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PubMed1987

The characterization of atrial natriuretic peptide (ANP) expression by primary cultures of atrial myocytes using an ANP-specific monoclonal antibody and an ANP messenger ribonucleic acid probe.

The biochemical and morphological characteristics of primary neonatal rat atrial myocytes were examined in order to establish a model system for future studies of the biosynthesis and secretion of atrial natriuretic peptide (ANP). Preliminary studies demonstrated that the quantity of immunoactive ANP/microgram protein within rat atria increased as a function of age from 2 ng/micrograms in 19 day prenatal animals to 400 ng/micrograms in the adult. Gel filtration, reversed phase HPLC, and ion exchange HPLC indicated that there were similar quantities of immunoactive ANP in the right and left atria at various ages, and that the major molecular form of the peptide in the heart is chromatographically indistinguishable from ANP(1-126). Cultures of dissociated cells were prepared from pooled left and right atria derived from 1 day postnatal animals. A complete serum-free medium was developed which resulted in the maintenance of high levels of immunoactive ANP in the cultures. As determined by RIA, the cellular content of ANP increased in the cultures as a function of time through 7 days in vitro. The quantity of immunoactive ANP in the cultures increased approximately 2- to 3-fold between days 3 and 7. When the cultures that had been maintained for 7 days were submitted to immunocytochemistry using an ANP-specific monoclonal antibody, distinct colonies of spindle-shaped cells stained positively. In situ hybridization, utilizing an 35S-labeled ANP messenger RNA probe, demonstrated that these colonies of myocytes expressed the ANP message. Using quantitative dot-blot hybridization it was shown that the ANP mRNA level increased approximately 50-fold between days 1 and 7 in culture. These studies indicate that the serum-free culture medium allows continued accumulation of both ANP and the ANP message in culture and will provide a useful model system to characterize factors that regulate the biosynthesis and secretion of this hormone.

Aging↗

PubMed2006

Factors affecting tyramine production in Enterococcus durans IPLA 655.

The decarboxylation of tyrosine by certain lactic acid bacteria leads to the undesirable presence of tyramine in fermented foods. Tyramine is the most frequent biogenic amine found in cheese and is also commonly found in other fermented foods and beverages. The tyramine-producing strain Enterococcus durans IPLA 655 was grown in a bioreactor under different conditions to determine the influence of carbon source, tyrosine and tyramine concentrations, and pH on tyramine production. The carbon source appeared to have no significant effect on the production of tyramine. In contrast, tyrosine was necessary for tyramine production, while the presence of tyramine itself in the growth medium inhibited such production. pH showed by far the greatest influence on tyramine synthesis; tyramine was produced in the greatest quantities at pH 5.0, although this was accompanied by a reduced growth rate.

Biogenic Amines↗

PubMed1994

Optimization of subtractive hybridization in construction of subtractive cDNA libraries.

The efficiency of subtraction, integrity of residual single-stranded cDNA, and efficient recovery of nanogram quantities of double-stranded cDNA are the three most important factors affecting quality of subtractive hybridization reactions prior to subtractive cDNA library construction. Techniques for efficient isolation of single-stranded cDNA, after subtraction, have greatly improved from early protocols based on hydroxylapatite chromatography to phenol-chloroform extraction of biotin-streptavidin-crosslinked polynucleotides or oligo(dA)-cellulose affinity chromatography. Factors affecting mRNA stability at the hybridization step, however, also have consequences that directly affect the complexity of the library and the length of cDNAs recovered. We have optimized the subtractive hybridization step in subtractive cDNA library construction to ensure that single-stranded cDNAs survive hybridization as near to full length as possible. These improvements have enabled successful construction of subtractive cDNA libraries from the nanogram quantities of single-stranded cDNA remaining after extensive liquid hybridization to high calculated C(o)t values.

DNA, Complementary↗

PubMed1998

STAT6 is required for the anti-inflammatory activity of interleukin-4 in mouse peritoneal macrophages.

