Effect of pancreatic DNAse on the nuclear DNA topology of Candida tropicalis yeasts

It has been shown that pancreatic DNAase added to the nutritious medium caused the change in the nuclear DNA topology of asporogenic Candida tropicalis yeast. DNA conformative changes are due to the unwinding of supertwisted molecules as a result of single-strand ruptures formation which induce DNA synthesis acceleration, cell growth and division.     

Dynamics of proteins in a culture broth growing beta-hemolytic streptococci group C

The dynamic study of the protein spectrum of culture fluid during the growth of beta-hemolytic streptococcal strain H46A has been carried out by the methods of electrophoresis and isoelectrofocusing in polyacrylamide gel. Changes in the protein spectrum have phasic character and, on the whole, reflect the state of the microbial population, the presence of fractions corresponding to streptokinase and streptolysins being detected at all phases of growth. The electrophoretic mobility of streptokinase perceptibly changes at the end of the logarithmic phase; as shown by electrofocusing, at all stages of the population growth the heterogeneity of streptokinase is observed.     

Identification of chemical elements as pollutants and their primary distribution in steppes of the southern Minusinsk depression

The chemical composition of solid and liquid phases of snow samples collected at different distances from various sources of anthropogenic emissions was studied. The main chemical elements polluting geosystems during the production of aluminum were determined and the halos of their spatial scattering were revealed. The total pollution index and the load of pollution were calculated. Based on quantitative indicators in comparison with maximum permissible concentration values, zones of ecological and geochemical risk were identified. A comparative analysis of the levels of the selected association of elements in technogenic aerosols of the area of the greatest risk and in background soil was carried out.     

An engineered ultra‐high affinity Fab‐Protein G pair enables a modular antibody platform with multifunctional capability

Engineered recombinant antibody‐based reagents are rapidly supplanting traditionally derived antibodies in many cell biological applications. A particularly powerful aspect of these engineered reagents is that other modules having myriad functions can be attached to them either chemically or through molecular fusions. However, these processes can be cumbersome and do not lend themselves to high throughput applications. Consequently, we have endeavored to develop a platform that can introduce multiple functionalities into a class of Fab‐based affinity reagents in a “plug and play” fashion. This platform exploits the ultra‐tight binding interaction between affinity matured variants of a Fab scaffold (Fab^S) and a domain of an immunoglobulin binding protein, protein G (GA1). GA1 is easily genetically manipulatable facilitating the ability to link these modules together like beads on a string with adjustable spacing to produce multivalent and bi‐specific entities. GA1 can also be fused to other proteins or be chemically modified to engage other types of functional components. To demonstrate the utility for the Fab‐GA1 platform, we applied it to a detection proximity assay based on the β‐lactamase (BL) split enzyme system. We also show the bi‐specific capabilities of the module by using it in context of a Bi‐specific T‐cell engager (BiTE), which is a therapeutic assemblage that induces cell killing by crosslinking T‐cells to cancer cells. We show that GA1‐Fab modules are easily engineered into potent cell‐killing BiTE‐like assemblages and have the advantage of interchanging Fabs directed against different cell surface cancer‐related targets in a plug and play fashion.     

The VD1 neutralizing antibody to vascular endothelial growth factor-D: binding epitope and relationship to receptor binding

Vascular endothelial growth factor-D (VEGF-D) is a secreted protein that promotes tumor growth and metastatic spread in animal models of cancer. Expression of VEGF-D in prevalent human cancers was reported to correlate with lymph node metastasis and patient outcome—hence, this protein is a potential target for novel anticancer therapeutics designed to restrict tumor growth and spread. Here, we define the binding site in VEGF-D of a neutralizing antibody, designated VD1, which blocks the interaction of VEGF-D with its cell surface receptors vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 and is being used for the development of therapeutic antibodies. We show by peptide-based mapping and site-directed mutagenesis that the VD1 binding site includes the five residues ₁₄₇NEESL₁₅₁ and that immunization with a synthetic peptide containing this motif generates antibodies that neutralize VEGF-D. The tertiary structure of VEGF-D indicates that the ₁₄₇NEESL₁₅₁ epitope is located in the L2 loop of the growth factor, which is important for receptor binding. Mutation of any of these five residues influences receptor binding; for example, mutations to E148, which abolished binding to VD1, impaired the interaction with VEGFR-2 but enhanced binding to VEGFR-3. This structure/function study indicates that the VD1 binding epitope is part of the receptor binding site of VEGF-D, identifies a region of VEGF-D critical for binding of receptors and explains why VD1 does not bind other members of the VEGF family of growth factors.     

