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PubMed1990

Ribonuclease in different chromosomal species of the mole rat, superspecies Spalax ehrenbergi: concentration in the pancreas and primary structure.

Ribonucleases are found in considerable quantities in the pancreas of a number of mammalian taxa and a few reptiles. The ribonuclease content varies greatly in different species. Large quantities are found in ruminants and species that have a ruminant-like digestion and in a number of species with coecal digestion. This is a response to the necessity of digesting large amounts of RNA derived from the microflora of the stomach of ruminants or species with ruminant-like digestion or of the coecum of species with coecal digestion. The amino acid sequence of pancreatic ribonuclease from the chromosomal species 2n = 60 of the mole rat, superspecies Spalax Ehrenbergi was determined. From the comparison of the sequence with those of other mammalian species we found that Spalax diverged from the myomorph rodent branch before the divergence of the Muridae (mouse, rat) from the Cricetidae (hamster, muskrat). Spalax ribonuclease shares several amino acid residues with other myomorph rodent species. These are not or only rarely observed outside this rodent suborder. Although the ribonuclease content varies greatly in different mammalian species, the variation in content between individuals within a species is small. Spalax is an exception to this with ribonuclease contents varying over more than an order of magnitude in different individuals. Ribonucleases isolated from the chromosomal species 2n = 52, 2n = 58 and 2n = 60 have identical elution positions on reversed-phase HPLC. The enzyme from the 2n = 54 species, however, elutes at a slightly earlier elution position. No amino acid sequence differences have been found hitherto between the ribonucleases of the four chromosomal species of Spalax ehrenbergi occurring in Israel. However, due to lack of material we were unable to determine more than about 20% of the sequence of the enzyme from the 2n = 54 species, which is the oldest offshoot.

Amino Acid Sequence↗
PubMed1994

[Recent biological techniques for the diagnosis of mycobacterial infections].

More rapid and more sensitive techniques have replaced traditional methods of direct examination and culturing for diagnosing mycobacterial infections. Among the most recent methods are Isolator blood culture, radiometric detection, hybridization and amplification, each with its advantages and disadvantages. The Isolator blood culture system improves the sensitivity of mycobacteria detection, especially for Mycobacterium avium-intracellulare. In terms of cost and culture time, this system is similar to traditional methods, although it is limited to blood cultures. Radiometric detection, associated with culture in a liquid medium, greatly improves diagnostic power. The main advantage lies in the reduced delay before detection, 13.4 days compared with 21.7 days on solid media (for M. tuberculosis) and 5.1 days instead of 18.6 days for other mycobacteria. A culture is considered to be negative after 6 weeks without growth compared with 12 weeks for the Löwenstein-Jensen medium. The disadvantages of this system include the cost of equipment, personnel and radio-active products. In addition, as for all culture methods, a sufficient bacterial population is required in the sample. Hybridization techniques rely on DNA or RNA probes which recognize mycobacterial sequences. The Gen-Probe recognizes M. tuberculosis, M. avium, M. intracellulare and M. gordonae ribosomal RNA. Syngene probes recognize M. tuberculosis and M. avium-intracellulare DNA sequences. Rapid identification in less than 2 hours is a major advantage. At the present time 50 pg of DNA are required, a quantity which is not usually present in clinical samples. Combining this technique with primary culture in liquid medium or amplification techniques improves sensitivity. Polymerase chain reaction amplifies a specific sequence of double strand DNA. This method provides a means of identifying mycobacterial DNA in a clinical sample within 24-48 hours, a major improvement over traditional culture. Cost however may be a barrier although the cost/benefit ration remains to be determined.

Humans↗
PubMed2004

Investigation of the Bernoulli model for RNA secondary structures.

Within this paper we investigate the Bernoulli model for random secondary structures of ribonucleic acid (RNA) molecules. Assuming that two random bases can form a hydrogen bond with probability p we prove asymptotic equivalents for the averaged number of hairpins and bulges, the averaged loop length, the expected order, the expected number of secondary structures of size n and order k and further parameters all depending on p. In this way we get an insight into the change of shape of a random structure during the process 1 p -->0. Afterwards we compare the computed parameters for random structures in the Bernoulli model to the corresponding quantities for real existing secondary structures of large subunit rRNA molecules found in the database of Wuyts et al. That is how it becomes possible to identify those parameters which behave (almost) randomly and those which do not and thus should be considered as interesting, e.g., with respect to the biological functions or the algorithmic prediction of RNA secondary structures.

