Spore and crystal formation inBacillus thuringiensis var.thuringiensis during growth in cystine and cysteine

The effect of the addition of different concentratons of cystine and cysteine on sporulation and parasporal crystal formation inBacillus thuringiensis var.thuringiensis was studied. The effect was well pronounced when the cystine/cysteine additions were made after the stationary phase. Heat stable spores and crystals were formed when the culture was provided with a low concentration of cystine/cysteine (0.05 per cent w/v). At a moderate concentration of cystine or cysteine (0.15%), only heat labile spores were formed without the production of the crystal. When the cystine/cysteine concentration was high (0.25%), spore and crystal formation were completely inhibited. Partial reversal of inhibition of sporulation was brought about by sodium sulphate or Zinc sulphate and lead, copper, cadmium or cobalt acetate at 0.2 mM or at 0.2% of sodium or potassium pyruvate, citrate, cisaconitate, oxalosuccinate, ∞ -keto-glutarate, succinate, fumarate, malate, or oxalacetate. Glutamate (0.2%) overcame the inhibitory effect of cystine/cysteine completely. The structural changes observed using phase contrast microscopy were dependent upon the concentration of cystine/cysteine.     

Isolation of 3-(2-carboxyethyl)thymine following in vitro reaction of β-propiolactone with calf thymus DNA

3-(2-Carboxyethyl)thymine (3-CET) was synthesized from β-propiolactone (BPL) and dThd5′P at pH 9.0–9.5 via the intermediate 3-(2-carboxyethyl)thymidine-5′-monophosphoric acid (3-CEdThd5′P). 3-CEdThd5′P was converted to 3-CET by hydrolysis in 1.5 N HCl at 100°C for 2 h. The structure of 3-CET was assigned on the basis of UV spectra, electron impact (EI) and isobutane chemical ionization mass spectra and the EI mass spectrum of a trimethylsilyl derivative of 3-CET. BPL was reacted in vitro with calf thymus DNA at pH 7.5. 100 A units of BPL-reacted DNA yielded, following perchloric acid hydrolysis and preparative paper chromatography, 3 A units of 3-CET. Reaction of BPL with the phosphodiester thymidylyl-(3′-5′)thymidine gave 3-(2-carboxyethyl)thymidylyl-(3′-5′)-3-(2-carboxyethyl)thymidine (～3%). Phosphotriester formation was not detected.     

Investigation of the pH dependence of proton uptake by porcine heart mitochondrial malate dehydrogenase upon binding of NADH

The pH dependence of proton uptake upon binding of NADH to porcine heart mitochondrial malate dehydrogenase (l-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) has been investigated. The enzyme has been shown to exhibit a pH-dependent uptake of protons upon binding NADH at pH values from 6.0 to 8.5. Enzyme in which one histidine residue has been modified per subunit by the reagent iodoacetamide (E. M. Gregory, M. S. Rohrbach, and J. H. Harrison, 1971, Biochim. Biophys. Acta253, 489–497) was used to establish that this specific histidine residue was responsible for the uptake of a proton upon binding of NADH to the native enzyme. It has also been established that while there is no enhancement of the nucleotide fluorescence upon addition of NADH to the iodoacetamide-modified enzyme, NADH is nevertheless binding to the modified enzyme with the same stoichiometry as with native enzyme. The data are discussed in relation to the involvement of the essential histidine residue in the catalytic mechanism of “histidine dehydrogenases” recently proposed by Lodola et al. (A. Lodola, D. M. Parker, R. Jeck, and J. J. Holbrook, 1978, Biochem. J.173, 597–605) and the catalytic mechanism of “malate dehydrogenases” recently proposed by L. H. Bernstein and J. Everse (1978, J. Biol. Chem.253, 8702–8707).     

A requirement for three cell types for in vitro generation of specific secondary cell-mediated cytotoxic response against SV40-induced tumor-associated antigens in mice

The present study demonstrates that a collaborative interaction among three cell types, namely, two distinct subsets of T cells and macrophages is needed for in vitro generation of specific secondary cell-mediated cytotoxicity against syngeneic SV40-transformed cells in mice. These data suggest a central role of three cell types in the generation of efficient antitumor immune response in syngeneic tumor systems.     

Antimicrobial defense mechanisms in the horseshoe crab, Limulus polyphemus: preliminary observations with heat-derived extracts of Limulus amoebocyte lysate.

Heat-derived (60°C) extracts of Limulus amoebocyte lysate (LAL) were found to contain potent “broad-spectrum” antimicrobial activity. Additional heating of the LAL extracts to 100°C for 30 min completely inactivated the antimicrobial activity and served as a control. Antimicrobial activity was observed over a temperature range of 0° to 37°C (higher temperatures not tested) with greatest activity at 37°C. Antimicrobial activity of LAL extracts was variable when tested against Gram-negative bacteria of the family Enterobacteriaceae. A twofold concentration of the extracts resulted in a significant decrease in antimicrobial effectiveness. Dialysis of single- and double-strength LAL extracts against deionized water produced a marked and significant enhancement of antimicrobial activity against both resistant and sensitive species, confirming the presence of a dialyzable inhibitor(s). Dialyzed LAL extracts were active against 13 of 14 species of Enterobacteriaceae tested. Two strains of Pseudomonas aeruginosa were susceptible as were two of three Gram-positive cocci tested. Highly sensitive bacterial species were rapidly killed with a greater than 90% reduction in viable counts occurring within the first 30 min of reaction time. Dialyzed LAL extracts also possessed considerable antifungal activity. The role of the Limulus polyphemus amoebocyte in defense against microbial invasion and dissemination is discussed.     

