Biomedical subjects

G Wu

Publications and source records attributed to G Wu.

At least 145 records · Page 8Linked to original sources

PubMed2000

Inhibition of lipoprotein lipase by alkanesulfonyl fluorides.

A number of alkanesulfonyl halides (chlorides and fluorides) and esters were synthesized and their effect on the activity of lipoprotein lipase (LPL) was studied. Sulfonyl fluorides proved to be efficient inhibitors of LPL when the enzyme was incubated with a 10-fold molar excess of the inhibitors in a buffer containing bile salts (deoxycholate). Hexadecane- and dodecanesulfonyl fluorides caused 50% inhibition of LPL activity at concentrations of 10 to 20 microM.

Alkanesulfonates↗

PubMed2000

Rescue of contractile parameters and myocyte hypertrophy in calsequestrin overexpressing myocardium by phospholamban ablation.

Cardiac-specific overexpression of murine cardiac calsequestrin results in depressed cardiac contractile parameters, low Ca(2+)-induced Ca(2+) release from sarcoplasmic reticulum (SR) and cardiac hypertrophy in transgenic mice. To test the hypothesis that inhibition of phospholamban activity may rescue some of these phenotypic alterations, the calsequestrin overexpressing mice were cross-bred with phospholamban-knockout mice. Phospholamban ablation in calsequestrin overexpressing mice led to reversal of the depressed cardiac contractile parameters in Langendorff-perfused hearts or in vivo. This was associated with increases of SR Ca(2+) storage, assessed by caffeine-induced Na(+)-Ca(2+) exchanger currents. The inactivation time of the L-type Ca(2+) current (I(Ca)), which has an inverse correlation with Ca(2+)-induced SR Ca(2+) release, and the relation between the peak current density and half-inactivation time were also normalized, indicating a restoration in the ability of I(Ca) to trigger SR Ca(2+) release. The prolonged action potentials in calsequestrin overexpressing cardiomyocytes also reversed to normal upon phospholamban ablation. Furthermore, ablation of phospholamban restored the expression levels of atrial natriuretic factor and alpha-skeletal actin mRNA as well as ventricular myocyte size. These results indicate that attenuation of phospholamban function may prevent or overcome functional and remodeling defects in hypertrophied hearts.

1-Methyl-3-isobutylxanthine↗

PubMed2000

Glucosamine inhibits inducible nitric oxide synthesis.

Glucosamine is widely used in Europe for treatment of arthritis in humans. Based on recent findings that excess production of nitric oxide (NO) by inducible NO synthase (iNOS) mediates the pathogenesis of arthritis, we hypothesized that glucosamine may inhibit NO synthesis. To test this hypothesis, we used an in vivo rat model of lipopolysaccharide (LPS)-induced inflammation. Intravenous administration of d-glucosamine (0.5 mmol/kg) 6 h before, at the time of, and 6 h after intraperitoneal LPS injection (1 mg/kg) decreased urinary excretion of nitrate by 31 and 48%, respectively, at days 1 and 2 post LPS administration. When cultured macrophages were treated with LPS (1 microg/ml) to induce iNOS expression, addition of 0.1, 0.5, 1, and 2 mM d-glucosamine decreased NO production by 18, 38, 60, and 89%, respectively. Glucosamine had no effect on cellular arginine, NADPH or tetrahydrobiopterin concentrations, but dose-dependently suppressed iNOS protein expression. Similar decreases in iNOS protein occurred in spleen, lung, and peritoneal macrophages of glucosamine-treated rats. These studies demonstrate that glucosamine is a novel inhibitor of inducible NO synthesis via inhibition of iNOS protein expression, and provide a biochemical basis for the use of glucosamine in treating chronic inflammatory diseases such as arthritis.

Animals↗

PubMed2000

The binding of a glycoprotein 120 V3 loop peptide to HIV-1 neutralizing antibodies. Structural implications.

