Existence, localization and regulation of the biosynthesis of aminoendopeptidase in gram-negative bacteria. An enzyme capable of hydrolyzing the substrate L-alanine p-nitroanilide has been found in 18 gram-negative Enterobacteriaceae and Pseudomonas that we tested. This enzyme might be located near the cell surface. Contrastingly we have not detected this activity on colonies of the 8 gram-positive Bacillus that we tested, which suggest a taxonomic value for this test. The aminoendopeptidases found in the various gram-negative bacteria showed similar electrophoretic mobilities and immunological cross-reactivity when tested again Escherichia coli anti-aminoendopeptidase antiserum. The regulation of aminoendopeptidase biosynthesis by the endogenous level of inorganic phosphate that we previously demonstrated in E. coli has been found to exist in all gram-negative organisms tested.


Two forms of RNA polymerase B in yeast. Proteolytic conversion in vitro of enzyme BI into BII. Special care to prevent proteolysis during yeast RNA polymerase B purification leads to the appearance of two forms of enzymes, BI and BII, with different molecular weight (465 000) and 435 000, respectively). The two forms of enzyme can be separated by ion-exchange chromatography or polyacrylamide gel electrophoresis. Their subunit structures were compared by sodium dodecylsulfate gel electrophoresis, the only observed difference between the two enzymes is in the molecular weight of the heaviest subunit which is 220 000 for enzyme BI and 180 000 for enzyme BII. Otherwise, the two enzymes have seven common subunits of molecular weights 150 000, 45 000, 26 000, 22 500, 14 500, 12 500 and 9000. Two additional polypeptide chains of 32 000 and 16 500 Mr are dissociated from the enzyme upon polyacrylamide gel electrophoresis or DEAE Sephadex chromatography. The largest subunit of enzyme BI (Mr 220 000) can be specifically cleaved in vitro by a yeast protease extract, generating a polypeptide chain indistinguishable from the largest subunit of enzyme BII. This proteolytic cleavage of enzyme BI in vitro is inhibited by phenylmethylsulfonyl fluoride and does not significantly change the activity of the enzyme with single-stranded or double-stranded DNA as template. The precursor-product relationship of the different forms of class B RNA polymerases in eukaryotic cells is discussed.


Purification and properties of an N-formylmethionyl-tRNA hydrolase. The isolation and properties of a novel N-formylmethionyl-tRNA hydrolase (hydrolase II) from Escherichia coli are described. This enzyme is difficult to detect in crude extracts; purification, however, unmasks the activity. Sedimentation and gel filtration parameters of this enzyme differ from those of the previously described peptidyl-tRNA hydrolase (hydrolase I), and preparations can be obtained where the two activities are free of each other. A mutant of hydrolase I has wild-type levels of hydrolase II. These data indicate that hydrolase II is a different enzyme, or an altogether different form of hydrolase I. The bulk of the enzymic activity occurs in the ribosome-free cytoplasm; the remainder is found on intact or dissociated 70-S ribosomes. Purified preparations of hydrolase II analyzed by two-dimensional gel electrophoresis contain 2 protein bands. These 2 proteins do not coincide in electrophoretic mobility with any known ribosomal proteins. Analysis after mixing experiments verifies this conclusion. The purified enzyme (hydrolase II) is inhibited by ribosomes bearing bound N-formylmethionyl-tRNA. The inhibition is potentiated by sparsomycin and other antibiotics that block specifically peptide-bond synthesis. The relationship of this enzyme to other hydrolytic activities, including a newly described ribosome-dependent hydrolase, are discussed.


On the kinetics of the rifampicin-RNA-polymerase complex. Differences between crude and purified enzyme fractions. The antibiotic rifampicin forms a very tight complex with DNA-dependent RNA polymerase of Escherichia coli. The rate constants of association and dissociation of this complex have been measured and found to be dependent on the purity of the enzyme. Thus a crude RNA polymerase (fraction-3 enzyme) has rate constants different from those of an enzyme further purified by DEAE-cellulose chromatography (fraction-4 enzyme). The complex produced by the antibiotic and the fraction-3 enzyme is about ten times more stable and is formed about ten times more slowly than the complex with fraction-4 enzyme. It has been shown that the RNA present in the crude enzyme and removed by chromatography on DEAE-cellulose is the cause of the change in the kinetics of the complex. tRNA of rat liver and crude rat liver RNA added to purified RNA polymerase have a similar effect. Mg2+, which has no intrinsic influence, augments the effect of the nucleic acids, whereas monovalent cations do not. Since nucleic acids increase the stability of the complex, but at the same time decrease the rat of its formation, the equilibrium constant, Keq, remains almost the same. The possible effects of nucleic acids on the rifampicin binding site are discussed.


Immunochemical studies on lipopolysaccharides from wild-type and mutants of Escherichia coli K-12. Lipopolysaccharides from a number of mutants of Escherichia coli K-12 were investigated by means of chemical and serological methods. Inhibition of passive hemagglutination and inhibition of precipitation show that L-rhamnose is the immunodominant sugar in the lipopolysaccharide from wild-type E. coli K-12. The disaccharide rhamnosyl-KDO (where KDO is 3-deoxy-D-manno-octulosonic acid) was isolated and characterized after mild acid hydrolysis of the lipopolysaccharide. It is concluded that rhamnose is present in the innermost part of the core as a side-chain substituent on KDO. From crosses between an E. coli K-12 donor and E. coli O8, hybrids were obtained which contained either one or both of the donor rfa and rfb clusters. Serum absorption studies with lipopolysaccharides from these hybrids indicated that the histidine-linked rfb cluster is responsible for the presence of rhamnose in the K-12 core oligosaccharide. Using paper chromatography of 32P-labelled lipopolysaccharides we have found heterogeneous lipopolysaccharide in two strains as well as some differences between two wild-type strains. The latter difference is believed to be due to varying contents of KDO-linked ethanolamine phosphate. The overall results presented together with those described in the companion paper clearly show that the core oligosaccharide in E. coli K-12 has a structure different from the types previously described for other strains of E. coli (designed coli R1 to coli R4).


Cell-wall lipopolysaccharides of ampicillin-resistant mutants of Escherichia coli K-12. The lipopolysaccharides of ampicillin-resistant cell-wall-defective mutants of Escherichia coli K-12 were analyzed. From their lipopolysaccharides the respective core oligosaccharides were obtained. Following dephosphorylation,the core oligosaccharides were methylated and analyzed by gas chromatography/mass spectrometry. From core-defective mutants substructures of the K-12 core were obtained. Analysis of the lipopolysaccharide preparations from wild-type K-12 indicated the presence of several core structures with different degrees of completion. The lipopolysaccharide preparation was degraded and the oligosaccharide mixture was partially resolved by gel filtration chromatography. Methylation, gas chromatography and mass spectrometry of the oligosaccharides permitted the tentative formulation of the K-12 core structure. Alternative interpretations for this heterogeneity are discussed.


Luteinizing hormone and follicle stimulating hormone and the response to luteinizing hormone releasing hormone in relation to sex and age. Serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) before and after intravenous injection of luteinizing hormone releasing hormone (LHRH) were studied in 71 male and female subjects of various ages. Mean basal FSH and LH levels were not significantly different in the male groups, except that FSH was significantly higher in the very old subjects. Postmenopausal female subjects had much higher concentrations of both hormones than had premenopausal women. LH was higher in female subjects shortly after the menopause than in very old female subjects. The serum concentrations of both FSH and LH 30 min. after intravenous injection of 200 mug LHRH were not different from the 60 min. values. There was no significant difference in the response of LH in the male groups. The peak FSH concentration was higher in the very old male subjects. Postmenopausal women had a much higher peak concentration of both FSH and LH than had younger subjects. The increment of LH, but not of FSH, was larger in female subjects shortly after the menopause than in very old female subjects. In both sexes there was a significant correlation between the basal FSH/LH ratio and age. In younger male subjects there was a close positive correlation between basal LH and serum testosterone, in older male subjects this correlation was negative and significant.


Thermal properties of glyceraldehyde 3-phosphate dehydrogenase from Escherichia coli. The molecular properties of glyceraldehyde 3-phosphate dehydrogenase from E. coli have been evaluated by circular dichroism and fluorescence emission spectroscopy measurements, with the purpose of studying the structural properties which are relevant for a comparison with the enzyme from the obligate thermophile Bacillus stearothermophilus. The enzyme is moderately resistant to heat treatment, being pratically stable when treated for 10 min at 50 degrees C and completely inactivated when heating was performed at 60 degrees C. The secondary structure of the E. coli GPDH appears to be predominatly beta-structure as judged by circular dichroism, showing a negative band centered at about 219 nm. The emission fluorescence of the enzyme shows a maximum at 333 nm upon excitation at 295 nm. In the native E. coli enzyme the tryptophan residues seem to be buried in a hydrophobic region rather than exposed to a polar environment. The structure of the enzyme did not change up to about 50 degrees C, at which temperature thermal inactivation takes place. Upon denaturation the circular dichronic signal at 219 nm gradually decreases, and a red shift of the emission maximum from 333 nm to ca. 345 nm upon heating is indicative that the native structure of the enzyme is unfolded, the tryptophan being exposed to the solvent medium. Since it has been found that the E. coli GPDH closely resembles in many of its properties the B. stearothermophilus enzyme, this bacterial enzyme seems to be useful for comparision with the thermophilic enzyme in studies of its thermostability.


Comparative conformational properties of thermophilic and mesophilic 6-phosphogluconate dehydrogenase. The structural properties of 6-phosphogluconate dehydrogenase from the mesophilic bacterium E. coli and the thermophilic B. stearothermophilus are compared using circular dichroism and fluorescence emission spectroscopy. The enzymes appear to possess a similar structure which does not change on heating up to the respective temperature of stability of the enzyme. The thermostability of the two 6-phosphogluconate dehydrogenases as determined by activity measurements parallels that determined by CD with the melting profile method, indicating that the loss of biological activity in the enzymes is directly related to the unfolding of the protein molecule. The pattern of unfolding of the proteins by the action of 8 M urea suggests that a core of enhanced conformational stability exists in the B. stearothermophilus enzyme.


