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PubMed2006

Heat shock response and mammal adaptation to high elevation (hypoxia).

The mammal's high elevation (hypoxia) adaptation was studied by using the immunological and the molecular biological methods to understand the significance of Hsp (hypoxia) adaptation in the organic high elevation, through the mammal heat shock response. (1) From high elevation to low elevation (natural hypoxia): Western blot and conventional RT-PCR and real-time fluorescence quota PCR were adopted. Expression difference of heat shock protein of 70 (Hsp70) and natural expression of brain tissue of Hsp70 gene was determined in the cardiac muscle tissue among the different elevation mammals (yak). (2) From low elevation to high elevation (hypoxia induction): The mammals (domestic rabbits) from the low elevation were sent directly to the areas with different high elevations like 2300, 3300 and 5000 m above sea level to be raised for a period of 3 weeks before being slaughtered and the genetic inductive expression of the brain tissue of Hsp70 was determined with RT-PCR. The result indicated that all of the mammals at different elevations possessed their heat shock response gene. Hsp70 of the high elevation mammal rose abruptly under stress and might be induced to come into being by high elevation (hypoxia). The speedy synthesis of Hsp70 in the process of heat shock response is suitable to maintain the cells' normal physiological functions under stress. The Hsp70 has its threshold value. The altitude of 5000 m above sea level is the best condition for the heat shock response, and it starts to reduce when the altitude is over 6000 m above sea level. The Hsp70 production quantity and the cell hypoxia bearing capacity have their direct ratio.

Acclimatization↗
PubMed1992

A method for the direct measurement of mRNA in discrete regions of mammalian brain.

A rapid and nearly quantitative method for the direct analysis of steady-state mRNA levels in microgram quantities of frozen mammalian brain is described. Briefly, tissue punches 0.5-1.0 mm in diameter were sampled from 250-microns-thick cryostat sections of rat brain (approximately 50-200 micrograms tissue). The samples were homogenized in 50 microliters of a denaturing gel loading buffer and applied directly to a 2.2 M formaldehyde-agarose gel for electrophoresis and subsequent RNA blot analysis. The method is extremely rapid, results in excellent recovery of intact RNA, and allows the direct assay of mRNA levels in discrete subregions of the mammalian brain.

Animals↗
PubMed1988

Expression of an enzymatically active parasite molecule in Escherichia coli: Schistosoma japonicum glutathione S-transferase.

The NH2-terminal amino acid sequence of the Mr 26 000 glutathione S-transferase (EC 2.5.1.18) of Schistosoma japonicum (Sj26) has been deduced by RNA and protein sequence analysis. Using this information, a bacterial plasmid has been constructed that directs the synthesis of the entire Sj26 molecule in Escherichia coli. Recombinant Sj26 exhibits glutathione S-transferase activity and can be readily purified from bacteria in a one-step procedure under non-denaturing conditions. The availability of recombinant Sj26 in essentially unlimited quantities will aid its assessment as a candidate vaccine molecule in schistosomiasis and could eventually lead to the rational design of a drug targetted on schistosome glutathione S-transferases.

Amino Acid Sequence↗
PubMed2006

Stochastic transcription initiation: Time dependent transcription rates.

The noise in the central process such as transcription, replication and translation of the genomic DNA is very important since it can directly affect the phenotypic and behavioral aspects of an organism as well as the entire cellular function. Here we develop a model on the transcription process based on the assumption that the initiation of the transcription is a stochastic event and the transcription rates may be time dependent random quantities. We derive the central measure properties i.e. mean and the variance, of the distribution of the transcription rates. Our results show that the Fano factor which is a measure of deviation from the Poisson distribution associated with the fluctuations in the number of mRNA molecules deviates from unity due to the randomness in the transcription rates. However when the RNA polymerase molecule searches for the promoter sequences on the DNA lattice by random jumps, the Fano factor approaches the Poisson limit as the jump size associated with the RNA polymerase increases. Since the jump size associated with dynamics of RNAP molecule is positively correlated with the degree of super coiling of DNA, we argue that the super coiled or close-packed structure of DNA might have evolved to keep the noises at the transcriptional level in a minimum.

