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At least 1,603 records · Page 89Linked to original sources
PubMed1998

The expression of PDGF-B chain mRNA in lung tissue from rats repeatedly infected with mycoplasma pneumoniae.

In order to investigate the role played by platelet derived growth factor-BB (PDGF-BB) in the pathogenesis of pulmonary interstitial fibrosis in rats repeatedly infected with mycoplasma pneumoniae (MP), a rat MP infection model was developed by infecting rats with MP for 9 times during a period of 24 weeks with a technique of ultrasonic nebulizing inhalation. Then in situ hybridization was performed with PDGF-B chain cDNA probe and the results were quantitatively analyzed to measure the changes in PDGF-B chain mRNA expression in the lung tissue. The results showed that: (1) MP polymerase chain reaction (PCR) tests showed positive results in the bronchoalveolar lavage fluid (BALF) from all of the MP-infected rats (n = 4) while they were all negative in BALF from the control animals (n = 4, P < 0.05) and in BALF from those rats both infected with MP and, at the same time, treated with erythromycin (n = 4, P < 0.05). Bacterial cultures of the bronchial and lung tissue were negative in all three groups. The observation under a transmission electron microscope indicated that the interalveolar septa were widened with increased amount of collagen in the MP-infected rats while there were no obvious abnormalities in the other two groups. (2) Strong positive expression of PDGF-B chain mRNA was found in the plasma of monocytes and macrophages located in the locally widened interalveolar septa and alveolar spaces in the lung tissue from the MP-infected animals with the integral optical densities being 37.42 +/- 9.05 (n = 4) which was significantly higher than the values of control group (0.42 +/- 0.08, n = 4, P < 0.01) and of the group with MP-infection plus erythromycin treatment (1.62 +/- 0.40, n = 4, P < 0.01). These results suggest that PDGF-BB may be involved in the process of the development of pulmonary interstitial fibrosis caused by the repeated MP-infection. It may be an important growth factor for mediating the roles of monocytes and macrophages to promote the aggregation and proliferation of fibroblasts which can then secrete collagen in large quantity in the pulmonary interstitium.

Animals↗
PubMed1993

In vivo overexpression and purification of Escherichia coli tRNA(ser).

DNA fragments corresponding to the sequences of Escherichia coli tRNA(2ser) and amber suppressor tRNA(ser), were synthesized from overlapping oligonucleotides. These were interposed between a strong promotor and a synthetic transcriptional terminator to ensure the production of a transcript of the correct size. The genes of promotor, fragment and terminator were cloned into a conditional runaway replication plasmid. At temperatures below 37 degrees C this vector has a low copy number but, following a temperature shift to 42 degrees C, the copy number is no longer regulated. Using these constructs an overexpression of tRNA(ser) of about 20 times the level of the wild-type pool could be obtained (corresponding e.g. to 200 times the expression tRNA(2ser)). From these systems 10 mg quantities of tRNA(ser)s could be isolated with a serine acceptance of 1,100 pmol/A280 unit.

Base Sequence↗
PubMed1982

Intraneuronal alpha-fetoprotein and albumin are not synthesized locally in developing brain.

Alpha-Fetoprotein and albumin are found intraneuronally in developing mammalian brains. To investigate whether this results from local biosynthesis, the existence of the corresponding brain messenger RNAs and the ability of brain cultures to synthesize these proteins were studied in the mouse. The findings show that neither the proteins nor the mRNAs coding for them are made in sufficient quantities to account for the material detected immunologically, supporting an extracellular origin.

Animals↗
PubMed2005

Overexpression and purification of tagged Escherichia coli proteins using a chromosomal knock-in strategy.

The purification of recombinant proteins from Escherichia coli (E. coli) has become a standard procedure both for research purposes and in biotechnology. One common way by which this is accomplished is by subcloning the gene of interest into a suitable expression vector and purifying the overexpressed protein using an affinity tag. In some cases, however, subcloning into plasmid vectors can be problematic. An alternative method could be to overexpress the gene of interest from the chromosome. Here, I describe a strategy to juxtapose strong transcriptional and translational sequences in front of any E. coli gene by recombination, which allows the gene product to be expressed in large quantities in the cell, and purified as a tagged protein. An application of this method to create a recombinant strain overexpressing the HrpA protein is described.

