared to controls with and without hiatus hernia. Pepsin and acid secretory studies were done by isolating the esophagus. Barrett's esophagus was shown to produce pepsin by both chemical studies (2 cases) and agar gel electrophoresis at pH 5.7 (3 cases), and was also shown to produce acid. The mucosa contained either cathepsin or cathepsin and pepsinogens in all cases. Nissen's fundoplication was performed in all of the patients. Of 3 patients who were bleeding, 2 who consented for this operation stopped bleeding after the operation. It is to be noted that the usual clinical treatment of antacids, bedrest, and raising the head end of the bed failed in all of the patients. The follow-up of 9 months to 3 years postoperatively has shown persistence of Barrett's mucosa with no evidence for any reversion to normal esophageal type.


Comparison of methylated prostaglandin E2 analogues given orally in the inhibition of gastric responses to pentagastrin and peptone meal in man.

In 32 healthy male volunteers the effects on gastric secretion of three methyl analogues of prostaglandin (PG) E2 have been studied, namel, 15 (R) -15-methyl PGE2 methyl ester, 15 (S) -15-methyl PGE2 methyl ester, and 16, 16-dimethyl PGE2. Secretion was measured for 30 min and a PG analogue at doses ranging from 1.25 to 2.5 mug per kg or a placebo was administered. Gastric secretion was then stimulated either by an intravenous infusion of pentagastrin (2 mug per kg-hr) or by a peptone meal with acid secretion determined by intragastric titration technique. The tests were randomized and double blind. All three methyl PG analogues exhibited a profound and prolonged inhibitory action on gastric acid and pepsin secretion induced by pentagastrin. PG analogues caused almost complete inhibition of gastric acid response to a peptone meal accompanied by a significant reduction in the serum concentration of immunoassayable gastrin. Except with the highest dose of PG (S) -15-methyl PGE2 methyl ester, which caused abdominal discomfort and single episodes of diarrhea in some subjects, no symptoms or untoward biochemical effects were observed. It is concluded that these methylated PG analogues are very potent inhibitors of gastric acid and pepsin secretion stimulated by pentagastrin or a meal and may have clinical potential in the treatment of peptic ulcer.


Terminal redundancy heterozygotes involving the first-step-transfer region of the bacteriophage T5 chromosome.

Individual progeny of two-factor crosses between A1am and A2am T5 phages give rise to bursts containing more than one type of plaque. The simplest explanation for these mixed bursts is that the A1 and A2 genes are located within the terminally repeated portion of the T5 genome and that the mixed bursts are made by "terminal redundancy heterozygotes". The observation of genetic heterozygosity means that the A1 and A2 genes are repeated intact. This implies that the terminal segments of T5 are genetically interchangeable.


Deletions induced by heat treatment of E. coli K12 lysogenic for lambda prophages.

We have investigated the production of prophage deletions in heat-induced lambda lysogens of E. coli K12. Our results are indicative of a direct action of the heat-induced prophage in producing deletions. The temperature of 40 degrees used for the experiments may be critical to prove this effect. The phage function involved in deletion formation is not known.


Regulation of mating and meiosis in yeast by the mating-type region.

A supposed sporulation-deficient mutation of Saccharomyces cerevisiae is found to affect mating in haploids and in diploids, and to be inseparable from the mating-type locus by recombination. The mutation is regarded as a defective a allele and is designated a*. This is confirmed by its dominance relations in diploids, triploids, and tetraploids. Tetrad analysis of tetraploids and of their sporulating diploid progeny suggests the existence of an additional locus, RME, which regulates sporulation in yeast strains that can mate. Thus the recessive homozygous constitution rme/rm- enables the diploids a*/alpha, a/a*, and alpha/alpha to go through meiosis. Haploids carrying rme show apparent premeiotic DNA replication in sporulation conditions. This new regulatory locus is linked to the centromere of the mating-type chromosome, and its two alleles, rme and RME, are found among standard laboratory strains.


UV mutagenesis in radiation-sensitive strains of yeast.

The yeast Saccharomyces cerevisiae appears to possess a single mutagenic or "error prony" pathway for the repair of UV damage; rev1, rev2, rev3 (Lemontt 1971a), rad6, rad8, rad9 and rad18 (Lawrence et al. 1974; present results). Strains carrying rad6 are the most sensitive to the lethal effects of UV light in this group and double mutants carrying rad6 and either rev1, rev3, rad9 or rad18 are no more sensitive than this single mutant strain, rev3 rad6 doubl- mutant diploids failed to show any UV-induced reversion of the normally highly reversion of the normally highly revertible ochre allele cycl-9, even though a total of more than 2.5 X 10(9) viable cells was examined, suggesting that strains of this kind are entirely UV-immutable; spontaneous revertants could be recovered, however.-The rad6 and rev3 gene products would appear to be necessary for all kinds of mutagenic events at all sites within the genome, but the products of the other genes that act in the "error-prone" pathway have a more restricted role and are involved in the production of only some kinds of mutations. It is suggested that such selectivity arises from the interaction of some repair enzymes with specific nucleotide sequences.


Mutants of the killer plasmid of Saccharomyces cerevisiae dependent on chromosomal diploidy for expression and maintenance.

Mutants of the killer plasmid of Saccharomyces cerevisiae have been isolated that depend upon chromosomal diploidy for the expression of plasmid functions and for replication or maintenance of the plasmid itself. These mutants are not defective in any chromosomal gene needed for expression or replication of the killer plasmid.--Haploids carrying these mutant plasmids (called d for diploid-depen;ent) are either unable to kill or unable to resist being killed or both and show frequent loss of the plasmid. The wild-type phenotype (K+R+) is restored by mating the d plasmid-carrying strain with either (a) a wild-type sensitive strain which apparently has no killer plasmid; (b) a strain which has been cured of the killer plasmid by growth at elevated temperature; (c) a strain which has been cured of the plasmid by growth in the presence of cycloheximide; (d) a strain which has lost the plasmid because it carries a mutation in a chromosomal mak gene; or (e) a strain of the opposite mating type which carries the same d plasmid and has the same defective phenotype, indicating that the restoration of the normal phenotype is not due to recombination between plasmid genomes or complementation of plasmid or chromosomal genes.--Sporulation of the phenotypically K+R+ diploids formed in matings between d and wild-type nonkiller strains yields tetrads, all four of whose haploid spores are defective for killing or resistance or maintenance of the plasmid or a combination of these. Every defective phenotype may be found among the segregants of a single diploid clone carrying a d plasmid. These defective segregants resume the normal killer phenotype in the diploids formed when a second round of mating is performed, and the segregants from a second round of meiosis and sporulation are again defective.


Obesity and joint disease.

In male mice of strain C57Bl, obesity caused by intraperitoneal administration of aurothioglucose failed to alter the course of spontaneous osteoarthrosis. The distribution of osteoarthrosis of the knee joint and of spondylosis of the thoracic and lumbar spine was haphazard in regard to body weight, and levels of plasma and hepatic lipids. It is concluded that the arthrosis-promoting effects of fat-enriched diets are not mediated by the simultaneously occurring obesity.


The use of locally based triangular skin flaps for the repair of finger tip injuries.

This paper presents the results of a series of thirty-one finger tip injuries treated by local triangular advancement flaps. Criteria for selection of patients for this type of repair and several points of technique are discussed.


The effect of bacterial adjuvants on Trypanosoma lewisi infections in rats.

The effect of Escherichia coli endotoxin, B.C.G. vaccine and Bordetella pertussis vaccine on the parasitemia of Trypanosoma lewisi was studied in rats. It was found that in general, B. pertussis administration resulted in higher and more prolonged parasitemias than normal. In contrast E. coli endotoxin tended to reduce both the intensity and duration of parasitemia. Possible reasons for this phenomenon are discussed.


Scanning micrographs of the first legs and gnathosoma of two species of Myobiidae (Acarina: Trombidiformes).

The structure of legs I and of gnathosoma in Radfordia lemnina (Koch, 1835) and Protomyobia claparedei (Poppe, 1896) is described on the basis of SEM micrographs.


[Studies on proteinuria following kidney transplantation].

The method of discontinuous micro-disc-electrophoresis was used to characterize the proteinuria after kidney transplantation in man for the early diagnosis of acute rejection crisis. By daily analysis of proteinuria the increase of albuminuria to more than 1 g per 24 h was found to be a sure sign of a beginning rejection crisis. In one third of our cases the rejection crisis could be recognized 7 days and in 80% 3 days before clinical onset. Besides increased albuminuria the appearance of low molecular weight proteins in urine and in 50% gamma globulinuria are further criteria for the rejection crisis. The described method is also very useful for the differential diagnosis of rejection crisis and inflammation in addition to cytological examination of the urine.


[Evidences for the somatosis hypothesis of schizophrenia (author's transl)].