DNA-Directed RNA Polymerases↗
PubMed2005

Molecular cloning and characterization of murine caspase-12 gene promoter.

The activation of caspase-12 is involved in endoplasmic reticulum-mediated apoptosis. To investigate how caspase-12 is transcriptionally and translationally regulated, we isolated and sequenced the 5'-flanking region of mouse caspase-12 gene by a PCR-mediated chromosome-walking technique, using mouse genomic DNA as a template. Two DNA fragments of 3,221 and 800 bp were isolated and cloned into pGL3 promoterless vector upstream of the luciferase gene. The small DNA fragment contains the first intron sequence located downstream of the first exon and 27 bp from the second exon, whereas the large fragment contains the small fragment and the 5'-flanking region. Reporter constructs generated from these DNA fragments showed a substantial promoter activity in mouse NIH 3T3 or human embryonic kidney 293 cells grown in the presence of 10% serum. In the absence of serum, the luciferase activity was drastically reduced. However, the luciferase mRNA was higher in serum-starved cells than in control cells, suggesting that translation of luciferase mRNA was drastically inhibited. However, Western blot analysis revealed that the quantity of procaspase-12 is actually higher in serum-starved cells relative to that cultured in the presence of 10% serum. Progressive deletion analysis of the 3,221-bp sequence revealed that the highest luciferase activity was observed with the construct containing 700 bp upstream of ATG. The transcriptional initiation site was identified by 5' RACE techniques using total RNA from NIH 3T3 cells. Our results should facilitate studies on the mechanism regulating the expression of this important gene.

Animals↗
PubMed1993

Transcriptional analysis of the Aeromonas salmonicida S-layer protein gene vapA.

The vapA gene of Aeromonas salmonicida encodes the subunit of the surface protein array known as A-layer. Nucleotide sequence analysis of the 374 bp of DNA immediately upstream of vapA revealed two potential promoter sequences and other possible regulatory sequences. Sequencing and polymerase chain reaction analysis showed that the region was conserved in wild-type A. salmonicida. Primer extension and Northern (RNA) blot analysis showed that vapA transcription in A. salmonicida was directed predominantly by a distal promoter, P1, resulting in a 1.7-kb unit-length mRNA with an untranslated 181-nucleotide leader sequence which contained two predicted low-free-energy stem-loop structures. Northern analysis of cells grown at 15 degrees C showed that vapA transcript production peaked during the mid-log phase of growth (A600 = 0.25). At 15 degrees C, the half-life of the vapA mRNA was 22 min, while at 20 degrees C, the half-life was significantly shorter, 11 min. The amount of vapA transcript produced was reduced by growth in the presence of the DNA gyrase inhibitors nalidixic acid and novobiocin. Environmental factors such as growth temperature and atmospheric oxygen tension also affected the quantity of vapA mRNA. vapA transcript could not be detected in mutants which produced either low levels of full-length or truncated A protein or no detectable A protein.

Aeromonas↗
PubMed2001

Self-assembly of nucleocapsid-like particles from recombinant hepatitis C virus core protein.

Little is known about the assembly pathway and structure of hepatitis C virus (HCV) since insufficient quantities of purified virus are available for detailed biophysical and structural studies. Here, we show that bacterially expressed HCV core proteins can efficiently self-assemble in vitro into nucleocapsid-like particles. These particles have a regular, spherical morphology with a modal distribution of diameters of approximately 60 nm. Self-assembly of nucleocapsid-like particles requires structured RNA molecules. The 124 N-terminal residues of the core protein are sufficient for self-assembly into nucleocapsid-like particles. Inclusion of the carboxy-terminal domain of the core protein modifies the core assembly pathway such that the resultant particles have an irregular outline. However, these particles are similar in size and shape to those assembled from the 124 N-terminal residues of the core protein. These results provide novel opportunities to delineate protein-protein and protein-RNA interactions critical for HCV assembly, to study the molecular details of HCV assembly, and for performing high-throughput screening of assembly inhibitors.