Chromosomes, Bacterial↗
PubMed1990

Steroid hormone receptors in human adipose tissues.

Administration of glucocorticoid, estrogen, and progesterone is followed by changes in human adipose tissue distribution, morphology, and function. Therefore, specific receptors for these hormones were determined in different regions of human adipose tissue using ligand techniques, with separation of bound and free hormone by chromatography, absorption techniques, or isoelectric focusing, as well as protein quantitation with monoclonal antibodies against human estrogen and progesterone receptors. Furthermore, mRNAs were measured by solubilization hybridization technique with glucocorticoid, estrogen, and progesterone receptor cRNA probes for human receptors. Saturable specific cytosolic glucocorticoid binding was found. Quantitative analyses indicated more binding sites and mRNAs in intraabdominal than sc adipose tissue samples. In contrast, neither specific estrogen or progesterone binding, cytosolic or nuclear receptor protein, nor mRNAs for these receptors could be identified in abdominal, femoral, or omental adipose tissues. Parallel control experiments confirmed the presence of both estrogen and progesterone receptors in rat adipose tissues. It was concluded that while glucocorticoid receptors are clearly present in human adipose tissues, female sex hormone receptors are not present in quantities detectable with presently available methods. Effects of these hormones on human adipose tissue might, therefore, be mediated via a minute nondetectable quantity of receptors, the glucocorticoid receptor, or indirect mechanisms.

Adipose Tissue↗
PubMed1987

DNA probes for detection and identification of mycoplasmas (Mollicutes).

DNA probes are expected to prove a specific, sensitive, rapid and inexpensive means for diagnosis of mycoplasma infections, replacing procedures that depend on cultivation of the fastidious organisms. Probes made up of conserved genes, such as rRNA genes, do offer the advantage of identifying and distinguishing multiple species with a single labeled reagent. The mycoplasmal rRNA gene probe pMC5 was effective in detection and identification of mycoplasmas infecting cell cultures. However, use of pMC5 for detection of spiroplasmas and mycoplasma-like organisms (MLOs) in infected plants was hindered by hybridization of this probe with chloroplast rRNA genes. Moreover, for identifying species and strains by pMC5, a complex hybridization procedure--involving DNA purification, digestion, electrophoresis, and Southern blot hybridization--is required. More specific DNA probes, on the other hand, can identify specific Mollicutes by the much simpler, faster and more sensitive dot blot technique. Thus, a probe made of a cloned Spiroplasma citri plasmid could detect by this technique as little as 10 pg of S. citri DNA (equivalent to about 10(3) organisms) in infected plants and insects. DNA probes specific for Mycoplasma pneumoniae and M. genitalium were selected from genomic libraries and prepared in pUC13 by screening the libraries for inserts hybridizing only with DNA of the specific mycoplasma. The probes, labeled by nick translation with 32P-nucleotides, could detect as little as approximately 100 pg of the specific mycoplasmal DNA by dot blot hybridization. To eliminate radioactivity, the above DNA probes were labeled by biotinylation of sulfonation systems. Dot blot hybridization with these probes showed decreased sensitivity of detection by about one order of magnitude, and some nonspecific background reaction with large quantities of nonhomologous DNAs.

Animals↗
PubMed1990

Synthesis and processing of the nonstructural polyproteins of several temperature-sensitive mutants of Sindbis virus.

