[The pre-morbid personality of schizophrenics in the literature of the last hundred years (author's transl)]. A survey of the literature on the primary personality of schizophrenics shows that the essential concepts derive from Kraepelin (1893), E. Bleuler (who also introduced the technical term "schizoid", 1911) and C.G. Jung introversion; 1913). E Kretschmer (1921) adopted these preliminary findings and integrated them into his system of "Constitution" which greatly stimulated research in this field. Most of the following investigations confirmed his hypotheses on the basis of clinical impressions. A few only criticised his teaching, particularly as a theoretical concept. The musculo-somatotonic type apart, Sheldon's (1940) research agrees with Kretschmer to a large degree. Later investigators, however, could not sufficiently support Sheldon's constitutional biology on empirical grounds. No further pre-morbid types were developed in the following years, if one discards the somewhat rare "stormy" character (Arieti, 1955). All attempts to discover differences between the pre-morbid development of personality and the subheadings of schizophrenia failed. Pre-morbid sexual development is not typical, but the disturbances of contact are in the foreground. Whether schizophrenics differ later in their intelligence is still undecided. But it appears certain that, even before the start of their illness, they achieve a lower social status than their siblings. There exists a large experimental psychologic literature on relationships between the original personality, symptoms and psychophysiologic correlates in which it is confirmed again and again that bad pre-morbid adaptation corresponds closely with the severity of symptoms and loss of achievement. There are no consonant result of investigations into the frequency of schizoid structures in schizophrenics, in psychiatric control groups and in the normal population. Psychometric techniques have been used only rarely, but existing publications confirm in principle that schizophrenics are markedly more schizoid than control groups. As with schizophrenia, there are contrasting ideas as to the cause and pathogenesis of the pre-morbid personality. Psycho- and socio-genetic, behavioral, genetic and neurochemical hypotheses have their adherents. According to recent studies of adoption the genetic factor appears to predominate. The clinician relies on pre-morbid development of the personality in his search for a "kink in the life line" or in his assessment of the quality of remissions. There is general agreement that a schizoid primary personality has an unfavorable influence on the long-term prognosis. This is still true today, in spite of modern possibilities of treatment and rehabilitation. Prophylactic aspects have been discussed for a long time but have remained unhelpful clinically. Often it may be difficult to distinguish between schizoids and mild hebephrenias. In spite of this the clinical concept "schizoid" should never be used for "mild schizophrenia"...


[Identical psychosis of a monocygous twin set--a case report (author's transl)]. This is a report about a pair of identical twins with identical symptoms of a similar to schizophrenic psychosis. The course of the disease with special reference to the influence by each other and a similar and a dissimilar treatment is described. This disease is subgrouped to the symbiontic psychoses. Nosology, aetiology and psychodynamic aspects are discussed.


Gout and the regulation of purine biosynthesis. Overproduction of purine nucleotides de novo is the cause of hyperuricemia in a substantial portion of the gouty population. Specific enzyme abnormalities--deficiency of hypoxanthine-guanine phosphoribosyltransferase (an enzyme of the purine "salvage" pathway) and overactivity of 5- phosphoribosyl-1-pyrophosphate (PP-ribose-P) synthetase--result in hyperuricemia, and are associated with increased de novo purine synthesis and increased intracellular concentrations of PP-ribose-P. The latter is a common substrate for the first enzyme of the de novo pathway (phosphoribosyl amidotransferase) and the purine base salvage enzymes. Studies in cultured cells from patients, and in mutant cells derived from normal cell lines in vitro, suggest that elevated intracellular PP-ribose-P concentrations may increase the rate of de novo purine biosynthesis. This regulation can be explained in terms of the normal intracellular concentration of PP-ribose-P which is lower tthan the Km for the amidotransferase,  and by allosteric activation of this enzyme by PP-ribose-P. Feedback inhibition of the first step in the de novo pathway by exogenous purines can be explained either by end-product (nucleotide) inhibition of the amidotransferase, or by competition for PP-ribose-P by the salvage enzymes which have lower Km's for this substrate, or by a combination of these effects. Evidence for and against these mechanisms is discussed. Evidence is presented which suggests that exogenous purines exert a feedback effect, not only on the first step of the de novo pathway, but also at the distal branch point  in the pathway. Several potential regulatory mechanisms which might lead to excessive production of uric acid are discussed.


Transfer of substrates across the chloroplast envelope. The chloroplast represents a relatively autonomous metabolic compartment within the plant cell. It is surrounded by an envelope consisting of two membranes of which the inner membrane is the functional barrier. Utilizing the energy of light the chloroplast is able to synthesize dihydroxyacetonephosphate from carbon dioxide and water. To provide the cell with this substrate, inorganic phosphate is required. In the case of phosphate deficiency the product of CO2 fixation may be temporarily stored within the chloroplast as starch. Specific transport processes across the inner envelope membrane permit the transfer of metabolites between the chloroplast and the cytosol. The phosphate  translocator facilitates the export of dihydroxyacetone phosphate in exchange for inorganic phosphate. It also catalyzes a shuttle for inorganic phosphate with 3-phosphoglycerate, permitting the indirect transfer of reducing equivalents and of ATP from the chloroplast to the cytosol. The dicarboxylate carrier transporting various dicarboxylates may be suited for the transfer of reducing equivalents from the cytosol into the chloroplast. The ATP translocator, catalyzing a transport of ATP into the chloroplast in exchange for ADP, appears to be important for providing the chloroplast with ATP during the night phase, as required for the mobilization of starch.


Molecular aspects of electrical excitation in lipid bilayers and cell membranes. Several compounds of fungal or bacterial origin (EIM, alamethicin, monazomycin, DJ400B) can be incorporated into planar lipid bilayers where they form molecular channels and generate voltage-dependent ion conductances. When studied by voltage clamp, the kinetic and steady-state characteristics of these conductance changes are in every respect identical to those found in excitable cell membranes, and their major aspects can be quantitatively described by the Hodgkin-Huxley equations. Thus, the steady-state conductance is an expotential function of the membrane potential, the conductance rises with a sigmoid time course and decays exponentially, and the time constants of the conductance changes go through a maximum as a function of the potential. The conductances also show inactivation as seen in the sodium channels of nerve and the potassium channels of muscle. In addition, there appear for particular pulsing sequences certain kinetic transients that cannot be accounted for by the Hodgkin-Huxley equations but are also seen in identical form in nerve. Because the kinetics are identical in all excitable cell membranes and in these bilayers, it is likely that, in spite of the diverse chemical nature of the channel-forming molecules in the bilayers and the widely differing ion selectivities in the cellular systems, the mechanism by which the membrane opens and closes for the flow of ions is essentially the same in all cases. The kinetic data imply that a cooperative process is involved in the gating action. In principle, two different concepts could account for the kinetics--one involving an intramolecular configurational change within a complex permanent channel, the other, the assembly of a channel through the voltage-dependent aggregation of monomeric channel precursors. In the bilayers the high-order dependence of the steady-state conductance and of the gating time constants on the concentration of the channel formers suggests an aggregation mechanism in which the gating involves the voltage-induced insertion of all or part of the channel-forming molecules from the membrane surface into the hydrocarbon region and their subsequent aggregation into open channels by lateral diffusion. The mathematical description of this two-step insertion-aggregation mechanism accounts quantitatively for the entire conductancb-voltage kinetics including inactivation and other kinetic features which deviate from the Hodgkin-Huxley kinetics in the sense that the rate constants of the changes are dependent not only on the membrane potential but also on the value of the conductance and on time. The proposed mechanism is also in agreement with single-channel data for alamethicin which suggest that both the insertion and the aggregation rate constants are voltage-dependent...


Pancreatic lipase and colipase: an example of heterogeneous biocatalysis. The hydrolytic reactions catalyzed by pancreatic lipase represent a good example of heterogeneous catalysis. The particularity of this enzyme is provided by its preferential action on emulsified substrates. The first step of catalysis resides in a reversible adsorption of the enzyme to the oil-water interface. In fact, the formation of this adsorption complex is an obligatory step for the enzyme to display its full activity. Two principal but not necessarily exclusive hypotheses have been proposed to explain the observed interfacial activation: Either the interface confers new properties on the substrate which allow its subsequent hydrolysis, or the enzyme itself is modified by adsorption at the interface. Different approaches have recently been developed to clarify this point further. The results obtained by chemical modifications of lipase are consistent with the following hypothesis. The active site preexists in solution and becomes fully functional only by interaction of the interface with an additional site on the enzyme molecule which can be tentatively called the "interfacial activation site." Finally, a protein of low molecular weight, colipase, seems necessary for lipase to express its activity under physiological conditions. This protein enters specific interactions with bile salts micelles and is responsible for the reversal of the inhibition of lipolysis brought about by these detergents.


A quarter century of psychiatric care, 1950-1974: a statistical review. The authors describe the changing trends in psychiatric care over the last quarter century. Whereas in 1950 state and county mental hospitals were the principal site for psychiatric care, by 1974 community mental health centers and other types of mental health facilities had an important role in providing such care. There has been a marked shift from inpatient to outpatient care, and more individuals with diagnoses other than psychoses are being treated today.


Separation by column chromatography of cells active in delayed-onset hypersensitivities. Lymph node cells from guinea-pigs contact sensitive to 1-thiocyamo-2,4-dinitrobenzene have been fractionated by affinity chromatography over modified polyacrylamide beads. Cells mediating lymphokine release in response to active sensitizer were depleted only by chromatography over dinitrophyenyl (DNP) containing substrates and could be specifically eluted with DNP-glycine. DNP rosette-forming cells (RFC) were equally well depleted by chromatography using either DNP or trinitrophenyl containing materials but could not be eluted from the columns by DNP-glycine. While the antigen receptors of cells mediating the release of macrophage agglutination factor in response to DNP-containing antigens and of DNP-RFC were found to be hapten-specific, their specificity was shown to differ using chromatography over trinitrophenyl containing polyacrylamide.


Histochemical studies on fatty acid in lymphocyte-mediated immune reaction. Generation of fatty acids by cell-mediated immune reactions was demonstrated histochemically by Okamoto's method, which stains fatty acids and their alkaline earth salts red-brown. The red-brown deposits were observed more frequently on the mixed culture of immune lymph node cells of C3H/He and C57Bl/6 mice than on the culture of either of the pair alone. Similar evidence was observed in the reaction of immune lymph node cells with the corresponding allogeneic mouse tumour cells as well as on the immune lumphocytes of guinea-pig incubated in the presence of the soluble antigen PPD. In spite of the findings which indicated a possible generation of fatty acids in the immunological reaction of immune lymphocytes, we failed to demonstrate evidence of fatty acid generation in the ascites during rejection of allogeneic ascitic MM102 tumour cells, although accumulation of neutral lipid was evident. This unexpected contradicition was interpreted by finding that MM102 cells incubated with oleic acid did not contain fatty acid but did contain a remarkable amount of neutral lipid on histochemical examinations. This finding indicated that once generated, fatty acids would be converted to neutral lipid in the ascites where allogeneic tumour cells were rejected.


