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J Sun

Publications and source records attributed to J Sun.

At least 631 records · Page 35Linked to original sources
PubMed1994

Expressed swine VH genes belong to a small VH gene family homologous to human VHIII.

The sequences of 34 swine H chain V-regions expressed with either IgG, IgA, or IgM C regions in adult swine and newborn piglets are described and compared. Sixteen of these V-regions were cloned by using a specific leader primer whereas 18 were cloned by anchored PCR. According to the operational criterion of VH family classification, i.e., > 80% DNA sequence similarity, all sequences belong to a homogeneous VH gene family. A total of 31 additional VH-bearing C mu clones obtained by anchored PCR from colostrum-deprived newborn piglets hybridized equally with a probe for C mu and a pan-VH probe. When the consensus sequence of the expressed swine VH genes were compared with those of VH gene for humans (VHI, II, III), mouse (VHI, II, III), rabbit, and chicken, swine VH genes seem to have common ancestry with the human VHIII family, rabbit VH genes, and the single functional VH gene of the chicken. The leader peptides of all clones obtained by anchored PCR showed < 1% variability, and the deduced amino acid sequences from aa4 to aa25 in all 34 clones are identical. Framework (FR)1 and FR2 are conserved whereas FR3 shows greater variability. A total of 23 of 30 JH sequences were identical, suggesting preferential use of one JH. This frequent, putative JH sequence is not similar to any JH gene in humans. Identical genomic Southern hybridization patterns, each with 13 bands of differing intensity and regardless of stringency, were obtained when either a leader or a pan-specific VH probe was used. Analyses of individual genomic bands by single strand conformational polymorphism and sequence analysis suggest that the number of VHIII-related genes in the swine genome is < 20.

Amino Acid Sequence↗
PubMed1994

Identification of histidine 25 as the heme ligand in human liver heme oxygenase.

Electronic and resonance Raman spectroscopic studies are reported for the His25Ala mutant of human liver heme oxygenase (HO) and its complex with heme. In the oxidized (ferric) form of the enzyme.substrate complex, the heme is shown to be in a high-spin, five-coordinate state. This is distinct from the same complex in the wild-type enzyme in which the heme is six-coordinate, ligated to a proximal histidine and a water molecule in an environment reminiscent of aquometmyoglobin. The reduced (ferrous) form of the complex of the H25A heme oxygenase mutant has lost the very prominent resonance Raman band at approximately 217 cm-1 seen in the wild-type complex that has been unambiguously assigned to the proximal Fe-N(His) vibrational frequency [Sun et al. (1993) Biochemistry 32, 14151; Takahashi et al. (1994) Biochemistry 33, 1010]. The absence of this band in the spectrum of the mutant protein definitively identifies His 25 as the proximal ligand of the heme substrate. Furthermore, this ferrous heme-H25A HO complex exists as an equilibrium mixture between a five-coordinate, high-spin species and a four-coordinate, intermediate-spin species. Although the H25A mutant protein shows no heme oxygenase activity, the heme is competent to bind carbon monoxide. Studies of the CO adduct of the H25A HO complex show v(CO) and v(Fe-CO) frequencies at 1960 and 529 cm-1, respectively, that are characteristic of a hydrophobic carbon monoxide binding site on a heme with a weak proximal ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Monoxide↗
PubMed1994

Renin gene promoter activity in GC cells is regulated by cAMP and thyroid hormone through Pit-1-dependent mechanisms.

Transcriptional activity of human renin gene (hREN) 5'-flanking DNA sequences in pituitary cells is highly dependent on binding of the pituitary-specific transcription factor Pit-1. Pit-1 has been implicated in cAMP regulation of a number of pituitary genes and has also been shown to interact with thyroid hormone (T3) receptors in mediating T3 responsiveness of the rat growth hormone gene. In the present study we examine the effects of forskolin and T3 on the expression of luciferase hybrid genes containing hREN 5'-flanking DNAs (hREN.luc) transiently transfected into the pituitary cell line GC. Basal activities of all hREN.luc constructs transfected into cells grown in media containing serum stripped of hormones were low. Addition of forskolin stimulated expression up to 48-fold, depending on the hREN sequences present. The hREN sequence -148 to +18 was sufficient for both maximal expression and maximal stimulation by forskolin. Mutagenesis of the Pit-1 site between -82 and -58 reduced forskolin induction 4-5-fold. In addition to the Pit-1 site, the sequence between -148 and -98 was also required for maximal activity and forskolin induction. T3 on its own had no effect on hREN promoter activity in GC cells, but suppressed the effects of forskolin. Gel mobility shift and Western blot analyses indicated that forskolin treatment had no effect on Pit-1 DNA binding or Pit-1 levels. However, T3 reduced Pit-1 levels which was reflected in lower DNA binding under the conditions employed. Taken together, these findings emphasize the importance of cAMP-dependent mechanisms in directing renin gene expression.