The hypothesis of a hereditary mutative enzyme disturbance which becomes effective via biochemical, for peristatic influences susceptible cerebral mechanisms, is compatible with all findings. The evidences are stated. Schizophrenia is a predominantly hereditary disease. The findings of the family and twin research confirm this statement, in particular the difference of the concordance figures with monovular and binovular twins, furthermore the existence of substrate-close basic disturbances with their analogies to cerebro-organic symptomatology, the frequency and structure of pure residual syndromes and the results of clinical-encephalographic correlation examinations. Slightly marked internal brain atrophies which can be demonstrated by pneumo- and echoencephalogram can be correlated with the "pure defect" which is the most frequent residual syndrome of schizophrenic diseases. A geneticly conditioned cerebral enzyme defect can cause an atrophy in the region of the limbic system; however, a merely functional decompensation of a cerebral enzyme disturbance, without atrophy is imaginable in a part of the schizophrenia and cyclothymia. In certain active basic stages the EEG reveals abnormal rhythms ("parenrhythmiae"), which also allow a topical classification to functional structures of the limbic system. The non-characteristic organic "pure potential reduction" is irreversible, the typically schizophrenic syndromes potentially reversible. Basic disturbances and basic syndromes are in prodromes and outpost syndromes prior to manifestation of psychosis and after their remission the actually primary symptoms. The typically schizophrenic syndrome results from the amalgamation of the basal functional disturbance with the "anthropological matrix", partly provoked by - also non-specific - stress factors. The substrate-close basic symptoms: coenesthesias, perception disorders, zentral-vegetative dysregulations and cognitive primary disturbances can be seen as expression of a pathologically altered cerebral function in the region of the integrative system which is responsible for the regulation of the cerebral filter and protection processes.


[Radiation injury of the spinal cord].

The literature dealing with radiation myelopathy is reviewed. The following points are to be noticed:radiation myelopathy is a rare complication in the radiation therapy of extraspinal tumors, nevertheless the number of case reports is increasing during the last years; probably this is due to the increasing use of high energy therapy. Already a cord dose of 1000 rad may be dangerous; with an increasing dose the risk of radiation myelopathy is increasing too. Besides the total dose the incidence of radiation myelopathy depends on the rate of delivery, the over-all time of administration, the size of the individual fraction, the field size, the size of the volume irradiated, the type of irradiation, the use of hyperbaric oxygen and some other special conditions of radiation. But the incidence of radiation myelopathy depends not only on radiation technique but also on patients' variables. Individual variations in radiosensitivity are a well known fact; this may be partly due to an inherent biologic variation of response. Moreover the incidence of radiation myelopathy may be intensified by simultaneously existing diseases - above all by hypertension- and probably by some medicaments taken simultaneously. A dependence from age, sex, and the kind of the primary tumor seems not to exist. Radiation lesions of the cervical spinal cord have been reported much more frequently than lesions of the dorsal spinal cord; lesions of the lumbal spinal cord are a very rare event. There exist different conceptions of the pathogenesis: opinions differ as to whether the effect is primarily on the connective tissue and blood vessels or on nerve cells and their axons or if the different tissues are injured simultaneously; moreover an autoimmuno-hypothesis is discussed. The clinical signs of radiation myelopathy can be grouped into two major syndromes: the transient radiation myelopathy and the delayed or chronic radiation myelopathy, which usually develops gradually with a subsequent chronic progressive course but in some cases may occur acutely after the latent period; the course is not always progressive but may be undulating and remissions have been reported in some rare cases...


Clinical evaluation of a new sulfonylurea in maturity onset diabetes - glipizide (K-4024).

Glipizide, a new low dose sulfonylurea, was evaluated for its efficacy and toxicity in a double-blind controlled study. Forty adult-onset diabetics were treated with either chlorpropamide or glipizide. In ten out of twenty patients "excellent" to "good" control of hyperglycemia was achieved with glipizide and two patients evidenced "fair" control. In nine out of eighteen patients "excellent" to "good" control was achieved with chlorpropamide. Eight of the sixteen patients who were primary or secondary failures on the two drugs responded to glipizide and phenformin combination with "excellent" to "good" control. No therapeutic advantage was found in giving more than 25 mg/day glipizide in ten patients. Toxicity was low and side effects were uncommon over a period of 26 months.


Laboratory approaches to the identification of Enterobacteriaceae.

There are numerous approaches to the identification of the Enterobacteriaceae in the clinical laboratory. Accuracy in identifying organisms is related directly to the skill and experience of the user and to the number of tests inoculated. Many of the commercially available kits offer reproducibility, convenience, accuracy, and ease of storage, particularly for smaller hospital laboratories that have neither the facilities nor the expertise to prepare and exert quality control over microbiologic media. However, none of the kits offers the accuracy and few offer the flexibility available through the use of conventional media, the variety and duration of incubation of which are virtually unlimited. The proper use of any system requires training because accurate identification requires the concurrent examination of colonial morphology and biochemical reactions-manufacturer's flow charts, reaction tables, or printed recognition systems notwithstanding. Microbiologists should be wary of new systems before impartial evaluations have been performed and published in scientific journals; manufacturers' technical literature and salesmen's presentations are, for obvious reasons, seldom objective.


Isolation and identification of anaerobic bacteria.

Anaerobic bacteria make up a predominant part of the normal human flora. Adequate specimen collection must avoid contamination with this flora. Suitable methods include thoracentesis, transtracheal aspiration, needle and syringe aspiration of closed abscesses, and endocervical aspiration of intrauterine pus. Swabs are generally unsuitable. Sputum, voided urine, vaginal secretions, and specimens contaminated with feces are not cultured anaerobically. Specimens should be transported in an oxygen-free container. There are several efficient methods for culturing anaerobes, including the simple and inexpensive GasPak jar. The average clinical laboratory may not be able to afford the time, personnel, and equipment necessary for complete identification of all anaerobes isolated. However, the identification of certain ones, including Bacteroides fragilis and Clostridium perfringens, is relatively simple. Primary emphasis should be placed on the rapid recognition of the presence of obligate anaerobes in a culture and the immediate reporting of all available information to the clinician. Subsequent reports can provide as complete an identification as is possible for each laboratory.


Mechanical preparation of cells for chromosome studies.

A machine, performing hypotonic treatment and fixation of blood samples for chromosome analysis under standard conditions, is described. The machine has now handled specimens from 4,100 persons. 12 tubes are shaken at a rate of approximately 2,800/min, and simultaneously solutions for hypotonic treatment and fixation are added during 30-45 sec from a distribution chamber. The apparatus produces long, slender chromosomes suitable for staining by modern techniques which yield banded chromosomes.


Genetics of the +p9 syndrome.

An analysis of data about the +p9 syndrome revealed that this clinical entity may occur in some different genetic forms. The recurrence risk in cases with familial translocations is due to the type of meiotic segregation, 2:2 or 3:1. It was shown a nonrandomness of involvement of chromosomes 15 and 22 in translocations with chromosome number 9.


Mitogenic and adjuvant activity of a methanol extraction residue (MER) of tubercle bacilli on mouse lymphoid cells in vitro.

The mitogenic and adjuvant effect in vitro of MER (methanol extracted residue of tubercle bacilli), on Balb/C and nu/nu immunocompetent cells was examined and compared with the effect of PPD, LPS, DS, PHA and ConA. MER activated DNA synthesis in spleen cells of Balb/C and nu/nu mice and in blood cultures of Balb/C. The stimulation of DNA synthesis by MER in spleen cells was not macrophage dependent. Bone marrow and 1ymph node cells were slightly stimulated while thymus cells were not affected. Both MER and PPD enhanced  the in vitro immune response of Balb/C mice to SRBC and to TNP (trinitrophenyl) hapten. MER, LPS and PPD enhanced the immune response of nude spleen cells to SRBC in vitro.


On the physical-chemical effect of colostral antibody binding to Escherichia coli O 86.

Sensitization of Escherichia coli O 86 with colostrum or purified colostral sIgA antibody produces a physical-chemical change of the bacterial surface detectable as a change in partition in dextran-polyethylene glycol polymer two-phase systems. Sensitization with colostrum or sIgA reduces affinity for the dextran-rich phase. In contrast immune serum, IgG and complement increase the affinity for that phase. The results are compatible with the hypothesis that hydrophilic particles, e.g. certain strains of bacteria, are less liable to attachment to and phagocytosis by animal cells than hydrophobic particles, e.g. other bacterial strains. Sensitization with secretory IgA enhances the hydrophilicity thereby making possible an escape mechanism operating at the mucosal surfaces.


Estimation of hapten-specific antibody-forming cell precursors in microcultures.

The immune response of mouse spleen cells to hapten-conjugated polymer of flagellin (DNP-POL, NIP-POL) was studied using a microculture system. When increasing numbers of spleen cells were added to a 'filler' cell system, negative feedback effects became apparent and resulted in the generation of progressively lower numbers of plaque-forming cells (PFC) per input cell. This feedback inhibition was shown to be antigen-specific and mediated by factors released into the culture medium. The effect precludes calculation of the frequency of PFC precursors in cultures containing spleen cells alone and complicates the analysis of tolerance using in vitro assay systems. The addition of small numbers of spleen cells to a constant number of thymocytes provided a system in which Poisson analysis could be used to determine the frequency of PFC precursors capable of being activated by hapten-POL conjugates. This system was used to estimate the frequency of anti-NIP-PFC precursors in CBA spleen cells.


Separation of the mitogenic and antigenic responses to bacterial lipopolysaccharide.

An attempt was made to separate the antigenic and mitogenic properties of E. coli bacteria and bacterial lipopolysaccharide antigen inhibited the mitogenic response by the cultures but did not inhibit the induction of anti-LPS antibody or polyclonal antibody synthesis to SRBC. Dextran sulphate, acting as a B-cell mitogen, increased the mitogenic response in spleen cell cultures incubated with bacteria, but did not affect the production of anti-LPS antibody. Mild alkaline hydrolysis (0-1 N NaOH at 56 degrees) of LPS destroyed the mitogenic properties of the molecule, leaving the antigenic properties qualitatively intact. Harsher conditions of base hydrolysis destroyed both the mitogenic and antigenic properties of LPS, as determined by antigenicity in murine spleen cell cultures and haemagglutination inhibition tests.


