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H Yu

Publications and source records attributed to H Yu.

At least 649 records · Page 36Linked to original sources
PubMed1994

Purification and characterization of an elastinolytic metalloprotease from Aspergillus fumigatus and immunoelectron microscopic evidence of secretion of this enzyme by the fungus invading the murine lung.

Extracellular proteases have been suggested to be virulence factors in invasive aspergillosis. Since serine protease gene-disrupted mutants retain virulence, other proteases are suspected to be also involved in the degradation of lung structural material. An elastinolytic neutral metalloprotease was purified 320-fold from the extracellular fluid of Aspergillus fumigatus grown on elastin by affinity chromatography on bacitracin-Sepharose 4B and gel filtration on Sephadex G-75. The molecular mass was determined to be 43 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No carbohydrate was attached to this metalloprotease, and its first 22 N-terminal amino acids did not show any homology with the known metalloproteases. The enzyme was completely inhibited by EDTA, 1,10-phenanthroline, and phosphoramidon but not by inhibitors specific for serine, aspartate, and cysteine proteases. Zn2+ and, to a lesser extent, Co2+ reversed the inhibition caused by 1,10-phenanthroline. The protease hydrolyzed the peptide bonds His-Leu, Ala-Leu, Tyr-Leu, Gly-Phe, and Phe-Phe in the B chain of insulin. Synthetic substrate Abz-Ala-Ala-Phe-Phe-pNA could be used for the fluorimetric assay of the A. fumigatus metalloprotease. This enzyme had maximum activity in the pH range 7.5 to 8.0 and at 60 degrees C. It retained 50% of the protease activity when held at 60 degrees C for 1 h. Zn2+ and Co2+ at 1 mM did not inhibit the protease activity. The metalloprotease was able to hydrolyze elastin, and its elastinolytic activity was comparable to that of the serine protease from this organism. The presence of Zn2+ in the culture medium stimulated the metalloprotease production. Rabbit antibodies prepared against the enzyme severely inhibited the enzyme activity. Immunogold electron microscopy revealed that A. fumigatus invading neutropenic mouse lungs secretes this metalloprotease.

Amino Acid Sequence↗
PubMed1994

Analysis of promoters controlled by the putative sigma factor AlgU regulating conversion to mucoidy in Pseudomonas aeruginosa: relationship to sigma E and stress response.

Alginate overproducition by mucoid Pseudomonas aeruginosa is a critical pathogenic determinant expressed by this organism during chronic infections in cystic fibrosis. Conversion to mucoidy and a subsequent loss of mucoid character can occur via different mutations in the algU mucA mucB gene cluster. The algU gene encodes a 22.2-kDa putative alternative sigma factor required for expression of the critical alginate biosynthetic gene algD. In this work, algU transcription was studied by S1 nuclease protection analysis. Transcription from the promoter proximal to the algU coding region was found to be dependent on AlgU. The -35 and -10 sequences of this newly mapped promoter showed strong similarity ot the promoters of two other critical alg genes: algD and algR. The proximal promoter of algR was also shown to depend on algU. Interestingly, the putative -35 and -10 regions of all three promoters displayed striking similarity to the consensus sequence of the sigma E-dependent promoters in Escherichia coli and Salmonella typhimurium. This 24-kDa sigma factor, controlling genes participating in resistance to high temperatures and oxidative stress, has been previously biochemically characterized, but the gene for sigma E remained unidentified. To examine whether AlgU is related to sigma E, the effect of algU inactivation on the sensitivity of P. aeruginosa to killing by heat and reactive oxygen intermediates was tested. Two isogenic pairs of algU+ and algU mutant strains were compared. The algU mutants, irrespective of the mucoid status of the parental strains, displayed increased sensitivity to killing by paraquat, known to generate intracellular superoxide radicals, and heat. Further lgobal homology searches revealed the presence of a previously unrecognized E. coli gene with the predicted gene product showing a striking 66% identity to AlgU. The corresponding gene from S. typhimurium was cloned and sequenced, and it is displayed one amino acid substitution relative to its E. coli equivalent. AlgU and its close homologs in E. coli and S. typhimurium may be functionally related.

Adaptation, Physiological↗
PubMed1994

Cellular protein modulates effects of human immunodeficiency virus type 1 Rev.