Animals↗
PubMed1994

Five putative subclasses of swine IgG identified from the cDNA sequences of a single animal.

We report the sequences of more than 40 partial and complete swine C gamma cDNAs obtained by PCR cloning of first strand cDNA, and from a cDNA expression library, all from a single animal. These seem to represent five IgG subclasses, that can be grouped into two clusters; one contains IgG1 and IgG3 and the other, IgG2a, IgG2b and IgG4. IgG2a and IgG2b differ by only three amino acids, but single strand conformational polymorphism analyses of PCR-amplified IgG2-specific segments in animals of different breeds, argues for their putative subclass status. Major subclass differences are found in the hinge and C gamma 3, but differences in upper hinge length, associated with segmental flexibility in the IgGs of other species, are absent. All subclasses have identical middle hinge segments that can accommodate three interheavy chain disulfide bridges. The putative swine IgG subclasses have their greatest similarity with those of the human except for the near absence of hinge region variation. Swine subclasses such as ruminant IgG2a, have a lower hinge deletion which in human IgG1, contains one of the motifs believed necessary for interaction with Fc gamma Rs. IgG1 was the most frequently encountered subclass cDNA (25 of 43) and the single swine-mouse hybridoma tested had a sequence identical to IgG1. Partial sequence analyses of genomic clones identified one clone identical with the IgG1/IgG3 subclass cluster, two identical to the IgG2b/IgG4 subclass group, and two identical to each other but different from any of the expressed sequences reported here. Genomic blots suggest that up to eight C gamma genes are present in the genome.

Animals↗
PubMed1994

Divergent roles of RAS1 and RAS2 in yeast longevity.

Individual cells of the yeast Saccharomyces cerevisiae have a limited replicative life-span. The role of the genes RAS1 and RAS2 in yeast longevity was examined. Over-expression of RAS2 led to a 30% increase in the life-span on average and postponed the senescence-related increase in generation time seen during yeast aging. No life-span extension was obtained by overexpression of RAS1. However, deletion of RAS1 prolonged the life-span. These results suggest that RAS1 and RAS2 play reciprocal roles in determining yeast longevity. RAS1 and RAS2 mRNA and protein levels declined with replicative age, suggesting a diminishing impact on yeast longevity. The major known pathway through which Ras proteins function in yeast involves stimulation of adenylate cyclase. No evidence for a life-span-extending effect of elevated intracellular cAMP was found. Indeed, high intracellular cAMP was associated with curtailed life-span. A similar decrease in life-span was found on disruption of BCY1, which codes for the regulatory subunit of protein kinase A, the downstream target of cAMP. Importantly, overexpression of an effector domain mutant of RAS2, defective in stimulation of adenylate cyclase, prolonged life-span to the same extent as the wild-type gene, suggesting that the cAMP pathway is neither sufficient nor necessary for increased longevity.

Adenylyl Cyclases↗
PubMed1994

Tolerance to rat liver allografts. I. Differences between tolerance and rejection are more marked in the B cell compared with the T cell or cytokine response.

Liver allografts in the fully allogenic combination of LEW donor liver to DA recipient (LEW-->DA) are spontaneously tolerated (TOL) with no requirement for immunosuppression, while DA-->LEW allografts are rejected in 12-15 days (REJ). We investigated the mechanism of tolerance induction by identifying differences between TOL and REJ grafts from day 1 to day 9 after transplantation and in normal livers and syngeneic liver graft controls. Infiltrating cell populations were counted after immunohistochemical staining of liver graft sections. There were occasional minor differences between TOL and REJ grafts in the T cell or CD11b/c+ (monocyte/macrophage/granulocyte) infiltrate. In contrast, there was a major difference in B cell infiltrate between TOL and REJ liver grafts. Membrane IgD+ cells were significantly greater in TOL (1796 +/- 225) versus REJ (569 +/- 281) (P = 0.004) portal tracts, as were B220+ cells (1086 +/- 100 vs. 181 +/- 105, P = 0.0004) and CD45RC+ cells (2317 +/- 456 vs. 597 +/- 194, P = 0.004). IgG1, IgG2a, IgM, and IgD deposition in liver allografts, identified by immunohistochemical staining of tissue sections, revealed no IgG or IgD in normal rat liver and low levels of IgM. Deposition of IgG1 was observed in REJ but not in TOL liver on days 7 and 9. IgM was increased in both TOL and REJ liver and appeared to be associated mainly with hepatocytes in REJ and with infiltrate in TOL liver. There was a parallel increase in IgG1-expressing plasma cells in the spleen and lymph nodes of REJ but not TOL animals. Cytokine mRNA was analyzed by reverse transcription and semiquantitative polymerase chain reaction amplification of liver RNA. Increased levels of IL-2, IL-4, IL-6, IL-10, TNF-alpha, transforming growth factor-beta, and IFN-gamma were observed, with similar levels of expression in TOL compared with REJ liver. Cytokine mRNA in syngeneic grafts was not different from normal except for IL-6 and transforming growth factor-beta, which were increased. There is no major difference in the T cell component of the infiltrate or in the extent of upregulation of cytokine mRNA between TOL and REJ grafts. There is a major difference in the B cell compartment, with more B cells in TOL livers and deposition of IgG1 in REJ grafts.