Metabolism of prostaglandins in spontaneously hypertensive rats: NAD"-dependent 15-hydroxyprostaglandin dehydrogenase activity is decreased in kidney and increased in lung.

Renal, pulmonary and gastric NAD⁺-dependent 15-hydroxyprostaglandin dehydrogenase activities were determined in both spontaneously hypertensive and normotensive rats at 6 and 12 weeks of age. Renal enzyme activity in hypertensive rats was only 30–40% of that present in normotensive controls at both ages. In contract, pulmonary enzyme activity in hypertensive animals was twice as active as that in normal controls. There was no significant difference in gastric enzyme activity. NAD⁺-dependent 9-hydroxyprostaglandin dehydrogenase activity, the enzyme responsible for the conversion of vasoinactive PGF metabolites to PGE metabolites, also failed to show any difference in two types of rat kidneys. The results indicate that, in hypertension, prostaglandin inactivation is impaired in kidney but is facilitated in lung.     

Stimulation of gastrin secretion from the perfused rat stomach by somatostatin antiserum

The effect of a high capacity somatostatin antiserum on antral gastrin secretion was examined in an isolated vascularly perfused rat stomach preparation. Infusion of somatostatin antiserum diluted 1:1 and 1:9 with Krebs buffer solution produced significant increases in gastrin secretion throughout the period of infusion. Neither infusion of somatostatin antiserum diluted 1:99 nor infusion of control rabbit serum had any effect on gastrin secretion. The data indicate that antral somatostatin excercises a continous restraint on gastrin secretion in the basal state.     

Critical importance of microsome concentration in mutagenesis assay with V79 Chinese hamster cells.

For optimum mutagensis in V79 Chinese hamster cells, the amount of liver postmitochondrial fraction in the assay was found to be of critical importance, depending on the chemicals being tested. Benzo[a]pyrene (BP) required lower (1-5%) concentrations of the liver 15 000 X g supernatant (S15) from methylcholanthrene pretreated rats for a maximum induction of cytotoxicity and mutagenicity, as determined by 8-azaguanine- and ouabain-resistance. A sharp peak of mutagenicity and cytotoxicity was induced by 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (7,8-diol BP) at a concentration of 1% of the S15 fraction. Little or no response was induced by these compounds with the S15 concentrations of more than 10%. Similarly, aflatoxin B1 induced a sharp peak of mutagenicity and cytotoxicity at a concentration of 2% of the liver S15 fraction from Aroclor-pretreated rats. Under the same condition, non-carcinogenic aflatoxin G2 did not induce cytotoxicity and mutagenicity. Analysis of BP metabolites by high-pressure liquid chromatography indicates that with the 30% S15 fraction, more than 80% of BP was metabolized during the first 15 min, while with the 2% S15 fraction, 7,8-diol BP increased continuously throughout the 120-min incubation period, suggesting a strong metabolic competition to rapidly remove BP and 7,8-diol BP with a high concentration of the S15. In contrast with these compounds, N-nitrosodimethylamine induced mutagenicity and cytotoxicity which increased linearly in proportion to the increasing amount of the S15 fraction from phenobarbitone- and Aroclor-pretreated rats. Various nitrosamines with different lipophilicity were examined at a high (30%) and low (2%) concentration of the S15 fraction from Aroclor-pretreated rats, in which ratios of mutation frequencies at 30% and 2% correlated inversely with lipophilicity of the compound. This result suggests that the lipid solubility of test compounds may be one factor which determines the concentration of post-mitochondrial supernatant for optimum mutagenesis.     

The role of retinol in the initiation of sporangiophores of Phycomyces blakesleeanus

The initiation of sporangiophores of Phycomyces was analyzed under oxygen-limiting conditions. Mutants lacking -carotene have a higher oxygen threshold than the wild type depending on the residual amount of -carotene. The supersensitivity to low oxygen tension is specific for sporangiophore initiation and can be suppressed by addition of either retinal, retinol or retinol acetate to the medium. It is suggested that retinol is a natural regulator of differentiation in Phycomyces.     

Nuclear ultrastructure: Condensed chromatin in plants is species-specific (karyotypical), but not tissue-specific (functional)

Summary In contrast to mammalian cell nuclei those of plants display nearly an identical ultrastructure in all developmental stages and tissues. This indicates that the gross organization of chromatin is species-specific, but not tissue-specific and function-dependent. The species-specific nuclear ultrastructure is determined by the basic nuclear DNA content (2 C value). The higher the DNA content, the more the euchromatin remains in the condensed state during interphase, but to a lower coiling order than the heterochromatin.Some difficulties in the interpretation of electron micrographs of cell nuclei, and the possible role of repetitive DNA sequences in the karyotypical condensation of euchromatin in plants are discussed.