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PubMed1998

Peptones stimulate both the secretion of the incretin hormone glucagon-like peptide 1 and the transcription of the proglucagon gene.

Truncated glucagon-like peptide (GLP)-1 is a potent incretin. Its synthesis and secretion are modulated by food, but the influence of individual nutrients remains to be established. The hypothesis that protein hydrolysates (peptones) can directly regulate both GLP-1 secretion and proglucagon (PG) gene transcription was tested in this study, ex vivo in the isolated vascularly perfused rat intestine and in vitro in the murine enteroendocrine cell line STC-1. Peptones were albumin egg hydrolysate (AEH) and meat hydrolysate (MH). We demonstrate in these two models that peptones dose-dependently stimulate GLP-1 release, whereas isocaloric quantities of bovine serum albumin or of an amino acid mixture had no stimulatory effect. A strong and rapid increase of PG RNA level was observed in STC-1 cells treated with peptones (14-fold and 7-fold increase after 4 h of incubation with 3% wt/vol MH and AEH, respectively). Peptones also increased the PG RNA level in the colonic PG-expressing cell line GLUTag. In contrast, peptones did not modify the PG RNA level in two pancreatic glucagon-producing cell lines, namely, the RINm5F and INR1G9 cells. The peptone effect in STC-1 cells was completely abolished by blocking transcription before MH treatment. The stability of proglugacon transcripts was not modified by MH treatment, but nascent transcripts were more abundant in STC-1 cells preincubated with MH. Finally, MH treatment strongly stimulated (15-fold stimulation) the transcriptional activity of two PG gene promoter fragments (-1100 and -350 base pair) linked to the CAT reporter gene transiently transfected in STC-1 cells. Overall, peptones evoke an as yet undescribed release of GLP-1 when brought into contact with native intestinal L-cells or with STC-1 enteroendocrine cells. The increased transcription of the glucagon gene in the latter system suggests an important role of protein hydrolysates in the control of not only the secretion but also the synthesis of the incretin hormone.

Animals↗
PubMed2004

The structure and stability of biological metaphosphate, phosphate, and phosphorane compounds in the gas phase and in solution.

Density functional calculations of a series of metaphosphates, acyclic and cyclic phosphates and phosphoranes relevant to RNA catalysis are presented. Solvent effects calculated with three well-established solvation models are analyzed and compared. The structure and stability of the compounds are characterized in terms of thermodynamic quantities for isomerization and ligand substitution reactions, gas-phase proton affinities, and microscopic solution pK(a)() values. The large dataset of compounds allows the estimation of bond energies to determine the relative strengths of axial and equatorial P-O phosphorane single bonds and P-O single and double bonds in metaphosphates and phosphates. The relative apicophilicty of hydroxyl and methoxy ligands in phosphoranes are characterized. The results presented here provide quantitative insight into RNA catalysis and serve as a first step toward the construction of a high-level quantum database for development of new semiempirical Hamiltonian models for biological reactions

Catalysis↗
PubMed1986

[Protein synthesis and secretion and RNA and DNA synthesis in human skin fibroblasts in a medium with a low serum content].

Human skin fibroblasts, both postnatal and embryonic, were cultured in the stationary phase of growth for 6-10 days in the DMEM with bovine serum (BS), 0.1-0.5% fetal calf serum (FCS) or 1% human serum (HS). On the day 4 of culturing, a considerable increase was observed in the synthesis and secretion of protein by postnatal fibroblasts in the Eagle medium with 0.1-0.5% FCS, or with 0.5% BS, and in medium 199 with 0.1-0.5 BS, or with 0.1 FCS. Maximum synthesis and secretion of 14C-proline labeled protein was observed on day 2 of culturing of cells in the DMEM medium with 1% HS. In the DMEM medium with low serum content, protein synthesis being virtually unchanged, 75-80% of protein was secreted by cells into the culture medium with BS on days 2-4; in the medium with FCS such a high secretion of protein was observed only on day 4. High synthesis of protein by fetal fibroblasts in the DMEM medium with 0.1% BS and high protein secretion in all the media with 0.1% BS or 0.5% FCS were observed. The maximum level of secretion of protein by fibroblasts coincided with a considerable increase in both RNA and DNA syntheses. The data obtained suggest that cells in deep resting state actively react to the composition of the medium as well as to the quality and quantity of the serum. It may also be suggested that the mechanism of protein secretion has an important role in maintenance of the constant level of intracellular proteins in resting cells.