We have examined the synthesis and processing of nonstructural polyproteins by several temperature-sensitive mutants of Sindbis virus, representing the four known RNA-minus complementation groups. Four mutants that possess mutations in the C-terminal domain of nonstructural protein nsP2 all demonstrated aberrant processing patterns when cells infected with these mutants were shifted from a permissive (30 degrees) to a nonpermissive (40 degrees) temperature. Mutants ts17, ts18, and ts24 showed severe defects in processing of nonstructural polyproteins at 40 degrees, whereas ts7 showed only a minor defect. In each case, cleavage of the bond between nsP2 and nsP3 was greatly reduced whereas cleavage between nsP1 and nsP2 occurred almost normally, giving rise to a set of polyprotein precursors not seen in wild-type-infected cells at this stage of infection. The nsP1 produced by these mutants was unstable and only small amounts could be detected in infected cells at the nonpermissive temperature. Submolar quantities of nsP2 were also present. We suggest that nsP1 and nsP2 may function as a complex and that free nsP1, and possibly nsP2, is degraded. Cleavage between nsP3 and nsP4 appeared to be normal in the mutants except in the case of ts17, where upon shift to 40 degrees P34 was unstable and nsP4 accumulated. We propose that the change in the P34/nsP4 ratio upon shift is responsible for the previously observed temperature sensitivity of subgenomic 26 S RNA synthesis in ts17 and for the failure of the mutant to regulate minus strand synthesis at 40 degrees. Other mutations tested, including ts21, which is found in the N-terminal half of nsP2, ts11, which has a mutation in nsP1, and ts6, which has a mutation in nsP4, all demonstrated nonstructural polyprotein processing indistinguishable from that in wild-type-infected cells. These results support our conclusion, based upon deletion mapping studies, that the C-terminal domain of nsP2 contains the nonstructural proteinase activity.

Animals↗
PubMed2004

Cloning and characterisation of a channel catfish (Ictalurus punctatus) Mx gene.

A 2.5 kb full-length cDNA clone of a channel catfish (Ictalurus punctatus) Mx gene was obtained using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) from RNA extracted from the liver of poly I:C stimulated channel catfish. The gene consists of an open reading frame of 1905 nucleotides encoding a 635 amino acid protein. The predicted protein is 72.5 kDa and contains the dynamin family signature, a tripartite GTP binding motif and a leucine zipper, characteristic of all known Mx proteins. The catfish Mx protein exhibited 79% identity with perch Mx and between 71% and 74% identity with the three Atlantic salmon and the three rainbow trout Mx proteins. Mx mRNA was constitutively expressed in channel catfish ovary (CCO) cells, but in higher quantities in response to poly I:C treatment. Mx was induced in channel catfish following injection with channel catfish virus (CCV) and poly I:C.

Amino Acid Motifs↗
PubMed2001

Detection and quantification of depurinated benzo[a]pyrene-adducted DNA bases in the urine of cigarette smokers and women exposed to household coal smoke.

Polycyclic aromatic hydrocarbons (PAH) are metabolized to electrophiles that can bind to DNA bases and destabilize the N-glycosyl bond, causing rapid depurination of the adducted bases. Recent studies support depurination of DNA as a mechanism central to the genesis of H-ras mutations in PAH-treated mouse skin. Depurinating adducts account for 71% of all DNA adducts formed in mouse skin treated with benzo[a]pyrene (BP). This study analyzed urine of cigarette smokers, coal smoke-exposed women, and nonexposed controls for the presence and quantities of the depurinated BP-adducted DNA bases, 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua) and 7-(benzo[a]pyren-6-yl)adenine (BP-6-N7Ade). Since these adducted bases originate from reaction of the BP radical cation with double-stranded DNA and not with RNA or denatured DNA, their presence in urine is indicative of DNA damage. Urine samples were fractionated by a combination of SepPak extraction and reverse-phase HPLC, and then analyzed by tandem mass spectrometry and capillary electrophoresis with laser-induced fluorescence. BP-adducted bases were detected in the urine from three of seven cigarette smokers and three of seven women exposed to coal smoke, but were not detected in urine from the 13 control subjects. Concentrations were estimated to be 60-340 and 0.1-0.6 fmol/mg of creatinine equivalent of urine for coal smoke-exposed women (maximum possible BP intake of ca. 23 000 ng/day) and cigarette smokers (BP intake of ca. 800 ng/day), respectively, exhibiting a sensitive response to BP exposures. BP-6-N7Gua was present at ca. 20-300 times the concentration of BP-6-N7Ade in the urine of coal smoke-exposed women, but was not detected in the urine of cigarette smokers. This difference may be due to the remarkably different BP exposures experienced by the two groups of PAH-exposed individuals. These results justify more extensive studies of depurinated BP-adducted DNA bases as potential biomarkers of PAH-associated cancer risk.

