Possible mechanisms of the first step of the classical complement activation pathway: binding and activation of C1. Different immunoglobulin preparations of human monoclonal IgM, normal human and rat IgG, as well as purified rabbit antibodies were treated by various methods, fragmentation, aggregation and complexing with antigen. The ability of the treated and untreated preparations to fix isolated human C1, to activate the classical complement pathway (to consume C4 in normal human serum) were compared. It was found that the different methods affected the conformation of the immunoglobulin molecules in different ways and induced changes to a greater or lesser extent in the two capacities of the preparations tested. In the case of the monoclonal IgM preparation a strong C1-fixation was observed without measurable complement activation. Other preparations, interfacially aggregated human IgG, BSA-anti-BSA and OA-anti-OA immune complexes had a very weak C1-fixing but a marked complement activating capacity. Some preparations, e.g. heat-aggregated IgG, both fixed and activated C1 effectively, aggregates with a complement-activating capacity without C1-fixing effect were separated by gel-filtration. It was demonstrated further, that at a given time only a part of the activated C1 molecules could be found fixed to the immunoglobulins, the other part was released into the fluid phase after activation. On the basis of the results of this and previous studies a hypothesis is proposed suggesting three possible results of the interaction between C1 and the different preparations: (1) firm fixation and activation; (2) binding not followed by activation and (3) a transient binding leading to activation. The possible application of this hypothesis for the interpretation of the results of the different methods for detecting immune complexes is discussed.


Passage of immunoglobulins from plasma to the oral cavity in rhesus monkeys. The passage of immunoglobulin from plasma to the oral cavity was studied in rhesus monkeys. Immunoglobulins G, A and M were purified from pooled rhesus monkey serum, radiolabelled with 125I and injected intravenously into twelve monkeys. Sequential samples of oral fluids were taken over a 24 h period and were assayed for radioactivity. Radioactivity could be detected in crevicular fluid washings after 0.5 h in monkeys injected with IgG and IgA, and after 2 h in monkeys given IgM. Maximal levels were found after 4 h with each immunoglobulin. Radioactivity in parotid and mixed saliva could be detected in all animals after 30 min, reaching a maximal level after 4 h. Ultracentrifugation on sucrose density gradients revealed that most of the radioactivity in crevicular fluid washings was in the 7S zone in the animals given IgG and IgA, and in a 19S zone in animals given IgM. The radioactivity in partoid saliva did not represent intact immunoglobulin molecules, since all the activity was present in zones of low molecular weight in animals given IgG, IgA or IgM. In mixed saliva a small amount of radioactivity was found in the immunoglobulin zones. The results suggest that intact molecules of IgG, IgA and IgM can pass from plasma to the oral cavity via crevicular fluid, and could contribute to oral defence mechanisms particularly in the crevicular domain. The volume of crevicular fluid in the approximal space of deciduous molars of rhesus monkeys was estimated to be approximately 0.3 microliter.


Inhibition of lysosomal enzyme release from rat leukocytes by auranofin. A new chrysotherapeutic agent. Auranofin (SK&F D-39162), a new antiarthritic gold compound reported to be orally effective in animal (adjuvant rat) and human (rheumatoid) arthritic conditions, is a potent in vitro inhibitor of the release of lysosomal enzymes from phagocytizing rat leukocytes. Auranofin, at micromolar concentrations (1-10 microM), produced a dose-dependent reduction in extracellular levels of lysosomal enzyme markers (beta-glucuronidase and lysozyme) which are selectively released from rat leukocytes during phagocytosis of zymosan particles. The reduction in extracellular levels of lysosomal enzymes appears to be caused by inhibition of their selective cellular release, since effective concentrations of auranofin did not produce leukocyte cytotoxicity or inhibition of cell-free lysosomal enzyme activity. Morphologic and biochemical evidence indicated that auranofin also interferes with phagocytosis of zymosan particles. The potent in vitro activity of auranofin appears to result from its unique gold complex, since neither structurally related nongold compounds nor clinically used gold compounds (gold sodium thiomalate and gold thioglucose) were potent inhibitors of lysosomal enzyme release. The results of this investigation suggest that the antiarthritic activity of auranofin may be caused at least in part, by inhibition of lysosomal enzyme release and/or cellular processing of antigens.


Effect of auranofin, a new antiarthritic agent, on immune complex-induced release of lysosomal enzymes from human leukocytes. Auranofin, an oral chrysotherapeutic agent effective in the treatment of rheumatoid arthritis (RA), was found to be a potent, noncytotoxic inhibitor of IgG-RF immune complex-induced lysosomal enzyme release (LER) from human leukocytes. At a concentration of 1 microg Au/ml (5 microM), auranofin produced a marked reduction in beta-glucuronidase (100%), acid phosphatase (88%), and lysozyme (72%) release. In contrast, gold sodium thiosulfate (GST, an injectable gold compound) had no inhibitory activity on LER at equivalent gold concentrations (i.e., 1 microg Au/ml) and only modest activity (less than 36% inhibition) at concentrations as high as 40 microg Au/ml. The 50% inhibitory dose (LD50) of auranofin on LER was calculated to be 3-4 microM (0.6-0.8 microg Au/ml). Blood gold levels in auranofin-treated RA patients were found to be within the range required for in vitro inhibition of LER, and correlated with decreases in IgG, RF titers, and IgG-RF immune-complex formation in vitro. These results suggest that the therapeutic action of auranofin may be caused, at least in part, by inhibition of LER and/or decreases in immune-complex formation.


Immunopharmacology of gold sodium thiomalate and auranofin (SK&F D-39162): effects on cell-mediated immunity. The effects of gold sodium thiomalate (GST) and auranofin (SK&F D-39162) on cell-mediated immunity were investigated using oxazolone-induced contact sensitivity and delayed hypersensitivity to sheep red blood cells. C57Bl mice were sensitized to oxazolone on day 0 and challenged either 45 or 72 h later. The resulting paw edema was read plethysmographically 24 h after challenge. GST and auranofin both were capable of stimulating oxazollone-induced contact sensitivity which was compromised by using a shortened sensitization period (45 h). Auranofin but not GST stimulated the response to oxazolone in immunosuppressed mice, but neither agent significantly altered the uncompromised response in normal mice. The stimulatory effect of auranofin and GST on cell-mediated immunity was corroborated using SRBC to induce delayed hypersensitivity. Comparison of blood Au levels revealed that gold in the form of auranofin was approximately 4x more effective in stimulating cell-mediated immunity than was gold in the form of GST. These results were suggested to be due to the possible stimulation by gold of T effector as well as T suppressor lymphocytes, thus explaining the condition dependency of the immunoregulation.


The effect of cortisone on the accumulation, activation, and necrosis of macrophages in tuberculous lesions. Rabbits were injected intramuscularly with cortisone acetate (2 mg/kg) on alternate days. Six days after the first injection these rabbits and controls were injected intradermally in multiple sites with BCG (the vaccine strain of tubercle bacillus). Periodically, over the next 2 months, the resulting lesions were measured and surgically biopsied, and the animals were tuberculin-tested. Macrophage activation in the BCG lesions was evaluated histochemically by staining for beta-galactosidase activity. Both BCG lesions (and tuberculin reactions) in the cortisone-treated group were considerably smaller than those in the control group. Cortisone was highly effective in reducing the number of infiltrating mononuclear cells (MN), the amount of caseous necrosis and ulceration, and the percent of NM that were beta-galactosidase-positive. The decreased activation and reduced number of macrophages readily explains the increased susceptibility to tuberculosis found amoung patients receiving glucocorticosteroids. In the BCG lesions, the local decrease in the number and function of leukocytes probably explains the decreased tissue necrosis. Such antiinflammatory effects of corticosteroids may offset, in selected antimicrobial-treated cases, the hormone's detrimental effect on host resistance to infectious agents.


Ascorbate metabolism in the trained monkey. The metabolic fate of 14C-1-ascorbate was studied in trained monkeys (Macacca fasicularis) by measurement of the 14C in the expired breath, urine, and feces. To minimize stress, each monkey was placed in a restraining primate chair and trained to all experimental procedures prior to the isotope studies. The majority of the 14C-1-ascorbate was absorbed by the trained monkeys and ascorbate turnover increased with increasing plasma levels. The urine of trained monkeys was the major excretion route of 14C-1-ascorbate and its metabolites. less than 2.5% of the 14C-1-ascorbate was oxidized in 24 hr to 14CO2. The 14CO2 production was not changed by diet or the ascorbate nutritional status in the trained monkey. A significant increase in 14CO2 production was measured in an untrained monkey. The metabolic fate of 14C-1-ascorbate in man and the trained monkey was similar. Therefore, the trained monkey could be a study model for ascorbate metabolism in man.


Scanning electron microscopic studies of the zonular apparatus in human and monkey eyes. Scanning electron microscopic studies of the zonular apparatus in 10 human and 17 monkey eyes revealed two functionally different sets of zonular fibers: the "main fiber" and the "tension fiber" system. The two systems are connected, forming a broad, sagittally oriented "zonular plexus" within the pars plicata of the ciliary body. The zonular plexus is attached to the ciliary epithelium by the tension fibers, which leave the main system and run obliquely forward to the epithelium deep in the valleys of the ciliary processes. Anteriorly, the zonular plexus splits into the two branches of the "zonular fork," which run respectively to the anterior and posterior aspect of the lens. Thus the zonular plexus can act as a fulcrum. The three-dimensional architecture of the zonular apparatus is consistent with a new concept of accommodation.


Early lens changes in Macaca fascicularis monkeys under topical treatment with echothiophate or carbachol studied by slit-image photography. Cynomolgus monkeys were given topical treatment with echothiophate or carbachol, and the lens changes were followed by slit-image photography. Both drugs caused an early retrodisplacement of the anterior zone of disjunction due to swelling of the most superficial lens cortex. Deeper layers of the cortex did not swell. The exact cause of the swelling is not known, but there must be a true cholinergic link. Accommodation as such is not the cause, since (1) the effect was seen also in a monkey eye which had its ciliary muscle disinserted from the scleral spur and therefore had little accommodation left and (2) acute accommodation produced by systemic pilocarpine did not cause the effect. Anterior subcapsular opacities were quantified and appeared very early in the echothiophate-treated eyes but were not seen with the doses of carbachol used.


The effect of dopamine on renal cortical blood flow in baboons with experimentally induced obstructive jaundice. The effect of dopamine hydrochloride on renal blood flow in  bile duct ligated baboons was assessed using the 133Xe washout technique. Intrarenal infusions of dopamine in different concentrations did not significantly increase renal cortical blood flow. On the contrary, over the lower dose range, the normal dopamine-induced renal vasodilatation was abolished in the jaundiced state. An in vitro experiment that measured the perfusion pressure of an isolated rabbit kidney perfused with normal and jaundiced baboon plasma confirmed the in vivo results. These observations suggest that dopamine would not be of value in treating the renal failure that may develop in patients with obstructive jaundice.


