bluuebunny/crossref_metadata_2025 huggingface dataset:
Insulin modulation of human apolipoprotein B mRNA translation: studies in an in vitro cell-free system from HepG2 cells
Authors: Adeli Khosrow , Theriault Andre
Published: December 2009

Insulin modulation of apolipoprotein B gene expression was studied at the translational level by the use of a cell-free translation system from a hepatoma cell-line, HepG2. Extracts of HepG2 cells lysed with lysolecithin were found to have high in vitro protein synthesizing activity utilizing endogenous mRNA. The level of peptide chain initiation was high, as suggested by a significant inhibition of translation by edeine. The translation products of endogenous mRNA in HepG2 cell-free lysate were probed with anti-apolipoprotein B antibodies to investigate its synthesis. A 550 kilodalton (kDa) polypeptide was selected by a polyclonal antibody, as well as a monoclonal antibody, against the C-terminal end of apolipoprotein B molecule. This in vitro synthesized polypeptide was also found to compare well in size with the in vivo product. The HepG2 lysate was also shown to efficiently synthesize in vitro a number of other proteins including albumin, apolipoprotein E, apolipoprotein A1, and actin. The in vitro synthesis of polypeptides as large as 500 kDa was unexpected and has not previously been demonstrated in a cell-free system. The HepG2 translation system was used to investigate the effect of insulin on the in vitro translation of apolipoprotein B. Lysates prepared from HepG2 cells treated with insulin were found to have lower translational activity (by an average of 52.3%) for apolipoprotein B compared with lysates from control untreated cells. In vitro synthesis of actin and apolipoprotein E were unaffected under these conditions. The insulin-stimulated decline in in vitro apolipoprotein B synthesis was not due to a change in apolipoprotein B mRNA levels as determined by slot- and Northern-blot analyses, suggesting that the inhibitory effect of insulin may be exerted partly at the level of apolipoprotein B mRNA translation.Key words: apolipoprotein B, translation, cell-free system, HepG2, insulin.
l-O-Hexadecyl-2-O-methyl-sn-glycero-3-phosphocholine inhibits diacylglycerol kinase in WEHI-3B cells
Authors: Salari Hassan , Low Mervin , Howard Sandra , Edin Glenn , Bittman Robert
Published: December 2009

The effects of 1-O-hexadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-16-OCH3-GPC) and its metabolite 1-O-hexadecyl-2-O-methyl-sn-glycerol (AMG) on the activity of diacylglycerol kinase (DGK) in WEHI-3B cells were investigated. Treatment of WEHI-3B cells with 200 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 5 min leads to the activation of cytosolic DGK without significant effect on microsomal DGK. When these cells were first exposed to 50 μM ET-16-OCH3-GPC for 30 min prior to activation with TPA, the activity of DGK was inhibited by about 70%, as measured by the ability of enzyme to form [32P]phosphatidic acid ([32P]PA). Addition of either ET-16-OCH3-GPC or AMG to the preparation of enzyme in vitro also inhibited 1,2-dioleoyl-sn-glycerol (DG) phosphorylation in the presence of [γ-32P]ATP. The IC50 value for inhibition of cytosolic DGK by ET-16-OCH3-GPC and AMG were about 8.5 and 15 μM, respectively. ET-16-OCH3-GPC also inhibited the ability of guanosine 5′-O-(3-thiophosphate) (GTP-7S) to activate DGK in vitro. The potency of ET-16-OCH3-GPC at 10 μM in inhibiting DGK was greater than that of sphingosine at 50 μM, but less than that of R59022 (a specific DGK inhibitor) at 10 μM. The abilities of ET-16-OCH3-GPC and AMG to inhibit cytosolic DGK in intact WEHI-3B cells and enzyme preparations in vitro suggest that the cytotoxic activity of ether lipids may in part result from interference with this vital enzyme involved in the synthesis of phospholipids from DG and in cell-signaling systems.Key words: diacylglycerol, kinases, second messengers, ether lipids, cancer, WEHI-3B cells.
Halogenated general anesthetics in visual photoreceptor membranes as examined by static and dynamic properties of rhodopsin
Authors: Daigle Isabelle , Gilbert Monique , Boucher François
Published: December 2009

The visual pigment rhodopsin was used as an intrinsic probe to measure the effect of incorporation of enflurane into bovine rod outer segment disk membranes. At moderately low enflurane concentration, we find that, while extrinsic probes show little membrane perturbation, rhodopsin may experience large changes with respect to the native lipid–protein interactions which modulate some of its properties. Enflurane induces a small blue shift in the pigment λmax and a strong inhomogeneity in its photochemical behavior. As a function of enflurane concentration, there appears to be an increasing population of rhodopsin molecules for which the metarhodopsin I → II transition is blocked, while it is accelerated in the remaining population. Under these conditions, electrophoresis and partial membrane solubilization indicate that an increasing amount of rhodopsin does not behave like a free monomeric species. These results are all consistent with a reduction of the membrane order accompanied by lateral aggregation of rhodopsin in the presence of enflurane. By comparison with the perturbation probed by a spin label, the large molecular environment change experienced by the rhodopsin molecule suggests that enflurane may concentrate at the lipid-protein interface.Key words: anesthetics, flash photolysis, lateral aggregation, rhodopsin, visual membranes.
Comparative studies of CDP-diacylglycerol synthase in rat liver mitochondria and microsomes
Authors: Mok Amy Y. P. , McDougall Gordon E. , McMurray William C.
Published: December 2009