Adult↗
PubMed1966

Isolation and analysis of the protoplast membrane of Bacillus megaterium.

1. Optimum conditions were found for the lysis of Bacillus megaterium KM by lysozyme. The age of culture, density of suspension and concentration of lysozyme affected the rate of lysis. 2. Protoplast membranes were isolated by centrifugation of lysates and were exhaustively washed. 3. Treatment with chloroform removed some lipid from the membranes, but about half of the total membrane lipid could be extracted only after partial acid hydrolysis. 4. The defatted membranes consisted of protein together with variable amounts of RNA; carbohydrate was almost absent. 5. Lipid accounted for 23% of the weight of the membrane, and included both neutral lipid and phospholipid. In both classes, branched-chain C(15) acids made up about 80% of the total fatty acid. 6. The phospholipid was a kephalin, and contained small quantities of several amino acids.

Amino Acids↗
PubMed2001

Increased production of vascular endothelial growth factor in peritoneal macrophages of cirrhotic patients with spontaneous bacterial peritonitis.

Spontaneous bacterial peritonitis (SBP) is a common complication of cirrhotic patients with ascites that usually results in renal failure and death despite the efficacy of the current antibiotic therapy. The pathogenesis of these phenomena is poorly known but it has been related to the production of vasoactive cell mediators locally acting on the splanchnic vasculature. Because previous studies showed that peritoneal macrophages of cirrhotic patients may produce high quantities of vascular endothelial growth factor (VEGF), a powerful vessel permeabilizing agent, when stimulated by cytokines and bacterial lipopolysaccharide, the present study was aimed to seek whether peritoneal macrophages of SBP patients are induced to produce increased amounts of VEGF. Our results indicate that the production rate and the messenger RNA (mRNA) and protein expression of this substance are increased in macrophages of patients with SBP in comparison with those of noninfected cirrhotic patients. This characteristic feature is absent in circulating monocytes of these patients. Moreover, enhanced endothelial cell proliferation induced by conditioned medium of macrophages isolated from the ascites of patients with SBP is abolished by anti-VEGF antibody, and peritoneal tissue of cirrhotic patients expresses both VEGF receptors, Flt-1 and KDR. These results, therefore, are consistent with the concept that locally released macrophage-derived VEGF may result in increased vascular permeability and plasma leakage in the peritoneal vessels of cirrhotic patients with SBP.

Bacterial Infections↗
PubMed1992

Virus-specific proteins in cells infected with tomato black ring nepovirus: evidence for proteolytic processing in vivo.

The synthesis of proteins encoded by the RNA of tomato black ring virus (TBRV) in vivo was studied in protoplasts by direct labelling with [35S]methionine, and in protoplasts and plants by immunoblotting experiments with specific antisera. Comparison of the proteins synthesized in infected and mock-inoculated protoplasts suggested that proteins of M(r) 120K, 90K, 80K, 57K and 46K were virus-specific. The proteins derived from the RNA-1-encoded polyprotein detected by immunoblotting were a stable 120K protein and, only in protoplasts, small amounts of a 90K protein which contains the C-terminal part of the 120K protein and the polymerase domain. The results suggest that the polymerase and the adjacent protease function in vivo largely or solely when combined in a 120K protein. The proteins derived from the RNA-2-encoded polyprotein detected by immunoblotting were 59K and 57K proteins, which reacted with antiserum to TBRV particles, and a 46K protein. In extracts of infected Nicotiana clevelandii and Chenopodium quinoa made soon after inoculation, the 59K protein was more abundant than the 57K protein; later samples contained similar quantities of each protein. The 57K protein comigrated with protein extracted from virus particles. The results of amino acid sequencing suggested that the 57K protein is derived from the 59K protein by the loss of nine C-terminal amino acids. Antiserum to a peptide adjacent to the 57K protein in the 150K polyprotein detected a 46K protein in protoplasts and plant tissue. The results support the processing scheme for TBRV polyproteins proposed after analysis of the products of in vitro translation.

