Cloning and characterization of Schistosoma mansoni fructose-1,6-bisphosphate aldolase isoenzyme.

A Schistosoma mansoni cercarial cDNA expression library, constructed in lambda gt11, was screened using the IgG fraction of sera taken from rabbits vaccinated with irradiated cercariae. A positive cDNA clone (1,431 base pairs) was selected and characterized. The amino acid sequence predicted from the cDNA sequence identified a polypeptide of 363 amino acids that showed significant homology to different family members of the enzyme fructose-1,6-bisphosphate aldolase (EC 1.4.2.13). The identity was 66% and 65% with human C and A isoenzymes, respectively. Active sites and substrate-binding determinant analysis suggest that the isolated enzyme in terms of function resembles type A aldolase. The recombinant protein expressed in the vector pGEX-2T was found to be active enzymatically. Antibodies raised against the purified recombinant protein recognized a 40-kDa band in extracts from cercariae, schistosomula (5 and 25 days), adult worms, and eggs. Using immunocytochemistry, aldolase localized to the tegumental region of the adult worms.

Detection of hepatitis B virus DNA by in situ tissues hybridization in corneal of HBsAG positive donors.

HBV is a hepatotropic virus. The highest concentration found in the blood and liver with lower amount in saliva and semen. The virus was also detected in body fluids. Keratoplasty is an essential operation for the treatment of corneal blindness. At Ain Shams University International Eye Bank 10% of the collected corneas were from donors with HBs antigenemia. These corneas were rejected according to the Eye Bank Association of America. In this study 32 corneal tissues of 16 donors positive HBsAg were examined for,the presence of HBV by in situ DNA hybridization technique to detect HBV DNA in the corneal sections. This marker could not be seen in this work. This preliminary study could be an encouraging step for further researches to evaluate the possibility of the avascular cornea to carry HBV.

Application of Corynebacterium cutis lysate as an immune stimulant in cattle.

An ultrasonicated lysate of Corynebacterium cutis (Ultracorn, Virbac, France) was administered to 10-day-old calves, 5-month-old calves, and pregnant dams kept under Egyptian environmental conditions. Ninety-five calves and 50 dams were used in the study. All animals were treated with 2 ml/100 kg body weight of killed C cutis. Its effects on body weight gain and on calf mortality and morbidity were recorded. The results obtained showed that treated calves had greater weight gains, reduced susceptibility to common viral pathogens, and lower mortality. When given simultaneously with rinderpest vaccine, an immunopotentiating or adjuvant effect was seen. Thus, treated calves had higher neutralizing antibody titers to rinderpest as compared with untreated calves. When administered to pregnant cows in the last month of pregnancy, the offspring of these animals had higher birth weight, better weight gain, and reduced morbidity.

Expression of specific UDP-glucuronosyltransferase isoforms in carcinogen-induced preneoplastic rat liver nodules.

The expression of specific UDP-glucuronosyltransferase isoforms in 2-acetylaminofluorane-induced rat liver preneoplastic nodules was studied; livers from pair-fed littermates were used as controls. For comparison, liver and kidney from 3-methylcholanthrene-treated or untreated (control) rats were used. Steady-state UDP-glucuronosyltransferase mRNA levels were determined by Northern blot analysis or in situ hybridization of tissue sections using a 30-mer oligonucleotide specific for the 3-methylcholanthrene-inducible UDP-glucuronosyltransferase (which is active toward 4-nitrophenol) or a double-stranded cDNA probe specific for androsterone-UDP-glucuronosyltransferase. For 3-methylcholanthrene-inducible UDP-glucuronosyltransferase, the mRNA level was very low in control liver; there was a 15-fold increase after 3-methylcholanthrene treatment. This mRNA was present at relatively high concentration in the kidney and there was a threefold increase after 3-methylcholanthrene administration. In livers with preneoplastic nodules 1 mo after cessation of carcinogen administration, this mRNA concentration was approximately 15 times greater than in control liver. Similar changes in the level of the 3-methylcholanthrene-inducible UDP-glucuronosyltransferase were also observed by in situ hybridization of tissue sections. Immunocytochemical studies using an antiserum that recognizes the 3-methylcholanthrene-inducible UDP-glucuronosyltransferase showed a marked increase in the concentration of this isoform in preneoplastic nodules compared with the adjacent nonnodular liver.