Amino Acid Sequence↗
PubMed1992

Polymerase chain reaction analysis of renin in rat aortic smooth muscle.

Controversy exists whether vascular smooth muscle cells in vivo synthesize renin, thereby providing a critical component of the hypothesized vascular renin-angiotensin system. To examine this question, we enzymatically isolated and pooled the medial layer of thoracic aortas from Sprague-Dawley rats that were either untreated or enalapril treated for 3 days, isolated messenger RNA (mRNA), and performed Northern blot analysis with rat complementary DNA (cDNA) probes for renin, cathepsin D, and cathepsin E. Renin mRNA was detected in kidney but was not detected in aortic smooth muscle from the untreated or enalapril-treated groups. Cathepsin E mRNA was detected in enalapril-treated aorta and kidney, and cathepsin D mRNA was detected in all tissues examined. cDNA was synthesized and subjected to polymerase chain reaction analysis by using primers corresponding in sequence to regions conserved throughout the aspartic proteinases. Cathepsins D and E were amplified from kidney and aortic cDNA. Renin was less consistently amplified from the aortic cDNA and was much less abundant than cathepsin E or cathepsin D. These results suggest that 1) renin mRNA is present in aortic smooth muscle cells in vivo in quantities detectable only after multiple rounds of polymerase chain reaction amplification, 2) renin mRNA is not upregulated in aortic smooth muscle after converting enzyme inhibition, and 3) cathepsins D and E are the predominant aspartic proteinases in aortic smooth muscle.

Animals↗
PubMed1990

Changes in angiotensinogen messenger RNA in differentiating 3T3-F442A adipocytes.

Angiotensinogen messenger RNA (mRNA) has been identified in both brown and white adipose tissue. Recently we have shown that when 3T3-L1 cells were treated with isobutylmethylxanthine (IBMX) to accelerate differentiation, angiotensinogen mRNA increased markedly in adipocytes as compared with preadipocytes. To determine if a correlation existed between the regulatory events associated with the differentiation process, we compared the change in angiotensinogen mRNA in spontaneously differentiating 3T3-F442A cells with two established parameters of differentiation in adipocyte cell lines. Differentiation was assessed by visual examination of cells for lipid droplets, fluorescent staining of the F-actin fibers, and increases in glycerol phosphate dehydrogenase mRNA. F-actin fibers were highly structured in preadipocytes, becoming disassembled and very disorganized as cells differentiated into adipocytes. The quantity of angiotensinogen mRNA increased as the number of lipid-containing cells increased within a culture. Glycerol phosphate dehydrogenase mRNA accumulated in differentiated adipocytes to about the same extent as angiotensinogen mRNA. Thus, increases in angiotensinogen mRNA were associated with the morphological and biochemical changes that occur during the phenotypic modulation of 3T3-F442A cells.

Actins↗
PubMed1994

Expression of mRNA for the gap-junctional protein connexin43 in human colonic tissue is variable in response to beta-carotene supplementation.

