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Biomedical subjects

H Chao

Publications and source records attributed to H Chao.

At least 55 records · Page 3Linked to original sources
PubMed1996

Effect of type III antifreeze protein dilution and mutation on the growth inhibition of ice.

Mutation of residues at the ice-binding site of type III antifreeze protein (AFP) not only reduced antifreeze activity as indicated by the failure to halt ice crystal growth, but also altered ice crystal morphology to produce elongated hexagonal bipyramids. In general, the c axis to a axis ratio of the ice crystal increased from approximately 2 to over 10 with the severity of the mutation. It also increased during ice crystal growth upon serial dilution of the wild-type AFP. This is in marked contrast to the behavior of the alpha-helical type I AFPs, where neither dilution nor mutation of ice-binding residues increases the c:a axial ratio of the ice crystal above the standard 3.3. We suggest that the ice crystal morphology produced by type III AFP and its mutants can be accounted for by the protein binding to the prism faces of ice and operating by step growth inhibition. In this model a decrease in the affinity of the AFP for ice leads to filling in of individual steps at the prism surfaces, causing the ice crystals to grow with a longer c:a axial ratio.

Animals↗
PubMed1996

Effects of antifreeze proteins on red blood cell survival during cryopreservation.

Antifreeze protein (AFP) types, I, II and III were tested for their ability to protect red blood cells from lysis during warming, after cryopreservation in hydroxyethyl starch. All three types reduced hemolysis to 25% of control values at similar micromolar concentrations but enhanced lysis as the AFP concentration approached millimolar levels. Site-directed mutants of type III AFP with different thermal hysteresis activities were tested for their ability to protect the cryopreserved cells from lysis. Their relative efficacy in protecting the cells correlated closely with their thermal hysteresis activity. Cryomicroscopy indicated that the protection of red cells by type III AFP and the mutant forms was due to inhibition of ice recrystallization.

Animals↗
PubMed1995

Mixing antifreeze protein types changes ice crystal morphology without affecting antifreeze activity.

All three fish antifreeze protein types (I, II and III) inhibit the growth of ice to form hexagonal bipyramidal ice crystals of characteristic morphology. Mixtures of these different antifreezes produced ice crystals of hybrid shapes and dimensions, consistent with the different antifreeze types binding to the same ice surfaces. The activity of the mixtures was independent of the proportions of the iso-active antifreeze protein stocks present, indicating that the different antifreezes neither attenuated nor potentiated each other's activity. We suggest that antifreeze protein molecules are independently active and do not require protein-protein interactions for ice-binding.

Animals↗
PubMed1995

[Expression of MDM2 gene in acute leukemia].

In order to study expression of MDM2 gene, antagonist of tumor suppressor gene p53 in acute leukemia, forty acute leukemia patients were tested using RT-PCT to evaluate expression level of MDM2 gene. Over-expression of MDM2 gene was found in 52.5% patients (21/40). No significant relations were found between the level of MDM2 gene expression and FAB subtypes of acute leukemia. It is suggested that the over-expression of MDM2 gene may play a role in acute leukemia.

Adolescent↗
PubMed1995

[Late bladder complications following radiotherapy of carcinoma of the uterine cervix].

OBJECTIVE: To analyse the causes, clinical features, treatment and prevention of late bladder complications following radiotherapy of carcinoma of the uterine cervix. METHOD: This clinical retrospective study included 378 cases of irradiation cystitis following radiotherapy for carcinoma of the uterine cervix from February, 1958 to October, 1987. RESULTS: The incidence of irradiation cystitis was 3.2% and was not related to age, FIGO staging and the methods of intracavitary irradiation (conventional or afterloading). It was related to the external appearance of the local lesion; 66.5% of the tumors of patients with late bladder complications was of the exophytic type prior to radiotherapy. 51.1% of the patients developed late bladder complications in a period of 2-5 years after radiotherapy; 16.7% in 5-10 years and only 3.4% within the first years. The patients with irradiation cystitis had received a 10% higher dosage than that routine delivered, as found either in intracavitary irradiation of 39.4% of patients or in external irradiation of 31.6% of patients. Based on the clinical features and cystoscopic findings, late bladder complications can be divided into three types: (1) sudden and temporary hematuria (35.7%); (2) persistent hematuria (63.2%); and (3) vesico-vaginal fistula (1.1%). The 5-year survival rate of patients with irradiation cystitis was 90.4% which was higher than the survival rates of all patients treated by conventional intracavitary irradiation (65.7%) and all patients treated by afterloading intracavitary irradiation (70.4%). 3.7% of patients died of late bladder complications. CONCLUSIONS: To decrease the occurrence of late bladder complications and to obtain long term survival: (1) Dosage to the bladder should be adequate, and over dose to the bladder should by all means be avoided; (2) Patients are to be taught to observe indicated personal care during radiotherapy as well as in the follow-up period; (3) Early institution of proper conservative treatment of cystitis is beneficial.

