Assay for evaluation of rotavirus-cell interactions: identification of an enterocyte ganglioside fraction that mediates group A porcine rotavirus recognition.

A virus-host cell-binding assay was developed and used to investigate specific binding between group A porcine rotavirus and MA-104 cells or porcine enterocytes. A variety of glycoconjugates and cellular components were screened for their ability to block rotavirus binding to cells. During these experiments a crude ganglioside mixture was observed to specifically block rotavirus binding. On the basis of these results, enterocytes were harvested from susceptible piglets and a polar lipid fraction was isolated by solvent extraction and partitioning. Throughout subsequent purification of this fraction by Sephadex partition, ion-exchange, silicic acid, and thin-layer chromatography, blocking activity behaved as a monosialoganglioside (GMX) that displayed a thin-layer chromatographic mobility between those of GM2 and GM3. The blocking activity of GMX was inhibited by treatment with neuraminidase and ceramide glycanase but not by treatment with protease or heat (100 degrees C). Further purification of GMX by high-pressure liquid chromatography resulted in the resolution of two monosialogangliosides, GMX and a band which comigrated with GM1 on thin-layer chromatography. These data suggest that a cell surface monosialoganglioside or family of monosialogangliosides may function as an in vivo relevant receptor for group A porcine rotavirus and that sialic acid is a required epitope for virus-binding activity.     

Dermatoglyphics in hypertension: a review

Hypertension is a major contributor to the global burden of disease and mortality. A major medical advancement would be a better means to ascertain which persons are at higher risk for becoming hypertensive beforehand. To that end, there have been a number of studies showing that certain dermatoglyphic markers are associated with hypertension. This association could be explained if the risk toward developing hypertension later on in life is somehow connected with fetal development of dermatoglyphics. It would be highly valuable from a clinical standpoint if this conjecture could be substantiated since dermatoglyphic markers could then be used for screening out individuals who might be at an elevated risk of becoming hypertensive. The aim of this review was to search for and appraise available studies that pertain to the association between hypertension and dermatoglyphics.A systematic literature search conducted using articles from MEDLINE (PubMed), Trip, Cochran, Google scholar, and gray literature until December 2014. Of the 37 relevant publications, 17 were included in the review. The review performed according to the preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement.This review showed a fairly consistent finding of an increased frequency of whorl patterns along with a higher mean total ridge count in digital dermatoglyphic results in hypertensive samples compared to controls. However, it was imperative to discuss several limitations found in the studies that could make this association as yet unsettled.     

Phosphate-dependent glutaminase from rat kidney. Cause of increased activity in response to acidosis and identity with glutaminase from other tissues.

Immune serum was prepared against phosphate-dependent glutaminase purified from rat kidney and was used to investigate the cause of increased renal glutaminase activity in acidotic rats. Crude kidney homogenates from acidotic rats exhibited a fourfold greater specific activity for phosphate-dependent glutaminase. The glutaminase was solubilized initially by lyophilization of borate treated mitochondria with a 40–60% recovery and with maintenance of threefold difference in specific activity. Both preparations showed the same equivalence point in a quantitative precipitin experiment. To confirm these results, phosphate-dependent glutaminase was also solubilized by treatment of mitochondria isolated from normal and acidotic rat kidney cortex with 1% Triton X-100. The two preparations exhibited a fivefold difference in specific activity and again showed the same equivalence point in a quantitative precipitin experiment. These results indicate that the cause of increased phosphate-dependent glutaminase activity during acidosis is due to the presence of an increased amount of this enzyme. The antiserum prepared against the kidney phosphate-dependent glutaminase did not crossreact with glutaminase solubilized from rat liver mitochondria. But, rat brain mitochondria do contain a phosphate-dependent glutaminase that is immunologically identical to the enzyme from rat kidney.     