The structural and antigenic properties of a peptide ("CRK") derived from the V3 loop of HIV-1 gp120 protein were studied using NMR and SPR techniques. The sequence of CRK corresponds to the central portion of the V3 loop containing the highly conserved "GPGR" residue sequence. Although the biological significance of this conserved sequence is unknown, the adoption of conserved secondary structure (type II beta-turn) in this region has been proposed. The tendency of CRK (while free or conjugated to protein), to adopt such structure and the influence of such structure upon CRK antigenicity were investigated by NMR and SPR, respectively. Regardless of conjugation, CRK is conformationally averaged in solution but a weak tendency of the CRK "GPGR" residues to adopt a beta-turn conformation was observed after conjugation. The influence of GPGR structure upon CRK antigenicity was investigated by measuring the affinities of two cognate antibodies: "5023A" and "5025A," for CRK, protein-conjugated CRK and gp120 protein. Each antibody bound to all the antigens with nearly the same affinity. From these data, it appears that: (a) antibody binding most likely involves an induced fit of the peptide and (b) the gp120 V3 loop is probably conformationally heterogeneous. Since 5023A and 5025A are HIV-1 neutralizing antibodies, neutralization in these cases appears to be independent of adopted GPGR beta-turn structure.

Antibodies, Viral↗

PubMed2000

Induction of the BRCA2 promoter by nuclear factor-kappa B.

BRCA2 is a tumor suppressor gene that has been implicated in response to DNA damage, cell cycle control, and transcription. BRCA2 has been found to be overexpressed in many breast tumors, suggesting that altered expression of the BRCA2 gene may contribute to breast tumorigenesis. To determine how BRCA2 is overexpressed in tumors, we investigated the transcriptional regulation of the BRCA2 promoter. Deletion mapping of the BRCA2 promoter identified three regions associated with 3-fold activation or repression and one upstream stimulatory factor binding site associated with 20-fold activation. Gel shift and cotransfection studies verified the role of USF in regulation of BRCA2 transcription. Analysis of the -144 to -59 region associated with 3-fold activation identified a putative NFkappaB binding site. Cotransfection of the p65 and p50 subunits of NFkappaB up-regulated the BRCA2 promoter 16-fold in a luciferase reporter assay, whereas mutations in the binding site ablated the effect. Gel shift and supershift assays with anti-p65 and -p50 antibodies demonstrated that NFkappaB binds specifically to the NFkappaB site. In addition, ectopic expression of NFkappaB resulted in increased levels of endogeneous BRCA2 expression. Thus, NFkappaB and USF regulate BRCA2 expression through the BRCA2 promoter.

BRCA2 Protein↗

PubMed2000

Resonance Raman studies indicate a unique heme active site in prostaglandin H synthase.

Prostaglandin H synthase isoforms 1 and 2 (PGHS-1 and -2) catalyze the first two steps in the biosynthesis of prostaglandins. Resonance Raman spectroscopy was used to characterize the PGHS heme active site and its immediate environment. Ferric PGHS-1 has a predominant six-coordinate high-spin heme at room temperature, with water as the sixth ligand. The proximal histidine ligand (or the distal water ligand) of this hexacoordinate high-spin heme species was reversibly photolabile, leading to a pentacoordinate high-spin ferric heme iron. Ferrous PGHS-1 has a single species of five-coordinate high-spin heme, as evident from nu(2) at 1558 cm(-1) and nu(3) at 1471 cm(-1). nu(4) at 1359 cm(-1) indicates that histidine is the proximal ligand. A weak band at 226-228 cm(-1) was tentatively assigned as the Fe-His stretching vibration. Cyanoferric PGHS-1 exhibited a nu(Fe)(-)(CN) line at 446 cm(-1) and delta(Fe)(-)(C)(-)(N) at 410 cm(-1), indicating a "linear" Fe-C-N binding conformation with the proximal histidine. This linkage agrees well with the open distal heme pocket in PGHS-1. The ferrous PGHS-1 CO complex exhibited three important marker lines: nu(Fe)(-)(CO) (531 cm(-1)), delta(Fe)(-)(C)(-)(O) (567 cm(-1)), and nu(C)(-)(O) (1954 cm(-1)). No hydrogen bonding was detected for the heme-bound CO in PGHS-1. These frequencies markedly deviated from the nu(Fe)(-)(CO)/nu(C)(-)(O) correlation curve for heme proteins and porphyrins with a proximal histidine or imidazolate, suggesting an extremely weak bond between the heme iron and the proximal histidine in PGHS-1. At alkaline pH, PGHS-1 is converted to a second CO binding conformation (nu(Fe)(-)(CO): 496 cm(-1)) where disruption of the hydrogen bonding interactions to the proximal histidine may occur.