Interleukin-4 (IL-4) is an anti-inflammatory cytokine which inhibits many inducible macrophage functions. The present study demonstrates that the ability of IL-4 to inhibit interferon gamma (IFNgamma)-dependent gene transcription is dependent upon STAT6. IL-4 suppressed IFNgamma-induced expression of the MIG (monokine induced by IFNgamma) gene, a C-X-C chemokine, in mouse macrophages. IFNgamma-induced expression of MIG mRNA was abolished in peritoneal macrophages from Stat1-/- mice, and the suppression of MIG mRNA by IL-4 was abolished in macrophages from Stat6-/- mice. Transient transfection assays using a reporter gene containing the MIG gene promoter or the IFNgamma-responsive element (gammaRE) from the MIG gene revealed that the IFNgamma-dependent transcription was suppressed by IL-4, although IL-4 alone had no transactivating function. IFNgamma and IL-4 activated STAT1 and STAT6, respectively, and both proteins were able to bind the gammaRE motif. Furthermore, STAT6 was associated with the co-activator CREB-binding protein in RAW264.7 cells. These observations indicate that STAT6 is necessary for the IL-4-mediated suppression of IFNgamma-induced, STAT1-dependent transcription and suggest that STAT6 may directly suppress the STAT1-dependent transcription by competing with STAT1 for occupancy of the gammaRE motif and/or by competing with limiting quantities of the transcriptional coactivator.

Animals↗

PubMed2005

Cotranslational endoplasmic reticulum assembly of FcepsilonRI controls the formation of functional IgE-binding receptors.

The human high affinity receptor for IgE (FcepsilonRI) is a cell surface structure critical for the pathology of allergic reactions. Human FcepsilonRI is expressed as a tetramer (alphabetagamma(2)) on basophils or mast cells and as trimeric (alphagamma(2)) complex on antigen-presenting cells. Expression of the human alpha subunit can be down-regulated by a splice variant of FcepsilonRIbeta (beta(var)). We demonstrate that FcepsilonRIalpha is the core subunit with which the other subunits assemble strictly cotranslationally. In addition to alphabetagamma(2) and alphagamma(2), we demonstrate the presence of alphabeta and alphabeta(var)gamma(2) complexes that are stable in the detergent Brij 96. The role of individual FcepsilonRI subunits for the formation of functional, immunoglobulin E-binding FcepsilonRI complexes during endoplasmic reticulum (ER) assembly can be defined as follows: beta and gamma support ER insertion, signal peptide cleavage and proper N-glycosylation of alpha, whereas beta(var) allows accumulation of alpha protein backbone. We show that assembly of FcepsilonRI in the ER is a key step for the regulation of surface expression of FcepsilonRI. The ER quality control system thus regulates the quantity of functional FcepsilonRI, which in turn controls onset and persistence of allergic reactions.

Endoplasmic Reticulum↗

PubMed2002

Measurement of plasma and tissue relaxin concentrations in the pregnant hamster and fetus using a homologous radioimmunoassay.

A homologous hamster relaxin RIA was developed to evaluate plasma and tissue concentrations of relaxin in the latter half of pregnancy in this species. Relaxin protein and mRNA were localized using antibodies developed to synthetic hamster relaxin and gene-specific molecular probes, respectively. Molecular weight and isoelectric point of the synthetic and native hormones were identical by electrophoretic methods, and synthetic hamster relaxin was active in the mouse interpubic ligament bioassay. Synthetic hormone was used as tracer and standard with rabbit antiserum to the synthetic hormone in the RIA. Relaxin was assayed in blood samples recovered from the retro-orbital plexus on Days 6, 8, 10, 12, 14, 15, and 16 of gestation and on Days 1 and 5 postpartum. Relaxin was first detected on Day 8 of gestation (3.7 +/- 0.6 ng/ml), increased to reach a maximum in the evening of Day 15 (826.0 +/- 124.0 ng/ml), and decreased by Day 16 (day of parturition). Relaxin concentrations were assayed in aqueous extracts of implantation sites (Days 6, 8, and 10) and chorioallantoic placentae (Days 12, 14, and 15). Concentrations were low on Day 6 (0.02 +/- 0.001 microg/g tissue), increased to Day 15 (6.96 +/- 0.86 microg/g tissue), and subsequently declined by the evening of Day 15. Relaxin protein and mRNA were localized to primary and secondary giant trophoblast cells in the chorioallantoic placental trophospongium. However, relaxin protein was not localized in ovaries of pregnant animals or oviductal tissues of cycling animals. Significant quantities of relaxin were detected in the serum of fetal hamsters recovered on Day 15.