Bacterial endotoxin and impaired fetal development. Small doses of E. coli endotoxin given to pregnant mice on the 13th day of pregnancy caused only a mild maternal illness but induced resorption of approximately half the number of fetuses in each mouse. The remaining live fetuses developed normally and showed no evidence of retarded growth or malformations. The weights of their placentas and maternal spleens increased significantly. Endotoxin given on the 6th day of pregnancy caused a small reduction in fetal weights.


Attempt to alter antigenic structure of normal tissues by treatment with dimethyl-triazeno-imidazole-carboxamide in vivo. The antigenic structure of normal skin and spleen cells has been investigated following in vivo treatment with the compound DIC. In experiments involving skin grafting in the normal and sensitized host, cross sensitization with a DIC-antigenic lymphoma and 3H-thymidine incorporation by lymphocytes cultured with DIC-treated spleen cells, new antigens on DIC-treated tissues were not demonstrated.


Controlling protein evolution.

Controlling protein evolution. A method is presented for selecting, with a high degree of specificity, mutants for the enzyme alcohol dehydrogenase (ADH) of Saccharomyces cerevisiae having altered kinetics. The method depends on the facts that a) petite yeast strains (those unable to respire aerobically) have an absolute dependence on the presence of cytoplasmic alcohol dehydrogenase, and b) allyl alcohol is readily oxidized by yeast ADH to the highly poisonous product acrolein. If petite yeast are grown in the presence of allyl alcohol surviving mutants show alterations that can be traced to the ADH structural gene. Eight out of nine spontaneous mutant enzymes and five out of five mutants enzymes induced by nitrosoguanidine exhibited a slower electrophoresis mobility than wild type. There may therefore be some correlation between electrophoretic mobility and function. Of three mutant enzymes purified, one of spontaneous origin and two induced by nitrosoguanidine, each showed a different pattern of altered kinetics and a different specific activity. This strongly suggests that, despite the considerable specificity of the selective procedure, there are many ways in which an enzyme can be altered in response to an environmental stimulus. The implications of these results for the study of protein evolution and some directions for future research are briefly discussed.


DNA sequence arrangement and preliminary evidence on its evolution. Some recent measurements of the sequence arrangement and evolution of the eukaryotic genome are reviewed. The range of genome sizes and extent of sequence transcribed into nuclear and messenger RNA indicate that the majority of the single copy DNA is not made up of structural genes. The rate of base substitution in the single copy DNA among the primates is similar to that of the codons for certain rapidly changing amino acid residues. This leads to the hypothesis that there is a "basal" rate of change in the genome not strongly affected by selection. The DNA of most higher animals shows a large amount of short period interspersion of repetitive and single copy DNA sequences and a smaller amount of long repetitive regions. The sequence divergence among the short interspersed repetitive sequences is greater than that of the sequences in long repetitive regions. The long repetitive regions are most probably recent additions to the genome and the short interspersed repetitive sequences result from a history of base substitution and translocation. The process of sequence rearrangement appears to be a significant part of the evolution of the genome and may have a much greater effect on the evolution of the phenotype than sequence alteration by base substitution.


Delay of puberty and inhibition of reproductive processes in the rat by a gonadotropin-releasing hormone agonist analog. Studies were performed in immature and mature female rats to determine the effects of chronic administration of [D-Leu6,des-Gly-NH210, Pro-ethylamide9]-gonadotropin-releasing hormone (GnRH) upon maturation and maintenance of reproductive function. Treatment of immature rats inhibited normal overian growth and maturation as evidenced by arrest of uterine growth, delay of vaginal opening, and absence of normal cycling. The primary responses in the mature animal were cessation of cycling, atrophy of the ovaries to a "prepuberal" size, and uterine weight regression. In both groups termination of treatment was followed by prompt restoration of normal ovarian mass and function, including support of breeding processes. The results presented here suggest that chronic administration of the GnRH analog inhibits ovarian steroid output either by direct action of the analog on the ovary or indirectly through qualitative or quantitative changes in levels of luteinizing hormone and follicle-stimulating hormone.


Immunization of mice with a mutant of Cryptococcus neoformans. Characterization of the mutant, actively acquired resistance to experimental cryptococcosis in mice. In 69% of subdermally and 50% of subcutaneously immunized mice, immunity against Cryptococcus neoformans was obtained. The vaccine consisted of a gamma-irradiated mutant, which grew well at 5 degree C and at room temperature. Many subcultures and animal passages did not restore this mutant to the encapsulated, pathogenic wild type.


[Sperm agglutination, sperm immobilization and sperm immunofluorescence test in married couples with unexplained infertility (author's transl)]. Thirty-four married couples with clinically unexplained infertility and normal spermiograms in all male partners were investigated for sperm antibodies by means of the sperm agglutination, sperm immobilization and sperm immunofluorescence test. In 50% of the couples, sperm antibodies were found in at least one of the partners and with at least one of the tests, e.g. ten times in the female partner alone, six times in the male partner alone, and once in both partners. The correspondance of the three tests was astonishingly low; it is assumed, therefore, that they indicate different types of antibodies. On the basis of the present investigations it is not possible to assess which type of antibody is responsible for the infertility of the female or male partner respectively. This seems only possible when the decrease of certain antibody types can be determined after a period of no antigen contact (avoidance of sperm contact) and when conception has taken place. The significance of autoantibodies against sperm in clinically healthy men with normal spermiograms remains open.


Absence of an inhibitory effect of oestradiol on LH-RH induced release of LH in vitro caused by inhibition of protein synthesis. Pituitary glands of ovariectomized rats were incubated for four hours and the LH released into the medium was measured by radioimmunoassay at one-hour intervals. The effect of a maximally active dose of LH-RH (1000 mg/ml) on pituitary LH release was inhibited by oestradiol (15 ng/ml) added to the media. Although cycloheximide (25 mug/ml) and puromycin (54 mug/ml) in the medium did not affect LH-RH-induced LH release consistently, the inhibitory effect of oestradiol was prevented. It is concluded that this effect of oestradiol is dependent on de novo synthesis of protein.


Measurement of intravesical and rectal pressures simultaneously with electromyography of anal sphincter in children with myelomeningocele. This paper reports a technique for simultaneous measurement of intravesical pressure, intra-abdominal pressure, electromyographic activity of the external and sphincter and urinary flow. Using this technique during filling of the bladder and during micturition in 38 children with myelomeningocele and neurogenic bladder disturbance, it was possible to identify four main types of bladder dysfunction in these children. Once the type of bladder dysfunction is established for each child, adequate management can be planned far in advance of the detection of inherent upper urinary tract damage. This is particularly important in children with upper somatomotor lesions of bladder innervation in which detrusor-spincter dyssynergia may be present; this dyssynergia always causes some degree of obstruction at the level of the external urethral sphincter and is a constant threat to renal function.


Prophylaxis in urinary tract infection in children with myelomeningocele. In 39 infants and children with myelomeningocele, no lower incidence of urinary tract infection was obtained by a trial of prophylaxis treatment with nitrofurantoin. Instead, nitrofurantoin-resistant strains appeared in some children, with the accompanying higher risk of re-infection.


The pepsinogens of cat gastric mucosa and the pepsins derived from them. Fundic mucosal extracts of the cat were examined by chromatography using DEAE-cellulose at pH 5.3. The peaks of proteolytic activity located by this method were shown to be heterogeneous on electrophoresis. By electrophoresis of individual homogenous chromatographic fractions 9 separate zones of proteolytic activity were detected. One zone did not migrate from the origin and is possibly a non-pepsinogen gastric protease. Simultaneous electrophoresis of pepsinogens and pepsins indicated that each pepsinogen gave rise to one pepsin. One zymogen gives rise to a pepsin active with the synthetic substrate acetyl-L-phenylalanyl-L-diiodotyrosine (APDT).


In vitro secretion of gastrin, insulin, and glucagon in tissue cultures of pancreas from a child with neonatal intractable hypoglycemia. Small pieces of pancreatic tissue were obtained at surgery from a subtotal pancreatectomy performed in a 45-day-old child suffering from intractable neonatal hyperinsulinic hypoglycemia. Histological examination was performed using aldehyde fuchsin, Grimelius', and Hellerström-Hellman's stainings, and immunoperoxidase labeling of insulin and gastrin. The pancreatic tissue before explantation showed numerous and sometimes hyperplastic islets, together with isolated insulin-, glucagon-, and gastrin-containing cells scattered among the exocrine tissue, in aspects similar to "B cell nesidioblastosis." These features could be interpreted as an acinoinsular transformation and/or an embryonic malformation. Extralobular endocrine islet formation by budding from ductular structures was evoked, suggesting the persistency of embryonic properties. The pieces were cultivated on rooster plasma coagulum covered with culture medium. In vitro, endocrine cells survived for 43 days, with outgrowth from the explant and with retention of their secretory abilities. After each medium renewal, radioimmunoassays were performed on the culture medium; they showed that insulin and glucagon secretions decreased with time. On the contrary, secretion of immunoreactive gastrin progressively increased, and kept up to 43 days, with subcultures. Some explants developed in a peculiar way, outgrowing as epithelial layers rich in gastrin-secreting cells as indicated by radioimmunoassays performed after they were reexplanted.


Spontaneous peritonitis of cirrhosis due to Campylobacter fetus. An unusual case of spontaneous peritonitis of cirrhosis associated with Campylobacter fetus is reported. This microaerophilic gram-negative bacillus has been increasingly associated with human disease recently, particularly in patients with cirrhosis. The infection is characteristically relapsing in nature and requires protracted antimicrobial therapy.


A computerized questionnaire analysis of duodenal ulcer symptoms. The characteristics, daily routine of life, and symptom patterns of 100 patients with a radiologically proven duodenal ulcer were compared with 100 controls. This confirmed that ulcer pain occurred periodically with attacks and remissions, could be relieved by antacids, vomiting, and avoiding fatty food, but was made worse by worry. The anatomical site was usually epigastric but other sites were common. Pain was unaffected by diet or a change in the seasons and had no constant relationship to meals. It occurred during the day in all of the patients and at night in 88%.