Animals↗

PubMed2000

Effects of dopaminergic agents on carrageenan hyperalgesia in rats.

The present study explored the role of central dopaminergic transmission in a model of carrageenan-induced inflammatory pain by examining the effects of selective agonists and antagonists of dopamine receptors. The results were as follow: (1) LY171555 (trans-(-)-4aR-4,4a,5,6,7,8,8a,9-Octahydro-5-propyl-1H-pyrazolo[3, 4-g]quinoline hydrochloride), dopamine D(2) receptor agonist, produced anti-hyperalgesia or hypoalgesia in the inflamed hindpaws and non-inflamed hindpaws, respectively; spiperone hydrochloride (8-[4-(4-Fluorophenyl)-4-oxobutyl]-1-phenyl-1,3,8-triazaspiro[4, 5]decan-4-one hydrochloride), dopamine D(2) receptor antagonist, decreased the pain threshold of the non-inflamed hindpaws. (2) (+/-)-SKF38393 hydrochloride ((+/-)-1-Phenyl-2,3,4, 5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride), dopamine D(1) receptor agonist, produced anti-hyperalgesia or hypoalgesia when administered in a high dose (600 nmol), and decreased the pain threshold of non-inflamed hindpaws when administered in a low dose (150 nmol); R(+)-SCH23390 hydrochloride (R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4, 5-tetrahydro-1H-3-benzazepine hydrochloride), dopamine D(1) receptor antagonist, induced anti-hyperalgesia or hypoalgesia, respectively. The present study suggests that the dopaminergic system is involved in the central modulation of inflammatory hyperalgesia, and that the different effects are probably induced by the different receptors.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

PubMed2000

Solution structure of BmP01 from the venom of scorpion Buthus martensii Karsch.

From the venom of scorpion Buthus martensii Karsch,a short peptide (BmP01, 29 amino acid residues) was isolated and characterized as previously reported (Lebren, R. R., et al. (1997) Eur. J. Biochem. 245, 457-464). It was shown to reduce 33% outward K(+) channel (hippocampal neurons) currents at 10 microM. The solution structure of BmP01 was determined by 2D (1)H NMR spectroscopy. The NOEs, coupling constants, and H-D exchange obtained from NMR spectroscopy were used in structural calculations. The conformation of BmP01 is composed of a short alpha-helix (Cys 3-Thr 12) and a two-stranded antiparallel beta-sheet (Ala 15-Asp 20 and Lys 23-Pro 28). There are three disulfide bridges (Cys 3-Cys 19, Cys 6-Cys 24 and Cys 10-Cys 26) connecting the alpha-helix and beta-sheet. Asp 20 to Lys 23 form a type II turn linking the two strands. Structural and electrostatic potential comparison between BmP01 and its analogues are also presented.

Amino Acid Sequence↗

PubMed2000

The preparation and characterization of an immobilized l-glutamic decarboxylase and its application for determination of l-glutamic acid.