Albumin and collagen mRNA expression in normal and analbuminemic rodent liver: analysis by in situ hybridization using biotinylated probes.

We used in situ nucleic acid hybridization cytochemistry to examine cell types and subcellular sites expressing albumin (alb) or pro alpha 2 collagen (col) mRNA in livers from normal and analbuminemic rodents. Biotinylated cDNA or RNA probes were applied to aldehyde-fixed, non-frozen sections and the resulting DNA-RNA or RNA-RNA hybrids were subsequently visualized by enzymatic detection of either peroxidase or alkaline phosphatase conjugated to anti-biotin IgG or streptavidin. In normal rat liver, alb mRNA was expressed in all hepatocytes and was localized to discrete subcellular structures distributed as aggregates in the cytoplasm and in specific structures encircling the nucleus; these subcellular structures most likely represent the endoplasmic reticulum and nuclear envelope. In mouse liver, pro alpha 2 col mRNA was identified in a subpopulation of sinusoidal lining cells which have the morphological appearance of lipocytes. In liver from analbuminemic rats, a small number of hepatocytes, distributed throughout the hepatic lobule, expressed alb mRNA at high levels; the subcellular distribution of this alb mRNA was essentially identical to that observed in normal rat hepatocytes. Since non-radioactive in situ hybridization detected mRNA within the boundaries of individual cells and showed its precise subcellular location under conditions in which there was excellent preservation of tissue morphology, this procedure should be useful for a wide variety of histopathologic studies.

Molecular mechanisms for changes in hepatic protein synthesis induced by schistosomiasis infection in mice.

Mice infected with Schistosoma mansoni and littermate controls were evaluated serially for 12 weeks. Infected mice gained weight at the same rate as controls, but starting with the sixth week their livers became enlarged with granulomas and fibrous tissue, and they developed hypoalbuminemia. To evaluate the regulation of the albumin and type I collagen gene expression, total RNA was isolated from infected and control mice and translated in an mRNA-dependent rabbit reticulocyte lysate system. Protein synthesis was decreased 1.5-3-fold with RNA from infected vs. control liver. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell-free products showed a reduction in albumin but an increase in type I procollagen synthesis in infected mice. Immunoprecipitation of the cell-free product confirmed that albumin synthesis was reduced in greater proportion than other liver proteins in schistosome-infected mice. Hybridization of RNA from infected liver with cloned mouse albumin cDNA (pmalb-2) demonstrated a reduction in albumin mRNA to 37% of control, while hybridization with a chick type I pro alpha 2 collagen cDNA probe (pCg-45) revealed increased procollagen mRNA in infected liver beginning at 6 weeks postinfection. These results suggest that in murine schistosomiasis a reduction in biologically active albumin mRNA results in decreased albumin synthesis and may be responsible in part for hypoalbuminemia. In addition, increased collagen mRNA is associated with increased collagen synthesis during hepatic fibrosis.

Changes in collagen and albumin mRNA in liver tissue of mice infected with Schistosoma mansoni as determined by in situ hybridization.

We have employed in situ hybridization to evaluate the molecular mechanisms responsible for hypoalbuminemia and increased liver collagen content in murine schistosomiasis. Results were compared using a simplified method of hybridizing isolated hepatocytes from Schistosoma mansoni-infected and normal mouse liver with mouse albumin (pmalb-2) and chick pro-alpha 2(l) collagen (pCg45) probes. Whereas hepatocytes from infected mice showed significantly less albumin mRNA than hepatocytes from control, there were more grains of procollagen mRNA in hepatocytes from infected as compared with control liver. Hybridization of infected liver tissue sections with the collagen probe showed more grains per field in granulomas than in liver regions, whereas with the albumin probe there was more hybridization in liver tissue than in granulomas. These results suggest that in murine schistosomiasis a reduction in albumin mRNA sequence content may be associated with decreased albumin synthesis and ultimately leads to hypoalbuminemia. In addition, although the granuloma seems to be the primary source of type I collagen synthesis, hepatocytes are also capable of synthesizing collagen, especially under fibrogenic stimulation.

Use of in situ hybridization to identify collagen and albumin mRNAs in isolated mouse hepatocytes.

