Biomedical subjects

C W Sun

Publications and source records attributed to C W Sun.

At least 19 recordsLinked to original sources

PubMed2006

Platelet hyperreactivity generalizes to multiple forms of stimulation.

BACKGROUND: Although platelet hyperreactivity constitutes an important cardiovascular risk factor, standardized methods for its measurement are lacking. We recently reported that aggregometry using a submaximal concentration of epinephrine identifies individuals with in vitro platelet hyperreactivity; this hyperreactivity was reproducible on multiple occasions over long periods of time. OBJECTIVE AND METHODS: To better understand this aberrant reactivity, we studied in a large group of subjects (n = 386) the relationship between healthy individuals' platelet reactivity to epinephrine and their platelet phenotype as measured by other functional assays. RESULTS: Subjects with hyperreactivity to epinephrine were more likely to exhibit hyperfunction in each major aspect of platelet activity, including adhesion (response to low-dose ristocetin; P < 0.001), activation (surface P-selectin expression and PAC-1 binding after stimulation; P <or= 0.003) and aggregation to other agonists [no agonist, adenosine diphosphate (ADP), arachidonic acid, collagen, collagen-related peptide and ristocetin; P <or= 0.025] and to applied shear stress (PFA-100 and cone-and-plate viscometer; P < 0.05). These differences persisted after adjusting for demographic and hematologic differences between groups. We studied candidate genes relevant to epinephrine-mediated platelet activation and found that hyperreactivity to epinephrine was associated with a polymorphism on the gene (GNB3) encoding the beta-3 subunit of G proteins (P = 0.03). CONCLUSIONS: Robust aggregation to a submaximal concentration of epinephrine establishes a true hyperreactive platelet phenotype that is 'global' as opposed to agonist specific; detection of this phenotype could be useful for studying patients at risk for arterial thrombosis. The mechanisms underlying hyperreactivity to different types of platelet stimulation may share common signaling pathways, some of which may involve specific G protein subunits.

Adult↗

PubMed2004

Von Willebrand factor present in fibrillar collagen enhances platelet adhesion to collagen and collagen-induced platelet aggregation.

We examined the basis of the differences observed between different collagen preparations in their ability to aggregate platelets and support their adhesion under flow. As in previous studies, we found fibrillar collagen to be 10-fold more potent than acid-soluble collagen in inducing platelet aggregation and found that acid-soluble collagen did not support the adhesion of washed platelets under flow. Further, platelets in whole blood adhered to surfaces coated with either fibrillar or acid-soluble collagen, but thrombi formed faster and grew larger on fibrillar collagen. As a possible basis for this difference, we found that fibrillar collagen, but not acid-soluble collagen, contains a substantial quantity of von Willebrand factor (VWF), as demonstrated by enzyme-linked immunosorbent assay and by the ability of fibrillar collagen to support the adhesion of VWF antibody-coated beads and to agglutinate GPIb-IX-V complex-expressing Chinese hamster ovary cells. Supporting a role for VWF in collagen-induced platelet aggregation, aggregation induced by acid-soluble collagen was greatly enhanced by added VWF. Further, platelet aggregation by fibrillar collagen was partially blocked by a GPIbalpha antibody that inhibits the GPIb-VWF interaction. Taken together, these results suggest that much of the difference in prothrombotic potency of different collagens is directly related to their differences in VWF content. This probably accounts for the different conclusions made regarding the relative importance of different direct and indirect collagen receptors in collagen-dependent platelet functions and further emphasizes the close synergistic roles of the GPIb-IX-V complex and the collagen receptors GPVI and alpha2beta1 in supporting platelet adhesion.

Cells, Cultured↗

PubMed2001

Distinguishing malignant from normal oral tissues using FTIR fiber-optic techniques.

Oral tissue samples were studied using mid-IR fiber-optic attenuated total reflectance spectroscopy and other spectral techniques. The 1745 cm(-1) band, which is assigned to the ester group (C==O) vibration of triglycerides, is a reliable marker that is present in normal tissues but absent or a weak band in malignant oral tissues. Other bands such as C--H stretching bands and the amide bands are also helpful in distinguishing malignant tissues from normal tissues. Subtraction spectra confirmed the above conclusion. In addition, Raman spectroscopic measurements were in agreement with the results observed from FTIR spectra.

