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Biomedical subjects

B Huang

Publications and source records attributed to B Huang.

At least 145 records · Page 8Linked to original sources
PubMed1996

Splase: a new class IIS zinc-finger restriction endonuclease with specificity for Sp1 binding sites.

A new restriction endonuclease, named Splase, was constructed by genetically fusing the DNA-cleavage domain of the restriction endonuclease Fok1 with the zinc-finger DNA-binding domain of the transcription factor Sp1. The resulting protein was expressed in Escherichia coli., partially purified, and shown to selectively digest plasmid DNA harboring consensus Sp1 sites. Splase was also shown to selectively digest the long terminal repeat of the HIV-1 DNA at Sp1 sites. Splase recognizes a 10-bp DNA sequence and hydrolyzes phosphodiester bonds upstream of the binding sequence. The binding specificity of Splase makes this a "rare cutter" restriction enzyme which could be valuable in creating large DNA fragments for genome sequencing projects. The result also presents the opportunity to create other restriction enzymes by altering the binding specificity of the zinc-finger recognition helix.

Base Sequence↗
PubMed1996

Preconcentration of trace elements from natural water with palladium precipitation.

Palladium salts can be used as a coprecipitation carrier for the preconcentration of trace elements from natural water prior to their measurement by atomic spectrometry (AAS). The palladium is subsequently reduced by the introduction of hydrogen gas into the sample solution. The procedure is applied to the determination of Cu, Pb and Cd in seawater (enrichment factor 50) and synthetic water samples. Operating conditions have been optimized for the analysis of real samples. With the technique established an enrichment factor (500 fold) is feasible in synthetic samples. The recoveries of Cu, Cd and Pb from seawater are 95, 103 and 100%, respectively. This simple and rapid method can be applied in a wide pH-range and with complex matrices.

Journal Article↗
PubMed1996

Comparison of bladder rupture pressure after intestinal bladder augmentation (ileocystoplasty) and myomyotomy (autoaugmentation).

OBJECTIVES: To compare the risk of bladder rupture of bladder augmentation using ileocystoplasty versus that of autoaugmentation with myomyotomy in a rat model. METHODS: Bladder rupture pressure and volume of three groups of female Sprague-Dawley rats were determined by cystometry. The first group of 11 rats had undergone ileocystoplasty using a detubularized 1 -cm segment of ileum. A second group of 9 rats had undergone autoaugmentation with myomyotomy. One month after surgery the animals were studied cystometrically to determine the bladder rupture pressure, then killed. A third group, consisting of 10 nonoperated rats, was studied and served as controls. RESULTS: Nonoperated, control rat bladders were able to sustain 154 +/- 43 mm Hg pressure and 2.5 +/- 2.0 mL volume prior to bladder rupture. Conventional ileocystoplasty was noted to increase bladder capacity to 4.0 +/- 1.9 mL, but decrease rupture pressure to 111 +/- 49 mm Hg. Myomyotomy resulted in a mean bladder rupture volume of 1.2 +/- 0.4 mL, with a rupture pressure of 101 +/- 13 mm Hg. The rupture pressure after myomyotomy is significantly lower than that of the native bladder (P < 0.001), whereas the rupture volume after myomyotomy is significantly lower than either after the ileocystoplasty or with the native bladder (P < 0.001). Bladder rupture occurred at the augmented ileal bladder dome in 7 of 11 ileocystoplasty animals and at the anastomotic suture line in 4 animals. Bladder rupture occurred at the area of bladder diverticulum in all 9 myomyotomy animals. Among controls, no specific site pattern of bladder rupture was noted. CONCLUSIONS: Bladder augmentation with myomyotomy increases vulnerability to urinary extravasation, evidenced by a significantly reduced rupture pressure and bladder volume at rupture when compared to the native bladder.

Animals↗
PubMed1996

Structural basis for IL-4 receptor phosphopeptide recognition by the IRS-1 PTB domain.