Alcoholism: its effect on hypothalamic pituitary gonadal function. Both as the result of liver disease and of alcoholism per se, chronic alcoholics develop infertility, sterility, gonadal atrophy, hypoandrogenization, and feminization. The hypothalamic-pituitary abnormalities associated with alcoholism include hyperprolactenemia-increased estrogen-stimulated neurophysin levels, suppressed secretion of plasma gonadotropins, and loss of gonadotropin reserve. Several of the possible mechanisms potentially responsible for the development of these endocrine abnormalities have been discussed. The rational for suspecting that alcohol might interfere with either vitamin A metabolism of alter the redox state of the testes, thus affecting germ cell proliferation and steroidogenesis, has been presented. A possible mechanism for the sexual changes observed in chronic alcoholic men has been proposed. Much work remains to be done in this area before a complete understanding of the pathogenesis of these phenomena is obtained. The omission of any consideration of the effects of alcohol on sexual function in women is an admission of gross ignorance greatly in need of rectification. The necessity for couching a description of even the natural history of the syndrome in alcoholic men in conditional terms is a reflection of the limited state of the art. Nevertheless, it behooves the gastroenterologist, who is frequently called upon to be the primary physician for alcoholic men, to keep abreast of the nongastrointestinal medical consequences of alcohol addiction so that they can be recognized early and incorporated into long range medical planning designed to care for the chronic alcoholic patient.


Bacterial variants: etiologic agent in Crohn's disease? Bacterial variants of Pseudomonas maltophilia and Pseudomonas-like bacteria were recovered from tissues removed during the surgical treatment of three successive patients with Crohn's disease and from one patient with clinical and pathological features of both Crohn's disease and chronic ulcerative colitis. Bacterial variants were not cultured from colonic specimens of one patient with classical features of chronic ulcerative colitis and two patients with adenocarcinoma of the colon. The findings suggest that a relationship exists between variant bacteria and the pathogenesis of Crohn's disease.


[Circulatory disturbances in the embryo (author's transl)]. Circulatory disturbances in human embryo in the phase of organogenesis are frequent, as shown by the pathological-anatomical findings and the results of experimental embryology. There is edema and hemorrhage around the blood vessels and emboli of necrotic tissue (chiefly from the chorion) are found. The embryonic heart, which shows peristaltic movements, has a very small reserve of performance. Since there is no peripheral regulation of circulation, every damage to the vascular system results in a higher cardiac load. During organogenesis the extraembryonic vascular plexus is considerably larger than the intraembryonic plexus. Lesions of the chorion, the barrier between embryo and environment, can result in chronic or acute cardiac insufficiency. This is presumably one of the reasons why the mortality in this developmental stage is particularly high.


[Report of clinical testing of BCG vaccine (Strain "1331 Copenhagen") for vaccination of newborns against tuberculosis (author's transl)]. BCG vaccination with the vaccine "1331 Copenhagen" in various concentrations was carried out in 129 newborns. Control examinations were conducted to establish the complication rate and the percentage of tuberculin-positive children. Complications, either locally or in regional lymphatic areas, did not appear. The conversion rate increased with the number of viable units pro dose of vaccine. A vaccine with a concentration of about 250,000 units of vaccine pro dose is recommended for additional clinical testing.


Detecting and treating arteriosclerotic renovascular hypertension. Renovascular hypertension is the most prevalent form of surgically remediable hypertension. With appropriate selection of operative candidates, a high rate of cure or improvement may be anticipated. Favorable results justify revascularization in hypertensive patients with focal arteriosclerotic renal artery disease. The absence of advanced arteriosclerosis provides the most valid basis for predicting long-term survival following revascularization. Patients with clinically demonstrable diffuse arteriosclerotic disease are preferably treated medically; we consider them candidates for surgery only if drug therapy cannot control blood pressure. Renal revascularization does not appear to alter the inexorable progression of concomitant generalized arteriosclerosis. Revascularization rather than nephrectomy is the primary surgical therapy for renal artery stenosis in properly selected patients.


Delayed flexor tendon repair in the digital sheath with end-to-end suture and fascial graft. The results of treatment of nine divided tendons in scarred fingers by secondary suture and reconstruction of a new sheath by fascial graft is reported and discussed.


The by-pass operation for arthritis at the first carpometacarpal joint. The By-Pass operation provides a less destructive alternative to arthroplasty or arthrodesis for fixation of the first carpometacarpal joint.


Effect of phytohaemagglutinin on primary immune response in the rat. PHA pretreatment if given as a single stimulus exerted a stimulatory effect on SRBC haemolysin production rats. If administered repeatedly, it proved to be immunosuppressive.


Lymphocyte immunological patterns in leukaemia: a review. Investigation of the cell S--Ig in acute lymphocytic leukaemia (ALL), at the onset of relapse of the disease, shows quite marked differences from patient to patient according to the extent of the immunofluorescent-positive cells. They may vary from 0.5 to 25% or more. When these Ig-positive cells are treated with trypsin and then incubated "in vitro" for six hours, many of them are no longer Ig-positive, i.e. they do not synthesize Ig. It might be possible, that the membrane-Ig observed before trypsinization does not represent true Ig-determinants of mature B-cells (antibodies attached to leukaemia-specific determinants?). The extent of these features decrease in remission until their disappearance. Relationship between the cell immunological patterns and the treatment response in ALL could exist. In a group of ALL-patients under the same treatment, that is, vincristine and prednisone, the correlation between the course of the disease after the above-mentioned therapy showed quick and complete remission in patients with low percentage of Ig-positive cells (below 10%) and poor improvement (often without complete remission) in patients with higher percentage of Ig-positive cells. Among the most important B-lymphocyte abnormalities in chronic lymphocytic leukaemia (CLL) are the following: (a) fluorescence intensity may vary not only from patient to patient, but also from cell to cell in the same patient; (b) the Fc-receptor can be lacking; (c) the C3b-receptor is not always present, or it is from 2 to 20-folds less frequent than the C3d-receptor, whereas normal human lymphocytes do not show any outstanding differences between the number of EAC rosette-forming cells either when tested with mouse complement (C3d-receptor) or with human complement C3b-receptor); (d) the traffic capacity of peripheral-blood B-lymphocytes in CLL is quite defective. Results of the observations on lymphocytes in CLL, taken as a whole, suggest that CLL is in general given by the expansion of an abnormal clone of cells of B origin, arrested in their maturative development, non-responsive to the mitogen stimulation, accumulating in the peripheral-blood for a traffic deficiency. On the contrary, the T-cells class is apparently normal, and the T-cell extent in CLL-peripheral blood can be even greater than normal when taken as absolute value.


Leptospiral antibodies in wild living animals from North Tyrol. The authors examined serologically 623 wild living animals (22 species) from North Tyrol for the incidence of leptospirosis. Positive reactions (MAL) in the titre 1: 100 and more were found in 4.5% of the animals examined; the serotypes concerned were these: icterohaemorrhagiae, sorex-jalna, castellonis or arboreae, grippotyphosa, bratislava, pomona, sejroe, saxkoebing. Positive reactions were obtained with the sera of Sorex araneus, Erinaceus europaeus, Putorius putorius, Vulpes vulpes, Cervus elaphus, Capreolus capreolus, Rupicapra rupicapra, Apodemus sp., Clethrionomys glareolus. The serotypes forming natural foci of leptospirosis in North Tyrol are these: sorex-jalna, grippotyphosa, bratislava, saxkoebing and, apparently, jalna. Synanthropic foci are formed by icterohaemorrhagiae or copenhageni and sejroe.


Loss of inducible D-galactose transport by baker's yeast after osmotic treatment. The ability of Saccharomyces cerevisiae to transport D-galactose and related sugars with an axial hydroxyl group at C-4, acquired by induction with D-galactose, was lost either by exposing early exponential-phase cells to an osmotic shock involving incubation in 0.6M NaC1O4, 0.66M sucrose and 1mM histidine and transfer to 5mM Tris-HC1 with 2mM dithiothreitol, or simply by transferring them to distilled water. The total amount of protein thus released was 0.1--0.35 and 0.1 mg per mg dry wt., respectively. The shock fluid contained at least six proteins, among them a galactose-binding component. L-Arabinose transport could not be restored by adding the concentrated shock fluid to depleted cells but cells remained viable after the shock and resynthesized the transport system if incubated in a galactose-containing growth medium.


Role of post-replication and excision repair mechanism in the induction of Trp+ revertants of UV-irradiated Escherichia coli. Both the post-replication and the excision repair mechanism participate in the induction of Trp+ revertants in Escherichia coli B/r Hcr+ thy trp after a UV-irradiation. At low radiation doses (surviving cell fraction greater than 10(-1) most Trp+ reversions are due to post-replication repair mechanism while at high doses (surviving cell fraction less than 10(-1)) the Trp+ reversions arise probably as the result of an inaccurate excision repair. The absolute accuracy of repair processes decreases with increasing radiation dose.


The effect of substitution of diaminopimelic acid by 4-hydroxy-diaminopimelic acid on the synthesis and degradation of murein in Escherichia coli 173-25. Defects in the formation of the septum and gradually autolysis of cells occur when the dap-dependent mutant of Escherichia coli is grown in a medium with 4-hydroxy-diaminopimelic acid. When the culture grown in the presence of the labelled analogue is supplemented with the non-radioactive diaminopimelic acid a portion of the TCA-soluble radioactivity is released from the cells during 20 min after the addition of diaminopimelic acid. During this time interval the elongated forms formed in the presence of the analogue divide, however, only on the condition that the above forms are not irreversibly damaged. The increased concentration of the analogue in the medium substantially suppresses the irregularities in the development of the septum as well as the degradation of analogue containing cell wall. However, the growth rate in the presence of the analogue is always slightly lower than that in the presence of diaminopimelic acid. The cell wall pulse-labelled with diaminopimelic acid or its analogue for a time interval shorter than 1/4 of the generation time exhibits the same or only slightly higher rate of diaminopimelic acid is probably utilized less effectively for the synthesis of murein than diaminopimelic acid. However, its incorporation into the wall does not result in pronounced damage of the cell.


Insulin release and the microtubular system of the islets of Langerhans: effects of insulin secretagogues on microtubule subunit pool size. An assay system was devised to estimate the pool size of microtubule subunits in islet cells, and to study the importance of the equilibrium between polymerised microtubules and their subunits in the regulation of insulin release. The assay was based on the observation that colchicine binds specifically and quantitatively to microtubule protein subunits, but not to intact microtubules. Vinblastine and cold treatment, which have been shown to cause disaggregation of microtubules into subunits and to inhibit insulin release from islets, increased the number of colchicine binding subunits. D2O, which promotoes stability of microtubules and increases their number in islet cells, caused a decrease in subunit concentration. These results suggest that changes in the equilibrium between polymerised microtubules and their subunits could be studied by measuring the size of the subunit pool. When insulin release was stimulated, by incubating islets in high glucose concentrations or by increasing the intracellular concentration of cyclic AMP there was a reduction in the content of subunit protein. Conversely, when insulin release was inhibited by removal of calcium from the incubation medium there was a shift in the microtubule subunit equilibrium to give an increase in the number of subunits assayed. These results indicate that changes in the equilibrium between subunits and microtubules may play an important role in regulating rates of insulin secretion.