Increased expression of the gap-junctional protein connexin43 (Cx43) is reported to be increased in mouse and human dermal fibroblasts in vitro in response to beta-carotene treatment. In the present study, we determined the level of Cx43 mRNA expression in colonic mucosa from normal subjects and subjects with a prior history of colonic polyps or cancer before and after three months of administration of a placebo or beta-carotene. RNA was reverse transcribed and used in a polymerase chain reaction assay employing primers selected from the human Cx43 gene sequence. Cx43 mRNA expression was normalized on the basis of beta 2-microglobulin mRNA expression. The beta-carotene concentration in colonic mucosa, as well as serum and diet, was also determined to establish a correlation between Cx43 expression and beta-carotene concentration. In an initial analysis of samples collected from 10 subjects before supplementation, the quantity of Cx43 mRNA was variable and did not correlate with beta-carotene intake or the concentration of beta-carotene in tissue or serum. In samples collected at zero and three months from eight subjects who were controls or received a placebo, there was no correlation between Cx43 mRNA level and tissue or serum beta-carotene concentration. In samples collected from subjects before and after three months of beta-carotene supplementation, there was a significant increase in tissue and serum beta-carotene concentration in all subjects and an increase in Cx43 mRNA expression after supplementation relative to baseline in four of six samples. The high variability in Cx43 expression indicates that induction of Cx43 mRNA expression is not solely dependent on the concentration of beta-carotene in diet, serum, or tissue. However, the results from subjects supplemented with beta-carotene suggest that induced expression may occur in colonic tissue of some individuals. some individuals.

Base Sequence↗
PubMed2001

Expression of telomeric repeat binding factor 1 and 2 and TRF1-interacting nuclear protein 2 in human gastric carcinomas.

Telomeric repeat binding factor 1 (TRF1) and 2 (TRF2) may play key roles in the maintenance of telomere function. TRF1 negatively regulates telomere elongation, while TRF2 protects the chromosome ends by inhibiting end-to-end fusions. We examined the expression of TRF1 and TRF2 in 20 gastric carcinomas by reverse transcription polymerase chain reaction and then analyzed the relation with telomerase activity and other telomerase components such as human telomerase reverse transcriptase (TERT), human telomerase RNA component (hTR), human telomerase-associated protein (TEP1) and TRF1-interacting, ankyrin-related ADP-ribose polymerase (tankyrase) as well as TRF1-interacting nuclear protein 2 (TIN2). Of 20 gastric carcinomas examined, 10 (50%) and 12 (60%) expressed TRF1 and TRF2 at higher levels than did non-neoplastic mucosa, respectively. No obvious correlation was observed between TRF1 expression and telomerase activity or expression of TERT, hTR and TEP1. Carcinomas with high TRF1 expression expressed significantly higher levels of tankyrase and TIN2 than did those with low TRF2 expression (p<0.05). The telomerase activities and the levels of TERT, hTR and TEP1 showed tendency to be lower in tumors expressing TRF1 at low levels, although it was not significant. On the other hand, carcinomas with short telomere length (shorter than 2 Kbp) expressed significantly stronger telomerase activities and higher TRF1 expression (p<0.05) and tended to express TRF2 and TIN2 at higher levels than those with long telomere length. The results suggest that gastric carcinomas with short telomeres need high levels of telomerase activity and large quantity of TRFs and TIN2, whereas those with long telomeres do not require high levels of telomerase activity and telomere associated proteins.

Aged↗
PubMedDate not supplied

Histochemical characteristics of the anal sacs of the cat.

The histochemistry of polysaccharides and proteins and the distribution of RNA were investigated in the glands and mature secretion of the anal sacs of the cat. Glycogen was extremely abundant in the epithelial lining of the lumen and efferent duct of the anal sac and also in ducts of sebaceous glands of the sacs constantly formed in the place of degenerating acini. Little glycogen was found in the unchanged sebaceous acini. The apocrine glands of the anal sacs were substantially richer in glycogen than the apocrine sweat glands of the skin and were in an active state. A high concentration of acid mucopolysaccharides was present in the mature secretion, and their source was the apocrine glands. The phases of the secretory cycle alternated in different cells and secretory portions of the apocrine glands highly asynchronously. Carboxylated acid mucopolysaccharides were more abundant than sulfated. Sialomucin, hyaluronic acid, and chondroitin sulfate could be identified in the secretion. Neutral and acid mucopolysaccharides were present in the mature secretion in the form of complexes with one another in approximately equal proportions; neutral polysaccharides consisted chiefly of glycogen. Besides polysaccharides, the apocrine glands produce large quantities of protein, in which the mature secretion was rich. This protein was predominantly basic, and the secretion contained very little acid protein. Maturing cells of the sebaceous acini, and also other specific sebaceous glands, were characterized by a much higher protein concentration than in ordinary sebaceous glands.