CDP-diacylglycerol for polyglycerophosphatide biogenesis can be synthesized within rat liver mitochondria. Contamination by microsomal membranes cannot account for the CDP-diacylglycerol synthesis found in the mitochondria. Phosphatidic acid from egg lecithin was the best substrate for the synthesis of CDP-diacylglycerol in both subcellular fractions. Concentration curves for CTP and Mg2+ differed for the two subcellular fractions. Microsomal CDP-diacylglycerol synthase was specifically stimulated by the nucleotide GTP; this stimulatory effect by GTP was not observed in the mitochondrial fraction. By comparison, the microsomal enzyme was more sensitive towards sulfhydryl inhibitors than the mitochondrial enzyme. The enzymes could be solubilized from the membrane fractions using 3-[(cholamidopropyl)dimethylammonio]-1-propanesulfonate, and the detergent-soluble activity could be partially restored by addition of phospholipids. Based on the differences in properties, it was concluded that there are two distinct enzyme localizations for CDP-diacylglycerol synthesis in mitochondria and microsomes from rat liver.Key words: CDP-diacylglycerol, synthase, phosphatidic acid, mitochondria, microsomes, solubilization.
NAD+-specific glutamate dehydrogenase of Neurospora crassa: cloning, complete nucleotide sequence, and gene mapping
Authors: Kapoor M. , Vijayaraghavan Y. , Kadonaga R. , LaRue K. E. A.
Published: December 2009

The NAD+ -specific glutamate dehydrogenase (NAD-GDH) of the filamentous fungus Neurospora crassa is a tetrameric enzyme, regulated by catabolite repression. The amino acid sequence of this enzyme had been published several years ago. With the object of investigating the molecular mechanism of catabolite repression, the nucleotide sequence of genomic clones containing the coding region, along with 5′- and 3′-flanking noncoding segments of the NAD-GDH transcription unit, was obtained. The gdh structural gene was shown to code for a polypeptide of 1047 residues, with a calculated molecular mass of 118 280 daltons. The coding sequence is interrupted by two short introns located close to the N- and C-terminal domains of the polypeptide. Consensus intron boundaries and internal splice sequences resemble closely those of other N. crassa genes. A comparison of the amino acid sequence deduced from the nucleotide sequence with the previously published sequence showed several discrepancies between the two. Nucleotide sequence corresponding to a gap in the amino acid sequence was located in the genomic clone. Genetic mapping by restriction fragment length polymorphism analysis localized the gdh gene close to the loci trp-1 and con-7 on the right arm of linkage group III.Key words: Neurospora, NAD-specifïc glutamate dehydrogenase, sequences, gene map.
Molecular cloning of G1 phase mRNAs from a subtractive G1 phase cDNA library
Authors: Wu Gin , Su Shiawhwa , Kung Tzyy-Yun Tzeng , Bird R. Curtis
Published: December 2009

Many G1-phase-specific mRNAs have been identified from various normal or transformed cells based on serum induction and re-entry into the cell cycle from quiescence. However, these mRNAs may not represent some important genes expressed during G1 phase in continuously cycling cells. The eukaryotic cell cycle possesses two cdk (cyclin-dependent kinase) dependent regulatory gates through which cells pass during late G1 phase and G2 phase of each cycle. Subtractive hybridization was employed to synthesize a high R0t fraction cDNA library enriched in sequences expressed during G1 phase prior to passage through the G1-phase gate. To prepare G1-phase cells from continuously cycling cell populations, G1-phase HeLa cells were collected by centrifugal elutriation and highly synchronous S phase cells were obtained by double thymidine block followed by centrifugal elutriation. A G1-phase subtractive cDNA library was prepared by subtracting G1-phase cDNA with a 10-fold excess of S-phase mRNA. Single-stranded, G1-phase cDNAs were isolated by oligo(dA) chromatography. The library was screened with a high R0t fraction subtractive probe population. Following two rounds of screening, 20 positive clones were obtained. Northern blot analysis indicated that six of these clones were enhanced in expression level during G1 phase when compared with S phase. Nucleotide sequence comparison of each clone with the GenBank data base revealed that hG1.11 was highly homologous (99%) to the apoferritin light chain gene and clones hG1.6, hG1.10, hG1.17, and hG1.18 represented new G1-phase-enriched members of four human ribosomal protein gene families (71–95% homology). The last clone, hG1.1, encoded a highly charged polypeptide not previously identified. Additional study of these G1-phase-enriched mRNAs will be required to determine their role in cell cycle progression and the G1-phase gateway through which cells transit as they proceed through the cell cycle.Key words: cell cycle, G1 phase, subtractive hybridization, cDNA cloning, ribosomal proteins, apoferritin light chain, HeLa cells.
Calcium ions reverse a latency period induced by bovine serum albumin in the time course of phospholipase A2 action on 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomes
Authors: Pacheco Fermín , Mejía Ricardo , Fernández Marta S.
Published: December 2009