Hydrogen Bonding↗
PubMed1992

Cloning of noncultivatable human rotavirus by single primer amplification.

A novel, sequence-independent strategy has been developed for the amplification of full-length cDNA copies of the genes of double-stranded RNA (dsRNA) viruses. Using human (Bristol) group C rotavirus as an example, a single amino-linked modified oligonucleotide (primer 1) was ligated to either end of each dsRNA genome segment by using T4 RNA ligase. Following reverse transcription, annealing, and repair of cDNA strands, amplification of the viral dsRNA genome was accomplished by polymerase chain reaction using a single complementary oligonucleotide (primer 2). Northern (RNA) hybridization of cDNA to virus dsRNA indicated that it was possible to generate cDNA representing the complete genome from very small clinical samples. This technique was used to determine the complete nucleotide sequence (728 bp) and coding assignment of gene 10, which revealed an open reading frame of 212 amino acids with limited homology to NS26 from human group A rotavirus. In contrast to previous tailing methods, the addition of one defined primer allowed unequivocal identification of terminal nucleotides and should be generally applicable to viruses with segmented dsRNA genomes and especially for analysis of clinical samples, for which very limited quantities of biological material are available.

Amino Acid Sequence↗
PubMed1996

Expression and processing of peptidylglycine alpha-amidating monooxygenase messenger RNA in rat prostate.

The prostate gland contains neuroendocrine cells and amidated neuroendocrine peptides whose presence has been related to aggressive forms of prostate cancer. The enzyme peptidylglycine alpha-amidating monooxygenase (PAM) is critical to the bio-synthesis of amidated peptides and is commonly present in neuroendocrine cells. By northern blot hybridization analysis, PAM mRNA was detected in similar quantities in dorsolateral and ventral prostates of 3-month-old and 13-month-old rats. Multiple forms of PAM mRNA were present whose size distribution was more similar to PAM mRNAs found in pituitary than atrium. Alternative splice sites in PAM mRNA were investigated by reverse-transcriptase polymerase chain reaction. Similar alternatively spliced forms of PAM mRNA were found in both prostate lobes, pituitary, and atrium. However, the distribution of forms in the prostate most resembled that of pituitary. Multiple forms of PAM mRNA are present in prostate and may serve as markers of neuroendocrine differentiation.

Alternative Splicing↗
PubMed2005

Development of a semi-quantitative real-time RT-PCR for the detection of measles virus.

Real-time detection of polymerase chain reactions allows convenient detection and quantification of virus-derived nucleic acids in clinical specimens. We have developed a real-time RT-PCR assay for the detection of measles virus (MV) genomic RNA, and compared it to a well-established conventional RT-PCR assay. Based on a serial dilution of the live-attenuated MV Edmonston Zagreb vaccine, the detection limits were approximately 0.1 and 0.02 cell culture infectious dose 50% units (CCID50) per test for the conventional and TaqMan RT-PCR assays, respectively. Furthermore, tissue materials spiked with known quantities of MV were equally well detected in both assays. The TaqMan assay was linear within a range of 10(4.4) to 10(-0.6)CCID50/ml, with an intra-assay variability lower than 3% and an inter-assay variability ranging from 1.5% at 10(4.4)CCID50/ml to 8.7% at 10(-0.6)CCID50/ml. The TaqMan assay could detect representative wild-type viruses from the currently active MV clades, and could detect MV genome in clinical specimens obtained from measles patients. Finally, quantification of MV RNA in peripheral blood mononuclear cells or broncho-alveolar lavage cells from cynomolgus macaques collected at different time points after experimental infection showed a good correlation with virus isolation data. In conclusion, the TaqMan assay developed is specific, sensitive, rapid and reproducible, and can be of use for diagnostic purposes or for studies on the pathogenesis of measles.