Effect of ovariectomy and estradiol benzoate treatment on LH-RH induced LH and FSH release in the female rabbit. Synthetic LH-RH (0.75 and 2.5 mug/animal) was administered to mature intact and castrated female rabbits. The animals were injected for 3 consecutive days with either 40 mug estradiol benzoate (EB) dissolved in 0.5 ml propylene glycol 60%, water 40% or with the solvent alone. LH-RH was injected i.v. on the 4th day. In intact rabbits EB priming increased the pituitary LH response while LH basal levels were lowered. In the unprimed castrated rabbits, basal FSH and LH levels and the cumulative responses to LH-RH were higher than in intact control animals; EB priming lowered basal LH levels and cumulative responses to the values of intact control rabbits while both basal FSH levels and the cumulative responses were less influenced by the treatment. It is concluded that estrogen acts at both hypothalamic and pituitary levels to modify the LH responses to endogenous LH-RH, and that the complete inhibition of FSH hypersecretion in the ovariectomized rabbit probably needs another ovarian factor.


The National Health Planning Act: significance for mental health agencies. In examining the National Health Planning and Resources Development Act, the authors identify nine areas that have special meaning for those responsible for planning, administration, and delivery of mental health services. Believing that mental health planning can be done more effectively by mental health agencies, they recommend a series of steps that can be taken by mental health administrators to maintain responsibility for planning their programs while ensuring close coordination with general health planning activities. It is their conviction that mental health authorities should commit themselves to the planning process and put their commitment to work by recruiting knowledgeable and skilled planning staffs.


Growth and characterization of human skin epithelial cell cultures. In 129 of 140 attempts, human skin cells were successfully cultured on the dermal collagen bed of sterile, dead pigskin. Diploid epithelial cells grew selectively on the collagen bed; fibroblasts grew on the glass surfaces of the culture dishes. The cultures could be subdivided physically up to six times at a 1:2 split ratio, but at least 24 to 48 cell generations were produced over the months the cells could be carried. Much of the cell multiplication resulted in maturation into distinct basal, squamous, granular, and keratinized cell layers. The cultured cells were considered epithelial because of their shape, possession of intercellular bridges, desmosomes and tonofibrils, and because they formed maturating epithelium in vitro and upon transplantation back to the original human donor. As the cells grew they digested the pigskin collagen, thus producing clear zones that could be used to monitor and quantitate cell growth. Multiplication of epilthelial cells, rather than migration, was indicated by mitotic figures in colchicine-treated cultures and by DNA synthesis.


The effect of fetal bovine serum on polyene macrolide antibiotic cytotoxicity and antifungal activity. The relationships between fetal bovine serum (FBS) concentration and polyene macrolide antibiotic cytotoxicity to animal cells and to fungi were evaluated. The toxicity of amphotericin B (AB) and its derivative, amphotericin B methyl ester (AME), toward KB cells was found to be directly related to fetal bovine serum concentration. At higher FBS levels, increased concentrations of AB and AME were required to reduce 72-hr KB viable cell numbers of 50% of control values. Similarly, polyene macrolide antibiotic levels required to inhibit the growth of Saccharomyces cerevisiae to 50% of controls, and for obtaining minimum fungicidal concentrations (MFC), were greater when higher levels of FBS were used. In addition, AME was less toxic than AB toward KB cells grown in media containing 2, 5, 10, 15 or 20% FBS, whereas the antifungal activities of AB and AME were similar. AME was also capable of eliminating Candida albicans, Saccharomyces cerevisiae, Aspergillus niger or Fusarium moniliforme from KB cultures at antibiotic levels which exhibited less cell toxicity than did the concentrations of AB required for a similar response. These findings indicate that AME may be a potentially useful antifungal antibiotic for tissue culture systems.


Simple method for simultaneous analysis of different fibroblast strains on one slide. Microscopic slides were mounted with paraffin on Teflon plates that contain 18 holes of 6 mm diameter each. This arrangement is reversible and yields 18 individual cell culture wells on one slide, thus allowing a simultaneous culture of different cell lines under identical conditions. The glass-paraffin junction can be easily detached and the paraffin sticks exclusively to the Teflon.


Identification of group a streptococci by reverse passive hemagglutination. A method for preparing sensitized human O erythrocytes with specific antibody is reported. Whole sera or 40% ammonium sulfate insoluble  globulin fractions did not satisfactorily sensitize erythrocytes to agglutinate in the presence of either group specific polysaccharide of group A beta-hemolytic strepticocci or antigen produced during colony formation. Antibody purified by affinity chromatography sensitized the erythrocytes to rapidly agglutinate in the presence of either antigen. No spontaneous or "pseudo-immune" agglutination occured when the sensitized erythrocytes were suspended in human serum. Such sensitized human cells, while more difficult to prepare than sensitized staphylococci, should be suitable for detecting bacterial or viral antigens in vivo.


Resemblance of blast responses of C3H/HeJ mouse and rabbit spleen lymphocytes to B-cell mitogenic components of E. coli cell walls. Nude mice lymphocytes were stimulated by heat-killed E.coli bacterial cells as well as by various components of their cell walls such as lipopolysaccharide, peptidoglycan and lipoprotein. Only bacterial cells, peptidoglycan and lipoprotein induce a high stimulation of C3H/HeJ mouse and rabbit lymphocytes. These data show the resemblance of mutant C3H/HeJ mice to rabbits with regard to blast response induced by various mitogenic components of E.Coli cell walls.


Fate of 51Cr-labeled lipopolysaccharide in tissue culture cells and livers of normal mice. Livers of normal mice trapped over 80% of intravenously injected 51Cr-labeled lipopolysaccharide after 1 h. Liver fractionation studies showed that nearly 45% of the labeled endotoxin was associated with cell nuclei, 20% with the mitochondrial-lysosomal fraction, and approximately 30% with the cell sap. Analysis of the distribution of 51Cr-labeled lipopolysaccharide among parenchymal and Kupffer cells showed that over 75% of the in vivo-trapped counts were parenchymal cell associated. Cell populations were approximately 65% parenchymal cells and 35% nonparenchymal cells. Further, six non-reticuloendothelial system tissue culture cell lines were tested for their ability to internalize labeled lipopolysaccharide. In all cells studied, 1 to 4% of the labeled lipopolysaccharide was taken up after 3 h, with greater than 80% of the counts localized in the nuclear fraction. The data show that non-reticuloendothelial system cells can sequester endotoxin both in vivo and in vitro and suggest that parenchymal cells as well as Kupffer cells remove circulating endotoxin from the blood.


Chemiluminescence response of phagocytizing human monocytes. Chemiluminescence (CL) occurs as a consequence of phagocytosis of bacteria or inert particles by human polymorphonuclear neutrophils. This phenomenon appears to be related to the production of singlet oxygen and/or electronically excited carbonyl groups which relax with light emission. We report that human monocytes also produce CL after phagocytosis of opsonized bacteria, fungi, or zymosan particles. Monocytes produce approximately one-third of the CL produced by polymorphonuclear neutrophils for all three particles, reflecting possible metabolic differences between cells of these types. Addition of exogenous superoxide dismutase reduced the peak production of CL by 65% for cells of both types, whereas raising the temperature of the cultures from 25 to 37 C approximately doubled the peak CL responses.


T and B cell population changes in young and in adult rats infected with Plasmodium berghei. Malaria infection in young rats is characterized by high parasitemia, severe anemia, and death. Parasitemia is lower in older rats, and the rats usually survive. This study was designed to investigate the immunological basis of this difference. T cell numbers in the thymuses and spleens of young (4 weeks old) and in adult (18 weeks old) infected and control rats were determined by killing with anti-theta serum and complement. The number of complement receptor lymphocytes (B cells) in spleens was determined after these cells had formed rosettes with sensitized, complement-coated sheep erythrocytes. Infection in young rats was characterized by progressive and severe thymic involution and by decreasing numbers of T and B cells in the spleen. In 18-week-old rats, T cell numbers in the spleen were slightly below those of controls early in infection but exceeded normal values by day 15. Progressive thymic involution was not a feature of infection in adult rats. The number of complement receptor lymphocytes in the spleens of adult rats decreased dramatically early in infection but were nearly normal by day 15. Severity of malarial infection in young rats is related to the inability of their lymphocytes to respond to Plasmodium berghei antigens early in infection in a way that leads to immunity.


Adoptive transfer of immunity to Plasmodium berghei with immune T and B lymphocytes. Immunity to malarial infection may be transferred with immune lymphocytes. This study was designed to determine which lymphocyte type is responsible for the adoptive transfer of immunity to malarial infection. In one set of experiments, the ability of immune T and B lymphocytes, separated by passage through nylon-wool columns, to transfer immunity to infection was determined. In another experiment, the effect of killing T lymphocytes with anti-theta serum on the transfer of immunity was determined. The effect on the ability of immune lymphocyte suspensions to transfer immunity after B lymphocytes were removed from such suspensions by centrifugation on Ficoll-Hypaque gradients, after they had formed rosettes with sensitized, complement-coated sheep erythrocytes, was also determined. The ability of lymphocyte suspensions to adoptively transfer resistance to malarial infection was greatly impaired by the removal from the suspensions of differentiated B-type lymphocytes. Our results indicate that it is the differentiated B cell, most probably an antibody-producing cell, which lacks both theta antigen and the complement receptor that is responsible for conferring immunity to malaria.


