Experimental model for studying the primary cilia in tissue culture cells.

In HeLa, PK, 3T3, PtK1 cells and rat embryo fibroblasts (REF), antibodies against acetylated tubulin stained centrioles, primary cilia, some cytoplasmic microtubules and microtubule bundles of the mid-body. The primary cilia were stained more intensively than cytoplasmic microtubules and could easily be distinguished. This makes it possible to detect the primary cilia in cultured cells and to estimate their number by light microscopy. The four cultures studied had 1/4 to 1/3 of interphase cells with detectable primary cilia, and only in HeLa cells the primary cilia were very rare. Comparison of electron microscopic and immunofluorescence data showed that the frequencies of occurrence of the primary cilia in four tissue cultures determined by these two methods were the same. Therefore, antibodies against acetylated tubulin can be used to study the primary cilia. In synchronized mitotic fibroblasts (3T3 and REF) the primary cilia appeared first 2 h after the cells had been plated on coverslips, which is 1 h after the cells had entered the interphase. Four hours after plating the number of ciliated cells reached the average level for nonsynchronous population. This model can be used for further studies of the expression of primary cilia.     

Extracellular metabolites of hydrocarbon-oxidizing bacteria as a substrate for sulfate reduction

The relationship between bacterial oxidation of hydrocarbons and sulfate reduction was studied in the experimental system with liquid paraffin was used as a source of organic compounds inoculated with silt taken from a reservoir. Pseudomonads dominated in the hydrocarbon-oxidizing silt bacteriocenosis. However, Rhodococcus and Arthrobacteria amounted to not more than 3%. Arthrobacteria dominated the microbial association formed in the paraffin film of the model system. Sulfate-reducing bacteria were represented by genera Desulfomonas, Desulfotomaculum, and Desulfovibrio. The growth of sulfate-reducing bacteria in media containing with paraffin, successive products of its oxidation (cetyl alcohol, stearate, and acetate), and extracellular metabolites of hydrocarbon-reducing bacteria was studied. The data showed that sulfate-reducing bacteria did not use paraffin or cetyl alcohol as growth substrates. However, active growth of sulfate-reducing bacteria was observed in the presence of stearate and extracellular water-soluble or lipid metabolites of Arthrobacteria.     

New species of the genus Nocardia and its antibiotic properties]

A culture of a new species of Nocardia, i.e. N. indigoensis producing an antibiotic close to celicomycins was isolated from a soil sample of Kazakhstan plated on the selective medium with kanamycin. By a number of chemical properties and biological activity the antibiotic differed from celicomycins. Probably it is a new natural substance.     

EB1 and EB3 control CLIP dissociation from the ends of growing microtubules

EBs and CLIPs are evolutionarily conserved proteins, which associate with the tips of growing microtubules, and regulate microtubule dynamics and their interactions with intracellular structures. In this study we investigated the functional relationship of CLIP-170 and CLIP-115 with the three EB family members, EB1, EB2(RP1), and EB3 in mammalian cells. We showed that both CLIPs bind to EB proteins directly. The C-terminal tyrosine residue of EB proteins is important for this interaction. When EB1 and EB3 or all three EBs were significantly depleted using RNA interference, CLIPs accumulated at the MT tips at a reduced level, because CLIP dissociation from the tips was accelerated. Normal CLIP localization was restored by expression of EB1 but not of EB2. An EB1 mutant lacking the C-terminal tail could also fully rescue CLIP dissociation kinetics, but could only partially restore CLIP accumulation at the tips, suggesting that the interaction of CLIPs with the EB tails contributes to CLIP localization. When EB1 was distributed evenly along the microtubules because of overexpression, it slowed down CLIP dissociation but did not abolish its preferential plus-end localization, indicating that CLIPs possess an intrinsic affinity for growing microtubule ends, which is enhanced by an interaction with the EBs.     