Demonstration of acid phosphatase in Eimeria spp.: partial characterization of the enzyme in E. vermiformis

Sporozoite extracts of E. vermiformis, E. stiedai, and E. tenella are rich in acid phosphatase activity. They contain specific enzyme activities equal to or greater than those reported for other highly virulent protozoan parasites. The absolute amount of enzyme activity per oocyst dramatically increases during sporulation of E. stiedai and E. vermiformis. Partial characterization of the acid phosphatase activity of E. vermiformis indicates that sporozoites account for greater than 92% of the total activity in sporulated oocysts, that the enzyme is resistant to inhibition by tartrate, and that it can be separated into two forms by anion exchange chromatography.     

A fluorescamine assay for submicrogram quantities of protein in the presence of Triton X-100

The sensitivity of the fluorescamine assay is increased by hydrolysis of the protein sample in 2 n NaOH. The assay is linear from 0.125 to 10 μg of protein and can measure concentrations as low as 0.5 μg/ml. The presence of several detergents including Triton X-100 in both the protein sample (up to 10%) and in the fluorescamine assay itself (up to 0.33%) does not interfere.     

Implications of Storing Urinary DNA from Different Populations for Molecular Analyses

Background

Molecular diagnosis using urine is established for many sexually transmitted diseases and is increasingly used to diagnose tumours and other infectious diseases. Storage of urine prior to analysis, whether due to home collection or bio-banking, is increasingly advocated yet no best practice has emerged. Here, we examined the stability of DNA in stored urine in two populations over 28 days.

Methodology

Urine from 40 (20 male) healthy volunteers from two populations, Italy and Zambia, was stored at four different temperatures (RT, 4°C, −20°C & −80°C) with and without EDTA preservative solution. Urines were extracted at days 0, 1, 3, 7 and 28 after storage. Human DNA content was measured using multi-copy (ALU J) and single copy (TLR2) targets by quantitative real-time PCR. Zambian and Italian samples contained comparable DNA quantity at time zero. Generally, two trends were observed during storage; no degradation, or rapid degradation from days 0 to 7 followed by little further degradation to 28 days. The biphasic degradation was always observed in Zambia regardless of storage conditions, but only twice in Italy.

Conclusion

Site-specific differences in urine composition significantly affect the stability of DNA during storage. Assessing the quality of stored urine for molecular analysis, by using the type of strategy described here, is paramount before these samples are used for molecular prognostic monitoring, genetic analyses and disease diagnosis.     

The role of extra-hepatic tissues in the receptor-mediated plasma clearance of glycoproteins terminated by mannose or N-acetylglucosamine.

The mannose- and N-acetylglucosamine-specific pathway for the clearance of mammalian glycoproteins has been characterized by using 125I-labelled neoglycoproteins, glycosidase-treated orosomucoid and lysosomal glycosidases (beta-glucuronidase and beta-N-acetylglucosaminidase) as probes. There are two components to this pathway in vivo; one liver-dependent and the other extrahepatic or liver-independent. Cells that mediate clearance by the latter component of the pathway are present in spleen, bone and in elements of the reticuloendothelial system, but not in the kidney. Glycoproteins that possess terminal mannose, glucose or N-acetylglucosamine residues, including various lysosomal enzymes, are rapidly cleared from plasma via this pathway. Glucose-terminated glycoproteins are recognized by two pathways in the intact animal; the hepatic galactose-specific pathway and the mannose/N-acetylglycosamine-specific pathway, which is present in liver and in peripheral tissues. Following removal of the liver by surgical evisceration, glucose-terminated glycoproteins are cleared whereas glycoproteins bearing galactose are not cleared. Uptake of 125I-labelled neoglycoproteins and agalacto-orosomucoid by isolated alveolar macrophages closely mimics clearance in vivo by the mannose/N-acetylglucosamine pathway. Neoglycoproteins terminated by mannose, glucose or N-acetylglucosamine all compete with 125I-labelled agalacto-orosomucoid for uptake by receptor-mediated pinocytosis. The extent of substitution of the neoglycoproteins is a critical determinant of their inhibitory potency. It is proposed that mononuclear phagocytes are in important component of the clearance pathway in vivo. The mannose/N-acetylglucosamine pathway may be important in the regulation of extracellular levels of various glycosylated macromolecules, including lysosomal hydrolases.     

