Whole-genome random sequencing and assembly of Haemophilus influenzae Rd.

An approach for genome analysis based on sequencing and assembly of unselected pieces of DNA from the whole chromosome has been applied to obtain the complete nucleotide sequence (1,830,137 base pairs) of the genome from the bacterium Haemophilus influenzae Rd. This approach eliminates the need for initial mapping efforts and is therefore applicable to the vast array of microbial species for which genome maps are unavailable. The H. influenzae Rd genome sequence (Genome Sequence DataBase accession number L42023) represents the only complete genome sequence from a free-living organism.

The minimal gene complement of Mycoplasma genitalium.

The complete nucleotide sequence (580,070 base pairs) of the Mycoplasma genitalium genome, the smallest known genome of any free-living organism, has been determined by whole-genome random sequencing and assembly. A total of only 470 predicted coding regions were identified that include genes required for DNA replication, transcription and translation, DNA repair, cellular transport, and energy metabolism. Comparison of this genome to that of Haemophilus influenzae suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms.

Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii.

The complete 1.66-megabase pair genome sequence of an autotrophic archaeon, Methanococcus jannaschii, and its 58- and 16-kilobase pair extrachromosomal elements have been determined by whole-genome random sequencing. A total of 1738 predicted protein-coding genes were identified; however, only a minority of these (38 percent) could be assigned a putative cellular role with high confidence. Although the majority of genes related to energy production, cell division, and metabolism in M. jannaschii are most similar to those found in Bacteria, most of the genes involved in transcription, translation, and replication in M. jannaschii are more similar to those found in Eukaryotes.

Interaction of bovine erythrocyte N-glycolylneuraminic acid-containing gangliosides and glycoproteins with a human Hanganutziu-Deicher serum.

A human Hanganutziu-Deicher (H-D) serum reacted with N-glycolylneuraminic acid (NeuGc)-containing gangliosides and glycoproteins isolated from bovine erythrocyte membranes. Three populations of H-D antibodies were identified in the human H-D serum. One population very likely recognized the NeuGc-Gal sequence; a second population appears to recognize additional sugars in the oligosaccharide sequence, e.g. NeuGc-Gal-GlcNAc; while a third population may also recognize polypeptide determinants in addition to the NeuGc-Gal-GlcNAc sequence. The H-D serum distinguished two high mol. wt glycoproteins (HMGP I and II) present in crude extracts of bovine erythrocyte membranes. These glycoproteins were separated by repetitive fractionation on Sephacryl S-1000 in the presence of urea and their composition determined.

Bone scintigraphy in lung cancer: a reappraisal.

The prognostic significance of bone scintigraphy was investigated by following 587 consecutive patients with lung cancer, in whom this investigation had been performed, for up to 9 years, or until death. Survival was unrelated to age, sex or cell type. However, pain and abnormal bone scintigraphy were both independently associated with a significantly reduced survival compared with those who were free of pain or who had normal bone scintigraphy. These factors were cumulative. The association remained equally valid for all cell types. Claims that a single metastasis is not prognostically significant are unfounded. It is suggested that the results of some chemotherapy trials must be reconsidered in the light of present findings, because of the lack of adequate control groups; the results could be construed to show a beneficial effect only in patients with bone metastases and a poor prognosis, but little or no effect in patients with normal bone scintigraphy. As judged by clinical and radiological follow-up and post-mortem examination, skeletal scintigraphy in patients with lung cancer had a sensitivity of 0.89, a non-specificity (false positives/true negatives) of 0.00 and an accuracy of 0.78. With existing radiopharmaceuticals there is an irreducible residue of false negatives due to deposits which provoke little or no osteoblastic response. Bone scintigraphy is, thus, indicated in any patient with lung cancer with unexplained symptoms and whenever staging is required, because of the prognostic implications. It should precede other staging investigations because the high detection rate may render other tests unnecessary.