Animals↗
PubMed1994

Cloning and sequencing of a cDNA encoding the murine vitamin K-dependent protein S.

A cDNA clone encoding the vitamin K-dependent protein S was isolated from a murine liver cDNA library. Sequencing revealed an open reading frame encoding a 634 amino acid mature protein. Comparison to human and bovine sequences showed a high degree of similarity and the conservation of key residues between the species.

Amino Acid Sequence↗
PubMed1994

Effect of a bradykinin receptor antagonist, HOE 140, against bradykinin- and vagal stimulation-induced airway responses in the guinea-pig.

We have investigated the effect of inhaled HOE 140, a novel bradykinin B2 receptor antagonist, against bradykinin- and vagal stimulation-induced airway microvascular leakage and bronchoconstriction in anesthetized guinea-pigs. Lung resistance was measured for 6 min after challenge, followed by measurement of extravasation of Evans blue dye into airway tissues, used as an index of airway microvascular leakage. Bradykinin was given by inhalation (1 mM, 45 breaths) and bilateral vagus nerves were stimulated electrically during a 5-min period (3 and 10 Hz, 5 V, pulse width of 5 ms), both of which caused a significant increase in lung resistance and leakage of dye in the airway. HOE 140 (20 and 200 microM, 60 breaths) completely abolished both the airway effects induced by bradykinin, whereas even the higher dose of HOE 140 had no effect against those induced by electrical vagal stimulation. In conclusion, airway microvascular leakage and bronchoconstriction induced by inhaled bradykinin are mediated by activation of bradykinin B2 receptors in the guinea-pig. In contrast, mechanisms via bradykinin B2 receptors do not play an important role in the acute airway responses induced by vagal stimulation.

Administration, Inhalation↗
PubMed1994

Distribution of intracerebral ventricularly administered neurotrophins in rat brain and its correlation with trk receptor expression.

To assess the potential effectiveness by which injected neurotrophins can diffuse throughout the brain, we used autoradiographic and immunohistochemical techniques to examine the brain distributions of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) after a single injection into the lateral cerebral ventricle (ICV) in rats. As described previously, ICV-injected NGF labeled cholinergic neurons in the basal forebrain. Injection of BDNF resulted in few or no labeled neurons in the basal forebrain or in the substantia nigra. However, very intense labeling was associated with the ventricular walls and immediate parenchyma. The distribution of NT-3 after ICV injection was intermediate between that of NGF and BDNF. In the basal forebrain, similar neurotrophin distributions were observed in neonate versus adult animals. Our in situ hybridization analysis has shown that mRNA encoding the BDNF receptor(s) (trkB) is highly expressed by ependymal cells as well as by many neurons and glia. On the other hand, expression of the high-affinity NGF receptor (trkA) is restricted to cholinergic neurons in basal forebrain and striatum. In addition, staining with antisera specific for the trkA or trkB receptors demonstrated that their expression patterns closely reflect their mRNA distributions. Taken together, these data suggest that the presence of the trkB receptor on the ependymal layer of the ventricle and its expression throughout the brain parenchyma represents a significant impediment to the adequate diffusion of ICV-injected BDNF into the brain for delivery to target neurons.

Animals↗
PubMed1994

A Pit-1 binding site in the human renin gene promoter stimulates activity in pituitary, placental and juxtaglomerular cells.

