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Biomedical subjects

X Zhao

Publications and source records attributed to X Zhao.

At least 541 records · Page 30Linked to original sources
PubMed1995

A double mutant of sperm whale myoglobin mimics the structure and function of elephant myoglobin.

The functional, spectral, and structural properties of elephant myoglobin and the L29F/H64Q mutant of sperm whale myoglobin have been compared in detail by conventional kinetic techniques, infrared and resonance Raman spectroscopy, 1H NMR, and x-ray crystallography. There is a striking correspondence between the properties of the naturally occurring elephant protein and those of the sperm whale double mutant, both of which are quite distinct from those of native sperm whale myoglobin and the single H64Q mutant. These results and the recent crystal structure determination by Bisig et al. (Bisig, D. A., Di Iorio, E. E., Diederichs, K., Winterhalter, K. H., and Piontek, K. (1995) J. Biol. Chem. 270, 20754-20762) confirm that a Phe residue is present at position 29 (B10) in elephant myoglobin, and not a Leu residue as is reported in the published amino acid sequence. The single Gln64(E7) substitution lowers oxygen affinity approximately 5-fold and increases the rate of autooxidation 3-fold. These unfavorable effects are reversed by the Phe29(B10) replacement in both elephant myoglobin and the sperm whale double mutant. The latter, genetically engineered protein was originally constructed to be a blood substitute prototype with moderately low O2 affinity, large rate constants, and increased resistance to autooxidation. Thus, the same distal pocket combination that we designed rationally on the basis of proposed mechanisms for ligand binding and autooxidation is also found in nature.

Amino Acid Sequence↗
PubMed1995

Calpain inhibitors reduce depolarization induced loss of tau protein in primary septo-hippocampal cultures.

We studied the effects of a 6-min potassium depolarization injury produced by 60 mM KCl and 1.8 mM or 5.8 mM extracellular CaCl2 on tau protein levels in primary rat septo-hippocampal cultures. One day after injury, Western blot analyses revealed a calcium dependent loss of tau protein of approximately 50% of control values. Loss of tau protein was associated with calpain 1 mediated breakdown products to alpha-spectrin. Calpain inhibitors 1 and 2, applied immediately after depolarization injury and available to cultures for 24 h reduced depolarization induced degradation of tau protein to approximately 35% or 25% of control values, respectively. We propose that brief potassium depolarization causes degradation of tau protein, possibly due to calpain activation. Thus, calpain inhibitors could represent a viable strategy for preserving the cytoskeletal structure of injured neurons.

Animals↗
PubMed1995

Calcium-induced troponin flexibility revealed by distance distribution measurements between engineered sites.

The contraction of vertebrate striated muscle is regulated by Ca2+ binding to troponin C (TnC). This causes conformational changes which alter the interaction of TnC with the inhibitory protein TnI and the tropomyosin-binding protein TnT. We have used the frequency domain method of fluorescence resonance energy transfer to measure TnT-TnC and TnT-TnI distances and distance distributions, in the presence of Ca2+, Mg2+, or EGTA, in TnC.TnI.TnT complexes. We reconstituted functional, ternary troponin complexes using the following recombinant subunits whose sequences were based on those of rabbit skeletal muscle: wild-type TnC; TnT25, a mutant C-terminal 25-kDa fragment of TnT containing a single Trp212 which was used as the sole donor for fluorescence energy transfer measurements; Trp-less TnI mutants which contained either no Cys or a single Cys at position 9, 96, or 117. Energy acceptor groups were introduced into TnC or TnI by labeling with dansyl aziridine or N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine. Our results indicate that the troponin complex is relatively rigid in relaxed muscle, but becomes much more flexible when Ca2+ binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, releasing the inhibition of actomyosin ATPase activity and allowing the muscle to contract. This is the first report of distance distribution measurements between troponin subunits.

Adenosine Triphosphatases↗
PubMed1995

Rescue of injury-induced neurofilament loss by BDNF gene transfection in primary septo-hippocampal cell cultures.

We employed primary septo-hippocampal cell cultures to determine the ability of liposome-mediated BDNF gene transfection to facilitate recovery of neurofilament loss caused by depolarization injury. After BDNF gene transfection in uninjured cultures, RT-PCR and immunohistochemical staining confirmed increases in BDNF mRNA and protein in transfected cells. Three days after depolarization injury, Western blot and immunohistochemical analyses detected significant loss of neurofilament proteins in non-transfected cultures, while BDNF transfection produced marked increases in neurofilament proteins following either pre-injury transfection or transfection 24 h following injury. Immunohistochemical studies also detected enhanced immunolabeling of BDNF and total neurofilament protein (phosphorylated and non-phosphorylated) in injured neurons following BDNF transfection or administration of exogenous BDNF protein, compared to untransfected, injured controls.