Adhesive properties of Vibrio cholerae: nature of the interaction with intact mucosal surfaces. Two companion papers in this series have characterized the interaction between Vibrio cholerae and the surfaces of eukaryotic cells. The present paper reports studies of the association between vibrios or Salmonella enteritidis and intact slices of intestinal tissue. A significant number of differences were noted in the characteristics of bacterial adhesion in these systems. The results are interpreted to indicate the presence of at least two receptors for vibrio adhesion on the mucosal surface of the rabbit small intestine. The receptor mediating the adhesion of salmonella appeared to be distinct from these. A primary role for bacterial motility in the process of adhesion of vibrios to mucosal surfaces could not be demonstrated in the assay systems studied. Rather, loss of motility in mutant vibrios appeared to be correlated with the simultaneous loss of adhesive factors (adhesins) from the bacterial surface. The inhibition of vibrio adhesion to slices of intestinal tissues by antibody to the heat-stable antigens of V. cholerae occurred in the absence of bacterial agglutination. Agglutination in this assay system appeared to be an artifact in that it could be observed only in experiments where extremely high concentrations of vibrios were used. We speculate that such high vibrio concentrations are not likely to be present in humans at the time of infection and that agglutination in the lumen of the intestine might therefore play only a minor role in prophylactic immunity against natural cholera and other enteric infections of humans.


Increased antibacterial activity against Escherichia coli in bovine serum after the induction of endotoxin tolerance. Small amounts of endotoxin injected intramuscularly into cows induced endotoxin pyrogenic tolerance and an increase in the rate at which the serum killed a strain of Escherichia coli. Most of the difference between normal serum and serum from the endotoxin-tolerant animal was shown to be due to a bentonite-adsorbable factor other than lysozyme or beta-lysin. The antibacterial activity was not completely removed from either type of serum after bentonite adsorption. Electron microscope studies and measurement of the rate of release of radioactively labeled cytoplasmic contents showed that the bentonite-adsorbable factor was important in the final breakdown of the cell membrane and release of cellular contents. The antibacterial system was totally dependent on complement, and the importance of antibodies could not be entirely ruled out because adsorption at O C with homologous cells eliminated the killing activity.


Effect of systemic BCG infection in syrian golden hamsters. The response of Syrian golden hamsters to systemic infection with several doses of Mycobacterium bovis (strain BCG) was assessed. Large numbers of organisms (10(7)), injected intravenously, were lethal for hamsters, whereas all animals survived infection with 10(4) colony-forming units of BCG. Animals responded immunologically to purified protein derivative as assessed by increased footpad swelling and splenic lymphocyte proliferation. The immediate cause of death was a diffuse granulomatous interstitial pneumonia.


Depression of lymphocyte response to concanavalin A in rabbits infected with Treponema pallidum (Nichols strain). Peripheral blood lymphocytes isolated from rabbits in the early stages of Treponema pallidum infection responded poorly when exposed to concanavalin A in vitro. Maximal depression of blastogenesis occurred when lymphocytes were cultured in the presence of autologous serum in comparison with fetal calf or normal homologous rabbit serum.


Experimental Candida albicans infection in conventional mice and germfree rats. Swiss-Webster white mice were intravenously infected with various doses of Candida albicans, and the viable units in their spleens, livers, lungs, and kidneys were determined at various intervals after challenge. The results showed that C. albicans multiplied to a greater extent in the kidneys of mice than in their spleens, lungs, or livers. The infection in mice was chronic; increasing numbers of C. albicans were observed in their kidneys until about 17 to 24 days postchallenge. Clearance of C. albicans from infected kidneys was not symmetrical, since the number of viable C. albicans in one kidney did not coincide with the viable counts observed in the opposite kidney of that same animal. Male and female mice did not differ in their overall susceptibility (50% lethal dose test) or in the number of viable C. albicans in the kidneys at various time intervals after infection. C. albicans also multiplied in the kidneys of germfree rats; however, the peak of the C. albicans infection in their kidneys occurred earlier than in those of conventional mice.


Inhibition of phagocytosis by cryptococcal polysaccharide: dissociation of the attachment and ingestion phases of phagocytosis. The effects of cryptococcal polysaccharide and selected serum factors on (i) the attachment of Cryptococcus neoformans to macrophages and (ii) the subsequent ingestion of yeast cells by the macrophages were investigated. Percent attachment was measured after incubation of yeast cells with macrophages at 4 C. Percent engulfment was determined after incubation of yeast cells and macrophages at 37 C. Nonencapsulated yeast cells readily attached to macrophages at the low temperature and were engulfed at a high rate at 37 C, whereas encapsulated yeast cells attached to macrophages at low rates and were engulfed at low rates. Addition of varying doses of purified cryptococcal polysaccharide to nonencapsulated yeast cells inhibited attachment at approximately the same concentration of polysaccharide required for inhibition of engulfment. Nonencapsulated yeast cells that attached to macrophages at 4 C were eluted from the macrophages by addition of purified cryptococcal polysaccharide to the incubation medium. Heat-labile opsonins were not required for attachment of yeast cells to macrophages, but they were necessary for maximal initial rates of phagocytosis. Heat-stable components of serum facilitated attachment of cryptococci, but their most important function appeared to be triggering the ingestion of attached yeast. Specific antiserum had no effect on the attachment and engulfment of nonencapsulated cryptococci, and the antiserum produced only a small enhancement of the engulfment of encapsulated cryptococci.


Suckling mouse model for detection of heat-stable Escherichia coli enterotoxin: characteristics of the model. Although the suckling mouse assay is widely used for the detection of heat-stable Escherichia coli enterotoxin (ST), few data have been published concerning the reproducibility, optimal growth, and test conditions of this assay. Four strains of toxigenic E. coli known to elaborate both heat-labile enterotoxin and ST or ST alone were used to study these parameters. ST activity after heat treatment and the effect of purified choleragen were also examined. ST production was optimal in Casamino Acids-yeast extract media, but both Trypticase soy and brain heart infusion broths resulted in several false negative reactions. Growing cultures in roller tubes was the most reliable method of ST production. Shaking-flask cultures and stationary-grown cultures resulted in suboptimal ST production in several strains. Optimal mouse incubation time was 3 h, and fluid secretion did not rise thereafter. Adequate toxin production occurred after 16 to 24 h of incubation. The coefficient of variation of various toxins tested on many occasions varied between 10.5 and 15.7%. Toxin activity was stable for 6 months when frozen at - 20 C. There was no decrease in ST activity when heated at 65 C for 15 min, but a small decrease was observed in two of four strains after heating at 100 C for 30 min. Choleragen, tested at various doses and at multiple times, gave uniformly negative results. These studies indicate that when done under the proper conditions, the suckling mouse assay is a simple, rapid, and reproducible assay for E. coli ST.


[The importance of serum components, particularly complement factors, properdin and transferrin, in inhibition of bacterial growth by human serum (author's transl)]. In this study human serum was broken down of column chromatography into its protein components, which were than examined singly and in comnination for antibacterial activity. The bactericidal effect of serum was retained to an apperciable extent in only one of six fractions. Antibacterial inhibitory activity could be restored fully by combining the fractions however. There was no correlation with the specific antibody titers. Complement had an essential part in this unspecific defence. In the absence of C'9 all other combinations of the remanining complement components exhibited no appreciable inhibitory effect. Not all complement componets are required, however, for unspcific bacterial growth inhibition. In contrast to this all complement components are necessary in the antigen-antibody reaction. Futhermore, properdin displays bactericidal activity which is related to the presence of complements. A great deal of the antibacterial activity of human serum thus arises only from the combined action of several components. These do not include proteins with a molecular weight between 100,000 and 70,000, whereby transferrin plays a part.


Parameters of acquired resistance and their role in the evaluation of new chemotherapeutic drugs. Acquired resistance can be defined as a qualitative alteration of the genetic material of a cell which is phenotypically correlated with a measurable decrease of the cell's sensitivity against one or several chemotherapeutic agents. There are two basic genetic mechanisms which can lead to the emergence of resistance: mutation and the acquisition of additional genetic material from another cell. Both forms of resistance play an important role in clinical situations: the emergence of resistance by mutation occurs in tumor cells and can also lead to therapeutic problems in antimicrobial chemotherapy. In bacteria, however, acquisition of resistance plasmids represents the dominating mechanism which is responsible for most therapeutic problems in the clinical environment. The different genetic mechanisms involved in the emergence of resistance are paralleled -- at least in bacteria -- by two principally different groups of biochemical mechanisms implementing resistance. Mutations lead to alterations of single cell constituents such as the cell membrane or cellular receptors necessary for the binding of the antimicrobial agent. This form of resistance is biochemically characterized by the inaccessibility of the cell interior for a particular compound or by the modification of an intracellular binding site which loses its affinity for the chemotherapeutic agent. Resistance plasmids on the other hand code for enzymes which inactivate the antibiotic (beta-lactamases, aminoglycosideinactivating enzymes, chloramphenicol-acetyltransferase); In some cases, they direct the synthesis of proteins which affect cell permeability (tetracycline) or isoenzymes which have a lower affinity for the inhibitor (trimethoprim). Resistance against antibiotics can be inducible; In these cases the regulatory mechanisms involved are stable genetical traits as resistance itself; Using chloramphenicol, beta-lactam-antibiotics and aminoglycosides as examples, it is demonstrated that resistance data gathered early in the development of a new drug are of little value in estimating the clinical potential of a new compound. Information on the rate at which resistance develops, on the pattern according to which it emerges ("single step" or "multi step") and on cross-resistance patterns is important in the characterization of a new drug but is often invalidated by later findings obtained in the clinical environment; The problem appears somewhat simpler if a new drug is a member of an already known class of compounds, e.g. a beta-lactam or an aminoglycoside. In such cases our knowledge of frequent enzymatic inactivation mechanisms provides a basis not only for the evaluation of an existing drug, but also for the synthesis of new derivatives.


Selective suppression of T-cell activity in tumor-bearing mice and its improvement by lentinan, a potent anti-tumor polysaccharide. The cellular site of immunosuppression in Ehrlich tumor-bearing mice was analysed with particular reference to the T- and B-cell activities. The  B-cell activity as measured by the anti-dinitrophenyl (DNP) antibody responses to DNP-thymus-independent carriers (TID) was not impaired in tumor-bearing mice as compared with normal mice, whereas the anti-DNP antibody response to DNP-thymus-dependent carriers (TD) and the development of helper T-cell activity to TD were markedly suppressed in tumor-bearing animals or mice pretreated with cell-free cancerous ascitic fluid. The selective suppression of T-cell response was not mediated by the generation of suppressor cell activity toward TD, which may depress the manifestation of developed helper T-cell activity. A marked suppression of T-cell response was observed when the animals were inoculated with tumor cells or injected with cancerous ascitic fluid prior to antigenic stimulation, but not when the animals were rendered tumor-bearing by such treatments after the immunization. The suppression of T-cell activity in both sarcoma 180 tumor-bearing mice and cell-free Ehrlich cancerous ascitic fluid-treated mice was prevented by treatment with lentinan, a potent anti-tumor polysaccharide. The applicability of this experimental system to the search for immunopotentiators relevant to tumor immunotherapy is discussed in the light of the preventive effect of lentinan on the suppression of T-cell response in tumor-bearing animals.