Is GIP a glucagon cell constituent?

Is GIP a glucagon cell constituent? "Gastric inhibitory peptide" or "glucose-dependent insulin-releasing peptide" (GIP) is a member of the gut hormone family. Its physiological action is thought to be related to its insulinotrophic effect. The occurrence and distribution of GIP was studied by immunohistochemistry. In all species examined including man, GIP immunoreactivity was found to reside in the glucagon cells of the pancrease and gut. Three pancreatic glucagonomas were found to contain numerous cells displaying GIP and glucagon immunoreactivity. The GIP antiserum used did not cross react with either pancreatic-type or gut-type glucagon (GLI).


Hematotropic parasites of translocated raccoons in the southeast. Ninety-four raccoons (Procyon lotor) from 6 southeastern states were examined for hematotropic parasites, to evaluate the carrier potential of these animals as they relate to translocation and release for hunting purposes. Four species of hematotropic parasites were identified, viz, Babesia procyonis, Haemobartonella procyoni, Hepatozoon procyonis, and Trypanosoma cruzi. Lesions in infected raccoons were associated only with H procyonis. Babesia procyonis was enzootic in populations of raccoons used as sources for exportation and in raccoons resident in typical release areas. Haemobartonella procyoni, H procyonis, and T cruzi were prevalent only in locales of raccoon origin. Due to the potential pathogenicity of H procyonis and T cruzi in raccoons or other hosts, the practice of translocating raccoons from enzootic to nonenzootic areas was considered biologically hazardous.


CC-1065 (NSC-298223), a new antitumor antibiotic. Production, in vitro biological activity, microbiological assays and taxonomy of the producing microorganism. A new antitumor antibiotic is produced in fermentation liquors of Streptomyces zelensis sp.n. The antibiotic is biologically active at extremely low concentrations. At 40 pg/ml, it inhibited 90% of the growth of L1210 cells in culture in tube dilution assays. The minimal inhibitory concentrations against Gram-positive bacteria is between 1 approximately 10 ng/ml, while these values for Gram-negative bacteria and fungi are mostly under 1 microgram/ml. A microbiological assay with Bacillus subtilis can detect concentrations of 1 approximately 2 ng/ml.


The synthesis of highly phosphorylated nucleotides, RNA and protein by Streptomyces aureofaciens. During the sudden decrease in RNA synthesis in Streptomyces aureofaciens, i.e. around the 6th hour of cultivation, synthesis of adenosine and guanosine tetraphosphates and pentaphosphates begins. The synthesis of these nucleotides is highest during the onset of chlortetracycline production, around the 20th hour of cultivation and continues. During this phase of growth of S. aureofaciens, RNA and protein synthesis are reduced by about one order of magnitude as compared to the rate which can be observed at the beginning of cultivation, but the synthesis is not inhibited by exogenous CTC.


Effect of 6-(p-hydroxyphenylazo)-uracil on the homologous and heterologous transduction processes in Bacillus subtilis. We have studied the effect of 6-(p-hydroxyphenylazo)-uracil on the recombination processes that operate in the homologous and heterologous transduction mediated by PBS1 and SP10 phages of Bacillus subtilis. The results obtained demonstrate that the process of heterologous genetic exchange is sensitive to this compound, whereas the homologous process is not. The present data, along with those of our previous work (U. Canosi, A. G. Siccardi, A. Falaschi, and G. Mazza, J. Bacteriol. 126:108--121, 1976), suggest that the DNA polymerase III is involved in the recombination process that operates in transformation and heterologous transduction, whereas homologous transduction follows a partially independent pathway not involving this protein.


Tight-binding repressors of the lac operon: selection system and in vitro analysis. The isolation and characterization of altered repressors of the lac operon which have an increased affinity for an operator should give useful clues about the molecular basis for the very tight and specific interaction between repressor and operator. A selection system has been devised which allows the isolation of such repressor mutants. This system selects for mutant repressors which can overcome lac operator-constitutive (Oc) mutations. By using in vivo assays, 24 candidates were obtained which, compared with wild type, have an increased trans effect of their repressor on one or several Oc operators. Three of these candidates have been investigated in vitro; the affinity of their repressor for inducer was unchanged, whereas the affinity for wild-type operator was increased 15-, 86-, and 262-fold, respectively.


Effect of levulinic acid on pigment biosynthesis in Agmenellum quadruplicatum. When levulinic acid was added to a growing culture of the cyanobacterium (blue-green alga) Agmenellum quadruplicatum PR-6, delta-aminoelevulinic acid accumulated in the medium and chlorophyll a synthesis and cell growth were inhibited, but there was a small amount of c-phycocyanin synthesis. The amount of delta-aminolevulinic acid produced in the treated culture did not fully account for the amount of pigment synthesized in the untreated control. Levulinic acid and either sodium nitrate or ammonium chloride were added to nitrogen-starved cultures of PR-6, and delta-aminolevulinic acid production and chlorophyll a and c-phycocyanin content were monitored. When ammonium chloride was added as a nitrogen source after nitrogen starvation, the cells recovered more rapidly than when sodium nitrate was added as a nitrogen source. In cultures recovering from nitrogen starvation, synthesis of c-phycocyanin occurred before synthesis of chlorophyll a.


Carbon and nitrogen repression of arginine catabolic enzymes in Bacillus subtilis. Specific activities of arginase and ornithine aminotransferase, inducible enzymes of arginine catabolism in Bacillus subtilis 168, were examined in cells grown with various carbon and nitrogen sources. Levels of these enzymes were similar in arginine-induced cultures whether glucose or citrate was the carbon source (in contrast to histidase), suggesting that carbon source catabolite repression has only limited effect. In media with combinations of nitrogen sources, glutamine strongly repressed induction of these enzymes by proline or arginine. Ammonium, however, only repressed induction by proline and had no effect on induction by arginine. These effects correlate with generation times in media containing these substances as sole nitrogen sources: growth rates decreased in the order glutamine-arginine-ammonium-proline. Similar phenomena were observed when glutamine or ammonium were added to arginine- or proline-grown cultures, or when arginine or proline were added to glutamine- or ammonium-grown cultures. In the latter cases, an additional feature was apparent, namely a surprisingly long transition between steady-state enzyme levels. The results are compared with those for other bacteria and for eucaryotic microorganisms.


Transcription and translation in a pleiotropic streptomycin-resistant mutant of Escherichia coli. The role of the ribosomal protein S12 (streptomycin protein) in ribosome function and in other metabolic processes in the cell has been investigated. A spontaneous streptomycin-resistant strain of Escherichia coli (SM3) carrying a mutation in the rpsL gene is deficient in its ability to induce the synthesis of the enzyme bets-galactosidase. It was demonstrated that the reduced rate of enzyme synthesis results from deficiencies in both the transcription of the lactose operon and translation of the lactose operon mRNA. The transcription deficiency was in part due to increased catabolite repression and could therefore be partially suppressed by the addition of cyclic AMP. Streptomycin also appeared to partially suppress catabolite repression. In the SM3 mutant strain, the translation of the lactose operon mRNA was only about 60% as efficient as in the parental control, and addition of streptomycin did not alter the translation efficiency. In contrast, both transcription and translation of ribosomal protein mRNA were equally efficient in the two strains. These observations imply that mutational alterations in the ribosomal protein S12 either directly or indirectly alter (i) the extent of catabolite repression, (ii) the efficiency of transcription of the lactose operon even in the absence of catabolite repression, and (iii) the efficiency of translation of some but not all mRNA species in the cell.


Inhibition of Bacillus subtilis growth and sporulation by threonine. A 1-mg/ml amount of threonine (8.4 mM) inhibited growth and sporulation of Bacillus subtilis 168. Inhibition of sporulation was efficiently reversed by valine and less efficiently by pyruvate, arginine, glutamine, and isoleucine. Inhibition of vegetative growth was reversed by asparate and glutamate as well as by valine, arginine, or glutamine. Cells in minimal growth medium were inhibited only transiently by very high concentrations of threonine, whereas inhibition of sporulation was permanent. Addition of threonine prevented the normal increase in alkaline phosphatase and reduced the production of extracellular protease by about 50%, suggesting that threonine blocked the sporulation process relatively early. 2-Ketobutyrate was able to mimic the effect of threonine on sporulation. Sporulation in a strain selected for resistance to azaleucine was partially resistant. Seventy-five percent of the mutants selected for the ability to grow vegetatively in the presence of high threonine concentrations were found to be simultaneously isoleucine auxotrophs. In at least one of these mutants, the threonine resistance phenotpye could not be dissociated from the isoleucine requirement by transformation. This mutation was closely linked to a known ilvA mutation (recombination index, 0.16). This strain also had reduced intracellular threonine deaminase activity. These results suggest that threonine inhibits B. subtilis by causing valine starvation.


Specificity and biological distribution of coenzyme M (2-mercaptoethanesulfonic acid). The specificity of the growth requirement of Methanobacterium ruminantium strain M1 for a new coenzyme, 2-mercaptoethanesulfonic acid (HS--CoM), was examined. A variety of derivatives, analogs, and potential biosynthetic precursors of coenzyme M were tested; only a restricted range of thioether, thioester, and thiocarbonate derivatives of the cofactor were found to replace the HS--CoM requirement. Bromoethanesulfonic acid (BrCH2CH2SO3-), a halogenated analog of HS--CoM, potently inhibited the growth response. No coenzyme was detectable in a wide range of nonmethanogenic eucaryotic tissues and procaryotic organisms. However, all methanogens available in pure culture exhibited high levels of coenzyme M which ranged from 0.3 to 16 nmol/mg of dry weight.


Chemotaxis by Pseudomonas aeruginosa.

Chemotaxis by Pseudomonas aeruginosa. Chemotaxis by Pseudomonas aeruginosa RM46 has been studied, and conditions required for chemotaxis have been defined, by using the Adler capillary assay technique. Several amino acids, organic acids, and glucose were shown to be attractants of varying effectiveness for this organism. Ethylenediaminetetraacetic acid was absolutely required for chemotaxis, and magnesium was also necessary for a maximum response. Serine taxis was greatest when the chemotaxis medium contained 1.5 X 10(-5) M ethylenediaminetetraacetic acid and 0.005 M magnesium chloride. It was not necessary to include methionine in the chemotaxis medium. The strength of the chemotactic responses to glucose and to citrate was dependent on prior growth of the bacteria on glucose and citrate, respectively. Accumulation in response to serine was inhibited by the addition of succinate, citrate, malate, glucose, pyruvate, or methionine to the chemotaxis medium. Inhibition by succinate was not dependent on the concentration of attractant in the capillary. However, the degree to which glucose and citrate inhibited serine taxis was dependent on the carbon source utilized for growth. Further investigation of this inhibition may provide information about the mechanisms of chemotaxis in P. aeruginosa.


