Turbulent flow evaluation of the venous needle during hemodialysis

Arteriovenous (AV) grafts and fistulas used for hemodialysis frequently develop intimal hyperplasia (IH) at the venous anastomosis of the graft, leading to flow-limiting stenosis, and ultimately to graft failure due to thrombosis. Although the high AV access blood flow has been implicated in the pathogenesis of graft stenosis, the potential role of needle turbulence during hemodialysis is relatively unexplored. High turbulent stresses from the needle jet that reach the venous anastomosis may contribute to endothelial denudation and vessel wall injury. This may trigger the molecular and cellular cascade involving platelet activation and IH, leading to eventual graft failure. In an in-vitro graft/needle model dye injection flow visualization was used for qualitative study of flow patterns, whereas laser Doppler velocimetry was used to compare the levels of turbulence at the venous anastomosis in the presence and absence of a venous needle jet. Considerably higher turbulence was observed downstream of the venous needle, in comparison to graft flow alone without the needle. While turbulent RMS remained around 0.1 m/s for the graft flow alone, turbulent RMS fluctuations downstream of the needle soared to 0.4-0.7 m/s at 2 cm from the tip of the needle and maintained values higher than 0.1 m/s up to 7-8 cm downstream. Turbulent intensities were 5-6 times greater in the presence of the needle, in comparison with graft flow alone. Since hemodialysis patients are exposed to needle turbulence for four hours three times a week, the role of post-venous needle turbulence may be important in the pathogenesis of AV graft complications. A better understanding of the role of needle turbulence in the mechanisms of AV graft failure may lead to improved design of AV grafts and venous needles associated with reduced turbulence, and to pharmacological interventions that attenuate IH and graft failure resulting from turbulence.     

Discovery of a second human molar and cranium fragment in the late Middle to Late Pleistocene cave of Ma U'Oi (Northern Vietnam)

In November 2002, during the second season of work by a Vietnamese-French-Japanese team, we discovered a human molar and a fragment of an occipital bone in the late Middle to Late Pleistocene cave of Ma U'Oi (Bacon et al., Geobios. 37 (2004) 305). The layer from which this material comes is the same as that in which a human lower molar was found in 2001. Both molars can be attributed to archaic Homo, and both exhibit archaic and modern traits.     

Expression of 2-deoxy-<Emphasis Type="Italic">scyllo</Emphasis>-inosose synthase (<Emphasis Type="Italic">kan</Emphasis>A) from kanamycin gene cluster in <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

An actinomycetes expression vector (pIBR25) was constructed and applied to express a gene from the kanamycin biosynthetic gene cluster encoding 2-deoxy-scyllo-inosose synthase (kanA) in Streptomyces lividans TK24. The expression of kanA in pIBR25 transformants reached a maximum after 72 h of culture. The plasmid pIBR25 showed better expression than pSET152, and resulted in the formation of insoluble KanA when it was expressed in Escherichia coli. This strategy thus provides a valuable tool for expressing aminoglycoside-aminocyclitols (AmAcs) biosynthetic genes in Streptomyces spp.     

Structure-function relationships of purple acid phosphatase from red kidney beans based on heterologously expressed mutants

Purple acid phosphatases are binuclear metalloenzymes, which catalyze the conversion of orthophosphoric monoesters to alcohol and orthophosphate. The enzyme from red kidney beans is characterized with a Fe(III)-Zn(II) active center. So far, the reaction mechanisms postulated for PAPs assume the essentiality of two amino acids, residing near the bimetallic active site. Based on the amino acid sequence of kidney bean PAP (kbPAP), residues H296 and H202 are believed to be essential for catalytic function of the enzyme. In the present study, the role of residue H202 has been elucidated. Mutants H202A and H202R were prepared by site-directed mutagenesis and expressed in baculovirus-infected insect cells. Based on kinetic studies, residue H202 is assumed to play a role in stabilizing the transition state, particularly in charge compensation, steric positioning of the substrate, and facilitating the release of the product by protonating the substrate leaving groups. The study confirmed the essentiality and elucidates the functional role of H202 in the catalytic mechanism of kbPAP.     

