The quaternary structure of the sheaths of defective phages similar to PBS X. The contractile sheaths of five defective, PBS X-like bacteriophages from Bacillus subtilis and B. licheniformis were investigated by electron microscopy, dodecylsulphate gel electrophoresis and immunodiffusion. Electron microscope images of the extended and contracted sheaths were of similar appearance, although their lengths were different. The surface lattices of both the extended and the contracted sheaths were determined by optical diffraction. This showed that the quaternary structure of the sheaths of all five defective phages originated from identical surface lattices, which could be approximately expressed by the selection rules L = -2n' + 3m and L = 9N' + 17M for the extended and contracted sheaths respectively, in which 6n' = n with n = 0 or an integer multiple of 6. These results indicated that the packing of the protein subunits in these sheaths differed from those of other bacteriophages, for example T4 and millimicron [Amos and Klug, J. Mol. Biol. 99, 51--73 (1975); Admiraal and Mellema, J. Ultrastruct. Res. 56, 48--64 (1976)]. The molecular weight of the main sheath protein of the defective phages, as determined by dodecylsulphate gel electrophoresis, was approximately 50000. This value differed from that for T4, but was similar to that of millimicron [Admiraal and Mellema, J. Ultrastruct. Res. 56, 48--64 (1976); King and Laemmli, J. Mol. Biol, 75, 315--337 (1973)]. The results of immunodiffusion experiments, however, pointed to a chemical difference between the sheath proteins of the defective phages and millimicron, in addition to T4.


Ribosomal subunits from Tetrahymena pyriformis. Isolation and properties of active 40-S and 60-S subunits. Tetrahymena pyriformis ribosomal subunits were obtained by incubation of post-mitochondrial supernatant in the presence of 0.2 mM GTP and 0.1 mM puromycin for 45 min at 28 degrees C, followed by sucrose density gradient centrifugation. Isolated 40-S subunits were able to reassociate in vitro in the presence of 5 mM MgCl2 and 50 mM KCl and to perform poly(U)-dependent protein synthesis. The 60-S subunit carries the peptidyl transferase activity. The number of proteins in T. pyriformis ribosomal subunits was determined by two-dimensional polyacrylamide gel electrophoresis. The 40-S subunit contains 30 different protein species (including two acidic proteins). The 60-S subunit contains 35 different protein species (including two acidic proteins). The proteins were numbered following the system of Kaltschmidt and Wittmann.


Involvement of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system in regulation of transcription of catabolic genes. Synthesis of catabolite-sensitive enzymes is repressed in mutants defective in the general proteins (enzyme I and HPr) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system (ptsI and ptsH mutations). To elucidate the mechanism of this phenomenon we constructed isogenic strains carrying pts mutations as well as different lesions of regulation of the lac operon or mutations affecting adenylate cyclase activity (cya mutation) and synthesis of cyclic AMP-receptor protein (crp mutation) Measurements of the differential rate of beta-galactosidase synthesis in these strains showed that the repressive effect of pts mutations was revealed in lac+, lacI, lacOc and cya bacteria, but it was lost in lacP and crp strains. It was concluded that mutational damage to the general components of the phosphoenolpyruvate-dependent phosphotransferase system diminishes activity of the lac promoter. The results obtained led to the conclusion that pts gene products (apparently phospho approximately HPr) are necessary for the initiation of transcription of catabolite-sensitive operons in E. coli.


Isolation and characterization of RNA polymerase B from the larval fat body of the tobacco hornworm, Manduca sexta. DNA-dependent RNA polymerase B has been extensively purified from the larval fat body of the tobacco hornworm (Manduca sexta) by employing chromatography on ion-exchange columns of DEAE-Sephadex, DEAE-cellulose and phosphocellulose and centrifugation on glycerol gradients. The isolated enzyme after electrophoresis on acrylamide gels shows one main band and one minor band, both having enzyme activity sensitive to alpha-amanitin. The catalytic and physicochemical properties of the enzyme are similar to those of other eucaryotic B-type RNA polymerases. The enzyme has an apparent molecular weight of 530000, is inhibited 50% by alpha-amanitin at 0.04 microgram/ml and shows maximum activity on denatured DNA at 5 mM Mn2+ and 100 mM ammonium sulfate. An antibody was obtained that cross-reacts with the pure enzyme and forms a precipitin line. This antibody does not cross react with either Escherichia coli RNA polymerase or with wheat germ RNA polymerase but does react with one of the B polymerases isolated from wing tissue of the silkmoth, Antheraea pernyi.


The phenoloxidases of the ascomycete Podospora anserina. Structural differences between laccases of high and low molecular weight. In order to investigate the extent of the relationship between the three copper-containing glycoproteins, laccases I, II and III (Mr70000, 80000 and 390000 respectively) of Podospora anserina, the following experiments were carried out on laccases II and III: (a) determination of amino acid composition; (b) determination of N-terminal and C-terminal amino acid; (c) determination of sugar composition; (d) dissociation studies on native and denatured laccases and also after removal of copper from the enzymes; (e) digestion of the carbohydrate moieties with the aid of glycosylhydrolases. A comparison between the results of these experiments and data previously obtained with laccase I allows the following conclusions to be drawn. 1. Laccases II and III are not identical. 2. Neither of these low molecular weight laccases are as complete molecules subunits of the oligomeric laccase I. 3. The possibility of partial identity of amino acid sequences of laccases I and III can not be excluded. 4. Laccase II possibly consists of subunits of Mr37000 whereas laccase III does not. 5. Digestion of 50% of the carbohydrate content leads to complete loss of serological specificity (serological reaction and cross reaction). This finding is discussed with regard to the possible role of the carbohydrate moiety as antigenic determinants and thus as the reason for the immunological relationship. As a consequence, at least three independent structural genes for laccases must be assumed.


Anti-H-Y responses of H-2b mutant mice.

Anti-H-Y responses of H-2b mutant mice. Two strains of H-2b mutant mice, H-2ba and H-2bf, in which the mutational event took place at H-2K, make anti-H-Y cytotoxic T cell responses which are H-2-restricted, Db-associated and indistinguishable in target cell specificity from those of H-2b mice. Thus, alteration of the H-2K molecule affects neither the Ir gene controlling the response, nor the associative antigen. On the other hand, one H-2Db mutant strain, H-2bo, although it makes a good anti-H-Y cytotoxic response, shows target cell specificity restricted to its own Dbo antigen(s), and neither H-2b, H-2ba or H-2bf anti-H-Y cytotoxic cells kill H-2bo male target cells. Thus, the alteration of the H-2Db molecule does not affect the Ir gene of H-2b mice, but it does alter the H-2Db-associative antigen.


Differential sensitivity of memory cell subpopulations to anti-immunoglobulin and complement. Evidence is presented for the unique sensitivity of memory cells bearing surface immunoglobulin G1 (IgG1) to functional elimination with anti-immunoglobulin (anti-Ig) sera and complement (C). Treatment of cells for adoptive transfer with C and anti-gamma1, anti-kappa, or anti-Ig significantly reduces the number of plaque-forming cells (PFC) of only the IgG1 isotype found in adoptive recipients. An increase in PFC of other isotypes accompanies the decrease in IgG1 PFC; there is no net change in the total PFC response. The depletion of IgG1 PFC requires treatment of transferred cells with both specific antisera and C; antisera directed against other isotypes show no significant effects. The maintenance of the magnitude of PFC response, compensation, is discussed.


[Distribution of Pseudomonas aeruginosa in experimentally infected mice (author's transl)]. The distribution of experimentally administered Pseudomonas aeruginosa was studied in germfree CF no. 1 mice, barrier-sustained DDD mice with or without oral treatment of aminobenzyl-penicillin and conventional DDD mice. On day 1 after oral administration of 8 X 10(7) organisms, more than 10(8) of organisms/g were recovered from the feces of ex-germfree mice and the number was maintained during the experiment. On the other hand, from barrier-sustained mice with antibiotic treatment and those without the treatment, 10(4-7) and 10(3) organisms were recovered, respectively. In all of monoassociated mice and some antibiotic-treated barrier-sustained ones the organisms were recovered also from the lungs, liver, spleen and kidneys. In conventional mice, however, no organisms were recovered from the internal organs throughout the experiment, while some were detectable from the intestinal tracts. The distribution of the organisms in naturally infected mice appears to be similar to that of experimentally infected animals with antibiotics.


Interceptive activity of azastene in rhesus monkeys. Azastene is an orally effective "luteolytic" agent in rhesus monkeys. In nonpregnant monkeys it reverses the human chorionic gonadotropin-stimulated increase in progesterone production and delay in the onset of menstruation, and, in inseminated monkeys, it prevents pregnancy if given for 5 days beginning on day 24 of the menstrual cycle. The drug is also effective in terminating pregnancy if given for 5 days beginning on approximately day 26, day 50, or day 80 of gestation. Concurrent progesterone administration prevents the interceptive action of the drug. Although azastene inhibits gonadal and placental progesterone production, it has no effect on cortisol production in monkeys and is devoid of apparent hormonal activity.


[Anterior pituitary function in patients with cerebrovascular diseases with special reference to TSH secretion in response to TRH administration (author's transl)]. TSH secretion in response to TRH was studied in patients with cerebrovascular diseases in order to elucidate the influence of cerebrovascular lesions on the hypothalamus-anterior pituitary function. Blood specimens were obtained before and at intervals of 10, 20, 30, 40, 60, 90 and 120 minutes after the intravenous administration of 500 microgram of TRH. Serum TSH was measured using the RIA method. In 20 normal subjects, the serum TSH level before TRH administration was 1.0 +/- 1.4 microunits/ml (MEAN +/- SD). Following the intravenous administration of TRH, serum TSH increased and reached the maximum level of 9.0 +/- 2.3 microunits/mil at 30 minutes and returned near to the original level at 120 minutes. The response was the same for both male & female patients. In 17 patients with cerebral hemorrhage, the response of serum TSH to TRH was variable, including the types of excess, delayed or low response besides the normal response. In severe cases, cases of acute phase and male patients, a marked variability in the response was observed. In 8 patients with cerebral infarction, a low response of serum TSH to TRH was observed in all cases. There was no difference of the response with regard to severity of the diseases, duration after onset or sex difference of the patients.


