Biomedical subjects

J Zhao

Publications and source records attributed to J Zhao.

At least 271 records · Page 15Linked to original sources

PubMed2000

Steroid-dependent up-regulation of adipose leptin secretion in vitro during pregnancy in mice.

Circulating leptin levels are elevated during the later stages of pregnancy in mammals, suggesting that maternal leptin may play a role in maintenance of pregnancy and/or preparation for parturition and lactation. The regulation and source of circulating leptin during pregnancy remains undetermined, but leptin mRNA levels increase in adipose tissue during this time in some species. Considerable controversy exists whether placenta is also a leptin-secreting tissue during pregnancy. Here, we directly demonstrate that leptin secretion rates from mouse adipose tissue in vitro are decreased during early pregnancy and up-regulated during late pregnancy and lactation. Changes in leptin secretion rates in vitro paralleled those of circulating leptin in vivo during gestation. Subcutaneous implants of estradiol or corticosterone into lactating mice for 48 h stimulated adipose leptin secretion rates in vitro to the level of that in pregnant mice. However, corticosterone, but not estradiol, increased leptin secretion when added to isolated adipose tissue in vitro. Placentae obtained at two stages of pregnancy did not secrete leptin in vitro, either when acutely isolated or when dissociated into cells for long-term cultures. Placental tissue (or cells) secreted progesterone, however, demonstrating placental viability. We conclude that hyperleptinemia during late pregnancy in mice primarily results from corticosterone-dependent up-regulation of leptin secretion from adipose tissue, and that the placenta does not contribute to leptin secretion. The initial decrease in leptin secretory rates from adipose tissue during early pregnancy may facilitate energy storage for the subsequent, increased metabolic demands of later pregnancy and lactation.

Adipose Tissue↗

PubMed2000

Purification, characterization, and molecular cloning of the gene of a seed-specific antimicrobial protein from pokeweed.

A small cysteine-rich protein with antimicrobial activity was isolated from pokeweed (Phytolacca americana) seeds and purified to homogeneity. The protein inhibits the growth of several filamentous fungi and gram-positive bacteria. The protein was highly basic, with a pI higher than 10. The entire amino acid sequence of the protein was determined to be homologous to antimicrobial protein (AMP) from Mirabilis jalapa. The cDNA encoding the P. americana AMP (Pa-AMP-1) and chromosomal DNA containing the gene were cloned and sequenced. The deduced amino acid sequence shows the presence of a signal peptide at the amino terminus, suggesting that the protein is synthesized as a preprotein and secreted outside the cells. The chromosomal gene shows the presence of an intron located within the region encoding the signal peptide. Southern hybridization showed that there was small gene family encoding Pa-AMP. Immunoblotting showed that Pa-AMP-1 was only present in seeds, and was absent in roots, leaves, and stems. The Pa-AMP-1 protein was secreted into the environment of the seeds during germination, and may create an inhibitory zone against soil-borne microorganisms. The disulfide bridges of Pa-AMP-1 were identified. The three-dimensional modeling of Pa-AMP-1 indicates that the protein has a small cystine-knot folding, a positive patch, and a hydrophobic patch.

Amino Acid Sequence↗

PubMed2000

Clausa, a tomato mutant with a wide range of phenotypic perturbations, displays a cell type-dependent expression of the homeobox gene LeT6/TKn2.

Class I knox genes play an important role in shoot meristem function and are thus involved in the ordered development of stems, leaves, and reproductive organs. To elucidate the mechanism underlying the expression pattern of these homeobox genes, we studied a spontaneous tomato (Lycopersicon esculentum) mutant that phenotypically resembles, though is more extreme than, transgenic plants misexpressing class I knox genes. This mutant was found to carry a recessive allele, denoted clausa:shootyleaf (clau:shl)-a newly identified allele of clausa. Mutant plants exhibited abnormal leaf and flower morphology, epiphyllus inflorescences, fusion of organs, calyx asymmetry, and navel-like fruits. Analysis by scanning electron microscopy revealed that such fruits carried ectopic ovules, various vegetative primordia, as well as "forests" of stalked glandular trichomes. In situ RNA hybridization showed a peculiar expression pattern of the class I knox gene LeT6/TKn2; expression was restricted to the vascular system and palisade layer of mature leaves and to the inner part of ovules integuments. We conclude that CLAUSA regulates various aspects of tomato plant development, at least partly, by rendering the LeT6/TKn2 gene silent in specific tissues during development. Considering the expression pattern of LeT6/TKn2 in the clausa mutant, we suggest that the control over a given homeobox gene is maintained by several different regulatory mechanisms, in a cell type-dependent manner.