Seasonal changes in plasma dehydro-epiandrosterone (DHEA) levels of domestic geese

Changes in plasma DHEA, testosterone (T) and 17-B-oestradiol (E2) levels were examined in domestic geese of both sexes in the fall and winter. The levels of steroid hormones were determined in blood plasma by means of radio-immunoassay (RIA). A so-called second (autumn) cycle was induced in geese via a dark-room preparation and natural keeping conditions. The plasma levels of DHEA showed a minor peak at onset of the autumn breeding and a major one prior to the more intense spring reproduction in both sexes. The seasonal curves of plasma DHEA appeared fairly similar in ganders and layers and without considerable differences between the absolute values. In ganders, plasma DHEA peaks preceded the elevations in T levels in the fall and spring alike. With layers, in turn, the autumn and spring peaks of plasma DHEA appeared after the peaks in E2 levels. With ganders, the concentration of plasma T seemed to predominate between the two androgens throughout the experimental period. With layers, in turn, the concentration of DHEA surpassed the level of plasma E2 at the time of the peak periods and other times during the study, as well. In domestic geese, DHEA is probably involved in the autumn physiological processes and the induction of reproduction during fall and early spring periods, alike.     

Activation of the Ca(2+)/calcineurin/NFAT2 pathway controls smooth muscle cell differentiation

Cellular mechanisms controlling smooth muscle cells (SMCs) phenotypic modulation are largely unknown. Intracellular Ca2+ movements are essential to ensure SMC functions; one of the roles of Ca2+ is to regulate calcineurin, which in turn induces nuclear localization of the nuclear factor of activated T-cell (NFAT). In order to investigate, during phenotypic differentiation of SMCs, the effect of calcineurin inhibition on NFAT2 nuclear translocation, we used a culture model of SMC differentiation in serum-free conditions. We show that the treatment of cultured SMC with the calcineurin inhibitor cyclosporine A induced their dedifferentiation while preventing their differentiation. These findings suggest that nuclear translocation of NFAT2 is dependent of calcineurin activity during the in vitro SMC differentiation kinetic and that the nuclear presence of NFAT2 is critical in the acquisition and maintenance of SMC differentiation.     

Expression and characterization of a low molecular weight recombinant human gelatin: development of a substitute for animal-derived gelatin with superior features

Gelatin is used as a stabilizer in several vaccines. Allergic reactions to gelatins have been reported, including anaphylaxis. These gelatins are derived from animal tissues and thus represent a potential source of contaminants that cause transmissible spongiform encephalopathies. We have developed a low molecular weight human sequence gelatin that can substitute for the animal sourced materials. A cDNA fragment encoding 101 amino acids of the human proalpha1 (I) chain was amplified, cloned into plasmid pPICZalpha, integrated into Pichia pastoris strain X-33, and isolates expressing high levels of recombinant gelatin FG-5001 were identified. Purified FG-5001 was able to stabilize a live attenuated viral vaccine as effectively as porcine gelatin. This prototype recombinant gelatin was homogeneous with respect to molecular weight but consisted of several charge isoforms. These isoforms were separated by cation exchange chromatography and found to result from a combination of truncation of the C-terminal arginine and post-translational phosphorylation. Site-directed mutagenesis was used to identify the primary site of phosphorylation as serine residue 546; serine 543 was phosphorylated at a low level. A new construct was designed encoding an engineered gelatin, FG-5009, with point mutations that eliminated the charge heterogeneity. FG-5009 was not recognized by antigelatin IgE antibodies from children with confirmed gelatin allergies, establishing the low allergenic potential of this gelatin. The homogeneity of FG-5009, the ability to produce large quantities in a reproducible manner, and its low allergenic potential make this a superior substitute for the animal gelatin hydrolysates currently used to stabilize many pharmaceuticals.