Animals↗
PubMed1995

In vitro assembly of cowpea chlorotic mottle virus from coat protein expressed in Escherichia coli and in vitro-transcribed viral cDNA.

The small spherical plant virus, cowpea chlorotic mottle virus (CCMV), provides an ideal system to examine spherical virus assembly. We have modified the CCMV in vitro assembly system to produce virions from coat protein expressed in Escherichia coli and viral RNA transcribed in vitro from full-length cDNAs. Examination of the in vitro-assembled particles with cryoelectron microscopy and image reconstruction techniques demonstrates that the particles are indistinguishable from plant purified particles at 2.5 nm resolution. Mutational analysis of the coat protein N- and C-terminal extensions demonstrate their respective roles in virus assembly. The N-terminus is required for assembly of RNA containing particles but not for the assembly of empty virions. The C-terminus is essential for coat protein dimer formation and particle assembly.

Base Sequence↗
PubMed1995

Preparation and characterization of a deoxyoligonucleotide 49-mer containing a site-specific thymidylyl-(3',5')-deoxyadenosine photoproduct.

Irradiation of d(GTATTATG) with 254 nm light gave rise to four major photoproducts, two of which were readily identified by NMR as the cis-syn cyclobutane dimer and the (6-4) photoproduct of the central TT site. Analysis of the NMR data for the other two photoproducts indicated that they were not any of the other known photoproducts of a TT site and might be TA* photoproducts [Bose, S. N., et al. (1983) Science 220, 723-725]. In support of this possibility, the fluorescence spectra of the products of acid hydrolysis of the two photoproducts were very similar to that reported for the hydrolysis product of the TA* photoproduct of TpdA. Only one of the two TA*-containing octamers could be ligated at both ends to form a 49-mer oligonucleotide in the presence of a complementary oligonucleotide scaffold, suggesting that the TA* photoproduct had formed between T5 and A6. The position of the TA* photoproduct was confirmed by mapping the arrest sites for 3'-->5' exonucleolytic degradation of the 49-mer by T4 DNA polymerase and for primer extension opposite the 49-mer by exonuclease deficient Klenow fragment (KF) and Sequenase Version 2.0. The TA* product could also be bypassed by both polymerases, but it was less of a block to KF. Treatment with 1 M aqueous piperidine at 100 degrees C led to a maximum of about 34% cleavage of the DNA at the site of the TA* product.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

Physical and genetic localization of a Shab subfamily potassium channel (KCNB1) gene to chromosomal region 20q13.2.

A human delayed rectifier K+ channel gene has been localized to the long arm of human chromosome 20q13.2 by fluorescence in situ hybridization of genomic P1 clones from this locus. A polymorphic (GA) microsatellite repeat was identified in one of the P1 clones. The new SSR marker (D20S436) was genotyped in four CEPH pedigrees. Two-point linkage analysis indicated linkage of this marker to a PCR marker, D20S109, with a maximum lod score of 9.32 at theta = 0.001. The assignment of this K+ channel gene to 20q13.2 eliminates it as a candidate for the gene associated with benign familial neonatal convulsions (BFNC), which has been localized to 20q13.3. Genetically, the K+ channel gene maps more than 30 cM proximal to the BFNC locus.

Base Sequence↗
PubMed1995

Inhibition of proliferation and induction of apoptosis by abrogation of heat-shock protein (HSP) 70 expression in tumor cells.

Tumor cells often express elevated levels of heat-shock protein (HSP) 70. The present study was designed to investigate the role of HSP70 in the proliferation and survival of tumor cells in the human system. When Molt-4 and other tumor cells were treated in vitro with HSP70 antisense oligomer, they displayed propidium-iodide-stained condensed nuclei (intact or fragmented). A ladder-like pattern of DNA fragments was observed with HSP70 antisense-oligomer-treated tumor cells in agrose gel electrophoresis, which was consistent with internucleosomal DNA fragmentation. Flow cytometry analysis revealed the hypodiploid DNA peak of propidium-iodide-stained nuclei in the antisense-oligomer-treated cells. The apoptosis induced by HSP antisense oligomer was dose- and time-dependent. The antisense oligomer induced apoptosis mainly in tumor cells at G1 and S phase, resulting in an inhibition of cell proliferation. HSP70 antisense oligomer caused DNA-sequence-specific inhibition of HSP70 expression, which preceded apparent apoptosis. These results indicate that HSP70 antisense treatment inhibits the expression of HSP70, which in turn inhibits cell proliferation and induces apoptosis in tumor cells and suggest that HSP70 is required for tumor cells to proliferate and survive under normal condition.