We present the NMR structure of the PTB domain of insulin receptor substrate-1 (IRS-1) complexed to a tyrosine-phosphorylated peptide derived from the IL-4 receptor. Despite the lack of sequence homology and different binding specificity, the overall fold of the protein is similar to that of the Shc PTB domain and closely resembles that of PH domains. However, the PTB domain of IRS-1 is smaller than that of Shc (110 versus 170 residues) and binds to phosphopeptides in a distinct manner. We explain the phosphopeptide binding specificity based on the structure of the complex and results of site-directed mutagenesis experiments.

Amino Acid Sequence↗
PubMed1996

Differential expression of voltage-gated K+ channel genes in left ventricular remodeled myocardium after experimental myocardial infarction.

Left ventricular (LV) remodeling after experimental myocardial infarction (MI) is associated with hypertrophy of noninfarcted myocardium and electrophysiological alterations. We have recently shown that post-MI hypertrophied LV myocytes have prolonged action potential duration (APD) and generate triggered activity from early afterdepolarizations. The prolonged APD was attributed to decreased density of the two outward K+ currents, I(to)-fast (I(to)-f) and I(to)-slow (I(to)-s), rather than changes in the density and/or kinetics of the L-type Ca2+ current. The changes in ionic current density may be related to alterations in the expression and levels of ion channel proteins. To test this hypothesis, rats underwent either left anterior descending coronary artery (LAD) ligation (post-MI group [n = 10]) or sham surgery (sham group [n = 10]). Three weeks later transcripts from the noninfarcted LV myocardium in the post-MI group (n = 6) and LV myocardium of the sham group (n = 6) were analyzed by RNase protection assay. Expressions of five K+ channel subunit mRNAs (Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.2) reported in the rat ventricle were analyzed. Compared with the sham group, expressions of Kv1.4, Kv2.1 (putative I(to)-s), and Kv4.2 (putative I(to)-f) channel subunit mRNAs were significantly decreased by 60% (P < .03), 54% (P < .005), and 53% (P < .002), respectively, in the post-MI group. There was no significant change in the Kv1.2 and Kv1.5 mRNA levels. Western blotting demonstrated a similar decrease in the Kv2.1 and Kv4.2 immunoreactive protein levels (43% [P < .03] and 67% [P < .003], respectively [n = 4]) and no significant change in Kv1.5 immunoreactive protein level. Our results strongly correlate with the electrophysiological findings in this model and show that transcriptional regulation in the post-MI remodeled rat LV is distinct for each voltage-gated K+ channel subunit. These findings provide, at least in part, the molecular basis for the electrophysiological alterations observed in this model.

Animals↗
PubMed1996

Evidence of Na Current Contribution to the Transient Outward Current in Cardiac Ventricular Myocytes.