Adult↗
PubMed1994

Structure-function relationship in the globular type III antifreeze protein: identification of a cluster of surface residues required for binding to ice.

Antifreeze proteins (AFPs) depress the freezing point of aqueous solutions by binding to and inhibiting the growth of ice. Whereas the ice-binding surface of some fish AFPs is suggested by their linear, repetitive, hydrogen bonding motifs, the 66-amino-acid-long Type III AFP has a compact, globular fold without any obvious periodicity. In the structure, 9 beta-strands are paired to form 2 triple-stranded antiparallel sheets and 1 double-stranded antiparallel sheet, with the 2 triple sheets arranged as an orthogonal beta-sandwich (Sönnichsen FD, Sykes BD, Chao H, Davies PL, 1993, Science 259:1154-1157). Based on its structure and an alignment of Type III AFP isoform sequences, a cluster of conserved, polar, surface-accessible amino acids (N14, T18, Q44, and N46) was noted on and around the triple-stranded sheet near the C-terminus. At 3 of these sites, mutations that switched amide and hydroxyl groups caused a large decrease in antifreeze activity, but amide to carboxylic acid changes produced AFPs that were fully active at pH 3 and pH 6. This is consistent with the observation that Type III AFP is optimally active from pH 2 to pH 11. At a concentration of 1 mg/mL, Q44T, N14S, and T18N had 50%, 25%, and 10% of the activity of wild-type antifreeze, respectively. The effects of the mutations were cumulative, such that the double mutant N14S/Q44T had 10% of the wild-type activity and the triple mutant N14S/T18N/Q44T had no activity. All mutants with reduced activity were shown to be correctly folded by NMR spectroscopy. Moreover, a complete characterization of the triple mutant by 2-dimensional NMR spectroscopy indicated that the individual and combined mutations did not significantly alter the structure of these proteins. These results suggest that the C-terminal beta-sheet of Type III AFP is primarily responsible for antifreeze activity, and they identify N14, T18, and Q44 as key residues for the AFP-ice interaction.

Antifreeze Proteins↗
PubMed1993

The nonhelical structure of antifreeze protein type III.

Antifreeze proteins (AFPs) are present in the blood of some marine fishes and inhibit the growth of ice crystals at subzero temperatures by adsorption to the ice lattice. The solution structure of a Type III AFP was determined by two-dimensional nuclear magnetic resonance spectroscopy. These measurements indicate that this 66-residue protein has an unusual fold in which eight beta strands form two sheets of three antiparallel strands and one sheet of two antiparallel strands, and the triple-stranded sheets are packed orthogonally into a beta sandwich. This structure is completely different from the amphipathic, helical structure observed for Type I AFPs.

Amino Acid Sequence↗
PubMed1993

Use of proline mutants to help solve the NMR solution structure of type III antifreeze protein.

To help understand the structure/function relationships in antifreeze proteins (AFP), and to define the motifs required for ice binding, a Type III AFP suitable for two-dimensional (2D) NMR studies was produced in Escherichia coli. A synthetic gene for one of the Type III AFP isoforms was assembled in a T7 polymerase-directed expression vector. The 67-amino acid-long gene product differed from the natural AFP by inclusion of an N-terminal methionine but was indistinguishable in activity. The NMR spectra of this AFP were complicated by cis-trans proline isomerization from the C-terminal sequence YPPA. Substitution of this sequence by YAA eliminated isomer signals without altering the activity or structure of the mutant AFP. This variant (rQAE m1.1) was selected for sequential assignment and the secondary structure determination using 2D 1H NMR spectroscopy. Nine beta-strands are paired to form two triple-stranded antiparallel sheets and one double-stranded antiparallel sheet. Two further proline replacements, P29A and P33A, were made to delineate the role of conserved prolines in Type III AFP. These mutants were valuable in clarifying ambiguous NMR spectral assignments amongst the remaining six prolines of rQAE m1.1. In contrast to the replacement of the C-terminal prolyl residues, the exchange of P29 and P33 caused some structural changes and significantly decreased protein solubility and antifreeze activity.

Amino Acid Sequence↗
PubMed1993

Solute/solvent interaction corrections account for non-ideal freezing point depression.