A larger perspective on the Flexner report. This paper is an attempt to place the Flexner Report of 1910 in the larger perspective of dynamics of American society in the years surrounding the turn of the 19th century. It looks at medicine as an integral part of society affected by the contradictions affecting the larger society. The Flexner Report is viewed as an attempt by the capitalist class to ameliorate those contradictions affecting medicine. The events preceding the publication of the Flexner Report are described in detail, and some problems with conventional accounts of the origins of the Flexner Report are discussed. In addition, the paradigm of medicine that was institutionalized with the acceptance and implementation of the Flexnerian recommendations is discussed.


The effect of food and clopamide on the absorption of pindolol in man. Plasma levels and urinary excretion of pindolol were measured in each of two groups of eleven subjects in order to compare the absorption of the drug when administered alone in the fasting state, and either with food or Binaldix. The data were analyzed according to a one-compartment model with first order absorption. No significant differences in absorption of pindolol were obtained in each study. Food appeared to increase the speed of absorption of pindolol leading to a slightly earlier and higher maximum plasma concentration, but this observation appears to be of no clinical relevance.


Postcardiotomy delirium: an overview.

Postcardiotomy delirium: an overview. Any one of a number of psychologic patterns may appear cardiotomy: (1) Some patients may be elated and confident after awakening from anesthesis and have no severe changes of affect or neurologic deficit. Denial seems to be for them an adequate defense against anxiety. (2) Others are disoriented and manifest neurologic disturbance immediately after awakening, without a lucid interval. The sensorium begins to clear five days after surgery. (3) Some patients go into delirium after being lucid for as long as a week and have hallucinations, illusions, and motor excitation for a few days-or over several weeks. Pathologic brain changes that are apparently anatomical correlates of neurologic deficits in delirium include anoxic lesions of the hippocampus, and infarcted foci. Physiologic factors that contribute to this reaction include: long periods of extracorporeal circulation, arterial hypotension during surgery, emboli, and low postoperative cardiac output. Age, and the type and severity of heart impairment are also factors. Psychologic factors to be taken into account include preexisting psychopathology and the failure of denial under the stress of physical symptoms or hospitalization. Delirium is fostered by sensory overload (or deprivation) in the recovery room and intensive care unit, and by staff tension. Modification of the intensive care unit environment, the administration of antipsychotic drugs, and metabolic correctives are recommended. Preoperative psychologic evaluation, with therapy as needed, preliminary familiarization with perioperative procedures, as well as collaboration between psychiatrist and surgeon, can do much to prevent post-cardiotomy delirium.


The treatment of enuresis: an overview.

The treatment of enuresis: an overview. The treatment of enuresis has had a long and colorful history in world medical literature. Although some reasonable approaches to the problem exist, there are many conflicting theories about its etiology and consequently there are many recommended therapies. A perspective of literature encompassing the diverse fields of psychoanalysis, behavioral psychology, urology, pharmacology and sleep physiology is provided for the psychiatrist consultant dealing with the problem of enuresis.


Group therapy for kidney transplant patients. A short-term, open membership group therapy program for kidney transplant patients and their families is described. The content and process of this group is related to the ten curative factors described by Yalom for psychiatric patients. The primary benefits for the patients seemed to be the opportunity to observe others cope with similar problems and to learn of the adaptive strategies used by others. The patients were able to offer advice to others, to overcome their tendency toward seclusion and in general, experience a feeling of hope for the future. The family members gained more from a sense of group cohesiveness than did the patients. Both family and patients utilized the meetings to ventilate their anger and frustrations associated with chronic illness and to learn more about transplantation. A more realistic expectation of the future was provided. The group did not seem long enough to measurably improve the interpersonal relations between patients and their families nor did the group members comment upon maladaptive behaviors exhibited between family members within the meetings.


Late somatic effects in syngeneic radiation chimaeras. III. Observations on animals repopulated with irradiated marrow. Lethally-irradiated mice received a graft of bone-marrow cells obtained from intact syngeneic animals or from donors pre-irradiated with 200 or 400 rad. The numbers of haemopoietic cells injected were sufficient to give a high percentage of recipients which survived short-term bone-marrow failure. Epidemiological and pathological observations on long-term surviving animals showed an increased incidence of nephrosclerosis and of systemic or solid tumours following different relationships with the radiation dose given to the cells. These data are discussed within the framework of an experimental model system which might allow estimates of incidence of some neoplastic diseases according to cellular hypotheses of tumour induction.


Observations on late effects in mice exposed to 400 MeV neutrons. Life-long observations on mortality and pathology at death were carried out on groups of mice irradiated with 250 kV X-rays or exposed to a 400 MeV neutron beam, both directly and after attenuation corresponding to the maximum dose build-up region, at comparable dose-rates. Doses up to 84 rad of 400 MeV neutrons and up to 200 rad of X-rays showed no effect on the longevity of the animals, which suggest an upper limit to the r.b.e. for life-shortening of approximately 2-5. Similar conclusions were drawn from the data on all types of leukemias. For all other neoplasms, the age-specific death-rate showed a similar shortening of the latency times for groups of mice irradiated with 0-84 rad of 400 MeV direct neutrons and 0-400 rad of X-rays, also suggesting an upper limit to the r.b.e. slightly higher than that previously indicated for life-shortening. No definite effect was observed after exposure to the attenuated neutron beam at the doses used in these experiments.


The treatment of endomorphous and psychogenic depressions with a fixed combination of amitriptyline/flupenthixol (Lu 7410). The clinical efficacy of a fixed combination of 10 mg amitriptyline and 0.5 mg flupenthixol (Lu 7410) was studied in 30 (15 endomorphous and 15 psychogenic) depressive patients over 4 weeks. Stepwise titration of the drug resulted in an optimal daily dosage of 6 tablets (1 table, morning, 1 at noon, and 4 in the evening). Based on the ECDEU Global Score the overall symptomatology improved significantly as early as in the first week and was best in the third week. Evaluation of the detailed psychopathology by means of the AMP-system showed, that aside from thymoleptic properties, Lu 7410 has a stimulatory-activating-effect (improvement of drive) as well as an anxiolytic-sedative component (improvement of suicidal tendencies, agitation and sleep disturbances). While the former is seen predominantly in low doses and in endomorphous depressions, the latter is prominent in higher doses and in psychogenic depressions. There was a lack of extrapyramidal side effects as well as of alterations in blood count, blood chemistry and ECG. Psychological tests supported the clinical observations, inasmuch as a significant increase of extroversion and aggressivity was seen in the FPI of endomorphous depressives, while psychogenic depressives revealed decreases in extroversion and sociability. Moreover, a decrease in the Taylor anxiety score and increase in concentration was noted, based on the AD test. The results will be discussed.


An automated diagnostic process (PDA) in clinical psychopharmacology. An exemplification of its use in a sulpiride versus haloperidol comparative trial. One of the main unsolved problems, and one which produces divergent results in clinical psychopharmacology, is that concerning the selection of patients and their diagnostic definition. An automated diagnostic procedure (PDA) was set up in order to classify each patient into one nosographic category on the basis of a cross-sectional examination of his mental state. Such diagnostic procedure appears particularly suitable for multicenter drug trials, since it gives a profile and a diagnostic definition of patients, assessed by investigators from different areas and with different cultural, and clinical backgrounds. In a multicenter trial (sulpiride versus haloperidol) PDA offered a chance to re-examine and analyze the characteristics of each patient and therefore to control the criteria followed for the sample selection in the various experimental settings. The agreement between clinician and computer diagnosis was 78.9%; this agreement rises to 85.5% if the computerlabelled schizo-affective syndromes are considered within the schizophrenic group. Moreover, and attempt has been made to relate psychopathological patterns to drug responses.


Effects of the intracoronary and intraventricular injections of a commonly available vs. a newly available contrast medium. Two contrast media (Renografin 76 and Isopaque 370) were used in a double-blind study for coronary angiography and left ventriculography. Fifty-eight patients with presumed coronary artery disease were studied. The effects of the contrast media upon the myocardium and upon ventricular function of patients with and without coronary artery disease were assessed. Pre and post angiography myocardial enzymes were obtained. Cardiac output was determined before and after the angiograms. Hemodynamic and electrocardiographic changes were continuously monitored during the procedure. The study showed that both contrast media produced similar changes; however, the "maximum" decrease in T wave amplitude was more marked in the normal group when Renografin 76 (as compared with Isopaque 370) was injected (p = 0.05) on the right. Renografin 76 injection into the left coronary artery produced a higher median T wave amplitude in lead II relative to Isopaque 370 only in the abnormal group (p = 0.01). The heart rate dropped more severely in the normal patients when the left coronary artery was injected with Renografin 76 than with Isopaque 370 (p = 0.05).


[Pemphigus].

[Pemphigus]. This review stresses particularly the role that "intercellular" antigens and antibodies, complement and lymphocytic dysfunction may play in pemphigus. In addition evidences in the literature in favour of exogenic factors inducing pemphigus are surveyed. Further an attempt is made to summarize the possibilities for the etiology and the blister formation in this disease.


[Results and progress of scanning- and analytical electron microscopy in dermatology]. This is a review of the achievements of scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM) and ultrastructural x-ray microanalysis in Dermatology. Eight years after its introduction, the scanning electron microscope opened new possibilities for qualitative and semi-quantitative ultrastructural analysis of human skin, nails and hairs and provided new valuable information in clinical dermatology and dermatopathology. Considerable work has been done on the spatial architecture of normal skin, hair, hyperkeratotic conditions, psoriasis, hair abnormalities, fungus infections, dermal collagen in normal and diseased skin, and the surfaces of cutaneous vascular endothelia. Recently, x-ray microanalysis has been applied for the first time in dermatological research. Subcellular particles, the products of cytochemical reactions and tracer substances, such as heavy metals, can now be analyzed by SEM and STEM techniques. Keratohyaline granules do show a sulfur peak by means of this technique and lipoid droplets fail to demonstrate the peaks of sulfur and chloride. X-ray-microanalysis of ATPase and AChE-reactions in human skin facilitates the indentification and the localization of the reaction product in tissue, whereas the penetration of mercury compounds can be followed more precisely by this technique.