Anal Sacs↗
PubMed2004

Isolation of nascent messenger RNA from mouse preimplantation embryos.

The expression of the zygotic genome starts at the late one-cell stage in mouse embryos, and its regulation changes dynamically until the late two-cell stage. To understand this process, it is important to accumulate the profiles of the genes transcribed at any given instant at each stage of development. However, because large amounts of maternal mRNA accumulate in embryos to sustain early development, it is difficult to determine the profile of newly synthesized mRNA just after gene activation. To overcome this difficulty, we established a novel method of isolating nascent mRNA from the large pool of preexisting mRNA. Briefly, the procedure was as follows. Embryos were electrically permeabilized and loaded with 5-bromouridine-5'-triphosphate (BrUTP). Nascent mRNA with incorporated BrU was isolated by immunoprecipitation with an antibody recognizing BrU. The cDNA was synthesized from the isolated mRNA, and its abundance was evaluated using semiquantitative real-time PCR. Using this method, we examined the amounts of newly synthesized eIF-1A, MuERV-L, and cyclin-A2 transcripts in two-cell mouse embryos and compared them with the quantities of these transcripts present in the total mRNA pool. The amount of each transcript in the nascent mRNA fraction and in the total mRNA pool changed differently over time, demonstrating that this method can be used to obtain profiles of genes transcribed during development.

Animals↗
PubMed2002

Transient expression of TIP60 protein during early chick heart development.

Screening of an embryonic chick cDNA library revealed a gene product termed chick TIP60 (cTIP60) due to its homology with human TIP60, a founding member of the "MYST" family of proteins that possess functional motifs, including chromo, zinc finger, and histone acetyltransferase domains. cTIP60 expression was assessed during early chick embryogenesis, at the RNA level by using reverse transcriptase-polymerase chain reaction (RT-PCR) and at the protein level by using Western blotting and immunohistochemistry. RT-PCR indicated that cTIP60 transcripts in whole embryos are present as early as Hamburger-Hamilton (HH) stage 5, diminishing after HH10. Western blotting of total embryonic protein revealed that cTIP60 was present in uniform quantities between HH3 and HH25. By contrast, Western blotting of protein from isolated hearts revealed that cTIP60 protein was strongly expressed at the earliest stages of heart development (HH11-13), diminishing thereafter. This finding was corroborated by immunohistochemistry, which revealed that cTIP60 protein was selectively expressed at high levels in the myocardium between HH 10-14. Considered in the context of its functional domains, these findings suggest that cTIP60 modulates transcriptional processes which regulate terminal cell differentiation, proliferation, or both, during early myocardial development.

Acetyltransferases↗
PubMed1993

Quantitation of IL-4 expression in small numbers of cells from mice.

Interleukin-4 (IL-4) is an important T cell and mast cell product that participates in allergic and cytotoxic responses, as well as functions as a growth factor for B, T, and inflammatory cells. Studies of the expression of IL-4 by T cells present in inflammatory reactions would be facilitated by using polymerase chain reaction (PCR) coupled to reverse transcription of mRNA to amplify the small quantity of mRNA present in these cells. In order to use this method in a quantitative manner, a plasmid was constructed that contained a modified form of mouse IL-4 cDNA. This plasmid was transcribed to produce cRNA for this modified sequence. The cRNA was used as an internal standard for the reverse transcription and amplification of IL-4 transcripts in RNA samples from mouse thymocytes. Amplification of reverse-transcribed native IL-4 mRNA produced a 286 bp PCR product. Amplification of the reverse-transcribed standard RNA produced a 155 bp product, which reflected a deletion introduced into the original IL-4 cDNA sequence. Comparison of the amount of the 286 bp native product to the amount of 155 bp standard product enabled the quantitative determination of IL-4 expression in each sample. This method was used to demonstrate that platelet activating factor increases the expression of IL-4 in mouse thymocytes and in a mouse T cell line. The expression of IL-4 by thymocytes exposed to platelet-activating factor (PAF) may reveal an important link between inflammation and the maturation of T cells in the thymus.