Comparison of the immune response potential of newborn mice to T-dependent and T-independent synthetic polypeptides.

Newborn mice do not, in general, produce antibodies during the 1st week of life; this inability to respond immunologically has been attributed to lack of functional macrophages and T cells. To determine whether B cells of newborn mice are functionally mature and therefore capable of producing antibodies to thymus (T) independent antigens, the response of 1-9-day-old C3H/HeJ mice injected with a thymus-independent polypeptide, poly(DTyr,DGlu)-polyDPro- -polyDLys was compared to that of their littermates injected with a thymus-dependent immunogen, poly(LTyr,LGlu)-polyLPro- -polyLLys. No antibodies were detected in 1- or 2-day-old mice immunized with the T-dependent antigen, as revealed by haemagglutination and haemolytic plaque-forming cell assays, performed 6 days after injection of the antigen. Injection of 3-day-old animals with the thymus-dependent immunogen resulted in significant immune responses which increased with age. In contrast, 1- and 2-day-old mice responded to the T-independent immunogen with high antibody levels, however, in 3-day-old injected mice, the levels were lower. When 3-day-old nude mice were injected with this antigen, no decrease in the immune response was observed. Thus, newborn mice respond immunologically to a thymus-independent antigen injected at the first 2 days after birth and the antibody levels decrease with maturation of the thymus.


Immunization by an insoluble fraction extracted from Brucella melitensis: immunological and chemical characterization of the active substances.

A peptidoglycan-containing fraction called fraction P.I. (phenol insoluble), extracted from Brucella melitensis and previously described by some of us, had immunogenic and protective properties and did not produce any allergic reactions. Since it is well known that bacterial peptidoglycans studied so far have immunoadjuvant properties, the isolation of the active factor(s) of Brucella was undertaken. By successive enzymatic and chemical treatments, a new, much more purified fraction, called "4A" (approximately 5% of fraction P.I.), is obtained, retaining the same properties as P.I. and giving better protection against infection by Brucella. Immunogenicity, immunoadjuvant activity, allergizing capacity, and specific and nonspecific protective effects of fractions P.I. and 4A are compared. Chemically, fraction 4A is constituted by a lipoprotein covalently linked to peptidoglycan and by a few (lipo)proteins that could be solubilized by hot sodium dodecyl sulfate. Intrinsic properties of peptidoglycan could not be studied, but it does not seem to be essential for the activity. In conclusion, fractions P.I. and 4A are not agglutinogenic and, since fraction 4A induces better protection against infection by Brucella, it could advantageously replace fraction P.I. as a vaccine for humans.


Role of the rfa locus in the immunogenicity of common enterobacterial antigen.

Escherichia coli O14 is one of the few strains of Enterobacteriaceae which in the rabbit engender antibodies in high titers against the common enterobacterial antigen (CA). It is a rough strain that does not possess a specific side chain in its lipopolysaccharide structure (rfa+rfb-). An rfa mutant (rfa-rfb-) was isolated from this parental strain. Chemical analysis revealed the absence of one glucose and of one galactose molecule in the core polysaccharide of the mutant. The incomplete synthesis of the core was accompanied by loss of immunogenicity of CA. In the mutant, in contrast to the wild type, CA was not bound to the core; hence, the core lipopolysaccharide might exert immunosuppressive activity on CA. A protein fraction of normal rabbit serum was shown to destroy the erythrocyte-coating capacity of CA. This fraction also displayed immunosuppressive activity on free CA but not on CA bound to the core polysaccharide.


Effects of Trypanosoma brucei gambiense infections in Microtus montanus on susceptibility to Ehrlich's tumors.

Trypanosoma brucei gambiense infections in the field vole Microtus montanus increased susceptibility to Ehrlich's tumor growth. Whereas uninfected voles were totally resistant to intraperitoneal Ehrlich's ascites tumor cell challenge, over 78% of the animals infected with the trypanosomes developed tumors after challenge. Likewise, when Ehrlich's ascites cells were injected subcutaneously to induce solid tumor formation, only 7% of uninfected controls developed tumors, whereas over 82% of trypanosome-infected animals exhibited malignancies after Ehrlich's cell challenge. Finally, when solid tumors grown in albino CD-1 mice were implanted subcutaneously into uninfected voles, the tumor mass rapidly diminished in size and could not be found when animals were examined 2 weeks postimplant. However, in trypanosome-infected voles, implanted tumors exhibited pronounced expansion, and viable, solid tumors were recovered from over 70% of the challenged voles at 2 weeks postimplant. The implications of trypanosome-induced immunosuppression, especially toward susceptibility to neoplastic growth, are discussed.


Antigens of group A streptococci involved in passive hemagglutination reactions.

Antibodies to streptococcal extracellular products were detected in rabbit sera by passive hemagglutination tests as early as 1 week after intravenous injection of live group A streptococci (strain C203S). Using these antibodies in immunoadsorbent columns, we prepared an antigenic fraction from crude concentrates of streptococcal extracellular products. The specific activity of this fraction in sensitizing glutaraldehyde-treated erythrocytes for passive hemagglutination tests and in absorbing passive hemagglutination antibodies from immune sera was increased by 80-fold and at least 24-fold, respectively, as compared with crude extracellular products. The fraction has been found to contain at least three serologically active antigens, which are of considerable interest because: (i) they gave rise to early and consistent immune responses both in humans and in experimental animals; (ii) they appear so far to be produced only by beta-hemolytic streptococci; and (iii) antibodies to these antigens are present in high titers in patients with acute rheumatic fever. Our data suggest that passive hemagglutination antigens may be distinct from those extracellular enzymes and hemolysins ordinarily employed in streptococcal serological studies.


Effect of estradiol on immune competence: in vivo and in vitro studies.

The administration of a single dose of 2.5 mug of microcrystalline estradiol-17 beta from 1 day before and up until 3.5 days after the administration of 3 X 10(5) heat-killed Escherichia coli significantly increased numbers of splenic anti-E. coli antibody-producing cells in male mice sacrificed 4 days after receiving anitgen. Administration early in the proliferative phase of antibody production, i.e., 1 day before or 1 day after the antigen, appeared to increase numbers of antibody-producing cells more than when it was administered at a later time. When given 2 days before the antigen or 2 h before sacrifice no effect was observed. Spleen cells harvested from male animals injected 3 days before with 5 X 10(6) heat-killed E. coli were incubated for 24 h in vitro with estradiol in concentrations ranging from 5 pg to 20 ng/ml. With concentrations of 500 pg to 5,000 pg/ml, significant increases in antibody-producing cells occurred, whereas at concentrations of 20 ng/ml some decrease was observed. The increase in antibody-producing cells was blocked by a mitotic inhibitor. Significant changes in numbers of antibody-producing cells were not observed after a 2-h incubation period. Uptake of titrated thymidine was increased in thymic and spleen cells incubated for 24 h with 500 pg of estradiol per ml; a concentration of 20 ng/ml slightly (but insignificantly) decreased uptake. Findings suggest that estradiol, in concentrations that approximate physiological serum levels in females, enhances mitosis of immunocompetent cells. This phenomenon may have bearing on the better immunological responsiveness of females than males.


Suppression of cell-mediated immunity after infection with attenuated rubella virus.

The effects of attenuated rubella virus infection upon cell-mediated immunity of human volunteers were studied. The volunteers received the vaccine either by nose drops or by the subcutaneous route. Changes in cell-mediated immunity in terms of delayed cutaneous sensitivity to recall antigens, phytohemagglutination stimulation, and spontaneous migration inhibitory factor-like activity were studied at various time periods after infection. Spontaneous migration inhibitory factor-like activity was studied on supernatants of the lymphocytes obtained from the volunteers and incubated for 72 h in the absence of any antigens. A significant proportion of the volunteers showed suppression of one or more parameters of cell-medicated immunity tested by week 2 of infection compared to the control; however, there was no correlation between suppression of the various parameters studied. No difference was noticed in the incidence of cell-mediated immunity suppression between nose drops and subcutaneous route groups.


Systemic Mycobacterium lepraemurium infection in mice: differences in doubling time in liver, spleen, and bone marrow, and a method for measuring the proportion of viable organisms in an inoculum.

Counts of acid-fast bacilli were made on homogenates of whole liver, whole spleen, and two femurs of CBA mice killed at various time intervals after intravenous infection with Mycobacterium lepraemurium. The growth curves so obtained showed that the bacillus multiplied faster in bone marrow than in liver or spleen. No evidence of redistribution during the early part of infection was obtained. The time of appearance of significant numbers of bacilli (10(7)) in the bone marrow was used to make estimates of viability of M. lepraemurium suspensions. Several applications of the techniques described are discussed.


Conditional virulence of a p-aminobenzoic acid-requiring mutant of Aspergillus fumigatus.

The induced auxotrophy for p-aminobenzoic acid (PABA) resulted in a complete loss of virulence of Aspergillus fumigatus for normal as well as cortisone-treated mice. The PABA-requiring mutant of A. fumigatus survived in vivo for 4 to 7 days without causing any infection. However, it showed conditional virulence in animals receiving PABA in very small quantities. Repeated inoculations of the viable spores of the avirulent mutant strain gave favorable results in building immunity against intravenous challenge of the virulent strain. The immunogenicity of the PABA-requiring mutant was comparable with that of a wild strain of the fungus in agar gel double-diffusion tests using clinical and hyperimmune sera and in skin tests on patients with allergic bronchopulmonary aspergillosis.


Changes in lactoferrin, immunoglobulin G, bovine serum albumin, and alpha-lactalbumin during acute experimental and natural coliform mastitis in cows.