Strain-related differences in immunosuppressive effects of Enterobacteriaceae and their lipopolysaccharides on production in rabbits of antibody to enterobacterial common antigen. Certain polysaccharides have been shown to inhibit the antibody response of rabbits to the common enterobacterial antigen (CA). The present investigation revealed that striking differences exist in the immunosuppressive effects of enteric bacteria and their lipolysaccharides (lps), depending upon CA production by the strains. Mixtures of immunogenic strains (Escherichia coli F2378 [R4], E. coli F470 [R1], or Shigella boydii F3140 [R]) and non-immunogenic CA-producing strains, such as E. coli O1, E. coli O113, Salmonella montevideo, and S. minnesota, as well as the R mutants E. coli F614 (R1), E. coli F757 (R1), and S. typhimurium his 642 (Ra), failed to elicit CA antibodies. In contrast, mixtures of the immunogen and CA-negative strains S. typhimurium his 386 (Ra) and S. minnesota P595 (Re) or R555 (Ra) yielded antibodies in titers similar to those elicited by the immunogen alone. Further, LPS of CA-positive but not of CA-negative strains exerted this immunosuppressive effect. Quantitative studies revealed that LPS of S. minnesota in amounts of 100 mug/ml was strongly immunosuppressive, in amounts of 20 mug/ml slightly effective, and in amounts of 4 mug/ml ineffective. It is postulated that hitherto unknown differences exist, either in composition or in configuration, between LPS obtained from different microorganisms to account for the strain-related differences in immunosuppressive effects and, further, that the immunosuppressive LPS interacts with immunogenic CA.


Occurrence and characteristics of enterotoxigenic Escherichia coli isolated from calves with diarrhea. Of 1,004 isolates of Escherichia coli obtained during the spring of 1975 in seven different states from calves with diarrhea, 124 isolates were enterotoxigenic based upon ability to cause distention of the calf ligated intestinal segment. Isolates of enterotoxigenic E. coli (ETEC) were obtained from calves in six of the seven states. ETEC were detected in calves in 118 of 355 herds in Montana during the 1974 and 1975 spring beef calving seasons. The occurrence and serotypes of ETEC isolated from calves in states outside Montana were similar to ETEC isolated from calves in Montana. One hundred and fourteen of the 124 isolates of ETEC were placed in one of six different groups upon agglutination in OK antiserum. Serotyping of 35 of the 124 isolates of ETEC indicated the following serotype for isolates in each group: group 1, O9:K35; group 2, O101:K30; group 3, O8:K85; group 4, O20:K?; group 5, O8:K25; and group 6, O101:K28. Determination of the presence of K99 antigen indicated that 28 of 35 isolates of ETEC had K99 antigen, whereas the antigen was not detected in any of the 10 isolates of non-ETEC studied.


Interaction of Nocardia asteroides with cultured rabbit alveolar macrophages. The interaction between virulent and less virulent strains of Nocardia asteroides and cultured rabbit alveolar macrophages was studied. It was shown that cells of the less virulent strain (N. asteroides 10905) were rapidly phagocytized and destroyed. However, some cells were able to avoid being killed, and they persisted within the macrophage in an altered, gram-negative form. These variants apparently increased in numbers after several days within the macrophage population, so that at 9 days postinfection more colony-forming units per macrophage were recovered than at 3 h. Little or no extracellular growth was observed in the tissue culture medium. During the increase at 9 days, both transitional-phase variants and L-forms of N. asteroides were isolated from the macrophages but not from the medium. Gram-positive bacterial cells were never observed in 9-day infected macrophages. In contrast, cells of the more virulent strain (N. asteroides 14759) were not destroyed after being ingested. After 6 h postinfection, it was observed that the number of colony-forming units per macrophage had increased significantly. There was no corresponding increase in extracellular organisms observed in the culture medium. Therefore, cells of N. asteroides 14759 were able to grow rapidly within cultured rabbit alveolar macrophages. Upon continued incubation of the infected cells (24 h postinfection), it was shown that this strain of Nocardia grew out of the macrophages as acid-fast branching filaments. From these data, it is clear that the initial interaction between N. asteroides and unstimulated, nonimmune alveolar macrophages depends upon the relative virulence of the nocardial strain.


Role of host factors in the pathogenesis of Salmonella-associated arthritis in rats. To evaluate the roles of the infectious agent and the host in the pathogensis of Salmonella-associated arthritis (SAA), 1O(3) to 10(6) heat-killed Salmonella enteritidis were injected directly into involved carpal joints in normal and actively immunized rats and in hosts adoptively immunized by the intravenous transfer of spleen cells from syngeneic donors with ongoing SAA. As many as 10(6) living Salmonellae invariably failed to generate more than a transient inflammatory response in normal rats. The regression of acute joint swelling was accelerated in both types of immunized hosts. The intensity and duration of acute inflammation evoked in normal rats by 10(6) and 10(9) heat-killed Salmonellae did not exceed the response elicited by 10(3) living organisms. In sharply contrasting results, however, a chronic arthritis became established in a significant number of actively and adoptively sensitized rats after the intra-articular injection of 10(9) heat-killed organisms. No Salmonellae were recovered from these adoptively sensitized rats although small numbers of organisms has been present among the spleen cells in the transfer inocula. Taken together, these results indicate the obligatory involvement of host factors in the mediation of this chronic arthritis and virtually eliminate any likelihood that joint damage in SAA is due to the directly destructive effects of intra-articular infection.


Evidence for an extrinsic immunogen in effective ribosomal vaccines from Salmonella typhimurium. Previous work has indicated that ribosomes isolated from Salmonella typhimurium were highly immunogenic and afforded excellent protection against homologous challenge. Effective protection was obtained also when ribonucleic acid ( RNA) extracted from these ribosomes was used as a vaccine. In this investigation ribosomes prepared by another method and washed repeatedly in 1 M NH1Cl lost much of their prophylactic potency and yielded poorly protective RNA. The high-salt wash of the ribosomes was immunogenic. The RNA and the protein isolated from the salt wash of the ribosomes were effective vaccines. No intrinsic component of the ribosomes was removed by the NH4Cl wash, since the ability of both "crude" and "clean" ribosomes to function equally well in an in vitro protein synthesizing system was demonstrated. The presence of a component with toxic properties similar to those of endotoxin was found in active vaccines but not in weak ones. This was shown by the ability of effective vaccines to kill lead acetate-sensitized mice and to induce tolerance to endotoxin.


Growth of Mycobacterium lepraemurinum in the mouse bone marrow: an ultrastructural study. The ultrastruct of the mouse bone marrow during the first 8 weeks after intravenous inoculation of animals with 10(9) Mycobacterium lepraemurium is described. The bacteria were almost exclusively in macrophages, which became converted to epithelioid cells after 8 weeks, at which time they were very heavily infected. The nature of the exceptionally rapid increase in numbers of bacteria in the bone marrow compared with other tissues early in the infection is discussed. It is concluded that a short doubling time of bacteria situated in the marrow is a more probable explanation than recruitment from elsewhere in the animal.


Experimental mycoplasma mastitis in mice. Thirteen strains of mycoplasma representing six different species, Acholeplasma laidlawii, Mycoplasma dispar, M. bovirhinis, M. bovigenitalium, M. agalactiae subsp. bovis, and M. mycoides subsp. mycoides, were inoculated into the mammary glands of mice, and the number of mycoplasmas present in the glands three days after inoculation was determined. The predominant response included involution of inoculated glands and a neutrophil infiltration. With the exception of M. dispar, the pathogenicity of the six species for mice was found to be similar to their pathogenicity for cattle.


Colonization of porcine small intestine by Escherichia coli: ileal colonization and adhesion by pig enteropathogens that lack K88 antigen and by some acapsular mutants. Seven K88-negative porcine enteropathogenic Escherichia coli, representing three different serogroups, caused severe diarrhea and characteristically colonized the ileum, but not the jejunum, of intragastrically exposed newborn pigs. Bacterial counts of intestinal contents and wall, fluorescence, and scanning electron microscopy all suggested that these strains colonized the ileum by adhesion to the villous epithelium. However, in ligated intestinal loops, these enteropathogenic E. coli strains adhered to jejunal epithelium as well as to ileal epithelium. Acapsular (K-) mutants, derived from one of the principal strains, retained their colonizing and adhesive abilities, whereas K- mutants from three other enteropathogenic E. coli strains did not. It is suggested that: (i) these K88-negative enteropathogenic E. coli colonize the ileum by adhesion, and (ii) the adhesion of some K-88-negative strains is mediated by surface factors other than, or in addition to, the polysaccharide K antigen.


Multiplication of bovine parvovirus in two cell strains. Bovine parvovirus has been found to infect two cell strains: buffalo lung fibroblasts and embryonic bovine tracheal cells. Infectivity titers were best determined by plaque assays. Immunofluorescence studies revealed intranuclear staining, whereas complement fixation tests confirmed bovine parvovirus antigen production. These cell strains represent economical and convenient cell types which can be used in studies on bovine parvovirus.


Acquired immunity to Aspergillus fumigatus. In mice preinfected with Aspergillus fumigatus for greater than or equal to days, administration of cortisone failed to stimulate mycelial growth. The liver and heart of mice preinfected for 14 days resisted infection after a cortisone + conidia challenge.