Animals↗

PubMed2004

Biologically active neutrophil chemokine pattern in tonsillitis.

To gain an insight into the mechanisms of chronic and acute inflammation, the production of neutrophil chemokines in different types of tonsillitis - hyperplastic tonsillitis (HT), recurrent tonsillitis (RT) and peritonsillar abscesses (PA) - was investigated. The chemokines interleukin-8 (IL-8), growth-related oncogene-alpha (GRO-alpha), epithelial cell-derived neutrophil attractant-78 (ENA-78) and granulocyte chemotactic protein-2 (GCP-2) were detected and shown to have different biological activities. With respect to the biological properties of CXC chemokines, the biological activity of the chemokines was identified using a three-step high-performance liquid chromatography (HPLC) technique, a bioassay involving measurement of neutrophil chemotaxis in a single Boyden chamber in tissue of HT, RT and PA. Using reverse transcription-polymerase chain reaction (RT-PCR), the chemokine concentrations were determined in the different tonsillitis entities. The chemokine pattern was dominated in PA by IL-8 and GRO-alpha and in RT by GRO-alpha. Hyperplastic tonsils of patients without a history of infection generated about five times lower IL-8 than PA. A protein concentration of GCP-2 was induced in PA and RT, whereas ENA-78 remained the same in all entities. In conclusion, it would appear that IL-8 was up-regulated in acute inflammation, whereas GRO-alpha dominated in chronic inflammation. ENA-78 seems not to play a pivotal role in inflammatory processes in tonsils. GCP-2 may serve as a substitute chemokine in certain inflammatory conditions as its quantity of mRNA and protein was higher in RT and PA than in HT.

Acute Disease↗

PubMed2001

Identification and function of glucose transporter 1 in human mesangial cells.

OBJECTIVE: To evaluate the role of glucose transporter 1 (GLUT1) in the glucose uptake of glomerular mesangial cells. METHODS: Cultured human glomerular mesangial cells were used. The expressions of glucose transporter 1 mRNA and protein were detected with reverse transcription-polymerase chain reaction (RT-PCR), flow cytometry and immunofluorescence staining. Glucose uptake was determined with 2-deoxy-[3H]-D-glucose uptake assay. RESULTS: The expressions of GLUT1 mRNA and proteins were detected in human mesangial cells. Glucose uptake and its kinetics assay showed that GLUT1 is a functional glucose transporter in cultured human mesangial cells, and that its function could be inhibited by the specific inhibitor, Phloretin. CONCLUSION: GLUT1 is the predominant glucose transporter in human mesangial cells, which has the kinetic characteristics of high affinity and low capacity for D-glucose. This suggests that in order for mesangial cells to take up excessive quantities of glucose, as in diabetes, changes in glucose transporter expression, translocation or activity may be required.

Glomerular Mesangium↗

PubMedDate not supplied

[Shewanella genus bacteria isolated from the Black sea water and molluscs].

Six (6) strains of bacteria isolated from the Black Sea water and mollusks Mytilaster lineatus have been assigned to Shewanella genus on the basis of phenotypical and molecular-genetic study. Partial sequence of 16S rRNA gene revealed 100% similarity of three of these strains to S. baltica, although they had some phenotypical differences. Three more strains were genetically related to S. colwelliana and S. affinis (99-100% of 16S rRNA sequences similarity), but they differed from these species as well as from each other in some phenotypical properties, so the question about their taxonomic status still remains open. In aerobic conditions the Black Sea strains of S. baltica adsorbed significant quantities of Fe III from the media. They reduced it to Fe (II) in anaerobic conditions. Strains isolated from mollusks were not able to reduce Fe (III).