The time course of hydrolysis of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposomes in the gel phase catalyzed by porcine pancreatic phospholipase A2 was studied at 1 mM NaCl and variable CaCl2 concentrations, in the presence of delipidated bovine serum albumin. It has been found that the duration of the latency induced by bovine serum albumin shows an inverse dependence with CaCl2 concentration. As we showed previously, the induction of a lag phase by bovine serum albumin is related to its ability to sequester the fatty acid newly released by hydrolysis. Based on this and on our observation that there is an inverse dependence between the length of the latency period and the interfacial calcium ion concentration, it is interpreted that, while a direct effect of bovine serum albumin is the diminution of the liposome negative surface charge density by sequestration of the fatty acid released during hydrolysis, an indirect effect could be the decrease in the surface Ca2+ concentration. This, in turn, should diminish the enzyme binding to the lipid–water interface. The appearance of a latency phase seems to be the final consequence of these events.Key words: phospholipase A2, latency period, calcium ions, serum albumin.
Characterization of parvalbumin cDNA clones and gene expression in normal and dystrophic mice of strain 129 ReJ
Authors: Chatterjee A. , Butler A. M. , Blum M. , Warner A. H.
Published: December 2009

Parvalbumin is a calcium-binding protein found in fast-twitch skeletal muscles and selected cells in the brain. In several dystrophic mutants in the mouse, the parvalbumin content of skeletal muscles and brain is reduced and this deficiency appears to correlate with the inability of these mice to handle enhanced calcium uptake associated with the dystrophic process. In this study, two overlapping cDNA clones of 392 and 1268 base pairs were isolated from a mouse cDNA library in λgt11, characterized, and used as probes to study the involvement of the parvalbumin gene and its expression in various tissues of dystrophic mice of strain 129 ReJ. Southern blot analyses of restriction fragments of genomic DNA from normal and dystrophic mice indicate the same number and size of parvalbumin-specific gene fragments observed by other researchers, suggesting that the size of the Pva gene is the same in both normal and dystrophic mice of strain 129 ReJ. Northern blot analyses of total RNA from hind-limb muscles using cloned parvalbumin cDNA as probes revealed an abundant 800-nucleotide mRNA with lesser amounts of a 1000-nucleotide mRNA transcript in both normal and dystrophic mice of strain 129 ReJ. The amount of these mRNAs was reduced by 65–77% in dystrophic muscles preparations and was similar to the levels of β-actin mRNA in these animals. These results suggest that parvalbumin gene expression is not down regulated in dystrophic mice of strain 129 ReJ.Key words: parvalbumin, Pva gene, dystrophy, dystrophic mice.
The intricacies of β-globin gene expression
Authors: Andrin Christi , Spencer Charlotte
Published: December 2009

Gene expression is an extremely complicated process in which several mechanisms are involved. Owing to its developmental and tissue-specific expression, the β-globin gene is an excellent model for studying gene expression. β-Globin gene expression involves an interplay between several different mechanisms. Chromatin structure is thought to be altered by the locus control region (LCR) located far upstream of the β-globin gene locus. As well, multiple transcription factors come into play both in the LCR and in the individual promoters and enhancers of the β-globin genes. The interaction between these then allows for delicate regulation of β-globin gene expression. In the following review the elaborate system of β-globin gene expression will briefly be examined.Key words: β-globin, gene expression, chromatin, GATA-I, NF-E2, developmental regulation.
Heterogeneous glycosylation of cationic peanut peroxidase
Authors: Wan Lianglu , Gijzen Mark , Huystee Robert B. Van
Published: December 2009

Cationic peanut peroxidase (CPrx) from a cell suspension culture is N-glycosylated at Asn60, Asn144, and Asn185. All three N-glycans are complex type and galactose rich, and show heterogeneity in length and ConA (concanavalin A) binding property. The glycan heterogeneity causes a polymorphism of the enzyme. Based on its behavior on ConA columns, CPrx can be grouped into two fractions: nonbinding (CPrx−) and binding (CPrx+) types. A synchronously cosecreted β-galactosidase has been discovered in the culture medium; there are two isozymes of 60 kDa (pI 7.3) and 66 kDa (pI 7.6). This β-galactosidase has been partially purified by a combination of ion-exchange and size-exclusion chromatographies and preparative isoelectrofocusing. In vitro experiments indicate that the cosecreted β-galactosidase is able to convert peroxidase from CPrx− to CPrx+ and may, to some extent, contribute to the glycan heterogeneity of peroxidase in the cell culture.Key words: peroxidase, peanut, glycan, heterogeneity, β-galactosidase.
The role of clusterin in tissue injury
Authors: Silkensen John R. , Schwochau Gary B. , Rosenberg Mark. E.
Published: December 2009

Clusterin is a widely distributed glycoprotein with a wide range of biologic properties. A prominent and defining feature of clusterin is its marked induction in such disease states as glomerulonephritis, cystic renal disease, renal tubular injury, neurodegenerative conditions, atherosclerosis, and myocardial infarction. The expression of clusterin in these states is intriguing given the apparent incongruity of the conditions listed, the variety of stimuli eliciting such expression, and the multiple proposed roles once induced. This review will outline the conditions associated with clusterin induction and speculate on its role in disease.Key words: clusterin, cell interactions, complement, injury, lipoprotein.
Expression of tenascin during carcinogenesis and involution of hormone-dependent tissues
Authors: Vollmer Günter
Published: December 2009