This paper is to study the preparation and characterization of an immobilized L-glutamic decarboxylase (GDC) and develop a sensitive method for the determination of L-glutamate using a new biosensor, which consists of an enzyme column reactor of GDC immobilized on a novel ion exchange resin (carboxymethyl-copolymer of allyl dextran and N.N'-methylene-bisacrylamide CM-CADB) and ion analyzer coupled with a CO(2) electrode. The conditions for the enzyme immobilization were optimized by the parameters: buffer composition and concentration, adsorption equilibration time, amount of enzyme, temperature, ionic strength and pH. The dynamic response of Na(2)HPO(4)-citric acid buffer system selected is much better than that of the others, 0.10 M HAc-0.10 M NaAc and 0.10 M sodium citrate-0.10 M citric acid. The initial rate of the enzyme reaction v(0) in this buffer system is 1.76 mol. l(-1) min(-1), moreover, the rate of the enzyme reaction appears linear in the first 4 min. The optimum adsorption equilibrium time is around 6 h. The amount of enzyme adsorbed on CM-CADB resin affects the response to substrate L-glutamic acid, the widest range of linearity is obtained with over 30 mg (GDC)/g(resin). The GDC activity immobilized on CM-CADB reaches a maximum when the immobilization temperature was kept around 40 degrees C. pH was kept at 4.4 when measuring the activity of the immobilized GDC. No variation of the activity of immobilized GDC is observed when the capacity is over 2.5 meq/g.(CM-CADB resin). The properties of the immobilized enzyme on CM-CADB were characterized. No significant improvement can be achieved when the substrate concentration exceeds 12.00 mmol/l, where the activity of immobilized GDC is equal to 1.58 mmol/l.min.g. The optimum pH is found to be 5.2, which changes 0.2 unit, comparing with that of the free GDC (5.0). The optimum temperature is found to be around 48 degrees C, which is lower than that of free GDC (55 degrees C). The critical temperature of the free GDC and the immobilized GDC is approximately 50 degrees C and 45 degrees C, respectively. The half-life of the activity is 127 days when the immobilized enzyme was stored in the cold (4 degrees C). An immobilized GDC enzyme column reactor matched with a flow injection system-ion analyzer coupled with CO(2) electrode-data collection system made up the original form of the apparatus of biosensor for determining of L-glutamic acid. The determination conditions are that the buffer solution is 0.10 M Na(2)HPO(4)-0.05 M citric acid at pH 4.4 and t = 37 degrees C. The limit of detection is 1.0 x 10(-)(5) M. The linearity response is in the range of 5 x 10 (-2) - 5 x 10 (-5) M. The equation of linear regression of the calibration curve is y = 43.3x + 181.6 (y is the milli-volt of electrical potential response, x is the logarithm of the concentration of the substrate of L-glutamic acid). The correlation coefficient equals 0.99. The coefficient of variation equals 2.7%.

Journal Article↗

PubMed2000

Epsilon protein kinase C in pathological myocardial hypertrophy. Analysis by combined transgenic expression of translocation modifiers and Galphaq.

The epsilon isoform of protein kinase C (PKC) has a critical cardiotrophic function in normal postnatal developing heart as demonstrated by cardiac-specific transgenic expression of epsilonPKC-selective translocation inhibitor (epsilonV1) and activator (psiepsilonRACK) peptides (Mochly-Rosen, D., Wu, G., Hahn, H., Osinska, H., Liron, T., Lorenz, J. N., Robbins, J., and Dorn, G. W., II (2000) Circ. Res. 86, 1173-1179). To define the role of epsilonPKC signaling in pathological myocardial hypertrophy, epsilonV1 or psiepsilonRACK were co-expressed in mouse hearts with Galpha(q), a PKC-linked hypertrophy signal transducer. Compared with Galpha(q) overexpression alone, co-expression of psiepsilonRACK with Galpha(q) increased epsilonPKC particulate partitioning by 30 +/- 2%, whereas co-expression of epsilonV1 with Galpha(q) reduced particulate-associated epsilonPKC by 22 +/- 1%. Facilitation of epsilonPKC translocation by psiepsilonRACK in Galpha(q) mice improved cardiac contractile function measured as left ventricular fractional shortening (30 +/- 3% Galpha(q) versus 43 +/- 2% psiepsilonRACK/Galpha(q), p < 0.05). Conversely, inhibition of epsilonPKC by epsilonV1 modified the Galpha(q) nonfailing hypertrophy phenotype to that of a lethal dilated cardiomyopathy. These opposing effects of epsilonPKC translocation activation and inhibition in Galpha(q) hypertrophy indicate that epsilonPKC signaling is a compensatory event in myocardial hypertrophy, rather than a pathological event, and support the possible therapeutic efficacy of selective epsilonPKC translocation enhancement in cardiac insufficiency.