Immunity and neoplasia in Mollusks.

Immunity and neoplasia in Mollusks. The Mollusca is one of the largest of invertebrate phyla. Two major classes, the Gastropoda and the Pelecypoda, have been the subject of numerous studies on immunity and neoplasia. Investigations of immunity have dealt with cellular and humoral aspects, phagocytosis and encapsulation, and rejection of foreign tissue grafts. Work on humoral responses has focused on lysozyme, the hemagglutinins (especially in the oyster), and the clearance of certain antigens. Neoplasms in these animals resemble certain cancers in vertebrates, but it is not clear whether a relationship exists between the invertebrate immune system and the development of neoplasia. Studies of immunity and neoplasia in invertebrates may reveal how these two phenomena of living systems have evolved.


[Preliminary psychological evaluation of a group of children before and after renal transplantation]. Eighteen children who have received kidney transplants within the last three years at the Hopital des Enfants Malades in Paris provide material for this survey. We try to find out how these transplants effect the way the children live, and the main psychological problems that they create. This new way of life is characterized by an explosion of vitality, a certain liberation from prior restraint and the possibility of activities which up to then had been difficult or forbidden. However, the necessity for the child to be watched regularly, to attend the hospital for examination, to follow a special diet, to take immunodepressive drugs remind him that he is not cured. Consequences of corticotherapy and the growth retardation remain essential problems for some of them. The child will have to cope with this complex situation which also creates difficulties to acquire independence.


The localization of enzymes in tissue sections by immuno-histochemistry. Conventional antibody and mixed aggregation techniques. Methods for detecting enzymes in tissue sections by antibody techniques are reviewed. In all these techniques, sections are first incubated with antibody. The bound antibody is visualized in one of four ways: identifying a label such as fluorescein linked to the antibody; using a labelled anti-antibody; employing complement and labelled anti-complement; or making use of a mixed aggregation immuno-cytochemical method. The last technique consists of three steps. A section is first incubated with antiserum, and secondly with the soluble enzyme under investigation. Thirdly the desired enzyme is "stained" using a conventional cytochemical method. The method is specific since, for example, the soluble enzyme used in the second step can bind only to antigenic determinants which are identical to those of the enzyme localized in the tissue. Thus purification of antigen and antibody sources is simplified, and chemical modifications of the antigen and antibody are avoided. Antibody also acts as a selective fixative for tissue antigen. It will inhibit the catalytic activity of its antigen and, in this way, permit the enzyme activity arising after the reaction of tissue enzyme-antibody complex with soluble enzyme to be amplified selectively. The mixed aggregation immuno-cytochemical technique has been used successfully with membrane-bound enzymes and cytoplasmic enzymes and for the demostration of catalytically inactive enzyme precursors.


Comparison of three techniques for intestinal anastomosis in Equidae. Thirteen horses and ponies were used to evaluate an anastomotic technique utilizing a modified Gambee suture pattern. The modified Gambee technique was compared with a single-row everting technique and a double-row inverting technique. The double-row inverting technique predisposed to intestinal obstruction and resulted in minimal adhesions. The everting technique predisposed to leakage of intestinal contents and subsequent peritonitis. Adhesions were formed at the site of the anastomosis to such an extent as to jeopardize the proper position of the bowel. The technique did not cause any luminal constriction initially, but the ensuing adhesions did tend to limit the expansibility of the bowel. The modified Gambee technique was associated with minimal constriction of the bowel lumen. Excessive adhesions and leakage were not found to be problems associated with the technique. It was concluded that, in Equidae, the modified Gambee technique for intestinal anastomosis results in near normal lumen diameter, minimal adhesions, and adequate strength, thus being safe and effective.


Mutant of Escherichia coli that instantaneously loses the ability to adsorb lambda bacteriophage upon exposure to high temperature. lad (lambda adsorption), an Escherichia coli mutant that loses the ability to adsorb lambda phage immediately after a shift to high temperature (e.g., 42 C), was isolated. This property for phage adsorption is irreversible and has been observed with phage lambda and 21 but not with phages 434, phi 170, and phi 80. A crude receptor preparation, extracted from lad cells will cholate-ethylenediaminetetraacetic acid by the procedure of Randall-Hazelbauer and Schwartz (1973), inactivated the phage lambda only at low temperature.


Amino acid starvation in Escherichia coli K-12: characteristics of the translation process. Some characteristics of the translation process during amino acid starvation in Escherichia coli have been examined. Once starvation has been established, premature termination of polypeptides is negligible and complete proteins are formed. There is some preference for the synthesis of shorter proteins. The number of ribosomes involved in protein synthesis appears to decline to about half during amino acid-starvation. The assembly time of proteins during amino acid starvation is increased to only about fourfold, though protein synthesis maintained by turnover is reduced to 10%. To explain these observations, a model has been proposed for the course of events that possibly take place from the onset of starvation.


Genetic and biochemical characterization of some missense mutations in the lacZ gene of Escherichia coli K-12. Some preparations of beta-galactosidase from strains of Escherichia coli carrying point mutations in their lacZ genes did not precipitate with antibody as effectively as wild-type enzyme, but did not appear to be chain-terminating mutations as judged by polarity measurements and suppression. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extracts of induced Lac+ strains revealed that the monomer of beta-galactosidase ran as a band uncontaminated by other cellular proteins. This method was used to identify missense mutations in the alpha and beta portions of the lacZ gene. Six of 13 mutations investigated were judged to be missense by this criterion. Measurement of the degree of polarity, the ability to complement a nonsense mutation at the operator-distal extremity of the gene (omega-complementation), and suppressibility by 12 nonsense suppressors allowed the assignment of six other mutations as either number or ochre. The protein figments produced by these six nonsense mutations appeared to be degraded in vivo. One mutation that could not be classified was either a missense mutation whose protein product was degraded or a very leak nonsense mutation. Two lacZ alleles were suppressed by the ochre suppressors supM and supN, although they were missense by other criteria. The ability of supM to suppress both nonsense and missense mutations can be explained if it is derived from a tyrosine transfer ribonucleic acid with a modified base in the first position of the anticodon. The mutations assigned to the missense class were not suppressed by the missense suppressors supH, supQ, glyV, glyU, or glyT. Our results suggest that the criteria used in the past to distinguish between nonsense and missense mutations may not be conclusive even when used together.


Genetic mapping of xthA, the structural gene for exonuclease III in Escherichia coli K-12. The genes xthA, pncA, and pabB were ordered relative to others by two- and three-factor transductional crosses with bacteriophage P1. The genes studied span 2 min (2%) of the genetic map of Escherichia coli K-12 in the clockwise sequence pheS-pfkB-xthA-pncA-gap-pabB-fadD. Eleven independently derived xth mutations were examined; all were known to affect exonuclease III and its associated endonuclease II activity, and all were mapped in the xthA region. pncA mutations were found to confer resistance to 6-aminonicotinamide, whereas some pheS mutations are known to specify resistance to p-fluorphenylalanine. xth mutations were readily transferred into other strains by selecting for these co-transducible drug resistance markers.


Metal ion content of Escherichia coli versus cell age. The potassium, calcium, magnesium, and zinc ion content of cells in exponential and synchronously growing cultures of Escherichia coli B/r was determined with an X-ray fluorescence spectrometer and an atomic absorption spectrophotometer. Cellular potassium, calcium, and magnesium content increased smoothly during the cell cycle, but cellular zinc showed a steplike increase about 10 to 15 min after cell division in a culture having a doubling time of 47 min. The possible role of cellular zinc in the control of cell division is discussed.


Mutants in three genes affecting transport of magnesium in Escherichia coli: genetics and physiology. Mutants in three genes affecting two Mg2+ transport systems are described. System I, for which Co2+, Mn2+, and Mg2+ are substrates, is inactive in corA mutants corB mutants express system I after growth on high (10 mM) Mg2+ but not low (0.1 mM) Mg2+. Both corA and corB mutants are resistant to Co2+ or Mn2+. corA mutants are sensitive to CA2+. Transport system II is specific for Mg2+ and is repressed by growth on 10 mM Mg2+. mgt mutations inactivate system II. Growth on mgt mutants in normal except on very low (1 muM) concentrations of Mg2+, corA mgt strains exhibit no high-affinity, energy-dependent transport of Mg2+ and require 10 mM Mg2+ for optimal growth. The three genes are not linked. The corA locus is contransducible with ilv at 75 min, corB is cotransducible with pyrB at 85 min, and mgt is cotransducible with malB and mel at 81 min on the genetic map.


Properties of the entry and exit reactions of the beta-methyl galactoside transport system in Escherichia coli. The Km, Vmax, and Ki of the entry reaction were determined for three substrates of the beta-methyl galactoside transport system: D-galactose, D-glycerol-beta-D-galactoside, and beta-methyl-D-galactoside. Although the data for D-galactose and D-glycerol-beta-D-galactoside followed simple Michaelis-Menten kinetics, the results for beta-methyl-D-galactoside deviated from Michaelis-Menten kinetics in that the Ki for beta-methyl-D-galactoside inhibition of both of the other two substrates was 10-fold greater than the Km for beta-methyl-D-galactoside entry. Furthermore, two partial mgl- strains retain 56% of the parental level of the beta-methyl-D-galactoside entry reaction, but only 12% of the parental level of transport of the other two substrates. The exit reaction of beta-methyl-D-galactoside was shown to be first order. It was stimulated sixfold when the cells were provided with an energy source. This stimulation required adenosine 5'-triphosphate or a related compound. The exit reaction was not altered by mutations in any of the three cistrons which inactivate the beta-methyl-D-galactoside entry reaction, was not increased by growth in the presence of inducers of the entry reaction, and was not repressed by growth on glucose. The striking differences between the entry and exit reactions suggest that they either use different carriers or that none of the three cistrons which are currently known to code for components of the beta-methyl galactoside transport system code for its membrane carrier.