An experimentally induced Escherichia coli infection of a bovine mammary gland resulted in a 30-fold increase in lactoferrin (Lf) concentration in the mammary secretion by 90 h postinoculation and a 4-fold increase in total daily production of Lf by 264 h postinoculation in the infected quarter. A simultaneous rise and fall of bovine serum albumin (BSA) and immunoglobulin G (IgG) concentrations occurred during the acute phase of the infection. Peak BSA and IgG levels were reached 36 h before peak Lf levels. BSA concentrations declined rapidly after the acute phase, whereas IgG and Lf levels remained elevated and decreased slowly as the infection subsided. A decline in alpha-lactalbumin concentration by 48 h postinoculation indicated decreased synthetic capability. The increased Lf production may be a result of a specific response of secretory tissue to inflammatory agents and thus the infectious process. Analogous changes in Lf, IgG, and BSA were observed during a natural coliform infection. Sephadex G-200 chromatography of mastitis skim milk showed that Lf approximated the monomer (molecular weight 77,100) early in infections progressed and abated, the apparent molecular weight of Lf increased to approximately that of the trimer and subsequently decreased to about 1.5 times that of the monomer.


Cutaneous granulomatous response to BCG cell walls with reference to cancer immunotherapy.

A chronic inflammatory response produced by cell walls of Mycobacterium bovis strain BCG associated with microscopic oil droplets (BCG CW-O) was studied with reference to the tumor-regressing ability of this preparation. When BCG CW-O was injected intradermally in the footpads of guinea pigs, intense inflammation developed at the site of injection and in the draining popliteal lymph node. This was characterized histologically as granulomatous inflammation. The intensity of the response was related to the dose of BCG CW-O. Cell walls without oil produced the same type of inflammation but much less in degree. Data are presented that demonstrate the immunological nature of this response and classify it as a cell-mediated immune reaction. Ramifications of this chronic inflammatory reaction occurring at the site of a tumor are discussed.


Restoration of T-cell responsiveness by thymosin: development of antituberculous resistance in BCG-infected animals.

T-cell-depleted (adult thymectomized, lethally irradiated, bone marrow-reconstituted [THXB]), sham-thymectomized (XB) and normal control mice were injected daily with 3 mg of calf thymosin for 16 days. On day 8 of the treatment, the mice, together with untreated controls, were infected intravenously with 4 X 10(6) viable Mycobacterium bovis (BCG Montreal). Growth of the BCG in the lungs and spleens was compared quantitatively for up to 100 days. Thymosin treatment reversed the progressive weight loss seen in BCG-infected THXB mice and prevented their death due to the ongoing mycobacteriosis that developed in the T-cell-depleted animal. There was a late-developing anti-mycobacterial response in the thymosin-treated THXB mice, which resulted in a progressive decline in viability for the lung and spleen populations over the 40- to 80-day period, when the corresponding counts for the untreated THXB mice remained relatively constant. The histopathology of the lung and the increased antibacterial activity seen in the thymosin-treated THXB mice correlated with decreased [3H]deoxyribonucleic acid levels seen in the lungs and spleen compared with that present in the T-cell-depleted controls.


[Old and new data on diarrhetic diseases in childhood I. Etiology and pathophysiology].

Etiology and Pathophysiology. The present article is a comprehensive review of recent research results in the field of acute diarrhea. The most important new idea in contrast to older views is that almost all acute diarrheas are associated with a disturbance of intestinal bacterial homeostasis: overgrowth of the small intestine with apathogenic or pathogenic organisms is followed by changes in intestinal metabolism with increase in intestinal water and electrolyte secretion. Anaerobic organisms cause by enzymatic deconugation and dydroxylation of bile acids secretion of fluid into the small intestine and inhibition of fluid absorption from the large intestine. 10-OH-fatty acids, which are formed intraluminally by enzymatic hydroxylation of long-chain unsaturated fatty acids from the diet by similar anaerobic organisms, produce profuse secretion of fluid into the small intestine. The ability of numerous strains of E. coli to produce enterotoxin, which has a qualitatively similar action to cholera toxin, is now considered to be a major cause of infantile diarrhea. The separation of two completely different pathophysiologic mechanisms of E. Coli, the enterotoxic and the enteroinvasive action which are determined by extranuclear chromosomal material, is an important result of recent research. Overgrowth of the small intestine with different bacteria is followed by loss of actiivity of lactase, and later of all disaccharidases in the intestinal mucosa.


Dermatologic radiotheraphy: selection of radiation qualities and treatment techniques.

The penetration of x-rays should be correlated the depth of the pathological process. The introduction of the "half-value depth" (D1/2) as a guidline in the selection of radiation qualities has greatly simplified cutaneous radiotherapy. Modern dermatologic x-ray units with beryllium windows operated between 10 and 50 (or 100 kV, offer the widest spectrum of radiation qualities suitable for dermatologic therapy, although older types of radiation equipment are still useful for most dermatology indications. Previous risks in dermatologic radiation therapy have been reduced to a large degree by the introduction of new sophisticated safety devices and by a deliberate reduction of unnecessary variations in the selection of physical factors. Beryllium window machine can be operated without changing milliamperage, target-skin distance or dose rate for different indications. The recommended standardization of physical factors offers a safer and easier alternative to previous techniques.


William Farr: founder of modern concepts of surveillance.

Considerable confusion surrounds the use of the term surveillance in the context of public health but the principles underlying all uses are as old as epidemiology itself. William Farr has contributed more than anyone else to the development of these principles and to the demonstration of their value in practice on a large scale. This paper re-examines the basic tenets which guided him and the methods and techniques which he developed in the hope of reviving insights and resetting standards that modern epidemiologists might, with benefit, strive to emulate.


Cancer surveillance with particular reference to the uses of mortality data.

Cancer surveillance serves several purposes related to prevention, control and treatment. Morbidity information from cancer registration and mortality data based on death certification complement each other and both are necessary for surveillance. In many respects mortality data are sufficiently accurate and specific to reveal major differences and trends. The importance of recognizing even small aggregations of rare tumours is stressed.


Health surveillance and planning for health care in the Netherlands.

This paper begins by describing the historical background to health surveillance and planning in the Netherlands from their beginnings in the late Middle Ages. It goes on to discuss the present organization of health care and recent attempts to obtain more reliable and comprehensive information on which to base health care planning. The rudiments of a general health information system already exist and a feasibility study for establishing such a system on a regional basis has been proposed.


The movement pattern of oral tardive dyskinesia in relation to anticholinergic and antidopaminergic treatment.

24 hospitalized psychiatric patients with neuroleptic-induced tardive dyskinesia were treated with alpha-methyl-para-tyrosine (AMPT), 4 g daily for 3 days, and biperiden, 12 mg daily for 3 weeks. The results were evaluated blindly by means of videotape technique. The frequency of tardive dyskinesia was significantly reduced by AMPT and significantly increased by biperiden. The amplitude was reduced by AMPT in ten cases, unchanged in 12 cases and increased in two cases. Biperiden significantly increased the amplitude. The duration of each separate tongue protrusion and/or mouth opening was significantly increased by AMPT and reduced or unchanged by biperiden. It is concluded that a reduced dopaminergic activity (pharmacological or organic) may constitute the primary pathogenetic background for tardive dyskinesia, but that dopaminergic hypersensitivity and/or cholinergic hypofunction is necessary before the hyperkinetic element of the movement disturbances can minifest itself.


Application of the neuroleptic pimozide (ORAP-R 6238) for tranquilizer indications in a controlled study.

The effect of the neuroleptic pimozide at doses of 2 mg/day was compared with that of diazepam at doses of 3 x 5 mg. This double-blind study involved 50 outpatients with psychovegetative syndromes and anxiety neuroses. At these doses and under the described test conditions both preparations were virtually equally effective. Pimozide can therefore be considered an alternative preparation for 'genuine' tranquilizers. Above all, its application seems to be useful in patients that are prone to drug abuse, and therefore, a priori, endangered by habit formation.


Mianserin versus amitriptyline. A double-blind-trial evaluated by the AMP system.

Amitriptyline and mianserin were compared in a double-blind trial. Most of the depressive symptoms were influenced by both drugs, though quantitative and qualitative differences became evident. Amitriptyline predominantly influenced anxiety, despair and suicidal tendencies, while mianserin was particularly effective in psychomotor inhibition and the reduction of vegitative complaints. Mianserin did not show anticholinergic side effects.


5-Hydroxytryptophan (5-HTP) and a MAOI (nialamide) in the treatment of depressions. A double-blind controlled study.

Through a controlled double-blind study in 30 hospitalized patients affected with endogenous depression, the antidepressant action of the combination of nialamide+l-5-HTP has been evaluated and compared with a control group which only received nialamide (+ placebo). The patients treated with nialamide + l-5-HTP achieved a fuller recovery than those who were treated with nialamide alone. The treatment with nialamide + l-5-HTP proved to have a shorter delay of onset. Side effects showed no marked differences except for the orthostatic hypotension which was less apparent in those patients treated with nialamide + l-5-HTP.


Scanning electron microscopy of postoperative M-K and moist chamber-stored corneas.

In the present study, using the in vivo test situation which all corneal donor material must meet, we performed penetrating keratoplastics utilizing corneal tissue stored for five days in either M-K medium or conventional moist chambers. Cat corneas stored for five days in M-K medium had a higher incidence of postoperative graft clarity along with an intact endothelial cell layer as determined by scanning electron microscopy than corneas stored in a conventional moist chamber. The endothelium of corneas immediately after moist chamber storage revealed minimal surface alteration which contrasted to the more severely stressed endothelium immediately after M-K storage. However, the M-K stored endothelium survived the trauma of penetrating keratoplasty much better than cells stored in moist chambers.