Hydrolysis of native poly(hydroxybutyrate) granules (PHB), crystalline PHB, and artificial amorphous PHB granules by intracellular and extracellular depolymerases.

Native poly(hydroxybutyrate) (PHB) granules, purified PHB and artificial amorphous PHB granules were examined as putative substrates for hydrolysis by the intracellular depolymerase system of Rhodospirillum rubrum and the extracellular depolymerase of Pseudomonas lemoignei. The R. rubrum depolymerizing system requires pretreatment of granules with a heat stable 'activator' fraction; the activator can be replaced by mild trypsin treatment. Artificial granules were prepared with a cationic detergent, cetyltrimethylammonium bromide (CTAB) and an anionic detergent, (sodium cholate). Cholate and CTAB PHB granules were hydrolyzed by both enzyme systems; however, some differences were noted. Cholate granules were hydrolyzed in the absence of the R. rubrum activator fraction. Activator was required for the hydrolysis of CTAB granules but could be replaced by heparin in the extracellular depolymerase system but not in the intracellular depolymerase system. A Triton X-114 extract of native PHB granules inhibited the hydrolysis of trypsin-activated granules by the intracellular depolymerase. The inhibition was reversed by the activator fraction. Detergent extracts of granules activated with the R. rubrum activator were unable to inhibit the hydrolysis of trypsin-activated granules. These data suggest that the activator acts to modify an inhibitor present on native granules.

Alterations in glycosphingolipid patterns in a line of African green monkey kidney cells infected with herpesvirus.

The major glycosphingolipids (GSLs) of a line of African green monkey kidney cells (BGM) were characterized as glucosylceramide, lactosylceramide, galactosyl-galactosyl-glucosylceramide, and N-acetylgalactosaminyl-galactosyl-galactosyl-glucosylceramide. Neutral GSLs accounted for approximately 80% of the total GSLs isolated. The predominant gangliosides were N-acetylneuraminyl-galactosyl-glucosylceramide, N-acetylgalactosaminyl-N-acetylneuraminyl-galactosyl- glucosylceramide, and galactosyl-N-acetylgalactosaminyl-N-acetylneuraminyl -galactosyl-glucosylceramide. The incorporation of labeled galactose into GSLs was compared in mock-infected and herpes simplex virus type 1-infected BGM cells. Herpes simplex virus type 1 infection resulted in a three- to four-fold increase in galactose incorporation into glucosylceramide and a decrease in galactose incorporation into galactosyl-galactosyl-glucosylceramide and N-acetyl-galactosaminyl-galactosyl-galactosyl-glucosylceramide. The virus-induced alteration in the GSL labeling pattern occurred early in infection, before the release of infectious virus, and was not prevented by the presence of cytosine arabinoside. Treatment of uninfected BGM cells with cycloheximide resulted in alterations in the GSL pattern which were similar to those observed in herpes simplex virus type 1-infected cells. These observations suggest that an early virus function such as inhibition of host cell protein synthesis is responsible for the observed alterations of GSL metabolism. Experiments with a syncytium-producing strain of herpes simplex virus type 1, herpes simplex virus type 2, and pseudorabies virus indicated that other herpes viruses altered GSL metabolism in a manner similar to herpes simplex virus type 1.

Identification of propionate as an endogenous CO2 acceptor in Rhodospirillum rubrum and properties of purified propionyl-coenzyme A carboxylase.

A heat-stable endogenous CO(2) acceptor has been found in extracts of Rhodospirillum rubrum grown photoheterotrophically on acetate. Evidence is presented which suggests that this factor is propionic acid. Thus, paper and gas chromatographic analyses have indicated that propionic acid is present in boiled extracts prepared from R. rubrum cells. The products of (14)CO(2) fixation obtained with either the boiled extract or propionic acid as the CO(2) acceptor were identical and were identified as methylmalonic acid and succinic acid by paper chromatography. The enzyme which catalyzes the carboxylation of propionyl-coenzyme A (propionyl-CoA carboxylase) was purified from R. rubrum cells grown on acetate and its properties were studied. The enzyme is similar to propionyl-CoA carboxylases isolated from mammalian sources.