Cytotactin/tenascin/hexabrachion, now referred to as tenascin-C (TN-C), is a hexameric glycoprotein of the extracellular matrix of mesenchymal tissue constituents. A high expression was found in embryonic development and during carcinogenesis of almost all organs. TN-C expression by the mesenchyme thereby appears to be induced by paracrine-acting, epithelial-derived (growth) factors. In normal adult organs there is little, if any, TN-C expression. In the human endometrium for instance, tenascin expression is low in the normal proliferative endometrium and undetectable in the normal secretory endometrium. In this paper, the appearance and expression of TN-C in hormone-dependent tissues, regressing hormone-dependent tissues, and tumors of the endometrium, breast and prostate is reviewed. Further, the regulation of TN-C expression is summarized and possible functions of TN-C during regression and carcinogenesis of hormone-dependent tissues are discussed.Key words: apoptosis, tenascin-C, extracellular matrix, prostate, mesenchyme, regression.
Active cell death: role in hepatocarcinogenesis and subtypes
Authors: Bursch Wilfried , Grasl-Kraupp Bettina , Ellinger Adolf , Török Ladislaus , Kienzl Harald , Müllauer Leonhard , Schulte-Hermann Rolf
Published: December 2009

Active cell death, the genetically programmed self-destruction of a cell, is now recognized to be a widespread phenomenon in biology that counterbalances mitosis to preserve tissue homeostasis. It is subject to the control of the growth regulatory networks in tissues. Close examination of the morphology of dying cells in liver and other tissues suggests that there are a number of morphological types of active cell death, ranging from forms dominated by nuclear changes and without signs of autophagy ("classical" apoptosis), e.g., in thymocytes and the liver, to those dominated by autophagic degradation of cytoplasm, e.g., in the mammary gland. The induction of gene expression in these diverse types of cell death is anticipated to be different. Here we review the data regarding the regulation of apoptosis in the liver by liver tumor promoter, transforming growth factor β-1 and related peptides as well as nutrition. In the course of hepatocarcinogenesis, initiated cells as well as preneoplastic and neoplastic cell populations showed enhanced cell replication, but also enhanced apoptosis. Tumor promoter shift the balance between birth and death by increasing the rate of cell replication and by decreasing the rate of apoptosis. Thereby, liver tumor formation is accelerated. Food restriction exhibits the opposite effect and consequently, provides protection from carcinogenesis.Key words: apoptosis, carcinogenesis, tumor therapy, growth factors, food restriction.
Identification of phosphatidate nonlamellar phases on liposomes by flow cytometry
Authors: Baeza Isabel , Aguilar Leopoldo , Ibáñez Miguel , Wong Carlos , Alvarado-Alemán Francisco , Soto Carolina , Escobar-Gutiérrez Alejandro , Mondragón Ricardo , González Sirenia
Published: December 2009

This study is the first report that demonstrates nonlamellar arrangements, or lipidic particles, of phosphatidate inserted in the lipid bilayer of liposomes using polyclonal antibodies from mice and flow cytometry. Sera immunoreactivity was analyzed using liposomes that displayed smooth bilayers or phosphatidate particles, as shown by electron microscopy. This cytofluorimetric analysis showed that immune mice sera have a specific immunoreactivity with the phosphatidate particles formed by Mn2+, which also cross-reacted with those formed by Ca2+ and with the cardiolipin particles formed by Mn2+. In addition, these immune sera hardly reacted with smooth bilayered liposomes, independently of the lipid composition studied. Thus, this new methodology can be applied to demonstrate nonlamellar molecular arrangements of lipids in biological membranes.Key words: nonlamellar lipid immunogenicity, lipidic particles immunogenicity, liposomes, flow citometry, anti-phospholipid antibodies.
Morphogenesis and mechanical instability of a prestressed tissue
Authors: Totafurno John , Bjerknes Matthew
Published: December 2009

We consider the issue of whether purely mechanical properties of biological systems can, in principle, play a significant role in morphogenesis. As a simple example, we model a spherically arranged epithelium that is symmetrically prestressed under the action of cytoskeletal elements. A three-dimensional exact bifurcation analysis indicates the existence of a critical radius beyond which, for a physiologically attainable prestress, the spherical organoid is mechanically unstable and will buckle. We conclude that the purely mechanical aspects of biological tissues may indeed play a role in morphogenesis.Key words: buckling of sphere, morphogenesis, pattern formation, nonlinear elasticity, developmental biology.
Structure and expression of ribosomal protein genes inXenopus laevis
Authors: Amaldi Francesco , Camacho-Vanegas Olga , Cecconi Francesco , Loreni Fabrizio , Cardinali Beatrice , Crosio Claudia , Pellizzoni Livio , Pierandrei-Amaldi Paola , Mariottini Paolo
Published: December 2009

In Xenopus laevis, as well as in other vertebrates, ribosomal proteins (r-proteins) are coded by a class of genes that share some organizational and structural features. One of these, also common to genes coding for other proteins involved in the translation apparatus synthesis and function, is the presence within their introns of sequences coding for small nucleolar RNAs. Another feature is the presence of common structures, mainly in the regions surrounding the 5′ ends, involved in their coregulated expression. This is attained at various regulatory levels: transcriptional, posttranscriptional, and translational. Particular attention is given here to regulation at the translational level, which has been studied during Xenopus oogenesis and embryogenesis and also during nutritional changes of Xenopus cultured cells. This regulation, which responds to the cellular need for new ribosomes, operates by changing the fraction of rp-mRNA (ribosomal protein mRNA) engaged on polysomes. A typical 5′ untranslated region characterizing all vertebrate rp-mRNAs analyzed to date is responsible for this translational behaviour: it is always short and starts with an 8–12 nucleotide polypyrimidine tract. This region binds in vitro some proteins that can represent putative trans-acting factors for this translational regulation.Key words: ribosomal proteins, snoRNA, translational regulation, Xenopus laevis.
The accuracy center of a eukaryotic ribosome
Authors: Liebman Susan W. , Chernoff Yury O. , Liu Rong
Published: December 2009

