Evidence from cell-free systems for differences in the sterol biosynthetic pathway of Rhizoctonia solani and Phytophthora cinnamomi.

Cell-free preparations of both Rhizoctonia solani, a sterol-synthesizing fungus, and Phytophthora cinnamomi, a non-sterol-synthesizing fungus, incubated in the presence of [2(-14)C]mevalonate and iodacetamide, converted the mevalonate into labelled mevalonate 5-phosphate, mevalonate 5-pyrophosphate and isopentenyl pyrophosphate. In the absence of iodoacetamide, but under anaerobic conditions, the same preparations converted the mevalonate into labelled geraniol, farnesol and squalene, the first two compounds presumably as their pyrophosphates. When cell-free preparations of both organisms were incubated aerobically in the presence of [1(-14)C]isopentenyl pyrophosphate, only labelled geraniol, farnesol and squalene were recovered from the P. cinnamomi reaction mixture, whereas labelled geraniol, farnesol, squalene, squalene epoxide, lanosterol and ergosterol were present in the R. solani reaction mixture. When these same preparations were incubated in the presence of 14C-labelled squalene, labelled squalene epoxide, lanosterol and ergosterol were recovered from the R. solani reaction mixture. In contrast, the P. cinnamomi preparation was unable to convert the squalene into products further along the sterol pathway; instead, a portion of the labelled squalene was converted into water-soluble products, indicating the possible existence of a squalene-degradation process in this organism. It appears that the block in the sterol biosynthetic pathway of P. cinnamomi occurs at the level of squalene epoxidation.     

Formamide denaturation of chromosomal DNA for in situ hybridization and C-band preparation in the guinea pig, Cavia porcellus

Cytological preparations were incubated in 0.07 N NaOH at room temperature or 90% formamide (final salt concentration 2 × SSC) at either 65 °C or 37 °C for 2.5 h to denature guinea pig chromosomes. Chromosomes treated with NaOH or formamide at 65 °C showed a large amount of DNA loss, while chromosomes treated with formamide at 37 °C showed little or no DNA loss. Repeated sequences were isolated from guinea pig DNA and [³H]cRNA was transcribed with Escherichia coli RNA polymerase for in situ hybridization. Localization of the [³H]cRNA occurred in the centromeric regions and C-band positive short arms of almost all of the chromosomes in the NaOH preparations. Chromosomes treated with formamide at 65 °C showed the same grain distribution with a decrease in the number of grains/cluster. Slides incubated in formamide at 37 °C showed localization in only a few chromosomes and the number of grains/cluster was greatly diminished. Thermal denaturation of isolated chromatin indicated that incubation of chromosomes in formamide at 37 °C did not fully denature the DNA. C-bands could be induced by treating slides in formamide at either 65 °C or 37 °C when followed by a “reassociation” in 2 × SSC at 65 °C for 16 h. If the “reassociation” step was omitted, C-bands were found in the 65 °C formamide slides but not the 37 °C formamide slides.     

Circular forms of Uukuniemi virion RNA: an electron microscopic study.

Because the ribonucleoprotein forms of the segments of the Uukuniemi virus genome have previously been characterized as circular, we examined the isolated RNAs by electron microscopy under conditions of increasing denaturation. After spreading under moderately denaturing conditions (50 or 60% formamide), 50 to 70% of the molecules were circular. Increasing the formamide concentration to 70 and 85% decreased the number of circular forms, and only linear forms were observed after incubation of the RNA at 60 degrees C for 15 min in 99% formamide. When spread from 4 M urea-80% formamide--another condition known to denature RNA--only 5 to 30% circular molecules were observed. Pretreatment of the RNA with 0.5 M glyoxal at 37 degrees C for 15 min prior to spreading from 50% formamide gave less than 5% cirucular forms. Length measurement of the molecules showed that they were not significantly degraded by any of the methods employed. The circular molecules were destroyed by treatment with pancreatic RNase, but were unaffected by DNase or proteinase K treatment. After complete denaturation of the RNA, the circles could be reformed under reannealing conditions. We conclude that the three size classes of RNA that comprise the Uukuniemi virus genome are circular molecules probably maintained in that form by base pairing between inverted complementary sequences at the 3'' and 5'' ends of linear molecules.     