The mechanism of immunoinhibition by arachidonic and linoleic acid: effects on the lymphoid and reticulo-endothelial systems. Subcutaneous injection with certain polyunsaturated fatty acids (PUFA), in particular linoleic acid (C18:2), has recently been shown to prolong the survival of skin allografts in mice and to reduce both primary and secondary cytotoxic responses by isolated spleen cells. In this study we have examined changes in the lymphoid and reticulo-endothelial systems of both grafted and ungrafted mice treated according to the schedules shown to prolong allograft survival. Many of the changes in the lymphoid and reticulo-endothelial systems which can be produced by allografting could also be produced in ungrafted animals by PUFA treatment. These changes included: increased 125IudR uptake by spleen, lymph nodes, and bone marrow, increased spleen and lymph node weight, increased proportion of red pulp and granulocyte precursors in the spleen, and reticulo-endothelial activation as shown by an increased rate of carbon clearance. The combined effects of allografting and C18:2 on peripheral lymph node weight were greater than the effect of either treatment alone, but fell short of the sum of both treatments. Allografting increased 125IudR uptake/unit organ weight, not only in the peripheral lymph nodes, but also in bone marrow, thymus and spleen. When allografted animals were C18:2 treated, this increase was considerably reduced, uptake being little different from that in C18:2 treated ungrafted controls. The number of theta-positive cells in the spleen was markedly increased by a tumor allograft, but such an increase could not be seen in allografted animals when treated with C18:2. Whilst prolonged C18:2 treatment led to destructive changes in the spleen, immunoinhibition could still be demonstrated after shorter treatments with C18:2 when no gross histological evidence for tissue destruction was apparent. Partial reversibility of inhibition of cytotoxic activity of isolated spleen cells could be domonstrated when treatment was discontinued.


Induction of adjuvant arthritis in the rat by various bacterial cell walls and their water-soluble components. A number of purified cell walls of various gram-positive bacteria had arthritogenic activity in the rat. The water-soluble adjuvant-active component(s), which were isolated from some of these cell walls by utilizing a peptidoglycan-degrading enzyme, did produce severe adjuvant arthritis. However, the components obtained by digestion with glycan-degrading enzymes failed to produce arthritis. Thus, the present finding indicates the importance of a peptidoglycan portion, especially its intact glycan chain, for induction of adjuvant arthritis.


Cellular and humoral immune response to guinea pig infected with Treponema pallidum. Guinea pigs infected intradermally with Treponema pallidum Nichols strain were examined for the presence of lesions and for cellular and humoral response. Of the 26 guinea pigs (77%) demonstrated darkfield positive lesions between 6 and 20 days after infection. Animals divided into six groups of 4-5 in each were sacrificed between 2 and 24 weeks. The peritoneal exudate cells, obtained 4 days after injection of oil, were examined by the direct and the cells from lymphoid organs by the indirect capillary leukocyte migration method in the presence of 10 and 30 mug/ml of Reiter antigen. The peritoneal exudate cells responded with enhancement of leukocyte migration, reaching significant values between 6 and 24 weeks after injection. The cells from the lymphoid organs showed enhancement of leukocyte migration until the 4th week of infection. Treponemal antibodies, found in all animals, started to appear at the 4th week reaching a maximum titer at the 12th week after infection. The appearance of the antibodies correlated well (p less than 0.05) with the appearance of the enhancement of leukocyte migration. Wassermann antibodies were not detected throughout the 24 weeks of infection. The unique immune responses are discussed in view of the available data concerning natural acquired and experimental syphilis.


Treatment of Burkitt's lymphoma: randomized clinical trial of single-agent versus combination chemotherapy. A randomized clinical trial designed to compare the effectiveness of cytoxan (CTX) alone versus a combination consisting of CTX, vincristine (Oncovin) and methotrexate (COM) in the treatment of Burkitt's lymphoma (BL) was carried out. Nineteen patients were selected at random to receive CTX alone while 21 received COM. The two treatment regimens were equally effective in inducing remissions, and complete response rates of 83.3% and 84.3% were observed for CTX- and COM-treated patients, respectively. The relapse frequencies were also equal but the pattern of relapse was clearly different. Seven out of 8 (87.5%) in the CTX group relapsed with systemic and central nervous system (CNS) tumor, while 8 out of 10 (80%) in the COM group relapsed with CNS disease only. This difference is highly significant p = 0.008. The remission durations and survival to date are the same.


A histopathologic study of striated muscle biopsies in leprosy. Histopathologic changes in striated muscle biopsies in 50 cases of leprosy were studied; 40 being the lepromatous type and 10 the nonlepromatous type. All the biopsies were obtained from midportions of normal looking biceps muscles and paraffin embedded. Sections cut in transverse and longitudinal planes were stained by hematoxylin and eosin, Masson's trichrome, Mallory's PTAH, Gomori's silver impregnation, and Ziehl-Neelsen's technic. Lepromas, focal or confluent, in the endomysium, perimysium, muscle fibers and perineurally, constituted the most common pathological lesion, being observed in 34% of all cases with a higher frequency in the lepromatous type. Acid-fast bacilli could be demonstrated in some of these lepromas. These nodules were observed even in younger patients and increased in frequency as the age of patient advanced. Three cases of nonlepromatous leprosy showed granulomas. Other changes noted in varying proportions were loss of striations, hyaline change, fatty change, sarcolemmal changes, along with endomysial thickening, muscle necrosis and fibrosis. Bacillemia in leprosy and the possible route of muscle invasion resulting in subsequent production of leprous nodules with associated degenerative changes, indpendent of nerve involvement, have been postulated.


[Sensitivity to dapsone, sulfamethoxypyridazine and ethionamide of mycobacterium leprae taken from patients treated by the drugs]. Suspensions of M. leprae from skin biopsies of patients treated with dapsone (DDS) (four cases), sulfamethoxypyridazine (SMP) (six cases), and ethionamide (ETH) (seven cases), were inoculated into mouse foot pads and their sensitivity for the different drugs determined. Two strains were DDS resistant. Resistance appeared after 13 and 14 years respectively after the start of treatment. Five strains were isolated from patients treated with SMP. Relapses during sulfonamide treatment are considered to be due to the low effective serum concentrations reached by SMP, a situation which is aggravated by irregularities in drug intake. Fortunately all strains were sensitive to SMP and DDS as well. Four strains were ETH resistant. ETH resistance at the present moment reaches 4% and appeared in two cases six years after the start of treatment. It is concluded that SMP is not indicated for the treatment of multibacillary leprosy and that ETH can be used only in association with other drugs during the introductory phase of treatment of multibacillary forms of leprosy.


The presence of systemic and local activated macrophages in mice infected in the foot pad with Mycobacterium leprae and M. marinum. A highly sensitive assay for quantitating the presence of activated macrophages was employed to show that foot pad infection of mice with neither M. leprae nor M. marinum induced a population of activated peritoneal macrophages. In vitro attempts to demonstrate local activated macrophages in the foot pads of M. leprae infected mice failed, but, because of the technical problems encountered, do not preclude their presence. Additional studies are being carried out.


Evaluation of cell mediated immunity in the histopathologic spectrum of leprosy using lymphocyte transformation test. Lymphocyte transformation in response to PHA, M. leprae and BCG with the use of measurements of 3H-thymidine uptake, was studied in 10 normal controls, and 71 leprosy patients of different types histologically classified according to the Ridley-Jopling scale. Lepromin and PPD tests were done in each one of them. It was found that there was a generalized depression of immune responsiveness to PHA in all forms of leprosy. The immune responsiveness of lymphocytes to M. leprae continuously and gradually increases from the lepromatous (LL) towards the tuberculoid (TT) end of the spectrum. The loss of immune response of lymphocytes from lepromatous patients to M. leprae is specific as evidenced by their capacity to respond selectively to BCG when PPD-positive, irrespective of their place in the leprosy spectrum.


Growth of macrophages obtained from various sources. Techniques to obtain macrophages from various sources of the mouse were reported. The following sources were included: peritoneal exudate, alveolar lavage, blood leucocytes, bone marrow, spleen, liver, lungs, lymph nodes, thymus, thyroid, heart muscle, kidney, and subcutaneous cover glass implants. Human blood macrophages were also included. Long-term cinemicrographic studies revealed sustained good growth of these macrophages. Cell multiplication was detected in all of these cultures except those obtained from the peritoneal exudate. Pure cultures of macrophages were obtained from blood of the mouse and human. Macrophages obtained from other sources were accompanied by some growth of fibroblasts. Methods to eliminate the fibroblasts in cultures were discussed.


Cell walls of Mycobacterium leprae.

Cell walls of Mycobacterium leprae. Walls were prepared from M. leprae isolated from livers and spleens of infected armadillos. These contained mycolic acids and arabinogalactan, but the composition of the peptidoglycan differed from that of the other mycobacteria that have been studied, since it contained substantial amounts of glycine. The relationship of M. leprae to the other mycobacteria remains obscure.


Treatment of hyperhidrosis with topical methenamine. Idiopathic palmar and plantar hyperhidrosis is a relatively common disorder of eccrine sweat gland function. Treatment with glutaraldehyde of formaldehyde, although successful, may cause undesirable side effects. Methenamine is a polycyclic organic compound which release ammonia and formaldehyde at acid pH. Five per cent methenamine in a firm stick gel, applied daily to one palmar or plantar surface of 109 patients with hyperhidrosis, resulted in significantly less sweating after one month. No patients were sensitized to formaldehyde.


Isolation of Icterohaemorrhagiae from Microtus monte belli trapped at the Nagaoka area, Niigata Prefecture in Japan. Three strains of leptospira were isolated from field rodents on the occasion of scrub typhus survey on the Nagaoka, Kurojo, Kamikawanishi area, Niigata Prefecture in September 1961. Out of 10 Microtus montebelli 3 strains of leptospira by means of heart puncture in febrile stage from guinea pigs. They were identified as icterohaemorrhagiae by cross-agglutination-lysis and a cross absorption tests. It was the first report of icterohaemorrhagiae in Microtus montebelli in Japan.


Reflected fascial flaps for the repair of large abdominal defects. Three cases of large abdominal defects that were successfully closed with reflected fascial flaps from contiguous fascia have been presented. In each case the defect was repaired without evidence of breakdown or compromise of the blood supply. No disability was detected at the donor site. This procedure is recommended in conditions where rigid fixed borders are present and potential infection is feared.


In vitro demonstration of cytotoxic antibodies and their possible role in corneal graft rejections. Rabbit corneas were exposed to heterologous guinea pig anti-rabbit antibodies in the presence of complement and cytotoxic damage to the endothelium was demonstrated using para nitroblue tetrazolium. The possible role of such antibodies as "blocking antibodies" and as cytotoxic antibodies in the corneal graft rejection process are discussed.


The effect of splenectomy on corneal graft rejection. Recent studies have suggested that the spleen may be essential for the "immunologic privilege" enjoyed by corneal grafts. We tested this hypothesis in rabbits by performing 38 exchange penetrating keratoplasties between 19 normal rabbits and 19 rabbits that had undergone splenectomy. No significant difference was found in the number of corneas rejected or the mean graft survival time between the normal and splenectomized rabbits.