Amino Acid Sequence↗
PubMed1977

[Polysomal apparatus as a reflection of function interactions between translation and transcription processes during protein synthesis inhibited by cycloheximide].

Distribution of optical density and radioactivity in sucrose density gradient was studied for 14C-RNA and 3H-protein polysomal components isolated from normal rat liver 1, 3, 6 12 and 24 hrs after cycloheximide (CHI) treatment (sublethal dose--0,3 mg/100 g body weight). Soon after the antibiotic injection (1--3 hrs), when protein biosynthesis was strongly inhibited (more than 90%), the total quantity of "heavy" polyribosomes and 14C-mRNA associated with this fraction significantly increased. At the same time the amount of free ribosomes showed a decrease. On the contrary, after 6 hrs the amount of polysomes sharply decreased and that of ribosomes and their subunits was increased. In the middle period and at the end of experiment (12--24 hrs) when protein biosynthesis was continuously restored, the polysomes sedimentation pattern was restored as well. Specific radioactivity of 14-C-rRNA's and 3H-proteins in this fraction was also increased. The restoration of optimal protein biosynthesis level after its inhibition by a single cycloheximide injection (sublethal doses) is thought to be a complex process of consecutive interactions between translation and transcription cell systems.

Animals↗
PubMed1997

Osteoarthritic lesions: involvement of three different collagenases.

OBJECTIVE: To assess the presence of fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8), and collagenase 3 (MMP-13) in osteoarthritic (OA) cartilage, with particular emphasis on areas of macroscopic cartilage erosion. METHODS: Messenger RNA (mRNA) levels were assessed by reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and Northern blot analysis. RESULTS: MMP-1 and MMP-13 were expressed at higher levels by OA chondrocytes than by normal chondrocytes. In addition, mRNA for MMP-8 was present in OA cartilage but not normal cartilage by PCR and Northern blot analyses. Chondrocytes from areas surrounding the OA lesion expressed greater quantities of MMP-1 and MMP-13 compared with normal chondrocytes, suggesting local modulation by mechanical and inflammatory factors. Tumor necrosis factor alpha stimulated the expression of all 3 collagenases. Retinoic acid, an agent which induces autodigestion of cartilage in vitro, stimulated only the expression of MMP-13. CONCLUSION: These findings suggest a key role of MMP-13 and MMP-8, as well as MMP-1 in osteoarthritis.

Aged↗
PubMed2000

Prostanoids in human colonic mucosa: effects of inflammation on PGE(2) receptor expression.

Although the tissue concentration of PGE(2) is heightened 3-fold or more during mucosal inflammation, the cellular targets of prostanoids in human mucosa and the resulting changes in cell physiology have not been fully explored. We used a panel of immunoglobulin and mRNA probes in order to localize and quantitate the four member EP family of prostanoid receptors for binding PGE(2) to cells of histologically normal and inflamed human colonic mucosa, and then examined prostanoid-induced changes in mucosal lymphocyte function. Prostanoid receptors were selectively expressed on a limited number of human colonic mucosal cells; EP(4) alone was expressed on lamina propria mononuclear cells. Dual immunostaining in situ identified the CD3(+) T lymphocyte as a major EP(4) receptor-bearing cell in normal mucosa. Flow cytometry of isolated cells showed that 19.2% of lamina propria mononuclear cells were EP(4)(+), and almost 30% of these were CD3(+). In situ hybridization with digoxygenin-labeled RNA probes largely confirmed this localization. During inflammation, mucosal T lymphocytes showed a significant enhancement in EP(4) immunoreactive receptor protein. Computer-assisted densitometry of single cells demonstrated an increase in fluorescence intensity from 4.8 +/- 1.8 to 8.6 +/- 1.8 (p < 0.04). The effects of PGE(2) included a 35% reduction in T lymphocyte IL-2 secretion. COX 1(+) lamina propria cells nearly doubled in number during inflammation; expressed a T lymphocyte marker; but retained an unchanged quantity of immunoreactive COX 1 protein per cell. The number of newly appeared COX 2(+) lymphocytes remained <50% that of COX 1(+) cells. A major perturbation in the number and distribution of PGE(2) receptors and enzymes for prostanoid synthesis occurs in chronic inflammation of the colon, with consequences for mucosal T lymphocyte function.