A new highly accurate curve-fitting technique for looking at freezing-point depression data was proposed by Fullerton et al. (Biochem. Cell Biol., in press). The method involve plotting mass solvent to mass solute ratio (Mw/M(s)) vs. 1/delta T (i.e. the inverse change in freezing point). A measured molecular weight and a solute/solvent interaction parameter (called I value) are inferred from the resultant linear plot. The accuracy of the molecular weight method was first demonstrated with the monomers of ethylene glycol, glycerol, propanol, mannitol, glucose and sucrose to show a mean molecular weight error of 0.02% with root mean square (RMS) error 0.9%. The RMS error (0.9%) is our best estimate of the molecular weight measurement accuracy for the method applied to a monomer. This error is consistent with the experimental precision (approximately 1%) which implies no systematic error. Non-ideality is described with a single constant, I. Polyethylene glycol (PEG) polymers of increasing length (vendor designation 200 to 10,000 Da) were analyzed to show monotonically increasing non-ideality (I values of 0.12 to 3.67) with increasing molecular weight. The measured molecular weights agreed with the end-point titration value for the three smallest polymers (where the number of polymeric units was less than or equal to 7). The method underestimates the vendor molecular weights for longer polymers. This disagreement is assigned to segmental motion (internal entropy) of longer, more flexible, PEG molecules.

Alcohols↗
PubMed1992

Amino acid sequence of the unique protamine from yellow perch.

By the criteria of gel electrophoresis, ion-exchange chromatography, and reverse-phase HPLC, yellow perch protamine behaves as a single component. This observation was confirmed by automated Edman degradation which gave a single unambiguous amino acid sequence PRRRRHAARPVRRRRRTRRSSRVHRRRRAVRRRR. Yellow perch protamine has 34 amino acids, including 21 arginines. It has two histidines, neither of which interrupts an arginine tract. It is unusual among fish protamines in not having a serine or threonine N-terminal to the second arginine tract, and is unique in not being a mixture of components.

Amino Acid Sequence↗
PubMed1991

Ultrafast spectroscopy of the visual pigment rhodopsin.

We report on time-resolved absorption studies of the bovine visual pigment rhodopsin with subpicosecond resolution at room temperature. Our data show that bathorhodopsin, rhodopsin's early photoproduct, is photochemically formed in 3.0 +/- 0.7 ps. The data suggest that bathorhodopsin formation is kinetically preceded by two species along the rhodopsin-to-bathorhodopsin reaction coordinate. The first is identified with the vertically excited Franck-Condon state. This decays with an approximately 200-fs lifetime to an intermediate, which then decays to bathorhodopsin in 3.0 ps. We assign this intermediate to be an excited state transient near 90 degrees along the 11-12 torsional coordinate of rhodopsin's chromophore. Exchange of rhodopsin's exchangeable protons for deuterons does not affect the observed dynamics. These observations are both qualitatively and quantitatively consistent with molecular dynamics calculations, which model the rhodopsin to bathorhodopsin phototransition as a cis-trans isomerization along the 11-12 torsional coordinate of rhodopsin's chromophore.

Animals↗
PubMed1991

Effects of relief of biliary obstruction on mononuclear phagocyte system function and cell mediated immunity.

Obstructive jaundice causes depression of immune system function but it is unclear at present how rapidly immune function recovers after relief of biliary obstruction. To address this issue, we studied 218 Sprague-Dawley rats with common bile duct obstruction. Mononuclear phagocyte function, cell mediated immune function, portal-systemic shunt fraction, liver function tests, and liver histology were evaluated in normal (sham) rats, obstructed rats, and at weekly intervals after relief of biliary obstruction. Hepatic uptake of radiolabelled bacteria was 82 per cent in sham rats and 66 per cent in rats 21 days after CBD obstruction (P less than 0.05). Phagocytic activity returned to normal within 7 days after choledochoduodenostomy. Cell mediated immunity, measured by skin graft rejection, was significantly prolonged in the obstructed group (P less than 0.05) but had returned to normal 7 days after biliary diversion. Return of hepatocellular function, as measured by liver function tests, paralleled recovery of immune function. This study demonstrates prompt recovery of the immune system after internal biliary drainage for obstructive jaundice. This finding is in contrast to previous studies that demonstrated persistent immune suppression months after biliary diversion. These data may have implications concerning the usefulness of internal biliary drainage before surgery in patients with obstructive jaundice.

Animals↗
PubMed1990

Prolonged survival and decreased mucosal injury after low-dose enteral allopurinol prophylaxis in mesenteric ischemia.