Apoptosis↗
PubMed1995

Cardiovascular and renal effects of alpha-trinositol in ischemic heart failure rats.

Previous studies have demonstrated that alpha-trinositol (D-myo-inositol-1.2.6-trisphosphate; PP56) may act as a functional neuropeptide Y (NPY) inhibitor. Because NPY is known to be a potent vasoconstrictor, the effects of alpha-trinositol on renal function, vascular responses and the potentiating effects of NPY were investigated in rats with congestive heart failure (CHF) induced by ligation of the left coronary artery. Incremental doses of alpha-trinositol were given to conscious rats (bolus 2, 4 or 10 mg/kg i.v. followed by a 15-minute infusion 20, 40 and 100 mg/kg/h, respectively). Urinary volume, sodium and potassium excretions were significantly increased in both CHF and sham-operated control animals after alpha-trinositol administration compared with saline. Diuresis and natriuresis were observed also during co-administration of alpha-trinositol with NPY but not with norepinephrine (NE). In the pithed CHF rats, threshold doses of NPY potentiated the pressor effects of endothelin-1 (ET-1) and angiotensin II (AII), but not preganglionic nerve stimulation or phenylephrine administration. Alpha-trinositol antagonized both the pressor response to NPY and the potentiation by NPY of pressor responses to effects of ET-1 and AII. Our data show that alpha-trinositol exhibis diuretic and natriuretic effects as well as vascular antagonistic effects on NPY in normal and CHF rats. These effects of alpha-trinositol may be due to an interaction with NPY mediated antidiuresis and antinatriuresis.

Animals↗
PubMed1995

The influence of dietary fibre on body composition, visceral organ weight, digestibility and energy balance in rats housed in different thermal environments.

The present study was undertaken to provide detailed information on the effect of dietary fibre (DF) level on body composition, visceral organ weight, nutrient digestibility and on energy and protein metabolism of rats housed in cold (16 degrees), warm (24 degrees) or hot (32 degrees) thermal environments. High- or low-fibre diets (257 v. 56 g DF/kg dry matter (DM)) were studied in a 6-week balance experiment (initial body weight about 100 g). Heat production was measured using open-air circuit respiration chambers. Pea fibre and pectin were used to adjust the DF level in the high-fibre diet. The ranking order of daily gain of rats kept in different environments was: 24 degrees > 16 degrees > 32 degrees, while the ranking order for carcass protein was: 16 degrees > 24 degrees > 32 degrees. Rats on the high-DF diet had a lower daily gain than those on the low DF diet, and more protein in DM of empty body weight (EBW) and less fat. The relative weights (g/kg EBW) of liver, heart and kidney decreased when increasing the environmental temperature. The relative weight of the heart was highest in rats on the high DF level, while liver and kidney weights were unaffected by DF. Per kg EBW, the stomach, small intestine, caecum and colon and the length of colon were significantly greater in rats consuming the high-fibre diet compared with those on the low-fibre diet. Rats kept at low temperature had a significantly heavier gastrointestinal (GI) tract than those kept at the highest temperature. Digestibility of protein, DM and energy was lowest for rats fed on the high-fibre diet. Heat production (HP) of fed rats as well as fasting HP decreased significantly as environmental temperature increased. HP as a proportion of metabolizable energy (ME) was significantly lower for rats at 24 degrees compared with the other environmental temperatures. The proportion of energy retained as protein was slightly higher in rats fed on the high-fibre than on the low-fibre diet. Based on the results of the present study the authors measured a net energy value of 5.4 kJ/g DF fermented; approximately 50% of the DF came from peas. Possible implications of the present findings are discussed.

Animals↗
PubMed1995

Mapping quantitative trait loci controlling milk production in dairy cattle by exploiting progeny testing.

We have exploited "progeny testing" to map quantitative trait loci (QTL) underlying the genetic variation of milk production in a selected dairy cattle population. A total of 1,518 sires, with progeny tests based on the milking performances of > 150,000 daughters jointly, was genotyped for 159 autosomal microsatellites bracketing 1645 centimorgan or approximately two thirds of the bovine genome. Using a maximum likelihood multilocus linkage analysis accounting for variance heterogeneity of the phenotypes, we identified five chromosomes giving very strong evidence (LOD score > or = 3) for the presence of a QTL controlling milk production: chromosomes 1, 6, 9, 10 and 20. These findings demonstrate that loci with considerable effects on milk production are still segregating in highly selected populations and pave the way toward marker-assisted selection in dairy cattle breeding.