Base Sequence↗

PubMed2000

Identification of G protein-coupled receptor kinase 2 phosphorylation sites responsible for agonist-stimulated delta-opioid receptor phosphorylation.

Agonist-induced receptor phosphorylation is an initial step in opioid receptor desensitization, a molecular mechanism of opioid tolerance and dependence. Our previous research suggested that agonist-induced delta-opioid receptor (DOR) phosphorylation occurs at the receptor carboxyl terminal domain. The current study was carried out to identify the site of DOR phosphorylation during agonist stimulation and the kinases catalyzing this reaction. Truncation (Delta15) or substitutions (T358A, T361A, and S363G single or triple mutants) at the DOR cytoplasmic tail caused 80 to 100% loss of opioid-stimulated receptor phosphorylation, indicating that T358, T361, and S363 all contribute and are cooperatively involved in agonist-stimulated DOR phosphorylation. Coexpression of GRK2 strongly enhanced agonist-stimulated phosphorylation of the wild-type DOR (WT), but Delta15 or mutant DOR (T358A/T361A/S363G) failed to show any detectable phosphorylation under these conditions. These results demonstrate that T358, T361, and S363 are required for agonist-induced and GRK-mediated receptor phosphorylation. Agonist-induced receptor phosphorylation was severely impaired by substitution of either T358 or S363 with aspartic acid residue, but phosphorylation of the T361D mutant was comparable with that of WT. In the presence of exogenously expressed GRK2, phosphorylation levels of T358D and S363D mutants were approximately half of that of WT, whereas significant phosphorylation of the T358/S363 double-point mutant was not detected. These results indicate that both T358 and S363 residues at the DOR carboxyl terminus are capable of serving cooperatively as phosphate acceptor sites of GRK2 in vivo. Taken together, we have demonstrated that agonist-induced opioid receptor phosphorylation occurs exclusively at two phosphate acceptor sites (T358 and S363) of GRK2 at the DOR carboxyl terminus. These results represent the identification of the GRK phosphorylation site on an opioid receptor for the first time and demonstrate that GRK is the prominent kinase responsible for agonist-induced opioid receptor phosphorylation in vivo.

Amino Acid Sequence↗

PubMed2000

Molecular cloning, characterization, and differential expression of a glucoamylase gene from the basidiomycetous fungus Lentinula edodes.

The complete nucleotide sequence of putative glucoamylase gene gla1 from the basidiomycetous fungus Lentinula edodes strain L54 is reported. The coding region of the genomic glucoamylase sequence, which is preceded by eukaryotic promoter elements CAAT and TATA, spans 2,076 bp. The gla1 gene sequence codes for a putative polypeptide of 571 amino acids and is interrupted by seven introns. The open reading frame sequence of the gla1 gene shows strong homology with those of other fungal glucoamylase genes and encodes a protein with an N-terminal catalytic domain and a C-terminal starch-binding domain. The similarity between the Gla1 protein and other fungal glucoamylases is from 45 to 61%, with the region of highest conservation found in catalytic domains and starch-binding domains. We compared the kinetics of glucoamylase activity and levels of gene expression in L. edodes strain L54 grown on different carbon sources (glucose, starch, cellulose, and potato extract) and in various developmental stages (mycelium growth, primordium appearance, and fruiting body formation). Quantitative reverse transcription PCR utilizing pairs of primers specific for gla1 gene expression shows that expression of gla1 was induced by starch and increased during the process of fruiting body formation, which indicates that glucoamylases may play an important role in the morphogenesis of the basidiomycetous fungus.