Metabolism of poly- -hydroxybutyrate: effect of mild alkaline extraction on native poly- -hydroxybutyrate granules.

Mild alkaline extraction of native poly-beta-hydroxybutyrate (PHB) granules results in the solubilization of a protein fraction. Both the solubilized protein fraction and the extracted granules are essentially devoid of PHB synthetase activity unless recombined. The protein fraction has been separated by chromatography into two components (A-I and A-II). A-I but not A-II can be recombined with extracted granules to give rise to PHB synthetase activity. Extracted granules no longer require pretreatment with activator or trypsin but are directly susceptible to hydrolysis by Rhodospirillum rubrum depolymerase. Addition of A-II or A-I prevents the direct hydrolysis by depolymerase. The inhibition is reversed by activator or trypsin. We conclude that native granules are associated with a protein inhibitor which prevents the hydrolysis of PHB by depolymerase unless the protein is destroyed by trypsin, removed by alkaline extraction, or modified by activator.

Extracellular enzyme secretion by Pseudomonas lemoignei.

The ability of succinate to repress the secretion of Pseudomonas lemoignei poly-beta-hydroxybutyrate depolymerase was a function of pH. Repression only occurred when the pH of the medium was 7.0 or less. At a higher pH, lack of sensitivity to succinate concentration may have been due to a limited ability to transport succinate. Actively secreting cultures (at pH 7.4) continued to secrete enzyme for approximately 30 min after the pH was rapidly decreased to pH 6.8, even though sufficient succinate was present to repress enzyme synthesis. Similarly, after the addition of rifampin to secreting cultures, there was a 30-min delay before secretion was inhibited. Evidence is presented which suggests that continued secretion may be the result of depolymerase messenger ribonucleic acid accumulation within the cells. Studies with chloramphenicol indicated that de novo protein synthesis is necessary for the secretion of poly-beta-hydroxybutyrate depolymerase and that exoenzyme is not released from a preformed pool. Studies with various inhibitors of protein synthesis indicated that synthesis of exoenzyme is 5 to 10 times more susceptible to inhibition than is the synthesis of cell-associated proteins.

Observations on the natural history of Paget's disease.

Whole-body skeletal scintigraphy was performed in 4700 adult patients from south-east Scotland. In 3831 with proven or suspected malignant disease the prevalence of osteitis deformans in males is 0.006 in those aged 15-54 years, 0.026 in the age group 55-84 years and 0.24 over the age of 84 years. The corresponding figures in females are 0.002, 0.021 and 0.15. Previous estimates of prevalence should be increased by at least 25% to take account of peripheral and monostotic disease, which is more common than hitherto recognised. The increase in prevalence with age may not be linear. Possible associations with environmental and genetic factors which would account for such a distribution are considered. It is suggested that the conventional distinction between 'active' and 'burned out' Paget's disease may be incorrect.

Effect of oral immunization with Pseudomonas aeruginosa on the development of specific antibacterial immunity in the lungs.

Oral administration of Pseudomonas aeruginosa to rats elicited a systemic and mucosal antibody response in the intestinal and respiratory tracts. The antibody response did not influence the course of the disease when immunized animals were subsequently challenged by the introduction of viable bacteria into the lungs.

Characterization of the Hanganutziu-Deicher (serum-sickness) antigen as gangliosides containing n-glycolylneuraminic acid.

Gangliosides that possess the same sugar sequence but differing in the type of sialic acid (N-acetyl- or N-glycolylneuraminic acid) were compared for their reactivity with Hanganutziu-Deicher ('serum sickness') antibodies by double-diffusion gel precipitation tests. Only N-glycolylneuraminic acid containing gangliosides formed precipitation lines with Hanganutziu-Deicher antibodies, thus suggesting that Hanganutziu-Deicher antigens are gangliosides that contain N-glycolylneuraminic acid.