Animals↗
PubMed2002

A history of poly A sequences: from formation to factors to function.

Biological polyadenylation, first recognized as an enzymatic activity, remained an orphan enzyme until poly A sequences were found on the 3' ends of eukarvotic mRNAs. Their presence in bacteria viruses and later in archeae (ref. 338) established their universality. The lack of compelling evidence for a specific function limited attention to their cellular formation. Eventually the newer techniques of molecular biology and development of accurate nuclear processing extracts showed 3' end formation to be a two-step process. Pre-mRNA was first cleaved endonucleolytically at a specific site that was followed by sequential addition of AMPs from ATP to the 3' hydroxyl group at the end of mRNA. The site of cleavage was specified by a conserved hexanucleotide, AAUAAA, from 10 to 30 nt upstream of this 3' end. Extensive purification of these two activities showed that more than 10 polypeptides were needed for mRNA 3' end formation. Most of these were in complexes involved in the cleavage step. Two of the best characterized are CstF and CPSF, while two other remain partially purified but essential. Oddly, the specific proteins involved in phosphodiester bond hydrolysis have yet to be identified. The polyadenylation step occurs within the complex of poly A polymerase and poly A-binding protein, PABII, that controls poly A length. That the cleavage complex, CPSF, is also required for this step attests to a tight coupling of the two steps of 3' and formation. The reaction reconstituted from these RNA-free purified factors correctly processes pre-mRNAs. Meaningful analysis of the role of poly A in mRNA metabolism or function was possible once quantities of these proteins most often over-expressed from cDNA clones became available. The large number needed for two simple reactions of an endonuclease, a polymerase and a sequence recognition factor, pointed to 3' end formation as a regulated process. Polyadenylation itself had appeared to require regulation in cases where two poly A sites were alternatively processed to produce mRNA coding for two different proteins. The 64-KDa subunit of CstF is now known to be a regulator of poly A site choice between two sites in the immunoglobulin heavy chain of B cells. In resting cells the site used favors the mRNA for a membrane-bound protein. Upon differentiation to plasma cells, an upstream site is used the produce a secreted form of the heavy chain. Poly A site choice in the calcitonin pre-mRNA involves splicing factors at a pseudo splice site in an intron downstream of the active poly site that interacts with cleavage factors for most tissues. The molecular basis for choice of the alternate site in neuronal tissue is unknown. Proteins needed for mRNA 3' end formation also participate in other RNA-processing reactions: cleavage factors bind to the C-terminal domain of RNA polymerase during transcription; splicing of 3' terminal exons is stimulated port of by cleavage factors that bind to splicing factors at 3' splice sites. nuclear ex mRNAs is linked to cleavage factors and requires the poly A II-binding protein. Most striking is the long-sought evidence for a role for poly A in translation in yeast where it provides the surface on which the poly A-binding protein assembles the factors needed for the initiation of translation. This adaptability of eukaryotic cells to use a sequence of low information content extends to bacteria where poly A serves as a site for assembly of an mRNA degradation complex in E. coli. Vaccinia virus creates mRNA poly A tails by a streamlined mechanism independent of cleavage that requires only two proteins that recognize unique poly A signals. Thus, in spite of 40 years of study of poly A sequences, this growing multiplicity of uses and even mechanisms of formation seem destined to continue.

Adenoviridae↗
PubMed1999

Identification of IPNV-specified components released from productively infected RTG-2 cells following massive cytopathic effect.

