Valery A. Rasskazov

Differential effects of triterpene glycosides, frondoside A and cucumarioside A2-2 isolated from sea cucumbers on caspase activation and apoptosis of human leukemia cells

Frondoside A is a pentaoside having an acetyl moiety at the aglycon ring and xylose as a third monosaccharide residue. Cucumarioside A2‐2 is a pentaoside having glucose as a third monosaccahride unit. We compared the effects of frondoside A and A2‐2 for cell death‐inducing capability with close attention paid to structure–activity relationships. Both frondoside A and A2‐2 strongly induced apoptosis of leukemic cells. Frondoside A‐induced apoptosis was more potent and rapid than A2‐2‐induced apoptosis. A2‐2‐induced but not frondoside A‐induced apoptosis was caspase‐dependent. This suggests that holothurians may induce apoptosis of leukemic cells caspase‐dependently or ‐independently, depending on the holothurian structure.

A Highly Active Alkaline Phosphatase from the Marine Bacterium Cobetia

An alkaline phosphatase with unusually high specific activity has been found to be produced by the marine bacterium Cobetia marina (strain KMM MC-296) isolated from coelomic liquid of the mussel Crenomytilus grayanus. The properties of enzyme, such as a very high specific activity (15000 DE U/1 mg of protein), no activation with divalent cations, resistance to high concentrations of inorganic phosphorus, as well as substrate specificity toward 5′ nucleotides suggest that the enzyme falls in an intermediate position between unspecific alkaline phosphatases (EC 3.1.3.1) and 5′ nucleotidases (EC 3.1.3.5).

cDNA cloning and structural characterization of a lectin from the mussel Crenomytilus grayanus with a unique amino acid sequence and antibacterial activity.

An amino acid sequence of GalNAc/Gal-specific lectin from the mussel Crenomytilus grayanus (CGL) was determined by cDNA sequencing. CGL consists of 150 amino acid residues, contains three tandem repeats with high sequence similarities to each other (up to 73%) and does not belong to any known lectins family. According to circular dichroism results CGL is a β/α-protein with the predominance of β-structure. CGL was predicted to adopt a ß-trefoil fold. The lectin exhibits antibacterial activity and might be involved in the recognition and clearance of bacterial pathogens in the shellfish.

Molecular Characterization and Therapeutic Potential of a Marine Bacterium Pseudoalteromonas sp. KMM 701 α-Galactosidase

An α-galactosidase capable of converting B red blood cells into the universal blood type cells at the neutral pH was produced by a novel obligate marine bacterium strain KMM 701 (VKM B-2135 D). The organism is heterotrophic, aerobic, and halophilic and requires Na+ ions and temperature up to 34°C for its growth. The strain has a unique combination of polysaccharide-degrading enzymes. Its single intracellular α-galactosidase exceeded other glycoside hydrolases in the level of expression up to 20-fold. The α-galactosidase was purified to determine the N-terminal amino acid sequences and new activities. It was found to inhibit Corynebacterium diphtheria adhesion to host buccal epithelium cell surfaces with high effectiveness. The nucleotide sequence of the homodimeric α-galactosidase indicates that its subunit is composed of 710 amino acid residues with a calculated Mr of 80,055. This α-galactosidase shares structural property with 36 family glycoside hydrolases. The properties of the enzyme are likely to be highly beneficial for medicinal purposes.

Carbohydrate-binding motifs in a novel type lectin from the sea mussel Crenomytilus grayanus: Homology modeling study and site-specific mutagenesis.

The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) was shown to represent a novel family of lectins and to be characterized by three amino acid tandem repeats with high (up to 73%) sequence similarities to each other. We have used homology modeling approach to predict CGL sugar-binding sites. In silico analysis of CGL-GalNAc complexes showed that CGL contained three binding sites, each of which included conserved HPY(K)G motif. In silico substitutions of histidine, proline and glycine residues by alanine in the HPY(K)G motifs of the Sites 1-3 was shown to lead to loss of hydrogen bonds between His and GalNAc and to the increasing the calculated CGL-GalNAc binding energies. We have obtained recombinant CGL and used site-specific mutagenesis to experimentally examine the role of HPK(Y)G motifs in hemagglutinating and carbohydrate binding activities of CGL. Substitutions of histidine, proline and glycine residues by alanine in the HPYG motif of Site 1 and Site 2 was found to led to complete loss of CGL hemagglutinating and mucin-binding activities. The same mutations in HPKG motif of the Site 3 resulted in decreasing the mucin-binding activity in 6-folds in comparison with the wild type lectin. The mutagenesis and in silico analysis indicates the importance of the all three HPY(K)G motifs in the carbohydrate-binding and hemagglutinating activities of CGL.