Electron microscopic study of the chitosan effect on the intracellular accumulation and the state of tobacco mosaic virus particles in tobacco leaves

Influence of chitosan on the accumulation and state of tobacco mosaic virus (TMV) in the mesophyll cells of Nicotiana tabacum L. var Samsun leaves in early period of infection development (3 days after infection of leaves) has been studied. The virus accumulated in the cells of the leaves treated for 24 h before infection with chitosan to a lesser degree than in the control cells. The chitosan affected the formation of TMV-specific granular and tubular inclusions which are known to consist of the viral replicase components. Three days after infection of the leaves treated with the chitosan, a typical sign of the infection development was the predominant formation of granular inclusions which are known to appear at the early stages of TMV replication. The infected cells of the leaves untreated with chitosan contained mainly tubular inclusions which had been shown previously to be formed from granular ones at the last stages of the infection process. This indicates that chitosan treatment of the leaves leads to a delay of the development of infection. In phosphotungstic acid-stained suspensions obtained from the infected leaves, abnormal (swollen and "thin") TMV particles were observed along with normal ones. The appearance of abnormal virus particles seems to be caused by virus-induced activation of intracellular lytic processes. The most lytic activity in the infected cells as well as the highest number of abnormal viral particles was observed under the chitosan action. Therefore, it appears that chitosan-mediated stimulation of lytic processes causing destruction of TMV particles may be one of the protective mechanisms limiting virus accumulation in cells.     

Production of Penicillium funiculosum 433 Glucose Oxidase and Its Properties

A method for isolation of extracellular glucose oxidase from Penicillium funiculosum 433 and its purification is proposed. The enzymatic preparation was produced with a yield of 56% and a specific activity of 3730 AU per 1 mg protein. The enzyme studied displayed a high thermostability, resistance to metal ions, and performance in a wide pH range and was equal in its properties to foreign analogues.     

Presynaptic 5-HT1A receptors in immunomodulation

It is shown that a selective agonist of 5-HT1A receptors 8-OH-DPAT in a low dose (0.1 mg/kg), which is known to affect mainly the presynaptic 5-HT1A receptors increased the immune response at the peak of reactions (the forth or fifth day after immunization with sheep red blood cells - SRBC) in CBA mice and Wistar rats. Treatment of the animals with the drug 15 min prior to antigen injection raised the number of plaque-forming cells (lgM-PFC) and rosette-forming cells (RFC) in the spleen. The preliminary blockade of 5-HT1A receptor with a selective antagonist of 5-HT1A receptors WAY-100635 (0.1 mg/kg) prevented the immunostimulating effect of 5-HT 1A receptors agonist 8-OH-DPAT, whereas WAY-100635 administration alone in the same dose didn't change the immune response. Activation of 5-HT1A receptors under conditions of electrical lesion of 5-HTergic neurons of the nucleus raphe was unable to enhance the immune reactions, as it did in sham-operated rats. The data obtained indicate that the somatodendric 5-HT1A autoreceptors are involved in immunomodulation.     

Protective action of glutamate antibodies on increased expression of genes of programmed death of rat brain cells induced by injection of a β-amyloid fragment (25–35)

Glutamate antibodies intranasally administered to Wistar rats at a dose of 300 μg/kg reduced the elevated levels of expression of Aifml, Casp3, and Parp1 genes in the prefrontal cortex and Aifml and Casp3 genes in the hippocampus on the third day after administration of the β-amyloid fragment Aβ_25–35 into the Meynert nuclei of the brain. Changes in Aifm1, Bax, Casp3, and Parp1 gene expression were not found in the hypothalamus, and changes in Bax gene expression were not found in the brain structures studied. The discovered features of gene expression in the prefrontal cortex and hippocampus are considered in terms of development of various cell-death programs, which are modulated by glutamate antibodies.     

Evidence for key enzymatic controls on metabolism of Arctic river organic matter

Permafrost thaw in the Arctic driven by climate change is mobilizing ancient terrigenous organic carbon (OC) into fluvial networks. Understanding the controls on metabolism of this OC is imperative for assessing its role with respect to climate feedbacks. In this study, we examined the effect of inorganic nutrient supply and dissolved organic matter (DOM) composition on aquatic extracellular enzyme activities (EEAs) in waters draining the Kolyma River Basin (Siberia), including permafrost‐derived OC. Reducing the phenolic content of the DOM pool resulted in dramatic increases in hydrolase EEAs (e.g., phosphatase activity increased >28‐fold) supporting the idea that high concentrations of polyphenolic compounds in DOM (e.g., plant structural tissues) inhibit enzyme synthesis or activity, limiting OC degradation. EEAs were significantly more responsive to inorganic nutrient additions only after phenolic inhibition was experimentally removed. In controlled mixtures of modern OC and thawed permafrost endmember OC sources, respiration rates per unit dissolved OC were 1.3–1.6 times higher in waters containing ancient carbon, suggesting that permafrost‐derived OC was more available for microbial mineralization. In addition, waters containing ancient permafrost‐derived OC supported elevated phosphatase and glucosidase activities. Based on these combined results, we propose that both composition and nutrient availability regulate DOM metabolism in Arctic aquatic ecosystems. Our empirical findings are incorporated into a mechanistic conceptual model highlighting two key enzymatic processes in the mineralization of riverine OM: (i) the role of phenol oxidase activity in reducing inhibitory phenolic compounds and (ii) the role of phosphatase in mobilizing organic P. Permafrost‐derived DOM degradation was less constrained by this initial ‘phenolic‐OM’ inhibition; thus, informing reports of high biological availability of ancient, permafrost‐derived DOM with clear ramifications for its metabolism in fluvial networks and feedbacks to climate.