Animals↗
PubMed1994

Dexamethasone increases jejunal glutamine synthetase expression via translational regulation.

Glutamine provides energy and precursors for nucleotide biosynthesis for the gut mucosa, and it is essential for intestinal metabolism and function. During stress states, glutamine uptake of circulating and luminal glutamine may be diminished, but the ability of the gut mucosa to synthesize glutamine de novo in response to this decreased delivery remains undefined. Since the glucocorticoids play an important role in regulating interorgan glutamine metabolism during catabolic states, we hypothesized that these hormones induce the expression of gut mucosal glutamine synthetase (GS), the enzyme that catalyzes the intracellular biosynthesis of glutamine. Adult rats were treated with dexamethasone (DEX, 0.5 mg/kg intraperitoneally) or saline (controls). At various times after treatment (4, 12, 24, 48, and 72 hours), jejunal mucosal GS-specific activity was assayed, and total RNA was extracted. GS transcripts were detected by Northern blot analysis, using a radiolabeled rat GS cDNA probe. Transcripts were quantitated by phospho-imaging and normalized to beta-actin. An anti-GS polyclonal antibody was used to quantitate GS protein concentrations by Western blot analysis. The relative quantities of GS translated were measured using a cell-free protein-synthesizing system (reticulocyte lysate assay). Data were analyzed using analysis of variance and were considered statistically significant for p < 0.05. DEX increased GS activity by 45% 12 hours after administration. Western blot analysis revealed an increase in the concentration of the GS protein in the jejunum of DEX-treated animals. Northern blot analysis demonstrated no significant change in GS mRNA levels after DEX treatment, indicating the possibility of post-transcriptional regulation. In vitro translational experiments demonstrated that the quantity of GS translated was increased by 25% after the administration of DEX. These data suggest that glucocorticoids may increase jejunal mucosal GS levels by accelerating protein translation. This adaptive response could provide glutamine for the gut mucosa during stress, when exogenous glutamine supplies may be rate limiting.

Animals↗
PubMed2003

Inhibition of proteasome function leads to NF-kappaB-independent IL-8 expression in human hepatocytes.

Breakdown of cellular proteins is a highly regulated process, and the ubiquitin-proteasome pathway is the major proteolytic system in the cell. It regulates the levels of numerous proteins that control gene expression and cell division, as well as responses to stress and inflammation. Recent studies have reported abnormalities in proteasome function in alcoholic liver disease (ALD). Moreover, a direct relation has been reported between impaired proteasome function and oxidative stress in experimental models of ALD. Neutrophil infiltration is a hallmark of ALD, and activated neutrophils are thought to play a role in the pathology of ALD. As a potent neutrophil chemoattractant and activator, interleukin 8 (IL-8) likely plays a key mechanistic role in many forms of liver injury. In this study, we evaluated the effects of inhibition of proteasome function on expression and release of IL-8 by human fetal hepatocytes and hepatoma cells. Our data demonstrate that inhibition of proteasome function in hepatocytes leads to apoptotic cell death. Decreased hepatocyte survival coincides with enhanced expression of IL-8, both at the protein and the messenger RNA (mRNA) levels. This increase in IL-8 is independent of nuclear factor kappaB (NF-kappaB) activation and is associated with an increase in c-Jun N-terminal kinase (JNK) and activator protein-1 (AP-1) activity. In conclusion, hepatocytes dying because of inhibition of proteasome function produce massive quantities of the proinflammatory chemokine IL-8, possibly resulting in neutrophil infiltration, increased inflammation, and liver injury.

Acetylcysteine↗
PubMed1997

Time course for the modulation of hepatic cytochrome P450 after administration of ethylbenzene and its correlation with toluene metabolism.