Replication of human immunodeficiency virus type 1 requires expression of the viral trans activator Rev. Rev binds to a highly structured RNA, the Rev response element, which is present in singly spliced and unspliced genomic viral RNAs. Although Rev helps to transport these transcripts from the nucleus to the cytoplasm, the mechanism(s) involved is not fully understood. Using the yeast two-hybrid system, we isolated a murine protein (YL2) that interacts with the basic domain of Rev, which is essential for the function of Rev in vivo and for the inhibitory splicing activity of Rev in vitro. YL2 has 92% identity to a human 32-kDa protein (p32), which copurifies with alternative splicing factor SF2/ASF. Furthermore, we found that whereas expression of YL2 greatly potentiated the activity of Rev, antisense YL2 transcripts blocked the effects of Rev in mammalian cells. YL2 also increased the activities of Rex on the Rex response element and of hybrid Rev proteins fused to Tat and the coat protein of bacteriophage MS2 on their respective RNAs. Thus, YL2 or p32 is a cellular protein that modulates the function of human immunodeficiency virus type 1 Rev.

Amino Acid Sequence↗
PubMed1994

Dietary sodium and central vs. peripheral ouabain-like activity in Dahl salt-sensitive vs. salt-resistant rats.

To assess the possible contribution of brain ouabain-like activity (OLA) to the pressor effects of high-sodium intake in Dahl salt-sensitive (Dahl S) rats, we assessed the effects of high (8%) on blood pressure (BP) and peripheral and brain OLA in Dahl on blood pressure (BP) and peripheral and brain OLA in Dahl S and Dahl salt-resistant (Dahl R) rats. On regular sodium intake, Dahl S and R had similar BP; however, by 7 wk of age adrenal and plasma OLA were 15-30% higher in Dahl S vs. R, whereas central OLA remained similar. On high-sodium intake, in Dahl S both peripheral and central OLA increased within 1 wk with additional increases after 3 wk. These increases preceded the rise in BP. In Dahl R rats, high sodium did not increase BP. However, 3 wk of high sodium did increase peripheral as well as central OLA, the latter to a lesser extent compared with Dahl S and not in the hypothalamus. These results are consistent with the concept that central OLA may be involved in the pressor responses to high sodium in Dahl S. Circulating OLA may play a role in the regulation of renal function to excrete excess sodium in both strains.

Adrenal Glands↗
PubMed1994

Actions of vasoactive intestinal peptide and neuropeptide Y on the pacemaker current in canine Purkinje fibers.

We have investigated the actions of vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) on the pacemaker current (I(f)) in canine Purkinje fibers. On voltage pulses to the middle of the I(f) activation range, VIP reversibly increases I(f), whereas NPY reversibly decreases I(f). A three-pulse voltage protocol suggests that VIP shifts I(f) activation in the positive direction and that NPY shifts I(f) activation in the negative direction on the voltage axis without changing maximal I(f) conductance. These effects of VIP and NPY on I(f) are exerted through their specific peptide receptors, since the effects are blocked by VIP and NPY receptor antagonists. VIP and NPY are colocalized in cardiac parasympathetic and sympathetic nerve endings, respectively, and can be released preferentially on high and long-lasting nerve stimulation. Given this colocalization and frequency-dependent release, these results suggest a role for these neuropeptides in controlling cardiac I(f) and consequently heart rate.

Animals↗
PubMed1994

Evidence for direct local effect of angiotensin in vascular hypertrophy. In vivo gene transfer of angiotensin converting enzyme.

In vitro studies have demonstrated that angiotensin (Ang) II directly stimulates vascular smooth muscle cell (VSMC) growth. However, it is still unclear if Ang II exerts a direct effect on vascular hypertrophy in vivo independent of its effect on blood pressure. In vivo gene transfer provides the opportunity to assess the effects of increased activity of the vascular angiotensin system in the intact animal while avoiding an increase in circulating angiotensin or in blood pressure. Accordingly, we transfected the human angiotensin converting enzyme (ACE) vector into intact rat carotid arteries by the hemagglutinating virus of Japan-liposome method. 3 d after transfection, we detected increased ACE activity in the transfected artery. Immunohistochemistry localized immunoreactive ACE in the medial VSMC as well as in the intimal endothelial cells. The increase in vascular ACE activity was associated with a parallel increase in DNA synthesis as assessed by BrdU (bromo-deoxyuridine) index and vascular DNA content. This increase in DNA synthesis was abolished by the in vivo administration of an Ang II receptor-specific antagonist (DuP 753). Morphometry at 2 wk after transfection revealed an increase in the wall to lumen ratio of the ACE-transfected blood vessel as compared with control vector transfected vessels. This was accompanied by increases in protein and DNA contents without an increase in cell number. Local transfection of ACE vector did not result in systemic effects such as increased blood pressure, heart rate, or serum ACE activity. These morphological changes were abolished by the administration of the Ang II receptor antagonist. In this study, we used in vivo gene transfer to increase local expression of vascular angiotensin converting enzyme and provided proof that increased autocrine/paracrine angiotensin can directly cause vascular hypertrophy independent of systemic factors and hemodynamic effects. This approach has important potentials for defining the role of autocrine/paracrine substances in vascular biology and hypertension.