Effect of acetone fixation on rabies immunofluorescence in glycerine-preserved tissues. Rabies immunofluorescence in glycerol-preserved tissues is more intense without acetone fixation. Glycerol preservation of suspected rabid tissue should be avoided whenever possible.


Shigella flexneri 6 biotypes: a review.

Shigella flexneri 6 biotypes: a review. Except in gas production, S. flexneri 6 is generally similar to other S. flexneri. S. flexneri 6 contains both aerogenic and anaerogenic strains which are identical serologically. It is not surprising that S. flexneri 6 is not separable from other S. flexneri types on the basis of DNA relatedness (overall genetic makeup) (4). The Manchester biotype is seen in the United States, and is easily separable from other Enterobacteriaceae by biochemical tests. One can no longer assume à priori that any given biochemical reaction rules an organism out of Shigella. This is particularly true for the production of gas from glucose, but also applies to the fermentation of lactose and the production of H2S. Clinical laboratory personnel should be alert to these possibilities.


Nuclear cardiology in the evaluation of therapy and ventricular function in ischemic heart disease. The advent of nuclear cardiology has resulted in a veritable explosion of new techniques for evaluating coronary artery disease. Most of these new techniques for detecting myocardial performance, myocardial perfusion patterns, shunt detection, and infarct detection can be done in sequential noninvasive fashion so that patient may be selected earlier in their disease when the benefits of surgical and medical therapy may be restorative rather than reparative. It is hoped this new medical technology will satisfy the desire of Mackenzie for "comparative evaluation of medical remedies and different modes of treatment of disease by the lynx-eyed scrutiny of dispassionate analysis."


Orally active 7-phenylglycyl cephalosporins. Structure-activity studies related to cefatrizine (SK&F 60771). The synthesis of a series of related broad-spectrum 7-phenylglycyl cephalosporins with 3-heterocyclicthiomethyl substituents is described. The effects of benzene-ring hydroxylation and 3-substituent variation on the in vitro antibacterial activity, height and duration of mouse serum levels, and effectiveness in protecting against bacterial infection in the mouse are examined. Included for comparison are cephalexin, cephaloglycin and their ortho-, meta- and para-hydroxy derivatives. The biological properties examined were influenced by the position of the hydroxyl group and by the nature of the 3-substituent. The 7-(p-hydroxyphenylglycyl)-3-heterocyclicthiomethyl analogs were found to produce significantly higher serum levels on oral administration to mice than their unhydroxylated counterparts. This effect was not observed with the 7-(m-hydroxyphenylglycyl)-3-heterocyclicthiomethyl cephalosporins, nor with the p-hydroxyphenylglycyl analog of cephalexin. While m- and p-hydroxylation had little effect on in vitro activity and o-hydroxyphenylglycyl cephalosporins tested had very low antibacterial activities and were not examined further. One derivative, 7-[R-2-amino-2-(4-hydroxyphenyl)acetamido]-3-(1H-1, 2, 3-triazole-4(5)-ylthiomethyl)-3-cephem-4-carboxylic acid (SK&F 60771) was found to have outstanding in vitro and in vivo activities along with oral and subcutaneous serum levels in the mouse that were significantly higher than those obtained with cephalexin. This derivative which has been given the generic name cefatrizine was selected for extensive additional biological evaluation.


C-2801X, a new cephamycin-type antibiotic II. Isolation and characterization. Two new species of Streptomyces, S. heteromorphus and heteromorphus and S. panayensis, were found to produce a new atibiotic named C-2801X together with cephamycins A and B. The antibiotics were separated from each other by column chromatography on Amberlite XAD-2 and isolated in pure form as mono-sodium salts. C-2801X mono-sodium salt has a molecular formula C25H28N3O12SNa, and exhibits antibacterial activity against Gram-positive and Gram-negative bacteria including those insensitive to cephamycins A and B. From its physicochemical and biological properties, C2801X was considered to be a new cephamycin-type antibiotic.


Mechanism of action of bicyclomycin.

Mechanism of action of bicyclomycin. Microscopic examination of cultures of Escherichia coli exposed to bicyclomycin revelaed elongated or spheroplast-like cells. At the lethal level, bicyclomycin was shown to inhibit the synthesis of RNA and protein in the growing cells of E. coli 15 THU, whereas DNA and lipid synthesis were not significantly affected. However, the antibiotic did not block RNA and protein synthesis in vitro. Bicyclomycin was observed to inhibit the synthesis of envelope proteins more markedly than that of cytoplasmic proteins. The synthesis of two major envelope proteins was more sensetive to bicyclomycin than that of the other envelope proteins. One (peak I), which was inhibited to the greatest extent, seemed to be identical with a bound form of lipoprotein, and the other (peak V) with a free form of lipoprotein. Bicyclomycin exhibited inhibitory effects on the exclusive biosynthesis of the lipoprotein in histidine-starved cells of E. coli 15 THU. The biosynthesis of the bound form of lipoprotein was more profoundly inhibited by bicyclomycin than that of the free form. These results indicate that the primary action of bicyclomycin may be due to the interference with the biosynthesis of lipoprotein, and its assembly to peptidoglycan.


The structure of the calcium complex of A23187, a divalent cation ionophore antibiotic. The crystal and molecular structures of the calcium complex of A23187 has been determined by X-ray diffraction studies.


Light-absorbing properties, stability, and spectral stabilization of indocyanine green. The absorption spectrum of indocyanine green depends on the nature of the solvent medium and on the dye concentration. Binding to plasma proteins causes the principal peaks in the absorption spectrum to shift about 25 nm toward the higher wavelengths. The much greater influence on the spectrum of the dye concentration results from progressive aggregate formation with increasing concentration. Indocyanine green solutions therefore do not follow Lambert-Beer's law above 15 mg-I-1 (in plasma). Indocyanine green solutions in plasma and concentrated (1,000 mg-I-1) solutions in distilled water are stable for at least 4 h. In long-term experiments the optical density of indocyanine green solutions in plasma as well as in distilled water generally diminishes, even in the dark. On the 7th day a new absorption maximum starts to appear at gamma=900 nm, possibly caused by further aggregate formation leading to much larger particles. Spectral stabilization after injection of a concentrated solution into the blood is most rapid when the dye is dissolved in distilled water. Spectral stabilization slows down with decreasing temperature. As rapid spectral stabilization is essential in quantitative dye dilution studies, the practice of adding a albumin and/or isotonic saline solution to the injectate should be discontinued. When a 10 g-1(-1) aqueous solution of indocyanine green is used, spectral stabilization takes less than 1.5 a (at 37 degrees C), which is sufficiently fast for almost any application.


Changes in the fluid compartments and dry weights of reimplanted dog lungs. We studied changes in fluid compartments and dry weights of reimplanted lungs of dogs during the first 6 wk after operation. Excision and reimplantation caused a large increase in lung weight. The increase in weight was maximal 3 days after operation, was proportionately greater in the upper middle lobe than in the lower lobe, and was principally due to increased extravascular water. Dry weight and noncirculating red cells (measured by subtracting 51Cr-labeled red cell mass from total red cell mass as measured by hemoglobin extraction) also increased within 3 days after operation. Lung weight and extravascular water decreased progressively after 3 days and were normal in 6 wk. Chromated blood mass remaining within excised, passively drained lobes did not change at any stage postoperatively. Three days after operation, frozen lung sections showed minimal alveolar fluid but large amounts of peribronchial and perivascular edema which was occasionally blood tinged; submucosal edema was present in a few bronchi. Using intraalveolar Evans blue dye, we confirmed that several bronchial lymphatics close within 6 h and refenerate during the first postoperative week. Many of observed functional changes in freshly reimplanted lungs are temporally related to changes in extravascular water.


Salmonella typhimurium SA host specificity system is based on deoxyribonucleic acid-adenine methylation. We have determined the nature of the deoxyribonucleic acid (DNA) modification governed by the SA host specificity system of Salmonella typhimurium. Two lines of evidence indicate that SA modification is based on methylation of DNA-adenine residues. (i) The SA+ locus of Salmonella was transferred into Escherichia coli B, a strain that does not contain 5-methylcytosine in its DNA; although the hybrid strain was able to confer SA modification, its DNA still did not contain 5-methylcytosine. (ii) the N6-methyladenine content of phage L DNA was measured after growth in various host strains; phage lacking SA modification contained fewer N6-methyladenine residues per DNA. We also investigated the possibility, suggested by others (32), that SA modification protects phage DNA against restriction by the RII host specificity system. Phages lambda, P3, and L were grown in various SA+ and SA- hosts and tested for their relative plating ability on strains containing or lacking RII restriction; the presence or absence of SA modification had no effect on RII restriation. In vitro studies revealed, however, that Salmonella DNA is protected against cleavage by purified RII restriction endonuclease (R-EcoRII). This protection is not dependent on SA modification; rather, it appears to be due to methylation by a DNA-cytosine methylase which has overlapping specificity with the RII modification enzyme, but which is not involved in any other known host specificity system.


Isolation and genetic characterizaion of Escherichia coli K-12 mutations affecting bacteriophage T5 restriction by the ColIb plasmid. A mutant derivative of Escherichia coli K-12 has been isolated which is permissive for bacteriophage T5 infection even when harboring a wild-type ColIb plasmid. The fully permissive phenotype was the result of two mutations that are located near the rpsL-rpsE region on the E. coli chromosome and are recessive to the wild-type alleles. These mutations had little or no effect on induction of colicin synthesis and did not affect the expression of antibiotic resistance by the resistance plasmids R64drd11 or R1drd19. Cells harboring the mutant alleles grew more slowly than isogenic wild-type derivatives in either minimal or complete media.


Evidence against the involvement of chemotaxis in swarming of Proteus mirabilis. Nonswarming and nonchemotactic mutants of Proteus mirabilis were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine or ultraviolet light. These mutants were used in experiments to determine if chemotaxis is involved in the swarming of P. mirabilis. Nonchemotactic mutants failed to form chemotactic bands in a semisolid casein hydrolysate medium, yet they swarmed on the same medium containing 1.5% agar. Nonswarming mutants were attracted towards individual amino acids and components of tryptose. In cross-feeding experiments, no evidence was obtained to indicate the production of a diffusable chemical repellent. In studies with the wild-type P. mirabilis, no clear-cut negative chemotaxis was seen even though three different assays were used and numerous chemicals were tested. Additional evidence against the involvement of chemotaxis in swarming comes from finding that dialysis does not interfere with swarming; swarm cells will swarm immediately when transferred to fresh media, and swarm cells will swarm on an agar-water medium supplemented with a surfactant. These data indicate that chemotaxis is not involved in the swarming of P. mirabilis.