Animals↗

PubMed1996

Effect of protein restriction on the messenger RNA contents of bone-matrix proteins, insulin-like growth factors and insulin-like growth factor binding proteins in femur of ovariectomized rats.

It has been reported that loss of ovarian oestrogen after menopause or by ovariectomy causes osteoporosis. In order to elucidate the effect of dietary protein restriction on bone metabolism after ovariectomy, we fed ovariectomized young female rats on a casein-based diet (50 g/kg diet (protein restriction) or 200 g/kg diet (control)) for 3 weeks and measured mRNA contents of bone-matrix proteins such as osteocalcin, osteopontin and alpha 1 type I collagen, insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) in femur. Ovariectomy decreased the weight of fat-free dry bone and increased urinary excretion of pyridinium cross-links significantly, although dietary protein restriction did not affect them. Neither ovariectomy nor protein restriction affected the content of mRNA of osteopontin and osteocalcin; however, ovariectomy increased and protein restriction extensively decreased the alpha 1 type I collagen mRNA content in bone tissues. Ovariectomy increased IGF-I mRNA only in the rats fed on the control diet. Conversely, protein restriction increased and ovariectomy decreased the IGF-II mRNA content in femur. Furthermore, the contents of IGFBP-2, IGFBP-4 and IGFBP-5 mRNA increased, but the content of IGFBP-3 mRNA decreased in femur of the rats fed on the protein-restricted diet. In particular, ovariectomy decreased the IGFBP-2 mRNA content in the protein-restricted rats and the IGFBP-6 mRNA content in the rats fed on the control diet. These results clearly show that the mRNA for some of the proteins which have been shown to be involved in bone formation are regulated by both quantity of dietary proteins and ovarian hormones.

Animals↗

PubMed1985

Nonspecific immunostimulation against viruses.

A trypsinized preparation from chromogenic selected strain of Mycobacterium phlei (NSI) stimulated the recipient immune system non-specifically against a variety of viruses viz. Rabies virus (RNA virus). Marek's disease (DNA virus) and Foot and Mouth disease virus (RNA virus) in phylogenetically different hosts like mice, chicks and guinea pigs respectively. Investigation into mechanisms of such nonspecific immunostimulation revealed that there was induction of strong cell mediated immune response (CMIR) against both specific as well as to nonspecific viral antigens as evinced by LMIT, lymphokine (LyIF) assay and lymphocyte transformation test etc. There was induction of appreciable quantity of "Infectivity inhibiting substance(s)" (IIS) which was not the classical antibody (Ig) in the serum of NSI inoculated animals and birds. This substance(s) neutralized FMD, IBR, rabies and Newcastle disease virus in cell culture, mice and in embryonated eggs. Electrophoretic separation of the NSI induced serum revealed an increase in beta fraction of globulin.

Adjuvants, Immunologic↗

PubMed1999

The use of nocodazole in cell cycle analysis and parasite purification from Theileria parva-infected B cells.

Theileria parasites transform bovine leukocytes and induce uncontrolled lymphoproliferation only in the macroschizont stage of their life cycle. The isolation of highly purified stage-specific parasite RNA and proteins is an essential prerequisite when studying the Theileria-host relationship. We therefore improved a protocol based on the cytolytic bacterial toxin aerolysin by taking advantage of the microtubule inhibitor nocodazole. In this report we describe that nocodazole-mediated separation of the parasite from the host cell microtubule network was used with success to improve quantity and quality of purified parasites. We furthermore show that nocodazole is a useful tool to study cell cycle checkpoints due to its capacity to induce reversible cell cycle arrest in Theileria-infected B cells.

Animals↗

PubMed1992

Cytokine activity after allogeneic bone marrow transplantation. IV. Production of mRNA for IL-3 and GM-CSF by mitogen-stimulated circulating mononuclear cells.

