Detection of insulin-antibody binding on a solid surface using imaging ellipsometry

Imaging ellipsometry (IE) was used to detect the binding of insulin to its antibody on a solid surface. The modification of a gold surface with 11-mecaptoundecanoic acid (11-MUA), the adsorption of protein G, and antibody immobilization onto the protein G layer were confirmed by surface plasmon resonance. Ellipsometric images and ellipsometric angles of the surface antibody were acquired using the IE system by off-null ellipsometry. Ellipsometric images of antigen binding to the antibody were acquired, and their mean optical intensities estimated. Changes in mean optical intensity indicated that the detection range for insulin was from 10 ng/ml to 100 microg/ml.     

Phospholipase C-delta1 rescues intracellular Ca2+ overload in ischemic heart and hypoxic neonatal cardiomyocytes

Ischemia and simulated ischemic conditions cause intracellular Ca2+ overload in the myocardium. The relationship between ischemia injury and Ca2+ overload has not been fully characterized. The aim of the present study was to investigate the expression and characteristics of PLC isozymes in myocardial infarction-induced cardiac remodeling and heart failure. In normal rat heart tissue, PLC-delta1 (about 44 ng/mg of heart tissue) was most abundant isozymes compared to PLC-gamma1 (6.8 ng/mg) and PLC-beta1 (0.4 ng/mg). In ischemic heart and hypoxic neonatal cardiomyocytes, PLC-delta1, but not PLC-beta1 and PLC-gamma1, was selectively degraded, a response that could be inhibited by the calpain inhibitor, calpastatin, and by the caspase inhibitor, zVAD-fmk. Overexpression of the PLC-delta1 in hypoxic neonatal cardiomyocytes rescued intracellular Ca2+ overload by ischemic conditions. In the border zone and scar region of infarcted myocardium, and in hypoxic neonatal cardiomyocytes, the selective degradation of PLC-delta1 by the calcium sensitive proteases may play important roles in intracellular Ca2+ regulations under the ischemic conditions. It is suggested that PLC isozyme-changes may contribute to the alterations in calcium homeostasis in myocardial ischemia.     

Matrix metalloproteinase-2 and -9 secretion by the equine ovary during follicular growth and prior to ovulation

Profound hormonally controlled tissue remodelling occurs in the equine ovary for follicle growth and development, and also for the alteration in follicle shape directed towards the ovulation fossa, the site where ovulation occurs. The aim of this study was to examine the spatial and temporal regulation of matrix metalloproteinases (MMP)-2 and MMP-9, important enzymes in tissue remodelling, during follicle growth, and ovulation. Using gelatin substrate zymography, we measured these MMPs in follicular fluid of large anovulatory follicles collected during spring transition, early dominant follicles (> 23 mm), and at oestrus in follicles approximately 3 days prior to ovulation, and post-hCG treatment when ovulation was predicted in approximately 4 h. The most abundant activity detected in follicular fluid was MMP-2, although there were no changes in secretion or activation in association with ovulation. The activity of MMP-9 was detected in lower amounts, with no changes prior to ovulation, although it decreased significantly (P < 0.05) post-hCG treatment. At oestrus, when different regions of the ovary were maintained in explant culture for 24 h, there were no significant changes in either MMP-2 or MMP-9 secretion by stromal tissues collected at the ovarian fossa, adjacent to the preovulatory follicle but away from the fossa, and a further site remote from the preovulatory follicle. Over this same time period, follicular progesterone (P < 0.01) and oestradiol (P < 0.05) increased significantly, although oestradiol tended to decrease after hCG administration. These findings indicate that MMP-2 and MMP-9 are not key acute regulators for the changes in follicle shape immediately prior to ovulation.     

Two enzymes in one; two yeast peroxiredoxins display oxidative stress-dependent switching from a peroxidase to a molecular chaperone function

Although a great deal is known biochemically about peroxiredoxins (Prxs), little is known about their real physiological function. We show here that two cytosolic yeast Prxs, cPrxI and II, which display diversity in structure and apparent molecular weights (MW), can act alternatively as peroxidases and molecular chaperones. The peroxidase function predominates in the lower MW forms, whereas the chaperone function predominates in the higher MW complexes. Oxidative stress and heat shock exposure of yeasts causes the protein structures of cPrxI and II to shift from low MW species to high MW complexes. This triggers a peroxidase-to-chaperone functional switch. These in vivo changes are primarily guided by the active peroxidase site residue, Cys(47), which serves as an efficient "H(2)O(2)-sensor" in the cells. The chaperone function of these proteins enhances yeast resistance to heat shock.     