Wild-type and mutant in vitro products of an operon for ribonucleic acid polymerase subunits. An in vitro protein-synthesizing system can synthesize two ribonucleic acid (RNA) polymerase subunits of Escherichia coli, beta and beta', when a transducing phage deoxyribonucleic acid (DNA) template containing the rpoB region of the bacterial chromosome is added. Recombinant rpoB transducing phages were isolated that carry "nonsense" mutations of the class rpo-rifampin zero amber (formally referred to as rifoam). DNA was extracted from two of these phages. These DNAs are unable to direct the synthesis of beta subunits, whereas beta' synthesis is unaffected. Both mutations can be efficiently suppressed in vitro by the addition of suppressor transfer RNA. One of the mutations (rpoB115) produces a detectable nonsense (or restart) fragment of the beta protein in the absence of suppression. It is concluded that rpoB115 is an amber mutation within the structural gene for the beta subunit of RNA polymerase.


Altered properties of deoxyribonucleic acid polymerase I isolated from the membrane of bacteriophage T4-infected Escherichia coli. Previous studies have shown that a large fraction of the host cell deoxyribonucleic acid (DNA) polymerase I (EC 2.7.7.7) becomes associated with the cell membrane shortly after infection with bacteriophages T4 and T7. The present investigation of the bound enzyme revealed that the polymerase activity can be eluted from the membrane with chelating agents, and that the material thus obtained shows many properties that distinguish it from purified DNA polymerase I. These include its chromatographic behavior, sedimentation rate, sensitivity to anti-DNA polymerase I antiserum, and activity with synthetic and natural DNA primers. Several of these physical and biological parameters were shown to revert slowly during storage to those exhibited by the purified enzyme. Efforts to determine whether the unusual properties of the membrane enzyme resulted from its association with DNA failed to support that possibility. These observations suggest that either the cause or the result of membrane binding of DNA polymerase I is a transient change in conformation or structure of the enzyme, with a resultant change in its enzymatic activity.


Expression of a mutation affecting F incompatibility in the integrated but not the autonomous state of F. Previously we have described a mutant Hfr strain in which incompatibility between the integrated F factor and an autonomous F-prime (F') factor was abolished. The mutation (inc) was located in the integrated F factor. F-prime factors isolated from the mutant Hfr strain have the same incompatibility behavior as those isolated from normal Hfr strains. Reintegration of these F' factors into the chromosome restores the Inc- phenotype characteristic of the mutant Hfr. The inc mutation thus affects incompatibility between integrated F and autonomous F(Fi-Fa incompatibility) but not incompatibility between two autonomous F factors (Fa-Fa incompatibility). The implications of this finding for the mechanism of plasmid incompatibility are discussed.


Bromodeoxyuridine 5'-monophosphate incorporation into yeast nuclear and mitochondrial deoxyribonucleic acid. Standard laboratory yeast strains can be enriched for thymidine 5'-monophosphate (TMP) uptake derivatives that generate only a low percentage of respiratory-deficient colonies (petites) under inhibition of TMP biosynthesis. Such mutants incorporated bromodeoxyuridine 5'-monophosphate (BrdUMP) into both nuclear and mitochondrial deoxyribonucleic acid (mtDNA); however, they showed a selectivity for TMP over BrdUMP incorporation. The preferential incorporation of [3H]TMP or BrdUMP into mtDNA was strain dependent. The density increments after growth in the presence of BrdUMP reached 50 mg/ml for nuclear DNA and 22 mg/ml for mtDNA in CsCl gradients. Density shifts corresponding to 4% bromouracil substitution were easily detected. Preliminary density transfer experiments confirm that mtDNA does not replicate in synchrony with nuclear DNA.


Novel genotypes among transductants made with bacteriophage P1 lysates from an F14 merogenote strain of Escherichia coli K-12. Among P1 transductants in Escherichia coli K-12 that were selected for the proximal and distal markers from the large F14 merogenote, a variety of unusual genotypes were found. As earlier workers had found, one class of these could transfer the proximal genes (argH, metB) and distal genes (ilvEDAC) of the F14 during conjugation. These F14 genes could be transferred into RecA recipients, indicating that they were carried on an F-merogenote rather than on an Hfr chromosome. The transduced F-merogenotes could transfer other F14 genes (metE, rha) as well. Transfer kinetic analysis showed that all of the latter transduced F-merogenotes that were examined were indistinguishable from the parental F14 in the order of transfer and the genetic distance between proximal and distal markers. This suggests that the whole F14 had been received somehow by the primary transductional recipients, a remarkable possibility since the F14 is much larger than the largest deoxyribonucleic acid segment normally transduced by P1. The mechanism of this phenomenon is not yet known. Many of the transductants did not transfer any of the F14 markers tested. Some of these transductants segregated certain F14 genes, indicating they were carried on self-replicating genetic elements, but others were not cured of F14 markers, even by acridine orange. Cotransductional analysis of this group was consistent with the hypothesis that the F14 markers in some of these strains had integrated into the chromosome in the expected manner, since in these latter the F14 alleles were linked to the expected chromosomal genes. Other strains among the stable transductants had acquired new linkages in that genes previously separated by several minutes could now be cotransduced. These latter included the novel cotransductional linkages of rbs-ilv-argH, rbs-ilv-argH-metB, and ilvD-argH-purD. Such strains might have been formed as a result of insertion into the chromosome of small circles derived from F14.


Regulation of beta-glucuronidase synthesis in Escherichia coli K-12: constitutive mutants specifically derepressed for uidA expression. All methyl-beta-D-galacturonide-positive mutants isolated from Escherichia coli K-12 carry constitutive mutations for beta-glucuronidase (UID) synthesis. Most of these mutants are specific for UID synthesis and are distributed in three classes according to the derepression level of UID. Each specific mutant carries a mutation(s) near uidA, the structural gene for UID, at min 30.5 of the E. coli K-12 linkage map. The expression of UID synthesis in F-merodiploid strains carrying these mutations permits discrimination between dominant and recessive constitutivity over the wild-type allele. The first kind of mutation (dominant) should affect the operator site uidO of the structural gene uidA; the second type of mutation (recessive) should affect a regulatory gene, uidR, operating through a negative control. The isolation of mutants bearing at this locus superrepressed mutations, which can revert to produce a constitutive phenotype, confirms the occurrence of such a regulatory gene. The partially derepressed uidR mutants of the first class are normally inducible and remain constitutive at low temperature; their UID has the same thermal sensitivity as in the wild-type strains. The occurrence of similar regulatory gene mutants has been recently described in the lactose system (Shineberg, 1974).


Regulation of beta-glucuronidase synthesis in Escherichia coli K-12: pleiotropic constitutive mutations affecting uxu and uidA expression. Among the beta-glucuronidase (UID)-constitutive mutants obtained by growth on methyl-beta-D-galacturonide, some strains are also derepressed for the two enzymes of the uxu operon: mannonate oxidoreductase (MOR) and mannonate hydrolyase (HLM). By conjugation and transduction experiments, two distinct constitutive mutations were separated in each pleiotropic mutant strain. One of them was specific for uidA gene expression and was characterized as affecting either uidO or uidR sites. The second type of mutation was mapped close to the uxu operon and was found to be responsible for the pleiotropic effect revealed in the primary mutants: after separation such a mutation still fully derepresses MOR and HLM synthesis but weakly derepresses UID synthesis. The pleiotropic effect of this mutation was maintained even though the activity of the structural genes was altered. This rules out the occurrence of an internal derepressing interaction between these enzymes. In merodiploid strains, uxu-linked constitutive mutations were recessive to the wild-type allele, suggesting that these mutations could affect a regulatory gene. The uxuR gene is probably a specific regulatory gene for a very close operon, uxu. Moreover, it has a weak effect on uidA expression. Thus, UID synthesis would be negatively controlled through the activity of two repressor molecules that are synthesized by two distinct regulatory genes, uidR and uxuR. These two repressing factors are antagonized, respectively, by phenyl-thio-beta-D-glucuronide and mannonic amide and could cooperate in a unique repression/induction control over uidA expression. Constitutive mutations affecting the control sites of uidA gene probably characterize two distinct attachment sites in the operator locus for each of the repressor molecules.


R factor-mediated polarized chromosomal transfer in Escherichia coli C. Five transferable drug resistance factors (R factors) with the ability to bring about chromosomal transfer in Escherichia coli C were investigated with respect to the direction and origin of chromosome transfer. They were found to constitute two groups, both with a clockwise direction of transfer. One group has its origin of transfer between arg and pro, and the other group has its origin of transfer between try and man on the E. coli C chromosome. With one R factor in particular, the rapid increase in the production of recombinants was followed by a decrease in numbers as mating was prolonged.


Ultrastructural visualization of anionic sites on mycoplasma membranes by polycationic ferritin. Anionic sites on mycoplasma membranes were visualized in the electron microscope by a polycationized ferritin derivative. The technique of thin sectioning was used. Staining prior to fixation led to clustering of ferritin granules on the mycoplasma cell surface. On glutaraldehyde-fixed Mycoplasma mycoides subsp. capri, M. gallisepticum, M. pneumoniae, and Acholeplasma laidlawii, the anionic sites were uniformly distributed over the entire membrane surface. M. hominis did not bind the polycationic ferritin label. Chemical and enzymatic treatments of the mycoplasmas indicated that the anionic sites may be lipid phosphate groups. Isolated M. mycoides subsp. capri membranes were labeled exclusively on only one membrane surface, presumably the outer one. Liposomes prepared from diphosphatidylglycerol and phosphatidylcholine were also labeled by the polycationic ferritin.


Mutants of Salmonella typhimurium deficient in an endoprotease. Three bands of hydrolytic activity toward the chromogenic protease substrate N-acetyl-DL-phenylalanine beta-naphthyl ester (NAPNE) can be observed after gel electrophoresis of crude extracts of Salmonella typhimurium or Escherichia coli. Mutants deficient in one of these three activities have been isolated using a staining procedure that identifies colonies that show reduced ability to hydrolyze NAPNE. These mutants lack the strongest of the three bands of activity. The Salmonella mutations (designated apeA) are all co-transducible with purE, and the order (pro)-apeA-Hfr K17 origin-purE has been established. Strains carrying apeA mutations have wild-type doubling times. None of the apeA mutants isolated gains an auxotrophic requirement as a result of loss of the apeA gene product. The rates and extents of protein degradation during starvation for a carbon source or during growth after exposure to the amino acid analogue canavanine do not seem to be affected by apeA mutations. Revertants of apeA mutations (selected by screening for clones that have regained the ability to hydrolyze NAPNE) frequently contain a new enzymatic activity not found in wild-type cells.