Isolation and characterization of conjugation-deficient mutants of Escherichia coli K-12. Conjugation-deficient mutants (Con-) of Escherichia coli K-12 have been isolated by a variety of indirect selective techniques. Mutants with mutations conferring ampicillin resistance, fosfomycin resistance, an alanine requirement, and a failure of ferment a number of carbohydrates were selected because the impaired functions occur in association with cell wall and cell membrane defects. The integrity of these catalytic or structural elements is postulated to have a role in conjugation. The mutants could be divided into at least six general categories corresponding to their defectiveness in the following postulated recipient cell functions: (i) specific-union formation, (ii) effective-union formation, (iii) deoxyribonucleic acid transfer, (iv) plasmid establishment, (v) plasmid maintenance, and (vi) recombination. The availability of these mutants should contribute to the description of the molecular events involved in each of these conjugation steps and the elucidation of these genetic control over the inheritance of conjugationally transferred deoxyribonucleic acid.


Suppression of an Escherichia coli dnaA mutation by the integrated R factor R.100.1: Change of chromosome replication origin in synchronized cultures. We have followed, by deoxyribonucleic acid-deoxyribonucleic acid hybridization, the order of replication of three chromosomal markers during a synchronous round of replication in three strains of Escherichia coli carrying a dnaAts mutation: one strain in which the F-like R factor R.100.1 was established as a plasmid and two strains in which the dnaA mutation was suppressed by the integration of R.100.1 into the chromosome. In the R+ strain at 30C, replication of the plasmid took place simultaneously with the initiation of chromosome replication at the normal origin. In the integratively suppressed Hfr strains, at 42.5 C, chromosome replication was initiated preferentially from the integrated plasmid; little or no initiation occurred at the normal origin. Similar results were obtained for the one strain tested at 30 C. For both Hfr strains at 42.5 C, the data suggest that at least part of the population replicated bidirectionally. This conclusion had been confirmed using an autoradiographic procedure. Both types of experiment indicate a wide variation in the rate of travel of individual replication forks within the population.


Activity of three murein hydrolases during the cell division cycle of Escherichia coli K-12 as measured in toluene-treated cells. The specific activities of three murein hydrolases, carboxypeptidase I, carboxypeptidase II, and amidase were studied with respect to cell division in toluene-treated cells of Escherichia coli K-12. Carboxypeptidase I and amidase activities were constant throughout the division cycle in cells of D11/lac+pro+. Detectable carboxypeptidase II activity varied and was highest at the time of division by a factor of three. Carboxypeptidase II specific activity was also correlated with cell division in BUG 6, a temperature-sensitive mutant (J.N Reeve, D.J. Groves, and D.J. Clark, 1970). Fifteen minutes after shifting BUG 6 from 42 C (nondividing conditions) to 32 C (dividing conditions), there was a rapid resumption of cell division, accompanied by a 10-fold increase in the specific activity of carboxypeptidase II. These results demonstrate a correlation between detectable carboxypeptidase II activity and cell division as reflected by activity in toluene-treated cells. The subcellular location of carboxypeptidase II, a soluble enzyme was found to be periplasmic since it was released by tris(hydroxymethyl)-aminomethane-ethylenediaminetetraacetate treatment and osmotic shock, two methods known to release periplasmic enzymes.


Cross-resistance between bacteriophages and colicins in Escherichia coli K-12. Cross-resistance between bacteriophages and colicins was studied using collections of bacteriophage- and colicin-resistant mutants of Escherichia coli K-12. No new examples were found of highly specific one-to-one cross-resistance of the type suggestive of common receptors. However, several groups of mutants showed tolerance to colicins and resistance to bacteriophages. Mutants known to be very defective in lipopolysaccharides composition were found to commonly show tolerance to certain colicins in addition to their bacteriophage resistance. Another group of mutants showed varying patterns of resistance to colicins E2, E3, K, L, A, S4, N, and X and bacteriophages E4, K2, K20, K21, K29, and H+. However, many bacteriophage-resistant mutants were fully colicin sensitive, and most colicin-resistant mutants were fully sensitive to bacteriophages.


Uni-directional growth of F-actin.

Uni-directional growth of F-actin. In was shown that the growth of F-actin occurs uni-directionally in vitro; that is, actin monomers (G-ADP-actin) polymerize onto only one of the two ends of a pre-existing seed in the absence of ATP. Fragments of the F-actin-heavy meromyosin complex prepared by sonication were used as seeds. By studying a large number of electron micrographs, we concluded that growth occurs on the tail end of the arrow-head structure of the seed. This result suggests that the thin filament grows in vivo from the center of the sarcomere towards the Z-line. It was further shown that the fragments of F-actin combine with both ends of the seed with nearly equal probability. These results are well interpreted according to the theory developed by Oosawa (1,2).


Fluorescent probes for antibody active sites. I. Production of antibodies specific to the N-methyl-2-anilinonaphthalene-6-sulfonyl group in rabbits and some fluorescent properties of the hapten bound to the antibodies. 1. Rabbits were immunized with N-methyl-2-anilinonaphthalene-6-sulfonyl (MANS) bovine serum albumin (MANS-BSA) prepared by the reaction of bovine serum albumin with N-methyl-2-anilinonaphthalene-6-sulfonyl chloride to obtain IgG fraction containing anti-MANS antibodies. 2. Both N-methyl-2-anilinonaphthalene-6-sulfonate (MANSate) and N-methyl-2-anilinonaphthalene-6-sulfonamide (MANSamide), which are virtually non-fluorescent in aqueous solution, became strongly fluorescent, with a blue shift of about 100 nm, when they were specifically bound to the antibodies against the hapten group contained in the IgG fractions. 3. IgG fractions were obtained from five immunized rabbits at intervals after the primary immunization. The fluorescence characteristics of the hepten were carefully examined with preparations of IgG fraction differing in source and time of bleeding. One of the rabbits showed a very interesting immune response. The rabbit produced two groups of anti-MANS antibodies which were significantly different in their effect on the fluorescence properties of the hapten group. One of the groups was exclusively produced at the early stage in the immune response. The other group appeared at the late stage. It is suggested that the two groups differ as regards the structure of the hepten combining sites. 4. The above findings indicate that the MANS group can be successfully used as a hapten with useful properties as a fluorescent probe.


Mechanism of polyadenylate-polyuridylate synthesis by RNA polymerase holoenzyme II of Escherichia coli. Poly(A)-poly(U) synthesis in the absence of template DNA is a unique reaction catalyzed by the RNA polymerase [EC 2.7:7.6] holoenzyme II of Escherichia coli. As one approach to investigating the physiological role of the enzyme, the molecular mechanism of poly(A)-poly(U) synthesis was studied. Streptolydigin, an inhibitor of the elongation of RNA chains, was shown to inhibit poly(A)-poly(U) synthesis, and the inhibition was released by a streptolydigin-resistant mutation on the beta subunit. These observations indicate that the active site for the reaction might be located on the beta subunit. Another antibiotic, rifampicin, which is known  to be a specific inhibitor of the initiation of DNA-dependent RNA synthesis, effectively inhibited both initiation and elongation steps in poly(A)-poly(U) synthesis. This suggests that the enzyme conformation during the chain elongation reaction might be different in this case from that in DNA-dependent RNA synthesis. Analysis of the products formed during the initiation reaction indicated that the rate-determining reaction in poly(A)-poly(U) synthesis was the formation of primers of short chain length, and that holoenzyme I was unable to form the first phosphodiester bond in this reaction. Functional properties of holoenzyme II are discussed in connection with these observations.


Fluorometric investigation of the local conformation of 16S rRNA in the regions of the 3'- and 5'-ends. The conformational changes of ribonucleic acid induced by heat treatment were studied by measurements of fluorescence polarization, circular dichroism, and ultraviolet absorption. The fluorescence polarization of proflavine covalently bound to the 3'-end of 16S ribosomal RNA of Escherichia coli and that of ethenoadenosine conjugated to the 5'-end of the RNA indicated that a conformational difference existed between heat-treated and untreated samples of the RNA in the regions near the 3'- and 5'-ends. Comparison of the fluorescence polarization data with other optical measurements indicated that the local conformation around the 3'- and 5'-ends of the RNA was converted to a more rigid and stable form by the heat treatment, while the gross conformation did not appear to change.


Pantothenate synthetase of Escherichia coli B. I. Physicochemical properties. Using a new apparatus for preparative polyacrylamide gel electrophoresis, pantothenate synthetase (D-pantoate: beta-alanine ligase (AMP-forming), [EC 6.3.2.1] was purified about 500-fold from Escherichia coli B. It was found to be homogeneous in analytical disc gel electrophoresis and sedimentation ultracentrifugation (so20, w=4.9). From sedimentation equilibrium ultracentrifugation, a molecular weight of 70,100 was obtained, which is in good agreement with the value obtained by the Sephadex G-150 gel filtration method (69,000); the diffusion constant was calculated to be 5.88X10(-7) cm2/sec. The minimum molecular weight calculated from the amino acid composition of this enzyme protein was 19,700, a value in reasonable accord with molecular weight of the enzyme subunit, 18,000, obtained by gel electrophoresis in the presence of sodium dodecylsulfate. The partial specific volume, v, was calculated to be 0.71 cm3/g. The enzyme had an amino-terminal glycyl residue and a Leu-Ala-Ser-OH sequence at the carboxyl end. Electrophoresis of the enzyme with carrier ampholine gave an isoelectric point of pH 4.6.


The nucleotide sequence in the promoter region of the gene for an Escherichia coli tyrosine transfer ribonucleic acid. The sequence of the first 29 nucleotides in the promoter region of a tyrosine tRNA gene has previously been determined (Sekiya, T., van Ormondt, H., and Khorana, H.G. (1975) J. Biol. Chem. 250, 1087-1098). This work has now been extended to give the sequence of a total of 59 nucleotides; the sequence is as follows: (see article). The general approach used in the determination of the sequence involved the DNA polymerase I-catalyzed elongation of synthetic deoxyribopolynucleotide primers hydridized to the l-strand of phi80psu+III DNA at the appropriate site. Sequencing of the newly added nucleotides was facilitated by the use of a number of techniques including (a) elongation of the primer with the use of all of the four nucleoside 5'-triphosphates but limiting the concentration of one of the triphosphates, (b) insertion of ribonucleotide units at appropriate sites so as to permit subsequent specific cleavages by pancreatic RNase, and (c) two-dimensional fingerprinting of the oligonucleotides in conjunction with partial exonucleolytic degradation, comprehensive nearest neighbor analyses, and the determination of pyrimidine tracts.