BACKGROUND: To study the transient outward current (I(to)) investigators often use sodium-free external solution to minimize the possible contamination of I(to) by sodium current. Removal of extracellular sodium creates reversal of sodium gradient and thus possibly contributing to I(to) mainly at positive potentials. METHODS AND RESULTS: To address this issue, whole-cell I(to) was recorded in sodium-free choline chloride and cobalt solutions, from rat ventricular myocytes known to exhibit a prominent I(to). Depolarizing pulse to 40 mV from -100 mV holding potential every 10 seconds elicited a fast activating and time-dependent inactivating components. The activation of I(to) was fast and complete within 10 ms at 40 mV, and the decay was rapid over the first 100 ms of the pulse and slower thereafter. External superfusion of the cell with 50 µM tetrodotoxin reversibly reduced I(to) amplitude by 25% from 1.47 +/- 0.2 to 1.1 +/- 0.3 nA (P <.04, n = 9). When sea anemone toxin (ATXII), known to selectively enhanced I(Na) by causing a delay in the inactivation gate, is applied to the cell, I(to) amplitude increased in a time- and dose-dependent manner (EC(50) =.86.4 nM). ATXII (100 nM) dramatically increased I(to) amplitude at all voltages between -20 and 60 mV (from 1.51 +/- 0.4 to 3.35 +/- 0.8 nA at 40 mV, P <.003, n = 12). Superfusion of cells with 5 mM 4-AP resulted in 82% reduction in I(to) amplitude at 40 mV (from 1.95 +/- 0.5 to 0.37 +/- 0.2 nA, P <.02, n = 8). Addition of ATXII to 4-AP containing solution increased peak I(to) by 965% (from 0.37+/-0.2 to 3.95 +/- 0.9, n = 8, P <.0003). However, in 11 other cells, addition of tetrodotoxin (50 µM) to the ATXII-containing solution blocked ATXII-induced outward current (from 3.51 +/- 0.64 nA to 1.60 +/- 0.17 nA, P <.05). The conductance (G(Ito)) was calculated by dividing peak I(to) by (Vm-E(K)), with an E(K) of -75 mV. G(Ito) was increased at all voltages (greater than -40 mV). Normalized G(Ito) was fitted by Boltzmann equation and ATXII did not significantly modify V(0.5) and k (from -20.5 +/- 3.9 to -17.0 +/- 3.5 mV for V(0.5), and 12.2 +/- 2.6 to 13.4 +/- 2.1 mV for k, n = 4). Also, atropine (1 µM) did not have any significant effect on I(to) (from 1.92 +/- 0.15 nA to 1.85 +/- 0.25 nA, n = 5). CONCLUSIONS: The results showed that, in sodium-free external solution I(to) is tetrodotoxin but not atropine sensitive. ATXII-induced I(to) increase is 4-aminopyridine insensitive but tetrodotoxin sensitive. These data suggest that outward Na current due to reversal of Na gradient in the absence of external Na contributes to I(to). Caution must be taken when studying kinetics and pharmacology of I(to) in external sodium-free solutions.

Journal Article↗
PubMed1996

Impaired glucose tolerance, diabetes, and cardiovascular disease risk factor profiles in the elderly. The Honolulu Heart Program.

OBJECTIVE: The relationship between glucose tolerance status and other cardiovascular disease (CVD) risk factors was evaluated in a cohort of Japanese-American men (n = 3,741) ages 71-93 years who participated in the fourth examination of the Honolulu Heart Program in 1991-1993. RESEARCH DESIGN AND METHODS: In this cross-sectional study, subjects were classified by reported diabetes and glucose tolerance status using questionnaires and the World Health Organization (WHO) criteria, respectively. RESULTS: The prevalence of reported diabetes was 17%. Among the men who completed an oral glucose tolerance test and had no history of diabetes (n = 1,900), 23% were diagnosed as diabetic and 39% had impaired glucose tolerance (IGT) by WHO criteria. The CVD risk factor profiles of men with IGT and diabetes were significantly more adverse compared with men with normal glucose tolerance after adjustment for age. The rates of hypertension, mean levels of BMI, waist-to-hip ratio, triglycerides, and fasting insulin were higher in men with IGT and diabetes compared with normal subjects. Opposite trends were observed for HDL cholesterol. Two-hour insulin was significantly higher among men with IGT and previously undiagnosed diabetes. Men with known diabetes had a lower physical activity index and higher fibrinogen levels than normal subjects. No significant differences were observed for current smoking and alcohol intake. Differences in risk factor levels by glucose tolerance status remained after adjustment for age, physical activity, BMI, and waist-to-hip ratio. CONCLUSIONS: These findings show that among elderly men of Japanese ancestry, impaired glucose tolerance and undiagnosed and known diabetes are highly prevalent, and these conditions are associated with adverse CVD factor profiles.

Age Factors↗
PubMed1996

[Management of anterior vitreous by high gluey Healon].