Genetic and physiological regulation of intrinsic proteins of the outer membrane of Salmonella typhimurium. Four major outer membrane polypeptides, accounting for approximately 20% of the total protein of the outer membrane of Salmonella typhimurium, were induced by growth in minimal medium. The polypeptides were tightly bound membrane components. Physiological and genetic evidence indicates that the four polypeptides fall in two separate regulation groups. Synthesis of one of these groups was coordinately regulated by the concentration of iron in the medium, and a mutant strain has been identified in which there is constitutive synthesis of this group of major outer membrane proteins.


Comparative study of R1-specific chromosomal transfer in Escherichia coli K-12 and salmonella typhimurium LT2. High-frequency transfer of the chromosomal trp region by R1 observed in Escherichia coli (Pearce and Meynell, 1968) also occurs in Salmonella typhimurium. The reaction is recA independent in both species. The origin of transfer lies within a segment of the chromosome that is inverted in S. typhimurium relative to E. coli, and thus transfer occurs in a different direction in the two species. The character of R1 that is responsible, known as Tfa+, may be lost without affecting other properties of the R factor such as its own transfer.


Ultrastructure of paracrystals of a lipoprotein from the outer membrane of Escherichia coli. The highly purified lipoprotein of the outer membrane of Escherichia coli forms paracrystals. The ultrastructures of these paracrystals were examined by electron microscopy. The needle-shaped paracrystals show several different band patterns, depending on conditions of paracrystallization. Models are presented to explain possible arrangements of the lipoprotein molecules within the paracrystals.


In vitro spheroplast and L-form induction within the pathogenic nocardiae. Six strains of Nocardia asteroides, two strains of N. caviae, and two strains of N. braziliensis were grown in medium supplementted with glycine, lysozyme, D-cycloserine, glycine plus lysozyme, and glycine plus D-cycloserine. It was shown that three strains of N. asteroides, and two strains of N. caviae, readily formed spheroplasts and/or protoplasts when grown in the presence of glycine plus either lysozyme or D-cycloserine. This process was studied by both phase contrast microscopy and electron microscopy. The induced cultures were then plated on hypertonic medium for the isolation of L-forms. It was shown that the organisms differed greatly in their ability to produce spheroplasts and subsequently grew as L-forms or transitional-phase variants.


Morphogenic effects of alpha-factor on Saccharomyces cerevisiae a cells. Saccharomyces cerevisiae mating type a cells enlarged and elongated when exposed to alpha-factor, a sex pheromone produced by mating-type alpha cells. This morphogensis required exogenous-D-glucose, nitrogen, and phosphate, and cells in exponential phase responded better than stationary-phase cells. Morphogenesis was blocked by cycloheximide and by inhibitors of cell wall biosynthesis such as 2-deoxy-D-glucose, 2-deoxy-2-fluoro-D-glucose, and 2-deoxy-2-fluoro-D-mannose, but not by polyoxin D. One to two hours after addition of pheromone, a cells became more susceptible to lysis by glucanases, a change that was dependendent on the dose of alpha-factor and was blocked by drugs that block morphogenesis. On the other hand, treatment with alpha-factor did not increase susceptibility to attack by trypsin, subtilisin, or exo-alpha-mannanase. Radioactive label, incorporated into cell wall polysaccharides during treatment with alpha-factor, was not secreted into the medium during morphogenesis. Analysis of the labeled wall polymers showed that alpha-factor-treated cells contain more glucan and less mannan than control cells, and that the mannan of treated cells contains an increased proportion of shorter side chains and unsubstituted backbone mannose units. Thin-section electron microscopy of treated cells revealed that the cell wall possesses a diffuse outer layer in the extension and is thinner at the tip.


EcoRI restriction endonuclease map of the composite R plasmid NR1. A physical map of the composite R plasmid NR1 has been constructed using specific cleavage of deoxyribonucleic acid (DNA) by the restriction endonuclease EcoR-. Digestion of composite NR1 DNA by EcoRI yields thirteen fragments. The six largest fragments (designated A to F) are from the resistance transfer factor component that harbors the tetracycline resistance genes (RTF-TC). The seven smallest fragments (designated G to M) are from the r-determinants component that harbors the chloramphenicol (CM), streptomycin-spectinomycin (SM/SP), and sulfonamide (SA) resistance genes. The largest fragment of several RTF-TC segregants of NR1 that have deleted the r-determinants component is 0.8 X 10(6) daltons larger than fragment A of composite NR1. Only a part of fragment H of the r-determinants component is amplified in transitioned NR1 DNA in Proteus mirabilis, which consists of multiple, tandem sequences of r-determinants attached to a single copy of the RTF-TC component. Both of these changes can be explained by the locations of the excision sites at the RTF-TC: r-determinants junctions that are involved in the dissociation and reassociation of the RTF-TC and r-determinants components. The thirteen fragments of composite NR1 DNA produced by EcoRI have been ordered using partial digestion techniques. The order of the fragments is: A-D-C-E-F-B-H-I-L-K-G-M-J. The approximate locations of the TC, CM, SM/SP, and SA resistance genes on the EcoRI map were determined by analyzing several deletion mutants of NR1.


Isolation, by tetracycline selection, of small plasmids derived from R-factor R12 in Escherichia coli K-12. The examination, by agarose gel electrophoresis, of tetracycline-resistant colonies of Escherichia coli K-12 carrying R-factor R12 reveals the presence of smaller plasmid deoxyribonucleic acids (DNAs), incompatible with R12, in many of the clones. These plasmids are demonstrated to be homologous with R12 DNA by electron microscope heteroduplex experiments and by the production of consistent fragment patterns upon digestion with various restriction endonucleases. These autonomously replicating plasmids form a related series of covalently closed circular DNA molecules ranging in size from 3.6 X 10(6) to 61 X 10(6) daltons. Plasmids of molecular weight between 3.6 X 10(6) and 37 X 10(6) confer no antibiotic resistances, but when jointly present with R12 by nonetheless enhance the expression of the tetracycline resistance associated with this latter molecule.


Co-regulation of the phosphate-binding protein and alkaline phosphatase synthesis in Escherichia coli. In phosphate-starved cells of Escherichia coli, the synthesis of alkaline phosphatase and some additional periplasmic proteins is derepressed. One of these proteins, which does not appear in a phoS- constitutive strain, has been identified as well the periplasmic phosphate-binding protein.


Identification of the major penicillin-binding proteins of Escherichia coli as D-alanine carboxypeptidase IA. Penicillin-binding proteins 5 and 6 of Escherichia coli have been identified as d-alanine carboxypeptidase IA.


Partial characterization of nucleoids and nucleoid-plasmid complexes from Salmonella typhimurium. Nucleoids from Salmonella typhimurium strain LT2 consist of supercoiled deoxyribonucleic acid structures that are ribonuclease labile sedimenting at 1,700S. More than 90% of the covalently closed circular deoxyribonucleic acid of a cryptic plasmid harbored by this strain cosediments with the host's 1,700S nucleoids.


Fragility of plasma membranes in Saccharomyces cerevisiae enriched with different sterols. Saccharomyces cerevisiae NCYC 366, grown under strictly anaerobic conditions to induce requirements for an unsaturated fatty acid (supplied by Tween 80) and a sterol, contained free sterol fractions enriched to the extent of 67 to 93% with the exogenously supplied sterol (campesterol, cholesterol, 7-dehydrocholesterol, 22, 23-dihydrobrassicasterol, beta-sitosterol, or stigmasterol). Cells enriched in any one of the sterols did not differ in volume, growth rate, contents of free sterol, esters and phospholipids, or phospholipid composition. Cholesterol-enriched cells contained about 2% more lipid than cells enriched in any of the other sterols, which was largely accounted for by increased contents of triacylglycerols and, to a lesser extent, esterified sterols. Phospholipids were enriched to the extent of about 52 to 63% with C18:1 residues. Cells enriched in ergosterol or stigmasterol were slightly less susceptible to the action of a wall-digesting basidiomycete glucanase than cells enriched with any one of the other sterols. The capacity of the plasma membrane to resist stretching, as indicated by the stability and volume of spheroplasts suspended in hypotonic solutions of buffered sorbitol (particularly in the range 0.9 to 0.7 M), was greater with spheroplasts enriched in sterols with an unsaturated side chain at C17 (ergosterol or stigmasterol) than with any of the other sterols. Plasma membranes were obtained from spheroplasts enriched in cholesterol or stigmasterol and had free sterol fractions containing 70 and 71%, respectively, of the sterol supplied exogenously to the cells. The sterol-phospholipid molar ratios in these membranes were, respectively, 1:7 and 1:8.


Comparison of canavanyl-envelope proteins to normal envelope proteins. Electrophoretic analysis showed arginine- and canavanine-containing envelope proteins to be qualitatively the same. Quantitative differences may be due to rapid degradation of some canavanine-containing envelope proteins.


Selection for citrate synthase deficiency in icd mutants of Escherichia coli. Cultures of isocitrate dehydrogenase-deficient (icd) mutants were overgrown by double mutants (icd glt) lacking citrate synthase activity also. The icd mutants grew more slowly than wild-type cells or the double mutants because they accumulated an inhibitory metabolite (possibly citrate). Intracellular citrate levels were several hundred-fold higher in icd cells than in wild-type or icd glt cells. Final growth yields of the wild type and the icd mutant on limiting glucose were equivalent and greater than the growth yield of icd glt double mutants. The icd gene mapped between 60 and 74 min. icd mutants were resistant to nalidixic acid, but glt and icd glt mutants and wild-type cells were sensitive, indicating that resistance results from accumulation of isocitrate, citrate, or a derivative of these compounds.