The effect of imidazole on the disruption of the blood-aqueous barrier in the rabbit eye.

The aqueous flare (AF) of an intact rabbit eye was measured by a photoelectrical instrument and the intraocular pressure by vibration tonometry. Prior treatment with imidazole given intraperitoneally noticeably inhibited the disruption of the blood-aqueous barrier in rabbit eyes induced by topical prostaglandin E2 (PGE)2, topical arachidonic acid (AA), infrared irradiation of the iris, endotoxin of Proteus mirabilis given intravenously, and alpha-melanocyte-stimulating hormone (alpha-MSH) given subcutaneously. Prior treatment with imidazole given topically had no effect on the disruption of the blood aqueous barrier caused by topical PGE2, topical AA, infrared irradiation of the iris, or endotoxin of P. mirabilis given intravenously, but facilitated and potentiated strongly the response to alpha-MSH given subcutaneously. Prior treatment with topical imidazole did not affect the histological changes of the anterior ciliary processes induced by alpha-MSH given subcutaneously.


The nuclear envelope in the crystalline lens fiber cell.

Rabbit lenses which have been fixed, dehydrated, and dried by a critical-point drying method, can be fractured through the cytoplasm of the differentiating lens fibers, exposing the cell nuclei. The fracture, under these conditions, causes a complete separation of the two membranes of the nuclear envelope from one another, thus exposing entire membrane surfaces (those which line the perinuclear space). These surfaces are not seen in their entirety in typical freeze-fracture or freeze-etch preparations, and consequently have not been described previously. The exposed membrane surfaces which line the perinuclear space have numerous convex structures of approximately 1,000 A, and some larger more irregularly shaped structures. These appear to be fragments of the nuclear pore complexes. Differences in these structures between young fibers and those nearing completion of differentiation is suggested.


Nocardiosis in Israel. A report of five cases.

Five cases of Nocardia infection are briefly described, four due to Nocardia asteroides and one to N. brasiliensis. The latter represents the first reported case of N. brasiliensis infection in Israel. In three of the patients, the infections were associated with an underlying disease process and with prolonged high-dosage therapy with steroids and/or cytotoxic drugs. In the other two patients, the disease was spontaneous.


Evaluation of a computerized Bayesian model for diagnosis of renal cyst vs. tumor vs. normal variant from urogram information.

The diagnostic problem of cyst/tumor/normal variant raised on an excretory urogram leads to a decision to do needle aspiration or renal arteriography. This decision depends critically upon the probability distribution for the three diagnoses. A computerized Bayesian model of a uroradiologist's diagnostic process in solving the problem was developed. The model was based on subjective probabilities supplied by an experienced uroradiologist. The model was evaluated in terms of its ability to decrease the cost of further diagnosis regarding aspiration versus arteriography. The model's output was compared with decisions made by unaided radiologists viewing the same panel of 50 urogram test cases. Results indicate that the model does not improve upon the decisions made by a radiologist highly experienced with this diagnostic problem. However, the decisions made by unaided, less experienced radiologists result in greater cost than those of the model.


A computer program for the localization of small areas in roentgenological images.

A method and associated algorithm are presented which allow a simple and accurate determination to be made of the location of small symmetric areas presented in roentgenological images. The method utilizes an operator to visually spot object positions but eliminates the need for critical positoning accuracy on the operator's part. The rapidity of measurement allows results to be evaluated on-line. Parameters associated with the algorithm have been analyzed, and methods to facilitate an optimum choice for any particular experimental setup are presented.


A modified membrana nictitians flap technique for the treatment of ulcerative keratitis in cattle.

A modified membrana nictitans flap technique was used successfully in 1,768 of 1,845 (95.8%) cattle with several types of ulcerative keratitis. The flap was easily applied and lasted 7 to 10 days.


Enterocin, a new antibiotic taxonomy, isolation and characterization.

Enterocin is a new antibiotic isolated from cultures of two strains of Streptomyces, which were given the names Streptomyces candidus var. enterostaticus WS-8096 and variant M-127 of Streptomyces viridochromogenes. Its elementary analysis and mass spectroscopic measurement suggest the molecular formula is C22H20010. The ultraviolet absorption gave two maximal peaks at 250 nm and 283 nm in methanol. Enterocin has static activities against gram-positive and gram-negative bacteria and no activity against fungi and yeast.


Identification of mycolutein and pulvomycin as aureothin and labilomycin respectively.

Two antibiotics isolated from a culture MA-2465 were identified as aureothin and labilomycin, the chemical structures of which had been reported previously. It was also concluded that mycolutein and pulvomycin isolated as antibiotics in 1955 and 1957 respectively are also identical with aureothin and labilomycin. A cursory study of MA-2465 indicates that it is distinctly different from the culture which was first observed to produce labilomycin and is probably different from the organism first noted to produce aureothin.


Antimycin A fermentation. I. Production and selection of strains.

Increase in antimycin A production was achieved through a parallel strain  and medium improvement program: a 125-fold augmentation (75 to 9,500 mug/ml) was obtained. The selective system included antimycin A productivity, conidiation, sensitivity to ultraviolet radiation, growth rate and yield, and absence of pigment and actinomycin D production. Among the original strains tested one natural isolate possessed high productivity and several of the above characteristics, and was selected for mutagenesis. Spontaneous and induced variability was then exploited in isolating high-producing strains. The first mutagen used was ultraviolet radiation; it was replaced by ethylenimine when it became no longer efficient in increasing variability. As new, high producers were isolated, the medium was modified to best suit their requirements for still higher productivity. The critical environmental factors were absence of phosphate and organic salts, concentration of the nitrogen source and ratio organic/inorganic nitrogen, ratio ammonium sulfate/calcium carbonate, and addition of slowly utilizable carbon sources, such as lactose and oil; optimum temperature and initial pH were 25 degrees C and 7.0. Aeration/agitation requirements of improved strains were high. Fermentation was characterized by abrupt pH changes which impaired rapid accumulation of the antibiotic. Antimycin A was produced during both the trophophase and idiophase.


Translocation of a discrete piece of deoxyribonucleic acid carrying an amp gene between replicons in Eschericha coli.

A number of well-characterized R plasmids (R1drd 19. K1.R100-1, R46, R55-1, R64-11, R388, and R751) will mobilize an integrated amp gene from the chromosome of Escherichia coli K-12. R391 will not act in this way. The process involves recombination, and in each case the mobilizing plasmid acquires an additional piece of deoxyribonucleic acid of molecular weight 4 X 10(6). Acquisition of this deoxyribonucleic acid may impair the transfer properties of the recombinant plasmid in some cases. The process will occur in a recA background.


Threonine deaminase from a nonsense mutant of Escherichia coli requiring isoleucine or pyridoxine: evidence for half-of-the-sites reactivity.

The mutant IP7 of Escherichia coli B requires isoleucine or pyridoxine for growth as a consequence of a mutation in the gene coding for biosynthetic threonine deaminase. The mutation of IP7 was shown to be of the nonsense type by the following data: (1) reversion to isoleucine prototrophy involves the formation of external suppression at a high frequency, as shown by transduction experiments; and (ii) the isoleucine requirement is suppressed by lysogenization with a phage carrying the amber suppressor su-3. Cell extracts of the mutant strain contain a low activity of threonine deaminase. The possibility that this activity is biodegradative was ruled out by kinetic experiments. The mutant threonine deaminase was purified to homogeneity by conventional procedures. The enzyme is a dimer of identical subunits of an approximate molecular weight of 43,000 (Grimminger and Feldner, 1974), whereas the wild-type enzyme is a tetramer of 50,000-dalton subunits (Calhoun et al., 1973; Grimminger et al., 1973). The mutant enzyme is not inhibited by isoleucine and does not bind isoleucine, as shown by equilibrium dialysis experiments. Pyridoxal phosphate enhances the maximum catalytic activity of the mutant enzyme by a factor of five, whereas the wild-type enzyme is not affected. In wild-type and mutant threonine deaminase the ratio of protein subunits and bound pyridoxal phosphate is 2:1. The activation of threonine deaminase from strain IP7 is due to a second coenzyme binding site, as shown by (i) spectrophotometric titration of the enzyme with pyridoxal phosphate and by (ii) measurement the pyridoxal phosphate content of the enzyme after sodium borohydride reduction of the protein. The observation of one pyridoxal phosphate binding site per peptide dimer in the wild-type enzyme and of two binding sites per dimer in the mutant strongly suggests that one of the potential sites in the wild-type enzyme is masked by allosteric effects. The factors responsible for the half-of-the-sites reactivity of the coenzyme sites appear to be nonoperative in the mutant protein.


Biosynthesis of membrane-bound nitrate reductase in Escherichia coli: evidence for a soluble precursor.

