Changes in end-to-end interactions of tropomyosin affect mouse cardiac muscle dynamics

The ends of striated muscle tropomyosin (TM) are integral for thin filament cooperativity, determining the cooperative unit size and regulating the affinity of TM for actin. We hypothesized that altering the alpha-TM carboxy terminal overlap end to the beta-TM counterpart would affect the amino-terminal association, which would alter the end-to-end interactions of TM molecules in the thin filament regulatory strand and affect the mechanisms of cardiac muscle contraction. To test this hypothesis, we generated transgenic (TG) mouse lines that express a mutant form of alpha-TM in which the first 275 residues are from alpha-TM and the last nine amino acids are from beta-TM (alpha-TM9aaDeltabeta). Molecular analyses show that endogenous alpha-TM mRNA and protein are nearly completely replaced with alpha-TM9aaDeltabeta. Working heart preparations data show that the rates of contraction and relaxation are reduced in alpha-TM9aaDeltabeta hearts. Left ventricular pressure and time to peak pressure are also reduced (-12% and -13%, respectively). The ratio of maximum to minimum first derivatives of change in left ventricular systolic pressure with respect to time (ratio of +dP/dt to -dP/dt, respectively) is increased, but tau is not changed significantly. Force-intracellular calcium concentration ([Ca2+]i) measurements from intact papillary fibers demonstrate that alpha-TM9aaDeltabeta TG fibers produce less force per given [Ca2+]i compared with nontransgenic fibers. Taken together, the data demonstrate that the rate of contraction is primarily affected in TM TG hearts. Protein docking studies show that in the mutant molecule, the overall carbon backbone is perturbed about 1.5 A, indicating that end-to-end interactions are altered. These results demonstrate that the localized flexibility present in the coiled-coil structures of TM isoforms is different, and that plays an important role in interacting with neighboring thin filament regulatory proteins and with differentially modulating the myofilament activation processes.     

Array CGH using whole genome amplification of fresh-frozen and formalin-fixed, paraffin-embedded tumor DNA

The ability to utilize formalin-fixed, paraffin-embedded (FFPE) archival specimens reliably for high-resolution molecular genetic analysis would be of immense practical application in the study of human disease. We have evaluated the ability of the GenomePlex whole genome amplification (WGA) kit to amplify frozen and FFPE tissue for use in array CGH (aCGH). GenomePlex gave highly representative data compared with unamplified controls both from frozen material (Pearson's R(2) = 0.898) and from FFPE (R(2) = 0.883). Artifactual amplification observed using DOP-PCR at chromosomes 1p, 3, 13q, and 16p was not seen with GenomePlex. Highly reproducible aCGH profiles were obtained using as little as 5 ng starting material from FFPE (R(2) = 0.918). This WGA method should readily lend itself to the determination of DNA copy number alterations from small fresh-frozen and FFPE clinical tumor specimens, although some care must be taken to optimize the DNA extraction procedure.     

Synthesis and preliminary biological evaluation of (2S,1'R,2'S)- and (2S,1'S,2'R)-2-(2'-phosphonocyclopropyl)glycines, two novel conformationally constrained l-AP4 analogues

Two novel constrained l-AP4 analogues, (2S,1'R,2'S)- and (2S,1'S,2'R)-2-(2'-phosphonocyclopropyl)glycines (7) and (8), were synthesized and evaluated as mGluR ligands. Compound 7 showed to be a group III mGluRs agonist with micromolar activity.     

Structure of an acidic polysaccharide from the marine bacterium Pseudoalteromonas flavipulchra NCIMB 2033(T)

An acidic polysaccharide was isolated from Pseudoalteromonas flavipulchra type strain NCIMB 2033(T) and found to consist of 6-deoxy-L-talose (L-6dTal), D-galactose and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The identities of the monosaccharides were ascertained by sugar analysis and 1D 1H and 13C NMR spectroscopy in conjunction with 2D COSY, TOCSY, ROESY and 1H, 13C HMQC experiments, which enabled determination of the following structure of the trisaccharide repeating unit of the polysaccharide:-->3)-alpha-L-6dTalp4Ac-(1-->3)-beta-D-Galp-(1-->7)-alpha-Kdop-(2-->.     