Rifampin-resistant mutants of Myxococcus xanthus defective in development. Rifampin, an antibiotic which is known to bind to and inhibit RNA polymerase, was used to probe the molecular regulation of development in Myxococcus xanthus. Rifampin-resistant mutants were screened for defects in fruiting-body formation. About 20% of the isolates in the initial screenings showed major defects in developmental aggregation or sporulation. Eleven independent mutants with wild-type growth rates and stable phenotypes were analyzed by transduction. In these strains, the rifampin-resistant and nonfruiting phenotypes showed cotransduction frequencies equal to or greater than 99.0 to 99.9%. The RNA polymerase activities were resistant to rifampin in vitro, indicating that the RNA polymerase is altered in these strains. Although their fruiting phenotypes are heterogeneous, these strains can be divided into two classes based on the level of aggregation. The results suggest that RNA polymerase plays a significant role in the regulation of development in M. xanthus since mutations which cause no apparent changes in vegetative growth result in striking defects in fruiting-body formation.


Control of heterocyst and nitrogenase synthesis in cyanobacteria. The development of the heterocyst by filamentous nitrogen-fixing cyanobacteria provides an attractive model system for studying cellular differentiation. Heterocyst synthesis is repressed by the presence of exogenous combined nitrogen. In this report, it is shown that the tryptophan analog, D,L-7-azatryptophan (Aza-T), is capable of relieving the repressive effect of exogenous NH4NO3 on heterocyst and nitrogenase synthesis. In nitrogen-fixing cultures, the presence of 20 micron Aza-T increases the heterocyst frequency twofold. The glutamate analog, L-methionine-D,L-sulfoximine (MSX), has also been shown to cause a derepression in the synthesis of heterocysts and nitrogenase. However, unlike MSX, Aza-T does not appear to exert its effects by inhibiting the activity of glutamine synthetase. Therefore, glutamine synthetase may not be the sole key to the derepression of heterocyst and nitrogenase development in the cyanobacteria. It is hoped that a study of Aza-T action may lead to the elucidation of a novel control mechanism.


Control of teichoic acid synthesis during phosphate limitation. The synthesis of teichoic acids was examined in Bacillus subtilis Marburg grown under conditions of phosphate limitation. The results indicate that the inhibition of polyglycerolphosphate synthesis observed under these conditions is the result of two processes. The first process is reversible and is independent of new protein synthesis; the second process is irreversible and requires the synthesis of new protein. During growth, under conditions of phosphate limitation, there is a slow decrease in the level of CDP glycerol pyrophosphorylase activity which is by itself not sufficient to account for the decrease in the rate of polyglycerolphosphate synthesis.


Isolation and characterization of an endogenous inhibitor of protein synthesis in Escherichia coli K-12. A low-molecular-weight factor was isolated from cell extracts of Escherichia coli K-12. The concentration of the factor in cells was dependent upon nutritional conditions, the concentration being higher in faster growing cells. Treatment of cells with colicin K caused an increase in concentration of the factor. The factor inhibited protein synthesis in E. coli. This inhibition was reversible, apparently because of metabolism of the factor. The inhibition of synthesis of beta-galactosidase lasted longer than the inhibition of protein synthesis; cyclic AMP eliminated this difference. The factor inhibited the synthesis of beta-galactosidase from preformed lac mRNA, indicating an inhibition of translation. Kinetic studies of the onset of inhibition of beta-galactosidase synthesis by the factor suggested that the factor may inhibit protein synthesis at the initiation of translation.


Cell wall teichoic acid as a reserve phosphate source in Bacillus subtilis. Although exponential growth of Bacillus subtilis 168 in a phosphate-limited medium halted with the exhaustion of inorganic phosphate, the bacteria continued to grow at a slower rate for a further 3 to 4 h at 37 degrees C. This postexponential growth in the absence of an exogenous phosphate supply was accompanied by a loss of teichoic acid from the cell walls of the bacteria. Quantitative analysis of walls and culture fluids showed that the phosphate loss from the walls could not be accounted for by an increase in phosphate-containing compounds in the medium, which implied that the cells were using their own wall teichoic acids to supply phosphate necessary for growth. Addition of exogenous teichoic acid to phosphate-starved cultures resulted in stimulation of growth and in the simultaneous disappearance of teichoic acid phosphate from the medium. It is proposed that teichoic acids, which can contain more than 30% of the total phosphorus of exponential-phase cells, can be used as a reserve phosphate source when the bacteria are starved for inorganic phosphate.


ATP sulfurylase from Penicillium chrysogenum: is the internal level of the enzyme sufficient to account for the rate of sulfate utilization? The in vivo rate of sulfate activation in Penicillium chrysogenum (wild-type strain ATCC 24791) was determined to be 0.19 +/- 0.09 mumol g(-1) (dry weight) min(-1) by the following methods. (i) The maximum growth of the organism in synthetic medium was a linear function of the initial Na(2)SO(4) concentration between 0 and 8 x 10(-4) Na(2)SO(4). The growth yield was 1.64 x 10(-2) g (dry weight) of mycelium per mumol of added sulfate, corresponding to a minimum sulfur requirement of 61 mumol/g (dry weight). Under these conditions (limiting sulfate) the minimum doubling time of P. chrysogenum in submerged culture was about 3.8 h, corresponding to a maximum exponential growth rate constant of 3.0 x 10(-3) min(-1). If all the sulfur in this mycelium passed through adenosine-5'-phosphosulfate, the rate of sulfate activation in vivo must have been 0.183 mumol min(-1) g(-1) (dry weight). (ii) In the presence of excess (35)SO(4) (2-), the total organic (35)S produced varied with the mycelial growth rate. However, until the culture approached maximum density, the product of [(growth rate constant) x (organic (35)S content)] was nearly constant at 0.24 to 0.28 mumol min(-1) g(-1) (dry weight). (iii) A sulfur-starved mycelium pulsed with 10(-4) M (35)SO(4) (2-) produced organic (35)S at a rate of about 0.10 mumol min(-1) g(-1) (dry weight) under conditions where the internal concentrations of ATP and sulfate would permit ATP sulfurylase to operate at about 70% of its V(max). Cell-free extracts of P. chrysogenum growing rapidly on excess sulfate contained 0.22 U of ATP sulfurylase per g (dry weight). Thus, in spite of the relatively low specific activity of homogeneous ATP sulfurylase (0.13 U/mg of protein, corresponding to an active site turnover of 7.15 min(-1)), the mycelial content of the enzyme was sufficient to account for the observed growth rate of the organism on inorganic sulfate as the sole sulfur source.


Isolation of an iron-binding compound from Pseudomonas aeruginosa. An iron-binding compound was isolated from ethyl acetate extracts of culture supernatant fluids of Pseudomonas aeruginosa and was purified by successive paper and thin-layer chromatographic procedures. The purified compound was characterized by UV, visible, infrared, and fluorescence spectroscopy. The compound possesses phenolic characteristics, with little or no similarity to dihydroxybenzoates and no indication of a hydroxamate group. P. aeruginosa synthesized the compound during active growth in culture media containing less than 5 X 10(-6) M added FeCl3. When added to iron-poor cultures of P. aeruginosa, the compound promoted the growth of the bacterium and also reversed growth inhibition by the iron chelator ethylenediamine-di-(o-hydroxyphenylacetic acid).


DNA repair in Bacillus subtilis: excision repair capacity of competent cells. Competent Bacillus subtilis were investigated for their ability to support the repair of UV-irradiated bacteriophage and bacteriophage DNA. UV-irradiated bacteriophage DNA cannot be repaired to the same level as UV-irradiated bacteriophage, suggesting a deficiency in the ability of competent cells to repair UV damage. However, competent cells were as repair proficient as noncompetent cells in their ability to repair irradiated bacteriophage in marker rescue experiments. The increased sensitivity of irradiated DNA is shown to be due to the inability of excision repair to function on transfecting DNA in competent bacteria. Furthermore, competent cells show no evidence of possessing an inducible BsuR restriction system to complement their inducible BsuR modification enzyme.


Effects of local anesthetics on bacterial cells. The membrane effects of chlorpromazine, nupercain, tetracain, and procain were studied using Bacillus cereus, B. megaterium, B. subtilis, and Streptococcus faecalis, protoplasts from S. faecalis, and isolated membranes from B. subtilis. Chlorpromazin, nupercain, and tetracain produced characteristic micromorphological alterations after treatment for 5 to 30 min at pH 7.0 and 20 degrees C; the membrane staining pattern changed from asymmetric to symmetric, complex mesosome-like structures appeared, and membrane fractures and solubilization occurred. Procain at concentrations up to 100 mM did not induce detectable alterations. Protoplasts were quickly lysed by 10 mM tetracain. A rapid and extensive leakage of K+ was induced by chlorpromazin, nupercain, and tetracain. Procain (100 mM) induced a slight K+ leakage. The membrane respiratory activity of intact B. cereus cells (as measured by the triphenyl tetrazolium reduction) and the succinic dehydrogenase activity of B. subtilis isolated membranes were found to be inhibited by the four local anesthetics. The concentrations that produced 50% inhibition of those activities are correlated with the hydrophobicities of the anesthetic molecules.


Ultrastructure and cytochemical localization of laccase in two strains of Leptosphaerulina briosiana (Pollaci) Graham and Luttrell. Substrate specificity tests were used to identify the presence of laccase in two strains of Leptosphaerulina briosiana (Poll.) Graham and Luttrell, an ascomycete which causes leaf spot in alfalfa. Cytochemical localization of monophenol monooxygenase (laccase) as well as the ultrastructures of the two strains were investigated. Laccase was observed in the outer layers of the cell walls of both strains. The ultrastructures of vegetative hyphae of both strains were typical of those found in most ascomycetes.


Homology of the gene coding for outer membrane lipoprotein within various Gram-negative bacteria. The mRNA for a major outer membrane lipoprotein from Escherichia coli was found to hybridize specifically with one of the EcoRI and one of the HindIII restriction endonuclease-generated fragments of total DNA from nine bacteria in the family Enterobacteriaceae: E. coli, Shigella dysenteriae, Salmonella typhimurium, Citrobacter freundii, Klebsiella aerogenes, Enterobacter aerogenes, Edwardsiella tarda, Serratia marcescens, and Erwinia amylovora. However, among the Enterobacteriaceae, DNA from two species of Proteus (P. mirabilis and P. morganii) did not contain any restriction endonuclease fragments that hybridized with the E. coli lipoprotein mRNA. Furthermore, no hybrid bands were detected in four other gram-negative bacteria outside the family Enterobacteriaceae: Pseudomonas aeruginosa, Acinetobacter sp. HO1-N, Caulobacter crescentus, and Myxococcus xanthus. Envelope fractions from all bacteria in the family Enterobacteriaceae tested above cross-reacted with antiserum against the purified E. coli free-form lipoprotein in the Ouchterlony immunodiffusion test. Both species of Proteus, however, gave considerably weaker precipitation lines, in comparison with the intense lines produced by the other members of the family. All of the above four bacteria outside the family Enterobacteriaceae did not cross-react with anti-E. coli lipoprotein serum. From these results, the rate of evolutionary changes in the lipoprotein gene seems to be closely related to that observed for various soluble enzymes of the Enterobacteriaceae.