Biomarkers, Tumor↗

PubMed2001

Leaf-specific upregulation of chloroplast translocon genes by a CCT motif-containing protein, CIA 2.

Chloroplasts are a major destination of protein traffic within leaf cells. Protein import into chloroplasts is mediated by a set of translocon complexes at the chloroplast envelope. Current data indicate that the expression of translocon genes is regulated in a tissue-specific manner, possibly to accommodate the higher import demand of chloroplasts in leaves and the lower demand of plastids in other tissues. We have designed a transgene-based positive screen to isolate mutants disrupted in protein import into plastids. The first locus we isolated, CIA2, encodes a protein containing a motif conserved within the CCT family of transcription factors. Biochemical analysis indicates that CIA2 is responsible for specific upregulation of the translocon genes atToc33 and atToc75 in leaves. Identification of CIA2 provides new insights into the tissue-specific regulation of translocon gene expression.

Amino Acid Motifs↗

PubMed2000

Human gamma-globin gene promoter element regulates human beta-globin gene developmental specificity.

The persistence of fetal hemoglobin in many patients with deletion type beta thalassemias and the expression patterns of human globin genes in transgenic mice suggest that gamma- to beta-globin gene switching results primarily from competition of gamma- and beta-globin genes for interaction with the beta-globin locus control region (LCR). To define regulatory sequences that are essential for the competitive advantage of the gamma gene at early developmental stages, stable transgenic mouse lines were produced with LCR gamma-beta constructs containing deletions of gamma 5'-flanking DNA. All constructs contained the full 22 kb LCR, a 4.1 kb beta-globin gene and a gamma-globin gene with 1348, 383, 202, 130, 72 or 52 bp of 5'-flanking sequence. Primer extension analysis of yolk sac, fetal liver and blood RNA from these lines demonstrated that a region between -202 and -130 of the human gamma-globin gene promoter was required to suppress beta-globin gene expression at early developmental stages. Four transcription factor binding sites within this region [GATA(p), Oct1, GATA(d) and CACCC] were mutated independently in LCR gamma-beta constructs and transgenic mouse lines were produced. Only the gamma CACCC box mutation resulted in high levels of beta-globin gene expression in early embryos. These results demonstrate that the CACCC box of the human gamma-globin gene plays a critical role in human beta-globin gene developmental specificity. The data also suggest that gamma CACCC box binding factors mediate LCR-gamma interactions which normally enhance gamma-globin and suppress beta-globin gene expression in fetal erythroid cells.

Animals↗

PubMed2000

Renal and cardiovascular actions of 20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids.

1. Arachidonic acid (AA) is metabolized by cytochrome P450 (CYP)-dependent pathways to epoxyeicosatrienoic acids (EET) and 20-hydroxyeicosatetraenoic acid (20-HETE) in the kidney and the peripheral vasculature. 2. The present short review summarizes the renal and cardiovascular actions of these important mediators. 3. Epoxyeicosatrienoic acids are vasodilators produced by the endothelium that hyperpolarize vascular smooth muscle (VSM) cells by opening Ca2+-activated K+ (KCa) channels. 20-Hydroxyeicosatetraenoic acid is a vasoconstrictor that inhibits the opening of KCa channels in VSM cells. Cytochrome P450 4A inhibitors block the myogenic response of small arterioles to elevations in transmural pressure and autoregulation of renal and cerebral blood flow in vivo. Cytochrome P450 4A blockers also attenuate the vasoconstrictor response to elevations in tissue PO2, suggesting that this system may serve as a vascular oxygen sensor. Nitric oxide and carbon monoxide inhibit the formation of 20-HETE and a fall in 20-HETE levels contributes to the activation of KCa channels in VSM cells and the vasodilator response to these gaseous mediators. 20-Hydroxyeicosatetraenoic acid also mediates the inhibitory actions of peptide hormones on sodium transport in the kidney and the mitogenic effects of growth factors in VSM and mesangial cells. A deficiency in the renal production of 20-HETE is associated with the development of hypertension in Dahl salt-sensitive rats. 4. In summary, the available evidence indicates that CYP metabolites of AA play a central role in the regulation of renal, pulmonary and vascular function and that abnormalities in this system may contribute to the pathogenesis of cardiovascular diseases.