Electron microscopic studies on the macronuclear development in the ciliate Chilodonella uncinata. The development of macronuclear anlage in the ciliate Chilodonella uncinata has been studied through electron microscopy. The ultrastructure of macro- and micronuclei is described for comparison. During the first stage of development, when the DNA content of the macronuclear anage increases from 2 C to 32 C, chromosomes are visible as distinct osmiophilic bodies inside the anlage. At the end of the initial polyploidization phase, the chromosomes despiralize, giving rise to long filamentous structures 25 to 50 nm in diameter. The latter show a singular banding pattern, with dense bands 12 to 25 nm thick alternating regularly with less dense interbands. Such an organization has not yet been observed in any other type of nucleus. These filamentous structures have been interpreted as highly despiralized oligotenic chromosomes. During the final stage of macronuclear development, these structures condense into thin chromatin strands and small dense granules; the number of granules increases progressively as the chromatin strands disappear. These small granules very likely fuse amongst themselves to form the chromatin granules of the vegetative macronucleus. No evidence has yet been found for a fragmentation of chromatin in this ciliate, but this problem needs further study. The old macronucleus maintains a normal ultrastructure until a late stage of development of the macronuclear anlage, becoming pycnotic only towards the very end of the latter process.


Demonstration of membrane-associated and oriented microfilaments in Amoeba proteus by means of a Schiff base/glutaraldehyde fixative. After fixation with a reaction product of glutaraldehyde and spermidine phosphate Amoeba proteus cells show a network of cortical microfilaments and oriented bundles of thick and thin filaments. The cortical filament network appears to be membrane-attached and extends beneath the whole cytoplasmic membrane surface. In the uroid region and in retracting pseudopods the cortical layer is thicker than in advancing cell regions. The filament bundles are located predominantly in the ectoplasmic tube within the cortical network. They strictly parallel the cell surface contours.


Comparative effects of two doses of glibenclamide upon metabolic rhythms in maturity-onset diabetics. Five maturity-onset diabetics have been studied during therapy with glibenclamide 2.5 mg and 5 mg by half-hourly blood sampling for twelve hours. All patients had lower mean blood glucose concentrations during therapy with 5 mg glibenclamide. There was no significant difference between serum insulin concentrations on the two doses, however, serum insulin/blood glucose ratio was higher during the larger dose of glibenclamide. Mean blood lactate, pyruvate and serum triglycerides were significantly lower, and blood glycerol, 3-hydroxybutyrate, and plasma non-esterified fatty acids were increased during therapy with 5 mg. In the individual patient the changes in blood glycerol and plasma non-esterified fatty acids were related to changes in circulating insulin concentration and did not appear to be a true extra-pancreatic effect of glibenclamide. The mechanism of any extra-pancreatic effect remains unclear.


Effect of oil-attached BCG cell-wall skeleton and thymectomy on the incidence of lung cancer and amyloidosis induced by chemical carcinogens in rabbits. Intravenous injection of oil-attached BCG cell-wall skeleton showed potent preventive activity for induction of lung cancer by the intrabronchial instillation of chemical carcinogens (3-methylcholanthrene, 4-nitroquinoline 1-oxide) in rabbits. On the other hand, the thymectomized rabbits developed lung cancer by instillation of chemical carcinogens at an 80% incidence, compared to 47.2% in the controls. There were no differences in the appearance of amyloidosis between thymectomized and BCG cell-wall skeleton-treated rabbits and the controls.


Adenosine deaminase and purine nucleoside phosphorylase activities in lymphocytes from patients with lung cancer. The activity of adenosine deaminase (EC 3.5.4.4.) was significantly lower in lymphocytes from patients with lung cancer than in those from healthy subjects, whereas the activity of purine nucleoside phosphorylase (EC 2.4.2.1.) was significantly higher in lymphocytes from the patients than in those from normal controls. When the one activity was plotted against the other, the plots for patients with lung cancer were all outside the frame formed by the lower and higher limits of the standard deviation of the mean of normal activities of the two enzymes. The ratio of adenosine deaminase activity to purine nucleoside phosphorylase activity was lower in patients with lung cancer than in controls. The possible effect of this ratio on the function of lymphocytes was briefly discussed. These enzyme activities were suggested to be useful measures of the immune responsiveness of patients with lung cancer.


Enhancing effect of coenzyme, Q10 on immunorestoration with Mycobacterium bovis BCG in tumor-bearing mice. Effect of the additional treatment with coenzyme Q10 on immunorestoration with Mycobacterium bovis BCG in tumor-bearing mice was investigated. Cell-mediated cytotoxicity in tumor-bearing mice against alloantigenic tumor cells was determined by 51Cr release assay using spleen cells of C57BL/6N mice which had been inoculated subcutaneously with syngeneic melanoma-B16 and immunized intraperitoneally with alloantigenic mastocytoma P815-X2 cells. The cell-mediated cytotoxicity against mastocytoma P815-X2 cells was gradually depressed with the growth of melanoma-B16. The depressed, cell-mediated cytotoxicity in tumor-bearing mice recovered slightly by the treatment with BCG. The recovery effect of BCG on the depressed, cell-mediated cytotoxicity was significantly enhanced by the additional treatment with coenzyme Q10. Coenzyme Q10 did not have an apparent effect on the depressed, cell-mediated cytotoxicity in tumor-bearing mice. These results show that coenzyme Q10 enhances the immunorestoration with BCG in tumor-bearing mice.


Carcinogenicity of 4-nitrosoquinoline 1-oxide and its possible role in carcinogenesis by 4-nitroquinoline 1-oxide. 4-Nitrosoquinoline 1-oxide induced malignant tumors at the subcutaneous site of injection in mice. It affected Escherichia coli to induce the so-called UV-type lesion in cellular DNA. DNA base-quinoline adducts produced by the treatment of mammalian cellular DNA with this carcinogen were proved to be identical with those obtained by the action of 4-nitroquinoline 1-oxide. Although this carcinogen was reactive enough to modify DNA chemically by itself, a different DNA modification took place in a chemical process from thos obtained in the in vivo process.


Difference in the induction of osteosarcoma in rabbit bone with single administration of three kinds of chemical carcinogens. A single intramedullary administration of each dose (15 approximately 20 mg) of 4-nitroquinoline 1-oxide, 3-methylcholanthrene, or 7,12-dimethylbenz[alpha]anthracene was applied to the mandible, diaphysis, or distal metaphysis of the femur of rabbits. The highest incidence in production of osteosarcoma was obtained from the group in which 4-nitroquinoline 1-oxide was applied to the distal metaphysis (75%, including one case of chondrosarcoma). Tumors hardly appeared in any of the groups when given 3-methylcholanthrene or 7,12-dimethylbenz[alpha]anthracene. Histologically, three kinds of entities were recognized from the quantitative difference of the reactive tissues which appeared around carcinogens. It is estimated that the condition of entity III induces the highest incidence of osteosarcoma if chemical carcinogens are given into the bone marrow of experimental animals.


Effects of graded doses of alcohol upon esophageal motor function. Twenty-nine normal volunteers were subjected to manometric studies before and after intraesophageal infusion of 100-proof alcohol at doses of 80, 120, 160, and 200 ml, corresponding to 31.6, 47.4, 63.2, and 79.0 g, respectively, administered over a 15-min interval on separate days. Corresponding mean serum concentrations of alcohol were 58.9 +/- 2.8, 77.0 +/- 4.8, 97.0 +/- 13.9, and 117.3 +/- 5.0 mg per dl, respectively. Both esophageal and lower esophageal sphincter function were unaffected at serum concentrations of alcohol less than 70 mg per dl. In contrast, when the serum alcohol concentration was 117 mg per dl, esophageal and lower esophageal sphincter function was altered significantly. The amplitudes of esophageal peristaltic waves were probably decreased in the proximal and distal segments. The maximal lower esophageal sphincter pressure response to pentagastrin was reduced from 35.4 +/- 4.6 to 17.3 +/- 6.1 mm Hg (P less than 0.01). In addition, the lower esophageal sphincter pressure response to a protein meal was inhibited significantly. Neither propagation velocities or esophageal contractions nor resting lower esophageal sphincter pressures were changed by the administration of alcohol at any of the doses tested.


Determination of prostaglandin synthetase activity in rectal biopsy material and its significance in colonic disease. A method is described for determining prostaglandin synthetase activity in milligram amounts of tissue. The procedure is based on the conversion of 14C-arachidonic acid to prostaglandin E2 and F2alpha-like substances. High levels of prostaglandin synthetase activity occurred in the inflamed mucosa of patients with ulcerative colitis and fell during successful drug therapy, but it is not yet known whether the cause of the inflammation first involves increased PG synthetase activity, or whether inflammation caused increase of PG synthetase.


Disinfection of upper gastrointestinal fibreoptic endoscopy equipment: an evaluation of a cetrimide chlorhexidine solution and glutaraldehyde. There is little information available on the bacteriological contamination of upper gastrointestinal fibreoptic endoscopes during routine use and the effects of 'disinfecting solutions'. A bacteriological evaluation was therefore made of cleaning an endoscope and its ancillary equipment with (1) water, (2) an aqueous solution of 1% cetrimide with 0.1% chlorhexidine, and (3) activated aqueous 2% glutaraldehyde. All equipment, but particularly the endoscope itself, was found to be heavily contaminated after use with a wide variety of organisms of which 53% were Gram positive. Cleaning the endoscope and ancillary equipment with water and the cetrimide/chlorhexidine solution alone or in combination was inadequate to produce disinfection but immersion in glutaraldehyde for two minutes consistently produced sterile cultures with our sampling technique. A rapid and simple method for disinfection of endoscopic equipment is therefore recommended and we think this is especially suitable for busy endoscopy units.


Role of bilirubin overproduction in revealing Gilbert's syndrome: is dyserythropoiesis an important factor? Gilbert's syndrome was diagnosed in 37 patients with unconjugated hyperbilirubinaemia without overt haemolysis or structural liver abnormality, who had a marked reduction in hepatic bilirubin UDP-glucuronosyltransferase activity (B-GTA) (as compared with that of 23 normal subjects). No significant correlation existed in these patients between serum bilirubin level and the values of B-GTA, thus suggesting that factors other than a low B-GTA must influence the degree of hyperbilirubinaemia in Gilbert's syndrome. Studies of 51Cr erythrocyte survival and 59Fe kinetics in 10 unselected patients demonstrated slight haemolysis in eight, whereas mild ineffective erythropoiesis was suggested in all from a low 24-hour incorporation of radioactive iron into circulating red cells. This overproduction of bilirubin resulting from mild haemolysis and perhaps dyserythropoiesis might reflect only an extreme degree of the normal situation. It certainly contributes to the hyperbilirubinaemia of Gilbert's syndrome and may play a major role in the manifestation of this condition.


Primary repair of flexor tendons in the hand without immobilisation-preliminary report. A new approach to the problem of flexor tendon repair within the fibro-osseous canal is presented. Using a technique of bevelling the tendon ends and suturing with a fine suture material, under magnificaiton, a sufficiently strong junction is obtained, which enables immediate active mobilisation without strangulation of the blood supply. The junction can resist gap formation up to tensions of 4 Kg. It is postulated that under these conditions tendon nutrition is minimally interfered with, adhesions do not form, and the tendon heals by its own intrinsic healing ability.


