Effects of some protein kinases, cyclic nucleotides and specific inhibitors of phosphorylation on the mitotic cycle of Physarum polycephalum

The true slime mould Physarum polycephalum was treated with various agents by spraying them upon the cell surface 4 hrs before the second synchronous mitosis. The onset of mitosis was considerably approximated after the plasmodium treatment with protein kinases from rat hepatoma or Ph. polycephalum at the late G2 phase. The catalytic and regulatory subunits of cAMP-dependent pig brain protein kinase caused retardation of mitosis, while the holoenzyme, casein kinase and alkaline phosphatase did not affect the timing of mitosis. The cyclic nucleotides and inhibitors of their metabolic enzymes were used to investigate the role of phosphorylation processes in the mitotic cycle.     

Hypophysiotrophic thyrotropin releasing hormone (TRH) synthesizing neurons. Ultrastructure, adrenergic innervation and putative transmitter action

The neuropeptide thyrotropin releasing hormone (TRH) is capable of influencing both neuronal mechanisms in the brain and the activity of the pituitary-thyroid endocrine axis. By the use of immunocytochemical techniques, first the ultrastructural features of TRH-immunoreactive (IR) perikarya and neuronal processes were studied, and then the relationship between TRH-IR neuronal elements and dopamine-beta-hydroxylase (DBH) or phenylethanolamine-N-methyltransferase (PNMT)-IR catecholaminergic axons was analyzed in the parvocellular subnuclei of the hypothalamic paraventricular nucleus (PVN). In control animals, only TRH-IR axons were detected and some of them seemed to follow the contour of immunonegative neurons. Colchicine treatment resulted in the appearance of TRH-IR material in parvocellular neurons of the PVN. At the ultrastructural level, immunolabel was associated with rough endoplasmic reticulum, free ribosomes and neurosecretory granules. Non-labelled axons formed synaptic specializations with both dendrites and perikarya of the TRH-synthesizing neurons. TRH-IR axons located in the parvocellular units of the PVN exhibited numerous intensely labelled dense-core and fewer small electron lucent vesicles. These axons were frequently observed to terminate on parvocellular neurons, forming both bouton- and en passant-type connections. The simultaneous light microscopic localization of DBH or PNMT-IR axons and TRH-synthesizing neurons demonstrated that catecholaminergic fibers established contacts with the dendrites and cell bodies of TRH-IR neurons. Ultrastructural analysis revealed the formation of asymmetric axo-somatic and axo-dendritic synaptic specializations between PNMT-immunopositive, adrenergic axons and TRH-IR neurons in the periventricular and medial parvocellular subnuclei of the PVN. These morphological data indicate that the hypophysiotrophic, thyrotropin releasing hormone synthesizing neurons of the PVN are directly influenced by the central epinephrine system and that TRH may act as a neurotransmitter or neuromodulator upon other paraventricular neurons.     

Mobilization of iron from ferritin by isolated mitochondria effects of species compatibility between ferritin and mitochondria and iron content of ferritin

Mitochondria mobilize iron from ferritin by a mechanism that depends on external FMN. With rat liver mitochondria, the rate of mobilization of iron is higher from rat liver ferritin than from horse spleen ferritin. With horse liver mitochondria, the rate of iron mobilization is higher from horse spleen ferritin than from rat liver ferritin. The results are explained by a higher affinity between mitochondria and ferritins of the same species. The mobilization of iron increases with the iron content of the ferritin and then levels off. A maximum is reached with ferritins containing about 1 200 iron atoms per molecule. The results represent further evidence that ferritin may function as a direct iron donor to the mitochondria.     

Estimation of population parameters and their application to the development of fishery management models in two African rivers

The dynamics of fish populations in two African rivers are being investigated with a view to developing fishery management models for tropical non-floodplain rivers. Preliminary work was carried out in the Rufiji River, East Africa, to establish growth and mortality rates along with L _∞ and K for six species in a river where fishing mortality is zero. These parameters, derived from scale-reading and length frequency analysis, also allowed the development of phased management plans for the fishery resulting from impoundment.
A subsequent investigation has begun on a West African river, the Taia in Sierra Leone, to pursue observations over four seasons, two of which are now complete. A systematic daily sampling programme is being carried out of the whole community, to accumulate similar but more extensive data to those obtained previously. In the Taia, however, fishing mortality must also be estimated from catch surveys, and analysis is carried out using the recently developed ELEFAN programs which can be compared with results obtained by other methods such as scale-reading.     

Inactivation of microbial glutamin-(asparagin-)ase by azaserine and 6-diazo-5-oxo-L-norleucine

The effect of substrate analogues on glutamin-(asparagin-)ase from Pseudomonas aurantiaca-548 has been studied. The enzyme was demonstrated to be highly sensitive to the the action of 6-diazo-5-oxo-L-norleucine and azaserine. L-isomers of glutamine, aspartate, glutamate and several other substrate analogues with free alpha-amino groups protected the enzyme against the inhibitory DON effect. Thus, thorough preliminary selection of appropriate inhibitors, their dosage and treatment duration is needed for the recommendation of combined enzyme-inhibitor application in anti-tumour chemotherapy.     

