Localization of the structural gene for botulinum neurotoxin type A using synthetic DNA probe for neurotoxin light chain

To obtain the information on the genetic control of toxin production in the botulism causative agents, the oligonucleotides were synthesized as the molecular probes by translation of the amino acid sequence of the botulinic type A neurotoxin. The optimal conditions for hybridization of botulinic DNA with the synthetic DNA probes were determined and the probes specificity was demonstrated. The DNA fragments homologous to the probes used were shown to belong to bacterial genome, but not to bacteriophage one.     

Carbon metabolism under conditions of regulated penicillin biosynthesis]

Carbon metabolism of P. chrysogenum under conditions of periodical addition of the nutrients was studied. It was found that a proper rate of the carbon source addition to the culture was of significant importance for intensive biosynthesis. The use of carbon for the energetic and constructive needs was not the same at different fermentation periods.     

Morphological,autoradiographic, immunochemical and cytochemical investigation of a cell strain with trisomy 7 from a spontaneous abortus

Summary A complex investigation of the phenotype of a stable strain of LHC—162 cells derived from a spontaneous abortus was carried out. The karyotype, of the strain was 47,XY,+C. The extra chromosome was identified by Giemsa staining as a No. 7. The strain was subjected to cytomorphological, autoradiographic, immunochemical and virological examination in comparison with diploid cell strains. The cells of the LHC—162 strain had no oncogenic activity. Their susceptibility to the three types of poliomyelitis virus did not differ from those of diploid strains. The growth of the LHC—162 cells was poorly organized, and they had a reduced capacity for the formation of histotypical structures. There was low collagen production, poor accumulation of lipid granules, high acid phosphatase, activity, and high glycogen content. Radioautographic investigation of cell cycle revealed lengthening of the mitotic cycle in the G₂, period which was twice as long as in diploid strains. Immunochemical investigation showed that fibroblasts of both trisomic and diploid strains synthesized proteins in ₁ and ₂ globulin zones. However, the synthesis of a protein in ₁ zone was considerably more intensive and in ₂ zone less intensive in the LHC—162 cells than in the diploid strain.The stability of these phenotypic features make it reasonable to assume that they represent a biological characteristic of the LHC—162 strain. It is suggested that this characteristic totality of phenotypical features can be considered as an expression of the C7 trisomic cell's differentiation confusion. It is suggested that the cellular syndrome, is not due only to this chromosomal aberration but also to other chromosomal aberrations.

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Zusammenfassung Es wurde eine Komplexuntersuchung, des Phenotypus der Zellen des stabilen Stammes LHC—162 (spontaner. Abortus) durchgeführt. Der Karyotyp der Zellen dieses Stammes ist 47,XY.+C. Das überzählige Chromosom wurde durch die Giemsa-Methode als C7 identifiziert. Der Karyotyp ist im Verlauf von 62 Passagen stabil geblieben., Die cytomorphologischen, autoradiographischen, immunochemischen Untersuchungen dieses Stammes wurden im Vergleich zu diploiden Stämmen, durchgeführt. Die Zellen dieses Stammes sind nicht krebsauslösend. Die Empfindlichkeit dieses Stammes gegenüber drei Polimyelitis-Virusstämmen ist nicht anders als die Empfindlichkeit des diploiden Stammes. Das Wachstum der Zellen des Stammes LHC—162 ist wenig organisiert; sie besitzen sehr wenig, Fähigkeit, eine histotypische Struktur zu formen; sie bilden wenig Kollagen, sie speichern zu wenig Lipidgranulan; in ihnen ist der Gehalt an Glykogen und die Aktivität von Phosphotasen zu hoch. Die radiographische Untersuchung des Zellcyclus hat gezeigt, daß die Zeit des mitotischen Cyclus der G₂-Periode vergrößert ist. Sie ist fast doppelt so lang wie bei dem diploiden Stamm. Bei der immunochemischen Untersuchung wurde entdeckt, daß der Stamm LHC—162 wie die diploiden Stämme Eiweiß-Komponenten in ₁- und ₂-Globulinzonen synthetisierte, aber der Stamm LHC—162 synthetisiert Eiweiß in der ₁-Globulinzone intensiver und Eiweiß in der Globulinzone ₂ schwächer als normale Zellen. Die Stabilität der, gefundenen phänotypischen Besonderheiten des Stammes LHC—162 läßt sie charakteristisch für diesen Stamm erscheinen. Diese charakteristische Gesamtheit der phänotypischen Eigenschaften kann man als Äußerung der gestörten Differenzierung der Zellen mit Trisomie C7 betrachten. Möglicherweise gibt es ähnliche Zellsyndrome auch für andere Chromosomenanomalien.