Amino Acid Sequence↗
PubMed1995

A reverse transcription-polymerase chain reaction technique to detect feline cytokine genes.

The ability to detect feline cytokine expression would allow further characterization of the feline immune system. Bioassays are currently available for the measurement of feline IL2, IL6 and TNF alpha but not for other biologically important cytokines. To detect the expression of other cytokines, a reverse transcription-polymerase chain reaction (RT-PCR) procedure was developed. Since feline cytokine gene sequences other than TNF alpha were not available, mammalian DNA and mRNA sequences for IL2, IFN gamma, IL4, IL6, IL10, IL12 and beta-actin, obtained from the Genbank database were compared and oligonucleotide primers chosen from consensus sequences. To validate the cytokine and beta-actin primers, peripheral blood mononuclear cells from specific pathogen free (SPF) cats were cultured in the presence of Con A for various periods of time (0-72 h). RNA was collected, reverse transcribed into cDNA, and the cDNA was amplified by PCR with each set of cytokine primer pairs. RT-PCR products were hybridized with specific 32P end-labeled internal oligonucleotide probes and then analyzed with the AMBIS imaging system to determine the kinetics of cytokine mRNA production. The beta-actin signal was used to control for sample to sample variation in the quantity of mRNA and variation in the RT and PCR reactions. Peak mRNA expression for most cytokines was found to occur between 2 to 4 h of Con A stimulation. mRNA expression was correlated with cytokine bioactivity for IL2 and IL6. Peak IL2 bioactivity occurred after 8 h of Con A stimulation, 4 h after the mRNA expression had peaked. Although IL6 mRNA expression peaked between 2 and 4 h of stimulation, bioactivity was not detected until 8 h of stimulation and continued to increase over the next 24-48 h.

Animals↗
PubMed1987

Human retinoblastoma susceptibility gene: cloning, identification, and sequence.

Recent evidence indicates the existence of a genetic locus in chromosome region 13q14 that confers susceptibility to retinoblastoma, a cancer of the eye in children. A gene encoding a messenger RNA (mRNA) of 4.6 kilobases (kb), located in the proximity of esterase D, was identified as the retinoblastoma susceptibility (RB) gene on the basis of chromosomal location, homozygous deletion, and tumor-specific alterations in expression. Transcription of this gene was abnormal in six of six retinoblastomas examined: in two tumors, RB mRNA was not detectable, while four others expressed variable quantities of RB mRNA with decreased molecular size of about 4.0 kb. In contrast, full-length RB mRNA was present in human fetal retina and placenta, and in other tumors such as neuroblastoma and medulloblastoma. DNA from retinoblastoma cells had a homozygous gene deletion in one case and hemizygous deletion in another case, while the remainder were not grossly different from normal human control DNA. The gene contains at least 12 exons distributed in a region of over 100 kb. Sequence analysis of complementary DNA clones yielded a single long open reading frame that could encode a hypothetical protein of 816 amino acids. A computer-assisted search of a protein sequence database revealed no closely related proteins. Features of the predicted amino acid sequence include potential metal-binding domains similar to those found in nucleic acid-binding proteins. These results provide a framework for further study of recessive genetic mechanisms in human cancers.

Amino Acid Sequence↗