Rainbow trout gonad cells (RTG-2) display a dramatic cytopathic effect and lysis following productive infection by infectious pancreatic necrosis virus (IPNV). In this study viruses were efficiently released into the growth medium together with low amounts of the monomeric free form of the structural protein VP3, heterodimers of VP2-VP3, aggregates of pVP2 and viral RNA associated with VP3. Ribonucleoprotein complexes of RNA-VP3 contained RNA equivalent to at the most 25% of full length viral genomes. Infectivity of material released into the growth medium late in infection was only associated with fully assembled viruses and isolated subviral RNA-VP3 complexes were not infectious. Upon purification of IPNV, viral hexa- and pentagonal particles of approx. 15 nm diameter were occasionally co-purified with the virus and then appeared in large quantities. Similar particle-like structures were seen as substructures of purified viruses that were treated with and partially disintegrated by CsCl of virus isodensity concentration.

Animals↗
PubMed1988

Anti-sense inhibition of tissue plasminogen activator production in differentiated F9 teratocarcinoma cells.

F9 teratocarcinoma cells secrete the serine protease, tissue plasminogen activator (t-PA), upon differentiation induced in vitro by retinoic acid (RA) or RA and dibutyryl cAMP (RA/dbcAMP). A recombinant plasmid capable of directing the production of t-PA anti-sense RNA was constructed and transfected into F9 stem cells in an attempt to create a hypomorphic phenotype for t-PA synthesis. Several colonies were isolated which contained anti-sense RNA and which showed greater than a 50% reduction in t-PA activity upon differentiation. One such colony, 3b4, exhibited a 75% reduction in t-PA activity and was analyzed further. Large quantities of t-PA anti-sense transcript were expressed in the stem cells which are characterized by the absence of t-PA gene expression. In the induced cells, which normally express t-PA, the amount of detectable anti-sense transcript was significantly decreased. The amount of t-PA mRNA in differentiated cells containing t-PA anti-sense RNA was comparable to that in differentiated control cells. Subcellular localization of the mRNA in induced 3b4 cells appeared to be the same as induced control cells. Expression of collagen type IV, another marker of differentiation, was also monitored and was unaffected by the presence of t-PA anti-sense RNA in RA/dbcAMP-treated cells. The inhibition of differentiation-specific gene expression by anti-sense RNA may be useful for further studies of developmentally regulated genes.

Animals↗
PubMed1991

High yield purification of active transcription factor IIIA expressed in E. coli.

Transcription factor IIIA (TFIIIA), a sequence-specific DNA-binding protein from Xenopus laevis, is a zinc finger protein required for transcription of 5S rRNA genes by RNA polymerase III. We describe the purification and characterization of recombinant TFIIIA (recTFIIIA) expressed in E. coli. RecTFIIIA was purified to greater than 95% homogeneity at a yield of 2-3 milligrams per liter of bacterial culture. This purified protein protects the internal control region of a 5S rRNA gene from DNase I digestion, yielding footprints on both strands identical to those produced by the ovarian protein (ovaTFIIIA). Quantitative analysis of binding data from gel retardation assays yielded a KD of about 0.4 nM for TFIIIA from either source. Using a quantitative TFIIIA-dependent in vitro transcription assay, we found that recTFIIIA is equivalent to ovaTFIIIA in supporting transcription of 5S rRNA genes. We conclude that recTFIIIA is functionally indistinguishable from the protein purified from Xenopus ovaries, and can be readily obtained in pure form and large quantity.

Amino Acid Sequence↗
PubMed2005

In vitro translation of RNA to lactase during postnatal development of rat intestine.

mRNA levels encoding lactase were detected by Northern blot analysis using two different probes in developing rat intestine. Probe I and probe II corresponding to second half of prolactase gene showed a 6.8 kb mRNA transcript in 7, 14, 21 and 30 day old rat intestine. There was no change in quantity of lactase mRNA detected using probe II, but hybridization with probe I showed a progressive decrease in mRNA transcript encoding lactase with age. At day 7 and 14 of postnatal development, the lactase mRNA was quite high, but it reduced upon weaning. The in vitro translation products of RNA detected by Western blot analysis using brush border lactase antibodies showed several isoforms of lactase antigen with molecular weight ranging from 100-220 kDa. Analysed at days 7 and 30 of postnatal development, lactase isoforms of molecular weight 130 kDa and 220 kDa were similar to those found in purified brush border membranes. The translation of RNA to 220 kDa lactase protein was high in 7 and 14 day old pups, but it was markedly reduced in 30 day old animals. These findings support the contention that translation of mRNA to lactase is impaired in weaned animals, which may also be responsible for the maturational decline in lactase activity in adult rat intestine.