Angiotensin I↗
PubMed1994

Differential regulation of brain angiotensin II in genetically hypertensive and normotensive rats after nephrectomy.

To investigate the role of tissue angiotensin II (Ang II) in the maintenance of hypertension after nephrectomy in spontaneously hypertensive rats (SHR), Ang II levels were measured in various tissues of both 12-week-old SHR and normotensive control, Wistar-Kyoto rats (WKY), 48 h after nephrectomy or sham operation. Ang II was determined by radioimmunoassay coupled with high performance liquid chromatography. Nephrectomy caused a decrease of plasma renin activity and plasma Ang II concentration in both SHR and WKY. Aortic Ang II levels were significantly lowered by nephrectomy only in WKY, and not in SHR. Ang II levels in hypothalamic block, brainstem and cerebellum of SHR increased after nephrectomy, whereas those of WKY were unchanged. Intracerebroventricular administration of ceronapril, an angiotensin converting enzyme inhibitor, significantly decreased sustained high blood pressure in SHR 48 h after nephrectomy compared with vehicle administration, whereas intravenous administration had no effect. These results suggest that in spite of the important role of the renal renin-angiotensin system in maintenance of high blood pressure in SHR, control mechanisms may switch to other systems after nephrectomy, and that the increased brain Ang II levels after nephrectomy may be related to these mechanisms.

Angiotensin II↗
PubMed1994

The water channel gene in human uterus.

The cDNA coding the water channel was isolated from a human uterus cDNA library template by a one-step polymerase chain reaction (PCR). The oligonucleotide primers corresponding to the 5' untranslated nucleotide sequence and complementary to the 3' untranslated nucleotide sequence of the cDNA coding the 28 kDa erythrocyte integral membrane protein (CHIP28) were synthesized and used to initiate the reaction. A 1340 bp cDNA coding the human uterine water channel (hUWC) was cloned and sequenced. The hUWC showed 99.8% and 99% identity with the nucleotide and amino-acid sequences of CHIP28, respectively. The deduced hUWC polypeptide is composed of 269 amino acid residues with a single amino acid variant from CHIP28 protein at position 45, where valine replaces alanine. The hUWC cDNA translated in a prokaryotic protein expression system produced a protein with an estimated Mr of 28 kDa, equivalent to the size of the human red cell CHIP28 protein. The present results suggest that the human uterus contains water channels that may play an important role in regulating water transport and imbibition in the uterus.

Amino Acid Sequence↗
PubMed1994

Bovine and rodent tamm-horsfall protein (THP) genes: cloning, structural analysis, and promoter identification.

We have isolated bovine and rodent cDNA and genomic clones encoding the kidney-specific Tamm-Horsfall protein (THP). In both species the gene contains 11 exons, the first of which is noncoding. Exon/intron junctions were analyzed and all were shown to follow the AG/GT rule. A kidney-specific DNase I hypersensitive site was mapped onto a rodent genomic fragment for which the sequence is highly conserved in three species (rat, cow, and human) over a stretch of 350 base pairs. Primer extension and RNase protection analysis identified a transcription start site at the 3' end of this conserved region. A TATA box is located at 32 nucleotides upstream of the start site in the bovine gene and 34 nucleotides upstream in the rodent gene. An inverted CCAAT motif occurs at 65 and 66 nucleotides upstream of the start site in the bovine and rodent genes, respectively. Other highly conserved regions were noted in this 350 bp region and these are likely to be binding sites for transcription factors. A functional assay based on an in vitro transcription system confirmed that the conserved region is an RNA Pol II promoter. The in vitro system accurately initiated transcription from the in vivo start site and was highly sensitive to inhibition by alpha-amanitin at a concentration of 2.5 micrograms/ml. These studies set the stage for the further definition of cis-acting sequences and trans-factors regulating expression of the THP gene, a model for kidney-specific gene expression.

Amino Acid Sequence↗
PubMed1994

[Sequence analysis of N--ras and p53 gene mutation in the human lung adenocarcinoma cell lines].

Gene mutation and abnormal expression of ras oncogenes and p53 gene have a direct bearing on the carcinogenesis. Polymerase chain reaction (PCR), sequencing technique of ds DNA cycle and sequencing system were used to detect the gene mutation of N-ras as well as the exon 5 and 7 of p53 gene in hLA and LTEP-a2 cell lines of human lung adenocarcinoma. The result showed that mutation of both cell lines occurred on the 154th codon in exon5 of p53 gene, where GGC was displaced by GTC resulting a substitution of Val for Gly, nevertheless, N-ras oncogene and the exon7 of p53 gene are normal.