Animals↗
PubMedDate not supplied

Molecular cloning and expression of cDNA for human granulocyte colony-stimulating factor.

Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of hormone-like glycoproteins that regulate haematopoietic cell proliferation and differentiation, and G-CSF almost exclusively stimulates the colony formation of granulocytes from committed precursor cells in semi-solid agar culture. Recently, Nomura et al. have established a human squamous carcinoma cell line (designated CHU-2) from a human oral cavity tumour which produces large quantities of CSF constitutively, and the CSF produced by CHU-2 cells has been purified to homogeneity from the conditioned medium. We have now determined the partial amino-acid sequence of the purified G-CSF protein, and by using oligonucleotides as probes, have isolated several clones containing G-CSF complementary DNA from the cDNA library prepared with messenger RNA from CHU-2 cells. The complete nucleotide sequences of two of these cDNAs were determined and the expression of the cDNA in monkey COS cells gave rise to a protein showing authentic G-CSF activity. Furthermore, Southern hybridization analysis of DNA from normal leukocytes and CHU-2 cells suggests that the human genome contains only one gene for G-CSF and that some rearrangement has occurred within one of the alleles of the G-CSF gene in CHU-2 cells.

Amino Acid Sequence↗
PubMed2000

Transmission of porcine endogenous retroviruses in severe combined immunodeficient mice xenotransplanted with fetal porcine pancreatic cells.

BACKGROUND: Xenotransplantation using pig organs or tissues may alleviate the human donor organ shortage. However, one concern is the potential transmission of pig pathogens to humans, especially pig endogenous retroviruses (PERV), which infect human cell lines in vitro. In this report, the cross-species in vivo transmission of PERV by xenotransplantation was studied using a severe combined immunodeficient (SCID) mouse model. METHODS: Twenty-one SCID mice were transplanted with fetal pig pancreatic cells and left for periods from three to 41 weeks before being killed. DNA and RNA were extracted from liver, spleen, and brain of these mice, and examined for PERV using nested polymerase chain reaction (PCR) and reverse transcriptase-PCR. The pig mitochondrial cytochrome oxidase II subunit gene (COII) was also amplified to monitor the presence of pig cell microchimerism in xenotransplanted tissues, and a housekeeping gene was included to monitor the DNA quality and quantity. RESULTS: Examination of 39 DNA samples from tissues of the 21 xenografted mice identified two mouse tissues (M4-liver and M19-spleen) that were positive for PERV but negative for COII. A total of 23 (59%) of the mouse tissues were positive for both PERV and COII, 6 (16%) were negative for both, and 8 (20%) were positive for COII only. PCR and direct sequencing of the PCR products identified three PERV variants, which were different from the PERV sequence detected by PCR direct sequencing from the pig donor cells. CONCLUSIONS: The PERV+/COII- results from M4-liver and M19-spleen indicated the presence of PERV transmission from pig to mouse tissue. The PERV variants detected in the mouse tissues indicated that different PERVs were transmissible from the pig to mouse tissue during xenotransplantation. The negative reverse transcriptase-PCR results for PERV from three mouse samples including M4-liver and M19-spleen suggest there was no active PERV transcription in the mouse tissues, although this would need to be studied further.

Animals↗
PubMed1990

Retroviral DNA integration directed by HIV integration protein in vitro.

Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a lambda DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

Animals↗