We present a simple and improved method for in situ localization of albumin and collagen mRNAs in isolated mouse hepatocytes. The cells were isolated by collagenase perfusion, mincing, and differential centrifugation. Nick-translated 3H-labeled mouse albumin cDNA (pmalb-2) and chicken pro-alpha 2(I) collagen cDNA (pCg45) probes were then hybridized with the cells in silane-treated microcentrifuge tubes. The cells were transferred and fixed to a microscope slide and hybridization was evaluated semiquantitatively by counting exposure of grains in autoradiographic emulsion placed over the cells. With this method of in situ hybridization, all hepatocytes appear to have significant, but highly variable, amounts of albumin mRNA. In addition, type I procollagen mRNA appears to be present at low abundance in hepatocytes. These results indicate that in situ hybridization can effectively demonstrate the presence of specific low- or high-abundance mRNAs in isolated well-differentiated eukaryotic cells.

Some biochemical effects of long term oral contraceptive therapy on Egyptian females living in rural areas.

PIP: The serum activity of glutamic oxaloacetic transaminase, glutamic pyruvic transaminase and alkaline phosphatase, serum nilirubin (total, direct and indirect), serum proteins (total, albumin, alpha 1, alpha 2, beta and gamma globulins), serum glycoproteins (glyco-albumin, alpha 1, alpha 2, beta and glycoglobulins) and serum lipoproteins (alpha,pre beta, beta and chylomicrons) were studied in 131 Egyptian females receiving either 1 of 2 types of oral contraceptives. These parameters were evaluated before and after 6 and 12 months of continuous use of either 1) mg lynestrenol + 0.05 mg ethinyl estradiol) or (0.5 mg lynestrenol). All these biochemical entities remained within normal limits except serum albumin which showed a significant decrease from the pretreatment level. This decrease could be attributed to a decreased rate of albumin synthesis. Since the liver is the main organ for albumin synthesis, the reduction in albumin might indicate a possible deleterious effect of the hormones on the liver. The determination of the albumin level in the serum may serve as an index to the effect of contraceptive hormones on the liver. Tables for the mean and standard deviation values of studied parameters for group 1 and group 2 before and after 6 and 12 months of continuous use of the combined pill and the minipill are included.^ieng

Effect of oral contraceptives on some biochemical aspects in bilharzial Egyptian females living in rural areas.

PIP: The serum activity of glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and alkaline phosphatase; serum proteins (total, albumin, alpha-1, alpha-2, B and gamma globulins); glycoproteins (glycoalbumin, alpha-1, alpha-2, B and gamma glycoglobulins) and lipoproteins (alpha-, pre B, B and chylomicrons) were studied in 42 bilharzial Egyptian females with hepatic or hepatosplenic affections. These parameters were evaluated before and after 6 and 12 months of continuous use of either a combined pill (0.05 mg ethinyl estradiol + 1 mg lynestrenol) or a minipill (0.5 mg lynestrenol). All these biochemical entities remained within the normal limits except serum albumin and total serum proteins which showed significcant decreases after 6 and 12 months use of either contraceptive. The decrease of serum albumin could be attributed to a decreased rate of its synthesis. No significant increase in globulins was observed. Therefore a significant decrease in total proteins occurred. The decrease of serum albumin during the use of either contraceptive had previously been recorded by the authors in normal females living under the same socioeconomic status as the cases in this study. The same decrease in serum albumin had previously been recorded in non Egyptian females. The determination of albumin level in the serum may serve as an index to the effect of contraceptive hormones on the liver. In contradistinction to the finding in normal cases, alpha-1 glycoprotein failed to increase in response to the contraceptive hormones. This finding may indicate an inadequate response of the liver in bilharzial females to the effect of these hormones.^ieng

Structure of N-terminal cyanogen bromide fragment of human plasma albumin.

The complete amino acid sequence of 87 residues of cyanogen bromide fragment CB1 (Asp), the N-terminal fragment of human plasma albumine molecule, has been established. The sequence was determined from the characterization of all tryptic peptides and of chymotryptic arginine-containing peptides in the fragment digested. Overlaps were obtained by tryptic and chymotryptic cleavage of the maleylated S-sulfo derivative of fragment CB1(Asp). Residue 34 is the only cysteine residue in the albumin molecule and it was determined in the form of S-carboxymethyl-cysteine. Edman and dansyl-Edman degradation were used for the sequential analysis.