Isolation of dicarboxylic acid- and glucose-binding proteins from Pseudomonas aeruginosa.

Inducible binding proteins for C4-dicarboxylic acids (DBP) and glucose (GBP) were isolated from Pseudomonas aeruginosa by extraction of exponential-phase cells with 0.2 M MgC12 (pH 8.5) and by an osmotic shock procedure without affecting cell viability. DBP synthesis was induced by growth on aspartate, alpha-ketoglutarate, succinate, fumarate, malate, and malonate but not by growth on acetate, citrate, pyruvate, or glucose. Binding of succinate by DBP was competitively inhibited by 10-fold concentrations of fumarate and malate but not by a variety of related substances. GBP synthesis and transport of methyl alpha-glucoside by whole cells were induced by growth on glucose or pyruvate plus galactose, 2-deoxyglucose, or methyl alpha-glucoside but not by growth on gluconate, succinate, acetate, or pyruvate. The binding of radioactive glucose by GBP was significantly inhibited by 10-fold concentrations of glucose, galactose, and glucose-1-phosphate but not by the other carbohydrates tested. The binding of glucose by GBP or succinate by DBP did not result in any chemical alteration of the substrates.

Classification of human heterophile antibodies.

A classification of heterophile antibodies is proposed, which is based on interactions with guinea pig kidney homogenate. The major groups of antibodies combining with guinea pig kidney encompass Hanganutziu-Deicher antibodies, Forssman antibodies, and antibodies to Newcastle disease virus. Antibodies which fail to combine with guinea pig kidney are primarily those of Paul-Bunnell variety.

Studies on Paul-Bunnell (P-B) antigen-antibody system. II. P-B antigens in extracts of lymphoma-leukemia spleens and pathologic sera.

Preparations obtained by the chloroform-methanol extraction procedure from spleen tissues of patients with Hodgkin's disease, lymphomas, and leukemias, as well as from peripheral blood buffy coat of infectious mononucleosis (IM) patients were studied for the presence of 2 Paul-Bunnell (P-B) antigens; BS antigen shared by bovine red blood cells (BRBC) and sheep red blood cells (SRBC) and another, B antigen characteristic for BRBC. Both BS and B antigens were demonstrated by means of agglutination inhibition tests in over 40% of these extracts. None of the extracts from spleens, tonsils, and buffy coat of apparently normal human beings contained these antigens. P-B antigens of lymphoma-leukemia extracts were further purified by DEAE-Sephadex column chromatography. The purified fractions of some of these spleen extracts formed a precipitation line with IM sera, which merged into a reaction of identity with the lines formed by P-B antigens of BRBC. In studying various pathologic sera, B antigen was detected in sera of 28% of lymphoma-leukemia patients, 15% of patients with carcinomas of internal organs, and 3% of patients with systemic lupus erythematosus. On the other hand, BS antigen was found in only 3% of lymphoma-leukemia sera. These results confirmed our previous observations and indicated that both BS and B antigens are expressed as neoantigens on the patient's spleen cells as a result of pathologic processes in lymphoreticular malignancies.

Studies on Paul-Bunnel (P-B) antigen-antibody system. I. Two distinct P-B antigens.