Animals↗

PubMed2000

Role of cGMP versus 20-HETE in the vasodilator response to nitric oxide in rat cerebral arteries.

This study examined the response to nitric oxide (NO) in rat middle cerebral arteries (MCA). NO donors increased the activity of a 205-pS K(+) channel recorded from vascular smooth muscle (VSM) cells isolated from MCA 10-fold. Blockade of guanylyl cyclase activity with 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ, 10(-5) M) did not alter the effect of NO on this channel. In contrast, adding 20-hydroxyeicosatetraenoic acid (20-HETE) to the bath (10(-7) M) abolished the response to NO. NO donors also increased the diameter of serotonin-preconstricted MCA to 85% of control. Blockade of K(+) channels with iberiotoxin or a high-K(+) medium reduced this response by 50%. ODQ (10(-5) M) reduced this response by 47 +/- 3%, whereas preventing the fall of 20-HETE levels reduced the response by 59 +/- 2% (n = 5). Blockade of both pathways eliminated the response to NO donors. These results indicate that activation of K(+) channels contributes 50% to vasodilator response to NO in rat MCA. This is mediated by a fall in 20-HETE levels rather than a rise in cGMP levels or a direct effect of NO.

Animals↗

PubMed1999

Altered renal hemodynamics and impaired myogenic responses in the fawn-hooded rat.

The present study examined whether an abnormality in the myogenic response of renal arterioles that impairs autoregulation of renal blood flow (RBF) and glomerular capillary pressure (PGC) contributes to the development of renal damage in fawn-hooded hypertensive (FHH) rats. Autoregulation of whole kidney, cortical, and medullary blood flow and PGC were compared in young (12 wk old) FHH and fawn-hooded low blood pressure (FHL) rats in volume-replete and volume-expanded conditions. Baseline RBF, cortical and medullary blood flow, and PGC were significantly greater in FHH than in FHL rats. Autoregulation of renal and cortical blood flow was significantly impaired in FHH rats compared with results obtained in FHL rats. Myogenically mediated autoregulation of PGC was significantly greater in FHL than in FHH rats. PGC rose from 46 +/- 1 to 71 +/- 2 mmHg in response to an increase in renal perfusion pressure from 100 to 150 mmHg in FHH rats, whereas it only increased from 39 +/- 2 to 53 +/- 1 mmHg in FHL rats. Isolated perfused renal interlobular arteries from FHL rats constricted by 10% in response to elevations in transmural pressure from 70 to 120 mmHg. In contrast, the diameter of vessels from FHH rats increased by 15%. These results indicate that the myogenic response of small renal arteries is altered in FHH rats, and this contributes to an impaired autoregulation of renal blood flow and elevations in PGC in this strain.

Animals↗

PubMed1999

Role of tyrosine kinase and PKC in the vasoconstrictor response to 20-HETE in renal arterioles.