Membrane-bound nitrate reductase of Escherichia coli consists of three subunits designated as A, B, and C, with subunit C being the apoprotein of cytochrome b, A hemA mutant that cannot synthesize delta-aminolevulinic acid (ALA) produces a normal, stable, membrane-bound enzyme when grown with ALA. When grown without ALA, this mutant makes a reduced amount of membrane-bound enzyme that is unstable and contains no C subunit. Under the same growth conditions, this mutant accumulates a large amount of a soluble form of the enzyme in the cytoplasm. Accumulation of this cytoplasmic form begins immediately upon induction of the enzyme with nitrate. The cytoplasmic form is very similar to the soluble form of the enzyme obtained by alkaline heat extraction. It is a high-molecular-weight complex with a Strokes radius of 8.0 nm and consists of intact A and B subunits. When ALA is added to a culture growing without ALA, the cytoplasmic form of the enzyme is incorporated into the membrane in a stable form, coincident with the formation of functional cytochrome b. Reconstitution experiments indicate that subunit C is present in cultures grown without ALA but is reduced in amount or unstable. These results indicate that membrane-bound nitrate reductase is synthesized via a soluble precursor containing subunits A and B, which then binds to the membrane upon interaction with the third subunit, cytochrome b.


Specific in vivo cleavage of D-serine deaminase and properties of tetrameric polypeptide aggregates of the fragments.

The primary D-serine deaminase (D-serine dehydratase, EC 4.2.1.14) of Escherichia coli K-12 is unstable within the cell. The protein, a single polypeptide chain, is cleaved at a lysine residue by a cellular proteolytic activity. Fragments containing the active site then aggregate into tetramers, which retain substrate affinity and show very low catalytic activity. Such degradations may represent an evolutionary mechanism for the generation of new enzymes.


Inactivation of membrane transport in Escherichia coli by near-ultraviolet light.

Evidence is presented that near-ultraviolet (near-UV) light can alter galactoside transport in Escherichia coli in several independent ways. It can inactivate the permease system per se, it can interfere with metabolic energy production or transfer, and it can cause an increase in the generalized permeability of the membrane. Earlier publications suggested that near-UV destroys cofactors needed for electron transport and thus places a limitation on energy reserves. In agreement, we found that the active accumulation of [14C]thiomethyl-beta-D-galactopyranoside is decreased after irradiation by a larger factor than that due to action directly on the permease system. The effect on the latter was measured by the decrease in the rate of o-nitrophenyl-beta-D-galactopyranoside (ONPG) transport. As evidence that energy supplies for this "downhill" process did not become rate limiting after irradiation, we found that carbonylcyanide-m-chlorophenyl-hydrazone did not stimulate ONPG transport of irradiated cells. Cells genetically deficient in functional permease or cells treated with formaldehyde still transport ONPG passively, although at much lower rates. With the use of such cells, it was found that high fluences (doses) made the cells leaky. Further evidence that the permease system and the metabolic energy system can be inactivated independently is also presented. It is shown that a photoproduct from the irradiation of chloramphenicol inactivates the permease system much more efficiently than the energy system. In addition, it is shown that thio-beta-D-digalactopyranoside protects the permease system, but not the energy system, both against direct inactivation by near-UV and against photosensitized inactivation in the presence of chloramphenicol.


Escherichia coli mutants altered in murein lipoprotein.

Mutants with alterations in the structure, biosynthesis, or assembly of murein lipoprotein were selected by a procedure based on radiation suicide of wild-type organisms by [3H]arginine under conditions where the radioactive arginine was preferentially incorporated into lipoprotein. Further screening for the potential mutants among the survivors of [3H]arginine suicide was carried out by using a sensitive immunodiffusion test, followed by radioactive double-labeling experiments. Three mutants were obtained and partially characterized.


Plasmid co-integrates of prophage lambda and R factor R100.

Single and tandem insertions of prophage lambda into R100 have been isolated. Insertions into the transfer genes, insertions into the transfer control gene finO, and insertions into regions that result in no detectable phenotypic change were found. From the last type, deletion mutants were isolated which established the sequence of antibiotic resistance genes as tet-cml-fus-str-sul-mer in R100. High frequency transducing phage preparations lambdamer, lambdasul str, and lambdasul str cml were also isolated from this type.


Temperature dependence of mating-pair formation in Escherichia coli.

The temperature dependence of mating-pair formation by Escherichia coli has been studied from 2 to 45 C by means of a physical assay. No mating-pair formation was found below 24 C. Between 30 and 41 C pair formation increased very rapidly, followed by an equally sharp decline between 41 and 45 C. The results are interpreted in terms of the pilus adsorption process of recipient cells and pili production by donor cells.


Effects of growth temperature on yield and maintenance during glucose-limited continuous culture of Escherichia coli.

The effects of growth temperature on the aerobic growth yield with respect to oxygen consumption (Y0-grams [dry weight] per gram-atom of O) and the rate of maintenance respiration (m0-milligram-atoms of O/gram [dry weight] per hour) are reported for Escherichia coli B cultivated continuously in the presence of oxygen with limiting glucose. During anaerobic continuous culture, YATP(max) (grams [dry weight] per mole of ATP corrected for maintenance) increases from 10.3 to 12.7 as the growth temperature is lowered from 37 to 25 C. Over this same range, Y0(max) (Y0 corrected for maintenance respiration) rises from 12.5 to 28.8 and remains at the higher value down to 17.5 C. From 37 to 32 C, m0 increases from 0.9 to 4.4 but then falls to 1.5 as the temperature is lowered to 17.5 C. The value of m0 sharply rises some 13-fold as the temperature is raised to 42 C without a significant change in the value of Y0(max). Changes of Y0(max) are consistent with a temperature-sensitive doubling of the efficiency of oxidative phosphorylation, but the reasons for the changes of the rate of maintenance respiration are not known.


Role of iron deposition in Sphaerotilus discophorus.

Various physiological aspects of the process of iron deposition in Sphaerotilus discophorus were examined to elucidate its role. The values of iron/protein ratios suggested that a direct relationship existed between the iron concentration of the media and the magnitude of final iron deposition. Saturation of the organism's iron deposition system occurred at a 2.0 mM iron concentration, at a value of 0.6 mg of ferric ion per mg of cell protein. Laboratory data indicated that the strain's very low capacity for iron deposition observed at low external iron concentrations makes it unlikely that it is significant in limiting iron in the natural milieu. Under optimal iron concentrations, however, strain SS1 caused precipitation of iron (adsorbed to cellular material) in broth cultures, which was 10 to 100 times that mediated by some "non-iron" microorganisms. The strain's iron requirement, which was found to be between 0.003 and 0.02 mM, is commensurate with that of other microbes. One hundred micrograms of Mn(II) per ml and possibly 10 mug of either Co(II) or  Ni(II) per ml could inhibit iron uptake in the deposition system. Sphaerotilus, when tested for its ability to withstand toxic concentrations of certain trace elements (Co, Cu, Mn, Ni, and Cd), demonstrated no exceptional resistance with respect to several other common microorganisms. Final cell yields were not affected by a varying iron concentration for Sphaerotilus growing under conditions of limiting carbon and nitrogen.


Regulation of purine utilization in bacteria. VI. Characterization of hypoxanthine and guanine uptake into isolated membrane vesicles from Salmonella typhimurium.

Uptake of hypoxanthine and guanine into isolated membrane vesicles of Salmonella typhimurium TR119 was stimulated by 5'-phosphoribosyl-1'-pyrophosphate (PRPP). For strain proAB47, a mutant that lacks guanine phosphoribosyltransferase, PRPP stimulated uptake of hypoxanthine into membrane vesicles. No PRPP-stimulated uptake of guanine was observed. For strain TR119, guanosine 5'-monophosphate and inosine 5'-monophosphate accumulated intravesicularly when guanine and hypoxanthine, respectively, were used with PRPP as transport substrates. For strain proAB47, IMP accumulated intravesicularly with hypoxanthine and PRPP as transport substrates. For strain TR119, hypoxanthine also accumulated when PRPP was absent. This free hypoxanthine uptake was completely inhibited by N-ethylmaleimide, but the PRPP-stimulated uptake of hypoxanthine was inhibited only 20% by N-ethylmaleimide. Hypoxanthine and guanine phosphoribosyltransferase activity paralleled uptake activity in both strains. But, when proAB47 vesicles were sonically treated to release the enzymes, a three- to sixfold activation of phosphoribosyltransferase molecules occurred. Since proAB47 vessicles lack the guanine phsophoribosyltransferase gene product and since hypoxanthine effectively competes out the phosphoribosylation of guanine by proAB47 vesicles, it was postulated that the hypoxanthine phosphoribosyltransferase gains specificity for both guanine and hypoxanthine when released from the membrane. A group translocation as the major mechanism for the uptake of guanine and hypoxanthine was proposed.


Dual regulation by arginine of the expression of the Escherichia coli argECBH operon.

The correlation between the level of messenger ribonucleic acid (mRNA) specific for the argECBH gene cluster (argECBH mRNA) measured by ribonucleic acid-deoxyribonucleic acid (RNA-DNA) hybridization and the rates of synthesis of N-acetylornithine deacetylase (argE enzyme) and of argininosuccinate lyase (argH enzyme) of Escherichia coli strain K-12 were determined for steady-state growth with and without added L-arginine and during the transition periods between these two states. During the transient period after arginine removal (transient derepression), the synthesis of enzymes argE and argH was initially three to five times greater than the steady-state derepressed rate finally reached 50 min later. The level of argECHB mRNA correlated well both quantitatively and temporally with the rates of enzyme synthesis during this transition. The level of in vivo charged arginyl-transfer RNA (tRNAarg), monitored simultaneously, was initially only 5 to 10% and gradually increased to a final level of 80% after 45 min. During the transient period after arginine addition (transient repression), the rates of synthesis of enzymes argE and argH decreased to almost zero and gradually reached steady-state repressed rates after about 180 min. The argECBH mRNA level remained constant at the steady-state repressed level throughout transient repression, revealing a discontinuity between the level of this mRNA and rates of enzyme synthesis. A similar discrepancy was noted during the transition after ornithine addition. In vivo charged tRNAarg remained constant at 80% during this transition. After removal of arginine, the zero-level transient enzyme synthesis developed after only 7.5 min of arginine deprivation and was maximum after 30 min. The results suggest an accumulation of a molecule regulated by arginine that plays a role in transient repression. Our data indicate that arginyl-tRNA synthetase is not this molecule since its synthesis was unaffected by arginine. The ratios of steady-state argE and argH enzyme synthesis without arginine to that with arginine were 12 and 20, respectively, whereas the similar ratio for argECBH mRNA was 2 to 3. The repressed level of argECBH mRNA was not affected by attempts to repress or derepress the ppc+ gene (carried on the DNA used for hybridization), and the repressed level of argECBH mRNA was lowered about 50% in cells carrying an internal argBH deletion. These data taken together indicate the presence of an excess of untranslated argECBH mRNA during both transient and steady-state repression by arginine. Thus, a second regulatory mechanism, not yet defined, appears to play an important role in arginine regulation of enzyme synthesis.


