Biomedical subjects

Y You

Publications and source records attributed to Y You.

At least 55 records · Page 3Linked to original sources

PubMed1997

Beta-adrenoceptor-coupled Gs protein facilitates the activation of cAMP-dependent cardiac Cl- current.

Here a comparison is made between adenosine 3',5'-cyclic monophosphate (cAMP)-activated Cl- current (ICl) density and activation time course in response to beta-adrenoceptor stimulation with isoproterenol and adenylyl cyclase activation with forskolin. Saturating concentrations of isoproterenol and forskolin failed to activate an ICl in guinea pig atrial as well as in rat and frog ventricular cardiomyocytes. In guinea pig ventricular cardiomyocytes, step application of 1 microM isoproterenol induced an ICl of -0.89 +/- 0.32 pA/pF (holding potential -40 mV, temperature 22 +/- 1 degrees C). ICl activation started after 3 +/- 1 s, was complete within 44 +/- 9 s, and was abolished after cell dialysis with the Rp diastereomer of adenosine 3',5'-cyclic monophosphothioate. Stimulation with increasing concentrations of forskolin (0.01-10 microM) increased ICl density and accelerated ICl activation. With 1 microM forskolin, ICl density was maximal (-0.57 +/- 0.30 pA/pF) but significantly smaller than that achieved with 1 microM isoproterenol. Although ICl density could not be further augmented by forskolin > 1 microM, current activation (latency 28 +/- 8 s, full activation after 112 +/- 8 s with 1 microM forskolin) was further accelerated by 3 and 10 microM forskolin. However, ICl activation with 10 microM forskolin was still slower than that with 1 microM isoproterenol. A low isoproterenol concentration (1 nM), which did not activate ICl by itself, accelerated the 1 microM forskolin-induced activation of ICl by 35%; this speeding up was abolished after cell dialysis with guanosine 5'-O-(2-thiodiphosphate). ICl deactivation after the washout of 1 microM forskolin or 1 microM isoproterenol followed a similar time course. After stimulation with 10 microM forskolin or 1 microM forskolin + 1 microM isoproterenol, but not with 1 microM forskolin + 1 nM isoproterenol, the decay of ICl was significantly delayed. These results indicate that both cAMP-dependent and cAMP-independent G protein pathways contribute to the regulation of guinea pig ventricular ICl.

Adrenergic beta-Agonists↗

PubMed1997

[Determination of fumaric acid in Sarcandra glabra (Thunb.) Nakai by HPLC].

A method for the determination of fumaric acid in Sarcandra glabra is reported. After the sample was pretreated with an anion exchange resin column, fumaric acid was determined on a Spherisorb C18 column, with methanol-0.02 mol/L monobasic potassium (20:80, pH2.5) as the mobile phase and detected at 210 nm. The method is simple and accurate.

Chromatography, High Pressure Liquid↗

PubMed1997

[Determination of docosyl ferulate in extract of Pygeum africanton Hook. by high performance liquid chromatography (HPLC)].

This paper reports a method for the determination of docosyl ferulate in the extract of bark of pygeum africanum Hook. by HPLC. After the sample was pretreated, the docosyl ferulate was well separated and determined on a Spherisorb C18 column (250 x 4.6mm, 5microm) using a mobile phase of methanol with a flow rate of 1mL/min. The column temperature was selected at 40 degrees C to avoid tailing of peak. UV detection was performed at 326nm. In order to confirm the docosyl ferulate separated from sample, the peak apex at 10.4 minute was scanned from 195nm to 360nm by photodiode array detector. Its spectrum showed the maxium absorption peak at 240nm and 326nm corresponding with the spectrum of docosyl ferulate. The linear correlation was observed from the 10mg/L to 100mg/L of docosyl ferulate (r = 0.9995). The average recovery was 98.4% +/- 1.98%. Three batches of sample were determined.

Chromatography, High Pressure Liquid↗

PubMed1996

Two isoxazolidines.

The crystal structures of trans-phenyl-1,6-dioxa-2-azaspiro[4,4]non- 3-yl ketone, C19H19NO3, and (3 alpha,3a alpha,6a alpha)-hexahydro-2-phenylfuro[3,2-d]isoxazol-3-yl phenyl ketone, C18H17NO3, are reported. In both compounds, the isoxazolidine rings adopt envelope conformations in which the O atom is bent out of the approximate plane of the other four ring atoms. Modest to negligible endo selectivities were confirmed in 1,3-dipolar cycloadditions of benzoylmethyleneaniline N-oxide with enol ethers.