A Novel Bifunctional Hybrid with Marine Bacterium Alkaline Phosphatase and Far Eastern Holothurian Mannan-Binding Lectin Activities

A fusion between the genes encoding the marine bacterium Cobetia marina alkaline phosphatase (CmAP) and Far Eastern holothurian Apostichopus japonicus mannan-binding C-type lectin (MBL-AJ) was performed. Expression of the fusion gene in E. coli cells resulted in yield of soluble recombinant chimeric protein CmAP/MBL-AJ with the high alkaline phosphatase activity and specificity of the lectin MBL-AJ. The bifunctional hybrid CmAP/MBL-AJ was produced as a dimer with the molecular mass of 200 kDa. The CmAP/MBL-AJ dimer model showed the two-subunit lectin part that is associated with two molecules of alkaline phosphatase functioning independently from each other. The highly active CmAP label genetically linked to MBL-AJ has advantaged the lectin-binding assay in its sensitivity and time. The double substitution A156N/F159K in the lectin domain of CmAP/MBL-AJ has enhanced its lectin activity by 25±5%. The bifunctional hybrid holothurians lectin could be promising tool for developing non-invasive methods for biological markers assessment, particularly for improving the MBL-AJ-based method for early detection of a malignant condition in cervical specimens.

The Distribution and Substrate Specificity of Extracellular Nuclease Activity in Marine Fungi

The distribution and specificity of extracellular nucleases produced by marine fungi belonging to eleven genera, namely: Alternaria, Aspergillus, Aureobasidium, Chaetomium, Fusarium, Gliomastix, Humicola, Penicillium, Scopulariopsis, Wardomyces, Periconia, have implied its important function in the organic phosphorus and nitrogen circle in the Ocean. The fungal nucleases of 64 isolates tested were more or less specific for single-stranded DNA with a high preferential specificity towards poly-U substrate with forming of 5’-phosphate mononucleotides. A couple of the nucleases were capable of RNA digesting. The highest level of extracellular nucleolytic ability was observed in Penicillium spp. isolates. The tight correlation found between extracellular nuclease activity and the rate of thymidine uptake by actively growing and sporulating marine fungus Penicillium melinii suggests that this nuclease is required for fulfilling the nucleotide pool of precursors of DNA biosynthesis during transformation of hyphae into the aerial mycelium and conidia in stressful environmental conditions.

Data supporting functional diversity of the marine bacterium Cobetia amphilecti KMM 296.

Data is presented in support of functionality of hyper-diverse protein families encoded by the Cobetia amphilecti KMM 296 (formerly Cobetia marina KMM 296) genome (“The genome of the marine bacterium Cobetia marina KMM 296 isolated from the mussel Crenomytilus grayanus (Dunker, 1853)” [1]) providing its nutritional versatility, adaptability and biocontrol that could be the basis of the marine bacterium evolutionary and application potential. Presented data include the information of growth and biofilm-forming properties of the food-associated isolates of Pseudomonas, Bacillus, Listeria, Salmonella and Staphylococcus under the conditions of their co-culturing with C. amphilecti KMM 296 to confirm its high inter-species communication and anti-microbial activity. Also included are the experiments on the crude petroleum consumption by C. amphilecti KMM 296 as the sole source of carbon in the presence of sulfate or nitrate to ensure its bioremediation capacity. The multifunctional C. amphilecti KMM 296 genome is a promising source for the beneficial psychrophilic enzymes and essential secondary metabolites.

Genetically modified proteins: functional improvement and chimeragenesis

This review focuses on the emerging role of site-specific mutagenesis and chimeragenesis for the functional improvement of proteins in areas where traditional protein engineering methods have been extensively used and practically exhausted. The novel path for the creation of the novel proteins has been created on the farther development of the new structure and sequence optimization algorithms for generating and designing the accurate structure models in result of x-ray crystallography studies of a lot of proteins and their mutant forms. Artificial genetic modifications aim to expand natures repertoire of biomolecules. One of the most exciting potential results of mutagenesis or chimeragenesis finding could be design of effective diagnostics, bio-therapeutics and biocatalysts. A sampling of recent examples is listed below for the in vivo and in vitro genetically improvement of various binding protein and enzyme functions, with references for more in-depth study provided for the readers benefit.

Regulation of Yersinia pseudotuberculosis Major Porin Expression in Response to Antibiotic Stress

The expression of the OmpF porin gene in Yersinia pseudotuberculosis in response to antibiotics of two different classes (kanamycin and nalidixic acid) was analyzed using a quantitative PCR and a fluorescence reporter system. Both antibiotics downregulated the expression of the ompF gene. The nalidixic acid significantly reduced the ompF expression, while kanamycin, for which porins are considered to be an alternative transport route, only slightly reduced the ompF level.