Mutations in yeast ribosomal proteins and ribosomal RNAs have been shown to affect translational fidelity. These mutations include: proteins homologous to Escherichia coli's S4, S5, and S12; a eukaryote specific ribosomal protein; yeast ribosomal rRNA alterations at positions corresponding to 517, 912, and 1054 in 16S E. coli rRNA and to 2658 in the sarcin–ricin domain of 23S E. coli rRNA. Overall there appears to be a remarkable conservation of the accuracy center throughout evolution.Key words: ribosomal RNA, ribosomal protein, yeast, translational accuracy.
Structural and functional implications in the eubacterial ribosome as revealed by protein–rRNA and antibiotic contact sites
Authors: Wittmann-Liebold Brigitte , Ühlein Monika , Urlaub Henning , Müller Eva-Christina , Otto Albrecht , Bischof Oliver
Published: December 2009

Contact sites between protein and rRNA in 30S and 50S ribosomal subunits of Escherichia coli and Bacillus stearothermophilus were investigated at the molecular level using UV and 2–iminothiolane as cross-linkers. Thirteen ribosomal proteins (S3, S4, S7, S14, S17, L2, L4, L6, L14, L27, L28, L29, andL36) from these organisms were cross-linked in direct contact with the RNAs, and the peptide stretches as well as amino acids involved were identified. Further, the binding sites of puromycin and spiramycin were established at die peptide level in several proteins that were found to constitute me antibiotic-binding sites. Peptide stretches of puromycin binding were identified from proteins S7, S14, S18, L18, and L29; those of spiramycin attachment were derived from proteins S12, S14, L17, L18, L27, and L35. Comparison of the RNA–peptide contact sites with the peptides identified for antibiotic binding and with those altered in antibiotic-resistant mutants clearly showed identical peptide areas to be involved and, hence, demonstrated the functional importance of these peptides. Further evidence for a functional implication of ribosomal proteins in the translational process came from complementation experiments in which protein L2 from Halobacterium marismortui was incorporated into the E. coli ribosomes that were active. The incorporated protein was present in 50S subunits and 70S particles, in disomes, and in higher polysomes. These results clearly demonstrate the functional implication of protein L2 in protein biosynthesis. Incorporation studies with a mutant of HmaL2 widi a replacement of histidine-229 by glycine completely abolished the functional activity of the ribosome. Accordingly, protein L2 with histidine-229 is a crucial element of the translational machinery.Key words: antibiotic-binding site, RNA–peptide-binding sites, protein–RNA interaction in ribosomes, functional role of protein L2.
Early steps in the biosynthesis of MUC2 epithelial mucin in colon cancer cells
Authors: McGuckin Michael A. , Devine Peter L. , Ward Bruce G.
Published: December 2009

Expression of the MUC2 mucin has been demonstrated in normal gastrointestinal and respiratory epithelium and in carcinomas of the gastrointestinal and respiratory tracts, breast, ovary, and bladder using RNA probes and (or) monoclonal antibodies reactive with peptide epitopes on the 23 amino acid tandem repeat. Mouse monoclonal antibodies 4F1 and 3A2 were previously obtained by immunization with mucin derived from the LS174T colon cancer cell line and a KLH conjugate of a synthetic MUC2 VNTR peptide. These antibodies react with distinct epitopes on synthetic VNTR peptides and with normal and malignant epithelial tissues. In the present study, we examined the biosynthesis of MUC2 in LS174T colon cancer cells, using these antibodies to immunoprecipitate labelled mucin. A very high molecular mass protein was immunoprecipitated following 1 min pulse labelling with [3H]threonine and [3H]proline. A slight increase in molecular mass was observed over the next 16 min; however, unlike the MUC1 mucin, there was no large difference in apparent molecular mass between the MUC2 protein precursor and fully processed mucin using separation by SDS–PAGE. O-Glycosylation began within 1 h of synthesis of the protein core. Mucin secretion into the culture medium was detected in the 2nd hour following synthesis and was largely completed within 4 h of synthesis. Secreted mucin was far less reactive with these monoclonal antibodies than the precursor protein.Key words: mucin, MUC2, biosynthesis, colon, cancer, glycosylation.
Developmental expression of the collagen-binding heat-shock protein GP46 and collagen types I and IV in rat tissues
Authors: Pak Brian J. , Wigle Dennis A. , Watson John D. , Pang Stephen C. , Cates George A. , Brickenden Anne M. , Ball Eric H.
Published: December 2009