Crystallography, X-Ray↗

PubMed1996

Construction of eukaryotic expression vector pBlacz and its expression both in vitro and in vivo.

A novel eukaryotic expression vector pBlacZ was constructed, which was transfected into the cell lines of NIH/3T3, COS-1, CHO and the primary culture of murine dermatic fibroblasts in vitro, and also into the murine subcutaneous layer and skeletal muscles of rats in vivo. It was detected that the gene expression vector could encode the E. Coli beta-galactosidase effectively in all these histocytes. The results suggested that pBlacZ, as a novel expression vector, might have certain value of application.

3T3 Cells↗

PubMed1996

Characterization of the cytoplasmic filament protein gene (cfpA) of Treponema pallidum subsp. pallidum.

Treponema pallidum and other members of the genera Treponema, Spirochaeta, and Leptonema contain multiple cytoplasmic filaments that run the length of the organism just underneath the cytoplasmic membrane. These cytoplasmic filaments have a ribbon-like profile and consist of a major cytoplasmic filament protein subunit (CfpA, formerly called TpN83) with a relative molecular weight of approximately 80,000. Degenerate DNA primers based on N-terminal and CNBr cleavage fragment amino acid sequences of T. pallidum subsp. pallidum (Nichols) CfpA were utilized to amplify a fragment of the encoding gene (cfpA). A 6.8-kb EcoRI fragment containing all but the 5' end of cfpA was identified by hybridization with the resulting PCR product and cloned into Lambda ZAP II. The 5' region was obtained by inverse PCR, and the complete gene sequence was determined. The cfpA sequence contained a 2,034-nucleotide coding region, a putative promoter with consensus sequences (5'-TTTACA-3' for -35 and 5'-TACAAT-3' for -10) similar to the sigma70 recognition sequence of Escherichia coli and other organisms, and a putative ribosome-binding site (5'-AGGAG-3'). The deduced amino acid sequence of CfpA indicated a protein of 678 residues with a calculated molecular mass of 78.5 kDa and an estimated pI of 6.15. No significant homology to known proteins or structural motifs was found among known prokaryotic or eukaryotic sequences. Expression of a LacZ-CfpA fusion protein in E. coli was detrimental to survival and growth of the host strain and resulted in the formation of short, irregular filaments suggestive of partial self-assembly of CfpA. The cytoplasmic filaments of T. pallidum and other spirochetes appear to represent a unique form of prokaryotic intracytoplasmic inclusions.

Amino Acid Sequence↗

PubMed1995

Trypsin and forskolin decrease the sensitivity of L-type calcium current to inhibition by cytoplasmic free calcium in guinea pig heart muscle cells.

A key feature of trypsin action on ionic membrane currents including L-type Ca2+ current (ICa) is the removal of inactivation upon intracellular application. Here we report that trypsin also occludes the resting cytoplasmic free Ca2+ ([Ca2+]i)-induced inhibition of peak ICa in isolated guinea pig ventricular cardiomyocytes, using the whole-cell patch clamp in combination with the Fura-2 ratio-fluorescence technique. The effectiveness of trypsin to guard ICa against [Ca2+]i-induced inhibition was compared with that of forskolin, as cAMP-dependent phosphorylation had been suggested to confer protection against [Ca2+]i-induced inactivation. Intracellular dialysis of trypsin (1 mg/ml) augmented ICa by 7.2-fold, significantly larger than the threefold increase induced by forskolin (3 microM). Forskolin application after trypsin dialysis did not further enhance ICa. An increase in [Ca2+]i from resting levels (varied by 0.2, 10, and 40 mM EGTA dialysis) to submicromolar concentrations after replacement of external Na+ (Na(o)+) with tetraethylammonium (TEA+) resulted in monotonic inhibition of control ICa, elicited from a holding potential of -40 mV at 22 degrees C. AFter trypsin dialysis, however, ICa became less sensitive to submicromolar [Ca2+]i; the [Ca2+]i of half-maximal inhibition (K0.5, normally around 60 nM) increased by approximately 20-fold. Forskolin also increased the K0.5 by approximately threefold. These and accompanying kinetic data on ICa decay are compatible with a model in which it is assumed that Ca2+ channels can exist in two modes (a high open probability "willing" and a low open probability "reluctant" mode) that are in equilibrium with one another. An increase in [Ca2+]i places a larger fraction of channels in the reluctant mode. This interconversion is hindered by cAMP-dependent phosphorylation and becomes nearly impossible after tryptic digestion.