Transfer of plasmid-mediated antibiotic resistance from streptococci to lactobacilli. The transmissible plasmid pAMbeta1, which codes for erythromycin and lincomycin resistance, was conjugally transferred from a Lancefield group F Streptococcus to a strain of Streptococcus avium. Both organisms served as pAMbeta1 donors for three strains of Lactobacillus casei. Introduction of pAMbeta1 into one of the L. casei strains caused the organism to lose its native 6.7 X 10(6)-dalton plasmid. Loss of the native plasmid produced no alterations in the organism's growth characteristics or fermentation pattern.


Effect of ethylenediaminetetraacetate on phospholipids and outer membrane function in Escherichia coli. Treatment of Escherichia coli K-12 strain S15, containing a normal amount of phospholipase A, with ethylenediaminetetraacetate (EDTA) resulted in an increase in sensitivity of the organism to actinomycin D. Strain S17, a mutant deficient in both detergent-resistant phospholipase A and detergent-sensitive phospholipase A, was considerably less sensitive to the antibiotic after the treatment. Both strains released lipopolysaccharide after EDTA treatment, indicating that this outer membrane component alone is not the barrier to actinomycin in these organisms. The phospholipase A-deficient strain released less alkaline phosphatase, a periplasmic enzyme. EDTA treatment of S15 resulted in the accumulation of free fatty acids, indicative of phospholipase A activation. Cells briefly treated with EDTA regained the barrier to actinomycin when incubated in growth media, and the cessation of the accumulation of free fatty acids was in approximate temporal agreement with restoration of the barrier. Cells in which phospholipase A was activated by brief exposure to EDTA synthesized relatively more phosphatidylethanolamine than did untreated cells in the initial period after dilution into growth media. These experiments suggest that the EDTA-induced loss of outer membrane barrier function of E. coli K-12 is mediated through the activation of phospholipase A.


Plasmid copy number control: isolation and characterization of high-copy-number mutants of plasmid pE194. A plasmid, pE194, obtained from Staphylococcus aureus confers resistance to macrolide, lincosamide, and streptogramin type B ("MLS") antibiotics. For full expression, the resistance phenotype requires a period of induction by subinhibitory concentrations of erythromycin. A copy number in the range of 10 to 25 copies per cell is maintained during cultivation at 32 degrees C. It is possible to transfer pE194 to Bacillus subtilis by transformation. In B. subtilis, the plasmid is maintained at a copy number of approximately 10 per cell at 37 degrees C, and resistance is inducible. Tylosin, a macrolide antibiotic which resembles erythromycin structurally and to which erythromycin induces resistance, lacks inducing activity. Two types of plasmid mutants were obtained and characterized after selection on medium containing 10 microgram of tylosin per ml. One mutant class appeared to express resistance constitutively and maintained a copy number indistinguishable from that of the parent plasmid. The other mutant type had a 5- to 10-fold-elevated plasmid copy number (i.e., 50 to 100 copies per cell) and expressed resistance inducibly. Both classes of tylosin-resistant mutants were shown to be due to alterations in the plasmid and not to modifications of the host genome.


Covalently closed circular DNAs in closely related unicellular cyanobacteria. Plasmids of Synechococcus cedrorum and two Anacytsis nidulans strains were characterized physically, and a probable instance of spontaneous "curing" is described.


Effect of light on the attachment of cyanophage AS-1 to Anacystis nidulans. The effect of illumination on the extent and kinetics of the adsorption of cyanophage AS-1 to the blue-green alga (cyanobacterium) Anacystis nidulans was studied by using 32P-labeled phage. The initial rate of adsorption was not significantly affected by light. However, at Na+ levels used ordinarily to culture the alga ([Na+] = 11.7 mM), the total amount of phage adsorbed was doubled in the illuminated cultures, as compared with the dark-grown ones, over a wide range of multiplicities of infection (0.05 to 20). Upon a 10-fold increase in Na+ concentration in the medium ([Na+] = 0.11 M), the dark adsorption of the phage increased to the level of light adsorption found in low Na+ medium. The effects on phage adsorption of high Na+ concentration and light were not additive.


Localization of membrane-derived oligosaccharides in the outer envelope of Escherichia coli and their occurrence in other Gram-negative bacteria. The glucose-containing, membrane-derived oligosaccharides of Escherichia coli are localized in the external envelope of that organism, most probably in the periplasmic space. The membrane-derived oligosaccharides appear to be generally occurring cell constituents of gram-negative (but not gram-positive) bacteria.


Monkey brain arylamidase. II. Further characterization and studies on mode of hydrolysis of physiologically active peptides. A large-scale purification of monkey brain arylamidase was carried out. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cystine. The amino terminal residue was determined to be alanine by dansylation. The enzyme was activated by sulfhydryl compounds. Dithiothreitol was more effective than beta-mercaptoethanol. Bestatin competitively inhibited the enzyme activity and the Ki value was calculated to be 2.5 x 10(-7) M, which was of the same order as that of puromycin. The inhibitions by puromycin and bestatin were reversible. The enzyme hydrolyzed di-, tri-, and oligopeptides including physiologically active peptides. Of physiologically active peptides, enkephalins and Met-Lys-bradykinin, which possess a neutral amino acid at the N-terminal position, were more rapidly hydrolyzed by the enzyme. Peptides such as LH-RH and TRH, which possess a pyrrolidonecarboxylyl group at the N-terminal position, and substance P and bradykinin, which possess a proline residue adjacent to the N-terminal residue, were not hydrolyzed by the enzyme. The Km values for various peptides indicate that the enzyme has higher affinity for oligopeptides than di- and tripeptides. The aminopeptidase activity of the enzyme was also competitively inhibited by puromycin and bestatin. Analyses of the hydrolysis products of various peptides by the dansylation method indicate that the enzyme has both kinin-converting activity and angiotensinase activity.


Effect of pyocin R1 on the glucose metabolism of sensitive cells of Pseudomonas aeruginosa. The effect of pyocin R1 on the glucose metabolism of sensitive Pseudomonas cells was investigated. Upon treatment with pyocin R1, although the rate of O2 uptake of the sensitive cells for glucose or gluconate was not very much affected at first, the final level of O2 uptake was greatly reduced. When 2-oxogluconate was used as a substrate, O2 uptake was immediately halted by pyocin. By determining the amounts of glucose, gluconate, and 2-oxogluconate before and after the reaction and the amount of O2 consumed, it was concluded that glucose was exclusively metabolized via the following pathway with quantitative accumulation of 2-oxogluconate after pyocin treatment. (Formula: see text). The possible mechanism of this change is discussed.


Adenylate cyclase in silkworm. Properties of the enzyme in pupal fat body. Adenylate cyclase was assayed in a sonicated preparation of silkworm pupal fat body. The adenylate cyclase was found mostly in the particulate fraction. The activity depended upon either Mg2+ or Mn2+, and the degree of stimulation by Mn2+ was 2 times greater than that by Mg2+ compared at the saturating concentrations. In the presence of Mg2+, the enzyme was inhibited by both EGTA and high concentrations of Ca2+, showing biphasical response to Ca2+. The enzyme was stimulated several-fold by NaF. The enzyme exhibited typical Michaelis-Menten kinetics and Km values were 0.13 mM for MgATP and 0.086 mM for MnATP.


X-ray analysis of ferredoxin from Spirulina platensis. II. Chelate structure of active center. A chloroplast-type ferredoxin from Spirulina platenis crystallized in an orthorhombic system, space group C2221, with cell dimensions a=62.32, b=28.51, and c=108.08 A. The electron density map at 2.8 A resolution was prepared by using the best phase angles determined by the single isomorphous replacement method coupled with the anomalous dispersion method. The chelating structure of the acitve center was revealed as follows. Of the six cysteinyl residues in the molecule, Cys 41, Cys 4k, Cys 49, and Cys 79 are involved in the active center. Cys 41 and Cys 46 are coordinated to one iron atom, and Cys 49 and Cys 79 to the other iron atom. Only one of these cysteinyl residues, Cys 79, is comparatively apart from the other three in the amino acids sequence of the molecule, as found in the case of bacterial ferredoxin. It appears that the NH....S hydrogen bonds are around the active center, as in other non-heme iron sulfur proteins.


Complete stabilization of water-soluble hydrogenase from Rhodospirillum rubrum under air atmosphere with a high concentration of chloride ions. Hydrogenase was easily solubilized from light-grown cells of R. rubrum with 10 mM Na ethylenediaminetetraacetate. The enzyme thus obtained was so stable that loss of its activity was undetectable during storage at room temperature for 6 months under air atmosphere, provided that NaCl, KCl or CsCl was present at greater than or equal to 0.7 M.


Isolation of characterization of a major outer membrane protein of Pseudomonas aeruginosa. Evidence for the occurrence of a lipoprotein. In the outer membrane of P. aeruginosa, a protein of apparent molecular weight 8,000 (protein I) is present as a major protein. Purification and chemical analysis of protein I were carried out. This protein was purified by essentially the same procedure as for the purification of the E. coli lipoprotein, which was developed by Inouye et al. (J. Bacteriol. (1976) 127, 555--563). The amino acid composition of protein I was determined. Protein I lacks proline, valine, isoleucine, phenylalanine, tryptophan, and half-cystine. Fatty acid analysis of the protein revealed that it contained 0.89 mol of fatty acids per mol of protein. Among the fatty acids hexadecanoic acid (C16:0) was predominant. In an in vivo labeling experiment, [2-3H]glycerol was incorporated into protein I. A protein with similar mobility to protein I on urea-SDS polyacrylamide gel electrophoresis was isolated from the purified peptidoglycan of P. aeruginosa by trypsin digestion. The amino acid composition of this protein was essentially the same as that of protein I. These results indicate that the outer membrane of P. aeruginosa contains a protein analogous to the E. coli lipoprotein, although considerable differences were observed in the amino acid composition and the fatty acid content.