Age Factors↗
PubMed1991

Inhibition of GFAP synthesis by antisense RNA in astrocytes.

Glial fibrillary acidic protein (GFAP) accumulation is a prominent feature of astrocytic gliosis. The inhibition or delay in GFAP synthesis might delay scar formation resulting from an insult such as spinal cord injury or central nervous system (CNS) demyelination. The delay in the formation of a physical barrier might allow the neurons and oligodendrocytes to reestablish a functional environment. We delivered antisense GFAP RNA complexed with Lipofectin (LF), a cationic liposome, into cerebral astrocytes in culture and tested the feasibility of inhibiting GFAP synthesis. Our results demonstrate that LF facilitated antisense RNA uptake into astrocytes. Astrocytes took up 3H-antisense GFAP RNA alone and reached an equilibrium of 7-8.8 eta g per mg protein after 2.5 hr. When complexed with LF, astrocytes could increase the uptake to 14 eta g per mg protein and the time for reaching this quantity was shortened to 10 min. This uptake level was further enhanced if experiments were carried out in HEPES buffered saline (HBS). All uptake studies were dose- and time-dependent. Dibutyryl cyclic AMP (dBcAMP) is known to induce an increase of GFAP content in cultured astrocytes. We studied the effect of LF/antisense GFAP RNA on the GFAP content in dBcAMP (0.25 mM)-treated astrocytes. Cultures of astrocytes treated with dBcAMP contained almost twice as much GFAP as untreated cultures after 2 days. Similar cultures treated with LF/antisense RNA in HBS did not show an increase but a 30-40% decrease in GFAP content 2 days after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The effects of vitamin K1 and warfarin on prothrombin expression in human hepatoblastoma (HepG2) cells.

Cultures of human hepatoblastoma (HepG2) cells were treated with vitamin K1 or warfarin and prothrombin antigen and mRNA levels were determined. With 3 and 6 h of 10 micrograms vitamin K1 treatment secreted prothrombin antigen levels, relative to total secreted protein levels, were increased 1.5-fold and 2.1-fold, respectively, over ethanol-treated control levels as determined by an enzyme-linked immunosorbent assay. Dose-response analysis with 3 h of 25 micrograms/ml vitamin K1 treatment demonstrated a maximal increase of 2.0-fold in secreted prothrombin antigen levels, relative to total secreted protein levels, over ethanol-treated control levels. Pulse-chase analysis with 35S-methionine and immunoprecipitation of 35S-labelled prothrombin demonstrated that, with vitamin K1 treatment (25 micrograms/ml, 3 h), the rate of prothrombin secretion increased approximately 2-fold and the total amount (intra- and extracellular) of prothrombin synthesized increased approximately 50% over ethanol-treated control levels. Warfarin treatment (1, 5, or 10 micrograms/ml, 24 h) resulted in decreases in secreted prothrombin antigen levels, relative to total protein levels to approximately 85%, 87% or 81% of ethanol-treated control levels. Analysis of total RNA isolated from these cultures by Northern and solution hybridization techniques demonstrated that prothrombin mRNA was approximately 2.1 kb and that neither vitamin K1 nor warfarin treatment affected the quantity of prothrombin mRNA (ranging from 240-350 prothrombin mRNA molecules per cell). These results demonstrate that vitamin K1 and warfarin, in addition to effects on gamma-carboxylation, affect prothrombin synthesis post-transcriptionally, perhaps influencing translation, post-translational processing and/or secretion mechanisms.

Antigens↗
PubMed1992

Calbindin-D9K gene expression in the lung of the rat. Absence of regulation by 1,25-dihydroxyvitamin D3 and estrogen.