Animals↗

PubMed1995

Physical map of the genome of Treponema pallidum subsp. pallidum (Nichols).

A physical map of the chromosome of Treponema pallidum subsp. pallidum (Nichols), the causative agent of syphilis, was constructed from restriction fragments produced by NotI, SfiI, and SrfI. These rare-cutting restriction endonucleases cleaved the T. pallidum genome into 16, 8, and 15 fragments, respectively. Summation of the physical lengths of the fragments indicates that the chromosome of T. pallidum subsp. pallidum is approximately 1,030 to 1,080 kbp in size. The physical map was constructed by hybridizing a variety of probes to Southern blots of single and double digests of T. pallidum genomic DNA separated by contour-clamped homogeneous electric field electrophoresis. Probes included cosmid clones constructed from T. pallidum subsp. pallidum genomic DNA, restriction fragments excised from gels, and selected genes. Physical mapping confirmed that the chromosome of T. pallidum subsp. pallidum is circular, as the SfiI and SrfI maps formed complete circles. A total of 13 genes, including those encoding five membrane lipoproteins (tpn47, tpn41, tpn29-35, tpn17, and tpn15), a putative outer membrane porin (tpn50), the flagellar sheath and hook proteins (flaA and flgE), the cytoplasmic filament protein (cfpA), 16S rRNA (rrnA), a major sigma factor (rpoD), and a homolog of cysteinyl-tRNA synthetase (cysS), have been localized in the physical map as a first step toward studying the genetic organization of this noncultivable pathogen.

Chromosome Mapping↗

PubMed1994

Modulation of calcium current density by intracellular calcium in isolated guinea pig ventricular cardiomyocytes.

This study describes quantitatively the relation between L-type calcium current (ICa) density and resting cytosolic free calcium concentration ([Ca2+]i) in isolated guinea pig ventricular cardiomyocytes. ICa was measured simultaneously with [Ca2+]i at 22 and 35 degrees C using the whole-cell patch-clamp method in combination with the Fura-2 ratio-fluorescence technique. At 22 degrees C, the increase in [Ca2+]i upon replacing external Na+ with tetraethylammonium resulted in a monotonic decrease in ICa density. Half-maximal inhibition of ICa density occurred at a [Ca2+]i value (K0.5) of 60 +/- 17 nM (mean +/- S.D., n = 59). At 35 degrees C, with a holding potential of -80 mV, a similar relation between [Ca2+]i and ICa was observed (K0.5 of 62 +/- 12 nM, n = 34). When the cells were depolarized from -40 mV, however, the initial decrease in ICa density with increasing [Ca2+]i was usually followed by transient stimulation (< or = 2-fold), which was again overcome by inhibition. Stimulation was not observed in the presence of broad spectrum protein kinase inhibitors. These data suggest that Ca2+i-dependent facilitation of cardiac ICa is mediated by a temperature-sensitive enzymatic pathway that ends in voltage-dependent phosphorylation of Ca2+ channels.

Animals↗

PubMed1994

The role of macronuclear DNA sequences in the permanent rescue of a non-mendelian mutation in Paramecium tetraurelia.

The paramecium tetraurelia mutant called d48 has a complete copy of the A surface protein gene in its micronuclei, but lacks the A gene in the macronucleus. Previous experiments have shown that microinjection of a plasmid containing the entire A gene or a large portion of the gene into the macronucleus of d48 rescued the cell line after formation of a new macronucleus (autogamy). Here we show that several different regions of the A gene can rescue d48, but 100% of the activity cannot be localized to a single, defined region. Inversion of a sequence contained within an A gene plasmid had no measurable effect on rescue efficiency and co-injection of two different plasmids results in enhancement of rescue activity despite the non-contiguous form of the DNA sequences. Both these results suggest that no specific product (RNA or protein) with defined end points is made from the rescuing fragment. A unique restriction site was created in the A gene and used to demonstrate that the injected DNA does not serve as a direct template for the synthesis of the new macronuclear DNA. Models to explain the action of the injected DNA are discussed.