Lipopolysaccharide-deficient, bacteriophage-resistant mutants of Escherichia coli K-12. Bacteriophage-resistant mutants isolated and classified in a previous study were examined for alterations in their lipopolysaccharide (LPS) composition, and properties likely to be affected by alterations in LPS composition were studied. It was found that many of the mutants of the Ktw (K2-resistance), Ttk (T2, T4, or K19 resistance), Bar (bacteriophage), Wrm (wide-range mutants), and miscellaneous resistance groups were altered in their response to a series of antibiotics and to two LPS-specific bacteriophages, C21 and U3. Furthermore, many of the bacteriophages to which these mutants were resistant adsorbed to LPS preparations. By direct sugar analysis of the mutant LPS preparations, it was shown that the mutants fitted into six distinct classes, which are readily derived from LPS core with a structure resembling that of Salmonella or Escherichia coli O100. A number of the mutants were shown to map between pyrE and mtl, which has been previously shown to be the site of a cluster of rfa genes in both Salmonella and E. coli. Outer membrane protein composition was studied in the above mutants using polyacrylamide gel electrophoresis. Some strains were shown to have alterations in the amount of major proteins. The nature of the bacteriophage receptors involved and the alterations leading to resistance are discussed.


Effect of nitrate reduction on the enzyme levels in carbon metabolism in Escherichia coli. The activities of twelve enzymes were measured in crude extracts from cells of Escherichia coli K-10 grown aerobically or anaerobically in a defined medium in the presence or absence of nitrate. The activities of isocitrate dehydrogenase, aconitate hydratase, 2-oxoglutarate dehydrogenase, malate dehydrogenase, malic enzyme, and D-lactate dehydrogenase (NAD+-independent) were found to be higher in cells grown in nitrate respiration than in those in fermentation, but lower than in those in respiration. This finding may explain the incomplete oxidation in nitrate respiration and, on the other hand, suggests the operation of the tricarboxylic acid even under these conditions. The activities of succinate dehydrogenase and alcohol dehydrogenase in relation to the formation of fermentation product were as high in cells grown in fermentation as in those in respiration and were low in those in nitrate respiration. However, that ratio of the activities in the latter case to the activities in respiration was the same as the ratio for most enzymes in the tricarboxylic acid cycle. The level of lactate dehydrogenase (NAD+-dependent) was not affected by nitrate respiration but its activity in the extract was inhibited by nitrate and nitrite. The absence of lactate in the anaerobic culture with nitrate may be due to this inhibition as well as NADH oxidation by nitrate. Levels of glucose-6-phosphate dehydrogenase and glutamate dehydrogenase were not altered by the growth conditions and that of pyruvate dehydrogenase was low only in cells grown  in fermentation.


Allocation of 15 RNase T1-resistant large oligonucleotides of MS2 RNA. Four large MS2 RNA fragments with the original 5'-end were obtained by limited RNase T1 digestion. The lengths of the fragments were 36, 30, 22, and 19% of the whole molecule. Whole MS2 RNA and the four fragments were completely digested with RNase T1 and analyzed by the two-dimensional "homochromatography fingerprint" technique. Sixteen oligonucleotides of MS2 RNA were separated and the oligonucleotides were assigned to the fragments. Fifteen of the sixteen oligonucleotides could be classified into three regions of the cistrons of bacteriophage MS2.


Cost of initial development of PLATO instruction in veterinary medicine. The cost of initial development of PLATO instruction during a 4-year period was $483,266. Of this amount, $220,765 were for equipment or renovation. The cost of lesson development for the entire 4 years averaged about $828 per hour of instruction. If 86 students used the lessons in each class at a maximum rate, over a 5-year period the cost per student-contact hour averages $1.93. With experience this figure should be reduced substantially. The median time required for an individual to program an hour of instruction for the entire 4 years was approximately 77 hours; however, under certain conditions a programmer may require as little as 36 1/2 hours. Programmer efficiency is based on a number of factors such as the quality and complexity of the lessons, experience of the programmer, and his knowledge of the subject matter. The University of Illinois College of Veterinary Medicine PLATO project has been developmental. The expense of continuing the project at this college or of initiating a similar project at another college should be much less as a result of this experience.


Partial amino acid sequences around the essential carboxylate in the active sites of the intestinal sucrase-isomaltase complex. A carboxylate group occurs in each of the active sites of the intestinal sucrase-isomaltase complex and can be labeled selectively by [3H]conduritol-B-epoxide (Quaroni, A., Gershon, E., and Semenza, G. (1974) J. Biol. Chem. 249, 6424-6433). After peptic digestion of the labeled, denatured, reduced, and cyanoethylated enzyme three radioactive peptides were isolated. The peptide originating from the sucrase subunit had the following sequence: Ile-Asp-Met-Asn-Gln-Pro-Asn-Ser-Ser; the other two, deriving from the isomaltase subunit, had the sequences: Asn-Gly2-Gln-Ile-Asp-Met and Ile-Asp-Met. The radioactive label was in each case bound to the beta carboxyl group of an aspartic acid residue.


Tryptophanyl transfer ribonucleic acid synthetase of Escherichia coli. Character of required thiol group and structure of thiol peptides. Native tryptophanyl-tRNA synthetase purified from Escherichia coli B has on each identical subunit a single thiol group which rapidly forms a mixed disulfide with a thionitrobenzoate moiety of 5,5'-dithiobis(2-nitrobenzoic acid). The reaction and the concomitant inactivation of the enzyme are both reversible by reductive removal of the thionitrobenzoate with dithiothreitol. Iodoacetamide and N-ethylmaleimide also react with the thiol group required for enzyme activity, but iodoacetic acid inactivates the enzyme through another mechanism. Three or 4 half-cystine residues/subunit were detected by amino acid analysis and by titration of the denatured enzyme with 5,5'-dithiobis(2-nitrobenzoic acid); no disulfide bonds were detected by borohydride reduction. Cleavage of the subunit (molecular weight 37,000) with 2-nitro-5-thiocyanobenzoic acid gave fragments of molecular weights 32,000, 27,000, and 9,500. Five carboxymethylated peptides were isolated from the trypsin products of the denatured enzyme after treatment with iodo[14C]lacetate. Three of these peptides represented unique sequences surrounding thiol groups in the enzyme. One cysteine-containing nonapeptide has a heptapeptide sequence homologous to a heptapeptide sequence in a cysteine containing decapeptide from the tryptophanyl-tRNA synthetase of human placenta. The nonapeptide appears to bear the thiol group required for enzyme activity.


Stabilization of an RNA molecule by 3'-terminal poly (A)-induced inhibition of RNase activity. The inhibitory properties of poly(A) on human spleen ribonuclease have been investigated. Hydrolytic activity has been shown to be strongly inhibited by poly(A) contained within RNAs isolated from a variety of natural sources. Furthermore, poly(A) segments of varying length have been covalently linked at the 3' terminus of Escherichia coli 5 S rRNA by polynucleotide phosphorylase in an attempt to construct an in vitro demonstration of the stabilization of RNA which contains poly(A). The extent to which these poly(A) tracts, varying from 4 to 132 nucleotides in length, could inhibit endonucleolytic attack on the 5 S rRNA to which they are linked was found to be dependent upon their length and upon small changes in spermidine concentration. The consequences of these findings are discussed in terms of a possible role for poly(A).


Interaction of bacterial initiation factor 2 with initiator tRNA. Interaction of bacterial polypeptide chain initiation factor 2 (IF-2) with Escherichia coli formylmethionyl-tRNA in the absence of free Mg2+ renders the fMet-tRNA adsorable to nitrocellulose membrane filters. This reaction does not require GTP and is strongly inhibited by low concentration (1 mM) of Mg2+ in the reaction mixture. The structural requirements of the tRNA for binary complex formation have been studied using modified fMet-tRNAfMet molecules and a series of N-blocked and normal aminoacyl-rRNAs. It has been observed that IF-2 will not blind either to free formylmethionine or to a short fMet-oligonucleotide, but will bind to any xRNA structure covalently attached to an N-blocked methionine group. The E. coli initiator and noninitiator methiionine tRNAs, which have many differences in primary structure, were found to bind identically. In addition, fMet-tRNAfMet molecules containing structural modidifications at 20 different sites had the same affinity for IF-2 as unmodified fMet-tRNAfMet. N-blocked eukaryotic initiator tRNAs were also found to bind strongly to the factor. Binary complex formation was readily reversible, f[14C]Met-tRNAfMet being competed out by addition of an equal amount of unlabeled fMet-tRNAfMet to the preformed complex. In contrast, deacylated tRNAfMet was a poor compeitor, a 30-fold excess being required for 50% inhibition of complex formation in the presence of limiting factor. Although tRNAs having an N-blocked methionine were found to have the greatest affinity for IF-2, specificity for the amino acid in binary complex formation was not absolute. Partial binding was observed with N-substituted tyrosine, valine, and phenylalanine tRNAs, and weak or no binding with N-subsituted lysine, alanine, and leucine tRNAs. In all cases, N-blocked derivatives had a higher affinity for IF-2 than the corresponding unsubstituted aminoacyl-tRNAs. These results indicate that IF-2 alone is not capable of distinguishing the nucleotide sequence of tRNAs and selects the initiator tRNA by recognizing the fMet moiety. The overall data suggest that the role of IF-2 in formation of the ribosomal initiation complex is to stablize the interaction of fMet-tRNAfMet with the ribosome at low Mg2+ concentrations by binding to both the ribosomal particle and the fMet group of the tRNA.


Limited proteolytic digestion of lac repressor by trypsin. Chemical nature of the resulting trypsin-resistant core. Tryptic digestion of Escherichia coli lac repressor under nondenaturing conditions readily removes 59 amino acids from the NH2-terminal end of the polypeptide chain. Longer digestion removes an additional 20 or more amino acids from the COOH terminus, leaving a highly trypsin-resistanct core molecule. The lac repressor tetrameric structure and inducer-binding activity are retained by the tryptic core. Operator-binding activity, however, is lost as the NH2-terminal end is degraded. Many or all of the possible trypsin cleavage sites in the NH2-terminal region are available to attack by the enzyme, indicating that this part of the polypeptide chain is exposed to the environment. Lac repressor, missing the NH2-terminal end, renatures efficiently from random-coil solvent to tetramers with full inducer-binding activity, indicating that the NH2-terminal region is not necessary for the appropriate three-dimensional folding of the polypeptide chains. Core which lacks both the NH2 and COOH termini renatures to tetramers with low efficiency.