Purification and partial characterization of three forms of alpha-glucosidase from the fruit fly Drosophila melanogaster. Three forms of alpha-glucosidase, I, II, and III, have been purified from the whole body extract of adult flies of Drosophila melanogaster in yields of 2.1, 5.3, and 6.7%, respectively. The purification procedures involved ammonium sulfate fractionation, Con A-Sepharose 4B affinity chromatography, DEAE-Sepharose CL-6B ion exchange chromatography, Sephacryl S-200 gel filtration, and preparative gel electrophoresis. Each purified enzyme showed a single band on polyacrylamide gel on both protein and enzyme activity staining. The molecular weights of alpha-glucosidases I, II, and III were estimated to be 200,000, 56,000, and 76,000, respectively, by gel filtration. SDS gels indicated that alpha-glucosidases II and III were each composed of a single polypeptide chain, whereas alpha-glucosidase I was composed of two identical subunits. Both alpha-glucosidases II and III hydrolyzed sucrose and p-nitrophenyl-alpha-D-glucoside (PNPG), but alpha-glucosidase I hydrolyzed PNPG to a much lesser extent than sucrose. For sucrose the pH optima of alpha-glucosidases I, II, and III were pH 6.0, 5.0, and 6.0 and the Km values were 13.1, 8.9, and 10 mM, respectively. For PNPG the pH optima of alpha-glucosidases II and III were pH 5.5 and 6.5 and the Km values were 0.77 and 0.21 mM, respectively.


Purification and characterization of two lectins from Aloe arborescens Mill. Two lectins have been isolated from leaves of Aloe arborescens Mill by salt precipitation, pH-dependent fractionation and gel filtration. One lectin (P-2) has a molecular weight of approximately 18,000, consists of two subunits (alphabeta) and contains more than 18% by weight of neutral carbohydrate. The smaller subunit (alpha) has a molecular weight of approximately 7,500 and the larger subunit (beta) a molecular weight of approximately 10,500. The other lectin (S-1) has a molecular weight of approximately 24,000, consists of two subunits (gamma2) with a molecular weight of approximately 12,000 and contains more than 50% by weight of neutral carbohydrate. An interesting feature of the amino acid compositions of these lectins is the high proportion of acidic amino acids, such as aspartic acid and glutamic acid, and the low proportion of methionine and histidine. S-1 has a strong hemagglutinating activity. On the other hand, P-2 has not only hemagglutinating activity but also mitogenic activity on lymphocytes, precipitate-forming reactivity with serum proteins, one of which is alpha2-macroglobulin, and complement C3 activating activity via the alternate pathway.


Physicochemical properties of pyocin F1. The physiochemical properties of pyocin F1 were studied. Pyocin F1 consists of flexuous rod-like particles homogenous in size. Each particle was composed of rod and fiber parts. The rod part was 105.5 +/- 9.5 nm long and 10.0 +/- 1.4 nm wide, and showed regular striations amounting to 23 layers. The fiber part was composed of several filaments; the length of the longest filament was 43.0 +/- 12.0 nm. The amino acid composition, the partial specific volume (0.720 ml/g), the sedimentation coefficient (S020,W = 35.1S), and the translational diffusion constant (0.94 +/- 0.01 x 10(-7) cm2/s) were determined. The particle weight was calculated to be 3.23 x 10(6) daltons.


Studies on human kininogens. I. Isolation, characterization, and cleavage by plasma kallikrein of high molecular weight (HMW)-kininogen. 1. Human high molecular weight (HMW)-kininogen was highly purified from human plasma by chromatographies on QAE-Sephadex A-50 and CM-Sephadex C-50. Human HMW-kininogen thus purified was a mixture of a single chain and a disulfide-linked pair of chains. Human HMW-kininogen is an acidic glycoprotein having a molecular weight of 120,000. The amino acid composition of human HMW-kininogen is quite similar to that of bovine HMW-kininogen. 2. We investigated whether the liberation of kinin from human HMW-kininogen by human plasma kallikrein was accompanied by liberation of histidine-rich fragments, as observed with bovine HMW-kininogen (Han et al. (1975) J. Biochem. 77, 55--68). After prolonged incubation of human HMW-kininogen and human plasma kallikrein followed by gel-filtration on Sephadex G-50, a fragment of molecular weight 8,000 was isolated together with bradykinin. However, the histidine content of the fragment was not as high as that in the bovine fragments. Most of the histidine in human HMW-kininogen was recovered in the kinin-free protein, and the light chain of kinin-free protein was found to be rich in histidine compared with the heavy chain. These results suggest that the histidine-rich sequence in human HMW-kininogen is not released by the action of human plasma kallikrein, but remains bound to the light chain of kinin-free protein.


Synthesis of levan by levansucrase. Some factors affecting the rate of synthesis and degree of polymerization of levan. The addition of levan as a so-called "acceptor" accelerated the rate of polymerization of levan catalyzed by levansucrase [EC 2.4.1.10]. However, this effect was seen only under conditions of low ionic strength. Under conditions of high ionic strength, only levan with an average degree of polymerization (DP) of 120 was synthesized with a reasonable yield. Incorporation of [14C]fructose residues into levan of high molecular weight (DP 1,200) added as an "acceptor" at high ionic strength was slight. It is suggested that the DP of levan synthesized is generally regulated by ionic strength, and that enzymic synthesis of levan occurs in the absence of an "acceptor."


Effects of alpha- and beta-D-glucose on germination of spores of Bacillus megaterium QM B1551. The effects of D-glucose anomers on the germination of dormant spores of Bacillus megaterium QM B1551 were studied, alpha-D-Glucose (1 mM) slightly initiated the germination of the dormant spores during 10 min incubation at 37 degrees C, while about 60% of the dormant spores became germinated with beta-D-glucose (1 mM) in the same conditions. From the above observations and the finding that only a trace amount of alpha- or beta-D-glucose may bind with the dormant spores, it is speculated that the beta-D-glucose-stereospecific receptor site for the germination exists on the surface of the dormant spores of the bacillus.


Biochemical properties of a new flexuous bacteriocin, pyocin F1, produced by Pseudomonas aeruginosa. Pseudomonas aeruginosa strain P15 produces not only pyocin R1 and phage PS10, but also a substance having a flexuous rod structure, the nature of which is so far unknown. A variant strain (P15--40) was obtained which produced these flexuous particles more effectively than the original strain, and the particles were purified to homogeneity and investigated. Several strains of P. aeruginosa were found to be killed by the particles. It was concluded that the flexuous rod-like particles are not related to pyocin R1 or phage PS10, but represent a new pyocin, which we have designated as pyocin F1. Pyocin F1 showed a different action spectrum and a different pattern on SDS-polyacrylamide gel electrophoresis from either pyocin R1 or phage PS10. The killing activity of pyocin F1 was of single-hit type. The activity was not affected by anti-R1, anti-R1-core or anti-PS10, or by DNase, RNase, pronase or trypsin, but was completely destroyed by treatment at 70 degrees C for 10 min. Some cofactor was required for the adsorption of this pyocin on sensitive bacteria. Another flexuous bacteriocin was also found and named pyocin F2.


Stimulation of glycogen synthase phosphorylation by calcium-dependent regulator protein. Phosphorylation of skeletal muscle glycogen synthase catalyzed by a protein kinase is stimulated up to 10-fold by the calcium-dependent regulator (CDR) protein. Half-maximal stimulation requires about 1 microgram of CDR/ml. Phosphorylation by the CDR-dependent synthase kinase is more rapid at pH 8.6 than at pH 6.8 and is blocked by ethylene glycol bis(beta-aminoethyl-ether)N,N'-tetraacetic acid and trifuloperazine. Approximately 60 to 70% of the phosphate is incorporated into the trypsin-insensitive region of glycogen synthase resulting in conversion of the a form to the b form of the enzyme. The CDR-dependent synthase kinase is not myosin light chain kinase, as this enzyme does not phosphorylate glycogen synthase. Furthermore, synthase phosphorylation by the cAMP-dependent protein kinase catalytic subunit is not affected by CDR. The possibility that CDR-dependent synthase kinase may be phosphorylase kinase is being investigated.


The polysaccharides from heterocyst and spore envelopes of a blue-green alga. Structure of the basic repeating unit. The polysaccharides from the envelopes of heterocysts and spores of Anabaena cylindrica consist of repeating units containing 1 mannosyl and 3 glucosyl residues, all linked by beta(1 yields 3) glycosidic bonds, with glycosidic bonds, with glucose, xylose, galactose, and mannose present in side branches. Degradation of the polysaccharides with specific glycosidases has permitted identification of the linkages to almost all of the branches. When the polysaccharides, from which all but two types of side branches had been cleaved, were digested with a beta(1 yields 3) endoglucanase, glucose, a tri-, and a pentasaccharide were produced. The oligosaccharide products were identified as (see article of journal). The backbones of the polysaccharides were sequenced from the reducing terminus by a modified Smith degradation. Analysis with NaB3H4 at each stage of the degradation showed that the backbones terminate in the sequence Man-Glc-Glc-Glc and are therefore presumed to have the structure (Man-Glc-Glc-Glc)n, and that they contain an average of from 128 to 150 sugar residues. From the information obtained, the repeating sequences of the original polysaccharides from the two types of differentiated cells of A. cylindrica could be largely deduced and appeared to be identical.


Preparation of homogenous NADPH cytochrome c (P-450) reductase from house flies using affinity chromatography techniques. NADPH-cytochrome c (P-450) reductase (EC 1.6.2.4) was purified to apparent homogeneity from microsomes of house flies, Musca domestica L. The purification procedure involves column chromatography on three different resins. The key step in the purification scheme is the chromatography of the enzyme mixture on an affinity column of agarose-hexane-nicotinamide adenine dinucleotide phosphate. The enzyme has an estimated molecular weight of 83,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 1 mol each of FAD and FMN per mol of enzyme. The enzyme exhibited a Bi Bi ping-pong kinetic mechanism with NADPH and cytochrome c. The Vmax and Km for cytochrome c were 42.3 mumol min-1 mg-1 and 12.7 muM, respectively. Turnover numbers based on micromoles of enzyme were 2,600 min-1. NADP+ and 2'-AMP both inhibited the reductases with apparent Ki values of 6.9 and 187 muM, respectively. These preparations of NADPH-cytochrome c reductase were found to reduce purified house fly cytochrome P-450 in the presence of NADPH.


Structure of the high mannose oligosaccharides of a human IgM myeloma protein. I. The major oligosaccharides of the two high mannose glycopeptides. The structures of the predominant high mannose oligosaccharides present in a human IgM myeloma protein (Patient Wa) have been determined. The IgM glycopeptides, produced by pronase digestion, were fractionated on DEAE-cellulonalysis shows that glycopeptide I contains Asn, Pro, Ala, Thr, and His and glycopeptide II contains Asn, Val, and Ser, which are the same amino acids found in the sequences around Asn 402 and Asn 563 respectively, to which high mannose oligosaccharides are attached in IgM (Patient Ou) (Putnman, F.W., Florent, G., Paul, C., Shinoda, T., and Shimizu, A. (1973) Science 182, 287-290). The high mannose glycopeptides in IgM (Wa) exhibit heterogeneity in the oligosaccharide portion. Structural analysis of the major oligosaccharides indicates that the simplest structure is: (see article of journal). The larger oligosaccharides present have additional mannose residues linked alpha 1 yields 2 to terminal mannose residues in the above structure. Glycopeptide I contains primarily Man5 and Man6 species, while glycopeptide II contains Man6 and Man8 species. The two Man6 oligosaccharides have different branching patterns.