Calbindin-D9K (CaBP9K) is classically considered to be the molecular expression of 1,25-dihydroxyvitamin D3. The hormone is known to regulate the rat CaBP9K gene in duodenal tissue at transcriptional and posttranscriptional levels. This study shows that the CaBP9K gene is expressed in the rat lung, and that this expression is probably not vitamin D- or estrogen-dependent. The CaBP9K gene is not expressed in alveolar macrophages, but CaBP9K messenger RNA (mRNA) was localized by in situ hybridization in alveolar epithelial cells. CaBP9K mRNA was detected as early as the 20th day of gestation. The quantity of CaBP9K mRNA gradually increased during growth, from 1-77 days after birth, whereas the CaBP9K concentration dramatically increased from day 19 to day 20 of gestation. Vitamin D-deficient male rats (8 weeks old) were given a single injection of 1,25-dihydroxyvitamin D3 (650 pmol/100 g body wt) and killed 1 h and 24 h after injection. The hormonal treatment resulted in a rise in duodenal CaBP9K mRNA, but no significant change in lung extracted CaBP9K mRNA. Mature ovariectomized rats were injected with 17 beta-estradiol (0.5 microgram/100 g body wt) and killed 24, 48, and 72 h later. The CaBP9K mRNA concentration in the uterus was markedly dependent on estrogen; that of the lung was not. The factors regulating the CaBP9K gene expression in the lung remain to be determined.

Aging↗
PubMed1997

Transcriptional characterization of the Rickettsia prowazekii major macromolecular synthesis operon.

Recent studies have demonstrated that Rickettsia prowazekii can regulate transcription of selected genes at the level of initiation. However, little information concerning the existence of operons and coordinate gene regulation in this obligate intracellular parasitic bacterium is available. To address these issues, we have focused on the rpoD gene linkage group (greA-open reading frame 23 [ORF23]-dnaG-rpoD), which includes the rickettsial analog (ORF23-dnaG-rpoD) of the major macromolecular synthesis operon (MMSO). The rickettsial MMSO consists of an ORF coding for a protein of unknown function the structural genes for DNA primase (dnaG) and the major sigma factor of RNA polymerase (rpoD). RNase protection assays (RPA) were used to determine if these genes are organized into an operon controlled by multiple promoters and the quantities of transcripts produced by these genes relative to each other. RPA with a probe spanning the 270-base greA-ORF23 intervening region identified a putative transcriptional promoter within the intervening sequence. Multiple RPA probes spanning the next 4,041 bases of the linkage group demonstrated the presence of a continuous transcript and thus the existence of an operon. A probe spanning the dnaG-rpoD region revealed that two additional mRNA fragments were also protected, which enabled us to identify additional putative promoters for rpoD within dnaG. Primer extension determined that the 5' ends of the three transcripts consist separately of adenine (located 227 bases upstream of ORF23) and uracil and adenine (located 336 and 250 bases upstream of rpoD, respectively). Quantitation of transcripts produced by the three ORFs determined the relative amounts of transcripts (ORF23 to dnaG to rpoD) to be 1:2.7:5.1.

Amino Acid Sequence↗
PubMed2004

Altered expression of CUG binding protein 1 mRNA in myotonic dystrophy 1: possible RNA-RNA interaction.

The triplet repeats mutation, which causes myotonic dystrophy 1 (DM1), is thought to have a dominant negative effect on RNA levels. In light of previous results using differential display analysis, the present study focused on the expression of CUG binding protein 1 (CUGBP1) mRNA. Northern blot analysis demonstrated that the quantity of CUGBP1 mRNA in three DM1 patients was approximately 70% of that observed in three normal controls (P < 0.05). In addition, a semi-quantitative RT-PCR assay showed that the relative amount of CUGBP1 mRNA was reduced in muscle biopsy samples from 10 DM1 patients compared to that from five normal individuals (P < 0.01) and 10 myopathic disease controls (P < 0.01). The amount of CUGBP1 mRNA was negatively correlated with the size of the CTG expansion (r = -0.85, P < 0.05). In vitro RNA-RNA binding experiments demonstrated that the incubation of expanded CUG repeats with CUGBP1 RNA generated a higher molecular weight band, which was digested by RNase III. The CUGBP1 mRNA was found to contain several CAG repeat sequences. These results suggest that the CUG expansion may bind to complementary sequences within the CUGBP1 mRNA and that this molecular interaction may affect CUGBP1 mRNA expression in DM1.