The goal of the present study was to examine the time course for changes in P450 expression and hydrocarbon metabolism after acute treatment with the simple aromatic hydrocarbon ethylbenzene (EB) and to correlate these alterations with the changes observed in alkylbenzene metabolism. Male Holtzman rats were treated with a single intraperitoneal injection of EB, and the effects on specific P450-dependent activities, immunoreactive P450 isozyme levels, and RNA levels were measured at various times after injection. Toluene was used as the test alkylbenzene for examination of the EB-mediated changes on in vitro hydrocarbon metabolism. In untreated rats, toluene was metabolized almost entirely by aliphatic hydroxylation (to benzyl alcohol); however, in EB-treated rats, significant quantities of benzyl alcohol, o-cresol, and p-cresol were produced. Interestingly, 5-10 h after EB treatment, there was a 40% decrease in benzyl alcohol production. By 24 h, rates of benzyl alcohol formation returned to control levels, whereas there was a 7-fold increase in o-cresol and a greater that 50-fold increase in p-cresol production. The changes in the disposition of toluene were then correlated with changes in particular P450 isozymes. Several P450 isozymes were induced after EB administration. P450 2B1/2-dependent testosterone 16 beta-hydroxylation and P450 2B1/2-immunoreactive protein were elevated 30-fold after EB administration, reaching maxima by 24 h and remaining elevated 48 h after exposure. Changes in P450 2B1 and 2B2 RNA preceded those of the proteins. Similar results were observed with P450 1A1. P450 2E1 RNA levels were elevated after a single EB injection. However, the elevation in P450 2E1-dependent activities and immunoreactive protein levels preceded the changes in RNA, suggesting that multiple steps are affected by EB exposure. In contrast to the increases in some isozymes, P450 2C11 protein was rapidly suppressed (within the first 2-10 h) after hydrocarbon exposure, suggestive of a destabilization of the protein. When comparing the changes in P450 isozymes to alterations in toluene metabolism, the immediate suppression in aliphatic hydroxylation of toluene (in the first 5-10 h) was consistent with the decrease in P450 2C11. Subsequent to this effect, P450 2B1/2 and 2E1 were induced, which elevated production of this metabolite to control levels. The increase in the aromatic hydroxylation of toluene to both o, and p-cresol was consistent with the induction of P450s 2B1/2, 2E1, and 1A1.

Animals↗
PubMed2005

LINE-1 retrotransposons: modulators of quantity and quality of mammalian gene expression?

LINE-1 (L1) retrotransposons are replicating repetitive elements that, by mass, are the most-abundant sequences in the human genome. Over one-third of mammalian genomes are the result, directly or indirectly, of L1 retrotransposition. L1 encodes two proteins: ORF1, an RNA-binding protein, and ORF2, an endonuclease/reverse transcriptase. Both proteins are required for L1 mobilization. Apart from the obvious function of self-replication, it is not clear what other roles, if any, L1 plays within its host. The sheer magnitude of L1 sequences in our genome has fueled speculation that over evolutionary time L1 insertions may structurally modify endogenous genes and regulate gene expression. Here we provide a review of L1 replication and its potential functional consequences.

Alleles↗
PubMed1989

Hepatitis B virus (HBV) promoters are regulated by the HBV enhancer in a tissue-specific manner.

The activities of the individual hepatitis B virus (HBV) promoters and the effects of the HBV enhancer on these promoters in several human cell types have been compared by measuring the activity and RNA levels of the linked reporter function chloramphenicol acetyltransferase. The relative promoter activities in the human HepG2 (liver), HeLa, and HS27 (fibroblast) cell lines are in the order precore greater than X greater than preS2 greater than preS1; thus, the promoters of the gene producing the largest quantity of viral proteins have relatively low activity. The juxtaposition of the HBV enhancer in either orientation increased the promoter activities only modestly (2- to 5-fold) in the nonliver cell lines, whereas it dramatically increased (20- to 100-fold) the promoter activities in the liver cell line. Thus, the HBV enhancer is especially active in liver cells. This may be one of the causes of hepatotrophicity of the virus.

Carcinoma, Hepatocellular↗
PubMed2001

Cytokine profiles in paraffin-embedded biopsy samples of lepromatous leprosy patients: semi-quantitative measure of cytokine mRNA using RT-PCR.