Amino Acid Sequence↗

PubMed2000

Reduction of GC --> TA transversion mutation by overexpression of MutS in Escherichia coli K-12.

Overexpression of the MutS repair protein significantly decreased the rate of lacZ GC --> TA transversion mutation in stationary-phase and exponentially growing bacteria and in mutY and mutM mutants, which accumulate mismatches between 8-oxoguanine (8-oxoG) and adenine residues in DNA. Conversely, GC --> TA transversion increased in mutL or mutS mutants in stationary phase. In contrast, overexpression of MutS did not appreciably reduce lacZ AT --> CG transversion mutation in a mutT mutant. These results suggest that MutS-dependent repair can correct 8-oxoG:A mismatches in Escherichia coli cells but may not be able to compete with mutation fixation by MutY in mutT mutants.

Adenine↗

PubMed2000

Identification of the active site of HetR protease and its requirement for heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120.

HetR is a serine-type protease required for heterocyst differentiation in heterocystous cyanobacteria under conditions of nitrogen deprivation. We have identified the active Ser residue of HetR from Anabaena sp. strain PCC 7120 by site-specific mutagenesis. By changing the S152 residue to an Ala residue, the mutant protein cannot be labeled by Dansyl fluoride, a specific serine-type protein inhibitor. The mutant protein showed no autodegradation in vitro. The mutant hetR gene was introduced into Anabaena strain 884a, a hetR mutant. The resultant strain, Anabaena strain S152A, could not form heterocysts under conditions of nitrogen deprivation even though the up-regulation of the mutant hetR gene was induced upon removal of combined nitrogen. The Anabaena strain 216, which carries a mutant hetR gene encoding S179N HetR and could not form heterocysts, also produced HetR protein upon induction. Sequence comparison shows that Ser152 is conserved in all cyanobacterial HetR. Immunoblotting was used to study HetR induction in both the wild-type and mutant strains. The amount of mutant HetR in strain S152A and in strain 216 increased continuously for 24 h after nitrogen step-down, while the amount of HetR in wild-type cells reached a maximum level within 6 h after nitrogen step-down. Our results show the Ser152 is the active site of HetR. The protease activity is required for heterocyst differentiation and might be needed for repression of HetR overproduction under conditions of nitrogen deprivation.

Anabaena↗

PubMed2000

Genotypic stability of cold-adapted influenza virus vaccine in an efficacy clinical trial.

An investigational live influenza virus vaccine, FluMist, contains three cold-adapted H1N1, H3N2, and B influenza viruses. The vaccine viruses are 6/2 reassortants, in which the hemagglutinin (HA) and neuraminidase (NA) genes are derived from the circulating wild-type viruses and the remaining six genes are derived from the cold-adapted master donor strains. The six genes from the cold-adapted master donor strains ensure the attenuation, and the HA and NA genes from the wild-type viruses confer the ability to induce protective immunity against contemporary influenza strains. The genotypic stability of this vaccine was studied by employing clinical samples collected during an efficacy trial. Viruses present in the nasal and throat swab specimens and in supernatants after culturing the specimens were detected and subtyped by multiplex reverse transcriptase (RT)-PCR. Complete genotypes of these detected viruses were determined by a combination of RT-PCR and restriction fragment length polymorphism, multiplex RT-PCR and fluorescent single-strand conformation polymorphism, and nucleic acid sequencing analysis. The FluMist vaccine appeared to be genotypically stable after replication in the human host. All viruses detected during the 2-week postvaccination period were shed vaccine viruses and had maintained the 6/2 genotype.

Cold Temperature↗

PubMed2000

Pyk2 and FAK differentially regulate progression of the cell cycle.