Adenocarcinoma↗
PubMed1994

[The pharmacokinetics of ciprofloxacin in patients with renal function of various degrees].

The pharmacokinetics of ciprofloxacin was investigated in 12 patients with renal function of various degrees. A single dose of 500 mg of ciprofloxacin was administered after overnight fasting. Serum and urine samples were collected serially at different intervals and subjected to bioassay and HPLC. Pharmacokinetic parameters were analyzed by assuming an open two-compartment model. Apparent difference was observed in pharmacokinetic parameters between patients with severe impairment of renal function and those with normal renal function. For the former group, the area under the curve of serum concentration versus time was tripled (42.22 +/- 12.48 vs. 14.55 +/- 6.66 mg.h-1.ml-1, the renal clearance of ciprofloxacin was reduced to one-tenth (22.43 +/- 12.08 vs. 193.13 +/- 65.14 ml/min), and the elimination half-life was prolonged twofold (9.28 +/- 2.42 vs. 4.72 +/- 1.35 h). The correlation between renal drug clearance and Ccr was highly significant (r = 0.935, P < 0.001). On the basis of these findings, it is suggested that a reduction of one half of the dose of ciprofloxacin or an increase of a double of the administration time interval may be indicated in chronic renal failure with GFR less than 25 ml/min.

Adult↗
PubMed1994

[Helicobacter pylori in the dental plaque].

Helicobacter pylori (Hp) were detected in the dental plaque in 40 patients with peptic ulcer, chronic gastritis or gastric cancer with rapid urease test, anti-Hp fluorescein-labelled antibody staining, bacterial culture and electronic microscopy. At the same time, biopsy specimens from the gastric antrum of these patients were studied with WS staining and rapid urease test to detect Hp. The results show that a great lot of Hps is present in most of the patients' dental plaque. The morphological, biochemical and immunological characteristics of the Hp in the dental plaque are similar to those Hp in the gastric mucosa. The above results reveal that the Hp in the dental plaque may be identical with those in the stomach.

Dental Plaque↗
PubMed1994

Effect of protein kinase C on cyclic 3',5'-adenosine monophosphate-dependent phosphodiesterase in hypertrophic cardiomyopathic hamster hearts.

"Cross-talk," or interaction between different signal transduction pathways, is known to exist in noncardiac cells, but it has not been demonstrated previously in mammalian hearts. We found that hypertrophic cardiomyopathic Syrian hamster (BIO 14.6, 6 months old) hearts were deficient in cyclic 3',5'-adenosine monophosphate (cAMP) [11.9 +/- 0.4 vs. 15.4 +/- 0.4 pmol/mg protein in age-matched control hamsters (BIO RB), n = 6, P = .0005] but not in cyclic 3',5'-guanosine monophosphate (1.23 +/- 0.10 vs. 1.34 +/- 0.18 pmol/mg protein in BIO RB, n = 6, P = N.S.). The reduction in cAMP was at least partly accounted for by an increase in the cytosolic phosphodiesterase (PDE) activity in BIO 14.6 hearts (1709 +/- 119 vs. 1341 +/- 113 pmol/min/mg protein in age-matched BIO RBs, n = 12, P = .006), suggesting that there is an increase in cAMP turnover in BIO 14.6 hearts. Protein kinase C (PKC) activities were also significantly elevated in BIO 14.6 hearts (77.9 +/- 2.1 vs. 54.6 +/- 3.3 pmol/min/mg protein in BIO RBs, n = 6, P < .001). Activation of PKC by phorbol 12-myristate 13-acetate (PMA, 10 microM) produced significant potentiation in PDE activities in BIO 14.6 but not in BIO RB hearts, and the PMA-induced increase in PDE activity could be blocked by the PKC-specific pseudosubstrate inhibitor peptide PKC(19-31).(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

[Preparation of antibodies against zip product and the distribution of zip protein in Drosophila embryos].

Zip gene is required during the late neurogenesis of Drosophila. Molecular analysis of zip gene revealed that it encodes an integral membrane glycoprotein, functioning as a cellular recognition and/or adhesion molecule. It has been cloned into an expression plasmid pWR 590. By expression of this plasmid in E. coli, a fusion protein of zip with lacZ has been purified and used to prepare polyclonal antibodies from rabbits. After being identified by West blotting, antibodies were used to react with the whole mount embryos of Drosophila. The results of this antibody labelling experiment showed that zip proteins are fundamentally synthesized after germ band shortening, supporting our previous predication that zip gene is involved in the late neurogenesis and that antibodies against zip recognize lateral neural fascicles as well as neurons in CNS, proving that zip protein expressed in CNS may play a role in the establishment and maintenance of neural fasciculation.