Animals↗

PubMed1993

Functional integrity of proximal tubule cells: effects of temperature and preservation solutions.

Electrophysiologic and morphologic changes during cooling and perfusion with preservation solutions in isolated perfused proximal straight tubules from Swiss white mice were investigated. In standard Ringer-substrate solution, cooling from 37 degrees C to 22 and 4 degrees C depolarized both transepithelial potential and basolateral cell membrane potential. Basolateral k+ transference number and cell membrane conductances were also significantly reduced. An increase in intracellular Na+ activity was observed only during cooling from 37 to 4 degrees C. No cell swelling was detected when tubules were perfused with Ringer-substrate solution at all three temperatures up to 1 h. Perfusion with Euro-Collins' (EC) solution at 37 degrees C resulted in rapid cell swelling, associated with rapid deterioration of transepithelial potential. Substitution of glucose with mannitol abolished the damaging effect of EC solution at 37 degrees C. EC perfusion at 22 degrees C also led to cell swelling and deterioration of transepithelial potential, but after a 10-min delay. In comparison, perfusion with University of Wisconsin (UW) solution at 22 or 37 degrees C had no effect on cell volume. Less damage to transepithelial potential was observed after the UW perfusion. It was concluded that EC solution is more damaging than UW solution to kidney tubules at 22 and 37 degrees C. The presence of EC solution in the renal interstitium during the rewarming phase may contribute significantly to reperfusion injuries in kidney transplantation.

Adenosine↗

PubMed1993

Brain-derived neurotrophic factor (BDNF) prevents the degeneration of medial septal cholinergic neurons following fimbria transection.

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, supports the survival of developing basal forebrain cholinergic neurons in vitro and is retrogradely transported by cholinergic neurons of the medial septum and diagonal band following intrahippocampal injections in vivo. To substantiate a potential role for BDNF in the maintenance of forebrain cholinergic neurons in the adult brain, we assessed the ability of BDNF to sustain the phenotype of medial septal cholinergic neurons following a unilateral transection of the fimbria. BDNF, NGF, or vehicle solutions were infused continuously in adult female rats either into the lateral ventricle (intracerebroventricularly) or directly into the septum for 2 weeks beginning at the time of the transection. In vehicle-infused animals, only 28% of the ChAT-immunoreactive neurons remained on the side ipsilateral to the lesion compared to the contralateral intact side. When infused intracerebroventricularly, both BDNF and NGF reduced the extent of the phenotypic loss, in that 44% and 68%, respectively, of the ChAT-immunopositive neurons remained on the lesioned side. Intraseptal infusion proved even more effective, in that following BDNF and NGF treatment 60% and 86%, respectively, of the normal complement of ChAT-immunopositive neurons were apparent on the side ipsilateral to the lesion. Similar results were obtained when an antibody to the low-affinity NGF receptor was used to identify the cholinergic neurons. To determine if the apparent greater efficacy of NGF compared to BDNF might be related to differences in delivery, we examined the patterns of distribution of radiolabeled BDNF and NGF injected into the lateral ventricle. 125I-BDNF showed only very little diffusion from the ventricles into the adjacent neural tissue and negligible retrograde labeling of the neurons within the basal forebrain. 125I-NGF, however, diffused readily into the brain, resulting in widespread retrograde labeling of basal forebrain neurons. A similarly limited distribution pattern was observed where BDNF was detected immunohistochemically in animals infused intracerebroventricularly (12 micrograms/d) for 2 weeks. In contrast, when delivered intraseptally, the same dose of BDNF exhibited a widespread diffusion within the surrounding neuropil and retrograde labeling of neurons in the medial septum and the vertical limb of the diagonal band. Thus, when delivered effectively, BDNF has a substantial capacity to rescue axotomized cholinergic neurons.

Animals↗

PubMed1992

Cloning and expression of an intron-less gene for AKAP 75, an anchor protein for the regulatory subunit of cAMP-dependent protein kinase II beta.