We have previously identified FAK and its associated signaling pathways as a mediator of cell cycle progression by integrins. In this report, we have analyzed the potential role and mechanism of Pyk2, a tyrosine kinase closely related to FAK, in cell cycle regulation by using tetracycline-regulated expression system as well as chimeric molecules. We have found that induction of Pyk2 inhibited G(1) to S phase transition whereas comparable induction of FAK expression accelerated it. Furthermore, expression of a chimeric protein containing Pyk2 N-terminal and kinase domain and FAK C-terminal domain (PFhy1) increased cell cycle progression as FAK. Conversely, the complementary chimeric molecule containing FAK N-terminal and kinase domain and Pyk2 C-terminal domain (FPhy2) inhibited cell cycle progression to an even greater extent than Pyk2. Biochemical analyses indicated that Pyk2 and FPhy2 stimulated JNK activation whereas FAK or PFhy1 had little effect on it, suggesting that differential activation of JNK by Pyk2 may contribute to its inhibition of cell cycle progression. In addition, Pyk2 and FPhy2 to a greater extent also inhibited Erk activation in cell adhesion whereas FAK and PFhy1 stimulated it, suggesting a role for Erk activation in mediating differential regulation of cell cycle by Pyk2 and FAK. A role for Erk and JNK pathways in mediating the cell cycle regulation by FAK and Pyk2 was also confirmed by using chemical inhibitors for these pathways. Finally, we showed that while FAK and PFhy1 were present in focal contacts, Pyk2 and FPhy2 were localized in the cytoplasm. Interestingly, both Pyk2 and FPhy2 (to a greater extent) were tyrosine phosphorylated and associated with Src and Fyn. This suggested that they may inhibit Erk activation in an analogous manner as the mislocalized FAK mutant (&Dgr;)C14 described previously by competing with endogenous FAK for binding signaling molecules such as Src and Fyn. This model is further supported by an inhibition of endogenous FAK association with active Src by Pyk2 and FPhy2 and a partial rescue by FAK of Pyk2-mediated cell cycle inhibition.

Animals↗

PubMed2000

The ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner.

Survivin, a human inhibitor of apoptosis protein (IAP), plays an important role in both cell cycle regulation and inhibition of apoptosis. Survivin is expressed in cells during the G(2)/M phase of the cell cycle, followed by rapid decline of both mRNA and protein levels at the G(1) phase. It has been suggested that cell cycle-dependent expression of survivin is regulated at the transcriptional level. In this study we demonstrate involvement of the ubiquitin-proteasome pathway in post-translational regulation of survivin. Survivin is a short-lived protein with a half-life of about 30 minutes and proteasome inhibitors greatly stabilise survivin in vivo. Expression of the survivin gene under the control of the CMV promoter cannot block cell cycle-dependent degradation of the protein. Proteasome inhibitors can block survivin degradation during the G(1) phase and polyubiquitinated derivatives can be detected in vivo. Mutation of critical amino acid residues within the baculovirus IAP repeat (BIR) domain or truncation of the N terminus or the C terminus sensitises survivin to proteasome degradation. Together, these results indicate that the ubiquitin-proteasome pathway regulates survivin degradation in a cell cycle-dependent manner and structural changes greatly destabilise the survivin protein.

Antineoplastic Agents↗

PubMed2000

A SXRF method for determining the relative concentration of trace elements in plasma protein affected by cisplatin.

This article presents an analytical method for the determination of the relative concentrations of trace elements in plasma protein by gel chromatography combined with SXRF (synchrotron radiation X-ray fluorescence). The fraction of plasma protein of male Kunming mice (body weight 24.2 +/- 0.3 g), treated with a cisplatin i.p. injection at a dose of 10 mg/kg, was obtained by the separation of a Sephadex G-50 column (buffered with ammonium acetate, pH 5.7). The SXRF experiments were performed at the Beijing Electron Positron Collider synchrotron radiation facility. The elements (Pt, S, Ca, Fe, Ni, Cu, Zn, Se, Br, and Sr) in the fraction of the plasma proteins (>22 kDa) were assayed using highly sensitive SXRF. The relative concentrations of elements were calculated by a normalization of Compton scattering intensity around 22 keV, after the normalization for the collection time of the X-ray spectrum and the counting of the ion chamber, and subtracting the contribution of the polycarbonate film for the supporting sample. The determination could prove that the element Pt in plasma was bound with macromolecular protein. Cu and S were present in the fraction of the protein in mice treated with cisplatin increase, and their ratios of treated/control were 1.66 +/- 0.06 and 1.78 +/- 0.33, respectively; Zn decreased to a ratio of 0.78 +/- 0.09. Our results are in agreement with others that cisplatin exposure leads to a marked loss of kidney copper and a moderate rise in kidney zinc. However, this article primarily describes one of the analytical methods used; it does not emphasize the results of the effect of cisplatin on trace elements in plasma protein.