The present study examined the hypothesis that activation of protein kinase C (PKC), components of the mitogen-activated protein (MAP) kinase pathway, or both contributes to the inhibitory effects of 20-hydroxyeicosatetraenoic acid (20-HETE) on K+-channel activity and its vasoconstrictor response in renal arterioles. 20-HETE (0.1 to 50 micromol/L) dose-dependently produced a 30% increase in PKC activity and a fivefold rise in the expression of active extracellular signal-regulated kinase 1 (ERK1) and ERK2 proteins in renal microvessels. 20-HETE (0.01 to 1 micromol/L) reduced the diameter of isolated perfused renal interlobular arterioles by 33+/-2%. Blockade of PKC activity with an N-myristoylated PKC pseudosubstrate inhibitor (Myr-PKCi, 100 micromol/L) or calphostin C (0.5 micromol/L) had no significant effect on the vasoconstrictor response to 20-HETE. In contrast, the tyrosine kinase inhibitors genistein (30 micromol/L) and tyrphostin 25 (10 micromol/L) reduced the response to 20-HETE by 76.5+/-2.1% and 67.5+/-1.8%, respectively. A specific inhibitor of mitogen-activated extracellular signal-regulated kinase (MEK), PD98059, had no effect on the vasoconstrictor response to 20-HETE. In cell-attached patches on renal vascular smooth muscle cells, 20-HETE reduced the open state probability of a large-conductance K+ channel (from 0.0026+/-0.0004 to 0.0006+/-0.0001). The Myr-PKCi (100 micromol/L) did not alter the inhibitory effects of 20-HETE on this channel. In contrast, the tyrosine kinase inhibitor genistein (30 micromol/L) blocked the inhibitory effects of 20-HETE on the large-conductance K+ channel. These data suggest that 20-HETE activates the MAP kinase system in renal arterioles and that the activation of a tyrosine kinase, which is proximal to MEK in this cascade, contributes to the inhibitory effects of 20-HETE on K+-channel activity and its vasoconstrictor effects in the renal arterioles.

Animals↗

PubMed1998

Nitric oxide-20-hydroxyeicosatetraenoic acid interaction in the regulation of K+ channel activity and vascular tone in renal arterioles.

The present study examined whether inhibition of P4504A enzyme activity and the formation of 20-HETE contributes to the activation of K+ channels and vasodilator effects of nitric oxide (NO) in renal arterioles. Addition of an NO donor to the P4504A2 enzyme that produces 20-HETE increased visible light absorbance at 440 nm indicating that NO binds to heme in this enzyme. NO donors also dose-dependently inhibited the formation of 20-HETE in microsomes prepared from renal arterioles. In patch-clamp experiments, NO donors increased the open-state probability of a voltage-sensitive, large-conductance (195+/-9 pS) K+ channel recorded with cell-attached patches on renal arteriolar smooth muscle cells. Blockade of guanylyl cyclase with [1H-[1,2,4]Oxadiazolo[4,3-a] quinoxalin-1-one] (ODQ, 10 micromol/L), or cGMP-dependent kinase with 8R,9S,11S-(-)-9-methoxycarbamyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cy-cloocta-(c ,d, e)-trinden-1-one (KT-5823) (1 micromol/L) did not alter the effects of NO on this channel. In contrast, inhibition of the formation of 20-HETE with 17-octadecynoic acid (1 micromol/L) activated this channel and masked the response to NO. Preventing the NO-induced reduction in intracellular 20-HETE levels also blocked the effects of NO on this channel. Sodium nitroprusside (SNP) increased the diameter of renal interlobular arteries preconstricted with phenylephrine to 80+/-4% of control. Blockade of guanylyl cyclase with ODQ (10 micromol/L) attenuated the response to SNP by 26+/-2%; however, fixing 20-HETE levels at 100 nmol/L reduced the response by 67+/-8%. Blockade of both pathways eliminated the response to SNP. These results indicate that inhibition of the formation of 20-HETE contributes to the activation of K+ channels and the vasodilator effects of NO in the renal microcirculation.

Animals↗

PubMed1998

Lovastatin reduces renal vascular reactivity in spontaneously hypertensive rats.

