Kinetic and inhibition studies of glutamine synthetase from the cyanobacterium Anabaena 7120

A number of biochemical parameters of glutamine synthetase (EC 6.3.1.2) isolated from the cyanobacterium Anabaena 7120 were determined. Apparent Michaelis constants for glutamate and ATP were found to be 2.1 and 0.32 mM, respectively; that for ammonia was found to be below 20 microM, significantly lower than that reported for glutamine synthetases from other species. Serine, alanine, glycine, cysteine, aspartic acid, methionine sulfone, and methionine sulfoximine were found to inhibit the enzyme. The enzyme is controlled neither by adenylylation nor by feedback inhibition by glutamine, mechanisms found in some other prokaryotes. It must therefore be regulated by a different mechanism, possibly a combination of feedback by alanine, serine, and glycine, metabolites which are especially effective in inhibiting Anabaena glutamine synthetase.     

Macrophage-T cell interactions. III. Production from T cells, under the influence of macrophages, of a factor which complexes Ig and antigen.

Soluble complexes of Ig and antigen have been detected in the serum of mice within 6 hr after immunization. Such complexes are taken up by a subpopulation of T cells. We present evidence which suggests that the complexes are formed through the mediation of a factor released from T cells, tentatively called Ig-antigen complexing factor or IACF. IACF is produced as a result of a macrophage/T-cell interaction, when macrophages are present in an optimal proportion in relation to T cells (4%). Particulate or aggregated substances stimulate macrophages to release a mediator which subsequently acts on Fc receptor-negative T cells to produce IACF. Free-SH groups are important for the activity of the macrophage mediator. Mercaptoethanol and l-cysteine can also release IACF from T cells in the absence of macrophages. Protein synthesis is necessary for the production of this factor, the activity of which is abolished by trypsin digestion. It is postulated that the complexes of Ig and antigen formed under the influence of IACF represent a mechanism of presentation of antigen to T cells.     

INFLORESCENCE DEVELOPMENT IN BRASSICA CAMPESTRIS L.

Vegetative seedlings of the Ceres strain Brassica campestris L., a quantitative, long-day plant, were induced to flower by exposure to a 16-hr, long-day cycle. Cytohistological and cytohistochemical changes associated with inflorescence development were examined. Developing shoot apices were classified in vegetative, transitional, and reproductive stages. The vegetative apex possessed a biseriate tunica, central zone, peripheral zone and pith-rib meristem. The transitional stage at 48 hr was marked by an increase in size and by a stratification of the upper cell layers of the shoot apex with a concurrent decrease of apical cytohistochemical zonation. The reproductive stage was initiated at 58 hr by periclinal cell divisions in the 3rd and 4th cell layers of the flank region. Cytohistochemical zonation in the vegetative apical meristem was restored in the floral apex. An “intermediate developmental” phase was not observed between the vegetative and reproductive stage.     

The degradation of paracetamol (4-hydroxyacetanilide) and other substituted acetanilides by aPenicillium species

A mould which was isolated from a solution of paracetamol was identified as aPenicillium species and was found to possess the ability to utilise a series of substituted acetanilides, including paracetamol (4-hydroxyacetanilide), phenacetin (4-ethoxyacetanilide) and metacetamol (3-hydroxyacetanilide) as sole carbon sources for growth. Studies with washed-cell suspensions indicated that growth of thePenicillium isolate in the presence of paracetamol induced the respective enzyme systems for the degradation of this compound. Manometric studies, measuring oxygen uptake rates, indicated that the mould was capable of degrading paracetamol to acetate and 4-aminophenol. Acetate was further metabolised whilst 4-aminophenol accumulated in the growth medium and was subsequently identified by UV spectroscopy and thin-layer chromatography. Similar experiments with phenacetin indicated metabolism by the mould to acetate and 4-ethoxyaniline which was isolated and identified by subsequent analysis of the growth medium. However, unlike 4-aminophenol and 4-ethoxyaniline, the degradation product (3-aminophenol) from metacetamol metabolism was further degraded by the mould.     