Production of the medicinal and prophylactic preparation enterobifidin usingBifidobacterium adolescentis MS-42

Production of Enterobifidin includes the stages of preparation of culture media, reparation of lyophilizedBifidobacterium adolescentis MS-42 culture, preparation of starters, cultivation of bacteria in fermenters, biomass conservation, and its biological control. The preparation contains physiologically active bifidobacterium cells with high activities of growth(μ = 0.7 h⁻¹,g = 1.0 h) and acid formation (titratable acidity is ∼120–140°T; acetate concentration, 0.50–0.75%; and lactate concentration, 0.33–0.50%). The antagonistic activity of these bacteria towardsEscherichia coli 08,E. coli 086,E. coli 015,E. coli 0115, andE. coli 0101 amounts to 98.2; toProteus vulgaris 102, to 87.2; andStaphylococcus aureus 209p, to 83.2%. The bifidobacteria (with a titer of ∼10⁹ CFU/ml) remained viable for two to five months.     

Involvement of the Lipopolysaccharides of Azospirilla in the Interaction with Wheat Seedling Roots

The present study was undertaken to comparatively investigate the attachment capacities of Azospirillum brasilenseSp245 and its lipopolysaccharide-defective Omegon-Km mutants KM018 and KM252, as well as their activities with respect to the alteration of the morphology of wheat seedling root hairs. The adsorption dynamics of the parent Sp245 and mutant KM252 strains of azospirilla on the seedling roots of the soft spring wheat cv. Saratovskaya 29 were similar; however, the attachment capacity of the mutant KM252 was lower than that of the parent strain throughout the incubation period (15 min to 48 h). The mutation led to a considerable decrease in the hydrophobicity of the Azospirillumcell surface. The lipopolysaccharides extracted from the outer membrane of A. brasilenseSp245 and mutant cells with hot phenol and purified by chromatographic methods were found to induce the deformation of the wheat seedling root hairs, the lipopolysaccharide of the parent strain being the most active in this respect. The role of the carbohydrate moiety of lipopolysaccharides in the interaction of Azospirillumcells with plants is discussed.     

Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies.

Bispecific single-chain diabodies (scDb) consist of the variable heavy and light chain domains of two antibodies connected by three linkers. The structure of an scDb in the V(H)-V(L) orientation is V(H)A-linkerA-V(L)B-linkerM-V(H)B-linkerB-V(L)A, with linkers A and B routinely chosen to be 5-6 residues and linker M 15-20 residues. Here, we applied display of scDb on filamentous phage to analyse the composition of optimal linker sequences. The three linkers were randomized in length and sequence using degenerated triplets coding for only six hydrophilic or aliphatic amino acids (Thr, Ser, Asp, Asn, Gly, Ala). Antigen-binding clones were then isolated by one to two rounds of selection on the two different antigens recognized by the bispecific scDb. Using an scDb directed against carcinoembryonic antigen (CEA) and beta-galactosidase (Gal), we found that monomeric scDb had a preferred length of 15 or more amino acid residues for the middle linker M and of 3-6 residues for the linkers A and B. No obvious bias towards a preferred linker sequence was observed. Reduction of the middle linker below 13 residues led to the formation of dimeric scDb, which most likely results from interchain pairing between all the V(H) and V(L) domains. Dimeric scDb were also formed by fragments possessing a long linker M and linkers A and B of 0 or 1 residue. We assume that these dimeric scDb are formed by intrachain pairing of the central variable domains and interchain pairing of the flanking variable domains. Thus, the latter molecules represent a novel format of bispecific and tetravalent molecules. The described strategy allows for the isolation of both optimized and minimal linker sequences for the assembly of monomeric or dimeric single-chain diabodies.     

Carcinogens can induce homologous recombination between duplicated chromosomal sequences in mouse L cells.

The ability of a series of DNA-damaging agents to induce homologous intrachromosomal recombination between duplicated genes in the chromosome of mouse cells was investigated. The target cells were the thymidine kinase-deficient mouse L-cell strain 333M, which contains a single integrated copy of a plasmid with two herpes simplex virus thymidine kinase (Htk) genes, each containing an 8-base-pair XhoI linker inserted at a unique site. Expression of a functional Htk enzyme requires a productive recombinational event between the two nonfunctional genes. The spontaneous rate of recombination in this strain is 3 per 10(6) cells per generation. The agents tested represent physical carcinogens (UV and ionizing radiation), a simple alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine), an alkylating cross-linking agent (mitomycin C), and a reactive metabolite of a polycyclic aromatic hydrocarbon ((+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene [BPDE] ). The background frequency of tk+ recombinants in the untreated population averaged 18 X 10(-6) +/- 5 X 10(-6). Ionizing radiation had little or no effect on recombination; exposure to mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, BPDE, or UV, at doses that lowered the survival to between 90 and 10% of the control, caused a dose-dependent increase in frequency of recombinants, reaching 50 X 10(-6) to 100 X 10(-6). No tk+ cells could be generated with a control cell line that contained only one mutant copy of the Htk gene. Molecular hybridization analysis showed that 85 to 90% of the tk+ recombinants retained the Htk gene duplication, consistent with nonreciprocal transfer of wild-type genetic information, gene conversion. In the rest, only a single copy of the Htk gene remained, reflecting a single reciprocal exchange within a chromatid or a single unequal exchange between sister chromatids. Each recombinant tested contained an XhoI-resistant (wild-type) Htk gene.