Adult↗
PubMed1991

Expression of alpha-subunits of G proteins in failing human heart: a reappraisal utilizing quantitative polymerase chain reaction.

A decrease in the density of the beta 1-adrenergic receptor and an increase in the functional activity of the G inhibitory protein Gi accompany human heart failure; however, the molecular and biochemical mechanisms responsible for these changes are unclear. We previously reported that the steady-state levels of the mRNAs encoding both alpha Gi-3 and alpha Gs were significantly increased in failing human heart. However, these results are not consistent with recent studies demonstrating that immunodetectable levels of alpha G proteins are not different in failing human hearts when compared with non-failing controls. In addition, analysis of the 5' flanking regions of alpha Gi and alpha Gs suggests that these two genes are unlikely to be co-regulated as their regulatory domains are quite different. Therefore, we hypothesized that the disparity between the measurements of alpha G protein gene expression and assessment of the actual levels of alpha G proteins might be due to technical limitations of the Northern blot technique utilized in previous studies for assessment of the mRNA levels; (i) cytoskeletal beta-actin mRNA was used as a standard for normalization; and (ii) only relative levels of alpha G mRNAs were measured. The recent application of the polymerase chain reaction to quantification of mRNA levels in small quantities of human heart provided the tool with which to test this hypothesis. When expressed in molecules of mRNA per microgram of total RNA, there were no differences in the levels of alpha Gi and alpha Gs mRNAs in failing human heart when compared with non-failing controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

Expression of tumor necrosis factor-alpha in mouse spermatogenic cells.

Enriched fractions of spermatogenic cells were isolated by unit gravity sedimentation and analyzed both for the presence of secreted tumor necrosis factor-alpha (TNF alpha) in vitro by bioassay and for the presence of TNF alpha mRNA by Northern blot analysis. Small quantities of bioactive TNF alpha were consistently detected in medium conditioned by round spermatid fractions. Both pachytene spermatocyte and round spermatid fractions contained RNA that hybridized with murine cDNA probes for TNF alpha, with pachytene spermatocytes containing a normal 1.9-kilobase (kb) transcript, while round spermatids contained principally an approximately 2.8-kb transcript. Both the normal size transcript and the larger haploid-specific transcript were enriched when total RNA from pachytene spermatocyte and round spermatid fractions was passed through an oligo(dT) column. The normal 1.9-kb transcript within pachytene spermatocytes could be induced by exposing the spermatogenic cells to lipopolysaccharides in vitro, yet the approximately 2.8-kb transcript within round spermatids appeared uninduced by LPS treatment. In situ hybridization for the TNF alpha message by using digoxigenin label antisense TNF alpha riboprobe labeled pachytene spermatocytes, round spermatids, and presumptive interstitial macrophages. Spermatogonia and elongating spermatids as well as other interstitial cells were unlabeled or very lightly labeled. Hybridization of 16-day-old prepuberal testis resulted in the labeling of spermatocytes and presumptive interstitial macrophages. RNA from Sertoli cells, but not pachytene spermatocytes or round spermatids, hybridized with human TNF alpha receptor p60 probe in Northern blot analysis. These results are consistent with the working hypothesis that spermatids release TNF alpha, which is detected by Sertoli cells and may serve as a paracrine factor, regulating an as yet unidentified process in spermatogenesis.

Animals↗
PubMed1990

[Effect of salvin on the synthesis of macromolecules in Staphylococcus aureus 209P].