Formamide chemistry and the origin of informational polymers

Formamide (HCONH2) provides a chemical frame potentially affording all the monomeric components necessary for the formation of nucleic polymers. In the presence of the appropriate catalysts, and by moderate heating, formamide yields a complete set of nucleic bases, acyclonucleosides, and favors both phosphorylations and transphosphorylations. Physico-chemical conditions exist in which formamide favors the stability of the phosphoester bonds in nucleic polymers more than that of the same bonds in monomers. This property establishes 'thermodynamic niches' in which the polymeric forms are favored. The hypothesis that these specific attributes of formamide allowed the onset of prebiotic chemical equilibria capable of Darwinian evolution is discussed.     

The influence of formamide on thermal denaturation profiles of DNA and metaphase chromosomes in suspension

Systematic photometric studies are presented to analyze the thermal denaturation behaviour with and without formamide of metaphase chromosome suspensions in comparison to DNA solutions. Temperature dependent hyperchromicity measurements at 256 nm and 313 nm were performed using an appropriately designed computer-controlled photometer device. Due to an upright optical axis, this allowed absorbance measurements with negligible sedimentation effects not only for solutions of pure DNA, but also for particle suspensions of isolated metaphase chromosomes. This device has a temperature resolution of +/- 0.5 degrees C and an optical sensitivity of 10(-3) to 10(-4) optical density. For calf thymus DNA the reduction of the melting point with the increase of formamide in the solution was measured at pH 7.0 and pH 3.2. The good correlation of the theoretical approximation to experimental data indicated the suitability of the apparatus to quantitatively describe DNA conformation changes induced by thermal denaturation. For metaphase chromosome preparations of Chinese hamster culture cells, absorbance changes were measured between 20 degrees C and 95 degrees C with a temperature gradient of 1 degrees C/min. These measurements were performed at pH 7.0 and at pH 3.2. The denaturation profiles (= first derivative of the absorbance curve) resulted in a highly variable peak pattern at 256 nm and 313 nm indicating complex conformation changes. A statistical evaluation of the temperature values of the peak maxima resulted in temperature ranges typical for chromosomal conformation changes during thermal treatment. Especially the range of highest temperature values was independent from pH modifications. For pH 3.2 the influence of formamide on the denaturation behaviour of metaphase chromosome preparations was analyzed. In contrast to pure DNA solutions, a reduction of the "melting point" (i.e. the maximum temperature at which a conformation change takes place) was not found. However, the denaturation behaviour depended on the duration of formamide treatment before the measurement.     

Formation of mixed hemagglutinin trimers during double infection with influenza virus strains of the same subtype

Formation of hemagglutinin spikes in the course of the mixed infection of cell culture by two influenza virus strains belonging to the same antigenic subtype or to different subtypes was studied by means of immunoprecipitation of [14C]-labelled hemagglutinins from cell lysates. The immunoprecipitates were further analysed by polyacrylamide gel electrophoresis. Lysates of separately infected cells mixed before lysis were used as control samples. The analysis of immunoprecipitates revealed the formation of chimeric hemagglutinin spikes in the cells infected by the strains possessing hemagglutinins of the same subtype but not in the cells infected by the strains of different subtypes (H1 and H3). The results are discussed in connection with the homology of amino-acid sequences of influenza virus hemagglutinins.     