PURPOSE: To investigate the technique of managing anterior vitreous by high gluey Healon. METHODS: In 15 eyes of 15 cases having undertaken in situ phacoemulsification vitreous loss, 12 eyes of 12 cases in ECCE, 3 eyes of 2 cases in Marfan Syndrome, 13 eyes of 13 cases in eye injury, tunnel incision was made and PC-IOL implantation was performed after high gluey Healon managing anterior vitreous on all the cases. RESULTS: High gluey Healon managing anterior vitreous in 43 eyes, post capsule injuries was not enlarge, and cortex or fragments of len nuclear not fall in vitreous cavity. Visual acuity of 0.5 or better at postoperative one week were 72.1% and 59.1% respectively. CONCLUSION: The technique of managing anterior vitreous by high gluey Healon was worthy to be spread due to minimum injury, vitreous loss, and fast restoration of visual acuity.

Adolescent↗
PubMed1996

[Comparison of human papilloma virus-DNA in condyloma acuminatum, cervical cancer and the female genital tract].

OBJECTIVE: To examine human papilloma virus (HPV) infection rate and types in several lesions of the genital tract and to observe the relation between HPV and host cells for differential handling of cases and for early diagnosis. METHODS: Tissues from 89 cases of condyloma acuminata, 76 cases of cervical cancer and swabs from 198 cases of normal vaginal were analysed by polymerase chain reaction for HPV infective rate. Main HPV types were compared and Southern blot was used to compare the physical pattern of HPV in condyloma acuminata and cervical cancer. RESULTS: The positive rates of HPV-DNA in condyloma acuminata and cervical cancer were respectively 98.9% and 94.7%. The primary types of the former were HPV 6, 11 and of the latter mainly 16 and 18. The latent infective rates in normal vagina was 37.4%, among which, HPV 6, 11, 16, 18 consisted of only 21.6%. HPV11-DNA in condyloma acuminata existed mainly in episodic form. Most of HPV16-DNA in cervical cancer integrates into the host cells with variation. CONCLUSIONS: The different types of HPV are related to particular morphology and characteristics of lesions. The relation of HPV-DNA to host cell and can offer some reference for the determination of the character of the lesion, and it may be award of malignant transformation.

Adolescent↗
PubMed1996

Gracilis muscle dynamic urethral sphincter myoplasty: rat model experience.

PURPOSE: Dynamic urethral sphincter myoplasty (skeletal muscle urinary sphincter reconstruction) using a neurovascularly intact gracilis muscle was investigated in a rat model. MATERIALS AND METHODS: In female Sprague-Dawley rats, a unilateral gracilis anticus muscle flap was dissected from the medial thigh, preserving the medial muscular insertion, vascular flow, and innervation. This muscle graft was used to completely encircle the urethra and was fixed in position. Urodynamic leak point pressure (LPP) and bladder volume at leakage were measured with cystometry after 1 month, using an 18 gauge catheter placed through the bladder dome with a constant infusion rate of 0.2 ml. per minute. In addition, the effect of electrical stimulation of the gracilis myoplasty (current parameters: 1 to 10 mA, 1 to 60 Hz, 0.05 to 1 msec. duration) on intravesical leak point pressure was noted during cystometry. RESULTS: The gracilis muscle measured 3.8 +/- 0.3 cm. in length, 0.5 +/- 0.1 cm. in width and 0.2 +/- 0.1 cm. in thickness. Blood flow rates to the grafted and contralateral gracilis myoplasty were similar at 43 +/- 26 and 51 +/- 30 g.cm.3, respectively (p = 0.46). The leak point pressure (LPP) of control, unstimulated gracilis myoplasty and gracilis myoplasty with electrical stimulation were 28 +/- 8, 32 +/- 12, and 85 +/- 27 mm.Hg (p < 0.01). Bladder volumes at LPP in the 3 respective groups were 0.5 +/- 0.2, 0.6 +/- 0.3 and 1.2 +/- 0.6 ml (p < 0.01). CONCLUSIONS: Gracilis myoplasty is not obstructive, as substantiated by unchanged leak point pressure and leak point capacity. Myoplasty with low current stimulation, however, significantly increased LPP and leak point capacity.