The effect of salvin and its active component, carnazolic acid, on the synthesis of macromolecular compounds in the cells of S. aureus 209P was studied. It was shown that the inhibitory action of salvin on the synthesis of peptidoglycane in the culture was defined by the presence of carnazolic acid in its composition. In the bactericidal concentration, carnazolic acid was twice as less active as salvin and inhibited incorporation of labeled precursors into RNA and protein. The findings grounded the conclusion that some nonidentified components of salvin with low antimicrobial activity contained in it in insignificant quantities had an additional inhibitory effect on the process. Comparative study of salvin and antibiotics with the known mechanisms of action such as benzyl penicillin or chloramphenicol revealed a certain similarity in the action of salvin and benzyl penicillin on incorporation of labeled precursors into the macromolecular compounds of S. aureus 209P.

Abietanes↗
PubMed1990

Corneal endothelium modulation factor released by polymorphonuclear leukocytes. Partial purification and initial characterization.

Polymorphonuclear leukocytes produce a polypeptide factor that is released into the medium. This factor is partially purified 83-fold by ammonium sulfate precipitation followed by chromatography on a DEAE-Sephadex or heparin-Sepharose column. The partially purified factor is trypsin-sensitive. This factor affects a population of rabbit corneal endothelial cells by modulating them to fibroblastlike cells and by further stimulating their growth, leading to the formation of colonies of multilayered modulated cells. There is a dose-dependent phenotypic modulation of corneal endothelial cells by the partially purified corneal endothelium modulation factor (CEMF); cell shape is changed and type I collagen synthesis is increased with greater concentrations of CEMF. Since the fully modulated endothelial cells have collagen phenotypes distinct from those of normal cells, collagen synthesized by the first-passaged cells (a mixture of normal and modulated cells) was determined by immunoblot analysis with antibodies specific against types I and IV collagens. The first-passaged cells, in the presence of CEMF, contained a large amount of type I collagen (modulated phenotype) and a dramatically reduced amount of type IV collagen (physiologic type), whereas the normal endothelial cells demonstrated strongly positive staining only with antibodies to type IV collagen. Using cloned cDNA probes, the relative quantities of the transcripts of these collagens were determined by slot-blot hybridization; the first-passaged cells contained type IV collagen RNA in an amount similar to the normal cells, but a slightly larger amount of type I mRNA. These results demonstrate a functional involvement of a protein factor released by polymorphonuclear leukocytes in modulating cell shape and collagen gene expression in corneal endothelial cells.

Animals↗
PubMed1994

[Transmission of hepatitis C by accidental needlestick injuries. Evaluation of the risk].

The risk of transmitting contagious diseases by accidental needle-stick injury has raised a considerable amount of concern among hospital staff. Before generalized vaccination in the early 80s, there was a high risk of hepatitis B transmission. More recently, the development of reliable techniques of detecting serum markers has made it possible to precisely evaluate the risk for hepatitis C. The risk of contamination by the hepatitis C virus by accidental needle-stick injury can be estimated at 0 to 3%, and can only reach a maximum of 10% when the patient is positive for hepatitis C RNA. The risk is thus less than for hepatitis B virus (7 to 30%). The low rate of transmission probably results from the quantity of viral material in blood and secretions. In populations of health personnel exposed to a risk of septic needle-stick injury, the prevalence of anti-hepatitis C virus antibodies has been observed in several studies at rates between 0 and 2%. This is similar to non-exposed populations and would be an argument suggesting that there is a low risk of hepatitis C virus transmission. Nevertheless, because hospital staff is frequently exposed to blood and because a significant number of patients are positive for anti-hepatitis C virus antibodies, adequate preventive measures must be taken. The Immunization Practice Advisory Committee (USA) recommends injection of polyvalent gammaglobulins when stick injury occurs with a needle used for a hepatitis C virus antibody positive patient, but the effectiveness of this protocol has not been demonstrated. Several preliminary studies suggest that treatment of hepatitis C in the acute phase could significantly reduce the rate of chronicity. When interferon has been authorized for this indication, and if effectiveness is confirmed, treatment might be recommended for health personnel with acute needle-stick transmitted HCV infection. Infected needle-stick victims might be followed by having their transaminases checked 4-12 weeks later. In case of positive results, early interferon therapy might be started.

Dentists↗