We have reported that lovastatin attenuates the development of hypertension in spontaneously hypertensive rats (SHR). The fall in arterial pressure is associated with an elevation in renal medullary blood flow, normalization of the pressure-natriuresis relationship, and diminished hypertrophy of renal arterioles. However, the mechanism by which lovastatin alters renal vascular tone is unknown. The present study examined the effects of lovastatin on renal vascular tone and the expression of G proteins. Four-week-old SHR were chronically treated with lovastatin (20 mg/kg/day) or vehicle by gavage for 4 weeks. At the end of the study, mean arterial pressure averaged 131 +/- 4 (n = 5) and 160 +/- 4 mm Hg (n = 6) in lovastatin- and vehicle-treated SHR, respectively. Renal arterioles isolated from lovastatin-treated SHR were significantly less responsive to norepinephrine and vasopressin than those obtained from vehicle-treated rats (ED50: 5.0 v 1.8 x 10(-7) mol/L for norepinephrine, and 8.0 v 5.2 x 10(-10) mol/L for vasopressin). The fall in renal vascular reactivity in lovastatin-treated SHR was associated with reduced levels of ras and rho proteins in renal arterioles, whereas the expressions of heterotrimeric G proteins (Gs Gq, Gi) were similar in renal arterioles from vehicle- and lovastatin-treated SHR. Overnight culture of renal arterioles with media containing lovastatin also diminished the expression of ras and rho proteins and the response to vasoconstrictors. These findings indicate that lovastatin diminishes the response to vasoconstrictors and the expression of small G proteins in the renal vasculature of SHR and suggest that a fall in the levels of ras and rho proteins in these vessels may contribute to the antihypertensive effects of lovastatin.

Animals↗

PubMed1998

Contribution of 20-HETE to the vasodilator actions of nitric oxide in renal arteries.

The present study examined the contribution of elevations in cGMP versus inhibition of cytochrome P-4504A enzymes and the production of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE) to the vasodilator actions of NO in renal arterioles. The NO donor sodium nitroprusside (SNP) at 10(-5), 10(-4), and 10(-3) M reduced the production of 20-HETE in microsomes prepared from renal arterioles to 80 +/- 2, 43 +/- 5, and 7 +/- 1% of control, respectively (n = 4). In other experiments, the vasodilator response to SNP (10(-7) to 10(-3) M) was examined in rat renal interlobular arteries (<90 micron ID), preconstricted with phenylephrine (1 microM) under control conditions and after blockade of the cGMP and P-4504A pathways. Inhibition of guanylyl cyclase with 1H-[1,2, 4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) (10 microM, n = 6) or of cGMP-dependent protein kinase with 8R,9S, 11S-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c, d, e)-trinden-1-one (KT-5823, 1 microM; n = 5) attenuated the vasodilator response to SNP by 26 and 30%, respectively. In contrast, inhibition of the endogenous production of 20-HETE with a suicide substrate, irreversible inhibitor [17-octadecynoic acid (17-ODYA), 1 microM, n = 5], or a selective, competitive inhibitor of 20-HETE formation (dibromo-dodecenyl-methylsulfimide, 25 microM, n = 5) markedly impaired the vasodilator response to SNP by 76 and 78%, respectively. Similarly, when 20-HETE levels were fixed at 100 nM (n = 6), the response to SNP was attenuated by 73%. Blockade of both pathways with ODQ and 17-ODYA completely abolished the response to SNP (n = 6). These results indicate that the vasodilator response to NO is largely cGMP independent and that inhibition of 20-HETE formation contributes to the cGMP-independent effects of NO in the renal microcirculation.

Alkaloids↗

PubMed1997

The bZIP transcription factor LCR-F1 is essential for mesoderm formation in mouse development.

LCR-F1 is a mammalian bZIP transcription factor containing a basic amino acid domain highly homologous to a domain in the Drosophila Cap 'N' Collar and Caenorhabditis elegans SKN-1 proteins. LCR-F1 binds to AP1-like sequences in the human beta-globin locus control region and activates high-level expression of beta-globin genes. To assess the role of LCR-F1 in mammalian development, the mouse Lcrf1 gene was deleted in embryonic stem (ES) cells, and mice derived from these cells were mated to produce Lcrf1 null animals. Homozygous mutant embryos progressed normally to the late egg cylinder stage at approximately 6.5 days post coitus (dpc), but development was arrested before 7.5 dpc. Lcrf1 mutant embryos failed to form a primitive streak and lacked detectable mesoderm. These results demonstrate that LCR-F1 is essential for gastrulation in the mouse and suggest that this transcription factor controls expression of genes critical for the earliest events in mesoderm formation. Interestingly, Lcrf1 null ES cells injected into wild-type blastocysts contributed to all mesodermally derived tissues examined, including erythroid cells producing hemoglobin. These results demonstrate that the Lcrf1 mutation is not cell autonomous and suggest that LCR-F1 regulates expression of signaling molecules essential for gastrulation. The synthesis of normal hemoglobin levels in erythroid cells of chimeras derived from Lcrf1 null cells suggests that LCR-F1 is not essential for globin gene expression. LCR-F1 and the related bZIP transcription factors NF-E2 p45 and NRF2 must compensate for each other in globin gene regulation.