Prostaglandin hydroperoxidase, an integral part of prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes. The highly purified prostaglandin endoperoxide synthetase from bovine vesicular gland microsomes had two still unresolved enzyme activities; the oxygenative cyclization of 8,11,14-eicosatrienoic acid to produce prostaglandin G1 and the conversion of the 15-hydro-peroxide of prostaglandin G1 to a 15-hydroxyl group, producing prostaglandin H1. The latter enzymatic reaction required heme and was stimulated by a variety of compounds, including tryptophan, epinephrine, and guaiacol, but not by glutathione. A peroxidatic dehydrogenation was demonstrated with epinephrine or guaiacol in the presence of various hydroperoxides, including hydrogen peroxide and prostaglandin G1. Higher activity and affinity were observed with the 15-hydroperoxide of eicosapolyenoic acid, especially those with the prostaglandin structure. Both the dehydrogenation of epinephrine or guaiacol and the 15-hydroperoxide reduction of prostaglandin G1 were demonstrated in nearly stoichiometric quantities. With tryptophan, however, such a stoichiometric transformation was not observed. The peroxidase activity as followed with guaiacol and hydrogen peroxide and the tryptophan-stimulated conversion of prostaglandin G1 to H1 were not dissociable as examined by isoelectric focusing, heat treatment, pH profile, and heme specificity. The results suggest that the peroxidase with a broad substrate specificity is an integral part of prostaglandin endoperoxide synthetase which is responsible for the conversion of prostaglandin G1 to H1.


Phaseolus vulgaris isolectin binding to human erythrocytes. The Phaseolus vulgaris isolectins L4,L3E1, L2E2, L1E3, and E4 were isolated by affinity and ion exchange chromatography. Pure isolectins were radiolabeled by the chloramine-T method with Na125IO4 and their binding to human erythrocytes was studied. A normal erythrocyte has approximately 8 times 10(5) receptor sites for each isolectin; however, the association constants (Ka) of binding increased from 1.1 times 10(7) M-1 to 3.8 times 10(8) M-1, with increasing number of E subunits per tetrameric isolectin molecule. Isolectin to erythrocyte binding reached equilibrium rapidly and was reversed by fetuin. All isolectins competed with 125I-E4 for erythrocyte binding sites, with a constant (KI) similar to the Ka calculated for each respective radiolabeled isolectin. When isolectin binding at 0 degrees C, 4 degrees C, or 8 degrees C was compared to that at 25 degrees C, there was no reduction in the number of binding sites per cell, but the Ka of E4 was reduced to 3 times 10(7) M-1. Fixed erythrocytes displayed similar isolectin binding characteristics.


Characterization of the extracellular lipase of Bacillus subtilis and its relationship to a membrane-bound lipase found in a mutant strain. Bacillus subtilis CMK33 is a mutant that is more osmotically fragile than the wild type when it is converted to the protoplast form. The protoplasts of this mutant contain a membrane-bound lipase, which is not found in protoplasts of the wild type. Hydrolysis of the membrane lipid of mutant protoplasts by the lipase is the cause of their fragility. A protein found in the wild type organism specifically inhibits the lipase (Kent, C., and Lennarz, W. J. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 2793-2797). This paper reports that cultures of both mutant and wild type cells contain an extracellular lipase which accumulates during the logarithmic phase of growth. The extracellular activity appears to be induced by a component of the growth medium. The membrane-bound lipase of the mutant has been partially purified and its properties have been compared to those of the extracellular lipase of the wild type. Their properties and sensitivity to the wild type inhibitor are similar, which suggests that the two molecules are closely related. The subcellular location of the lipase in the mutant has been investigated and compared to the location of the membrane-bound portion of the lipase inhibitor in the wild type. The lipase is located almost exclusively in the cytoplasmic membrane and not in mesosomal vesicles. In contrast, the lipase inhibitor is located in both types of membranes and is more concentrated in mesosomal vesicles. Under appropriate conditions, the appearance of new extracellular lipase activity in mutant cultures is paralleled by the loss of an equivalent amount of lipase activity from protoplasts prepared from the cells. This suggests that the membrane-bound lipase may be an intermediate in the secretion of the extracellular lipase. Because of the mutation in B. subtilis CMK33, which results in the absence of the lipase inhibitor, this intermediate can be found in protoplasts of the mutant, although it is not detectable in the wild type. Consequently, the mutant may be useful in studies of the mechanism of secretion of exoenzymes by Bacilli.


Purification and characterization of urinary choriogonadotropin from patients with hydatidiform mole. Human choriogonadotropin was isolated from urine of patients with hydatidiform mole by acid and salt precipitation, immunoaffinity, and DEAE-Sephadex chromatography. Polyacrylamide gel electrophoresis, immunodiffusion, immunoelectrophoresis, and NH2-terminal amino acid analysis showed that the product obtained is essentially homogeneous. This choriogonadotropin was found to resemble the choriogonadotropin from urine of normal pregnant women in amino acid composition but to differ from it in having a lower content of N-acetylglucosamine and mannose.


The primary structure of Lactobacillus casei thymidylate synthetase. I. The isolation of cyanogen bromide peptides 1 through 5 and the complete amino acid sequence of CNBr 1, 2, 3, and 5. Thymidylate synthetase from Lactobacillus casei was S-carboxymethylated and degraded by treatment with cyanogen bromide. Although the protein contains 6 methionine residues, only 5 cyanogen bromide peptides were obtained due to the presence of 1 methionine on the NH2 terminus and another adjacent to a threonine residue which was resistant to cleavage. The peptides were isolated by differential extraction, first with ammonium acetate, then pyridine acetate, and finally the residue was solubilized with 50% acetic acid. Each peptide was further purified to homogeneity by Bio-Gel chromatography. The size of the peptides from the amino to carboxyl end of the enzyme subunit was CNBr 1, 4,100; CNBr 2, 10,300; CNBr 3, 8,100; CNBr 4, 11,800; CNBr 5, 2,200. The sum of the amino acid residues of the peptides is equal to the sum of the residues in an enzyme subunit, indicating that all of the CNBr peptides have been isolated. The CNBr-resistant methionine was located in CNBr 2 and the 5-fluoro-2'-deoxyuridine 5'-monophosphate binding site in CNBr 4. The holoenzyme molecular weight, based on the residue weights of the amino acids in the two equivalent subunits, is equal to 73,176. The complete sequence of each of the CNBr peptides, except for CNBr 4, which is presented in the following paper, is described.


The primary structure of Lactobacillus casei thymidylate synthetase. II. The complete amino acid sequence of the active site peptide, CNBr 4. The 102 amino acid residues of CNBr 4, the largest of 5 cyanogen bromide peptides from the Lactobacillus casei thymidylate synthetase were completely sequenced by means of limited tryptic, tryptic, chymotryptic, and staphylococcal protease peptides. CNBr 4 contains both of the cysteines in an enzyme subunit, with the 5-fluorodeoxyuridylate-reactive cysteine at residue 198 and the other at residue 244.


Vitamin K-dependent gamma-glutamyl carboxylase activity in the chick embryonic chorioallantoic membrane. During embryonic development of the chick, the onset of calcium transport by the chorioallantoic membrane (CAM) is concomitant with the appearance of a calcium-binding protein (CaBP). The development-specific expression of the CaBP in the CAM is inhibited by vitamin K antagonism in ovo with the anticoagulant, warfarin. However, the CaBP remains immunologically detectable in the CAM of warfarin-treated embryos, suggesting the presence of a precursor form of the CaBP. Previously, we have demonstrated that CaBP expression in CAM organ cultures is inducible by vitamin K. Furthermore, the CaBP contains several residues of the modified amino acid, gamma-carboxyglutamic acid (gamma-CGlu), which has been shown to be formed by vitamin K-dependent carboxylation of glutamic acid in several plasma clotting proteins. This study reports the presence of a post-translational, vitamin K-dependent gamma-glutamyl carboxylase activity in the CAM. Our results show that explants of CAM incorporate H14CO3 in an age-specific and vitamin K-dependent manner. Incorporation of H14CO3 by the CAM is further potentiated by warfarin treatment of the embryos, presumably owing to an elevation of the amount of endogenous uncarboxylated protein precursor(s). Among the subcellular (nuclear, mitochondrial, microsomal, and soluble) fractions of the CAM, only microsomes exhibit specific incorporation of of H14CO3 into gamma-CGlu. The CAM microsomal carboxylation activity is post-translational, vitamin K-dependent, specific for prenylated homologs of vitamin K, sensitive to warfarin, and appears to be unrelated to the activities of biotin-dependent carboxylases or phosphoenolpyruvate carboxykinase. Optimal carboxylation activity occurs after incubation of the microsomes with H14CO3 for 60 min at 37 degrees C in the presence of over 100 microgram of vitamin K1/ml.


Purification and characterization of monkey salivary mucin. Highly purified mucin was prepared from monkey (Macaca arctoides) extraparotid saliva by sequential chromatography on Sephadex G-200 (followed by reduction and alkylation of void volume materials), Sepharose CL-2B with 6 M urea, and CM52 cellulose with 6 M urea. Purity was critically ascertained by anion exchange chromatography, ultracentrifugal analysis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide electrophoresis, and crossed immunoelectrophoresis. Use of crossed immunoelectrophoresis to examine mucin preparations has not been previously reported. This technique was useful for assessing purity and displaying charge and size microheterogeneity in the purified S-carboxymethylated mucin. Threonine and serine comprised 37.8% of the total amino acids while the oligosaccharide moiety contained N-acetyl-glucosamine, N-acetylgalactosamine, fucose, galactose, N-acetylneuraminic acid, and sulfate. Following alkaline borohydride treatment, the carbohydrate chains were found to be linked O-glycosidically between N-acetylgalactosamine and threonine (serine).


Rapid, sensitive and specific electron capture-gas chromatographic method for the quantitation of 6-chloro-2-(1-piperazinyl) pyrazine in biological fluids. A highly specific and sensitive gas chromatographic method for the determination of 6-chloro-2-(1-piperazinyl)pyrazine (MK-212), a central serotonin-like agent, in biological fluids is described. MK-212 and a related internal standard are extracted into benzene from an alkaline solution, back-extracted into acid and then re-extracted into benzene at an alkaline pH. The amines are converted to the trifluoroacetyl derivatives (characterized by gas-liquid chromatography-mass spectrometry), chromatographed and detected with a 63Ni electron capture detector. The sensitivity of the method is such that 10 ng of drug can be measured per aliquot of biological fluid. The precision and accuracy of the method are well within acceptable limits. Specificity of analysis was established by gas-liquid chromatography-mass spectrometry techniques.