The ability of circulating mononuclear cells from recipients of HLA identical sibling marrow transplants to generate messenger (m) RNA for the haemopoietic growth factors interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) upon mitogen stimulation was investigated. The amount of IL-3 mRNA per ml of blood and IL-3 mRNA per 10(7) mononuclear cells as well as the amount of GM-CSF mRNA per ml of blood was significantly lower in transplant recipients than in normal volunteers. Lower values for mRNA expression for both IL-3 and GM-CSF were associated with the use of immunosuppressive therapy post transplant. No correlation was found between the expression of message for either cytokine, or the presence or absence of acute graft-versus-host disease at the time of testing. While there was no correlation between the quantity of GM-CSF message produced and time elapsed post transplant, IL-3 message expression increased slightly with increasing time post transplant. These defects of haemopoietic regulators constitute another parameter of impaired cellular immunity occurring after allogeneic marrow transplantation.

Bone Marrow Transplantation↗

PubMed2003

Retrieval yield of total and messenger RNA in mesenchymal tissue ex vivo.

Mesenchymal neoplasms are a heterogeneous group of tumors comprising more than 200 benign entities and approximately 100 sarcomas. Large intraobserver and interobserver variability mandates improvements in diagnostic criteria. Gene expression microarrays are one tool in an evolving field of technology that permits the screening of tissue for massive amounts of information regarding its genetic composition. Such information may aid clinicians to diagnose and treat sarcomas. Complementary deoxyribonucleic acid microarrays, although very promising, are limited by the fact that messenger ribonucleic, the genetic messenger that permits deoxyribonucleic acid to encode for proteins and is the element retrieved from tumor samples ex vivo, is highly unstable, degrading quite readily. We found that even with optimal retrieval times and processing, total ribonucleic acid extraction from tumor tissue ex vivo is retrieved in adequate amounts to avoid amplification in 23% to 55% (mean 36%) of specimens. The percentage of high-grade tumors that yielded sufficient total ribonucleic acid was significantly higher than low grade and benign tumors. When adequate retrieval is achieved, the quantity and quality of messenger ribonucleic acid is robust. Surgeons, pathologists, and clinical intermediaries must be aware of issues surrounding messenger ribonucleic acid retrieval from surgical specimens to optimize collection.

Biopsy↗

PubMed1999

Mobilization of peripheral blood progenitor cells for human immunodeficiency virus-infected individuals.

Gene therapy is becoming one of the most promising modalities for the treatment of acquired immunodeficiency syndrome. The purpose of this study was to investigate the mobilization and collection of peripheral blood progenitor cells from human immunodeficiency virus (HIV)-infected individuals using granulocyte colony-stimulating factor (G-CSF). A total of 10 patients (9 male, 1 female; median age 36.5 years) with varying circulating CD4+ cell counts (13.9-1467/microL) were administered 10 microg/kg G-CSF daily for 6 days. Peripheral white blood cells (WBCs), CD34+ cell counts, lymphocyte subsets, and plasma viremia were monitored before each G-CSF injection. An average sixfold increase in WBCs was observed, which stabilized on day 4 or thereafter. The level of CD34+ cells was increased by 20-fold, and did not differ between days 5 and 6. Smaller increases in CD4+, CD8+, and CD4+CD8+ cells were observed. HIV viral load, as measured by RNA copy number in plasma, was not significantly altered by G-CSF administration. The leukapheresis product (LP), collected on day 7, contained an average of 6.25+/-4.52 (mean +/- standard deviation) x 10(10) WBCs and 3.08+/-2.98 x 10(6) CD34+ cells/kg. The levels of different CD34+ cell subsets were similar to those in the LPs of G-CSF-mobilized healthy individuals from an earlier study. Primitive hematopoietic cells (CD38- and CD38-HLA-DR+ cells) were detected in LPs (1.19+/-0.46% and 0.87+/-0.23%, respectively, of CD34+ cells). All parameters (WBC counts, lymphocyte populations, CD34+ cells, and HIV-1 RNA copies) measured 3 weeks after leukapheresis returned to baseline values. The administration of G-CSF was well tolerated by the HIV patients; side effects included bone pain, headache, flulike symptoms, and fatigue. There were no correlations between baseline CD4+ cell count and the WBCs, mononuclear cells, or CD34+ cells collected in the LP. Similarly, no correlation existed between baseline CD4+ and CD34+ cells, peak CD34+ cells, or days to achieve peak CD34+ cell counts after G-CSF mobilization. Our results showed that: (1) maximal mobilization can be achieved after 4 days of G-CSF administration; (2) therapeutic quantities of hematopoietic cells can be collected and used for gene therapy; and (3) G-CSF administration is well tolerated and does not cause a clinically significant increase in viremia.

