Inflammatory cytokines increase extracellular procathepsin D in permanent and primary endothelial cell cultures

The protease cathepsin D (Cath D) and its proteolytically inactive proform, procathepsin D (ProCath D), turned out to be multifunctional within and outside the cell. Elevated levels of ProCath D occur in malignant tumors and in organs under chronic inflammation. One important source for this increase of ProCath D might be endothelial cells. Here we examined the expression of Cath D in the human endothelial cell line EA.hy 926 and in primary endothelial cells isolated from human umbilical cord veins (HUVEC). After serum-free incubation with or without human interferon-gamma (hIFN-gamma) and/or human tumor necrosis factor-alpha (hTNF-alpha) immature and mature Cath D forms were examined in cell extracts and in cell-conditioned medium concentrates by Western blotting. Lysates of EA.hy 926 cells as well as of HUVEC contained active Cath D as two-chain form, but only negligible amounts of ProCath D and Cath D intermediates. Yet both endothelial cell cultures accumulated ProCath D in their conditioned media in the absence of any stimulus. The treatment with hIFN-gamma and/or hTNF-alpha had little effect on intracellular levels of Cath D, whereas the cytokine stimulation increased the extracellular presence of ProCath D in both endothelial cell cultures. The extracellular increase of ProCath D was not related to induction of apoptosis, as validated by cleaved caspase-3 in cell lysates. Acidification of cytokine-treated media converted ProCath D into Cath D, which was associated with cathepsin-like activity using a fluorogenic substrate-linked assay. We conclude, in vitro, endothelial cells are a cytokine-dependent source for extracellular ProCath D.     

Primary stability of cementless stem in THA improved with reduced interfacial gaps

Large interfacial gaps between the stem and the bone in cementless total hip arthroplasty may prevent successful bone ingrowth at the sites, and can also be a passage for wear particles. Furthermore, interfacial gaps between the stem and the bone are believed to compromise the primary stability of the implant. Thus, a broaching method that serves to reduce gaps is expected to give clinically preferable results. A modified broach system with a canal guide is introduced to enhance the accuracy of femoral canal shaping in comparison with the conventional broach system for a Versys fibermetal taper stem. The primary stability of the hip systems and the ratios of the stem surface in contact with the femur were measured in a composite femur model. With the conventional method, an average of 67% of the stem surface was shown to be in contact with the bone, and an average stem micromotion/migration of 35 microm 290 microm was observed under 1000 cycles of stair climbing loads. With the modified method, the stem-bone contact ratio significantly increased to 82% (p<0.05), and the average micromotion/migration reduced to 29 microm 49 microm, respectively (p<0.05 for migration). Our finite element models of the hip systems supported that the difference in micromotion could be attributed to the difference in interfacial contact. Interfacial gaps occurring with the conventional broach system were effectively reduced by the proposed method, resulting in improved primary stability.     

Impaired glucose-stimulated insulin secretion in the GK rat is associated with abnormalities in islet nitric oxide production

We investigated implications of nitric oxide (NO) derived from islet neuronal constitutive NO synthase (ncNOS) and inducible NOS (iNOS) on insulin secretory mechanisms in the mildly diabetic GK rat. Islets from GK rats and Wistar controls were analysed for ncNOS and iNOS by HPLC, immunoblotting and immunocytochemistry in relation to insulin secretion stimulated by glucose or l-arginine in vitro and in vivo. No obvious difference in ncNOS fluorescence in GK vs control islets was seen but freshly isolated GK islets displayed a marked iNOS expression and activity. After incubation at low glucose GK islets showed an abnormal increase in both iNOS and ncNOS activities. At high glucose the impaired glucose-stimulated insulin release was associated with an increased iNOS expression and activity and NOS inhibition dose-dependently amplified insulin secretion in both GK and control islets. This effect by NOS inhibition was also evident in depolarized islets at low glucose, where forskolin had a further amplifying effect in GK but not in control islets. NOS inhibition increased basal insulin release in perfused GK pancreata and amplified insulin release after glucose stimulation in both GK and control pancreata, almost abrogating the nadir separating first and second phase in controls. A defective insulin response to l-arginine was seen in GK rats in vitro and in vivo, being partially restored by NOS inhibition. The results suggest that increased islet NOS activities might contribute to the defective insulin response to glucose and l-arginine in the GK rat. Excessive iNOS expression and activity might be deleterious for the beta-cells over time.     

Substitution patterns in methylated potato starch as revealed from the structure and composition of fragments in enzymatic digests

The effect of the granule structure on the methylation of starch was investigated by comparing the substitution patterns of potato starch methylated in granular suspension and in solution to DS 0.3. Substitution patterns were analyzed by successive digestion with alpha-amylase and amyloglucosidase, fractionation of the resulting malto-oligosaccharide mixture by GPC on a preparative scale, and characterization of the fractions by GLC and MALDI-MS. The mass composition of fractions with intermediate and higher degree of polymerization was indicative of enhanced clustering of substituents in granular methyl starch. On the contrary, the composition of the smaller saccharides was governed by enzyme specificity, which was also reflected in strong deviations in their monomer composition. A sequencing study on selected 'pure' small saccharides confirmed and complemented previous conclusions on enzyme specificity.     