The diet and feeding behaviour of Cercopithecus aethiops tantalus. The feeding behaviour of representative groups of tantalus monkeys (Cercopithecus aethiops tantalus) was observed during 1974-1975 in three habitats in Cameroon. Observations took place during the wet and dry seasons in sahelian savannah, guinea savannah and farmed forest. The tantalus monkeys were similar to other races of C.aethiops that have been studied in that they were omnivorous and lacked any narrow specialisations of either diet or feeding techniques. The dietary range was similar in all three habitats, although considerable seasonal variation occurred. About one third of all feeding took place on the ground.


The nature of a primary feeding habit in different age-sex classes of yellow baboons (Papio cynocephalus). Two feeding habits of 30 baboons selected equally from five age-sex classes were studied at Mikumi National Park, Tanzania. The finding, gathering, and preparing of sedge corms and of seeds of tamarind fruit were described in detail. Adults obtained these foods faster than younger animals, although even small juveniles and weaned infants were efficient in gathering and preparing them. While gathering sedges or tamarinds, adult males sat in one place longer than others and obtained more food per sitting. Adults ate more pieces of food per minute than juveniles, but adult rates of eating did not differ by sex. The adult rate of food intake was inconsistent with the assumption of different food requirements for males and females of a species with pronounced sexual dimorphism.


[Conservative treatment of peripheral arterial occlusive diseases]. The therapeutical effect and tolerance of Defluina was studied in a selective open trial of 24 patients suffering from peripheral arterial diseases in Fontaine's stage I and II. All patients were treated with Defluina, 30 drops three times a day during a period of six weeks. From the statistically significant improvement of the different control parameters it can be concluded that peripheral arterial circulation has increased considerably in the course of the Defluina treatment. The preparation was always well tolerated, no side-effects were observed.


[Molsidomin and intraocular pressure. No effect in healthy subjects and in glaucoma patients]. Molsidomine is a new agent in coronary therapy. It does not influence the intra-ocular pressure in healthy volunteers or in glaucoma patients. This was tested in double-blind studies in short trials and in a long term study of 3 months. The outflow facility or the visual fields were also unchanged. These results correspond closely to those obtained previously with other coronary therapeutics. Molsidomine is not dangerous for glaucoma patients.


[Erythrocyte carbonic anhydrases in patients with hyperthyroidism (author's transl)]. Human erythrocyte carbonic anhydrase B (CA-B) and C (CA-C) were measured by a specific and immunological method and erythrocyte 2,3-diphosphoglycerate (2,3-DPG) concentrations were measured using a kinetical method. The levels of CA-B and CA-C were determined in normal subjects, patients with hyperthyroidism and patients with hypothyroidism, using the method of single radial immunodiffusion. A decrease of CA-B levels and an elevation of 2,3-DPG concentrations were noted in hyperthyroidism. The CA-C levels appeared to be essentially unchanged. Closely negative correlations were observed between CA-B levels and T3 resin sponge uptake T4, protein bound iodine levels, or 2,3-DPG concentrations. Simultaneously, the specific activity of CA-B and CA-C was determined using the immunoadsorbent method. The specific activity of CA-B elevated in hyperthyroidism two times higher than that of normal subjects. The levels of rabbit erythrocyte CA-B, CA-C and 2,3-DPG were measured in experimental hyperthyroidism by the methods employed for human erythrocytes. Thyroxin and 3,5,3'-triiodothyronine were administered orally for 30 days. A significant decrease in CA-B levels was observed after 30 days, although CA-C levels did not change significantly. These findings suggest that the steady state levels of CA-B is affected more readily than that of CA-C by thyroid hormone. An elevation of the specific activity of CA-B or the concentrations of 2,3-DPG was noted as seen in human hyperthyroidism. The effect of thyroid hormone on the biosynthesis of CA-B and CA-C was studied in vitro using rabbit reticulocyte lysates. The biosynthesis of CA-B was inhibited by the addition of thyroxin in this system, although no such an inhibitory effect was observed in the biosynthesis of CA-C. Th e clinical or pathological significances were discussed in relation to the changes in these isozymes and 2,3-DPG in red cells.


[Erythrocyte carbonic anhydrase isozymes in patients with iron deficiency anemia and their clinical significances (author's transl)]. The levels of erythrocyte carbonic anhydrase isozymes (CA-B and CA-C) were determined by a quantitative immunochemical technique in order to elucidate their clinical significances in patients with iron deficiency anemia. The specific antibodies against rabbit CA-B and CA-C were also obtained, and the levels of rabbit erythrocyte carbonic anhydrase B and C isozymes were assayed immunochemically in experimental hemolytic and bleeding anemia. Simultaneously carbonic anhydrase isozymes were synthesized in vitro using rabbit reticulocyte-lysate. 1 The levels of erythrocyte CA-B per gram hemoglobin, CA-B per ml erythrocyte and CA-C per gram hemoglobin all increased but CA-C per ml erythrocyte did not increased in iron deficiency anemia. Erythrocyte CA-B and CA-C expressed per gram hemoglobin were inversely correlated to hemoglobin levels. 2 In experimental bleeding anemia and hemolytic anemia of rabbits, erythrocyte CA-B increased with the progression of anemia and with increase in reticulocyte count. However the levels of CA-C did not increased with progression of anemia. 3 In experimental bleeding anemia, it was found that the level of erythrocyte CA-B was correlated to the esterase activity and erythrocyte zinc content. 4 Most of the rabbit erythrocyte CA-B was distributed in younger erythrocytes. 5 It was confirmed that CA isozymes were synthesized in rabbit reticulocyte-lysate. The content of CA-B synthesized in lysates was much higher than that of CA-C. 6 In iron deficiency anemia, the increase of erythrocyte CA is speculated to be compensatory mechanism, which brought about the transportation of CO2 and right shifts of O2 dissociation curve.


Prenatal development of acid beta-glycosidases in the rat liver, effect of triiodothyronine or cortisone administered to pregnant rats. We have found that acid beta-galactosidase, beta-glucuronidase and N-acetyl-beta-glucosaminidase in the rat fetal liver increase during the last week of pregnancy. These enzyme activities were influenced by treatment of pregnant rats (daily from day 16) with L-tri-iodothyronine (20 or 50 microgram/100 gm b.w.) or cortisone acetate (10 or 50 mg/100 gm b.w.) as studied in their fetuses obtained on day 22 by caesarian section.


Inhalation anthrax in a home craftsman.

Inhalation anthrax in a home craftsman. Inhalation anthrax with complicating subarachnoid hemorrhage due to simultaneous infection with two capsular biotypes of Bacillus anthracis of different virulence for the mouse is reported. The patient, a home craftsman, acquired his infection from imported animal-origin yarn.


Adjuvant effect of a peptidoglycan attached covalently to a synthetic antigen provoking anti-phage antibodies. The synthetic antigen denoted P2-A--L, comprising the fragment P2 of the coat protein of MS-2 coliphage attached to multichain poly-DL-alanine, served for the immunization of guinea-pigs. Immunization was carried out either in phosphate buffered saline (PBS) or in Freund's incomplete adjuvant (FIA) in the presence or absence of a small molecular weight peptidoglycan prepared from Bacillus megaterium, which was checked for its adjuvant effect. The various antisera were assessed by their capacity to neutralize MS-2 bacteriophage viability. When injected in PBS or FIA, P2-A--L did not elicit any measurable anti-phage activity. Addition of the peptidoglycan by simple mixing did not bring about a significant increase in antibody production. However, when the peptidoglycan was chemically linked to the P2-A--L conjugate, it had a marked adjuvant effect when the material was administered in FIA, almost identical to the extent of the effect of Freund's complete adjuvant.


[Clinical study on the concentration of free amino acids in plasma and urine during postaggression metabolism. I]. We studied the changes in the concentration pattern of free plasma amino acids in 30 surgical patients during standardized parenteral nutrition and exclusive water and electrolyte supply. Distinct reproducible differences were found in comparison to normal subjects. The concentration of branched-chain amino acids continuously increases during the post-operative period. The decrease of alanine is closely related. Phenylalanine also increases continuously due to insufficient transformation in the liver and increased release from the muscles. During post-aggression metabolism, cystine is probably not sufficiently formed from methionine and should be substituted. It may be that tyrosine is also essential for patients in the post-operative post-traumatic period. The glycine concentration of some amino acid mixtures is regarded as too high; the glycine content can be reduced in favour of serine supply.


[Side effects of parenterally administered carbohydrates during the postoperative phase]. The frequency of side effects occurring when administering carbohydrates in postoperative parenteral nutrition has been examined in 72 adult intensive-care patients. By keeping the dosage of 0.5 carbohydrate/kg body-weight/h at a relation of 2:1:1 (G:F:X), no significant deviations from the usual pattern were observed.


[Development of a pediatric amino-acid solution for premature and newborn infants following pharmacokinetic principles]. The blood level kinetics of an amino-acid solution following short-term infusions was measured in 25 premature and 5 hypotrophic newborns. From the calculated transfer rates it was possible to compose an amino-acid solution capable of avoiding imbalances. Clinical tests showed that the solution was tolerated well. Balance studies in 10 premature infants demonstrated a positive nitrogen balance from the first day of life on with a 55% retention of administered amino-acid nitrogen.


[Various metabolic effects in total parenteral feeding using a carbohydrate combination and amino-acid solution in the pre- and postoperative phase]. Ten metabolically healthy patients who had undergone gastric resection were fed intravenously from the preoperative to the 5th postoperative day. After the 24-hour preoperative infusion period the concentration of cholesterol, free fatty acids and alkaline phosphatase dropped significantly. So did the concentration of uric acid. During the postoperative period cholesterol and uric acid continued to fall. The concentration of free fatty acids remained low all the postoperative days. From the second postoperative day on the triglycerides rose continuously. Serum glutamic oxalacetic transaminase and serum lactic dehydrogenase reached their highest level on the first postoperative day. On the same day the alkaline phosphatase dropped to the lowest level. Five patients had a rise of total bilirubin up to 1.1 mg/100 at the end of the infusion period.


[Behavior of prealbumin, retinol-binding protein, transferrin and haptoglobin in the postoperative and post-traumatic phase]. The plasma proteins pre-albumin, retinol-binding protein, transferrin, and haptoglobin were determined in 3 different groups of surgical patients (8 patients after inguinal or incisional hernia operation, 12 patients with peritonitis due to insufficient anastomoses, 17 patients with skull-brain injuries) according to the radial immune diffusion method. The decrease in concentration of nutrition-dependent plasma proteins, pre-albumin, retinol-binding protein and transferrin, varied depending on the extent of postoperative or posttraumatic catabolism and respective nutritional regimen. The increase in concentration of the acute phase protein haptoglobin was not significantly dependent on the severity of trauma. There was, however, a further haptoglobin increase of significance due to postoperative peritonitis complication.