The temporal expression of protein and mRNA encoding the collagen-binding heat-shock glycoprotein, gp46, were determined in the heart, kidney, and lung during early rat postnatal development. The steady-state levels of collagen types I and IV mRNA expression were also examined to determine if gp46 and these collagen types are co-regulated during ontogenesis. Western blot analysis using a monoclonal antibody to gp46 revealed that gp46 levels are developmentally regulated. In heart and kidney, gp46 levels were high on days 3 and 8, reduced significantly on day 25, and low to undetectable on day 69. Protein levels of gp46 in the lung exhibited a similar temporal pattern except on day 3, when very low levels of gp46 were detected. mRNA expression of gp46 during early postnatal development did not correlate with gp46 protein accumulation in these tissues, suggesting a complex pre- and post-translational regulatory scheme. In the heart, protein levels of gp46 correlated well with collagen type I α1(I) mRNA expression but not with collagen type IV α1(IV). In contrast, gp46 protein levels closely paralleled α1(IV) expression in the kidney. Gp46 levels exhibited no apparent correlation with either α1(I) or α1(IV) levels in the lung. These results show that gp46 is developmentally regulated at both the protein and mRNA levels in a tissue specific manner. The relationship between gp46 and collagen α1(I) and α1(IV) chain mRNA expression also has been shown to be tissue specific.Key words: gp46, heat-shock proteins, development, molecular chaperone, collagen.
Dietary induction of pancreatic cholesterol esterase: a regulatory cycle for the intestinal absorption of cholesterol
Authors: Sasser Terry , Buddhiraju Chakradhar , Kumar Vijaya B. , Lopez-Candales Angel , Grosjlos Jackie , Scherrer Dave , Lange Louis G.
Published: December 2009

Atherosclerosis has a strong dietary basis without a proven molecular mechanism for cholesterol absorption. To investigate the potential role of pancreas in this process and its interaction with the two dietary forms of cholesterol (free and esterified), we undertook to study the role of pancreatic cholesterol esterase in cholesterol absorption. The results showed that (i) cholesterol esters contribute a disproportionately high fraction of absorbed dietary cholesterol, (ii) rates of intestinal cholesterol absorption are related to pancreatic cholesterol esterase activity, (iii) mRNA specific for pancreatic cholesterol esterase is induced 15-fold by dietary sterol esters and 10-fold by free sterol, (iv) the induction of cholesterol esterase mRNA is reversible, and (v) free cholesterol transport into cultured human intestinal cells is enhanced 300% by pancreatic cholesterol esterase. These data implicate pancreatic cholesterol esterase as pivotal in a metabolic loop under positive feedback control for the absorption of dietary cholesterol, whether free or esterified.Key words: cholesterol esterase, diet, transport, mRNA, induction.
Tin ethyl etiopurpurin significantly inhibits vascular smooth muscle cell proliferation in vivo
Authors: Coats Jr. William D. , Currier Jesse W. , Mejias Yvonne , Faxon David P. , Narciso Hugh L.
Published: December 2009

Smooth muscle cell proliferation is a major component of restenosis following angioplasty. Hematoporphyrin derivative and other photosensitive compounds inhibit proliferation by causing cellular necrosis upon light activation (photodynamic therapy). Other photosensitive compounds, such as benzoporphyrin derivative, have been suggested as having non-cytotoxic antiproliferative effects without photodynamic therapy, although other studies using benzoporphyrin derivative were negative. Inhibition of smooth muscle cell proliferation was examined in an in vivo rabbit model of vascular injury using a novel synthetic chlorin derivative, tin ethyl etiopurpurin, and benzoporphyrin derivative without photodynamic therapy. Tin ethyl etiopurpurin and benzoporphyrin derivative inhibited smooth muscle cell proliferation by 50–90% of control (p ≤ 0.05) without toxic side effects. These results suggest that tin ethyl etiopurpurin and benzoporphyrin derivative without photodynamic therapy may provide a novel and potent antiproliferative therapy that might be useful in the treatment of restenosis.Key words: vascular, hyperplasia, hematoporphyrin derivatives.
A novel immunosuppressive factor in bovine colostrum blocks activation of the interleukin 2 gene enhancer at the NFAT site
Authors: Sambasivarao Damaraju , Hooton Jonathan , Dost Axel , Paetkau Verner
Published: December 2009

A factor in bovine colostrum (colostrum inhibitory factor, CIF) inhibits interleukin 2 (IL2) production in activated T helper cells by blocking the accumulation of IL2 mRNA. To determine whether CIF blocks at the level of IL2 transcription, we introduced reporter plasmids into the human T leukemia cell line Jurkat by transient transfection. These contained the luciferase gene under the control of either the human IL2 upstream enhancer region (segments −326 to +45) or three repeats of the NFAT element contained within it (segments −255 to −285). Expression of luciferase in these cells was induced by phorbol myristate acetate plus a calcium ionophore. CIF inhibited induction of either construct as did cyclosporine, which is known to block activation of the NFAT element. CIF failed to inhibit several other enhancer elements. The NFAT-controlled luciferase gene system distinguishes CIF from other T cell inhibitory activities present in colostrum, in particular, TGFβ1and TGFβ2and the glucocorticoids. Stably transfected Jurkat cells behaved similarly to the transiently transfected ones with respect to inhibition by CIF and cyclosporine. The NFAT-luc assay is a useful technique for the rapid, sensitive measurement of CIF or other immunosuppressants with a similar mode of action.Key words: immunosuppression, cyclosporine, NFAT, reporter gene, transcriptional regulation.
Glycosaminoglycans in the three lobes of the rat prostate following castration and testosterone treatment
Authors: Terry Doris E. , Clark Albert F.
Published: December 2009