Extracts ofPhellinus gilvus andPhellinus baumii inhibit pulmonary inflammation induced by lipopolysaccharide in rats

Compared to saline-challenged rats, rats exposed to 50 microg intratracheal lipopolysaccharide showed an increase of total white cells (from 0.3 x 10(6) to 2.4 x 10(6)), neutrophils (from 0.09 x 10(6) to 1.8 x 10(6)), the levels of tumor necrosis factor (TNF)-alpha (from 200 pg ml(-1) to 1200 pg ml(-1)), and interleukin (IL)-1beta (from 220 pg ml(-1) to 650 pg ml(-1)) in the bronchial lavage fluid. However, after pretreatment with extracts of Phellinus gilvus and Phellinus baumii, the total white cells, neutrophils, and the level of IL-1beta in lipopolysaccharide-challenged rats were similar to those in saline-challenged rats, except for TNF-alpha. The results indicate that extracts of P. gilvus and P. baumii may be useful in preventing acute pulmonary inflammation in human diseases.     

Allelic loss at the GPx-1 locus in cancer of the head and neck

Glutathione peroxidase is a selenium-containing, antioxidant enzyme previously implicated in the risk and development of lung and breast cancer, in part the result of allelic loss at the GPx-1 locus. This study examined allelic loss at the same locus in squamous cell carcinomas of the head and neck. The frequency of a polymorphism at codon 198 resulting in either a leucine or a proline at that position was surveyed by comparing 133 DNA samples obtained from head and neck tumors and 517 samples obtained from cancer-free individuals. Tumor DNAs exhibited fewer pro/leu heterozygotes as compared to DNA obtained from the cancer-free population. Fewer GPx-1 heterozygotes were verified by determining the frequency of highly polymorphic alanine repeat sequences in the same gene. The analysis revealed an approximately 42% reduction in heterozygosity in the DNA from the tumor samples. In order to assess loss of heterozygosity (LOH) at the GPx-1 locus, DNA was genotyped from peripheral lymphocytes, tumor tissue, and microscopically normal tissues adjacent to the tumor, derived from the same patients. These studies indicated LOH at the GPx-1 locus in each of the three tumor/normal tissues sample sets examined. Furthermore, LOH in the microscopically normal tissues at the tumor margin occurred in two of the three sample sets examined. These data implicate GPx-1 in the development of squamous cell carcinoma the head and neck and suggest that allelic loss of this gene, or one tightly linked to it, is an early event in the development of this type of malignancy. Author to whom all correspondence and reprint requests should be addressed. These authors contributed equally to this work.     

Features of bacterial cellulose synthesis in a mutant generated by disruption of the diguanylate cyclase 1 gene of <Emphasis Type="Italic">Acetobacter xylinum</Emphasis> BPR 2001

The diguanylate cyclase 1 (DGC1) (dgc1) gene in Acetobacter xylinum BPR 2001—a bacterial cellulose (BC) producer—was cloned and sequenced, and a DGC1 gene-disrupted mutant, strain DD, was constructed. The production and structural characteristics of the BC formed by DD were compared with those of the parental strain BPR 2001. BC production by DD was almost the same as that by BPR 2001 in static cultivation and in shake flask cultivation. However, in a jar fermentor DD produced about 36% more BC than the parental strain. DD produced suspended particle materials that cannot aggregate owing to their random structural characteristics in static cultivation; more uniformly dispersed BC pellicles and smaller BC pellets are produced on average in a jar fermentor, as reflected by the higher BC production by DD than by the parental strain in a jar fermentor. Micrographs of BC produced by DD revealed that the width of cellulose ribbons assemblies decreased as a result of differences in the ultrastructure and mechanism of formation of BC between the two strains. These results reveal that disruption of the dgc1 gene, which catalyzes synthesis of c-di-GMP (an effector of BC synthase), is not fatal for BC synthesis, although it affects BC structure.     

Human ACAT-1 and -2 inhibitory activities of saucerneol B, manassantin A and B isolated from Saururus chinensis

The sesquineolignan, saucerneol B (1), and dineolignans, manassantin A (2), and manassantin B (3), were isolated from the methanol extracts of Saururus chinensis root and elucidated by their spectroscopic data analysis. Compounds 1-3 inhibited hACAT-1 and hACAT-2 with IC(50) values of 43.0 and 124.0 microM for 1, of 39.0 and 8.0 microM for 2, of 82.0 microM and only 32% inhibition at 1mM for 3, respectively. The EtOAc-soluble fraction, which contained compounds 1-3, of methanol extracts of S. chinensis exhibited strong cholesterol-lowering effect in high cholesterol-fed mice.