Animals↗
PubMed1995

Reemergence of the fetal pattern of L-type calcium channel gene expression in non infarcted myocardium during left ventricular remodeling.

The cardiac L-type voltage-dependent calcium channel (VDCC) is a critical component of cardiac action potential and excitation-contraction coupling. The objective of the present study was to examine the changes in expression in Motif IV, an alternatively spliced region of the alpha-1 subunit of the VDCC channel in postmyocardial infarction (MI) remodeled rat left ventricle. RNase protection assay was used to determine alteration in isoform expression in the noninfarcted hypertrophied ventricular myocardium 21 days post myocardial infarction. Our study demonstrates that cardiac hypertrophy is associated with significant increase in the mRNA level of the fetal isoform, with the reversion of fetal:adult isoform ratio to the fetal phenotype. Changes in isoform expression in the post-MI remodeled ventricle, not previously reported, is a pertinent genetic marker of cardiac hypertrophy.

Alternative Splicing↗
PubMed1995

Identification of an upstream region that controls the transcription of the human autocrine motility factor receptor.

We have isolated from a human placenta cosmid library a 0.7 kb genomic clone that contains the 5' terminal portion of the autocrine motility factor receptor (hAMFR) coding region. Chloramphenicol Acetyl Transferase (CAT) reporter gene assays have identified this region as the promoter of the hAMFR gene. A single transcription initiation site (+1) has been mapped to 129 bp upstream of the ATG start codon by primer extension. DNA sequence analysis and CAT assay revealed a TATA element at the position -485/-468 which was able to conduct only a marginal transcription (less than 5% of the total activity). The majority of the hAMFR promoter's activity is contributed by a transcription initiator (Inr) element overlapping the initiation site (+1) which independently controls the transcription of the hAMFR gene. Gel mobility shift assays showed that DNA-binding proteins in HeLa cells nuclear extract can bind specifically to both promoter's elements. DNA-binding proteins were found to be differentially expressed by sparse and dense cultured normal fibroblasts. The nuclear-binding protein expressed by sparse NIH-3T3 cells induced a DNA mobility shift similarly to the nuclear protein of HeLa cells, while a different DNA-protein complex size was observed with nuclear proteins extracted from dense cultured NIH-3T3 cells. Also CAT-reporter gene analysis revealed a significant lower activity in dense NIH-3T3 cells as compared with the sparse-cultured counterparts. These results help to explain the previously observed cell-cell contact regulation of AMFR expression in normal cells and its consecutive expression in tumor cells.

3T3 Cells↗
PubMed1995

Heritability and heteromorphic distributions of AluI chromosome banding variants in twins.

The heritability and heteromorphic appearance of chromosomal banding patterns induced through in situ digestion with the restriction enzyme AluI were studied by analyzing the chromosomes of 25 monozygotic and 25 dizygotic twin pairs selected at random from a juvenile twin registry. A total of 19 AluI banding variants were found to be heteromorphic, with the pericentromeric region of chromosome 3 and the satellites of chromosome 22 being most and least heteromorphic, respectively. As expected, the correlations of the semi-quantitative scores for each of the chromosomal variants were significantly higher between MZ twin pairs (ranging from 0.48 to 0.95) than DZ twin pairs (ranging from -0.02 to 0.69), suggesting that genetic factors play an important role in their appearance. This finding was confirmed in a model fitting analysis in which the heritabilities of the AluI-induced chromosome variants were found to range from 70 to 96% for 12/13 heteromorphisms studied. These consistent findings are significant in that these variants may be useful for family studies in clinical genetics.

Adolescent↗
PubMed1995

Phospholipase A2 engineering. Probing the structural and functional roles of N-terminal residues with site-directed mutagenesis, X-ray, and NMR.