Effect of arginine on the stability and size of argECBH messenger ribonucleic acid in Escherichia coli.

The chemical stability of argECBH messenger ribonucleic acid (mRNA) produced by Escherichia coli was found to be unaltered during steady-state repression by arginine. During extreme arginine deprivation, the increase in argECBH mRNA stability was related to general effects of amino acid starvation on mRNA stability. Thus a mechanism whereby argECBH gene expression is regulated by altering the decay rate of this mRNA is not consistent with our data. Sucrose gradient analysis followed by hybridization revealed that both heavy (14S) and light (8S) components of argECBH mRNA were produced by cells of E. coli K-12 grown without arginine, whereas predominantly light (8S) mRNA was produced by cells grown with arginine. A functional argR gene and the EC portion of the argECBH cluster were found essential for the arginine restriction of heavy-mRNA production. Experiments suggest that light argECBH mRNA did not arise from heavy message, and 8u% of both light and heavy mRNA was found bound to ribosomes. The data appear most consistent with the notion that a second site of control by arginine regulates the amounts of light and heavy arginine mRNA in the cell either by early termination of transcription or by endonucleolytic processing. Consideration of these data in conjunction with those of the accompanying report (Krzyzek and Rogers, 1976) permits the tentative conclusion that light argECBH mRNA is not translated into active enzymes and is thus responsible for the discrepancy between the high content of hybridizable mRNA and low rates of enzyme synthesis found during arginine repression.


Method for the isolation of Escherichia coli K-12 mutants deficient in essential genes.

We developed a general procedure for the induction and identification of mutations in chromosomal essential genes that are located in a diploid region of Escherichia coli K-12. The partial diploidy is conferred by an episome that is temperature sensitive for replication so that a mutant strain will form microcolonies at 42 C on complete media if an essential chromosomal gene in the diploid region is defective. Mutations identified by this procedure can be classified into cistrons by a complementation method devised for the purpose. To verify that the procedure works in practice, we fused an episome covering the rif region with an Ftslac+ and used the resulting temperature-sensitive episome to identify chromosomal mutations in essential functions near rif. As expected, a certain proportion of the mutations were in the rif gene, an essential gene that codes for the beta subunit of ribonucleic acid polymerase.


Molecular nature of two beta-lactamase-specifying plasmids isolated from Haemophilus influenzae type b.

The molecular nature of two beta-lactamase-specifying plasmids isolated from two separate ampicillin-resistant Haemophilus influenzae type b strains was examined. A 30 X 10(6)-dalton (30-Mdal) plasmid (RSF007) had a copy number of approximately 3 per chromosomal equivalent and a mole fraction guanine plus cytosine content of 0.39. By heteroduplex analysis the 30-Mdal plasmid was found to contain the entire ampicillin translocation DNA segment (TnA) found on R factors of enteric origin. A 3.0-Mdal plasmid (RSF0885) was found as a multicopy pool of approximately 28 copies per chromosomal equivalent, had a mole fraction guanine plus cytosine content of 0.40, and contained only about one-third of the transposable TnA sequence. RSF007 and RSF0885 appeared to be unrelated plasmids in that they share base sequence homology only within the confines of the TnA segment. The 3.0-Mdal Haemophilus plasmid was used to transform E. coli to ampicillin resistance but was found to be unstable in this host in the absence of antibiotic. The possibility that R-plasmids arose in Haemophilus by the translocation of TnA from a donor R-factor onto an indigenous H. influenzae plasmid is discussed.


Characterization of a mini-ColC1 plasmid.

An in vitro constructed plasmid, pVH15, consisting of the entire genome of the plasmid ColE1, the tryptophan operon of Escherichia coli, and regions of the bacteriophage PHI80pt190, spontaneously gave rise in E. coli to a mini-ColE1 plasmid consisting of approximately one-half of the ColE1 genome and a small segment of phi80pt190 DNA. This mini-ColE1 plasmid, designated pVH51, has a molecular weight of approximately 2.1 X 10(6) and possesses a single EcoRI restriction site. Heteroduplex analyses showed that about 90% of the pVH51 plasmid hybridizes to about 50% of the ColE1 plasmid. Phenotypically, pVH51 did not produce colicin E1 but conferred immunity to this colicin. The number of mini-ColE1 plasmid molecules per cell was maintained at a four- to fivefold higher level than normal ColE1. A mini-ColE1 hybrid plasmid, designated pML21 and consisting of pVH51 and the kan fragment of plasmid pSC105 inserted at the EcoRI restriction site of mini-ColE1, was maintained at a lower copy number level than pVH51. As in the case of normal ColE1, both pVH51 and pML21 continued to replicate in the presence of chloramphenicol. The promotion of conjugal transfer of pVH51 and pML21 by a self-transmissible plasmid was greatly reduced compared with normal ColE1.


Mode of replication of the conjugative R-plasmid RSF1040 in Escherichia coli.

Replicating deoxyribonucleic acid (DNA) molecules of plasmid RSF1040, a deletion mutant of the conjugative R plasmid R6K, appear in the electron microscope as partially supercoiled structures with two open circular branches of equal size, although open structures with three branches, two branching points and no supercoiled regions (theta structures) were also found at a lower frequency. The partially supercoiled molecules sediment more rapidly than native covalently closed circular DNA in neutral sucrose gradients and band at a position intermediate between covalently closed circular and open circular DNA in CsClethidium bromide gradients. Electron microscope measurements of the linear EcoRI-treated replicative intermediates indicate that replication can be initiated at two sites (origins) on the plasmid DNA molecule located at about 23% (alpha) and 39% (beta) of the total genome length from an EcoRI end designated arbitrarily as the "left-hand" end of the molecule. The overall replication of RSF1040 is asymmetrically bidirectional. Replication from the alpha origin proceeds first to the "right" to a unique termination site located some 55% of the total genome length from the left-hand end of the molecule. At this point replication proceeds from the alpha origin to the "left" (i.e., opposite to the original direction of replication) until replication of the molecule is completed. Replication also proceeds from the beta origin asymmetrically to the unique terminus site.


Mode of insertion of lipopolysaccharide into the outer membrane of escherichia coli.

A mutant of Escherichia coli that lacks uridine 5'-diphosphate galactose-4-epimerase makes lipopolysaccharide with less carbohydrate than the parent, unless galactose is present during growth. Carbohydrate is dense, and the outer membrane, which contains lipopolysaccharide, was found to be denser when isolated from cells grown with galactose then when galactose was omitted. Cells given galactose after growth in its absence rapidly formed dense regions within the outer membrane that disappeared when galactose was removed. These results indicate that lipopolysaccharide enters the outer membrane nonrandomly at a minimum of 10 to 22 discrete "insertion points." Isopycnic centrifugation provides a method for isolating these regions.


Formation of the formate-nitrate electron transport pathway from inactive components in Escherichia coli.

When Escherichia coli was grown on medium containing 10 mM tungstate the formation of active formate dehydrogenase, nitrate reductase, and the complete formate-nitrate electron transport pathway was inhibited. Incubation of the tungstate-grown cells with 1 mM molybdate in the presence of chloramphenicol led to the rapid activation of both formate dehydrogenase and nitrate reductase, and, after a considerable lag, the complete electron transport pathway. Protein bands which corresponded to formate dehydrogenase and nitrate reductase were identified on polyacrylamide gels containing Triton X-100 after the activities were released from the membrane fraction and partially purified Cytochrome b1 was associated with the protein band corresponding to formate dehydrogenase but was not found elsewhere on the gels. When a similar fraction was prepared from cells grown on 10 mM tungstate, an inactive band corresponding to formate dehydrogenase was not observed on polyacrylamide gels; rather, a new faster migrating band was present. Cytochrome b1 was not associated with this band nor was it found anywhere else on the gels. This new band disappeared when the tungstate-grown cells were incubated with molybdate in the presence of chloramphenicol. The formate dehydrogenase activity which was formed, as well as a corresponding protein band, appeared at the original position on the gels. Cytochrome b1 was again associated with this band. The protein band which corresponded to nitrate reductase also was severely depressed in the tungstate-grown cells and a new faster migrating band appeared on the polyacrylamide gels. Upon activation of the nitrate reductase by incubation of the cells with molybdate, the new band diminished and protein reappeared at the original position. Most of the nitrate reductase activity which was formed appeared at the original position of nitrate reductase on gels although some was present at the position of the inactive band formed by tungstate-grown cells. Apparently, inactive forms of both formate dehydrogenase and nitrate reductase accumulate during growth on tungstate which are electrophoretically distinct from the active enzymes. Activation by molybdate results in molecular changes which include the reassociation of cytochrome b1 with formate dehydrogenase and restoration of both enzymes to their original electrophoretic mobilities.