Previous studies demonstrating protective effects of allopurinol in intestinal ischemia have evaluated intravenous allopurinol (presently unavailable for human use) or enteral allopurinol at supranormal doses and, therefore, have questionable clinical relevance. To address this problem, we evaluated the protective effects of clinically used doses of enteral allopurinol in rats with intestinal ischemia. Forty male Sprague-Dawley rats (weighing 300 to 400 g) received enteral allopurinol (10 mg/kg) or water daily for 1 week. Rats were then subjected to superior mesenteric artery occlusion with interruption of collateral flow for 20 minutes to produce ischemic injury to the intestine. Segmental small bowel resections were performed in 10 control rats and 10 allopurinol-treated rats before and after reperfusion to identify histopathologic evidence of reperfusion injury. Mucosal injury was quantitated using a grading scale of 0 to 5 (5 being most severe). The remaining 20 rats (10 in each group) were observed for mortality (death within 7 days) after reperfusion. Mucosal injury after reperfusion was graded at 4.4 +/- 0.20 in controls versus 2.3 +/- 0.23 in the treated group (P less than .001). In addition, there was a significant increase in mucosal damage in the control group when postreperfusion specimens were compared with specimens taken before reperfusion (2.8 +/- 0.19 before and 4.4 +/- 0.20 after reperfusion, P less than .001). Injury score for the allopurinol-treated group did not significantly increase after reperfusion. Survival was 50% in the water-fed control group compared with 100% survival in allopurinol-treated rats (P = .016). We conclude that enteral allopurinol in the presently available form and dose is effective in reducing mesenteric reperfusion injury.

Administration, Oral↗
PubMed1990

A new model for intestinal ischemia in the rat.

Many models of intestinal ischemia in the rat have been described yielding mortality rates of 8-80% following superior mesenteric artery (SMA) occlusion for periods of 1-85 min. These results have been difficult to reliably reproduce in our lab. Based on our theory that these inconsistent findings are secondary to individual variability in collateral circulation, we have developed a new model for intestinal ischemia with reproducible and consistent mortality. Male Sprague-Dawley rats weighting 300-400 g underwent celiotomy and evisceration. Occlusion of the superior mesenteric artery just distal to the right colic artery was achieved. Collateral arcades from the right colic artery and the jejunal arteries proximal to the site of occlusion were ligated and the bowel was returned to the abdomen for the remainder of the ischemic period. SMA occlusion alone and SMA occlusion with interruption of collateral flow were evaluated and compared for severity of ischemic injury reflected by mortality and for reproducibility of ischemia and mortality. Quantitative measurements of blood flow for each technique were made using radiolabeled microspheres, and a survival curve for SMA occlusion with collateral ligation was constructed. SMA occlusion alone in the rat is not a reliable model for mesenteric ischemia because the resulting ischemic injury is inconsistent and not reproducible. SMA occlusion with collateral ligation produced more severe and reproducible ischemia with greater mortality than did SMA occlusion alone. This technique produced mortality rates that were reproducible and were more consistently related to duration of ischemia.

Animals↗
PubMed1990

Heparin-induced hyperkalemia.

Heparin-induced hypoaldosteronism leading to hyperkalemia is an uncommon adverse effect. It appears as though heparin blocks an enzymatic step in the synthesis of aldosterone, and reduced aldosterone levels may be evident as early as four days after initiation of therapy. Although all patients who receive heparin may have reduced aldosterone levels, most are able to compensate through increased renin production and therefore remain asymptomatic. However, patients on prolonged heparin therapy or those unable to adequately increase renin production (e.g., patients with diabetes or renal insufficiency) may exhibit signs of hypoaldosteronism, such as hyperkalemia.

Adult↗
PubMed1990

High dose versus low dose enteral allopurinol for prophylaxis in mesenteric ischemia.

Studies demonstrating protective effects of allopurinol in intestinal ischemia have been carried out using i.v. allopurinol (presently unavailable for human use) or enteral allopurinol at supra-normal doses and, therefore, have questionable clinical relevance. We evaluated the protective effects of clinically used doses of enteral allopurinol in rats with intestinal ischemia. One hundred nine male Sprague-Dawley rats (250-300 gm) received enteral allopurinol (5-30 mg/kg) or water daily for 1 week and were subjected to superior mesenteric artery occlusion for 20, 30, or 45 min. Mortality in water-fed controls after 20 min of mesenteric ischemia was 50%, but there was no mortality in rats pretreated with allopurinol (5, 10, and 20 mg/kg/day) in this group (P = 0.016). There was no reduction in mortality after allopurinol pretreatment at any dose in rats with 30 or 45 min of ischemia. We concluded that 1) prolonged intestinal ischemia causes lethal damage during the hypoperfusion phase that cannot be prevented by allopurinol pretreatment even at supra-normal doses, and 2) allopurinol at recommended enteral doses (5-10 mg/kg/day) can reduce morality from reperfusion injury when the phase of hypoperfusion is not, in itself, lethal. Allopurinol is effective in reducing reperfusion injury in the currently available enteral form in dose ranges that should not cause prohibitive side effects.

Allopurinol↗