Biomedical subjects

K Zhou

Publications and source records attributed to K Zhou.

At least 19 recordsLinked to original sources

PubMed2001

Direct pK(a) measurement of the active-site cytosine in a genomic hepatitis delta virus ribozyme.

Hepatitis delta virus ribozymes have been proposed to perform self-cleavage via a general acid/base mechanism involving an active-site cytosine, based on evidence from both a crystal structure of the cleavage product and kinetic measurements. To determine whether this cytosine (C75) in the genomic ribozyme has an altered pK(a) consistent with its role as a general acid or base, we used (13)C NMR to determine its microscopic pK(a) in the product form of the ribozyme. The measured pK(a) is moderately shifted from that of a free nucleoside or a base-paired cytosine and has the same divalent metal ion dependence as the apparent reaction pK(a)'s measured kinetically. However, under all conditions tested, the microscopic pK(a) is lower than the apparent reaction pK(a), supporting a model in which C75 is deprotonated in the product form of the ribozyme at physiological pH. While additional results suggest that the pK(a) is not shifted in the reactant state of the ribozyme, these data cannot rule out elevation of the C75 pK(a) in an intermediate state of the transesterification reaction.

Base Sequence↗

PubMed2001

Overexpression, biophysical characterization, and crystallization of ribonuclease I from Escherichia coli, a broad-specificity enzyme in the RNase T2 family.

We have constructed a strain that overproduces ribonuclease I of Escherichia coli and we have purified large quantities of the enzyme. Data from fluorescence, CD, and DSC measurements showed that it was a very stable protein. The conformation energy determined from urea and guanidine hydrochloride denaturation experiments was 11.5 kcal mol(-1) at pH 7.5. Thermal denaturation studies indicated that it had a T(m) of 64 degrees C at pH 4.0. RNase I belongs to the RNase T2/S-RNase group of endoribonucleases, but near the amino terminus it has an unusually long hydrophilic segment. Part of this was removed in the deletion construct, RNase I Delta(26-38). We have obtained crystals of both RNase I and of an enzyme-G2'p5'G complex in the P2(1) space group and oligonucleotide complexes with both wild type and mutant enzymes. The current study lays the groundwork for extensive investigation into the structure, function, and physical properties of this widely distributed group of ribonucleases.

Amino Acid Sequence↗

PubMed2001

Hepatitis C virus IRES RNA-induced changes in the conformation of the 40s ribosomal subunit.

Initiation of protein synthesis in eukaryotes requires recruitment of the 40S ribosomal subunit to the messenger RNA (mRNA). In most cases, this depends on recognition of a modified nucleotide cap on the 5' end of the mRNA. However, an alternate pathway uses a structured RNA element in the 5' untranslated region of the messenger or viral RNA called an internal ribosomal entry site (IRES). Here, we present a cryo-electron microscopy map of the hepatitis C virus (HCV) IRES bound to the 40S ribosomal subunit at about 20 A resolution. IRES binding induces a pronounced conformational change in the 40S subunit and closes the mRNA binding cleft, suggesting a mechanism for IRES-mediated positioning of mRNA in the ribosomal decoding center.

5' Untranslated Regions↗

PubMed2001

Mechanism of ribosome recruitment by hepatitis C IRES RNA.

Many viruses and certain cellular mRNAs initiate protein synthesis from a highly structured RNA sequence in the 5' untranslated region, called the internal ribosome entry site (IRES). In hepatitis C virus (HCV), the IRES RNA functionally replaces several large initiation factor proteins by directly recruiting the 43S particle. Using quantitative binding assays, modification interference of binding, and chemical and enzymatic footprinting experiments, we show that three independently folded tertiary structural domains in the IRES RNA make intimate contacts to two purified components of the 43S particle: the 40S ribosomal subunit and eukaryotic initiation factor 3 (eIF3). We measure the affinity and demonstrate the specificity of these interactions for the first time and show that the high affinity interaction of IRES RNA with the 40S subunit drives formation of the IRES RNA-40S-eIF3 ternary complex. Thus, the HCV IRES RNA recruits 43S particles in a mode distinct from both eukaryotic cap-dependent and prokaryotic ribosome recruitment strategies, and is architecturally and functionally unique from other large folded RNAs that have been characterized to date.

Animals↗

PubMed2001

Authentication of Chinese crude drug, Gecko, by allele-specific diagnostic PCR.