Animals↗

PubMed2000

NBS1 and TRF1 colocalize at promyelocytic leukemia bodies during late S/G2 phases in immortalized telomerase-negative cells. Implication of NBS1 in alternative lengthening of telomeres.

Nijmegen breakage syndrome, a chromosomal instability disorder, is characterized in part by cellular hypersensitivity to ionizing radiation. The NBS1 gene product, p95 (NBS1 or nibrin) forms a complex with Rad50 and Mre11. Cells deficient in the formation of this complex are defective in DNA double-strand break repair, cell cycle checkpoint control, and telomere length maintenance. How the NBS1 complex is involved in telomere length maintenance remains unclear. Here we show that the C-terminal region of NBS1 interacts directly with a telomere repeat binding factor, TRF1, by both yeast two-hybrid and in vivo DNA-coimmunoprecipitation assays. NBS1 and Mre11 colocalize with TRF1 at promyelocytic leukemia (PML) nuclear bodies in immortalized telomerase-negative cell lines, but rarely in telomerase-positive cell lines. The translocation of NBS1 to PML bodies occurs specifically during late S to G(2) phases of the cell cycle and coincides with active DNA synthesis in these NBS1-containing PML bodies. These results suggest that NBS1 may be involved in alternative lengthening of telomeres in telomerase-negative immortalized cells.

Abnormalities, Multiple↗

PubMed2000

Rapid determination of nitrite by reversed-phase high-performance liquid chromatography with fluorescence detection.

Measurement of nitrite and nitrate, the stable oxidation products of nitric oxide (NO), provides a useful tool to study NO synthesis in vivo and in cell cultures. A simple and rapid fluorometric HPLC method was developed for determination of nitrite through its derivatization with 2,3-diaminonaphthalene (DAN). Nitrite, in standard solution, cell culture medium, or biological samples, readily reacted with DAN under acidic conditions to yield the highly fluorescent 2,3-naphthotriazole (NAT). For analysis of nitrate, it was converted to nitrite by nitrate reductase, followed by the derivatization of nitrite with DAN to form NAT. NAT was separated on a 5-microm reversed-phase C18 column (150X4.6 mm, I.D.) guarded by a 40-microm reversed-phase C18 column (50x4.6 mm, I.D.), and eluted with 15 mM sodium phosphate buffer (pH 7.5) containing 50% methanol (flow-rate, 1.3 ml/min). Fluorescence was monitored with excitation at 375 nm and emission at 415 nm. Mean retention time for NAT was 4.4 min. The fluorescence intensity of NAT was linear with nitrite or nitrate concentrations ranging from 12.5 to 2,000 nM in water, cell culture media, plasma and urine. The detection limit for nitrite and nitrate was 10 pmol/ml. Because NAT is well separated from DAN and other fluorescent components present in biological samples, our HPLC method offers the advantages of high sensitivity and specificity as well as easy automation for quantifying picomole levels of nitrite and nitrate in cell culture medium and biological samples.

Animals↗

PubMed2000

Polymorphic CAG and GGN repeat lengths in the androgen receptor gene and prostate cancer risk: a population-based case-control study in China.