Essential genes in the metB-malB region of Escherichia coli K-12.

We isolated 25 independent mutants that are deficient in essential genes located in the metB-malB region of the Escherichia coli chromosome. The mutations were mapped within this region by using several F' factors and were also classified into 11 cistrons by complementation testing. There is clustering of mutations in essential genes within the metB-malB region.


Isolation and characterization of the specialized transducing bacteriophages phi80dargF and lambdah80cI857 dargF: specific cleavage of arginine transducing deoxyribonucleic acid by the endonucleases EcoRI and SmaR.

The directed transposition of argF to the tonB locus of the Escherichia coli chromosome and the subsequent isolation of the specialized transducing phage phi80dargF is described. The structure of this phage has been has been determined. A hybrid lambdah80cI857dargF phage has been constructed. Deoxyribonucleic acid isolated from these and their parent bacteriophages has been specifically cleaved by the endonucleases EcoRI and SmaR; the unique deoxyribonucleic acid fragments thus obtained have been resolved and analyzed by electrophoresis in agarose gel.


Relationship of structure to function in bacterial endotoxins. IX. Differences in the lipid moiety of endotoxic glycolipids.

Chemical, immunochemical, chromatographic, and endotoxic properties of five chromatographically pure glycolipids were compared. The preparations were extracted by chloroform-methanol from three Escherichia coli, one Salmonella minnesota, and one S. typhimurium Re heptoseless mutant strains. The local Shwartzman skin assay, the nonspecific resistance-enhancing effect, and the Limulus assays could not distinguish among the five glycolipids, all five being active in all three assays. Significant differences could be seen when the tumor resistance-enhancing effect of the glycolipids in mice was compared with the nonspecific TA3-Ha murine mammary adenocarcinoma growing in ascites form. Even greater variation was observed in the capacity of the preparations to enhance the nonspecific resistance of mice to virulent S. typhi 0901 infections. The data show that the five glycolipids are quite dissimilar in their biological effects. Similarly, thin-layer chromatography and molecular ratio determinations showed that differences exist in the chemical structure of the glycolipids. Accordingly, we claim that not only the polysaccharide but the lipid moiety as well may vary in various gram-negative endotoxin preparations.


Ultrastructure of septal complex in Filobasidiella neoformans (Cryptococcus neoformans).

Electron microscopy of Filobasidiella neoformans, the perfect state of Cryptococcus neoformans, revealed basidiomycete doliporesepta between hyphal cells and also between clamp connections and adjacent cells. The pore-occluding material was a heterogeneous flattened plate with dark margins and a lighter center, as seen in the species of Filobasidium. Representative basidiomycete parenthesomes were lacking, and endoplasmic reticulum was seen in the dolipore region.


Isolation and genetic analysis of amber uvrA and uvrB mutants.

Genetic properties of amber uvrA and uvrB mutants of Escherichia coli K-12 are described. The isolation of three amber uvrA and two amber uvrB mutants indicates that the products of these genes are proteins.


Methyl galactosidase activity: an alternative evolutionary destination for the ebgA0 gene.

Previous studies (Campbell et al., 1973; Hall and Hartl, 1974; Hall and Hartl, 1975) have shown that the ebgA0 gene, whose product does not hydrolyze lactose may evolve so that its product does hydrolyze lactose; i.e., lactase activity is one evolutionary destination of the ebgA0 gene. Beginning with a strain that synthesizes ebgA0 gene product constitutively and grows extremely slowly (doubling time, 30 to 50 h) on methyl-beta-D-galactopyranoside (MG), a derivative was selected capable of growth on MG at a moderate rate (doubling time, 5.9 h). Genetic evidence is presented showing that the gene that permits growth on MG is an allele of ebgA. A comparison among strains bearing several alleles of ebgA shows that the new allele, termed ebgAmg, synthesizes a product specific for MG and thus represents a true alternative evolutionary destination for the ebgA0 gene.


Active subunits of Escherichia coli glutamate synthase.

The large and small subunits of Escherichia coli glutamate synthase were isolated. The small subunit catalyzes the NH(3)-dependent synthesis of glutamate. The large subunit exhibits glutaminase activity.


Simplified method for interruption of conjugation in Escherichia coli.

A simplified procedure is described in which interruption of mating and recombinant selection both take place on plates containing selective medium plus nalidixic acid.


Threonine deaminase from Escherichia coli: feedback-hypersensitive enzyme from a genetic regulatory mutant.

A mutation, ilvA538, in the gene coding for the biosynthetic L-threonine deaminase of Escherichia coli K-12 has previously been demonstrated to have pleiotropic regulatory effects leading to low and invariant expression of some of the isoleucine-valine biosynthetic enzyme, and altered expression of the branched-chain aminoacyl-tRNA synthetases. Strain PS187, which carries the ilvA538 allele, has a partial growth requirement for L-isoleucine and is characterized by a sensitivity to growth inhibition by L-leucine. The experiments reported here demonstrate that the L-threonine deaminase produced by strain PS187 is hypersensitive to inhibition by the pathway end product L-isoleucine. In addition, L-leucine, which acts at relatively high concentrations in vitro as an inhibitor of L-threonine deaminase from the wild type, is a more potent inhibitor of the activity of the mutant enzyme. Forty-six derivatives of strain PS187 were isolated as spontaneous mutants resistant to the growth-inhibitory effects of L-leucine. Two of these, strains MSR14 and MSR16, produce an L-threonine deaminase that is more resistant than the wild type to L-isoleucine inhibition, and intermediate between the wild type and strain PS187 with respect to L-leucine inhibition. Strains MSR14 and MSR16 produce L-threonine deaminase and dihydroxyacid dehydrase, the ilvD gene product, at the low levels characteristic of the parent strain. Other L-leucine-resistant derivatives of strain PS187 produce higher levels of the feedback-hypersensitive L-threonine deaminase. Thus, the sensitivity to growth inhibition by L-leucine observed with strain PS187 appears to be related both to the hypersensitivity of L-threonine deaminase to inhibition of catalytic activity and to the low level of ilv gene expression. The results reported here indicated that L-threonine deaminase is structurally altered in strain PS187, and thus provide further support for the proposal that L-threonine deaminase participates as a genetic regulatory element for the expression of the branched-chain amino acid biosynthetic enzymes.


Comparative studies on membrane-associated, folded chromosomes from Escherichia coli.

Nuclear bodies were isolated from Escherichia coli spheroplasts by two different lysis procedures. Their lipid and protein content and the superhelix density was measured. The preparations differed mainly in respect to the amount of the attached membrane material, which seems to be an essential factor in maintaining the stability of the nuclear bodies. The superhelix density was found to be higher than that of naturally occurring, covalently closed, circular deoxyribonucleic acids. The influence of the preparative method on the superhelicity was comparatively small.


Mapping of two loci affecting the regulation of branched-chain amino acid transport in Escherichia coli K-12.

Two mutant loci resulting in derepression of, respectively, the L-leucine-specific transport system (lstR) and both the leucine-specific and the general branched-chain amino acid transport LIV-I systems (livR) were mapped by conjugation and transduction. Both livR and lstR were found to be closely linked to aroA at min 20 on the Escherichia coli genetic map. The merodiploid livR+/livR displayed wild-type regulation of L-leucine transport, indicating that the livR product is a diffusible, negative controlling element for high-affinity leucine transport systems. Isogenic strains carrying lstR, livR, and wild-type transport alleles were compared for leucine uptake kinetic parameters and leucine-binding protein levels. The higher levels of leucine transport in the mutant strains under repressing conditions were generally due to increased high-affinity systems, which were accompanied by striking increases in the level of leucine-binding proteins.


Selective methylation: an incorrect hypothesis.

"Selective methylation," a hypothesis proposed to explain the discrepancy found in the degree of methyl deficiency of transfer ribonucleic acid, cannot be explained on the basis of some biological phenomenon.


Enzymatic analysis of C27 sterol-accumulating yeast strains.

Several strain of bakers' yeast that accumulate only C27 sterols were analyzed for sterol methyltransferase activity, with no activity being found. Cholesta-5,7,22,24-tetraene-3beta-ol, one of the mutants' sterol products, was found to be an unacceptable substrate for in vitro transmethylation.


Extragenic suppression of two ribosomal protein cistrons lying near the rif locus in Escherichia coli.

The experiments reported here involve temperature-sensitive mutations in two of five cistrons encoding 50S ribosomal proteins that lie near the rif locus in Escherichia coli. I selected spontaneous TS+ mutants able to grow at elevated temperatures in which the TS+ event takes place outside this tract of cistrons near rif. Six distinct classes of extragenic suppressors were found, five of which have been mapped. Two of these suppressors lie near 64 min, a region known to be rich in cistrons ribosomal proteins (Dennis and Nomura, 1975). The remaining three extragenic suppressors were located near 16.5, 47, and 86 min.


Characterization of an Escherichia coli K-12 F-Con-mutant.

An Escherichia coli K-12 F-mutant defective in conjugation was isolated by means of a zygotic induction enrichment procedure. The recipient ability of the mutant was reduced about 50 times owing to a block in one of the first steps of the conjugation process. In the mutant, cell envelope alterations could not be observed. Sensitivity toward detergents, antibiotics, and phages was unaltered. The mutation appeared to be co-transducible with pyrD. The linkage order in the region of the mutation is origin KL 99-con-pyrD-aroA.