Adult↗

PubMed2003

MolMovDB: analysis and visualization of conformational change and structural flexibility.

The Database of Macromolecular Movements (http://MolMovDB.org) is a collection of data and software pertaining to flexibility in protein and RNA structures. The database is organized into two parts. Firstly, a collection of 'morphs' of solved structures representing different states of a molecule provides quantitative data for flexibility and a number of graphical representations. Secondly, a classification of known motions according to type of conformational change (e.g. 'hinged domain' or 'allosteric') incorporates textual annotation and information from the literature relating to the motion, linking together many of the morphs. A variety of subsets of the morphs are being developed for use in statistical analyses. In particular, for each subset it is possible to derive distributions of various motional quantities (e.g. maximum rotation) that can be used to place a specific motion in context as being typical or atypical for a given population. Over the past year, the database has been greatly expanded and enhanced to incorporate new structures and to improve the quality of data. The 'morph server', which enables users of the database to add new morphs either from their own research or the PDB, has also been enhanced to handle nucleic acid structures and multi-chain complexes.

Computer Graphics↗

PubMed1996

Human eosinophils express messenger RNA encoding RANTES and store and release biologically active RANTES protein.

Eosinophils synthesize and store various cytokines with potential autocrine activity. We hypothesized that eosinophils synthesize and store RANTES, a CC-chemokine with potent eosinophil chemotactic activity. Expression of RANTES mRNA in highly purified eosinophil populations was detected by reverse transcription followed by polymerase chain reaction analysis. In situ hybridization (ISH) with 35S-labeled RANTES-specific riboprobes showed that 6.8-10% of peripheral blood eosinophils obtained from atopic subjects expressed RANTES mRNA, increasing to 25% after incubation (16 h) with interferon (IFN)-gamma, but not ionomycin in vitro. Peripheral blood eosinophils also showed specific immunoreactivity with an anti-RANTES monoclonal antibody, consistent with translation of the mRNA. By enzyme-linked immunosorbent assay, blood eosinophils were shown to contain a median of 7300 pg (range 5200-8800) RANTES per 10(6) cells, of which a mean of 24% was released into culture supernatants after stimulation of the cells with serum-coated particles in vitro. These culture supernatants exhibited eosinophil chemotactic activity which was inhibited (mean 68%) by a specific anti-RANTES antibody. Sequential immunocytochemistry and ISH on biopsies obtained from allergen-induced late-phase cutaneous reactions showed that 55-75% of the infiltrating RANTES mRNA+ cells were EG2+ eosinophils. Allergen, but not diluent challenge, was also associated with a time-dependent increase in the number of cells showing RANTES immunoreactivity. Of these cells, 55% were identified as eosinophils by morphological criteria. Thus, human eosinophils have the capacity to synthesize, store and secrete physiologically relevant quantities of RANTES, and may therefore be an important source of this chemokine in allergic inflammation.

Allergens↗

PubMed1987

Inducible and tissue-specific expression of human C-reactive protein in transgenic mice.

C-reactive protein (CRP) is a major acute phase reactant in man but not in mouse. It is synthesized in abundant quantities by human hepatocytes during the course of several diseases, mainly acute inflammations. To investigate the regulation of CRP expression, the human CRP gene was introduced into fertilized eggs by microinjection and transgenic mouse lines were derived. The CRP gene is exclusively transcribed in the liver and expression is strictly dependent on experimental inflammation. The kinetics of induction both for RNA and protein synthesis is very fast; RNA is first detectable after 2 h in the liver, the protein after 6 h in the serum. Human CRP levels in the sera of transgenic mice are comparable to those observed in human diseases. Nuclear run-on experiments indicate that regulation is primarily at the transcriptional level.

Animals↗