Based on the sequences of the mitochondrial 12S rRNA gene fragment of 17 samples from Gekkonidae, Salamandridae, Agamidae and Hynobiidae, respectively, a pair of allele-specific primers was designed for differentiating the Chinese medicinal material Gecko from its adulterants by PCR. The results of amplification with the primers indicate that amplicons from the templates of Gekko gecko were clearly revealed by agarose gel electrophoresis, whereas no evident amplicons were found from other species. The primers were employed to identify crude drug samples from different sources. Among a total of 9 samples, 3 were diagnosed as genuine Gecko. This result is consistent with morphological identification and DNA sequence analyses.

Animals↗

PubMed2001

Gene expression profiling in human fetal liver and identification of tissue- and developmental-stage-specific genes through compiled expression profiles and efficient cloning of full-length cDNAs.

Fetal liver intriguingly consists of hepatic parenchymal cells and hematopoietic stem/progenitor cells. Human fetal liver aged 22 wk of gestation (HFL22w) corresponds to the turning point between immigration and emigration of the hematopoietic system. To gain further molecular insight into its developmental and functional characteristics, HFL22w was studied by generating expressed sequence tags (ESTs) and by analyzing the compiled expression profiles of liver at different developmental stages. A total of 13,077 ESTs were sequenced from a 3'-directed cDNA library of HFL22w, and classified as follows: 5819 (44.5%) matched to known genes; 5460 (41.8%) exhibited no significant homology to known genes; and the remaining 1798 (13.7%) were genomic sequences of unknown function, mitochondrial genomic sequences, or repetitive sequences. Integration of ESTs of known human genes generated a profile including 1660 genes that could be divided into 15 gene categories according to their functions. Genes related to general housekeeping, ESTs associated with hematopoiesis, and liver-specific genes were highly expressed. Genes for signal transduction and those associated with diseases, abnormalities, or transcription regulation were also noticeably active. By comparing the expression profiles, we identified six gene groups that were associated with different developmental stages of human fetal liver, tumorigenesis, different physiological functions of Itoh cells against the other types of hepatic cells, and fetal hematopoiesis. The gene expression profile therefore reflected the unique functional characteristics of HFL22w remarkably. Meanwhile, 110 full-length cDNAs of novel genes were cloned and sequenced. These novel genes might contribute to our understanding of the unique functional characteristics of the human fetal liver at 22 wk.

Cloning, Molecular↗

PubMed2001

SF/HGF-c-Met autocrine and paracrine promote metastasis of hepatocellular carcinoma.

AIM: To explore the role of SF/HGF-Met autocrine and paracrine in metastasis of hepatocellular carcinoma (HCC). METHODS: SF/HGF and c-met transcription and protein expression in HCC were examined by RT-PCR and Western Blot in 4 HCC cell lines, including HepG2, Hep3B, SMMC7721 and MHCC-1, the last cell line had a higher potential of metastasis. sf/hgf cDNA was transfected by the method of Lipofectin into SMMC7721. SF/HGF and c-met antibody were used to stimulate and block SF/HGF-c-met signal transduction. Cell morphology, mobility, and proliferation were respectively compared by microscopic observation, wound healing assay and cell growth curve. RESULTS: HCC malignancy appeared to be relative to its met-SF/HGF expression. In MHCC-1, c-met expression was much stronger than that in other cell lines with lower potential of metastasis and only SF/HGF autocrine existed in MHCC-1. After sf/hgf cDNA transfection or conditioned medium of MHCC-1 stimulation, SMMC7721 changed into elongated morphology, and the abilities of proliferation (P < 0.05) and mobility increased. Such bio-activity could be blocked by c-met antibody (P < 0.05). CONCLUSION: The system of SF/HGF-c-met autocrine and paracrine played an important role in development and metastasis potential of HCC. Inhibition of SF/HGF-c-met signal transduction system may reduce the growth and metastasis of HCC.

Autocrine Communication↗

PubMed2001

[Influence of scatter factor/hepatocyte growth factor on the growth and transmission of hepatocarcinoma SMMC 7721 cells].