A study of human leukocyte D locus related antigens in Graves' disease. An association between Graves' disease and the human leukocyte antigen (HLA) system has previously been reported. The disease was more strongly associated with the HLA D locus antigen Dw3 than with HLA B8. Products of the HLA D locus are determined by the interaction of test cells with standard typing lymphocytes, a technically difficult procedure. Recently, it has been possible to type serologically for D locus related (DRw) specificities on peripheral bone marrow-derived (B) lymphocytes. Blood B lymphocytes from 50 unrelated controls and 41 patients with Graves' disease were typed for seven HLA DRw specificities. 28 patients with Graves' disease (68%) were positive for DRw3, in contrast to 14 controls (28%); whereas only 21 patients (50%) were HLA B8 positive, compared with 13 (26%) controls. Thus, positivity for DRw3 afforded a relative risk for Graves' disease of 5.5, whereas that for HLA B8 amounted to 3.0. Additionally, a family with multiple cases of Graves' disease in which the disease was previously shown to be inherited with the haplotype, was linked to DRw2, which suggests that the susceptibility to the disease was inherited in association with that antigen. Two HLA B/glyoxalase recombination events were observed in this family; in both instances HLA DRw followed HLA B. This study thus demonstrates that the disease susceptibility gene for Graves' disease is in strong linkage disequilibrium with DRw3; however, it may be associated with other DRw specificities and inherited within family units in association with them.


Enzyme replacement therapy by fibroblast transplantation: long-term biochemical study in three cases of Hunter's syndrome. We have assessed the effectiveness of transplanted histocompatible fibroblasts as a long-lived source of lysosomal enzymes for replacement therapy in three patients with Hunter's syndrome, over periods ranging from 2.5 to 3.75 yr. The level of Hunter corrective factor excreted by all three patients increased after transplantation, as did the activity of alpha-L-idurono-2-sulfate sulfatase in serum, when measured directly with a radioactive disulfated disaccharide substrate. Sulfatase activity was also raised in leukocyte homogenates from the two patients that we were able to assess. These increases in enzyme activity were accompanied by corresponding increases in catabolism of heparan and dermatan sulfates, as shown by (a) a decrease in sulfate:uronic ratios of urinary oligosaccharides, (b) an increase in iduronic acid monosaccharide, and (c) a normalization of Bio-Gel P-2 gel filtration profiles. Both the increase in enzyme activity and increased catabolism were maintained during the period of study and were not affected by either a gradual decrease or total withdrawal of immunosuppressive therapy.


Aspirin inhibits development of coronary atherosclerosis in cynomolgus monkeys (Macaca fascicularis) fed an atherogenic diet. The effect of aspirin in the primary prevention of diet-induced atherogenesis in cynomolgus monkeys was studied. The diet consisted of 2% cholesterol and 10% butter by weight for 24 wk. Six monkeys received only the atherogenic diet and five monkeys received the diet plus aspirin, 81 mg/monkey per day. Aspirin did not affect plasma cholesterol levels or aortic atherosclerosis. Platelet aggregation to arachidonic acid was almost completely suppressed. Aspirin decreased significantly the number of coronary vessels with atherosclerotic involvement, and the number of coronary vessels narrowed by 20% or more. Thus, aspirin appears to exert a protective effect in the primary prevention of diet-induced coronary atherosclerosis in a primate model.


Turnover of human and monkey plasma kininogens in rhesus monkeys. The normal metabolic turnover of plasma kininogens was studied by measuring the disappearance of intravenously administered radiolabeled human and monkey plasma kininogens from the circulation of healthy adult rhesus monkeys. Curves obtained by plotting log radioactivity against time could be expressed as double exponential equations, with the first term representing diffusion, and the second, catabolism. No significant difference between the turnovers of human and monkey kininogens was observed. The difference between the t1/2 of high molecular weight kininogen (25.95 +/- 1.60 h) (mean +/- SEM) and that of low molecular weight kininogen (18.94 +/- 1.93 h) was only marginally significant (P  less than 0.05). In contrast, a highly significant (P less than 0.001) difference in their mean catabolic rates (1.12 +/- 0.08 d-1 for high molecular weight kininogen vs. 2.07 +/- 0.09 d-1 for low molecular weight kininogen) was observed.  These differences between the two kininogens were attributed to differences in their distribution between the intra- and extravascular pools. Studies of kininogen turnover will be useful in elucidating the in vivo functions of the various kininogens in health as well as during clinical illness.


Pitfalls in the use of artificial substrates for the diagnosis of Gaucher's disease. A patient with Gaucher's disease is described, in whom the disease could not be diagnosed enzymically in liver and leucocytes using artificial substrate 4-methylumbelliferyl beta-glucoside. Normal activity was found in the liver, and about 60% of control activity was determined in the patient's leucocytes. In contrast, when [14C]-N-stearoyl glucocerebroside was employed as a substrate, activity as low as 5% of control has been found in all the proband's tissues, and carrier levels were determined in the proband's parents and maternal uncle.


Assessment of the efficacy of interventions to limit ischemic injury by direct measurement of intramural carbon dioxide tension after coronary artery occlusion in the dog. Although numerous interventions have been shown to exert a salutary effect on the ischemic myocardium, the severity of ischemia generally has been measured by indirect techniques. In the present investigation the effect of ischemia on intramural carbon dioxide tension (PmCO(2)) was measured directly in the open-chest, anesthetized dog with a mass spectrometer during repetitive 10-min coronary artery occlusions separated by 45-min periods of reflow; simultaneously, regional myocardial blood flow in the ischemic area was measured by (127)Xenon washout. In all dogs the increase in PmCO(2) from before to 10 min after the first occlusion (DeltaPmCO(2)) exceeded that during subsequent occlusions. In those dogs not receiving an intervention (controls), DeltaPmCO(2) during the third occlusion was similar to that during the second occlusion. When propranolol, hyaluronidase, and nitroglycerin were administered to different groups of dogs before the third occlusion, each caused significantly smaller elevations in DeltaPmCO(2) than those occurring during the control second occlusion, and the combination of all three interventions induced the smallest increase in DeltaPmCO(2). Regional myocardial blood flow rose with hyaluronidase and was unchanged with propranolol, nitroglycerin, and the three drugs in combination. In contrast to these beneficial interventions, isoproterenol infused with the third occlusion caused a higher DeltaPmCO(2) than during the control second occlusion. It is concluded, first, that interventions that modify the severity of ischemia can be evaluated by measuring intramural carbon dioxide tension; second, that propranolol, hyaluronidase, and nitroglycerin reduce ischemic injury, whereas isoproterenol increases it; and third, that the combination of propranolol, hyaluronidase, and nitroglycerin exerts an additive beneficial effect on ischemia.


Studies of vasospasm. II. The use of nitroglycerin cigarettes. It was discovered that smoking a cigarette containing nitroglycerin produced identical action on the peripheral pulses and relieved angina pectoris in the same way as sublingual administration. A group of 73 patients with various vascular conditions was subjected to 121 acute tests using a cigarette containing 0.0012 Gm (1/50th grain) nitroglycerin. Thirteen patients smoked cigarettes containing 0.0004 Gm (1/150th grain) nitroglycerin six to 20 times daily for periods up to two years. This method of using the drug assures frequent application of small doses and gives a nearly continuous nitroglycerin effect.


An autoradiographic study of the efferent connections of the lateral hypothalamic area in the rat. The efferent connections of the lateral hypothalamic area (LHA) have been analyzed in a series of 30 rat brains with injections of 3H-amino acids into different parts of the area and the surrounding regions. Our findings indicate that all parts of the LHA contribute ascending and descending fibers to the medial forebrain bundle, and also project medially to certain of the adjoining hypothalamic nuclei. All levels of the LHA appear to send some fibers to a continuous group of structures that extends from the medial septal-diagonal band complex rostrally, through the lateral preoptic and lateral hypothalamic areas to the mammillary complex and the ventral tegmental area caudally. In addition, it is evident that cells at different levels within the LHA may have differential projections. Thus, the anterior and lateral parts of the LHA also appear to project substantially to the anterior hypothalamic area, the ventromedial and dorsomedial hypothalamic nuclei, the parataenial and paraventricular nuclei of the thalamus, and the medial part of the lateral habenular nucleus. Similarly, cells in the tuberal and posterior parts of the LHA project to the central gray, the longest projections from the posterior region reaching as far caudally as the central tegmental field, the parabrachial nucleus, the locus coeruleus, and the superior central and dorsal nuclei of the raphe. Viewed as a whole, the LHA is therefore well-suited to integrate inputs from the limbic system and brainstem and to relay them on the one hand to the medial zone of the hypothalamus and on the other to virtually every structure closely associated with the medial forebrain bundle and to the nuclei of origin of the major ascending monoaminergic systems.


The organization of feline entopenduncular nucleus projections: anatomical studies. The organization of entopeduncular nucleus (EPN) projections was studied in cats using autoradiographic and horseradish peroxidase (HRP) techniques. In autoradiographic studies, EPN axons were found to terminate in a J-shpaed region in the dorsal and medial part of the ventral anterior nucleus (VA) and the rostral portion of the adjacent ventral lateral nucleus (VL). EPN axons also terminated in the rostral portion of the centrum medianum (CM), the ventrolateral portion of the lateral habenular nucleus (LHB), and the pedunculopontine nucleus (PP). The VA included the largest terminal field although the LHB had the greatest density of terminals. Regardless of the region of EPN into which amino acids were injected, the terminal fields were the same: there was no localization within the EPN of the cells projecting to one region. HRP-containing cells were distributed throughout the EPN following injections into the VA, LHB, or PP, although many more cells were labeled following injections into either VA or LHB than PP. EPN cells containing HRP following injections into either VA or LHB were not morphologically different from those not containing HRP in the same respective animals. Following HRP injections into stria medullaris, only cells in the rostral part of the EPN were labeled, providing evidence that rostrally and caudally located EPN neurons have different paths to LHB. Although there may be a rostrocaudal organization of pathways to LHB, individual regions of the nucleus project to the same areas.


The effect of levamisole on DMBA-induced carcinogenesis in the hamster cheek pouch. The effect of levamisole on DMBA-induced carcinogenesis in the cheek pouch of the hamster was studied. Findings indicate that levamisole modified the response of the pouch to DMBA at six weeks, while this effect was not maintained after 12 weeks of DMBA application.


Serologic reactions of oral gram negative anaerobic bacilli. Serological reactions were performed using hyperimmune rabbit antisera and antigenic preparations of Leptotrichia buccalis, Fusobacterium fusiforme, and Fusobacterium polymorphum. All tests indicated that there was serologic cross reactivity between the two Fusobacterium species. No cross reactivity could be detected between the Fusobacterium species and L. buccalis. The findings suggest that F. fusiform and F. polymorphum are similar in their immunogenicity, and that the grouping of these two organisms as F. nucleatum may be justified.