Acquired cellular resistance, delayed hypersensitivity, and altered macrophage migration in Listeria monocytogenes-infected guinea pigs. A Listeria monocytogenes infection in guinea pigs was used to study the interrelationship between antigen-induced macrophage migration inhibition, delayed-type hypersensitivity, and acquired cellular resistance. Early after infection (at 2 and 7 days), very significant enhancement of macrophage migration was observed. Migration inhibition was detected beginning on day 14 and was uniformly observed only on day 21 of the infection, after which a shift again to enhancement was seen. The early detection (by day 2) of migration enhancement suggested that this assay may be more sensitive than assessment of delayed type hypersensitivity in vivo, which in this system was first detectable only on day 4. Acquired cellular resistance, as measured by enhanced survival following a high dose challenge with Listeria, was present from day 7 after infection until at least day 60. By splenic clearance studies, however, acquired cellular resistance was present only until day 14 after infection, suggesting that in this system splenic clearance was not a very reliable criterion for measuring acquired cellular resistance.


Suppression of lymphocyte transformation by plasma from owl monkeys acutely infected with Plasmodium falciparum. Plasma collected from owl monkeys during the acute phase of Plasmodium falciparum infection was shown to adversely affect several in vitro responses which are considered to be correlates of cell-mediated immune functions of normal monkeys. In the presence of acute-phase plasma, response of normal monkey peripheral blood lymphocytes to stimulation with phytohemagglutinin, concanavalin A, and pokeweed mitogen was severely reduced, as was the ability of peripheral blood lymphocytes to respond to allogenic and xenogenic histocompatible antigens. The transformation response of peripheral blood lymphocytes from normal humans to phytohemagglutinin and concanavalin A was also suppressed. Since acute-phase plasma was not cytotoxic for peripheral blood lymphocytes, decreased responsiveness did not result from cell destruction. Acute-phase plasma appears to block initial steps in lymphocyte transformation.


Antigenicity of purified glutaraldehyde-treated cholera toxoid administered orally. The antigenicity of orally administered glutaraldehyde-treated cholera toxoid was investigated in healthy volunteers. Fourteen volunteers ingested two or three 2-mg doses of toxoid with saline, with the doses spaced at 28-day intervals. Thirteen other volunteers received comparable toxoid doses with NaHCO3 and milk to neutralize gastric acid. Increments in circulating antitoxin levels were used to assay the antigenicity of oral toxoid. Antitoxin was measured by adrenal cell, rabbit skin permeability factor, and passive hemagglutination assays in sera collected on days 0, 28, 35, 56, 63, and 84 after primary immunization. Adrenal cell and rabbit skin assays exhibited identical sensitivity in detecting antitoxin rises in the 27 vaccinees (19/27) and were significantly more sensitive than passive hemagglutination (11/27) (P less than 0.03). Volunteers who ingested toxoid with NaHCO3 and milk had a higher rate of seroconversion (77%) than those who received toxoid with saline (64%); they also had earlier rises in antitoxin titer and consistently higher geometric mean titers on all days tested. These studies demonstrate that purified cholera toxoid is antigenic in humans after oral administration. The possible role of oral toxoid in enhancing the protective effect of killed whole-cell vaccines can now be investigated.


Experimental production of respiratory tract disease in cebus monkeys after intratracheal or intranasal infection with influenza A/Victoria/3/75 or influenza A/New Jersey/76 virus. A total of 28 cebus monkeys were inoculated intratracheally or intranasally with 10(6) 50% tissue culture infective doses of A/New Jersey/76 virus or 10(7) 50% tissue culture infective doses of A/Victoria/75 virus, and 8 additional monkeys received sterile allantoic fluid. Each of the animals became infected as evidenced by a serological response and/or shedding of the virus. Of the 10 animals inoculated intratracheally with A/Victoria/75 virus, 8 developed a systemic illness, and pulmonary infiltration was detected by X-ray in 7 of the 8.  Administration of A/New Jersey/76 virus intratracheally to 10 monkeys produced a mild systemic illness in 2 animals and an upper respiratory tract illness in 6, but no illness developed in the remaining 2 monkeys; none of the animals developed X-ray evidence of lower respiratory tract disease. Intranasal administration of either virus failed to induce any illness or produced, at most, mild illness confined to the upper respiratory tract. These studies demonstrate that cebus monkeys are susceptible to respiratory tract infection with influenza A viruses and that the development of pulmonary disease is reflected in the appearance of easily recognizable radiological changes.


Murine macrophage-lymphocyte interactions: scanning electron microscopic study. Light and scanning electron microscopic observations revealed murine macrophage-lymphocyte interactions involving the initial contact of peritoneal, spleen, or thymus lymphocytes with peritoneal macrophage processes or microprocesses followed by clustering of lymphocytes over the central nuclear area of the macrophages. Lymphocyte-lymphocyte clustering was not observed in the absence of macrophages. Attachment and subsequent clustering appeared not to require the presence of serum or antigen; the attachment of allogeneic or xenogeneic lymphocytes was comparable to that seen in the syngeneic system, but central clustering of these lymphocytes failed to occur. No attachment or clustering was observed when thymic lymphocytes were cultured with thymus derived fibroblasts rather than with peritoneal macrophages. Lymphocyte attachment to immune, antigen-activated, syngeneic macrophages occurred more rapidly than that to normal unstimulated syngeneic macrophages; however, lymphocytes attached to the "activated" macrophages appeared to be killed by a nonphagocytic mechanism. A similar increase in the rate of lymphocyte attachment to macrophages occurred in the presence of migration inhibitory factor. Subsequent lymphocyte clustering on macrophages was observed in the migration inhibitory factor-stimulated cultures. In addition, lymphocyte-macrophage interactions similar to those in vitro were observed to occur in vivo on intraperitoneally implanted cover slips.


Studies on gonococcus infection. XIV. Cell wall protein differences among color/opacity colony variants of Neisseria gonorrhoeae. Gonococci from colonies exhibiting optical opacity and dark coloration have surface proteins that are not visualized in isogenic transparent, light-colored colony forms. These "colony opacity-associated proteins" have apparent molecular weights varying from 24,000 to 30,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate; their apparent molecular weights are independent of that for their major outer membrane protein. The opacity-associated proteins are more susceptible to hydrolysis by trypsin than is the major outer membrane protein, but gonococci possessing the opacity-associated protein(s) also show enhanced susceptibility of their major outer membrane proteins to the action of trypsin. These conclusions were reached by comparing the electrophoretic patterns of whole-cell lysates from both "laboratory strains" and several recent clinical isolates of Neisseria gonorrhoeae.


Strain-dependent cytotoxic effects of endotoxin for mouse peritoneal macrophages. The cytotoxic effects of bacterial lipopolysaccharides (LPS) on mouse leukocytes have been examined in vivo and in vitro. Intraperitoneal injection of LPS into C57BL/6 mice greatly reduced the recovery of mononuclear cells; LPS was cytotoxic for macrophages, but had a mitogenic effect on lymphocytes. Similar effects of LPS on peritoneal leukocytes were observed in vitro. When monolayers of adherent peritoneal cells were studied in vitro, cytotoxicity was also observed, suggesting that the effect of LPS on macrophages is direct and does not require participation by lymphocytes. Entirely different results were obtained when peritoneal macrophages from LPS-resistant C3H/HeJ mice were studied. LPS failed to activate lymphocytes and was not cytotoxic for macrophages in vitro or in vivo. The effect of LPS on polymorphonuclear leukocytes appeared to be the same in all mouse stains studied. Lipid A was shown to be the most biologically active portion of the LPS molecule. Whereas polysaccharide-deficient endotoxins extracted from rough mutants of Salmonella typhimurium were cytotoxic for macrophages in vitro, polysaccharides that lacked esterified fatty acids did not exhibit this activity. Since LPS may mediate its effects through affinity for mammalian cell membranes, the cellular unresponsiveness of C3H/H3J mice to LPS may reflect an inability of cells from LPS-resistant strains to interact with LPS at the membrane level.


Plaque-forming cells in mice after experimental infection with Brucella abortus. Cells producing antibody to brucella lipopolysaccharide were detected in spleens of mice infected with Brucella abortus 19 by a hemolytic plaque assay. The appearance of immunoglobulin M-producing cells preceded humoral antibodies. The primary plaques were observed 5 days after inoculation, and they were still present by day 70.


Role of antibody and complement in opsonization of group B streptococci. A requirement for the classic complement pathway in opsonization of group B streptococci was observed by using both a chemiluminescence and a radiolabeled bacterial uptake technique. The classic pathway increased levels of opsonization for types Ia and II stock and wild strains and for some type III wild strains. In contrast, other type III wild strains and the type III stock strain had accelerated kinetics of uptake in the presence of an intact classic pathway, but the level of opsonization was unchanged from that with antibody alone. We could not demonstrate a significant role for the alternative pathway in opsonizing stock or wild strains of group B streptococci. Futhermore, electrophoretic and complement consumption analysis by hemolytic titration failed to reveal alternative pathway activation by the majority of strains of this group. Therapy aimed at supplying opsonins for these organisms will require the presence of type-specific antibody.


Inhibition of mycoplasma-induced lymphocyte activation by sodium aurothiomalate. Sodium aurothiomalate, at concentrations of 10 to 150 microgram/ml of culture, inhibited rat lymphocyte stimulation by Mycoplasma pulmonis mitogen in a dose-dependent manner.


Pseudomonas ribosomal vaccines: preparation, properties, and immunogenicity. The preparation, properties, and immunogenicity of ribosomal vaccines from Pseudomonas aeruginosa are described. These preparations, containing protein and RNA, were tested for immunogenicity by active immunization of mice and subsequent challenge with homologous, live bacteria. The results demonstrated that vaccines prepared from a majority of serotypes used were immunogenic, i.e., afforded 60 to 100% mouse protection against a challenge inoculum containing 8 to 50 50% lethal doses. In some cases vaccine doses as low as 1 microgram of RNA provided 100% mouse protection. Molecular sieve chromatography of a highly immunogenic ribosomal preparation on Sepharose 4B demonstrated the presence of two molecular weight fractions: (i) peak A, an excluded peak (thus having a molecular weight of at least 2 times 10(7)), and (ii) peak B, considerably retarded, with an elution position corresponding to a molecular weight of about 2.2 X 10(6), approximating that of typical 70S ribosomes. Both peaks A and B were immunogenic; however, the immunogenicity of peak A was greater (i.e., a smaller immunizing dose was required) than that of peak B. Peak A was shown to contain components of lipopolysaccharide in addition to protein and RNA (which comprised 80% of the dry weight of peak A). On the other hand, peak B was shown to be free of lipopolysaccharide, and 100% of its dry weight consisted of protein and RNA.