A reproducible technique for fixation of tissue, RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) analysis from paraffin-embedded leprosy biopsies, has been developed and used to study the mRNA profiles. This approach is valuable in retrospective analysis of gene expression, and the handling of infectious biopsy material is also minimized. Among the methods of RNA extraction compared, the most efficient method was found to be incubation of the tissue sections in digestion buffer followed by extraction with Trizol. The experimental conditions were optimized for first strand cDNA synthesis and PCR, and used to measure the quantity of cytokine transcripts in biopsy materials. Interleukin-10 (IL-10) mRNA was detectable in all cases examined, which correlates well with other earlier reports using frozen tissues. However, IL-5 transcripts were present in 60% of the biopsies, unlike the earlier reports which showed IL-5 mRNA in all LL cases. Transforming growth factor-beta (TGF-beta) mRNA was detected in 80% of the biopsies, and this confirmed earlier immuno-cytochemical data which showed TGF-beta protein in all cases. Tumor necrosis factor-alpha mRNA was found in about 60% of the biopsies; whereas interferon gamma mRNA was detected in 30% of the LL cases. In conclusion, the results obtained in our study confirm and extend earlier observations which examined cytokines in peripheral blood cells and dermal lesions of leprosy. The simplicity of this method would allow the examination of a large number of samples across the spectrum of leprosy.

Biopsy↗
PubMed1989

Synthesis and pathway of Luciola mingrelica firefly luciferase in Xenopus laevis frog oocytes and in cell-free systems.

Poly (A+)RNA was isolated from the lanterns of adult fireflies of L. mingrelica. The poly (A+)RNA was translated in a cell-free translation mixture from rabbit reticulocyte and from Krebs II mouse ascites cells and in Xenopus laevis frog oocytes. The synthesis of the enzymatically active firefly luciferase was demonstrated in all systems. In the cell-free systems a maximal quantity of luciferase is synthesized during the first 1-2 h, then the decreasing activity of luciferase is observed. The optimal concentration of mRNA for translation in the mouse ascites cell extract was found. The kinetics of the synthesis of the luciferase, its pathway and stability in X. laevis oocytes was studied. It was observed that luciferase is secreted from oocytes into the incubation medium.

Animals↗
PubMed2006

Down-regulation of Sox10 with specific small interfering RNA promotes transdifferentiation of Schwannoma cells into myofibroblasts.

Neural crest-derived cells are noted for the long lasting plasticity during lineage commitment process and the potential for transdifferentiation into other neural crest derivatives. Schwann cells in particular have been reported to transdifferentiate into melanocytes and myofibroblasts. Detailed studies of transdifferentiation at the molecular level have been hampered by difficulty in isolating sufficient quantity of primary cells or cellular materials. Here, we describe a robust in vitro system in which Schwannoma cells undergo an apparent transdifferentiation into myofibroblasts. Importantly, we induce the transdifferentiation by down-regulating a single transcription factor, Sox10, thereby identifying a key molecular event in this process. Myofibroblasts thus generated showed carbachol-stimulated contraction and calcium transients and express several established myofibroblast-specific genes. These results suggest that generating desired cell types based on "knock-down" of critical genes may be a viable strategy.

Actins↗
PubMed1985

Isolation of specific tRNAs using an ionic-hydrophobic mixed-mode chromatographic matrix.

The coating of a C18-reversed-phase high performance liquid chromatography support (octadecylsilyl-Hypersil) with a tetraalkylammonium salt (methyltrioctylammonium chloride) produces a chromatographic matrix with both ionic and hydrophobic character. Using this material oligonucleotides and tRNAs can be separated with high resolution. The observed resolution is in part due to the apparent lack of diffusion processes occurring during chromatography with this matrix. Some tRNAs can be obtained in high purity from a bulk tRNA mixture after a single chromatographic step. In general it is more efficient to use the matrix as the last step of a purification procedure for a particular tRNA. A two-step procedure is described which allows, in some cases, the isolation of small quantities of specific tRNA isoacceptors.

Carbon Radioisotopes↗
PubMed1985

Selective isolation of mercurated DNA by affinity chromatography on thiol matrices.