The A-Kinase Anchor Protein AKAP 75 (formerly designated bovine brain P75) is a particulate brain protein that avidly binds the regulatory subunit (RII beta) of cAMP-dependent protein kinase II beta (Bregman, D. B., Hirsch, A.H. and Rubin, C.S. (1991) J. Biol. Chem. 266, 7207-7213). The formation of stable AKAP 75.RII beta complexes provides a potential mechanism for targeting physiological signals carried by cAMP to specific effector sites within neurons and other brain cells. We have now cloned and characterized the AKAP 75 gene. Its coding sequence is novel and unexpectedly short (1284 base pairs) and contains no introns. When the AKAP 75 gene was transfected into HEK 293 cells, a new RII beta-binding protein with an apparent Mr of 75,000 accumulated. A high proportion (approximately 65%) of the AKAP 75 gene product was excluded from the cytoplasm and was recovered in the 40,000 x g pellet derived from disrupted transfected cells. In contrast, cells transfected with a construct encoding 249 amino acids from the central and C-terminal regions of AKAP 75 produced an RII beta-binding protein (apparent Mr = 45,000) that was exclusively cytosolic. AKAP 75 is a novel protein composed of only 428 amino acid residues (Mr = 47,878). A highly acidic C-terminal region mediates the binding of RII beta (and cAMP-dependent protein kinase II beta), whereas a positively charged N-terminal segment contains structural features that are essential for the association of AKAP 75 with the cytoskeleton and/or intracellular membranes.

A Kinase Anchor Proteins↗

PubMed1992

Two cellular proteins bind specifically to a purine-rich sequence necessary for the destabilization function of a c-fos protein-coding region determinant of mRNA instability.

The c-fos proto-oncogene mRNA is rapidly degraded within minutes after its appearance in the cytoplasm of growth factor-stimulated mammalian fibroblasts. At least two functionally independent sequence elements are responsible for the lability of c-fos mRNA. One of these determinants is located within a 0.32-kb sequence present in the protein-coding region. We demonstrate by gel mobility shift experiments and UV cross-linking that at least two protein factors specifically interact with a 56-nucleotide purine-rich sequence located at the 5' end of the 0.32-kb coding region determinant of mRNA instability (CRDI). One protein is predominantly associated with the polysomes, while the other is detected in the post-ribosomal supernatant. Sequence comparison of members of the fos gene family revealed that the high purine content of the protein-binding region is conserved through evolution. Deletion of this region from the 0.32-kb CRDI severely impedes its function as an RNA-destabilizing element. Our results suggest that binding of the two proteins to the purine-rich sequence may participate in the rapid mRNA decay mediated by this 0.32-kb c-fos CRDI.

3T3 Cells↗

PubMed1992

U-rich sequence-binding proteins (URBPs) interacting with a 20-nucleotide U-rich sequence in the 3' untranslated region of c-fos mRNA may be involved in the first step of c-fos mRNA degradation.

Rapid decay of the c-fos transcript plays a critical role in controlling transforming potential of the c-fos proto-oncogene. One of the mRNA instability determinants is a 75-nucleotide AU-rich element (ARE) present in the 3' untranslated region of the c-fos transcript. It appears to control two steps in the process of c-fos mRNA degradation: removal of the poly(A) tail, which does not require the AUUUA motifs, and subsequent degradation of deadenylated mRNA, which appears to be dependent on the AUUUA motifs. In this study, we report the identification of four U-rich sequence binding proteins (URBPs) that specifically interact with a 20-nucleotide U-rich sequence within the c-fos ARE. Gel mobility shift assay and competition experiments showed that these protein factors form three specific band-shifted complexes with the c-fos ARE. Binding activity of one of the protein factors, a 37-kDa protein, is significantly affected by serum induction and by pretreatment of cells with drugs known to stabilize many of the immediate-early gene mRNAs. Combining UV cross-linking with a new approach, designated sequential RNase digestion, we were able to better determine the molecular masses of these cellular proteins. The binding sites for the four proteins were all mapped to a 20-nucleotide U-rich sequence located at the 3' half of the c-fos ARE, which contains no AUUUA pentanucleotides but stretches of uridylate residues. Single U-to-A point mutations in each of the three AUUUA motifs within the c-fos ARE have little effect on formation of the mobility-shifted complexes. Our data indicate c-fos ARE-protein interaction involves recognition of U stretches rather than recognition of the AUUUA motifs. We propose that UTBP binding may be involved in the first step, removal of the Poly(A) tail, in the c-fos ARE-mediated decay pathway.