Electron Microscope Observations on the Effects of Polymixin B Sulfate on Cell Walls of Chlamydia psittaci

The effects of polymixin B sulfate on cell walls of mature elementary body (EB) and of immature developmental reticulate body (RB) of Chlamydia psittaci were investigated. When purified EB were treated with polymixin (10(4) units per ml or more) at 37 C for 60 min, about 70% of EB was found to be covered with a number of projections. Further incubation did not increase the percentage affected. The infectivity after treatment as assayed by the inclusion counting technique was reduced by 70% of the original titer. These results suggest that EB with the projections are no longer infective. The projections had obscure outlines and were 20 to 40 nm in diameter when seen in thin sections. In the negatively stained preparations, the projections were composed of aggregations of fine particles 4 to 5 nm in diameter. Treatment with sodium dodecyl sulfate at the same concentration used for cell wall isolation removed the projections completely, and the cell walls were converted to rather ragged forms apparently composed of outside and inside layers. When RB cell walls prepared from infected cells at 18 hr after infection were treated with polymixin at the same concentration, the projections having the same morphology with those seen on treated EB cell walls were observed only on the inside surface of cell wall.     

Epoxy derivatives of aromatic polycyclic hydrocarbons. The preparation and metabolism of epoxides related to benzo[a]pyrene and to 7,8- and 9,10-dihydrobenzo[a]pyrene

Products that appeared to be mainly benzo[a]pyrene 7,8-oxide and benzo[a]pyrene 9,10-oxide were synthesized and their chemical and biochemical properties were investigated. The oxides were unstable and readily rearranged to phenols. They were converted by rat liver homogenates and microsomal preparations into phenols and dihydrodiols, but glutathione conjugates were not formed in appreciable amounts. The dihydrodiols formed from benzo[a]pyrene 7,8- and 9,10-oxide by rat liver microsomal preparations were identical in their chromatographic and spectrographic properties with dihydrodiols formed when benzo[a]pyrene was metabolized by rat liver homogenates. 9,10-Dihydrobenzo[a]pyrene 7,8-oxide and 7,8-dihydrobenzo[a]pyrene 9,10-oxide were also synthesized. They were converted by rat liver homogenates and microsomal preparations into the related cis- and trans-dihydroxy compounds. Glutathione conjugates were formed from the oxides by rat liver homogenates. Both 7,8- and 9,10-dihydrobenzo[a]pyrene were metabolized by rat liver homogenates to mainly the trans-isomers of the related dihydroxy compounds. In experiments with boiled homogenates, the benzo[a]pyrene oxides were converted into phenols, whereas the dihydrobenzo[a]pyrene oxides yielded small amounts of the related dihydroxy compounds.     

Catabolism of D-gluaric acid to alpha-ketoglutarate in Bacillus megaterium

Crude cell-free extracts of d-glucarate-grown cells of Bacillus megaterium converted d-glucarate to alpha-keto-beta-deoxy-d-glucarate (KDG). Charcoal-treated cell-free extracts or partially purified enzyme preparations converted KDG to an intermediate which was isolated and identified as 2,5-diketoadipate (DKA). This compound was synthesized, and the cell-free extracts of d-glucarate grown cells were found to catalyze the reduction of nicotinamide adenine dinucleotide (NAD) in its presence. In the absence of NAD, the same enzyme preparation catalyzed the decarboxylation of the DKA to alpha-ketoglutarate semialdehyde (KGS), whereas in the presence of NAD the KGS was subsequently oxidized to alpha-ketoglutarate by alpha-ketoglutarate semialdehyde dehydrogenase. Since galactarate-grown B. megaterium contains a galactarate dehydrase forming KDG, the complete pathway for the metabolism of d-glucarate or galactarate to alpha-ketoglutarate and CO(2) is now known in a gram-positive bacterium.     