The length of the polymorphic CAG trinucleotide repeat in the polyglutamine region of the androgen receptor (AR) gene is inversely correlated with the transactivation function of the AR. Because increased androgenic activity has been linked to prostate cancer and because an ethnic variation exists in the CAG repeat length, this polymorphism has been suggested to explain part of the substantial racial difference in prostate cancer risk. We conducted a population-based case-control study in China to investigate whether CAG and other polymorphisms of the AR gene are associated with clinically significant prostate cancer in this low-risk population. Genomic DNA from 190 prostate cancer patients and 304 healthy controls was used for direct sequencing to evaluate the relationship of CAG and GGN (polyglycine) repeat length in the AR gene. Relative to western men, our study subjects had a longer CAG repeat length, with a median of 23 and only 10% of the subjects having a CAG repeat length shorter than 20. Men with a CAG repeat length shorter than 23 (median length) had a 65% increased risk of prostate cancer (odds ratio, 1.65; 95% confidence interval, 1.14-2.39), compared with men with a CAG repeat length of 23 or longer. For the GGN tract (GGT3GGG1GGT2GGCn), based on the sequencing results from 481 samples, we are the first to show that although GGC regions in the polyglycine tract are highly variable, there are no mutations or polymorphisms in the GGT and GGG regions. More than 72% of the subjects had a GGN repeat length of 23, and those with a GGN repeat length shorter than 23 had a 12% increased risk of prostate cancer (95% confidence interval, 0.71-1.78), compared with those with > or = 23 GGN repeats. Our study not only confirms that Chinese men do have a longer CAG repeat length than western men but also represents the first population-based study to show that even in a very low-risk population, a shorter CAG repeat length confers a higher risk of clinically significant prostate cancer. These results imply that CAG repeat length can potentially serve as a useful marker to identify a subset of individuals at higher risk of developing clinically significant prostate cancer. Larger studies are needed to evaluate the combined effect of CAG and GGN repeats. Because of the significance of AR in prostate cancer, investigation of factors that interact with the polyglutamine region of the AR gene to alter AR function and modulate prostate cancer risk is an important area for future research.

Aged↗

PubMed2000

Evidence of the synthesis of opioid receptor like 1 receptor in nociceptinergic neurons in rat brain suggests the existence of autoreceptor: a confocal double staining study.

Opioid receptor like 1 (ORL1) receptor is a novel member of the opioid receptor family, which was not bound by any of the typical opioid receptor ligands but bound by the recently discovered nociceptin (also termed orphanin FQ) with high affinity. By using double staining of fluorescent in situ hybridization and immunohistochemistry, we observed the expression of ORL1 receptor mRNA in nociceptin-like immunoreactive neurons in multiple areas in rat brain including the hippocampus, arcuate nucleus in the hypothalamus, ventralateral periaquiductal gray (PAG) and raphe nuclei in brain stem. The expression of ORL1 mRNA in nociceptinergic neurons suggests that these receptors mediate, at least in part, the presynaptic autoreceptor functions. Further anatomical and functional significance of the autoreceptor of nociceptinergic neurons remain to be elucidated.

Animals↗

PubMed2000

Localization of mGluR6 to dendrites of ON bipolar cells in primate retina.

We prepared antibodies selective for the C-terminus of the human mGluR6 receptor and used confocal and electron microscopy to study the patterns of immunostaining in retina of monkey, cat, and rabbit. In all three species punctate stain was restricted to the outer plexiform layer. In monkey, stain was always observed in the central element of the postsynaptic "triad" of rod and cone terminals. In monkey peripheral retina, stain was seen only in central elements, but in the fovea, stain was also observed in some dendrites contacting the base of the cone terminal. S-cone terminals, identified by staining for S opsin, showed staining of postsynaptic dendrites. These were identified as dendrites of the ON S-cone bipolar cell by immunostaining for the marker cholecystokinin precursor. The staining pattern suggests that all types of ON bipolar cells, despite their marked differences in function, express a single isoform of mGluR6. Ultrastructurally, mGluR6 was located not on the tip of the central element, near the site of vesicle release, but on its base at the mouth of the invagination, 400-800 nm from the release site. Thus, the mGluR6 receptors of ON bipolar cells lie at about the same distance from sites of vesicle release as the iGluR receptors of OFF bipolar cells at the basal contacts.