Reovirus sigma1 fiber incorporated into adenovirus serotype 5 enhances infectivity via a CAR-independent pathway

Adenovirus serotype 5 (Ad5) has been used for gene therapy with limited success because of insufficient infectivity in cells with low expression of the primary receptor, the coxsackie and adenovirus receptor (CAR). To enhance infectivity in tissues with low CAR expression, tropism expansion is required via non-CAR pathways. Serotype 3 Dearing reovirus utilizes a fiber-like sigma1 protein to infect cells expressing sialic acid and junction adhesion molecule 1 (JAM1). We hypothesized that replacement of the Ad5 fiber with sigma1 would result in an Ad5 vector with CAR-independent tropism. We therefore constructed a fiber mosaic Ad5 vector, designated as Ad5-sigma1, encoding two fibers: the sigma1 and the wild-type Ad5 fiber. Functionally, Ad5-sigma1 utilized CAR, sialic acid, and JAM1 for cell transduction and achieved maximum infectivity enhancement in cells with or without CAR. Thus, we have developed a new type of Ad5 vector with expanded tropism, possessing fibers from Ad5 and reovirus, that exhibits enhanced infectivity via CAR-independent pathway(s).     

An Escherichia coli strain producing a leaderless mRNA from the chromosomal lac promoter

A special Escherichia coli strain capable of producing a leaderless lacZ mRNA from the chromosomal lac promoter was constructed to study the mechanism of leaderless mRNA translation. The translation efficiency of this noncanonical mRNA is very low in comparison with the canonical cellular templates, but it increases by one order of magnitude in the presence of chromosomal mutations in the genes encoding the ribosomal S1 and S2 proteins. The new strain possesses obvious advantages over the commonly used plasmid constructs (first of all, due to the constant dosage of lacZ gene in the cell) and opens possibilities for investigation of the specific conditions for leaderless mRNA translation in vivo using molecular genetic approaches.     

Study of the Carbohydrate Specificity of Antibodies Against <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> Using the Library of Synthetic Mycoantigens

The carbohydrate specificity of antibodies obtained by immunizing laboratory animals with immunogens prepared from the Aspergillus fumigatus cell wall was analyzed using the library of biotinylated synthetic oligosaccharides. It has been shown that the main part of antipolysaccharide antibodies recognizes galactomannan in the studied immunogens with preference to its fragments containing more than two β-(1→5)-linked galactofuranosyl units. The data obtained can form the basis for the development of enzyme immunoassay for the detection of this dangerous fungal pathogen.     

Distinctive Features of the Functioning of Lactic Acid and the Alcohol Fermentation Enzyme in Sorghum and Pea Leaves under the Conditions of Oxygen Deficiency

Dynamic changes in the enzymatic activity of lactate dehydrogenase, L-lactate-cytochrome-c-coxidoreductase, and alcohol dehydrogenase were analyzed in the seedlings of sorghum, a plant resistant to oxygen deficiency, and peas, a plant vulnerable to oxygen deficiency, cultivated under the conditions of flooding. The subcellular localization and isoform composition of the enzymes were characterized. The mechanism underlying the adaptive reaction of cell metabolism was devised from analysis of the results obtained. The reaction is supposed to involve lactate dehydrogenase and L-lactate-cytochrome-c-oxidoreductase enzymes that suppress cytoplasm acidification in sorghum and pea roots in the case of flooding.     

Influence of light on the apoplastic ph in microwounded cells of <Emphasis Type="Italic">Chara corallina</Emphasis>

Microscopic wounding of plant cell walls by pathogens or by feeding insects triggers the defense responses, including a sharp rise in pH at the cell surface (pH_o). Using internodal cells of Chara corallina Klein ex Willd., we show here that the elevated pH_o in the area of cell wall microincision decreases in darkness and increases on illumination. These pH_o changes occurred specifically in cell areas affected by microincision and were lacking in intact areas with active pHotosynthesis (acid zones). Localized illumination of a remote cell region located upstream the cytoplasmic flow at a 1.5-mm distance from the analyzed area also caused a transient increase in pH_o in the area of microwounding but had no such effect in unwounded cell regions having weakly acidic pH_o. Apparently, the increase in pH_o after wounding is mediated by a metabolite released from illuminated chloroplasts, which is transported with the cytoplasmic flow for long distances. The transient pH_o increase in the area of cell wall incision after illumination of a distant cell region coincided with a temporal increase in chlorophyll fluorescence F’. This implies the concurrent influence of the transported reductant (presumably NADH) on light emission of chloroplasts and on the H⁺ flow across the plasmalemma. We suppose that the alkalinization of cell surface in the area of microincision arises from H⁺ consumption in the apoplast in association with the transmembrane electron transport from cytoplasmic reducing equivalents to molecular oxygen.