OBJECTIVE: To explore the influence of scatter factor/hepatocyte growth factor (SF/HGF) on the growth and transmission of hepatocarcinoma SMMC 7721 cells by SF/HGF cDNA transfection. METHODS: Gene transfection was operated by lipofectin method. In vitro, SF/HGF and c-met expression was tested by ELISA and Western blot. Cell proliferation and motility were compared by growth curves and wound healing assays. In vivo, cells before and after transfection were implanted subcutaneously into nude mice respectively to observe tumor growth and metastasis. RESULTS: After transfection, the expression of SF/HGF reached 694 pg/ml, compared with 0.26 pg/ml before transfection; however the expression of c-met protein did not change obviously. Growth curves showed that cells after transfection proliferated more rapidly than those before transfection and had an increased capability of motility together with enlarged morphological changes. In vivo experiment, tumors originated from SF/HGF(+) cells grew faster than those from SF/HGF(-) cells and had embolism formed inside and metastasis in the corresponding lung tissues, whereas no such findings in SF/HGF(-) cell originated tumors. CONCLUSIONS: High expression of SF/HGF can stimulate tumor cell proliferation and metastasis in hepatocellular carcinoma

Animals↗

PubMed2001

[Evaluation of dynamic enhanced fast multiplanar spoiling gradient recalled(FMPSPGR) in the diagnosis of small hepatocellular carcinoma].

OBJECTIVE: To analyze the features of small hepotocellular carcinoma (SHCC) by spin echo (SE) sequence and dynamic enhanced FMPSPGR, and to compare the sensitivity for detecting and the accuracy for characterization, further to discuss the value of FMPSPGR in the diagnosis of SHCC. METHODS: SE T(1)WI, T(2)WI and FMPSPGR dynamic imaging of the liver were performed for 58 patients with SHCC. The sensitivity for detecting and accuracy for characterization were statistically compared. RESULTS: Seventy-two lesions were found in 58 patients. In the detection of SHCC, the order of the sensitivity was dynamic enhanced FMPSPGR>SE T(2)WI>no-enhanced FMPSPGR>SE T(1)WI. Dynamic enhanced FMPSPGR was significantly higher than any of the others. There was significant difference between no-enhanced FMPSPGR and SE T(2)WI, but not significant difference between T(2)WI and no-enhanced FMPSPGR. In the characterization of SHCC, the accuracy of pre- and post-contrast FMPSPGR was significantly higher than that of SE (T(1)WI+T(2)WI) with remarkable statistical difference. CONCLUSIONS: FMPSPGR dynamic imaging is superior to SE in the detection and characterization of SHCC. The sensitivity for detecting and the accuracy for characterization will be improved when both FMPSPGR and SE are used.

Adult↗

PubMed2001

Antioxidant properties of phenolic diterpenes from Rosmarinus officinalis.

AIM: To investigate the inhibition capacities of carnosol, rosmanol, and epirosmanol, which are phenolic diterpenes from Rosmarinus officinalis, to oxidized low-density lipoprotein (LDL) formation in human blood and detect their scavenging activities to lipid free radical and superoxide anion in vitro. METHODS: The antioxidant activities which were expressed with the inhibilities to lipid free radicals in the membrane lipid of cell and oxidized LDL formation were evaluated by TBARS assay and ESR method. The inhibition on the Cu2+-mediated oxidization of apo B formation in LDL was investigated by fluorescence spectroscopy. RESULTS: Carnosol, rosmanol, and epirosmanol had an inhibitory activity to lipid peroxidation and oxidized apo B formation in human bloods LDL. The IC50 were 7-10 micromol/L. The antioxidant mechanism was related to the scavenging activities to lipid free radical. CONCLUSION: carnosol, rosmanol, and epirosmanol showed the activity in inhibiting LDL oxidation.

Abietanes↗

PubMed2001

Lack of correlation of vacA genotype, cagA gene of Helicobacter pylori and their expression products with various gastroduodenal diseases.