Amino Acid Sequence↗

PubMed2000

Impaired nitric oxide production in coronary endothelial cells of the spontaneously diabetic BB rat is due to tetrahydrobiopterin deficiency.

Endothelial cells (EC) from diabetic BioBreeding (BB) rats have an impaired ability to produce NO. This deficiency is not due to a defect in the constitutive isoform of NO synthase in EC (ecNOS) or alterations in intracellular calcium, calmodulin, NADPH or arginine levels. Instead, ecNOS cannot produce sufficient NO because of a deficiency in tetrahydrobiopterin (BH(4)), a cofactor necessary for enzyme activity. EC from diabetic rats exhibited only 12% of the BH(4) levels found in EC from normal animals or diabetes-prone animals which did not develop disease. As a result, NO synthesis by EC of diabetic rats was only 18% of that for normal animals. Increasing BH(4) levels with sepiapterin increased NO production, suggesting that BH(4) deficiency is a metabolic basis for impaired endothelial NO synthesis in diabetic BB rats. This deficiency is due to decreased activity of GTP-cyclohydrolase I, the first and rate-limiting enzyme in the de novo biosynthesis of BH(4). GTP-cyclohydrolase activity was low because of a decreased expression of the protein in the diabetic cells.

Animals↗

PubMed2000

Instrumentation for photocrystallographic experiments of transient species.

Instrumentation for stroboscopic time-resolved diffraction studies at low temperatures is described. Exciting laser light is delivered to the crystal through an optical fiber. During the diffraction experiment, fluorescence from the sample is focused onto a fiber optic bundle surrounding the laser-light fiber, and monitored by a photodiode. A rotating slotted disk produces a pulsed X-ray beam with pulse frequencies suitable for the study of molecular excited states with lifetimes of 10 micros or longer. Synchronization of the laser-pump/X-ray-probe pulses is achieved through a trigger signal from a photosensor mounted on the rotating disk, or from an X-ray sensitive photodiode inserted in the beamstop. For the study of shorter-lived species the time structure of the synchrotron beam is to be used. Equations are derived for the maximum and average fractional excited-state populations as a function of lifetime, pulse frequency and the fraction of molecules being excited by the laser pulse.

Journal Article↗

PubMed2000

Cloning and characterization of the murine pkd2 promoter.

Pkd2, the mouse homologue of PKD2, the gene responsible for the second form of autosomal dominant polycystic kidney disease, is highly expressed in fetal and adult mouse tissues. The expression of Pkd2 is developmentally regulated. To begin to dissect out the regulatory mechanism of Pkd2 expression, we characterized the basic features of the gene structure and identified potential cis-regulatory elements of Pkd2 transcription. Pkd2 spans 42 kb with a transcription start site 165 bp upstream of the translation start codon. Exon 1 of Pkd2 is 755 bp long, and the full-length transcript is 5215 bp. The Pkd2 promoter region is GC-rich and lacks a consensus TATA or CCAAT box. Consensus binding sites for the transcription factors Sp-1, NF-1, and Ap-2 lie in the 5' upstream region of Pkd2. The Sp-1 binding site is conserved in 5' upstream sequences of both the mouse and the human genes. The CAT activity of a series of upstream segments from +178 to -2749 was assessed in MDCK, LLCPK1, COS-7, and HEK293 cells. Deletion analysis identified a 409-bp fragment from position -221 to +178 responsible for basal promoter activity. A 922-bp fragment from -744 to +178 showed the highest level of CAT activity in the cell lines tested. These data define a functional promoter candidate region for Pkd2.

Animals↗