The hemagglutinating activity of bacteria in the genus Klebsiella and the morphofunctional characteristics of their fimbriae]

The hemagglutinating activity of 77 Klebsiella strains from the international collection, grown in a culture medium prepared on the basis of soy-bean flour enzymatic hydrolysate, was studied. These strains could be divided into four groups according to their capacity for synthesizing different types of hemagglutinins on their surface: 2 strains carried mannose-sensitive hemagglutinins, 18 strains had mannose-resistant K-type hemagglutinins, 48 strains exhibited the signs indicating the presence of both mannose-sensitive and mannose-resistant hemagglutinins, and 9 strains showed no hemagglutinating activity. The hemagglutinating activity of strains K-74, K-79, K-80, K-81 and K-82 was characterized. Of the reference strains under study, 22 strains were found to have mannose-resistant hemagglutinating activity with respect to fresh chick red blood cells. The occurrence of hemagglutinins in Klebsiella was shown to depend on the temperature of cultivation and the consistency of the culture medium. The formation of large-sized capsules in Klebsiella grown in the Werfel-Fergusson medium with a considerable content of saccharose was shown to cause the absorption of their fimbrial structures by the capsular substance and, as a consequence, the suppression of their hemagglutinating activity.     

Enzymes Produced by a Pseudomonas Species Which Inactivate Inhibitors of Certain Viral Hemagglutinins. I. Identification and Purification of a Proteinase and Phospholipase C

Filtrates from cultures of a psychrophilic Pseudomonas species, which inactivate serum inhibitors of certain viral hemagglutinins, were shown to contain both lecithinase (phospholipase C) and a proteolytic enzyme with elastase activity. The bacterium was cultivated under conditions favoring production of the respective enzymes, and the enzymes were purified by ammonium sulfate precipitation followed by column chromatography or by gel filtration. The elastase was obtained in crystalline form and was recrystallized. It has properties similar to those of a number of other bacterial elastases but is more heat-labile than most. Although a high degree of purification was achieved for the lecithinase, as evidenced by an increase in specific activity, it was not obtained in crystalline form. Partially purified preparations of the lecithinase had extremely high activity compared to that of commercial preparations of phospholipase C from Clostridium welchii.     

Inhibition of Clostridium botulinum types A and B hemagglutinins by sugars.

Chromatographically isolated hemagglutinins of Clostridium botulinum types A and B are serologically related but not identical. Of the sugars (5, 6, 12, 18 carbons, some derivatives, L and D forms) tested, only D-galactose and some of tis derivatives were inhibitors of these hemagglutinins. O-Nitrophenyl-beta-D-galactopyranoside and isopropyl-beta-D-thiogalactoside were the most potent inhibitors. The two hemagglutinins were bound tightly by p-aminophenyl-beta-D-thiogalactopyranoside coupled to CH-Sepharose 4B. The ligands to which these hemagglutinins bind were determined as the sugars which inhibited the hemagglutinating activity.     

Fate of 130 hemagglutinins from different influenza A viruses

In this study, we use our probabilistic models to analyze 130 hemagglutinins from different influenza A virus in order to gain the insight into their fate. The results provide three lines of evidence regarding the H5, H6, and H9 hemagglutinins: (i) the H5 hemagglutinins are more sensitive to mutations, this is the current state of the H5, H6, and H9 hemagglutinins; (ii) the H5 hemagglutinins had experienced more mutations in the past, this is the history of the H5, H6, and H9 hemagglutinins; and (iii) the H6 hemagglutinins has a bigger potential towards future mutations, this is the future of the H5, H6, and H9 hemagglutinins. Furthermore, this study gives two clues on the mutation tendency that is a degeneration process and the species susceptibility that is the chickens and ducks.     

Hemagglutinin activity in Nereis coelomic fluid

1. Nereis coelomic fluid agglutinates rat, mouse, chicken, guinea pig and rhesus monkey erythrocytes (RBC). 2. Lipid fractions of the particulate matter from coelomic fluid are hemagglutinins exhibiting different activity inhibition profiles with complex polysaccharides. 3. The high mol. wt hemagglutinin from coelomic fluid supernatant is not a protein and is inhibited by bovine submaxillary mucin (BSM), thyroglobulin, transferrin and their asialo derivatives. 4. Coelomic fluid supernatant has a population of low mol. wt protein hemagglutinins inhibited by BSM, fetuin, antiserum to coelomic fluid and some mannan preparations. 5. Hemagglutination by lipids characterized by RBC specificity and specificity for inhibition by carbohydrate is noteworthy and may be significant in studies of cellular interactions and immunity in invertebrates.     