Determination of the execution points of mutations in the nuclear replication cycle of Aspergillus nidulans.

Cultures of nuclear replication cycle mutants of Aspergillus nidulans were transferred to the nonpermissive temperature, and the fraction of nuclei still able to reach mitosis was determined. For the determinations, benomyl [methyl-1(butylcarbomoyl)benzimidazolecarbamate] was added to trap nuclei in mitosis, and these were detected by staining with aceto-orcein. The assumptions and controls required to relate the experimentally determined fractions to the points where a mutation blocks the nuclear cycle are discussed. Nine genetically distinct mutants were tested. Two of these were blocked early in the cycle, two in the middle, and five close to, or during, mitosis.     

Isolation and identification of products from alkylation of nucleic acids: ethyl- and isopropyl-purines.

Ethylation and isopropylation of guanine in alkaline solution, or of adenine in formic acid, by alkyl methanesulphonates gave the following products: 1-, N2-, 3-, O6-, 7- and 9-alkylguanines; 1-, 3-, 7- and 9-alkyladenines. The products were identified from their characteristic u.v-absorption spectra, by comparison with either known ethyladenines or with the corresponding known methyladenines, and were also characterized by mass spectrometry. Their chromatographic properties on paper, t.l.c. and various columns were determined. DNA was alkylated in neutral solution with 14C-labelled alkyl methanesulphonates and the ratios of the alkylpurines formed were obtained, and compared for alkylation by methyl, ethyl and isopropyl methanesulphonates and by N-methyl-N-nitrosourea. The extents of alkylation at O-6 of guanine relative to those at N-7 of guanine varied with the reactivity of the methylating agents according to the predictions of Swain & Scott (1953) relating nucleophilicity of the groups alkylated with the substrate constants of the alkylating agents. The relative extents of alkylation at N-3 of adenine did not follow this correlation.     

Bacteriological activity in unfiltered calf sera collected for tissue culture use

Summary Bacteriological tests were made on 24 lots of unfiltered calf serum collected for subsequent use as a component of tissue culture media. The examination included the isolation and identification of bacteria, assay of phages, and demonstration of endotoxin material. Only Gram-positive bacteria were isolated and 96% of the sera were contaminated with bacteria. The prevalent strains of bacteria found wereBacillus species and streptococci and 63% of the sera coagulatedLimulus amebocyte lysate. More than 90% of the lots contained phages demonstrable with the C-3000 strain ofEscherichia coli. Only one lot of the serum was found to be free from bacteria, phages, and endotoxin by the tests used.     

The role of octopamine and cyclic AMP in regulating hormone release from corpora cardiaca of the American cockroach

Incubation of corpora cardiaca from adult male Periplaneta americana in the presence of octopamine results in elevated tissue levels of cyclic AMP. The octopamine-induced elevation of cyclic AMP is partially blocked by phentolamine, gramine and cyproheptadine but not by propranolol. Dopamine and 5-hydroxytryptamine also increase cyclic AMP levels in the corpus cardiacum and additivity studies indicate that separate octopamine- and dopamine-binding sites are present within the tissue. Cyclic AMP levels in the corpus cardiacum also increase in response to electrical stimulation of nervi corporis cardiaci II (NCC II) and the electrically induced effect is eliminated in the presence of phentolamine.A factor, which causes elevated haemolymph trehalose levels when injected into adult cockroaches, is released from corpora cardiaca incubated in the presence of octopamine. The active factor is denatured by incubation in the presence of pronase. The hypertrehalosemic factor is also released when corpora cardiaca are incubated in the presence of dibutyryl cyclic AMP or 40 mM potassium chloride; however dopamine and 5-hydroxytryptamine fail to effect a marked release of the hypertrehalosemic factor.The results are discussed in light of the proposal that the release of hypertrehalosemic hormone from corpora cardiaca is regulated by octopaminergic neurones contained within NCC II.