One of the principal aims of our research is to determine the mechanisms which direct renin gene expression to different sites. We recently demonstrated that human renin (hRen) 5'-flanking DNA sequences -148 +/- 11 can drive the transient expression of a linked luciferase reporter gene transfected into pituitary GC cells. This activity was found to be dependent on the binding of Pit-1 to a site approximately 70 bp upstream from the transcription start site. Pit-1 is a pituitary-specific transcription factor which is involved in directing the cell-specific expression of growth hormone (GH) and prolactin (PRL) gene expression to somatotrope and lactotrope cells of the anterior pituitary. Thus, Pit-1 may be play a role in directing the expression of renin to primate lactotrope cells. Renin promoter-driven luciferase or CAT hybrid genes were found to be expressed following transfection into primary, or early passage cell cultures of placental chorionic membranes, and the renin-secreting renal tumor cell line As4.1. As with GC cells, deletion or mutagenesis of the Pit-1 site reduced activity several-fold in both placental and renal cells. These results suggest that members of the POU family of transcription factors, or some other closely related group such as the Hox proteins, participate in directing renin gene expression to placental and juxtaglomerular cells.

Binding Sites↗
PubMed1994

Multiplex polymerase chain reaction-based deletion analysis of spontaneous, gamma ray- and alpha-induced hprt mutants of CHO-K1 cells.

Independent Chinese hamster ovary (CHO)-K1 cell mutants at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus were isolated from untreated, 60Co gamma ray- and 212Bi alpha-exposed cells and the genetic changes underlying the mutation determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. In the 71 spontaneous mutants analyzed, 77.5% of the clones showed no change in exon number or size, 15.5% showed a loss of a single exon, 4.2% showed a loss of 2-8 exons, and 2.8% showed loss of all nine hprt exons (total gene deletion). Exposure to 6 Gy of gamma rays, which reduced survival levels to 10%, produced a significantly different deletion spectrum that was shifted toward deletions with 45% of the 20 mutants analyzed showing a loss of a single exon and 30% showing a loss of all nine exons. Exposure to 2 Gy alpha radiation from 212Bi, a 220Rn daughter, a dose which also reduced survival levels to about 10%, resulted in a deletion spectrum similar to the gamma-ray spectrum in that more than 75% of the 49 mutants analyzed were deletions. The alpha spectrum, however, was significantly different from both the spontaneous and gamma spectra with 55.1% of the alpha mutants showing a loss of all nine exons, 10.2% showing loss of a single exon, and 14.3% showing loss of 2-8 exons. Thus, alpha-radiation appears to produce larger intragenic deletions than gamma radiation. The results suggest that intragenic deletion size should be considered when low- and high linear energy transfer (LET) mutation spectra are compared.

Alpha Particles↗
PubMed1994

Depletion of T-cell subpopulations results in exacerbation of myocarditis and parasitism in experimental Chagas' disease.

The contribution of T-cell subpopulations to immunopathology in murine Trypanosoma cruzi infection was studied by using in situ localization of lymphocytes and in vivo depletion of T-lymphocyte populations. CD8+ T cells were the major lymphocyte population in the inflamed hearts of C3H/HeSnJ mice infected with the Sylvio X10/4 clone of T. cruzi at all time points of the acute and chronic phases of the infection examined. Depletion of CD8+ and/or CD4+ T cells beginning on day 20 of the infection resulted in a moderate decrease in the inflammation and an increase in parasite burden in the hearts of mice at day 30 of infection. Longer-term depletion, beginning at day 20 and extending as long as 200 days of infection, resulted in an increased inflammatory response in the heart. A large proportion of the inflammatory cells in the hearts of anti-CD8- or anti-CD4- and anti-CD8-treated mice were Thy1+ and CD4- CD8-. At 200 days of infection, the increased inflammation was accompanied by an increase in the parasite load in the heart. These results show that T-cell subset depletion does not prevent the inflammatory response associated with acute and chronic T. cruzi infection. The increased parasite load in T-cell-depleted mice also demonstrates the participation of these T-cell subsets in regulation of parasite load throughout the course of the infection. The increased inflammatory response despite T-cell depletion and in association with increased numbers of tissue parasites suggests that intracellular parasites are a driving force behind the inflammatory response in chronic murine T. cruzi infection.

Animals↗
PubMed1994

Analysis of human growth hormone gene 5' sequences in isolated growth hormone deficiency patients.

Human growth hormone (hGH) gene deletion (6.7 to 7.6 kb) is one of the causes of isolated growth hormone deficiency (IGHD), named IGHD IA. IGHD IA, however, only accounts for about 10% of the total IGHD patients. Most IGHD is caused by unknown mechanisms. Here, hGH gene 5' sequences in three IGHD patients without hGH gene deletion were analysed to see if there was any mutation hindering the expression of the hGH gene.

Base Sequence↗
PubMed1994

Contribution of intercellular-adhesion molecule-1 in allergen-induced airway hyperresponsiveness and inflammation in sensitised brown-Norway rats.