A theoretical analysis of the Ti(IV)-catalyzed glycoaminocyanation reaction

The chelation of the Lewis acid Ti(OiPr)(4) provides the necessary conformational control to induce pi-facial selectivity on the cyanation process. The steric hindrance imposed by the alpha-substituent restrains the addition for l-amino acid derivatives.     

The influence of extrusion on loss and racemization of amino acids

Summary. The influence of the operation conditions (temperature and residence time) of a thermic treatment on the total amount (free and protein-bound) of amino acid enantiomers of dry fullfat soya was investigated. Total amino acid content was determined using conventional ion-exchange amino acid analysis of total hydrolysates and chiral amino acid analysis was performed by HPLC after precolumn derivatization with o-phthaldialdehyde and 1-thio-β-D-glucose tetraacetate. Contrary to corn that was investigated previously, notable racemization was detected even at lower temperatures. At 140 °C the ratio of the D-enantiomer was 0.87% for glutamic acid, 2.81% for serine, and 1.92% for phenylalanine; at 220 °C the ratios of the D-enantiomer of the above amino acids were 1.43, 4.61, and 4.68%, respectively. The concentration of several L-amino acids decreased. At 220 °C there was 10% less L-glutamic acid, 17% less L-serine, 5% less L-phenylalanine, 6.6% less L-aspartic, acid and 21% less L-lysine than in the control; their loss can be assigned to different degrees of L – D conversion. While nearly complete transformation of L-phenylalanine can be attributed to racemization, the main cause of the loss of L-lysine is not racemization. The treatments in the same order of magnitude resulted in the formation of more D-amino acids and greater extent of racemization of amino acids in fullfat soya than that of maize. Authors’ address: J. Csapó, Faculty of Animal Science, Institute of Chemistry, University of Kaposvár, Guba S. u. 40., H-7400 Kaposvár, Hungary     

Specific regions within the embryonic midbrain and cerebellum require different levels of FGF signaling during development

Prospective midbrain and cerebellum formation are coordinated by FGF ligands produced by the isthmic organizer. Previous studies have suggested that midbrain and cerebellum development require different levels of FGF signaling. However, little is known about the extent to which specific regions within these two parts of the brain differ in their requirement for FGF signaling during embryogenesis. Here, we have explored the effects of inhibiting FGF signaling within the embryonic mouse midbrain (mesencephalon) and cerebellum (rhombomere 1) by misexpressing sprouty2 (Spry2) from an early stage. We show that such Spry2 misexpression moderately reduces FGF signaling, and that this reduction causes cell death in the anterior mesencephalon, the region furthest from the source of FGF ligands. Interestingly, the remaining mesencephalon cells develop into anterior midbrain, indicating that a low level of FGF signaling is sufficient to promote only anterior midbrain development. Spry2 misexpression also affects development of the vermis, the part of the cerebellum that spans the midline. We found that, whereas misexpression of Spry2 alone caused loss of the anterior vermis, reducing FGF signaling further, by decreasing Fgf8 gene dose, resulted in loss of the entire vermis. Our data suggest that cell death is not responsible for vermis loss, but rather that it fails to develop because reducing FGF signaling perturbs the balance between vermis and roof plate development in rhombomere 1. We suggest a molecular explanation for this phenomenon by providing evidence that FGF signaling functions to inhibit the BMP signaling that promotes roof plate development.     

Anaerobic methanethiol degradation and methanogenic community analysis in an alkaline (pH 10) biological process for liquefied petroleum gas desulfurization

Anaerobic methanethiol (MT) degradation by mesophilic (30 degrees C) alkaliphilic (pH 10) communities was studied in a lab-scale Upflow Anaerobic Sludge Bed (UASB) reactor inoculated with a mixture of sediments from the Wadden Sea (The Netherlands), Soap Lake (Central Washington), and Russian soda lakes. MT degradation started after 32 days of incubation. During the first 252 days, complete degradation was achieved till a volumetric loading rate of 7.5 mmol MT/L/day, and sulfide, methane, and carbon dioxide were the main reaction products. Temporary inhibition of MT degradation occurred after MT peak loads and in the presence of dimethyl disulfide (DMDS), which is the autooxidation product of MT. From day 252 onwards, methanol was dosed to the reactor as co-substrate at a loading rate of 3-6 mmol/L/day to stimulate growth of methylotrophic methanogens. Methanol was completely degraded and also a complete MT degradation was achieved till a volumetric loading rate of 13 mmol MT/L/day (0.77 mmol MT/gVSS/day). However, from day 354 till the end of the experimental run (day 365), acetate was formed and MT was not completely degraded anymore, indicating that methanol-degrading homoacetogenic bacteria had partially outcompeted the methanogenic MT-degrading archea. The archeal community in the reactor sludge was analyzed by DGGE and sequencing of 16S rRNA genes. The methanogenic archea responsible for the degradation of MT in the reactor were related to Methanolobus oregonensis. A pure culture, named strain SODA, was obtained by serial dilutions in medium containing both trimethyl amine and dimethyl sulfide (DMS). Strain SODA degraded MT, DMS, trimethyl amine, and methanol. Flow sheet simulations revealed that for sufficient MT removal from liquefied petroleum gas, the extraction and biological degradation process should be operated above pH 9.