OBJECTIVE: To investigate the correlation among vacA genotypes, cagA gene, VacA, serum CagA antibodies of Helicobacter pylori (H. pylori) and gastroduodenal diseases. METHODS: vacA genotypes and cagA gene of 62 H. pylori strains isolated from patients with chronic gastritis, peptic ulcer and gastric cancer were tested by polymerase chain reaction, and Hela cell assay for VacA activity in vitro. Serum CagA antibodies were measured by EIA method in the same patients. RESULTS: All 62 H. pylori strains possessed the vacA gene and vacA genotypes of all strains were type s1a/m2. Total positive rate of cagA gene was 56.45%; the positive rates of cagA gene of H. pylori strains isolated from patients with chronic gastritis, peptic ulcer and gastric cancer were 55.56%, 54.17% and 63.64%, respectively (P > 0.05). The total positive rate of VacA was 37.10%; the positive rates of VacA produced by H. pylori strains isolated from patients with chronic gastritis, peptic ulcer and gastric cancer were 33.33%, 29.17% and 63.64%, respectively (P > 0.05). The positive rates of CagA antibodies in patients with chronic gastritis, peptic ulcer and gastric cancer were 70.37%, 79.17% and 40.00%, respectively (P > 0.05). The total positive rate of CagA antibodies was 68.85%. CONCLUSION: There was no correlation among cagA gene and vacA genotypes of H. pylori, VacA, serum CagA antibodies and various gastroduodenal diseases.

Antibodies, Bacterial↗

PubMed2001

[A study on the relations of soluble interleukin-2 receptor and CD+25 cell with acute graft-versus-host disease].

OBJECTIVE: Acute graft-versus-host disease (aGVHD) is one of the major complications after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Prediction of human aGVHD may be of help in its diagnosis and therapy. METHODS: 22 patients undergoing HSCT were included in this study. After transplantation, serum concentrations of interleukin-6, interleukin-8, tumor necrosis factor-alpha, interferon-gamma and sIL-2R were measured periodically by using enzyme-linked immunosorbent assay. Immune reconstitution of CD(+)(3), CD(+)(4), CD(+)(8), CD(+)(25) and CD(+)(69) cells were analyzed with flow cytometry. RESULTS: All the patients achieved engraftment. 6 patients developed grade I GVHD and 4 patients developed grade III-IV GVHD. Patients with aGVHD demonstrated significantly higher sIL-2R levels than those without. The peak levels of grade III-IV GVHD were (420.3 +/- 59.8) U/L,and the peak levels of grade I GVHD were (221.5 +/- 38.8) U/L. The increase of sIL-2R level in serum preceded the clinical signs of aGVHD. The maximum levels of sIL-2R correlated significantly with the severity of aGVHD. The increase of CD(+)(25) cells was in accordance with that of sIL-2R. CONCLUSIONS: Analysis of serum cytokines and immune reconstitution after HSCT may provide predictive value for aGVHD.

Adolescent↗

PubMed2001

[Comparison of enhancement patterns of multi-phase scan of dynamic MRI and dynamic CT in small hepatocellular carcinoma].

OBJECTIVE: To study prospectively the enhancement features of multi-phase scan of dynamic MRI and spiral CT in the diagnosis of small hepatocellular carcinoma (SHCC) and to discuss the cause of dynamic MRI being superior to spiral CT. METHODS: Multi-phase contrast scanning of dynamic MR and spiral CT were done in 53 SHCC patients. The lesions were proved by surgery and pathology. The arterial-phase, portal venous-phase and delayed phase scan of spiral CT were carried out after the pre-contrast scanning of the entire liver. MRI was performed with SE sequence and FMPSPGR sequence dynamic multi-phase contrast scans. RESULTS: Seventy-six lesions were found in 53 patients. Sixty-nine lesions and 54 lesions were enhanced obviously in MR and spiral CT arterial-phase scans. The typical enhancement patterns of SHCC in the arterial-phase, portal venous-phase and delayed phase scan of MRI and spiral CT were hyper-hypo-hypointense (dense) and hyperintense (hyperdense), -isointense (isodense) and -hypointense (hypodense). Atypical enhancement patterns were hyperintense (hyperdense), -hyperintense (hyperdense), -hyperintense (hyperdense), -hyperintense (hyperdense), -isointense (isodense), -isointense (isodense) and -hypointense- (hypodense), -hypointense (hypodense) and -hypointense (hypodense). CONCLUSION: Both dynamic MRI and spiral CT multi-phase contrast scanning are able to demonstrate the enhancement features of SHCC, with arterial-phase scan of MRI being superior to spiral CT in reflecting the hypervascular characterization of SHCC. Combined with SE sequence in characterizing the SHCC, MRI is better than spiral CT.

Adult↗

PubMed2001

[Study on relationship between the reflux laryngitis and gastroesophageal reflux disease].