Structural studies of the pectic polysaccharide from duckweed Lemna minor L

The pectic polysaccharide of duckweed Lemna minor L. termed lemnan (LM) was shown to contain the ramified, "hairy" region. Using partial acid hydrolysis and Smith degradation followed by NMR spectroscopy of the fragments obtained, some structural features of the hairy region of LM were elucidated. Partial acid hydrolysis of LM afforded the crude polysaccharide fraction LMH that was separated into two polysaccharide fractions: LMH-1 and LMH-2. In addition, the oligosaccharide fraction LMH-3 contained 97% D-apiose was obtained from the supernatant. A further more rigorous acidic hydrolysis of LMH led to the crude polysaccharide fraction LMHR which was separated in to two fractions: LMHR-1 and LMHR-2. Smith degradation of LMH afforded the polysaccharide fragment LMHS differed in low contents of apiose residues. Unfortunately, NMR-spectroscopy failed to provide significant evidence concerning the structure of LMH-1 due to the complexity of the macromolecule. The structure of the 1H/13C-NMR spectroscopy including the correlation 2D NMR spectroscopy. As a result, alpha-1,4-D-galactopyranosyluronan was confirmed to be the main constituent of the LM backbone. In addition, the ramified, "hairy" region of the macromolecule appeared to contain segments consisting of residues of terminal and beta-1,5-linked apiofuranose, terminal and alpha-1,5-linked arabinofuranose, terminal and beta-1,3- and beta-1,4- linked galactopyranose, the terminal and beta-1,4-linked xylopyranose, and beta-1,4-linked 2-mono-O-methyl xylopyranose. Analytical and NMR-spectral data of LMHS confirmed the presence of considerable amounts of the non-oxidized of 1,4-linked D-galactopyranosyl uronic acid residues. Thus, some side chains of the ramified region of lemnan appeared to attach to D-galactopyranosyl uronic acid residues of the backbone.     

Structural and functional properties of Yersinia pestis Caf1 capsular antigen and their possible role in fulminant development of primary pneumonic plague

Yersinia pestis capsular antigen Caf1 is shown to be a beta-structural protein that in polymeric form possesses very high conformational stability. Different approaches show that a dimer is the minimal cooperative block of Caf1 adhesin. Caf1 dimer interacts effectively with IL-1 receptors of human macrophage and epithelial cells. The specificity of such interaction is confirmed by the inhibition of IL-1alpha binding by Caf1. The Caf1 role in pneumonic plague pathogenesis is discussed.     

Combined Vitamins B12b and C Induce the Glutathione Depletion and the Death of Epidermoid Human Larynx Carcinoma Cells HEp-2

The combination of hydroxocobalamin (vitamin B_12b) and ascorbicacid (vitamin C) can cause the death of tumor cells at the concentrationsof the components at which they are nontoxic when administeredseparately. This cytotoxic action on epidermoid human larynx carcinomacells HEp-2 in vitro is shown to be due to the hydrogen peroxidegenerated by the combination of vitamins B_12b and C. The drop inthe glutathione level preceding cell death was found to be the result ofcombined action of the vitamins. It is supposed that the induction of celldeath by combined action of vitamins B_12b and C is connected to the damageof the cell redox system.     

Atemonate and Imuteran: novel Russian monoclonal antibody-based therapeutic agents

The N. Blokhin National Cancer Research Center is one of the few Russian scientific institutions in which hybridoma technology of monoclonal antibody (mAb) production has been successfully established. Using this technology, several dozens of mAbs to various antigens of human leukocytes have been elaborated. These mAbs are widely used for immune status evaluation and for differential diagnostics of leukemias. Two mAbs were used to develop therapeutic drugs. Imuteran is a pharmaceutical form of mAb ICO-25 against a mucin-like antigen of human milk fat globules and proposed for treatment of epithelial cell-originating cancers (breast, intestinal, ovarian, lung cancer, etc.). ThePhase II clinical study of this agent is now nearly completed, and preliminary results suggest Imuteran to be a promising anticancer agent with tumor-stabilizing activity, but patients should be carefully monitored for signs of allergic reactions. mAb ICO-90 against the CD3 antigen of human T lymphocytes was used to develop the therapeutic agent Atemonate proposed for treatment of acute transplant rejection. At present, the Phase II clinical study of this agent is over, and the results confirm the drug safety and efficacy for this indication. The drug is being registered at the Ministry of Healthcare and Social Development, and transfer to serial production is expected shortly.     

Factors influencing the natural reproduction decline in the beluga (Huso huso,Linnaeus, 1758), Russian sturgeon (Acipenser gueldenstaedtii,Brandt & Ratzeburg, 1833), and stellate sturgeon (A. stellatus,Pallas, 1771) of the Volga–Caspian basin: A review

The review describes the changes in natural reproduction of three important sturgeon species in the Volga–Caspian basin: (a) the beluga (Huso huso Linneaus, 1758), (b) the Russian sturgeon (Acipenser gueldenstaedtii Brandt &Ratzeburg, 1833), and (c) the stellate sturgeon (Acipenser stellatus Pallas, 1771). Since the past 60 years, these species responded to severe influences of natural and anthropogenic factors. On the basis of original and published data, an analysis has been made of (a) the numbers of larvae migrating from spawning sites (according to plankton net survey), (b) fecundity and histological anomalies in gonad development, (c) the numbers of adult sturgeons in the Caspian Sea and of spawners migrating to the Volga River (according to trawl and beach seine survey), and (d) foraging resources for the sturgeons. The results show that their natural reproduction in the Volga–Caspian basin has declined drastically during the past decades under the impact of (a) fluctuations of the Caspian Sea level and flow discharge from the Volga River, (b) blockage of sturgeon migration routes and loss of spawning sites because of dam construction, (c) water pollution in the lower reaches of the Volga River and in the Caspian Sea, and (d) intensive and selective illegal and unreported fishing. The relative significance of these factors has been changing during the study period.