Peptidyl transfer RNA dissociates during protein synthesis from ribosomes of Escherichia coli. Growing cultures of mutant Escherichia coli with temperature-senstive peptidyl-tRNA hydrolase were shifted to nonpermissive 4o degrees. There followed a roughly linear increase in a fraction of isolated tRNA (over 50% after 20 min) whose amino acid-accepting activity was masked until treatment with active peptidyl-tRNA hydrolase. The ionophoretic mobility of amino acid label associated with this fraction could be altered by treatment with the hydrolase, trypsin, RNAse, and alkali. The rate of accumulation of this fraction could be altered by treating the growing cells with chloramphenicol, which reduced the rate, or erythromycin, which enhanced it. It is concluded that peptidyl-tRNA dissociates from ribosomes of the mutant cells during protein biosynthesis. The primary metabolic role of peptidyl-tRNA hydrolase is to prevent the accumulation of dissociated peptidyl-tRNA, which inhibits protein synthesis. The rate of dissociation of peptidyl-tRNA from ribosomes was estimated at between 1 per 90 and 1 per 2600 peptide elongation steps in the absence of antibiotics, depending on the level of inhibition of protein synthesis. After 20 min at 40 degrees, the size distribution of peptides found on tRNA was heterogeneous, with over 74% having a molecular weight greater than 8 X 10(2). The effect of erythromycin suggests that its mechanism of action is to destabilize the peptidyl-tRNA/ribosome interaction and thereby stimulate the dissociation of peptidyl-tRNA. The mechanism of inhibition of protein synthesis by accumulating peptidyl-tRNA and reasons why peptidyl-tRNA dissociates from ribosomes are discussed in terms of the current data.


Regulation of glutaminase B in Escherichia coli. II. Modulaltion of activity by carbosylate and borate ions. Glutaminase B is both activated and inhibited by L-glutamate, the product of the reaction. The activation process is time- and temperature-dependent. Activation by L-glutamate alters the Vmax, So.5, and shape of the substrate saturation curve. The activation decays as a first order process with time after separation of L-glutamate from glutaminase B. L-Glutamate inhibits both glutaminase B and the glutamate-activated enzyme. Like L-glutamate, borate activates and inhibits glutaminase B. Inhibition of the enzyme by glutamine and glutamate analogs is also examined and similarities between the glutamate activation and activation by warming at 23 degrees are noted.


Regulation of glutaminase B in Escherichia coli. III. Control by nucleotides and divalent cations. Glutaminase B from Escherichia coli is modulated by nucleotides and divalent cations. ATP and ADP inhibit glutaminase B whereas AMP and divalent cations activate it. Inhibition and activation required preincubation of the nucleotides with glutaminase B at 4 degrees. Mg2+, Mn2+, and Ca2+ activated the enzyme and prevented the inhibition by ATP. Dialysis in the presence of an activator ligand reversed the ATP inhibition of glutaminase B. The modulation of glutaminase B by energy charge is similar to that observed with other catabolic enzymes. We suggest that a pattern of reciprocal regulation of glutaminase B and glutamine synthetase by adenine nucleotides prevents the formation of a "futile cycle" of amide synthesis and degradation.


Evidence of the involvement of an outer membrane protein in DNA initiation. Protein D of molecular weight 80,000 is incorporated into the membrane of Escherichia coli B/r for a brief fraction of the cell cycle. The protein is incorporated into the outer membrane; its incorporation seems to reflect de novo synthesis and, once incorporated, it is stable. In synchronous acetate-grown cultures a 10-fold increase in the rate of protein D synthesis occurs toward the end of the cell cycle, near the end of one round of DNA synthesis and the start of the next. In synchronous glucose-grown cultures the protein appears approximately halfway through the cell cycle, shortly before DNA synthesis begins. The observed correlation between protein D synthesis and DNA initiation, approximately 15 min later, is strengthened by experiments with inhibitors and mutants. Inhibition of protein synthesis at any time during DNA synthesis delays the subsequent appearance of protein D and the initiation of the next round of DNA synthesis. The synthesis of both protein D and DNA is prevented by nalidixic acid; after its removal a sharp spike of protein D synthesis is followed by a wave of DNA synthesis. Experiments with DNA and DNAB mutants, with amino acid starvation of an auxotroph and with nutritional shift-up, all supported the coupling of protein D synthesis with the initiation of DNA synthesis. The addition of beta-lactam antibiotics which affect murein metabolism dramatically increases the amount of protein D. The cells are prevented from terminating protein D synthesis; different antibiotics have different kinetics of action. Protein D preferentially binds to double-stranded DNA in vitro. We suggest that this protein provides a metabolic link between murein synthesis, protein synthesis, and DNA initiation, and that it acts at a specific time in the cell cycle as an attachment site for DNA to the cell envelope.


Binding of substrates to Escherichia coli ribonucleotide reductase. Ribonucleoside diphosphate reductase from Escherichia coli consists of a 1/1 complex of two nonidentical subunits called proteins B1 and B2. The enzyme reduces the four common ribonucleoside diphosphates to the corresponding deoxyribonucleotides and is allosterically regulated by nucleoside triphosphates which influence its substrate specificity as well as its overall activity. The B1 subunit contains binding sites for the effectors while B2 contains iron and an organic free radical essential for catalytic activity. We now establish that only protein B1 binds substrates. Competition experiments support the presence of two identical substrate binding sites, distinct from the effector binding sites. The catalytic site of the enzyme thus is formed from both the B1 and B2 subunits. Dissociation constants for substrates ranged from 2 X 10(-5) to about 10(-3) M. In all cases effectors decreased these constants in agreement with their influence on the substrate specificity of ribonucleotide reductase, but did not induce cooperative effects. The increase in binding was pronounced at 20 degrees but only marginal at 0 degrees. Arrhenius plots of the influence of temperature on the catalytic activity of the enzyme showed sharp breaks at 12 degrees. The temperature effects can be interpreted as a conformational change occurring in the structure of protein B1 at the critical temperature.


Biosynthesis of riboflavin. Structure of the purine precursor and origin of the ribityl side chain. We studied the incorporation of 14C-labeled guanosine into riboflavin under conditions precluding the metabolic conversion of guanosine compounds to free guanine. For this purpose we isolated a mutant BM 2 of Salmonella typhimurium deficient in the enzymes IMP dehydrogenase, purine nucleoside phosphorylase, and purine nucleotide pyrophosphorylase. The mutant incorporated [ribose-14C]guanosine into riboflavin and GMP without dilution. The isolated compounds were exclusively labeled in the ribityl and ribosyl side chain, respectively. AMP and CMP were not labeled. [2-14C]Guanosine was incorporated into riboflavin and GMP without dilution. The isolated compounds were exclusively labeled in the isoalloxazine and guanine moiety, respectively. AMP and CMP were again unlabeled. We conclude that the ribose moiety of proffered guanosine is directly converted to the ribityl moiety of riboflavin. Thus the biosynthesis of the vitamin begins at the level of a guanosine compound. Guanine, ribose, ribitol, and the respective phosphates are not direct precursors of the vitamin.


Selective inhibition of the dnase activity of the recBC enzyme by the DNA binding protein from Escherichia coli. In the presence of the Escherichia coli DNA binding protein, single-stranded DNA is resistant to both the endo- and exonucleolytic activities of the recBC DNase. Linear duplex DNA, on the other hand, is unwound at a normal rate, but converted to large, single-stranded fragments which are resistant to further hydrolysis. Therefore, in the presence of the binding protein and linear duplex DNA, the recBC enzyme acts not as a DNase, but primarily as an ATP-dependent unwinding enzyme, able to generate large, single-stranded material. Duplex circular DNA containing short, single-stranded gaps is also resistant to the hydrolysis in the presence of the binding protein.


Lac Repressor. Fluorescence of the two tryptophans. By comparing the fluorescence emission properties of the wild type lac repressor with two lac repressors altered at tryptophan 190 and 209, respectively, we show that tryptophan residue 209 has its environment changed, either by its own motion, or that of the surrounding amino acids when an inducer molecule is bound. Substitution of this tryptophan with other amino acids results in lac respressor molecules with reduced affinity for inducer molecules, indicating that the geometry at residue 209 affects the geometry of the inducer-binding site. From the results of potassium iodide quenching of fluorescence from the tryptophans, and from attempts to react the native lac repressor with dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide and 2-hydroxy-5-nitrobenzyl bromide, we propose that tryptophan residue 209 is involved in a conformational change of the protein upon binding of inducer, but does not come in direct contact with inducer.


Ketopantoate hydroxymethyltransferase. I. Purification and role in pantothenate biosynthesis. A new enzyme, ketopantoate hydroxymethyltransferase (5,10-methylene tetrahydrofolate: alpha-ketoisovalerate hydroxymethyltransferase) has been purified 2400-fold to apparent homogeneity from Escherichia coli K12. It catalyzes the first committed step in pantothenate biosynthesis, the reversible formation of ketopantoate (2-keto-3,3-dimethyl-4-hydroxybutyrate) according to Equation 1, has low Km values for its substrates, and is abent (1) Methylenetetrahydrofolate + alpha-ketoisovalerate in equilibrium tetrahydrofolate + ketopantoate from a mutant of E. coli auxotrophic for ketopantoate. It thus appears to be the enzyme responsible for catalysis of ketopantoate formation in vivo. A previously described enzyme that catalyzes reaction 2 irreversible (McIntosh, E.N., Purko, M., and Wood, W.A. (1957) J. Biol. Chem. 228, 499-509) and does not require (2) HCHO + alpha-ketoisovalerate leads to ketopantoate tetrahydrofolate can be obtained free of ketopantoate hydroxymethyltransferase and is present in equal amounts in ketopantoate auxotrophs and wild type E. coli. We conclude that the latter enzyme is not involved in the normal biosynthetic pathway leading to pantothenate; its function is unknown.


Purification and properties of the alpha2beta2 complex of tryptophan synthetase of Proteus mirabilis. A procedure is described for the purification of the tryptophan synthetase alpha2beta2 complex from cell extracts of Proteus mirabilis. A 30-fold purification was achieved with an overall yield of about 23% and a specific activity of 1,600. The complex can be dissociated and the subunits isolated in a pure form. The complex can be reconstituted from the isolated subunits to regain the initial activity. The alpha and beta2 subunits of the tryptophan synthetase complex of P. mirabilis are not significantly different from those of Escherichia coli and other enteric bacteria as to their physical properties, amino acid compositions, and enzymic properties. Complementation studies indicate that the alpha subunit of P. mirabilis hybridizes well with the beta2 subunit from E. coli. Similarly, the beta2 subunit of P. mirabilis readily complexes with the alpha subunits from E. coli, Salmonella typhimurium, and Serratia marcescens. The hybrids formed are all effective in catalyzing the conversion of indoleglycerol phosphate plus serine into tryptophan and glyceraldehyde 3-phosphate. However, these hybrids have reduced or no activity in the other reactions, namely, the condensation of indole and serine to form tryptophan or the aldolytic cleavage of indoleglycerol phosphate.