Androgen dependence of glycosaminoglycans (GAGs) in the prostate was studied using tissue from intact (sham control), castrated, and androgen-treated castrated rats. GAGs from the ventral, dorsal, and lateral lobes of the prostate were isolated and characterized by cellulose electrophoresis using appropriate GAG standards and enzymatic digestion or nitrous acid hydrolysis. Androgen deprivation was initiated by castration and rats were sacrificed at various time intervals after 7 days castration. After castration, the total GAG content decreased in the three prostate lobes. At day 7 after castration, the total hyaluronic acid (HA) content decreased by 74% (ventral lobe) and 34% (lateral lobe) compared with the sham control. No effect was observed for HA content in the dorsal lobe. Castration decreased the total heparan sulphate (HS), dermatan sulphate (DS), and chondroitin sulphate (CS) contents in the three prostate lobes at 0 days of treatment, except for the CS content in the dorsal and lateral lobes. Androgen replacement increased the total GAG contents in the three prostate lobes. At 14 days of testosterone propionate treatment, there were 9-, 6.8-, 4.1- and 3.7-fold increases in HA, HS, DS, and CS, respectively, in the ventral lobe. These increases were more rapid and profound in the ventral than in the dorsal and lateral lobes. These findings indicate that all GAGs are regulated by androgen and there may be lobe-specific differences in their regulation. This could be a function of the heterogenous populations of cells in each lobe.Key words: castration, glycosaminoglycan, prostate, testosterone
Regulation of matrilysin in the rat uterus
Authors: Woessner Jr. J. Frederick
Published: December 2009

Matrilysin was first discovered in the involuting rat uterus; it has also been known as uterine metalloproteinase, putative metalloproteinase (Pump-1), and matrix metalloproteinase 7 (MMP-7). It is the smallest member (28 kDa) of a family of 15 MMPs that together are able to degrade most of the macromolecules of the extracellular matrix. This family is briefly reviewed; all members are zinc metalloproteinases that occur in zymogen form with the active site zinc blocked by cysteine. Matrilysin can degrade a wide range of gelatins, proteoglycans, and glycoproteins of the matrix and can activate several other MMPs including collagenase. With respect to the uterus, matrilysin is localized to epithelial cells and varies in amount with the estrus cycle and is found in high levels during postpartum involution. There is evidence for a role in the last stage of cervical ripening and immediately postpartum. Induction of premature delivery by onapristone and prostaglandin E2advances these changes in matrilysin. Regulation of the enzyme levels in the uterus are considered from four viewpoints: control of protein synthesis (particularly in response to hormones), activation of the proenzyme to functional protease, retention of enzyme by binding to matrix components such as heparan sulfate, and inhibition by natural inhibitors such as tissue inhibitor of metalloproteinases (TIMPs) and α2-macroglobulin.Key words: matrilysin, matrix metalloproteinases, TTMP, uterus, rat uterus.
Trends in the frequencies of ν(AsOxHx–1) [x = 2–4] in selected As(V)-containing compounds investigated using quantum chemical calculations
Authors: Adamescu Adrian , Gray Holly , Stewart Katherine M.E. , Hamilton I. P. , Al-Abadleh Hind A.
Published: December 2009

The application of computational chemistry to studies in geochemistry is increasingly becoming invaluable in explaining experimentally observed trends for surface interactions of pollutants with sorbents ubiquitous in the environment. We report computational results on factors that affect the force constant of AsOx bonds in As(V)-containing compounds relevant to geochemical environments. Geometries, atomic charges, and stretching frequencies of –AsOxHx–1 (x = 2– 4) moieties in these molecules were calculated using semi-empirical methods (PM3) and density functional theory (B3LYP) for both isolated (gas phase) molecules and hydrated complexes in which the molecules are surrounded by four water molecules. We found that the number of organic substituents has a relatively smaller effect on the force constant of AsOx bonds than protonation. The increase in resonance effect with deprotonation causes As–O bond lengths to increase, and the decrease in resonance in fully deprotonated species with increasing organic substitution causes As–O bond lengths to decrease. In the absence of the resonance effect in fully protonated species, As–O bond lengths increase with more organic substituents. Also, increasing organic substitution causes the charge on the central arsenic atom to decrease. Charges on oxygen atoms in As–OH bonds are more sensitive to deprotonation than to resonance relative to other oxygen atoms in As–O bonds. As expected, frequencies of ν(AsOx) show an inverse relationship with As–O bond lengths upon deprotonation and organic substitution. Our results have implication for the interpretation of infrared and X-ray absorption spectra of adsorbed As(V)-containing compounds.
Stress and reproduction inBoiga irregulariswith notes on the ultrastructure of the sexual segment of the kidney in squamates
Authors: Siegel D. S. , Aldridge R. D. , Clark C. S. , Poldemann E. H. , Gribbins K. M.
Published: December 2009

The reproductive tract of wild-caught male Boiga irregularis (Merrem, 1802) (Brown Treesnake) and the changes that result in these tissues from captivity are described. Wild-caught snakes were compared with snakes kept in captivity and sacrificed at approximately 10, 30, 50, and 60+ days. Snakes sacrificed after 10 days in captivity showed no differences in histological appearance or epithelial height in either the testis or sexual segment of the kidney (SSK) compared with wild snakes. Normal spermatogenic stages and SSK ultrastructure were observed in the testes and kidney from wild specimens and captive specimens after 10 days. In terms of the SSK, large electron-dense secretory vacuoles occupied the apices of the epithelial cells, while a basal nucleus, rough endoplasmic reticulum, and Golgi bodies filled the bases of these cells. All SSK cells were actively secretory in a mode that could most accurately be termed apocrine. At time of secretion, released vacuoles become diffuse and the apical membrane ruptures, spilling the SSK contents into the distal nephron lumen. Between 10 and 30 days in captivity, spermatogenesis and secretion in the SSK ceased and the epithelial height of both tissues decreased. At approximately 60 days, testicular epithelial height increased and spermatogenic activity was reestablished. The SSK, however, remained regressed at 60 days.
Demographic differences among populations of Northern Map Turtles (Graptemys geographica) in intact and fragmented sites
Authors: Bennett A. M. , Keevil M. , Litzgus J. D.
Published: December 2009