Formamide as the main building block in the origin of nucleic acids

The simplest molecules grouping the four most common elements of the universe H,C,O and N (with the exception of the biologically inert He) are isocyanate HNCO and formamide H2NCOH. Reasons for the availability of formamide on prebiotic Earth are presented. We review evidence showing that formamide in the presence of largely available catalysts and by moderate heating yields the complete set of nucleic bases necessary for the formation of nucleic acids. Formamide also favours the formation of acyclonucleosides and the phosphorylation and trans-phosphorylation of nucleosides, thus providing a plausible chemical frame for the passage from a simple one-carbon compound to nucleic polymers. Physico-chemical conditions exist in which formamide favours the stability of the phosphoester bonds in nucleic polymers relative to that of the same bonds in monomers. Starting from a formamide-laden environment subject only to the laws of chemistry, a hypothesis is outlined sketching the passage towards an aqueous world in which Darwinian rules apply.     

Isolation of plasma membrane glycoprotein from bovine thymocytes

Glycoprotein was isolated from a purified thymocyte membrane preparation by two methods: lithium diiodosalicylate-phenol extraction and hot 75% ethanol extraction. A higher yield of membrane sialic acid was obtained by the latter method. The preparations had similar apparent molecular weights on sodium dodecyl sulfate gel electrophoresis. Both had similar receptor activities against a panel of hemagglutinins, although the 75% ethanol extract was more active on a weight basis. However, there were significant differences in carbohydrate and amino acid compositions of the two thymocyte extracts. The lithium diiodosalicylate-extracted material had much more glucose, ribose, and glycine than the ethanol extract. The glycoprotein preparations from thymocytes were quite distinct from the glycoprotein of bovine erythrocytes in both composition and receptor properties.     

Sedimentation of ribosomal ribonucleic acid from ribonuclease treated and untreated ribosomes

The formation of a slowly sedimenting form of 23-S ribosomal RNA from E. coli has been investigated by analytical ultracentrifugation and thermal denaturation in aqueous solution and in formamide. Evidence is presented that the slow form of 23-S arises as a result of nucleate damage to the RNA in the 50-S ribosome. The 30-S ribosome (and 16-S RNA), is unaffected. The slow form of 23-S RNA cannot be demonstrated under conditions of complete denaturation in formamide, but only by partial denaturation in aqueous solution of low ionic strength (< 0.01M Na). Apparent maintenance of the integrity of 23-S RNA in formamide after nuclease treatment suggests that this may not be a simple linear molecule. An alternative model is suggested containing a circular element in the structure.     

Protective action of cytotoxic lymphocytes depending on the antigenic determinant composition of influenza virus hemagglutinin

The protective role of cytotoxic lymphocytes (CTL) in dependence on composition of antigenic determinants of hemagglutinin of influenza viruses H3N2 was studied. It was established that CTL do not exert protective effect under conditions of adoptive transfer, when there is one common antigenic determinant in hemagglutinins of the virus forming immunity. When all antigenic determinants in hemagglutinins of influenza viruses are identical, CTL-like antibodies represent one of the main factors of antivirus immunity.     

Biochemistry of Coxiella burnetii: Embden-Meyerhof pathway

Purified preparations of Coxiella burnetii were examined for enzymes of the glycolytic pathway. Glucose-phosphate isomerase, fructose-1,6-diphosphatase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase were shown to be present in C. burnetii extracts. Heat-killed C. burnetii purified with normal yolk sacs demonstrated no activity after disruption. Aldolase was shown to be of the class II type by complete inhibition of activity in the presence of 8 x 10(-3)m ethylenediaminetetraacetic acid. The host enzyme activity (normal and infected yolk sacs) was not affected by the same treatment. When cellulose acetate electrophoresis was performed on the extracts, aldolase from both normal and infected yolk sacs exhibited five isozyme bands, whereas aldolase from the C. burnetii extract appeared as a single band.