Animals↗

PubMed2000

Endemic goiter associated with high iodine intake.

OBJECTIVES: This study assessed the relation of iodine content of household water to thyroid size and urinary iodine excretion in an area with high iodine concentration in the water. METHODS: The iodine content of household water and indicators of iodine status (thyroid size and urinary iodine level) were assessed in selected villages in Jiangsu Province, China. RESULTS: Water iodine levels were positively correlated with urinary iodine levels and indicators of thyroid size at the township level. CONCLUSIONS: Excess iodine in household water was the likely cause of endemic goiter and elevated urinary iodine levels in the study area. This finding affects public health policy on the institution of universal salt iodization for the elimination of iodine deficiency disorders.

China↗

PubMed2000

Surgical treatment of comminuted intra-articular fractures of the distal humerus with double tension band osteosynthesis.

Intra-articular fractures of the distal humerus are usually treated with cancellous screws for the condylar metaphysis itself and two Y-shaped plates for fixation of the metaphysis to the shaft. Twenty-four patients with comminuted intra-articular fractures (class C on AO/ASIF classification) were treated with crossed K-wires for the condylar metaphysis and double tension band osteosynthesis for fixation of the metaphysis block to the shaft. Excellent or good results were achieved in 83% of patients. This technique is easier to perform and more cost effective than most other fixation methods. Additionally, it offers good stability and allows for earlier functioning of the elbow. Complication rates are comparable to other methods of internal fixation.

Adult↗

PubMed2000

[Information behavior of microsatellite loci in genome scanning].

OBJECTIVE: To determine the heterozygosity and polymorphism information content of 139 microsatellite loci in Han population. METHODS: Multiplex approach was used to analyze the 139 loci. The amplified fragments were subjected to electrophoresis in PAGE gel and analyzed with Genescan( TM) and Genotyper(TM). RESULTS: The heterozygosities are between 0.35 to 0.89, with the heterozygosities of 88% loci >0.60. PICs are in the range of 0.32 and 0.88, with PICs of 95% loci >0.50. CONCLUSION: The polymorphism information content of microsatellite loci is very high and their distribution varies in different races and populations.

Aged↗

PubMed2000

A polyphenolic fraction from grape seeds causes irreversible growth inhibition of breast carcinoma MDA-MB468 cells by inhibiting mitogen-activated protein kinases activation and inducing G1 arrest and differentiation.