Comparative ultrastructural study on the cell envelopes of Rickettsia prowazekii, Rickettsia rickettsii, and Rickettsia tsutsugamushi. Rickettsia tsutsugamushi differs from other rickettsiae in its cell envelope organization. The differences were made evident through a comparative study of the outer envelope of R. tsutsugamushi, R. prowazekii, and R. rickettsii by electron microscopy.


Surface proteins of typhus and spotted fever group rickettsiae. Six proteins, previously established as major constituents of intact organisms, were identified in cell envelopes obtained from intrinsically radiolabeled Rickettsia prowazekii. Extrinsic radioiodination of intact organisms conducted at 0.5 micronM iodide indicated that protein 4 was the most peripheral, although protein 1 also had reactive groups exposed on the surface of the organisms. A 10-fold increase in iodide concentration resulted in labeling of protein 2, and at 50 micronM iodide, all six major proteins were radiolabeled. Similar selective labeling was not achieved with R. conorii. Analysis of both typhus and spotted fever group organisms radiolabeled with galactose suggested that carbohydrate was associated with proteins 1, 3, and 4. Typhus soluble antigen included all major proteins except protein 2, which remained attached to particulate rickettsiae after ether extraction. Protein 4 appeared to be prominent in the surface topography of R. prowazekii, was a component of soluble antigen and may have an important role in rickettsiae-host interactions.


Role of nonagglutinating antibody in the protracted immunity of vaccinated mice to Pseudomonas aeruginosa infection. Effective immunization against infection with Pseudomonas aeruginosa is difficult to evaluate because agglutinin levels decline rapidly. Because fractionation of hyperimmune sera often yields more specific antibody than can be accounted for by direct agglutination tests, an immunoglobulin-specific assay based on antiglobulin augmentation was used to characterize antibody responses of C3H/HeJ mice vaccinated with P. aeruginosa type 2 lipopolysaccharide. Nonagglutinating antibodies, initially detected at 2 weeks post-primary vaccination, were predominantly immunoglobulin G after 5 weeks, and they remained elevated at levels usually 32-fold higher than the direct titer throughout the 4-month study period. The sequential production of immunoglobulin M, then immunoglobulin G, followed that found in orthodox immunological responses. Sera that contained nonagglutinating antibodies but not direct agglutinins (14 to 16 weeks) enhanced phagocytosis of P. aeruginosa type 2 by macrophages from unimmunized mice and passively immunized mice against lethal challenge doses; bactericidal activity of these sera was not demonstrated in the presence or absence of complement. When challenged with 1, 10, and 100 50% lethal doses at 16 weeks, survival rates of actively immunized mice were significantly higher than those of unvaccinated mice (P < 0.001). Thus, at a time when no direct agglutinins were detectable, the augmented system detected nonagglutinating antibodies that could confer protracted resistance in vaccinated mice to pseudomonas infection.


Effects of iron and culture filtrates on killing of Neisseria gonorrhoeae by normal human serum. Neisseria gonorrhoeae GC9, both colony types T2 and T4, were killed by normal human serum, although populations of colony type T4 were more susceptible. Ferric ammonium citrate prevented the killing of populations of both T2 and T4 colony types. Other iron compounds tested showed no protective effect, nor did ammonium citrate or the divalent cations magnesium or calcium. A filtrate from cultures of an N. gonorrhoeae strain grown in a liquid defined medium showed a similar protective effect in the serum assay. The filtrate appeared to chelate iron, as measured by decreased ability of iron-free transferin to bind iron in the presence of the filtrate. However, the two effects did not appear to be related. Neither ferric ammonium citrate nor the culture filtrate sufficiently inactivated complement to account for protection.


Comparison of antigens of pneumonia-associated mycoplasma species by gel diffusion. Comparison of fluorocarbon-extracted antigens of six mycoplasma species by double immunodiffusion and counterimmunodiffusion techniques revealed a close reciprocal relationship among Mycoplasma dispar, M. ovipneumoniae, and M. hyopneumoniae. A lesser degree of cross-reaction was also demonstrated between these three species and M. hyorhinis and M. bovoculi. The interrelationships were more clearly demonstrated by double immunodiffusion than by counterimmunodiffusion.


Antibody response to antigens distinct from smooth lipopolysaccharide complex in Brucella infection. The smooth lipopolysaccharide complex of the outer surface of smooth Brucella abortus cells is believed to be the antigenic component involved in serological tests routinely used for the diagnosis of brucellosis. Sera from cattle vaccinated or infected with B. abortus generally contain antibody directed toward the smooth lipopolysaccharide complex. The brucella organism contains a large number of other antigenically distinct components. The biological significance of some of these antigens has been demonstrated by showing that sera from infected cattle have precipitins to these components. These sera revealed up to seven distinct lines in immunoelectrophoresis with a protein-rich antigen mixture prepared from rough strain B. abortus 45/20, whereas sera from strain 19-vaccinated cattle did not reveal these lines at 4 or more months after vaccination. Monospecific antisera were prepared against six antigens in this mixture, and the purification of two of them by antibody affinity chromatography is described.


Three anorectic drugs: similar structures but different effects on brain and behavior. Neurochemical probes are starting to tease apart several neurotransmitter systems that are involved in suppressing feeding. Instead of a classical medial hypothalamic lesion, neurotoxins or neurochemical inhibitors can be used to produced animal models of the obese. These models make it clear that amphetamine and fenfluramine do not act in the same way. In addition to actions on brain synapses, these drugs may act directly or indirectly on the receptors that monitor energy availability. Phenylpropanolamine (PPA) apparently has anorectic action within the lateral hypothalamus, but this could be due to a number of actions such as: (a) an adrenergic synaptic effect, (b) a direct receptor effect, (c) an indirect effect on glucoreceptors caused by increasing glucose utilization, or (d) some combination of these. In addition, these drugs taken systemically could activate similar mechanisms peripherally, thereby prompting a barrage of cues to and from the brain. Throughout this discussion it has been emphasized that the therapist who uses any of these drugs, needs to be aware of the possible changes in mood, behavior and physiological signs that are thought to occur in animals. Eventually the therapist will be able to take advantage of these side-effects to coordinate the drug's style with that of the patient.


Improving the cost-effectiveness of obesity programs: three basic strategies for reducing the cost per pound. Empirical support is provided for three basic strategies of improving the cost-effectiveness of treatments for obesity. The importance of analyzing both the effectiveness and cost of different program components when attempting to improve cost-effectiveness is illustrated in the first two studies. First, the strength of relationships between changes in different eating behaviors and changes in obesity was assessed for a small sample of obese individuals. Change in only one of eight behaviors investigated was significantly and strongly correlated with change in obesity. Data also were collected on the subjective costs and benefits of changing 36 eating behaviors that are commonly included in treatments for obesity. Some eating behaviors were found to differ greatly in subjective cost and perceived benefit, as measured by clients' ratings of component difficulty and component usefulness. A final, quasi-experimental study demonstrated the importance of developing cost-effective systems for translating principles of treatment for obesity into actual treatment. The impact on cost-effectiveness in mind is illustrated in a comparison of dollars paid by clients in relation to percent reduction in obesity for two weight reduction programs. The studies are discussed as highlighting the importance of measuring the cost and effectiveness of alternative delivery systems for, and different components of, treatments for obesity.


Mortality of monkeys after exposure to fission neutrons and the effect of autologous bone marrow transplantation. In order to assess the risk of exposure to ionizing radiation in man, and to evaluate the results of therapeutic measures, the mortality of rhesus monkeys irradiated with X-rays and fission neutrons and the effect of autologous bone marrow transplantation have been investigated. The LD50/30d values for X- and neutron-irradiated monkeys amount to 525 and 260 rad respectively, resulting in an r.b.e. of approximately 2 for the occurrence of the bone marrow syndrome. Protection of the animals by autologous bone marrow transplantation was observed up to doses of 860 rad of X-rays and 440 rad of fission neutrons. After both fission-neutron irradiation and X-irradiation in the lowest range of lethal doses, the bone marrow syndrome was found to occur without the concurrent incidence of the intestinal syndrome. The studies indicate that, for humans accidentally exposed to what would otherwise be lethal doses of fast neutrons, bone marrow transplantation may be beneficial.


Hemolytic Streptococci in supramammary lymph nodes from healthy pigs from the Peruvian jungle. Hemolytic streptococci were isolated from 53 percent (18) supramammary lymph nodes from healthy adult pigs raised at the Peruvian jungle. The isolates were identified as Streptococcus zooepidemicus, Streptococcus infrequens, Streptococcus faecalis and Groups P and S. The possible significance of the findings are discussed.


Experimental pyelonephritis in the monkey. VI. Infection of infants versus adults. Pyelonephritis followed ureteral inoculation of bacteria in both infant and adult monkeys. Because of the frequency of reflux in infants this was done by bladder inoculation, although ureteral inoculation was necessary in adults. The longer duration of bacteriuria in infants may be attributable to a relative immunodeficiency.


Construction of hemibladders and cutaneous vesicostomies for bilateral renal function studies. A new technique in Macaca arctoides. A surgical technique for the construction of hemibladders and permanent cutaneous vesicostomies in Macaca arctoides is described. Serial studies of renal function were performed in sedated monkeys before and 1 or more months after division of the bladder. The results of the studies confirm our previous observation that renal function in monkeys is comparable to that in man and show that (i) the surgical procedure is successful in the majority of instances; (ii) simultaneous determinations of renal function of the right and left kidneys are comparable to function observed before dividing the bladder; and (iii) studies of function of the two kidneys may be repeated for 18 months or more and as often as three times a week. We conclude that the procedure provides an opportunity for prolonged observation of changes in renal function and morphology of a single experimentally manipulated kidney compared to (i) the normal function and morphology of the kidney before inducing disease and (ii) the function of a normal contralateral kidney.


Chronic pyelonephritis. An electron microscopic study in nonhuman primates. The rhesus monkey (Macaca mulatta) proved to be a successful model for the ultrastructural study of ascending nonobstructive pyelonephritis. The most severe kidney damage occurred where the inflammatory response was greatest, and these areas overlaid a dilated or clubbed calyx. The most prominent ultrastructural changes were in the renal tubular cells that showed swollen mitochondria with ruptured inner membranes, dilated endoplasmic reticulum, and flocculent and irregular basal laminae. No recognizable bacterial structures were seen.


Methylene blue and other agents as inhibitors of calcium oxalithiasis in vivo. Growth of calcium oxalate on an established calculus upon a zinc nucleus in the bladder of rats was studied. Animals were fed either a regular solid chow or chow containing a potential crystal inhibitor ad libitum, along with drinking water containing 0.75 per cent ethylene glycol. Chow containing 0.2 per cent methylene blue and 0.5 per cent vitamin C not only decreased the growth rate, but calculi were much softer than those in controls. Safranin O was the only other significant growth inhibitor identified. Ethylenediamonotetraacetic acid and ethylenebis (oxyethylenenitrilo)-tetraacetic acid transformed the additional growth from the mono- to the dihydrate form of calcium oxalate.