Animals↗
PubMed1995

Combined growth hormone (GH) and insulin-like growth factor-I (IGF-I) treatment is more effective than GH or IGF-I alone at enhancing recovery from neonatal malnutrition in rats.

The effect of hormonal therapy on enhancing recovery from neonatal malnutrition was assessed in rats. Malnutrition was induced by maternal food restriction (60% control intake) during lactation. On d 16 postpartum, restricted pups were refed by cross-fostering to control dams and were subcutaneously administered growth hormone (GH), insulin-like growth factor-I (IGF-I), GH + IGF-I or saline. At d 21, pups were weaned and continued hormonal treatments until d 39 postpartum. By d 39, body weight of GH (96% control) and GH + IGF-I- (98%) treated animals were normal, whereas IGF-I (88%) and saline- (85%) treated animals were still stunted. Hormone effectiveness was age dependent, with growth rates of GH + IGF-I-, IGF-I- and GH-treated pups being greatest between d 16 and 30, d 16 and 21 and after d 30, respectively. Protein accretion by d 39 was higher (P < 0.01) in GH and GH + IGF-I groups than saline- or IGF-I-treated groups. On d 39, serum IGF-I concentrations were as follows: GH + IGF-I > IGF-I > GH = saline (placebo), indicating that an elevated serum IGF-I was not required for GH-stimulated growth. Of the three hormonal treatments, GH + IGF-I therapy was most efficacious at promoting rapid and complete body weight recovery and supporting lean body mass accretion.

Animals↗
PubMed1995

Human growth hormone but not human insulin-like growth factor-I enhances recovery from neonatal malnutrition in rats.

The ability of hormonal therapy to enhance recovery from neonatal malnutrition was assessed in rats. Pups were malnourished via maternal food restriction (60% control intake). On d 16, restricted pups (n = 50) (62% control body wt) were refed until d 20 and were given growth hormone, insulin-like growth factor-I, destripeptide [1,3] insulin-like growth factor-I, or saline (placebo). Refed-placebo pups attained 80% of control body weight by d 20. Growth hormone treatment caused a greater weight gain than refeeding alone (P < or = 0.05). Treatment with insulin-like growth factor-I and destripeptide [1,3] insulin-like growth factor-I did not affect body weight. All three hormones increased spleen and kidney weights (P < or = 0.05) compared with the refed-placebo group, whereas only growth hormone increased muscle weight (P < or = 0.05). Malnourished pups had lower serum insulin-like growth factor-I (P < or = 0.05) and insulin-like growth factor binding protein-3 (P < or = 0.05), and higher serum insulin-like growth factor binding protein-2 (P < or = 0.05) and hepatic insulin-like growth factor binding protein-2 mRNA (P < or = 0.05) than controls. Refeeding increased serum insulin-like growth factor-I compared with restricted pups (P < or = 0.05), and insulin-like growth factor-I treatment caused a further increase in serum insulin-like growth factor-I compared with the refed-placebo group (P < or = 0.05). These results show that growth hormone was moderately effective at increasing body weight gain and muscle growth during recovery from malnutrition.

Animals↗
PubMed1995

Oestrogen-binding protein in Candida albicans: antibody development and cellular localization by electron immunocytochemistry.

Candida albicans, the most common fungal pathogen of humans, possesses an oestrogen (estrogen)-binding protein (EBP) that binds oestrogens with high affinity and specificity. The gene that encodes the EBP (CaEBP1) has been cloned and sequenced and shown to be structurally related to the old yellow enzyme from Saccharomyces cerevisiae. Here, we report the purification and the subcellular localization of the EBP from C. albicans. Using ion-exchange chromatography and an oestradiol affinity column, the EBP was purified from a strain of C. albicans (strain 422) which was selected because it constitutively expressed elevated levels of the binding protein. The purified protein displayed a subunit molecular mass of approximately 46 kDa when examined by denaturing gel electrophoresis, which is consistent with the size estimated from the sequence of the cloned CaEBP1 gene. An immunoaffinity column, prepared using a polyclonal antisera generated against EBP, depleted the oestrogen-binding activity from C. albicans cell extracts. Western blot analysis showed that the antisera specifically recognized the EBP from C. albicans. The antibodies also recognized the protein when the cloned CaEBP1 gene was expressed in S. cerevisiae and did not cross react with S. cerevisiae proteins. Using electron microscopy and antigen detection by immunogold staining, the EBP appeared to be primarily associated with vacuoles. However, when overexpressed in S. cerevisiae, the EBP was found diffusely throughout the cell. In conclusion, the EBP has been purified from C. albicans and antibodies generated against the protein were used to demonstrate that EBP is found associated with vacuoles in C. albicans.

Blotting, Western↗