We investigated the potential role of intercellular-adhesion molecule-1 (ICAM-1) in allergen-induced bronchial hyperresponsiveness (BHR) and inflammation in sensitised Brown-Norway rats. Rats were sensitised with ovalbumin (OA) intraperitoneally and 21 days later they were either exposed to 0.9% NaCl or 1% OA aerosol for 15 min. Rats exposed to OA aerosol were pretreated either with ICAM-1 antibody (3 mg/kg i.p. and i.v., 45 min prior to OA exposure) or with the diluent for the antibody. Eighteen to twenty-four hours after OA or 0.9% NaCl exposure, rats were anaesthetised, tracheostomised and mechanically ventilated, and airway responsiveness to acetylcholine (ACh) aerosol was measured as the provocative concentration of ACh needed to increase pulmorary resistance by 100% (PC100). Mean -log PC100 was increased in rats exposed to OA but pretreated with diluent (2.75 +/- 0.06) compared to rats treated with ICAM-1 antibody (2.51 +/- 0.08; < 0.05). However, only the former group showed significantly higher mean -log PC100 compared to the sensitised group exposed to 0.9% NaCl alone (2.22 +/- 0.12; p < 0.01). There was a significant increase in eosinophil and lymphocyte counts in bronchoalveolar lavage fluid at 24 h in rats pretreated with diluent compared to saline exposed rats. However, in ICAM-1 antibody-pretreated rats, eosinophil and lymphocyte counts were significantly different from diluent-treated ones. We conclude that ICAM-1 antibody inhibits BHR without reducing the influx of inflammatory cells.

Allergens↗
PubMed1994

Pituitary-specific transcription factor (Pit-1) binding site in the human renin gene 5'-flanking DNA stimulates promoter activity in placental cell primary cultures and pituitary lactosomatotropic cell lines.

Renin gene expression is limited to a number of specific tissues, including the kidney, adrenal glands, reproductive organs (of particular relevance to this study, the placenta), and the pituitary gland. In the present study, we investigated the human renin (hRen) 5'-flanking DNA sequences required to drive the expression of a luciferase reporter gene in placental and pituitary cells and in two cell lines, 293 and JEG-3, which have been proposed as model systems with which to study transcriptional regulation of renin genes. The activities of specific sequences in the hRen 5'-flanking DNA sequences in human placental cell primary cultures were very similar to those that we previously reported in pituitary cells, suggesting the involvement of common promoter elements and related transcription factors. Accordingly, the binding site for the pituitary-specific transcription factor (Pit-1) was the major determinant of renin promoter activity in both pituitary and placental cells. Gel mobility shift analysis showed a placental nuclear factor with a gel mobility different from that of Pit-1. However, Northern blot analysis failed to demonstrate abundant Pit-1-related mRNAs in renin-expressing cultures of chorionic and decidual cells, suggesting that the placental factor is not closely related to Pit-1. Although a factor from 293 cells also bound to the Pit-1 site, it had gel mobility shift characteristics different from Pit-1 and the placental factor. Moreover, the low promoter activity in 293 cells was independent of this site or, indeed, of sequences upstream from the TATA box. In JEG-3 cells, renin 5'-flanking DNA sequences showed virtually no transcriptional activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1994

Effects of prolonged repeated exposure to ovalbumin in sensitized brown Norway rats.

The effects of chronic exposure to ovalbumin (OA) aerosol were studied in Brown Norway rats following intraperitoneal injections with OA and AI(OH)3 and exposure to OA or saline aerosols, once or every third day for 3 to 8 wk. Measurements of airway responsiveness to acetylcholine (ACh) aerosol at 18 to 24 h after allergen exposure showed a significant increase in -logPC150, the concentration of ACh needed to cause a 150% increase in baseline lung resistance, in animals single-exposed or chronic OA-exposed for 3 wk, compared with saline-exposed control animals. The group receiving 8 wk of OA exposure demonstrated no difference from the control animals with -logPC150 lower than that of the two previous groups (p < 0.001). In all three groups, BAL fluid showed a significant increase in neutrophils, but a significant increase in eosinophils (p < 0.01) was only observed in the single-exposed group when compared with saline-exposed control animals. In the 8-wk exposed rats, there was a higher recovery of macrophages and lymphocytes (p < 0.01) compared with control animals and the other two groups. AHR, present after single or 3-wk repeated exposure, disappears by 8 wk of continuous allergen exposure. Both the enhancement and suppression of AHR may be linked to OA-induced immune and inflammatory mechanisms.

Acetylcholine↗