The N-terminal residues of phospholipase A2 (PLA2) are believed to be involved in the hydrogen-bonding network, the interfacial binding site, or the hydrophobic channel. Site-directed mutants of bovine pancreatic PLA2 with substitutions at positions 2, 3, 4, 5, 6, and 9 were constructed to test the roles of these residues in the structure and function of PLA2. Nonconservative mutations of Phe-5 and Ile-9, which are located inside the hydrophobic channel, led to significant perturbations in the conformation and conformational stability. Kinetic studies also indicated that mutations at Ile-9 and Phe-5 caused significant decreases in the rate of hydrolysis toward micellar and vesicle substrates. Scooting mode kinetic analysis showed that the binding step of the mutant enzymes to the DC14PM (1,2-dimyristoyl-sn-glycero-3-phosphomethanol) vesicle interface is not significantly affected and that the perturbations in catalysis occur mainly in kcat at the interface. The results taken together suggest that the residues Ile-9 and Phe-5 are important for both structure and catalysis. The mutant W3A (Trp-3 to Ala) also showed decreased rates of hydrolysis but to a lesser extent than Ile-9 and Phe-5 mutants. In addition, the binding affinity of W3A to the surface of the vesicles (i.e., the E to E* step) has been perturbed to the extent that hopping between anionic vesicles has been observed. On the other hand, the mutants of Gln-4 and Asn-6, which are located at or near the surface, displayed structural and kinetic properties similar to those of the wild-type PLA2 with the exception of the highly hydrophilic lysine mutant. The X-ray structure of the Q4E mutant indicates that the overall structure, the catalytic triad, and the link between residue 4 and Asp-99 via hydrogen bonding through Ala-1 and the structural water remain the same as in the WT. Substitutions for Leu at position 2 showed an acyl chain length discrimination toward different substrates, which may reflect the contacting position(s) of the substrate acyl chain with Leu-2.

Amino Acid Sequence↗
PubMed1995

Autonomic dysreflexia in a rat model spinal cord injury and the effect of pharmacologic agents.

The object of this study was to develop a spinal cord injury (SCI) rat model for autonomic dysreflexia (AD), assessing the effect of alpha-adrenergic and calcium channel blockade and to determine the relationship of detrusor-external sphincter dyssynergia (DESD) to the development of AD. A laminectomy was performed in male rats at the T4 or T10 level and a controlled 50 g cm blunt SCI was induced using an impounder. Four weeks after injury, changes in arterial blood pressure and heart rate were monitored while simultaneous cystometry (CMG) and pelvic floor electromography (EMG) were performed in vivo in sham (control) and spinal cord injured rats. The effects of terazosin (0.1 mg/kg), diltiazem (0.5 mg/kg), and oxybutynin chloride (0.1 mg/kg) on hemodynamic changes were assessed independently. Both T4 and T10 SCI rat displayed evidence of DESD (enhanced pelvic floor EMG activity at cystometric capacity) while control rats did not. Only T4 injured rats exhibited evidence of AD, with mean blood pressure elevations from 82.9 +/- 13.6 to 93.9 +/- 11.3 mm Hg (P < 0.01) and a mean heart rate decrease from 332.2 +/- 56.5 to 311.1 +/- 54.5 beats/min (P = 0.02) at cystometric capacity. The intravenous administration of terazosin or diltiazem abolished the AD response during CMG. The administration of oxybutynin exhibited the ability to increase bladder capacity and improve compliance in all 3 groups but did not blunt AD. The rat model of SCI effectively reproduced hemodynamic changes consistent with the AD complex in T4 level SCI but not T10 level SCI animals, despite incomplete lesions. Blockade with either an alpha-1 or a calcium channel antagonist effectively ablated the AD response to bladder distention. Anticholinergic agents had no effect on AD. DESD frequently accompanies autonomic dysreflexia, although the development of AD is not a prerequisite for DESD.

Adrenergic alpha-Antagonists↗