Unusually mild course of beta-glucuronidase deficiency in two brothers (mucopolysaccharidosis VII). Two brothers with oligosymptomatic mucopolysaccharidosis VII were observed from age 11 8/12 to 16 years, and 15 1/2 to 19 years, respectively. Asymptomatic thoracic kyphosis and mild scoliosis were the prominent clinical features. Herniae, hepatosplenomegaly, corneal clouding and shortness of stature were absent. Both had Alder type granulations in polymorphonuclear leukocytes and to a lesser degree in monocytes. Ultrastructural analysis of blood leukocytes revealed polymorphous inclusions of probably more than one class of organic substances. Radiological signs were mild, confined to the spine and consisted of irregularities of upper and lower vertebral plates, of vertebral flattening and some osteophytic changes. Both patients excreted excessive amounts of acid mucopolysaccharides in urine and also globoside. Cultured skin fibroblasts of both patients contained metachromatic granules, had only approx. 10% of normal beta-glucuronidase activity and degraded sulfated mucopolysaccharides at a slower than normal rate. Sera of the patients had none or minimal beta-glucuronidase activity, the mother's serum had subnormal and the father's serum low-normal activity. The older brother is the oldest known case of mucopolysaccharidosis VII. As this hereditary disorder may take a remarkably mild clinical course, beta-glucuronidase-deficient juveniles may exist undetected in the general population.


Influences of physical conditioning and deconditioning on coronary vasculature of dogs. To investigate the effects of physical conditioning and deconditioning on the coronary vasculature, eight dogs were exercised by treadmill running. Five dogs were deconditioned by confinement in cages following the conditioning period. A technique was developed and validated for measuring circumflex coronary artery diameter from magnified projections of standardized coronary angiograms. Myocardial capillary density, perimeter, and basement membrane thickness were determined from electron microscopy of serial ventricular septal biopsy samples. Physical conditioning caused a small but statistically significant increase in cross-sectional area of the circumflex artery. Although physical conditioning caused no statistically significant changes in the myocardial capillaries, trends were apparent for increases in density and perimeter of myocardial capillaries and a decrease in basement membrane thickness. Physical deconditioning caused statistically significant reductions in cross-sectional area of the circumflex artery and in myocardial capillary density but little change in perimeter or basement membrane thickness of myocardial capillaries. The results suggest that physical conditioning may be associated with an improvement in coronary vascular capacity which may regress rapidly with deconditioning.


A plethysmograph for long-term measurements of ventilation in unrestrained animals. An apparatus for quantitative measurement of ventilation in unrestrained small animals is described. The subject rests in an environmental chamber, and respiration is indicated by barometric pressure oscillations proportional to tidal volume. The chamber is purged continuously at a relatively high flow rate during studies. Thus, CO2 does not accumulate and long-term measurement can proceed without interruption. Respiratory control studies are especially facilitated since different gas mixtures can be rapidly passed through the chamber. An electronic device also is described which automatically calculates expired minute volumes (VE) from the pressure signal obtained from the plethysmograph.


beta-lactam antibiotics. II. Structure-activity relationships of 6-[alpha-(alpha'-ureido-acylamino) acylamino] penicillanic acids. The influence on the structure-activity relationships (S.A.R.) of the stereochemistry and various alkyl, aryl, aralkyl and heterocyclic substituents at the two chiral centres in the dipeptide side-chain of a new series of penicillins was examined. In many cases the effects of these changes had a pronounced influence on the degree of activity against Gram-positive and especially Gram-negative bacteria. Several compounds indicated that the size, shape and spatial disposition of a substituent were the parameters of importance in influencing activity, rather than it lipophilic or electronic character. The most active homologues in the series provided broad-spectrum penicillins which in terms of their in vitro antibacterial properties showed improvements over certain of the marketed penicillins. Thus 6-[D-alpha(alpha'-ureidoacyl-amino)acylamino]penicillanic acids were found which had a carbenicillin-like profile, with improvements against Pseudomonas aeruginosa, Klebsiella aerogenes, sensitive and beta-lactamase-producing Gram-positive cocci.


New antibiotics, enaminomycins A, B and C. II. Physico-chemical and biological properties. Physico-chemical characterization of enaminomycins revealed that these antibiotics are new members of the epoxy quinone family. From elementary analysis and mass spectroscopic measurements the molecular formulae of enaminomycins A, B and C appear to be C7H5NO5, C10H11N06 and C7H7NO5, respectively. They are very unique in their chemical properties, possessing various functions, such as epoxy, primary amine and carboxylic acid, in their small structural units. Enaminomycin A, the most potent component, has activity against Gram-positive and Gram-negative bacteria and shows cytostatic effect on L1210 mouse leukemia cells in vitro, but enaminomycins B and C are only weakly active against Gram-positive and Gram-negative bacteria.


Isolation of ekatetrone, a new metabolite of producing variants of Streptomyces aureofaciens. From a mixture of substances formed by producing strains of Streptomyces aureofaciens under conditions of submerged fermentation a new metabolite, ekatetrone, was isolated. Its isolation and basic physical and chemical data are described. Ekatetrone is a quinone derivative with a carboxamide group. In tests in vitro with cells of Ehrlich's ascites tumour evidence was provided that ekatetrone inhibits proteo- and nucleosynthesis.


The structure of ekatetrone, a metabolite of strains of Streptomyces aureofaciens. The structure of ekatetrone has been determined from physico-chemical data obtained using the natural compound, its derivatives and products of degradation reactions. Ekatetrone was found to be the lactone of 1,8-dihydroxy-2-(1'-hydroxy-2'-carbamoyl)ethyl-9,10-anthraquinone-3-acetic acid (I). It is proposed that ekatetrone is related, biogenetically, to protetrone.


Detergent-resistant variants of Bacillus subtilis with reduced cell diameter. Variants of Bacillus subtilis resistant to the detergent Triton X-100 may exhibit: (i) normal cell morphology, (ii) reduced cell diameter, or (iii) helical cell shape. One variant of type ii was studied in some detail. Triton resistance, cell diameter reduction, and poor sporulation all may have resulted from a single mutation. High concentrations of Triton caused rapid lysis of wild-type cells. B. subtilis adapted to low Triton concentrations such that, upon subsequent exposure to higher concentrations, growth continued, although it bacame inhibited at very high concentrations. The variant studied retained its sensitivity to Triton-induced lysis but, after adaptation, grew at very high Triton levels. In this strain, cell diameter and cross-sectional area were reduced to about 73 and 50%, respectively, of those of wild type, yet the cells grew at normal rates, and DNA/protein/RNA ratios were largely unaltered. Peptidoglycan content per unit of cell surface area was higher in the variant than in the wild type under at least certain growth conditions.


Independence of Bacillus subtilis spore outgrowth from DNA synthesis. The outgrowth of spores of Bacillus subtilis 168 proceeded normally in temperature-sensitive DNA mutants under restrictive conditions and in the absence of DNA synthesis. Two inhibitors of DNA synthesis, nalidoxic acid and 6-(p-hydroxyphenylazo)-uracil, inhibited spore outgrowth under some nutritional conditions; this inhibition of outgrowth however, though not that of DNA synthesis, could be reversed by glucose. The sensitivity of the outgrowing spores to nalidixic acid and 6-(p-hydroxyphenylazo)-uracil inhbition decreased as a function of outgrowth time. The cells became completely resistant to the inhibitors after 90 min. The development of this resistance occurred also in the absence of DNA synthesis. It was concluded that DNA synthesis is not needed for spore outgrowth, and that outgrowing cells and vegetative cells differ in their sensitivity to these inhibitors.


Arginine biosynthesis in Neisseria gonorrhoeae: enzymes catalyzing the formation of ornithine and citrulline. Many of the Neisseria gonorrhoeae strains isolated from patients require arginine for growth in a defined medium. As a basis for genetic studies of these Arg- strains, we examined two biosynthetic enzymes of Arg+ (nonrequiring) gonococci. Cell-free extracts contained (i) glutamate acetyltransferase, which catalyzes the formation of L-ornithine from alpha-N-acetyl-L-ornithine, and (ii) ornithine transcaramylase, which catalyzes the reaction between L-ornithine and carbamyl phosphate, yielding L-citrulline. Arg- strains were unable to utilze alpha-N-acetyl-L-ornithine for growth lacked significant activity of glutamate acetyltransferase, and activity was gained by Arg+ clones derived by DNA-mediated transformation. Some of the Arg- patient isolates were unable to use either alpha-N-acetyl-L-ornithine or L-ornithine in place of arginine, and two separate steps of genetic transformation were required to yield Arg+ cells. Extracts of these doubly auxotrophic cells lacked glutamate acetyltransferase activity, but, unexpectedly, they displayed normal ornithine transcarbamylase activity. This finding illustrates the importance of identifying the products specified by arg loci during genetic studies of arginine auxotrophy.


Composition of the fractions separated by polyacrylamide gel electrophoresis of the lipopolysaccharide of a marine bacterium. The sugar composition of lipopolysaccharide (LPS) isolated from whole cells of Alteromonas haloplanktis 214 (previously referred to as marine pseudomonas B-16, ATCC 19855), variant 3, of the lipid A, core, and side-chain fractions derived from it, and of the LPS fractions (LPS I, II, and III) obtained by subjecting it to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been determined. Conditions optimum for the release of constituent monosaccharides by hydrolysis were established. Sugars were quantitated by gas-liquid chromatography of their alditol acetate derivatives. Lipid A was detected by gel electrophoresis and by the spectral shift obtained with a carbocyanin dye. A comparison of the molar ratios of the various fractions suggest that LPS III is an LPS molecule lacking an O-antigenic side chain, whereas LPS I and II are LPS molecules differing in side-chain composition. LPS I may be a mixture of two LPS species. In double immunodiffusion experiments using anti-whole-cell serum, LPS I and II showed a homologous cross-reaction with isolated whole-cell LPS. LPS III as well as lipid A, core, and side-chain fractions failed to give rise to precipitin lines.


Phospholipid composition of Rickettsia prowazeki grown in chicken embryo yolk sacs. The phospholipid composition and phospholipid fatty acid composition of purified Rickettsia prowazeki were determined. The lipid phosphorous content was 6.8 +/- 1.3 microgram/mg of total rickettsial protein. The major phospholipid was phosphatidylethanolamine (60 to 70%); phosphatidylglycerol constituted 20%, and phosphatidylcholine constituted 15%. Small amounts of phosphatidylserine and cardiolipin were detected. The principal fatty acids were 18:1, 16:1, and 16:0. The fatty acid composition of the phosphatidylcholine in the rickettsial extracts was very different than that of the other rickettsial phosphatides and very similar to that of normal yolk sac phosphatidylcholine. The specific of the phosphatidylcholine of rickettsiae grown in the presence of 32P was markedly lower than that of phosphatidylethanolamine and phosphatidylglycerol. It is suggested that the phosphatidylcholine in the rickettsial extract is yolk sac derived and either tightly absorbed or exchanged into the rickettsial membrane.


