Genetic characterization and transmission cycles of Cryptosporidium species isolated from humans in New Zealand

Little is known about the genetic characteristics, distribution, and transmission cycles of Cryptosporidium species that cause human disease in New Zealand. To address these questions, 423 fecal specimens containing Cryptosporidium oocysts and obtained from different regions were examined by the PCR-restriction fragment length polymorphism technique. Indeterminant results were resolved by DNA sequence analysis. Two regions supplied the majority of isolates: one rural and one urban. Overall, Cryptosporidium hominis accounted for 47% of the isolates, with the remaining 53% being the C. parvum bovine genotype. A difference, however, was observed between the Cryptosporidium species from rural and urban isolates, with C. hominis dominant in the urban region, whereas the C. parvum bovine genotype was prevalent in rural New Zealand. A shift in transmission cycles was detected between seasons, with an anthroponotic cycle in autumn and a zoonotic cycle in spring. A novel Cryptosporidium sp., which on DNA sequence analysis showed a close relationship with C. canis, was detected in two unrelated children from different regions, illustrating the genetic diversity within this genus.     

Peroxynitrite resistance of sarco/endoplasmic reticulum Ca2+ pump in pig coronary artery endothelium and smooth muscle

We examined the effects of peroxynitrite pre-treatment on sarco/endoplasmic reticulum Ca(2+) (SERCA) pump in pig coronary artery smooth muscle and endothelium. In saponin-permeabilized cells, smooth muscle showed much greater rates of the SERCA Ca(2+) pump-dependent (45)Ca(2+) uptake/mg protein than did the endothelial cells. Peroxynitrite treatment of cells inhibited the SERCA pump more severely in smooth muscle cells than in endothelial cells. To determine implications of this observation, we next examined the effect of the SERCA pump inhibitor cyclopiazonic acid (CPA) on intracellular Ca(2+) concentration of intact cultured cells. CPA produced cytosolic Ca(2+) transients in cultured endothelial and smooth muscle cells. Pre-treatment with peroxynitrite (200 microM) inhibited the Ca(2+) transients in the smooth muscle but not in the endothelial cells. CPA contracts de-endothelialized artery rings and relaxes precontracted arteries with intact endothelium. Peroxynitrite (250 microM) pre-treatment inhibited contraction in the de-endothelialized artery rings, but not the endothelium-dependent relaxation. Thus, endothelial cells appear to be more resistant than smooth muscle to the effects of peroxynitrite at the levels of SERCA pump activity, CPA-induced Ca(2+) transients in cultured cells, and the effects of CPA on contractility. The greater resistance of endothelium to peroxynitrite may play a protective role in pathological conditions such as ischemia-reperfusion when excess free radicals are produced.     

CVT-4325: a potent fatty acid oxidation inhibitor with favorable oral bioavailability

New inhibitors of palmitoyl-CoA oxidation are based on the introduction of nitrogen heterocycles in the ‘Western Portion’ of the molecule. SAR studies led to the discovery of CVT-4325 (shown), a potent FOXi (IC₅₀ = 380 nM rat mitochondria) with favorable PK properties (F = 93%, t_1/2 = 13.6 h, dog).     

IL-6, RANKL, TNF-alpha/IL-1: interrelations in bone resorption pathophysiology

All osteogenic cells (osteoclasts, osteoblasts) contribute individually to bone remodeling. Their cellular interactions control their cellular activities and the bone remodeling intensity. These interactions can be established either through a cell-cell contact, involving molecules of the integrin family, or by the release of many polypeptidic factors and/or their soluble receptor chains. These factors can act directly on osteogenic cells and their precursors to control differentiation, formation and functions (matrix formation, mineralization, resorption...). Here, we present the involvement of three groups of cytokines which seem to be of particular importance in bone physiology: interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) (TNF-alpha)/IL-1, and the more recently known triad osteoprotegerin (OPG)/receptor activator of NF-kappaB (RANK)/RANK ligand (RANKL). The interactions between these three groups are presented within the framework of bone resorption pathophysiology such as tumor associated osteolysis. The central role of the OPG/RANK/RANKL triad is pointed out.     

Enhancing effect of low culture temperature on specific antibody productivity of recombinant Chinese hamster ovary cells: clonal variation

To understand the different responses of recombinant Chinese hamster ovary (rCHO) cells to low culture temperature regarding specific productivity (q), 12 parental clones and their corresponding amplified clones producing a humanized antibody were cultivated at 32 and 37 degrees C. The specific growth rate of all clones, including both parental and amplified clones, decreased by 30-63% at 32 degrees C, compared to rates at 37 degrees C. In contrast, their specific antibody productivity (qAb) was significantly enhanced at 32 degrees C. Furthermore, the degree of qAb enhancement at 32 degrees C varied a lot from 4- to 25-fold among the parental clones. At 32 degrees C, most of the amplified clones, regardless of methotrexate (MTX) levels, also showed enhanced qAb but to a lesser extent than their parental clones. However, clone 14 amplified at 0.32 microM MTX (clone 14-0.32) and clone 20 amplified at 1 microM MTX (clone 20-1.00), unlike their parental clones, did not show enhanced qAb at 32 degrees C. Thus, it was found that the enhancing effect of low culture temperature on q of rCHO cells depends on clones. Taken together, the results obtained here emphasize the importance of clonal selection for the successful application of low culture temperature to the enhanced foreign protein production in rCHO cells.     