OBJECTIVE: To investigate the relationship between reflux laryngitis and gastroesophageal reflux disease (GRED). METHOD: 130 Patients of refractory chronic laryngitis with upper endoscopy and ambulatory 24-hr esophageal pH-metry. The patients of gastroesophageal reflux disease were divided into two groups. Group A was treated with antacids and motile medications. Group B was control. RESULT: 45 (34.6%) gastroesophageal reflux disease were found in 130 cases. 21 cases(91.3%) were healed in group A. 3 of 22 cases (13.6%) were healed in control (P < 0.01). CONCLUSION: The gastroesophageal reflux disease was an important cause of the reflux laryngitis which symptoms and signs can be healed by antacids and motile medications.

Adult↗

PubMed2001

[Mitochondrial mechanisms of CPT-induced apoptosis in nasopharyngeal carcinoma cells].

OBJECTIVE: To understand whether CPT-induced apoptosis is related to the alteration of mitochondrial transmembrane potentials (delta psi m). METHOD: A low differentiation human nasopharyngeal carcinoma (NPC) cell line of CNE-2Z cells were used as in vitro model. After treatment with 2 mumol/L CPT for 24 h, the delta psi m was detected by the staining of rhodamine 123 (Rh123), while apoptosis was confirmed by sub-G1 cell content and the double staining of hoechst33258 and propidium iodide (PI). RESULT: CPT induced delta psi m collapse and apoptosis. With 2 mumol/L CPT treatment for 24 h, delta psi mlow cell was increased to (19.0 +/- 3.0)%, while in the DMSO control, delta psi mlow was (7.0 +/- 1.4)% P < 0.05. CONCLUSION: The disruption of the delta psi m may be one of the mechanisms of CPT-induced apoptosis in CNE-2Z cells.

Antineoplastic Agents, Phytogenic↗

PubMed2001

[Expression of the artificial cecropin CMIV-like gene of Chinese silkworm Bombyx mori in Laphygma exigua larvae].

The synthesized CMIV-like Gene was linked with the signal peptide gene of nature silkworm antibacterial peptide and was inserted into baculovirus expression vector pFastBac 1, construcing a recombinant transposing vector. The vector was transformed into DH10Bac competent E. coli cells. The recombinant Bacmid was obtained. The recombinant Bacmid was transfected into sf21 cells to get the recombinant virus. Laphygma exigua larvae were infected with the recombinant virus to express the antibacterial peptide. The hemolymph were tested to have antibacterial activity. The active antibacterial peptide was purified by acid polyacrylamide electrophoresis. The specific expression of mRNA of CMIV-like Gene was tested using Northern blotting.

Animals↗

PubMed2000

Authentication of an animal crude drug, Zaocys, by diagnostic PCR.

A pair of diagnostic primers for distinguishing the Chinese crude drug Zaocys (Zaocys dhumandes) from its substitutes was designed based on the sequence data of the original animal of the drug and substitutes. Total DNAs were extracted from genuine crude drug and 5 of its substitutes, as well as from 12 species of original animal of the snake crude drug. Diagnostic PCRs were performed using the primers with these total DNAs as a template, annealing at 60-65 degrees C. Positive amplifications were obtained from all DNA templates of Zaocys, whereas negative amplifications were obtained from that of others. The results indicate that Zaocys samples could be definitely distinguished from its substitutes by diagnostic PCR, and no incorrect discrimination was found under the same reaction conditions. The advantages of the method in the authentication of crude drugs are also discussed in the present paper.

Animals↗

PubMed2000

Fas activation reduces neutrophil adhesion to endothelial cells.

Polymorphonuclear neutrophils (PMN) express apoptotic markers and lose effector functions including adhesion, chemotaxis, and phagocytosis when cultured overnight. Although the loss of function correlates with apoptosis, it is not clear if functions are lost before an early marker of apoptosis, the display of phosphatidylserine (PS), targets PMN for removal by phagocytic cells. To address this question, freshly isolated PMN were treated with Fas-activating antibodies to induce apoptosis rapidly. Early markers of apoptosis and PMA-stimulated adhesion to endothelial cells were measured. After 1 h of Fas exposure, only 16% PMN had externalized PS. In contrast, Fas activation reduced PMA-stimulated adhesion between 68 and 27% depending on PMA concentration. The loss of adhesion was accompanied by a reduction in beta2 integrin expression and receptor clustering. These results indicate that the Fas-induced loss of adhesion may precede PS externalization and could limit participation in the inflammatory response before PS externalization targets PMN for removal.

Animals↗