Alleles↗

PubMed1997

A model for the evolution of polyubiquitin genes from the study of Arabidopsis thaliana ecotypes.

Polyubiquitin genes encode the highly conserved 76-amino acid protein ubiquitin that is covalently attached to substrate proteins targeting most for degradation. Polyubiquitin genes are characterized by the presence of tandem repeats of the 228 bp that encode a ubiquitin monomer. Five polyubiquitin genes UBQ3, UBQ4, UBQ10, UBQ11, and UBQ14, previously isolated from Arabidopsis thaliana ecotype Columbia [10] encode identical mature ubiquitin proteins, but differ in synonymous substitutions, nature of amino acids terminating the open reading frame, and in the number of ubiquitin repeats. The presence of these five genes in nine other Arabidopsis ecotypes was verified by polymerase chain reaction (PCR). Size differences in UBQ3 and UBQ11 amplified products from several ecotypes were observed, suggesting that alleles differ in ubiquitin repeat number. DNA sequence of UBQ11 alleles from each size class (ecotypes Be-0, Ler. and Rld-0) verified that PCR product size differences resulted from changes in the number of ubiquitin repeats. Nucleotide sequence between two UBQ11 alleles containing the same number of repeats was identical. Transcript size differences for UBQ3 and UBQ11 mRNAs between ecotypes Columbia and Landsberg indicated that repeat number changes did not inactivate these genes. Nucleotide sequence comparisons between UBQ11 repeats from different ecotypes suggest that first repeats are related to each other and last repeats are related to each other. We hypothesize that changes in UBQ11 ubiquitin repeat number occurred via the contraction and/or expansion of specific internal repeats or portions thereof by misalignment of alleles and recombination, most likely via unequal crossing-over events.

Arabidopsis↗

PubMed1997

Independent modulation of Arabidopsis thaliana polyubiquitin mRNAs in different organs and in response to environmental changes.

The highly conserved protein ubiquitin is encoded by five polyubiquitin genes in Arabidopsis thaliana ecotype Columbia that have been divided into two subtypes, the UBQ3/UBQ4 subtype and the UBQ10/UBQ11/UBQ14 subtype. Northern analysis using gene-specific oligonucleotides as hybridization probes and enzyme activity measurements from transgenic plants expressing beta-glucuronidase (GUS) under the control of individual polyubiquitin 5' flanking regions were used to determine the development and environmental regulation of polyubiquitin transcription and mRNA accumulation. Polyubiquitin mRNA levels within and between subtypes were independently modulated. UBQ3 mRNA levels were three-fold higher than UBQ4 mRNA levels in vegetative organs, but only two-thirds of the UBQ4 mRNA levels in flowers. UBQ3 mRNA was modulated by dark/light treatments, while mRNAs from UBQ and all members of the other subtype were unaffected. Similarly, within the UBQ10/UBQ11/UBQ14 subtype, UBQ11/UBQ14 mRNAs were modulated differently in seedlings after a two-hour heat-shock treatment. Among all the polyubiquitin genes, UBQ10 mRNA level was the most constant in all organs and environmental conditions examined. Transgenic plants transformed with a UBQ10 5' flanking region::GUS gene contained higher levels of GUS activity than transgenic plants expressing GUS under the control of UBQ3 5' flanking regions. In conclusion, the relative abundance of different Arabidopsis polyubiquitin mRNAs, even those produced from highly similar genes within a subtype, appears to be modulated independently in response to developmental and environmental cues.

Adaptation, Biological↗

PubMed1995

End joining of genomic DNA and transgene DNA in fertilized mouse eggs.