3T3 Cells↗

PubMed1992

Functional integrity of proximal tubule cells. Effects of hypoxia and ischemia.

Effects of warm hypoxia and ischemia on electrophysiologic properties of isolated perfused mouse proximal straight tubules were studied. Oxyrase (5 to 10 microliters/mL) was added to the hypoxic and ischemic solutions to lower the oxygen tension to 5 mm Hg. The ischemic solution also simulated acidosis, K+ and lactate accumulation, and substrate deprivation. Twenty-minute tubular perfusion with the hypoxic and ischemic solutions (lumen and bath) at 37 degrees C did not significantly alter basolateral membrane potential, basolateral K+ transference number, or intracellular Na+ activity from control values of -69 +/- 1 mV (N = 91), 0.71 +/- 0.01 (N = 15), and 15.2 +/- 0.8 mM (N = 12), respectively. However, the hypoxic and ischemic perfusions decreased transepithelial potential by 40% (hypoxia: -1.7 +/- 0.1 to -1.1 +/- 0.1 mV [N = 30; P < 0.001]; ischemia: -1.4 +/- 0.1 to -0.82 +/- 0.05 mV [N = 17; P < 0.001]). A similar extent of reduction in transepithelial resistance was observed (hypoxia: 14.3 +/- 1.0 to 9.2 +/- 1.1 omega.cm2 [N = 7; P < 0.005]; ischemia: 12.6 +/- 1.2 to 8.1 +/- 1.0 omega.cm2 [N = 6; P < 0.03]). In addition, neither apical (R(ap)) nor basolateral (Rbl) cell membrane resistances were significantly altered after the ischemic perfusion (control: R(ap) = 369 +/- 48 omega.cm2; Rbl = 92 +/- 11 omega.cm2 [N = 63]; reperfusion: R(ap) = 454 +/- 88 omega.cm2; Rbl = 101 +/- 16 omega.cm2 [N = 21]). It was concluded that tubular cells are able to maintain their electrogenic ionic transport after short-term exposure to hypoxic or ischemic conditions. However, cell-to-cell junctions are damaged by these insults, which could possibly increase leakage and decrease the efficiency of the active transport.

Animals↗

PubMed1991

Cysteine residue periodicity is a conserved structural feature of variable surface proteins from Paramecium tetraurelia.

The DNA sequences of the entire coding regions of the A and C type variable surface protein genes from Paramecium tetraurelia, stock 51 have been determined. The 8151 nucleotide open reading frame of the A gene contains several tandem repeats of 210 nucleotides within the central portion of the molecule as well as a periodic structure defined by cysteine residues. The 6699 nucleotide open reading frame of the C gene does not contain any identifiable tandem repeats or internal similarity but maintains a periodicity based on the cysteine residue spacing. The deduced amino acid sequences encoded by the two genes are most similar within the 600 amino-terminal and 600 carboxyl-terminal amino acid residues, the central portions show only limited sequence similarity. We conclude that internal repeats are not a conserved feature of variable surface proteins in Paramecium and discuss the possible importance of the regular pattern of cysteine residues.

Amino Acid Sequence↗

PubMed1991

Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia.

A previously isolated mutant cell line called d48 contains a complete copy of the A surface antigen gene in the micronuclear genome, but the gene is not incorporated into the macronucleus. Previous experiments have shown that a cytoplasmic factor made in the wild-type macronucleus can rescue the mutant. Recently, S. Koizumi and S. Kobayashi (Mol. Cell. Biol. 9:4398-4401, 1989) observed that injection of a plasmid containing the A gene into the d48 macronucleus rescued the cell line after autogamy. It is shown here that an 8.8-kb EcoRI fragment containing only a portion of the A gene coding region is sufficient for the rescue of d48. The inability of other A gene fragments to rescue the mutant shows that this effect is dependent upon specific Paramecium DNA sequences. Rescue results in restoration of the wild-type DNA restriction pattern in the macronucleus. These results are consistent with a model in which the macronuclear A locus normally makes an additional gene product that is required for correct processing of the micronuclear copy of the A gene.

Animals↗