Effects of Aroclor 1254 on the expression of rat placental PRL-family genes

This study was performed to investigate the effects of Aroclor 1254 (A1254), a commercial polychlorinated biphenyl mixture, on the expression of rat placental prolactin (PRL) family genes and reproductive activity. Placental lactogen-Iv and -II, and prolactin-like protein-A and -C mRNA levels were significantly decreased in the placentas of A1254-treated rats in a dose-dependent manner. The mRNA levels of Pit-1alpha and beta isotypes, which are involved in the regulation of PRL family gene expression, were also decreased in the A1254-treated rat placenta. In the rat placental junctional zone, high-dose A1254 (25 mg/kg B.W.) treatment reduced the number of spongiotrophoblasts, cells in which the PRL family genes are expressed. Finally, maternal exposure to A1254 was shown to have significant toxic effects on reproductive activity, including embryonic and placental growth retardation, delay of parturition, and reduction of the number of pups per litter. The results of the present study indicated that A1254 has an inhibitory effect on PRL family, Pit-1alpha, and beta gene expression in the rat placenta, leading to significant toxic effects on reproductive activity in rats.     

Acute and chronic leptin treatment mediate contrasting effects on signaling,glucose uptake,and GLUT4 translocation in L6-GLUT4myc myotubes

We have previously shown that in L6-GLUT4myc rat skeletal muscle cells, acute treatment with leptin reduced insulin-stimulated glucose uptake without altering insulin-stimulated GLUT4 translocation. In contrast, we show here that the ability of leptin to increase phosphorylation of its receptor and to reduce insulin-stimulated glucose uptake was lost in cells that were continuously exposed to leptin for 24 h. This desensitization correlated with an increase in expression of suppressor of cytokine signaling-3 (SOCS-3). Time course analysis demonstrated that the transition from acute to chronic effects of leptin occurs after 2 h. The desensitization of leptin action at 24 h was not reversed by 30 min washout period prior to re-exposing cells to leptin. However, despite insulin-stimulated glucose uptake being unaffected upon 24 h preincubation with leptin, a small but significant decrease (37%) in insulin-stimulated GLUT4 translocation and phosphorylation of Akt on T308 was detected. Insulin-stimulated phosphorylation of Akt on S473 or of p38 MAPK were unaffected. These results suggest that the chronic leptin treatment leads to desensitization of leptin signaling yet can simultaneously decrease the ability of insulin to phosphorylate Akt on T308 and translocate GLUT4. However, this does not manifest as a reduction in total glucose uptake into L6 myotubes.     

The role of the protein core in the inhibitory power of the classic serine protease inhibitor,chymotrypsin inhibitor 2

A synthetic cyclic peptide, reported to be a tight-binding inhibitor of serine proteases, is instead found to be a good substrate, as is the linear peptide of the same sequence. Both of the peptides, designed to mimic the binding loop of chymotrypsin inhibitor 2 (CI2), were cleaved by subtilisin primarily at the CI2 reactive-site Met-59-Glu-60 bond, revealing that the sequence, in the absence of the structural context of the inhibitor, provides sufficient specificity for hydrolysis of this bond. Insights from the crystal structure of the CI2/subtilisin complex, together with biochemical analysis of a CI2 Gly-83 deletion mutant, have allowed us to identify key features that make CI2 an effective inhibitor, while the cyclic and linear peptides are substrates.     

A mechanosensitive cation channel in endothelial cells and its role in vasoregulation

Ca2+ is an important intracellular second messenger in signal transduction of endothelial cells. It has long been recognized that a mechanosensitive Ca2+-permeable channel is present in vascular endothelial cells. The activity of this channel may increase intracellular Ca2+ level in endothelial cells. A recent finding is that the activity of this channel may be regulated by cGMP through a protein kinase G-dependent pathway. Inhibition of the channel by cGMP abolishes the Ca2+ influx elicited by flow. Several inhibitors of the cation channel including Gd3+, Ni2+, and SK&F-96365 also inhibit the Ca2+ influx due to flow stimulation. These data suggest that a mechanosensitive cation channel is the primary pathway mediating the flow-induced Ca2+ entry in vascular endothelial cells. Another important finding is that the opening of this mechanosensitive channel by KT5823 leads to endothelium-dependent vascular dilation. Therefore, it appears that this channel may play a crucial role in the regulation of vascular tone.