Habitat fragmentation is a leading cause of reptile declines worldwide. We examined demographic differences between populations of Northern Map Turtles, Graptemys geographica (Le Sueur, 1817), inhabiting intact and fragmented sites along the Trent–Severn Waterway (TSW) in Ontario over two field seasons. We examined population densities, sex ratios, body size, and growth rates in two control sites and two fragmented sites (where the waterway is disrupted by dams). We predicted that population density would be higher in control sites, and that sex ratios would differ among sites. Abundance was greater than expected, but densities could not be compared owing to a lack of recaptures. Sex ratios in control sites were slightly male-biased and were significantly female-biased in fragmented sites. Turtles in fragmented habitats were significantly smaller than those in control habitats. Estimated growth rates of juveniles and males were significantly reduced in fragmented populations compared with controls. We hypothesize that habitat alterations in fragmented sites are differentially affecting demographic classes because of the intersexual niche divergence observed in Map Turtles. We emphasize the need for further research to examine the interactions between habitat alteration and invasive species and the effects they are having on Northern Map Turtle populations.
The mating system of polar bears: a genetic approach
Authors: Zeyl E. , Aars J. , Ehrich D. , Bachmann L. , Wiig Ø.
Published: December 2009

Parentage analysis data for 583 individuals genotyped at 27 microsatellite loci were used to study the mating system of polar bears ( Ursus maritimus Phipps, 1774) in the Barents Sea area. We discriminated statistically between full and half-siblings identified through only one common parent. We document for the first time multiple paternity in polar bears. We demonstrated for both sexes low fidelity to mating partners over time. We did not detect any significant difference between the age distribution of adult males at capture and the age distribution of males siring cubs. This might indicate that the male’s age and size are less indicative of the reproductive success than previously thought. This is further supported by a rather long mean litter interval of 3.9 years for males siring several litters. The mating system of polar bears in the Barents Sea appears to be promiscuous, usually with a single successful father siring full siblings within a year, but with consecutive litters of a mother being fathered by different males. We discuss how population density, landscape characteristics, and adult sex ratio might influence the mating system of polar bears. This is of particular importance for management decisions such as, e.g., implementing sex ratios in hunting quotas.
Factors influencing large-scale distribution of two sister species of pine voles (Microtus lusitanicus and Microtus duodecimcostatus): the importance of spatial autocorrelation
Authors: Santos S. M. , Mira A. P. , Mathias M. L.
Published: December 2009

The Lusitanian pine vole ( Microtus lusitanicus (Gerbe, 1879)) and the Mediterranean pine vole ( Microtus duodecimcostatus de Selys-Longchamps, 1839) are sister species with burrowing habits and a restricted European distribution. Our aim was to assess the relative effect of environmental, soil, and spatial characteristics on the distribution of these species in Portugal, and obtain predicted occurrence maps for each species, particularly to identify areas of sympatry. We used spatial eigenvector mapping (SEVM) to describe the spatial autocorrelation in species data, and we partitioned the variance in species distributions to quantify the relative effects of environmental, soil, and spatial characteristics. The spatial variables explained the major part of variability in both species distributions and were more important than environmental or soil variables. The Lusitanian pine vole occurs in areas outside landscape units of grassland, higher rainfall, frost, and cambisols, with mostly acid soils, lower abundance of litosols, and presence of solonshaks. The Mediterranean pine vole is distributed in grassland areas within intermediate values of soil pH, dominated by litosols and luvisols, and lower rainfall, frost, and cambisols. Our results showed disjunct sympatric areas of small size and a parapatry boundary for the centre of Portugal, suggesting that contact zones are probably narrow.
The effects of feeding on cell morphology and proliferation of the gastrointestinal tract of juvenile Burmese pythons (Python molurus)
Authors: Helmstetter Cécile , Pope Robert K. , T’Flachebba Mathieu , Secor Stephen M. , Lignot Jean-Hervé
Published: December 2009

The gastrointestinal tract of Burmese pythons ( Python molurus (L., 1758)) exhibits large morphological and physiological changes in response to feeding and extended periods of fasting. In this study the mucosa of the stomach, small intestine, and colon were examined for changes in structure and cellular proliferation. The mucosa of fasting pythons exhibited low levels of cellular replication, but after feeding, cellular replication was evident as early as 12 h in the small intestine and colon and 24 h in the stomach. Replication peaked 3 days postfeeding for the small intestine and colon, but was still increasing at 6 days postfeeding in the stomach. Interestingly, cell proliferation was still evident after 45 days in the colon. In these tissues, a stock of “ready-to-use” primary lysosomes is found in the mucosal cells of fasting animals, whereas profound intracellular recycling is typical of animals that have been fed. These findings indicate that during the postprandial period, the intestinal mucosa undergoes extensive remodelling in anticipation of the next fasting and feeding period. One key adaptive factor for the python’s ability to cope with infrequent feeding is a well-prepared digestive system in fasting animals that can quickly start functioning again when food becomes available.