Glucocorticoid hepatopathy: effect on receptor-mediated endocytosis of asialoglycoproteins.

Histologic and electron microscopic examination of liver tissue from glucocorticoid-treated dogs (GT dogs) showed a markedly abnormal hepatocellular morphology which consisted of severe hepatocellular swelling, vacuolation, and peripheral displacement of subcellular organelles. The abnormal cell morphology was typical of that seen in clinical cases of canine Cushing's Syndrome. The hepatocyte isolation procedure used here works equally well for the preparation of viable hepatocytes from both normal and GT dogs even though GT dogs displayed a pronounced hepatopathy. Cell yields (10(9) cells from a 30-cm3 section of liver) are similar to those reported for rat hepatocytes using whole liver in situ perfusion and cell viability is routinely greater than 85%. The isolation procedure preserved the "abnormal" state or swollen morphology of the hepatocytes from GT dogs and thus can be used in pathophysiological studies of glucocorticoid-induced hepatopathy. The isolated hepatocytes were 3.2 times greater in cell volume than normal hepatocytes. We also observed over a 12.3-fold increase in alkaline phosphatase activity and the appearance in both the liver and the serum of GT dogs of the unique, corticosteroid alkaline phosphatase isozyme (CALP). In spite of the obvious abnormal liver morphology and elevated serum and liver alkaline phosphatase activities, the function of the hepatic cell surface carbohydrate binding protein, the Gal/GalNAc or asialoglycoprotein receptor, was not impaired. We found a trend of about a 1.5-fold increase in the initial rate of ligand uptake as well as 1.6-fold more receptors on GT dog hepatocytes compared to normal hepatocytes. The ligand binding affinity of these receptors, as well as the rate of ligand degradation, was identical in hepatocytes isolated from normal and diseased dogs. When intestinal alkaline phosphatase (IALP) is used as the ligand, approximately 25% was exocytosed intact following endocytosis. These results demonstrate that dogs with glucocorticoid hepatopathy possess a normally functioning Gal/GalNAc receptor. Furthermore, these data are consistent with the hypothesis that structurally related IALP and CALP isozymes may also be metabolically related through the Gal/GalNAc receptor endocytosis pathway. That is, a portion of the IALP normally endocytosed through the Gal/GalNAc receptor pathway in glucocorticoid-treated dogs may be recycled and converted (hyperglycosylated) to the abnormal serum CALP isozyme rather than being degraded.     

Sexual dimorphism in digital dermatoglyphic traits among Sinhalese people in Sri Lanka

Background

The purpose of this study was to evaluate gender-wise diversity of digital dermatoglyphic traits in a sample of Sinhalese people in Sri Lanka.

Findings

Four thousand and thirty-four digital prints of 434 Sinhalese individuals (217 males and 217 females) were examined for their digital dermatoglyphic pattern distribution. The mean age for the entire group was 23.66 years (standard deviation = 4.93 years). The loop pattern is observed more frequently (n = 2,592, 59.72%) compared to whorl (n = 1,542, 35.53%) and arch (n = 206, 4.75%) in the Sinhalese population. Females (n = 1,274, 58.71%) have a more ulnar loop pattern than males (n = 1,231, 56.73%). The plain whorl pattern is observed more frequently in males (n = 560, 25.81%) compared to females (n = 514, 23.69%).The double loop pattern is observed more frequently on the right and left thumb (digit 1) of both males and females. Pattern intensity index, Dankmeijer index and Furuhata index are higher in males.

Conclusions

Ulnar loop is the most frequently occurring digital dermatoglyphic pattern among the Sinhalese. All pattern indices are higher in males. To some extent, dermatoglyphic patterns of Sinhalese are similar to North Indians and other Caucasoid populations. Further studies with larger sample sizes are recommended to confirm our findings.