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The work was supported in part by research Grants HG-70/9684148 from Human Genetics Unit WHO.     

Interspecific recombinants of Bacillus thuringiensis x Bacillus cereus

The possibility of interspecies recombination was shown by using protoplast fusion method. The Bacillus thuringiensis var. galleriae strain 48S Thi Nic Gua Rifr Strr and 56R Gua Rifr, and also Bac. cereus carrying the plasmid pBC16 responsible for resistance to tetracycline (150 mcg/ml) were used. Recombinants were selected on the medium containing rifampicin and tetracycline. They were shown to combine the properties of both parents. The majority of recombinants were resistant to phages Tg4 and Td15 and represented the mean level of sensitivity to phages Tg12, Tg13 and Td14. Examination of the plasmid profiles of recombinants revealed that their resistance to tetracycline was due to the plasmid with mobility analogous to pBC16. It was concluded that the protoplast fusion method can be used to obtain recombinants between relatively remote species of microorganisms.     

Expression of calponin in rabbit and human aortic smooth muscle cells

Summary Polyclonal antibodies to chicken gizzard calponin were used to localize calponin and determine calponin expression in rabbit and human aortic smooth muscle cells in culture. Calponin was localized on the microfilament bundles of cultured smooth muscle cells. Early in primary culture,ccalponin staining was accumulated preferentially in the central part of the cell body. With time in culture, the number of calponin-negative smooth muscle cells increased while the distribution of calponin in calponin-positive cells became more even along the stress fibers. Calponin content and the calponin/actin ratio decreased about 5-fold in rabbit aortic smooth muscle cells during the first week in primary culture and remained low in proliferating cells. The same tendency in calponin expression was observed when human vascular smooth muscle was studied. On cryostat sections of human umbilical cord, calponin antibodies mainly stained vessel walls of both the arteries and veins, although less intensive labelling was also observed in non-vascular tissue. When primary isolates of human aortic intimal and medial smooth muscle cells were compared with corresponding passaged cultures, it was found that calponin content was reduced about 9-fold in these cells in culture and was similar to the amount of calponin in endothelial cells and fibroblasts. Thus, high calponin expression may be used as an additional marker of vascular smooth muscle cell contractile phenotype.     

Alkaline phosphatase activity of normal and transformed cultured human cells

A study was made of the relationship between the activity of alkaline phosphatase and the proliferation of cultured human cells with different replicative potentials. It is shown that alkaline phosphatase plays a role as one of endogenic stimulators of cellular proliferation. The ageing of diploid cells is accompanied by a decrease in the enzyme activity. Maximum activity was observed during a period of logarithmic cell growth. Addition of placental alkaline phosphatase to the synchronized diploid cells stimulated DNA synthesis in the S-phase of the cell cycle. Heteroploid cells with a high growth rate possessed a 30-100 times higher alkaline phosphatase activity than in the diploid cells. Under certain conditions alkaline phosphatase may presumably function as a proteinkinase.     

Intracellular mechanisms participating in the formation of neuronal calcium signals

Ion and metabolic processes in the endoplasmic reticulum, mitochondria, plasma membrane, etc. providing calcium signaling in the cells of excitable and nonexcitable tissues are discussed.Neirofiziologiya/Neurophysiology, Vol. 36, Nos. 5/6, pp. 405–417, September–December, 2004.This revised version was published online in April 2005 with a corrected cover date and copyright year.     

Novel peptide inhibitors of myosin light chain kinase suppress the hyperpermeability of vascular endothelium

The ability of novel cell-permeating peptide molecules derived from the peptide inhibitor of myosin light chain kinase (MLCK), L-PIK (Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys) to inhibit this kinase in vitro and attenuate the thrombin-induced hyperpermeability of endothelial cell monolayer in culture has been studied. It was found that compounds [N^αMeArg¹]-L-PIK and [Cit¹]-L-PIK possess inhibitory activity towards MLCK comparable to that of L-PIK and the ability to suppress the hyperpermeability of endothelium, whereas other modifications of L-PIK were less effective. Thus, among de novo synthesized peptides, [N^αMeArg¹]-L-PIK and [Cit¹]-L-PIK demonstrate the inhibitory properties of the original peptide L-PIK and additionally surpass it in stability in blood plasma. These peptides may be used in the design of novel antiedemic drugs.