Isolation of stable ribosomal subunits from spores of Bacillus cereus. Analyses of ribosomes extracted from spores of Bacillus cereus T by a dryspore disruption technique indicated that previously reported defects in ribosomes from spores may arise during the ribosome extraction process. The population of ribosomes from spores is shown to cotain a variable quantity of free 50S subunits which are unstable, giving rise to slowly sedimenting particles in low-Mg2+ sucrose gradients and showing extremely low activity in in vitro protein synthesis. The majority of the ribosomal subunits in spores, obtained by dissociation of 70S ribosomes and polysomes, are shown to be as stable as subunits from vegetative cells, though the activity of spore polysomes was lower than that of vegetative ribosomes. In spite of the instability and inactivity of a fraction of the spore's ribosomal subunits, the activity of the total population obtained from spores by the dry disruption technique was 32% of vegetative ribosome activity, fivefold higher than previously obtained with this species. The improvement in activity and the observed variability of subunit destabilization are taken as evidence for partial degradation of spore ribosomes during extraction.


Characterization of succinic dehydrogenase mutants of Bacillus subtilis by crossed immunoelectrophoresis. Eleven succinic dehydrogenase (SDH) mutants in Bacillus subtilis were analyzed by crossed immunoelectrophoresis with antiserum prepared against wild-type B. subtilis cytoplasmic membrane. A precipitate which stained for SDH was found in Triton X-100-solubilized wild-type membranes and in membranes from two of the SDH mutants. The remaining nine mutants did not show an SDH-staining precipitate. The respective mutations in these nine mutants all map in one locus, citF (Ohné et al., J. Bacteriol. 115:738-745, 1973). An SDH-specific antiserum was prepared by immunizing rabbits with the SDH precipitate obtained in crossed immunoelectrophoresis with solubilized wild-type membrane. Using this antiserum, it was shown that all of the nine citF mutants lack an SDH-specific antigen in the membrane but five of the citF mutants have a soluble SDH-specific antigen. No major differences were found in sodium dodecyl sulfatepolyacrylamide gels of membrane proteins from wild-type B. subtilis and from SDH mutants. A model for the organization of SDH in B. subtilis is proposed.


Deletion plasmids from transformants of Pseudomonas aeruginosa trp cells with the RSF1010-trp hybrid plasmid and high levels of enzyme activity from the gene on the plasmid. A RSF1010-trp hybrid plasmid which contained the tryptophan operon of Escherichia coli was introduced into Pseudomonas aeruginosa trp cells by transformation. From the Trp+ transformants several deletion plasmids were obtained, and their physical maps with restriction endonucleases were constructed. P. aeruginosa trp cells with these plasmids showed at first more than 100 times higher levels of tryptophan synthetase beta activity over that of the control P. aeruginosa wild-type cells, but these levels were drastically decreased by 1 week of successive transfers of cultures. This decrease in enzyme activity was found to be due to the change on the plasmids but not to the host cells. The production of E. coli tryptophan synthetase beta enzyme in P. aeruginosa cells was proved by immunological test.


Purification and characterization of additional low-molecular-weight basic proteins degraded during germination of Bacillus megaterium spores. Dormant spores Bacillus megaterium contained a group of low-molecular-weight (5,000 to 11,000) basic (pI greater than 9.4) proteins (termed D, E, F, and G proteins) which could be extracted from disrupted spores with strong acids. These proteins were distinct from the previously described A, B, and C proteins which are degraded during spore germination. However, the D, E, F, and G proteins were also rapidly degraded during spore germination, accounting for 10 to 15% of the protein degraded. Proteins similar to the D, E, F, and G species were also present in spores of other bacterial species. In B. megaterium, the D, E, F, and G proteins were low or absent (less than 15% of the spore level) in vegetative and young sporulating cells and appeared only late in sporulation. The D, E, F, and G proteins were purified to homogeneity, and all contained a high percentage of hydrophilic amino acids; one protein (G) contained 31% basic amino acids and also contained tryptophan. All four proteins were rapidly degraded in vitro by dormant spore extracts. Two proteins (D and F) were degraded in vitro by the previously described spore protease which initiates degradation of the A, B, and C proteins in vivo; the spore enzyme (s) degrading proteins E and G have not been identified.


Iron in Neisseria meningitidis: minimum requirements, effects of limitation, and characteristics of uptake. A simple defined medium (neisseria defined medium) was devised that does not require iron extraction to produce iron-limited growth of Neisseria meningitidis (SDIC). Comparison of this medium to Mueller-Hinton broth and agar showed nearly identical growth rates and yields. The defined medium was used in batch cultures to determine the disappearance of iron from the medium and its uptake by cells. To avoid a number of problems inherent in batch culture, continuous culture, in which iron and dissolved oxygen were varied independently, was used. Most of the cellular iron was found to be nonheme and associated with the particulate fraction in sonically disrupted cells. Nonheme and catalase-heme iron were reduced by iron starvation far more than cytochromes b and c and N,N,N',N'-tetramethylphenylenediamine-oxidase. The respiration rate and efficiency also decreased under iron limitation, whereas generation times increased. The iron-starved meningococcus took up iron by an energy-independent system operating in the first minute after an iron pulse and a slower energy-dependent system inhibited by respiratory poisons and an uncoupler. The energy-dependent system showed saturation kinetics and was stimulated nearly fourfold by iron privation. In addition, to determine the availability to the meningococcus of the iron in selected compounds, a sensitive assay was devised in which an iron-limited continuous culture was pulsed with the iron-containing compound.


Microbial oxidation of methane and methanol: isolation of methane-utilizing bacteria and characterization of a facultative methane-utilizing isolate. A methane-utilizing organism capable of growth both on methane and on more complex organic substrates as a sole source of carbon and energy, has been isolated and studied in detail. Suspensions of methane-grown cells of this organism oxidized C-1 compounds (methane, methanol, formaldehyde, formate); hydrocarbons (ethane, propane); primary alcohols (ethanol, propanol); primary aldehydes (acetaldehyde, propionaldehyde); alkenes (ethylene, propylene); dimethylether; and organic acids (acetate, malate, succinate, isocitrate). Suspensions of methanol-or succinate-grown cells did not oxidize methane, ethane, propane, ethylene, propylene, or dimethylether, suggesting that the enzymatic systems required for oxidation of these substrates are induced only during growth on methane. Extracts of methane-grown cells contained a particulate reduced nicotinamide adenine dinucleotide-dependent methane monooxygenase activity. Oxidation of methanol, formaldehyde, and primary alcohols was catalyzed by a phenazine methosulfate-linked, ammonium ion-requiring methanol dehydrogenase. Oxidation of primary aldehydes was catalyzed by a phenazine methosulfate-linked, ammonium ion-independent aldehyde dehydrogenase. Formate was oxidized by a nicotinamide adenine dinucleotide-specific formate dehydrogenase. Extracts of methane-grown, but not succinate-grown, cells contained the key enzymes of the serine pathway, hydroxypyruvate reductase and malate lyase, indicating that the enzymes of C-1 assimilation are induced only during growth on C-1 compounds. Glucose-6-phosphate dehydrogenase was induced during growth on glucose. Extracts of methane-grown cells contained low levels of enzymes of the tricarboxylic acid cycle, including alpha-keto glutarate dehydrogenase, relative to the levels found during growth on succinate.


Outer membranes of gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier. A method for separating the outer and inner membranes of Pseudomonas aeruginosa PAO1 in the absence of added ethylenediaminetetraacetic acid was devised. The method yields two outer membrane fractions which show the same protein pattern on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but differ substantially in their relative contents of phospholipids. One of these outer membrane fractions and the inner membrane fraction are less than 4% cross-contaminated, as judged by the content of typical inner and outer membrane markers. The outer membrane contains four major protein bands with apparent molecular weights of 37,000, 35,000, 21,000 and 17,000. Vesicles reconstituted from lipopolysaccharide and phospholipids were impermeable to all saccharides included in the vesicles during vesicle formation. When the vesicles contained outer membrane proteins, they fully retained only those saccharides of greater than 9,000 molecular weight, suggesting that the exclusion limit of the outer membrane of P. aeruginosa for saccharides is substantially larger than the figure (500 to 600 daltons) obtained for certain enteric bacteria. The advantages and potential disadvantages of having an outer membrane with a higher exclusion limit for hydrophilic substances are discussed.


Cell envelope alterations in antibiotic-sensitive and-resistant strains of Neisseria gonorrhoeae. The cell envelopes of antibiotic-resistant and -sensitive isogenic strains of Neisseria gonorrhoeae were analyzed to determine whether acquisition of genetic loci for altered antibiotic sensitivity was accompanied by alterations in cell envelope composition. No differences in the composition of phospholipids and lipopolysaccharides were noted. Acquisition of mtr-2, which results in low-level, nonspecific increased resistance to multiple antibiotics, dyes, and detergents, was accompanied by a sevenfold increase in the amount of a minor, 52,000-molecular-weight outer membrane protein and a 32% increase in the extent of peptidoglycan cross-linking. Subsequent addition of the nonspecific hypersensitivity loci env-1 or env-2 to a strain carrying mtr-2 resulted in reversal of the phenotypic resistance determined by mtr-2 and marked reduction in both the amount of the 52,000-molecular-weight outer membrane protein and the extent of peptidoglycan cross-linking. Introduction of penB2, which results in a fourfold increase in resistance to penicillin and tetracycline, was accompanied by the disappearance of the principal outer membrane protein of the wild-type strain (molecular weight, 36,900) and the appearance of a new species of the principal outer membrane protein (molecular weight, 39,400) in the transformant.


Survey of extrachromosomal DNA found in the filamentous cyanobacteria. Cleared lysates of 13 species of filamentous cyanobacteria were examined for the presence of extrachromosomal DNA by using agarose gel electrophoresis and ethidium bromide staining. Seven of the 13 species contained extrachromosomal covalently closed circular DNA, and all but 1 species contained multiple elements. There was no correlation between the presence of extrachomosomal DNA and either the range of metabolic activities found in the cyanobacteria or the differentiated cell types or structures elaborated by the morphologically complex filamentous cyanobacteria.


Absence of hydrogen peroxide production by or catalase action in Rickettsia prowazeki. Glutamic acid oxidation by Rickettsia prowazeki is not accompanied by hydrogen peroxide generation, nor is added hydrogen peroxide degraded, as measured by a manometric technique.