A method for isolating picomole quantities of nascent mercurated DNA from a mixture of cellular nucleic acids using affinity chromatography on thiol-agarose is described. Analysis of mercurated DNA (HgDNA) isolated in the presence of in vivo-labeled cellular RNA or in vitro-synthesized RNA showed a low level of RNA contamination, about 0.04-0.16%, in the HgDNA. Comparative binding studies on different thiol matrices showed that the efficiency of binding of HgDNA was related to the nature but not to the SH content of the matrix used. Another important parameter for binding was the structure of HgDNA. The recovery was 98% with large nascent HgDNA sedimenting at about 30 S, whereas for short pulse-labeled single-stranded HgDNA (20-50 nucleotides long), the maximum recovery was 60%. The effect of the structure of HgDNA on the binding to the thiol matrix was probed using a variety of well-defined mercurated structures obtained from phage DNA and their restriction fragments. For DNA containing one 5-mercuricytidine 5'-triphosphate (HgdCMP) residue at each 3'-end, short fragments (size range, 230-510 bp) were bound quantitatively. With larger fragments (size range, 490-1100 bp), the binding decreased progressively with increasing size. DNA fragments larger than 1060 bp did not bind to the matrix. Single-stranded DNA containing only one HgdCMP at one end did not bind to the matrix even in the size range 200-1100 nucleotides. In contrast, continuous stretches of HgdCMP residues in one strand or short stretches of HgdCMP residues at random in both strands permit quantitative binding irrespective of size.

Bacteriophages↗
PubMed1990

Schistosoma mansoni: biochemical characteristics of the antischistosomal effects of Ro 15-5458.

We evaluated a variety of biochemical parameters in Schistosoma mansoni isolated from mice up to 4 days after dosing with 15 mg/kg Ro 15-5458. While no drug effect could be demonstrated in the utilization of media glucose, glycogen content, gut pigment, or ATP levels of the parasites, a significant reduction (P less than or equal to 0.05) in parasite weight and protein content was observed. Possible drug actions that may contribute to the loss in parasite protein and perhaps ultimately result in parasite death have been investigated. We noted significant reduction in the incorporation of leucine and thymidine into acid-insoluble fractions of the parasites. The reduction in the incorporation of leucine into parasite proteins was nonspecific and preceded the effect of the drug on the uptake of the amino acid. Parasite and host liver RNA isolated after dosing were translated in vitro in a rabbit reticulocyte system. Drug-treated parasite mRNA, but not that of the host, was less effective than control mRNA in directing the incorporation of [35S]methionine. We propose a hypothesis that attributes the loss in protein content to a defect in the biosynthesis of parasite proteins as a result of a drug-induced reduction in the quantity of mRNA in the parasites This effect of Ro 15-5458 on the parasite may provide the basis for its schistosomicidal action.

Acridines↗
PubMed1989

Insulin-mediated regulation of epididymal fat pad malic enzyme.

The complex nature of insulin-mediated biological responses has made it difficult to interpret such data. Prior studies in our laboratory had characterized the insulin-mediated increases in hepatic malic enzyme activity in normal and diabetic rats (Drake et al., 1983; Drake and Mucenski, 1985). However, since insulin-mediated regulatory processes have been shown to be tissue specific, we decided to examine malic enzyme activity in the epididymal fat pads of normal, diabetic, and insulin-treated normal and diabetic rats. This data revealed that in direct contrast to the hepatic studies, the normal epididymal fat pad contained the low specific activity malic enzyme molecule. Insulin treatment of both normal and diabetic rats resulted in an increase in epididymal fat pad malic enzyme activity due to increases in both enzyme quantity and specific activity. In order to quantitate epididymal fat pad malic enzyme mRNA levels, we isolated a 2.4 kb malic enzyme specific cDNA which was designated pR ME 1. In both normal and diabetic rats, the observed increases in malic enzyme quantity were directly paralleled by increases in malic enzyme mRNA content. The Northern blot data revealed an apparent differential expression of the malic enzyme mRNA doublet between insulin-treated normal and diabetic epididymal fat pads. This study demonstrates that insulin modulates epididymal fat pad and hepatic malic enzyme activity in a tissue-specific manner utilizing a defined subset of insulin-sensitive parameters involving alterations in enzyme specific activity and/or quantity.

Adipose Tissue↗