A linear 5.2-kb HS2/beta-globin construct with an upstream KpnI terminus (4-nucleotide (nt) 3' protruding single strand, PSS) and a downstream SalI terminus (4-nt 5' PSS) was microinjected into fertilized mouse eggs. The injected DNA fragments integrated into the mouse genome primarily as a head-to-tail tandem array. Chromosome/transgene junctions were obtained from seven of eight transgenic animals. All of the junctions occurred in the proximity of a transgene KpnI end; a maximum loss of 8 nt from the transgene terminus was observed. Two of these junctions completely preserved the 4-nt KpnI 3' PSS. Transgene/transgene junctions from two animals were analyzed. SalI/KpnI junctions that completely preserved both the SalI 5' PSS and the KpnI 3' PSS were found in each animal. These are the first examples of complete nt preservation at junctions formed between a 5' PSS terminus and a 3' PSS terminus in transgenic mice. The data are consistent with the fill-in model of Thode et al. [Cell 60 (1990) 921-928] in which alignment proteins juxtapose 5' PSS and 3' PSS termini; DNA polymerase then utilizes the recessed 3'-OH of the 5' PSS terminus as a primer to synthesize DNA across the gap. This mechanism results in the formation of junctions with no loss of sequence. The results described in the present paper suggest that this mechanism may be involved in the formation of junctions in transgenic mice.

Animals↗

PubMed1995

Structure and evolution of genes encoding polyubiquitin and ubiquitin-like proteins in Arabidopsis thaliana ecotype Columbia.

The Arabidopsis thaliana ecotype Columbia ubiquitin gene family consists of 14 members that can be divided into three types of ubiquitin genes; polyubiquitin genes, ubiquitin-like genes and ubiquitin extension genes. The isolation and characterization of eight ubiquitin sequences, consisting of four polyubiquitin genes and four ubiquitin-like genes, are described here, and their relationships to each other and to previously identified Arabidopsis ubiquitin genes were analyzed. The polyubiquitin genes, UBQ3, UBQ10, UBQ11 and UBQ14, contain tandem repeats of the 228-bp ubiquitin coding region. Together with a previously described polyubiquitin gene, UBQ4, they differ in synonymous substitutions, number of ubiquitin coding regions, number and nature of nonubiquitin C-terminal amino acid(s) and chromosomal location, dividing into two subtypes; the UBQ3/UBQ4 and UBQ10/UBQ11/UBQ14 subtypes. Ubiquitin-like genes, UBQ7, UBQ8, UBQ9 and UBQ12, also contain tandem repeats of the ubiquitin coding region, but at least one repeat per gene encodes a protein with amino acid substitutions. Nucleotide comparisons, Ks value determinations and neighbor-joining analyses were employed to determine intra- and intergenic relationships. In general, the rate of synonymous substitution is too high to discern related repeats. Specific exceptions provide insight into gene relationships. The observed nucleotide relationships are consistent with previously described models involving gene duplications followed by both unequal crossing-over and gene conversion events.

Amino Acid Sequence↗

PubMed1995

[Calcium channel blockade and anti-free-radical actions of panaxadiol saponins Rb1, Rb2, Rb3, Rc, and Rd].

AIM: To identify the calcium channel blockade and anti-free-radical actions of panaxadiol saponins Rb1, Rb2, Rb3, Rc, and Rd. METHODS: On ventricular myocardiocytes of Wistar rats, single channel activities of L, T, and B type Ca2+ channels were recorded with the cell-attached configuration of patch-clamp technic, and free radical contents were measured with electron spin resenance method. RESULTS: Rb1, Rb2, Rb3, and Rc 200 mumol.L-1 shortened the open times, prolonged the close times, and reduced the openstate probabilities of calcium channels and 30 mumol.L-1 antagonized the increase of free radical content induced by xanthine 0.42 mmol.L-1-xanthine oxidase 5.3 nmol.L-1, but Rd in the same dose behaved none of the effects. CONCLUSION: Rb1, Rb2, Rb3, and Rc had both the calcium channel blockade and anti-free-radical actions.

Animals↗