One-carbon metabolism in methanogenic bacteria: analysis of short-term fixation products of 14CO2 and 14CH3OH incorporated into whole cells. Methanobacterium thermoautotrophicum, M. ruminantium, and Methanosarcina barkeri were labeled with 14CO2 (14CO2 + H14CO3- + 14CO32-) for from 2 to 45 s. Radioactivity was recovered in coenzyme M derivatives, alanine, aspartate, glutamate, and several unidentified compounds. The properties of one important structurally unidentified intermediate (yellow fluorescent compound) displayed UV absorbance maxima at pH 1 of 290 and 335 nm, no absorbance in the visible region, and a fluorescence maximum at 460 nm. Label did not appear in organic phosphates until after 1 min. 14CH3OH was converted by M. barkeri primarily into coenzyme M derivatives at 25 s. [2-14C]acetate was assimilated by M. thermoautotrophicum mainly into alanine and succinate during 2 to 240 s, but not into coenzyme M derivatives or yellow fluorescent compound. Cell-free extracts of M. thermoautotrophicum lacked ribulose 1,5-bisphosphate carboxylase activity. The data indicated the absence of the Calvin, serine, and hexulose phosphate paths of C1 assimilation in the methanogens examined and indicated that pyruvate was an early intermediate product of net CO2 fixation. The in vivo importance of coenzyme M derivatives in methanogenesis was demonstrated.


In vivo regulation of glycolysis and characterization of sugar: phosphotransferase systems in Streptococcus lactis. Two novel procedures have been used to regulate, in vivo, the formation of phosphoenolpyruvate (PEP) from glycolysis in Streptococcus lactis ML3. In the first procedure, glucose metabolism was specifically inhibited by p-chloromercuribenzoate. Autoradiographic and enzymatic analyses showed that the cells contained glucose 6-phosphate, fructose 6-phosphate, fructose-1,6-diphosphate, and triose phosphates. Dithiothreitol reversed the p-chloromercuribenzoate inhibition, and these intermediates were rapidly and quantitatively transformed into 3- and 2-phosphoglycerates plus PEP. The three intermediates were not further metabolized and constituted the intracellular PEP potential. The second procedure simply involved starvation of the organisms. The starved cells were devoid of glucose 6-phosphate, fructose 6-phosphate, fructose- 1,6-diphosphate, and triose phosphates but contained high levels of 3- and 2-phosphoglycerates and PEP (ca. 40 mM in total). The capacity to regulate PEP formation in vivo permitted the characterization of glucose and lactose phosphotransferase systems in physiologically intact cells. Evidence has been obtained for "feed forward" activation of pyruvate kinase in vivo by phosphorylated intermediates formed before the glyceraldehyde-3-phosphate dehydrogenase reaction in the glycolytic sequence. The data suggest that pyruvate kinase (an allosteric enzyme) plays a key role in the regulation of glycolysis and phosphotransferase system functions in S. lactis ML3.


Inhibition of Bacillus subtilis spore germination by various hydrophobic compounds: demonstration of hydrophobic character of the L-alanine receptor site. L-Alanine-initiated germination of Bacillus subtilis spores was inhibited by various kinds of hydrophobic compounds. Good correlation of inhibitory effect with hydrophobicity of the compound was demonstrated by using regression analysis in which the hydrophobic character was expressed by the partition coefficient in an octyl alcohol-water system. The correlation coefficient for 20 alcohols was 0.959, and that for 19 miscellaneous compounds was 0.906. Regression lines of the alcohols and other hydrophobic compounds were almost identical, showing that hydrophobic interaction played an important role in inhibition. Diphenylamine was one of the most effective inhibitors examined. n-Octyl, n-nonyl, and n-decyl alcohols were the most effective alcohols. The mode of inhibition by diphenylamine and n-octyl alcohol was a "mixed type" (competitive plus noncompetitive type) with respect to L-alanine; that by D-alanine was competitive inhibition. Sites for diphenylamine, n-octyl alcohol, and D-alanine may have overlapped. Inhibition was reversible by washing; heat resistance, stainability, and germination rate of the washed spores remained unaltered. Thus, we confirmed that the inhibition may occur before the initial trigger reaction of germination and that it may be due to the interaction between a hydrophobic compound and a hydrophobic region closely associated with the L-alanine receptor site on the spore.


Linkage map of Pseudomonas aeruginosa PAT. The locations of new markers relative to markers previously mapped on the chromosome of Pseudomonas aeruginosa strain PAT were defined by generalized transduction with phage F116L and F1083. Although the marker orders of the various marker groups were deduced mainly from the results of two-factor crosses, the locations of a number of markers were confirmed by three-factor crosses. A linkage map of the chromosome of P. aeruginosa PAT was constructed which shows the relative locations of 50 genes. From the available data, the linkage maps of P. aeruginosa strains PAO and PAT appear to be similar.


Synthesis and salvage of purines during cellular morphogenesis of Myxococcus xanthus. Intact cells of Myxococcus xanthus were examined for de novo purine synthesis and salvage utilization. The cellular uptake rates of radioactive glycine (de novo purine precursor), adenine, and guanine were measured, and thin-layer chromatography and radioautography were used to examine cell extracts for de novo synthesized purine nucleotides. Intact vegatative cells, glycerol-induced myxospores, and germinating cells of M. xanthus CW-1 were able to carry out de novo purine and salvage synthesis. Germinating cells and glycerol-induced myxospores were metabolically more active or as active as vegetative cells with respect to purine anabolism. We conclude that M. xanthus is capable of synthesizing purine nucleotides and salvaging purines throughout the glycerol version of its life cycle.


Occurrence of squalene and sterols in Cellulomonas dehydrogenans (Arnaudi 1942) comb. nov. Hester 1971. The neutral lipid fraction of the photochromogenic, coryneform bacterium Cellulomonas dehydrogenans (Arnaudi 1942) comb. nov. contains the sterol precursor squalene and at least two sterols, cholesterol and beta-sitosterol. The compounds were characterized by mass spectrometry and combination gas-liquid chromatography--mass spectrometry. De novo sterol biosynthetic ability was shown from incorporation of 14C from D-[U-14C]glucose into squalene and the sterol fraction. The squalene concentration approximated 0.002 to 0.005% of the total dry cell weight, and the sterols approximated 0.03 to 0.05%.


Relationship between catabolism of glycerol and metabolism of hexosephosphate derivatives by Pseudomonas aeruginosa. The relationship between catabolism of glycerol and metabolism of hexosephosphate derivatives in Pseudomonas aeruginosa was studied by comparing the growth on glycerol and enzymatic constitution of strain PAO with these characteristics of glucose-catabolic mutants and revertants. Growth of strain PAO on glycerol induced a catabolic oxidized nicotinamide adenine dinucleotide-linked glyceraldehyde-phosphate dehydrogenase and seven glucose-catabolic enzymes. The results indicated that these enzymes were induced by a six-carbon metabolite of glucose. All strains possessed a constitutive anabolic Embden-Meyerhof-Parnas pathway allowing limited conversion of glycerol-derived triosephosphate to hexosephosphate derivatives, which was consistent with induction of these enzymes by glycerol. Phosphogluconate dehydratase-deficient mutants grew on glycerol. However, mutants lacking both phosphogluconate dehydrogenase and phosphogluconate dehydratase were unable to grow on glycerol, although these strains possessed all of the enzymes needed for degradation of glycerol. These mutants apparently were inhibited by hexosephosphate derivatives, which originated from glycerol-derived triosephosphate and could not be dissimilated. This conclusion was supported by the fact that revertants regaining only a limited capacity to degrade 6-phosphogluconate were glycerol positive but remained glucose negative.


Classes of Anabaena variabilis mutants with oxygen-sensitive nitrogenase activity. Mutants of Anabaena variabilis deficient in the envelope glycolipids of heterocysts have no or very low nitrogenase activity when assayed aerobically. Revertants capable of aerobic growth on N2 have increased quantities of these glycolipids. Among mutants which require fixed nitrogen for growth in air and which have a normal complement of glycolipids, one expresses high nitrogenase activity at low oxygen tension. Three others show high nitrogenase activity only in the presence of dithionite and are therefore impaired in electron transfer.


Restriction endonucleases: general survey procedure and survey of gliding bacteria. Among 120 strains of gliding bacteria which were screened for restriction endonucleases, 27 were found positive. Additionally, three strains carried enzymes able to release the supercoiled state of closed circular DNA. By using a new rapid method, restriction endonuclease activity was released by stirring about 0.5 g of cells (fresh weight) in a motor-driven glass homogenizer in buffer containing Triton X-101, ethylenediaminetetraacetic acid, and mercaptoethanol. A yield from 60 to 80% of the total activity present in the cells was obtained with minimal destruction of the cells. The enzyme activity in the crude extract was measured semi-quantitatively by digestion of DNA and subsequent separation of the fragments on an agarose slab gel. The method appears to be generally applicable for the extraction of restriction endonucleases from gram-negative bacteria on an analytical scale and in a modified form for large-scale preparation of restriction enzymes.


Physiological and morphological observations on Thiovulum sp. Cell suspensions of Thiovulum sp., collected from enrichment cultures, were grown, maintained, and harvested for periods up to 7 months. In open-flow cultures run with aerated seawater, a continuous supply of hydrogen sulfide was provided by diffusion through a semipermeable membrane from either a live culture of Desulfovibrio esturaii, neutralized sodium sulfide, or a N2-H2S gas mixture. Attempts to grow Thiovulum in pure culture failed despite variation in concentrations of dissolved oxygen and hydrogen sulfide in stratified as well as in completely mixed systems. Uptake of 14CO2 and some organic compounds by purified cell suspensions was measured, and values were corrected for the activity of heterotrophic as well as autotrophic contaminants as determined in control experiments. Cell populations exhibited maximum uptake activities during formation of the characteristic veils. Substantial uptake of CO2 in air-saturated seawater was coincident with an optimal concentration of hydrogen sulfide of about 1 mM. Glutamate and a selection of vitamins (B12M biotin, and thiamine) did not significantly affect the uptake of CO2. No substantial uptake of carbon from acetate, glutamate, mannitol, and Casamino Acids was found. Within the range of error indicated, the data are consistent with acceptance of a chemolithotrophic nature of Thiovulum.


Chromosome-membrane association in Bacillus subtilis. IV. Further purification of DNA-membrane complex by using a combination of centrifugation and electrophoresis. We have developed a simple procedure to purify a DNA-membrane complex from Bacillus subtilis by using a combination of centrifugation and electrophoresis. Several unique proteins were detected in the purified complex.


Tunicamycin-resistant mutants and chromosomal locations of mutational sites in Bacillus subtilis. The types of tunicamycin-resistant mutants of Bacillus subtilis were analyzed, and their mutational sites on the chromosome were mapped. A type 1 mutation that simultaneously expressed hyperproductivity of extracellular alpha-amylase was located close to amy E. Type 2 mutations were near aroI.