Animals↗
PubMed1994

Localization of the ligand binding site of the neurokinin-1 receptor: interpretation of chimeric mutations and single-residue substitutions.

Previous studies have indicated that substitution of the third or fourth extracellular segment of the human neurokinin-1 receptor with the equivalent segment from the neurokinin-3 receptor affects the binding affinities of peptide agonists and/or nonpeptide antagonists. To elucidate the roles of the divergent residues within these domains in ligand binding, single-residue substitutions in these regions were analyzed. Neurokinin B affinity was increased by one single-residue substitution (E172K) in the third extracellular segment and one double-residue replacement (L279R+K280W) in the fourth extracellular segment, and the affinity for the antagonist L-703,606 was reduced by one single-residue substitution (Y272A) in the fourth extracellular segment. The effect of these three specific substitutions is consistent with the prediction of chimeric mutations. However, the substantial reduction in ligand binding affinity observed upon multiple-residue substitutions in the third extracellular segment (residues 176-183 or 187-195) has not been reproduced by eliminating potential electrostatic interactions or substituting with the corresponding residues from the neurokinin-3 receptor, suggesting that the reduction in ligand binding affinity observed with some chimeric receptors is not due to the loss of direct electrostatic interactions. These data indicate that other factors such as conformational effects may complicate the interpretation of data obtained with chimeric receptors, and they demonstrate the need to evaluate chimeric receptors along with single-residue substitutions in the same region to localize specific residues involved in ligand binding. Furthermore, the available data suggest that one major determinant of peptide selectivity in the neurokinin-1 receptor may be the conformational compatibility between a peptide and the receptor.

Amino Acid Sequence↗
PubMed1993

Derivatives of 2-(dipropylamino)tetralin: effect of the C8-substituent on the interaction with 5-HT1A receptors.

A series of 2-(dipropylamino)tetralin derivatives in which the C8 substituent is varied has been prepared and evaluated pharmacologically to explore the importance of the C8 substituent in the interaction of 2-aminotetralin-based ligands with serotonin (5-HT1A) receptors. Enantiopure derivatives were prepared by facile palladium-catalyzed reactions of the triflates of the enantiomers of 8-hydroxy-2-(dipropylamino)tetralin (8-OH-DPAT, 1). The affinity of the compounds for the 5-HT1A receptors was evaluated by competition experiments with [3H]-8-OH-DPAT in rat hippocampal and cortical tissue. In addition, the compounds were evaluated for central 5-HT and dopamine receptor stimulating activity in vivo by use of biochemical and behavioral assays in rats. With the exception of the carboxy-substituted derivative which is devoid of 5-HT1A receptor affinity, the compounds have moderate to high affinities (K(i) values range from 0.7 to 130 nM) for 5-HT1A receptors. Surprisingly, several of the derivatives do not produce any apparent effects in vivo although they have fairly high 5-HT1A receptor affinities. However, the methoxycarbonyl- and acetyl-substituted derivatives are potent 5-HT1A receptor agonists in vivo and exhibit in vitro affinities in the same range as the enantiomers of 1.

5-Hydroxytryptophan↗
PubMed1993

Sequential assignment and structure determination of spider toxin omega-Aga-IVB.

The solution structure of a peptide toxin isolated from funnel web spider venom, omega-Aga-IVB, was determined by 2D NMR methods. omega-Aga-IVB is a high-affinity specific blocker of P-type voltage-dependent calcium channels. Nearly all of the proton resonances of this 48-residue protein were assigned using conventional 2D homonuclear NMR experiments. The three-dimensional structure of the molecule was determined by simulated annealing. The distance and dihedral restraints used in the structure calculations were derived from NOESY and COSY-type experiments, respectively. Mass spectrometric analysis of omega-Aga-IVB suggests that the protein contains four disulfide bonds. In the absence of chemical data to identify the pattern of cysteine pairing, the disulfide bonds of the toxin are proposed from the NMR data and subsequent structural calculations. The structure of the toxin can be described as a three-stranded anti-parallel beta sheet connected by flexible loops. A striking feature of the structure is that the C-terminal 10 residues of this protein adopt random coil conformations. Several positively charged amino acid side chains are found localized on one face of the molecule, in close proximity to the C-terminal tail. This observation has led us to propose a speculative model of the toxins blockade mechanism.

Agatoxins↗