In recent years, significant emphasis is being placed on identifying naturally occurring cancer preventive and interventive agents. In this regard, a polyphenolic fraction isolated from grape seeds (hereafter referred as GSP) has recently been shown by us and others to prevent tumorigenesis in mouse skin models. Chemical analysis of GSP has shown that it is largely constituted with procyanidins that are strong antioxidants. Breast cancer is the most common invasive malignancy and the second leading cause of cancer-related deaths in United States women. Accordingly, here we investigated the effect of GSP on mitogenic signaling and regulators of cell cycle and apoptosis as molecular targets for the growth arrest, apoptotic death, and/or differentiation of estrogen-independent MDA-MB468 human breast carcinoma cells. Treatment of cells with GSP (at 25-, 50-, and 75-microg/ml doses for 1-3 days) resulted in a highly significant inhibition (90% to complete, P < 0.001) of constitutive activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase1/2 in a dose-dependent manner after 72 h of treatment. Whereas GSP treatment of cells did not show a conclusive effect on MAPK/ JNK1 activation, a moderate to highly significant inhibition (15-70%, P < 0.1-0.001) of constitutive activation of MAPK/p38 was also observed in a dose-dependent manner as early as 24 h of GSP treatment. GSP-treated cells also showed a strong induction (1.7-2.7 fold, P < 0.001) of cyclin-dependent kinase inhibitor Cip1/p21 and a decrease (10-50%, P < 0.1-0.001) in cyclin-dependent kinase 4. Consistent with these findings, GSP-treated cells resulted in their accumulation in G1 phase of the cell cycle in a dose-dependent manner. An irreversible growth inhibition (44-88%, P < 0.001) was also observed in 50 and 75 microg/ml GSP-treated cells in a time-dependent manner. Additional studies assessing the biological fate of GSP-treated cells showed that they do not undergo apoptotic death, as evidenced by a lack of DNA fragmentation, poly (ADP ribose) polymerase cleavage, and apoptotic morphology of the cells. A morphological change suggestive of differentiation was observed in GSP-treated cells that was further confirmed by a significant induction (1.7-2.6 fold, P < 0.001), in both a dose- and time-dependent manner, in cytokeratin 8 protein level, a marker of differentiation. An irreversible growth-inhibitory effect of GSP possibly via terminal differentiation of human breast carcinoma cells suggests that GSP and the procyanidins present therein should be studied more extensively to be developed as preventive and/or interventive agents against breast cancer in humans.

Animals↗

PubMed2000

[Expression of CPP32, Fas and Fas ligand and the relationship between their expression and viral antigens in chronic hepatitis].

OBJECTIVE: To study the significance of Fas-Fas ligand (FasL)-CPP32 mediated hepatocytic apoptosis and the relationship between their expression and hepatitis C virus (HCV) antigens in pathogenesis of chronic hepatitis C (CHC). METHODS: The expression of Fas and FasL proteins and CPP32 protease and their mRNAs was detected by immunohistochemistry and in situ hybridization respectively. Meanwhile, HCV core protein and nonstructural protein NS(3) and NS(5) were examined on the sequential liver tissue sections from 65 cases of patients with CHC and 5 cases of normal subjects. The positive staining was quantified by image analysis. RESULTS: In CHC liver tissues, CPP32 protease, Fas and their mRNAs were mainly expressed in the hepatocytes, especially in the HCV core protein-positive hepatocytes and their surroundings, but FasL protein and its mRNA were expressed dominantly in the infiltrating mononuclear cells in the portal area and hepatic sinusoid. According to the increase of histological activity index (HAI) in the CHC liver tissues, the expression of the three proteins (protease) and their mRNAs were upregrulated obviously. CONCLUSION: The results suggest i) that Fas-FasL-CPP32 system is associated with HAI of CHC, ii) that HCV core antigen may affect the expression of CPP32 and Fas hepatocytes apoptosis.

Caspase 3↗

PubMed2000

[Adaptation of insect cells(BTI-Tn-5B1-4) to serum-free culture].

The insect cells(BTI-Tn-5B1-4) were adapted, from the medium supplemented with 5% FBS, via 3%, 2%, 1% FBS, to grow in serum-free medium by sequential adaptation for about three months. After adaptation, the cells could successfully grow in the specifically designed serum-free medium with the viability above 90%. In addition, the effect of FBS concentrations and inoculum sizes on the insect cell growth was also investigated.

Adaptation, Physiological↗

PubMed2000

[On-line measurement of oxygen uptake rate in the cultivation of Vero cells using the dynamic method].

The oxygen uptake rate(OUR) during the cultivation of Vero cells in 1.5 L CelliGen bioreactor was on-line determined using the dynamic method. The results showed that the cell growth and metabolic state during the exponential growth phase was lineally related to the OUR. This implies that the on-line measurement of OUR can be used to promptly monitor the physiological state of cultured cells and to efficiently avoid contamination because of frequent sampling in the large-scale cultivation of mammalian cells.

Animals↗