The multiple nature of heterophile, Paul-Bunnel (P-B) antigen has been suggested by our previous studies as well as those of others. In the present study, two distinct antigens of P-B specificity were defined by means of hemagglutination and immunodiffusion tests in agarose gel; one antigen (BS) was shared by bovine (BRBC) and sheep red blood cells (SRBC) and another antigen (B) was limited to BRBC. Both anti-BS and anti-B antibodies were shown to be present in sera of 106 patients with infectious mono-nucleosis (IM) whereas they were virtually absent from the sera of the vast majority of patients with diseases other than IM and normal sera. Absorption and agglutination inhibition tests demonstrated the presence of BS and B antigens on horse erythrocytes. Goat erythrocytes were also shown to possess BS antigen, however, B antigen was found on erythrocytes of some but not all individual goats. By means of the previously established procedure, BS antigen was extracted from stromata of BRBC, SRBC and erythrocytes of horse and goat, and B antigen from BRBC and erythrocytes of some goats. BS and B antigens were also extracted from bovine lymphocytes and murine thymus and spleen tissues but not murine erythrocytes.

Adherence of Streptococcus mutans and Streptococcus sanguis to salivary components bound to glass.

Adherence of radiolabeled Streptococcus mutans and Streptococcus sanguis to saliva-treated glass surfaces was studied under conditions which minimized bacteria-glass interactions. Treatment of glass with an alkylsilane solution decreased nonspecific bacterial adherence and enhanced adsorption of radiolabeled salivary components to these surfaces. Addition of Triton X-100 to the bacterial suspensions also reduced nonspecific adherence to siliconized glass, but did not affect adherence to salivary components attached to siliconized glass. Calcium stimulated S. mutans adherence to saliva-free glass, but inhibited adherence to saliva-treated glass. S. sanguis adherence to either saliva-free or saliva-treated glass was inhibited slightly at high calcium ion concentrations. Adherence of streptococci to saliva-treated glass exhibited saturation kinetics, and the numbers of binding sites on the experimental salivary pellicle and the affinity constants for bacteria-saliva attachment were determined. Preincubation of the streptococci with whole saliva decreased their capacity to adhere to saliva-treated glass, but not to saliva-free glass. Bacteria adherent to saliva-treated glass surfaces were readily desorbed by washing with saliva. The addition of homologous antisera, ammonium sulfate-precipitated immunoglobulins, or Fab fragments to the bacterial suspensions inhibited cell adherence to saliva-treated glass.

Purification and properties of the periplasmic glucose-binding protein of Pseudomonas aeruginosa.

A glucose-binding glycoprotein (GBP) from the periplasm of Pseudomonas aeruginosa was purified to homogeneity as judged by polyacrylamide gel electrophoresis, molecular sieve chromatography, and double-diffusion gel precipitation. It had an average molecular weight of 44,500 and an isoelectric point of 4.7. One mole of glucose was bound per mole of GBP with a dissociation constant of 0.35 muM. The binding of radioactive glucose by GBP was not significantly inhibited by 10-fold-higher concentrations of other carbohydrates; however, a number of related compounds were found to compete at 100-fold-higher concentrations. Amino acid analyses revealed predominant amounts of alanine, glutamate, and glycine and a low content of sulfur-containing amino acids. The carbohydrate moiety of GBP, comprising nearly 16% of the total weight, contained galactosamine, glucosamine, fucose, galactose, glucose, and mannose. A GBP-deficient mutant, strain MB723, was found to be defective in both membrane transport and glucose chemotaxis. Strain MB724, a revertant to GBP-positive phenotype, simultaneously recovered normal levels of both membrane functions.

Anaerobiosis, type 1 fimbriae, and growth phase are factors that affect invasion of HEp-2 cells by Salmonella typhimurium.

The invasion of HEp-2 cells by Salmonella typhimurium was studied under various conditions. Anaerobiosis was shown to markedly affect the internalization of bacterial cells by HEp-2 cells. Anaerobically grown bacteria incubated with HEp-2 cells under anaerobic conditions markedly stimulated the rate of invasion. Anaerobiosis may therefore be a controlling factor in the invasion process. Cells obtained during the logarithmic phase of growth invaded at much higher rates than cells obtained during the stationary phase of growth. The presence of mannose-sensitive type 1 fimbriae on the bacterial surface also promoted invasion, and these fimbriae appear to play a role as an accessory virulence factor.