Comparative enamel histology of primate teeth. The use of nonhuman primates as experimental models has markedly increased during the past decade. However, very little is known about primate dental tissues, especially enamel. Comparative studies of the enamel from 25 primate species reveal a wide variation in (1) the thickness of enamel, (2) microstructure and ultrastructure, and (3) the species' capacity to respond to systemic stress. These data provide a baseline of information presently not available to the dental researcher.


Human dentition and experimental animals. A review of the recent literature concerning enamel maturation patterns following cessation of protein matrix secretion confirms that developing rodent enamel exhibits an overall mineral distribution different from that of developing enamel in man, monkey, dog, and swine. This seems to be a result of structural differences of the enamel as the pattern of crystal growth appears similar in these species. Whether the differences in enamel structure are reflected in significant differences in cellular events leading to enamel formation and mineralization remain unknown, as detailed radioautographic and ultrastructural studies almost always have dealt with rodent teeth. It is therefore concluded that such studies should be initiated in experimental animals having an enamel formation and mineralization pattern strictly comparable to that of man. Some requirements for such an experimental animal are suggested.


Planning, implementation, and evaluation of a fortification program. Control of vitamin A deficiency in the Philippines. In a three-year pilot project in the Philippines, the magnitude of vitamin A deficiency and its clinical manifestation (xerophthalmia) was determined, and three alternate programs for eliminating and preventing it in various ecologic zones were designed and implemented concurrently in separate areas in each ecologic zone. Results were evaluated, and costs and benefits of each program were determined. The results of the fortification program are reported. Monosodium glutamate (MSG) was selected as the ideal carrier was fortified at a level which provided 15,000 I.U. retinol palmitate to the average family each day. Significant increases in serum A, especially for children with more deficient vitamin A status, resulted. The program's economic benefits significantly outweighted the costs, and the MSG fortification program has been expanded to several additional pilot provinces in the Philippines.


Cross-modality: delivery of health services through non-professionals. The cross-modality (C-M) system of health care delivery was developed as one response to numerous societal requirements for better health care delivery. A pilot program is described which established C-M program in several institutions for the mentally retarded, using attendant staff as "primary deliverers of service." While the purpose of the program was to demonstrate the improved effectiveness of care, the additional cost-benefits of a C-M system appear significant. Dietetic literature supports the appropriateness of delivering certain dietetic services in selected environments through such a system.


Medical care evaluation studies in long-term care facilities. This report describes the selection, design, conduct, analysis, and application of medical care evaluation studies in long-term care facilities (skilled nursing homes) in a regional program in the Rochester region of upstate New York. Eight examples are presented to highlight methodologic approaches and problems. They are classified under four general headings: Administration Audits, Diagnosis-specific Studies, Care Modality-specific Studies, and General Outcome Indicators. The implementation of results and recommendations from the studies is discussed and an application of "tracer" methodology for assessing the components of care activities in long-term facilities is described. Problems and challenges in long-term quality care are outlined.


[Qualitative and quantitative studies on the lateral geniculate body in an ontogenic series of male Tupaia belangeri]. The cyto-, fibrillo- and myeloarchitectonics of the dorsal and ventral lateral geniculate body are described in an ontogenetic series of male Tupaia belangeri using light microscopy. The qualitative maturational changes show a heterochronical development. The ventral nucleus is developing earlier than the dorsal nucleus. A quantitative analysis of the fresh volume growth of the two brain regions also shows a heterochronical development. Two different kinds of growth curves were found. The dorsal lateral geniculate body has an overshooting growth which is approximated by the 6-parametric growth function: formula: (see text), The ventral lateral geniculate body shows a monotonously increasing s-shaped growth curve which is approximated by the generalized logistic growth function: formula: (see text), An analysis of the fresh volume growth of the six layers of the dorsal lateral geniculate body demonstrates all layers to take part in the overshooting growth type.


The specificity of the anti-haemagglutinin antibody response induced in man by inactivated influenza vaccines and by natural infection. The anti-haemagglutinin antibody response in adult human volunteers to inactivated whole virus or tween ether split influenza A/Victoria/75 (H3N2) and A/Scotland/74 (H3N2) virus vaccines was investigated using antibody absorption and single-radial-haemolysis (SRH) techniques. The concentrations of haemagglutinin (HA), nucleoprotein (NP) and matrix (M) antigens measured by single radial diffusion (SRD) and rocket immunoelectrophoresis were similar for both the whole virus and split vaccines. Whole virus and split vaccines induced crossreactive (CR) antibody in 87% of vaccinees. Strain specific (SS) antibody to A/Hong Kong/1/68 of the homologous virus was induced less frequently than CR antibody. Higher anti-haemagglutinin antibody titres were detected in persons receiving the split virus vaccines than in those receiving the whole virus vaccines. No antibody to the type-specific matrix protein was detectable, but 33% of volunteers developed an antibody rise to type-specific nucleoprotein antigen. The specificity of the anti-haemagglutinin antibody response in human adults to natural infection with A/Port Chalmers/73 (H3N2) virus was similar to that induced by inactivated vaccines in that a high proportion of subjects developed CR anti-haemagglutinin antibody, which reacted with A/Hong Kong/68 virus and the homologous A/Port Chalmers/73 virus, and SS antibody for A/Hong Kong/68 virus but SS antibody for A/Port Chalmers/73 virus was infrequently stimulated by natural infection.


Isolation of a polysaccharide antigen from Schistosoma mansoni eggs. A polysaccharide antigen was isolated from Schistosoma mansoni egg homogenates by the phenol procedure. The crude preparation (CPEA) contained at least two antigens. The more purified antigen (PEA) was isolated by sequential enzymatic treatment of CPEA with DNase, RNase, Pronase, and alpha-amylase. PEA was resistant to boiling, freezing and thawing, mild acid and alkali, and chloroform, but was destroyed with periodate. It gave a positive reaction with anthrone reagent. PEA was eluted in the wash fraction from a DEAE cellulose collumn and in the void volume of a Sephacryl 200 column. After immunoelectrophoresis and polyacrylamide electrophoresis there was little or no migration. Amino acid analysis failed to reveal ninhydrin-positive material in the a hydrolyzate of PEA. These resluts suggested that PEA is a neutral polysaccharide with a m.w. of more than 200,000 and contains no amino acids or hexosamine. Antibodies against PEA were detected in sera obtained from mice infected with S. mansoni. PEA is different from previously described antigens derived from schistosome eggs.


Comparisons of myeloma proteins from NZB and BALB/c mice: structural and functional differences of heavy chains. The N-terminal sequences from heavy variable regions of 47 myeloma proteins of the NZB mouse have been analyzed. Sixteen of these VH regions have unblocked alpha amino groups and have been analyzed over their N-terminal 20 residues by automatic sequence analysis. These sequence data along with the antigen-binding profiles and immunoglobulin class distribution are compared with comparable data from BALB/c myeloma proteins. These comparisons suggest that the NZB and BALB/c populations of myeloma proteins are distinct from one another. The genetic implications of this conclusion are discussed.


Expression of an unidentified immunoglobulin isotype on rabbit Ig-bearing lymphocytes. A non-IgM immunoglobulin molecule was found on most rabbit Ig-bearing lymphocytes isolated from mesenteric lymph nodes. Membrane bound immunoglobulin light chains and heavy chains were detected by immunofluorescence and by rosetting with antibody-coated erythrocytes on mesenteric lymph node cells stripped of IgM by anti-IgM allotype antibodies. The percentage of cells bearing these residual immunoglobulin molecules was similar to the percentage of cells bearing immunoglobulin before "stripping" with anti-IgM antibody. These residual immunoglobulin molecules were not IgA nor IgG and are believed to be the rabbit analogue of human IgD.


An interpretation of the apparent dual specificity of some murine myeloma immunoglobulins with inulinbinding activity. Five murine myeloma immunoglobulins have been studied for their binding with fructose-containing homopolysaccharides. The results indicate that the anti-(2 leads to 1)-D-fructofuranans are capable of binding (2 leads to 6)-D-fructofuranans with reduced affinity. An anti-(2 leads to 6)-D-fructofuranan (U10) was incapable of binding a (2 leads to 1)-D-fructofuranan. By using molecular models of fructofuranans of both linkage types, and a hypothetical molecular model of the combining region of E109, an anti-(2 leads to 1)-D-fructofuranan, a rationalization of these date can be proposed.


Bullous pemphigoid antigen. II. Isolation from the urine of a patient. This report concerns the isolation of bullous pemphigoid antigen from the nondialyzable urinary components of a patient with the disease. The isolation was accomplished by ion exchange chromatography and gel filtration. Pemphigoid antigen was found to be a basic glycoprotein that on SDS gel electrophoresis showed two major bands, one in the 18,000 m. w. region and the second with a m. w. of 74,000. Between these two bands, two additional bands appeared; one of 35,000 daltons and the other of 68,000 daltons. The 18,000 m. w. band was eluted from the gel and rerun on SDS gels. These gels showed the 18,000 m.w. band and also the appearance of the 35,000 and 74,000 m.w. bands. This finding indicates that urinary pemphigoid antigen may exist both as a single monomeric form and in polymeric aggregates.


Immunological cross-reactivity between a glycoprotein isolated from Trichophyton mentagrophytes and human isoantigen A. A glycoprotein isolated from the cell wall of Trichophyton mentagrophytes was assessed for its cross-reaction with human blood group isoantigens. Rabbit antiglycoprotein antibodies agglutinated human erythrocytes of blood groups A1 and A2, and precipitated Blood Group Substance A in agarose gels. Erythrocytes of blood group B were only slightly agglutinated, and O(Rho+) and O(Rho-) erythrocytes were not. Additionally, the glycoprotein was shown to specifically inhibit isoagglutination of erythrocytes of group A. Partial identity between the glycoprotein and a crude extract of the fungus was demonstrated by immunodiffusion. Analyses revealed the glycoprotein to be composed of approximately 17% protein and 80% carbohydrate. The glycoprotein was found by indirect immunofluorescence to be located in the mycelial cell wall. The possibility that cross-reacting antigens may lead to a chronic, spreading infection is discussed.


Antibody responses of human infants to three doses of group A Neisseria meningitidis polysaccharide vaccine administered at two, four, and six months of age. Infants were immunized with group A Neisseria meningitidis polysaccharide vaccine at two, four, and six months of age. Two lots of group A vaccine that differed in molecular weight were used; lot no. 1980A was of significantly larger molecular size than lot no. A-7. No significant adverse reactions occurred. The geometric mean concentration of serum antibody to group A N. meningitidis one month after the third dose of lot no. 1980A was 0.89 microgram/ml, nearly twice the concentration induced by lot no. A-7 (0.48 microgram/ml). When the infants were 12 months of age, concentrations of antibody in both groups had declined to similar levels, which were still greater than the mean concentration of antibody in unimmunized children. By 18 months of age, the antibody levels of immunized and unimmunized infants were the same. The three-dose schedule resulted in significantly lower concentrations of antibody than previously studied schedules of two doses given three to four months apart.