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Biomedical subjects

Hua He

Publications and source records attributed to Hua He.

48 records · Page 3Linked to original sources
PubMed2004

Effect of temperature on enantiomer separation of oxzepam and lorazepam by high-performance liquid chromatography on a beta-cyclodextrin derivatized bonded chiral stationary phase.

A reversed-phase high-performance liquid chromatography (HPLC) method with beta-cyclodextrin (beta-CD) derivatized as chiral stationary phase is used to directly separate oxazepam (Oxa) and lorazepam (Lor) enantiomers. The effect of temperature on the direct HPLC separation of Oxa and Lor enantiomers is studied for the commercially available beta-CD derivatized bonded chiral stationary phase. Chromatographic peak coalescence, appearing as a plateau between the resolved peaks, is observed at column temperatures of above 13 degrees C. Peak coalescence on the beta-CD derivatized bonded column is attributable to racemization of the Oxa enantiomer. By reducing the column temperature to 13 degrees C, the enantiomeric composition of Oxa and Lor could be determined on the chiral column. This method is expected to be useful for the resolution of 3-hydroxybenzodiazepines. At the same time, the separation mechanism is studied by calculating the thermodynamic parameters. The results reveal that the separation of Oxa and Lor enantiomer is a case of enthalpy-controlled separation, inclusion mechanism does not control the separation. The interaction between Oxa and beta-CD is an additionally strong pi-pi interaction or hydrogen bonding, but that between Lor or beta-CD derivatized is a weak pi-pi interaction or hydrogen bonding.

Anti-Anxiety Agents↗
PubMed2004

Characterization of corneal pannus removed from patients with total limbal stem cell deficiency.

PURPOSE: To determine the epithelial lineage of origin in corneal pannus tissue surgically removed from patients with total limbal stem cell (SC) deficiency. METHODS: The lineage of origin of the entire conjunctivalized pannus removed from eight corneas with a diagnosis of total limbal SC deficiency was characterized by anti-keratin (K)-3 and anti-K19 monoclonal antibodies. The protein and mRNA of epithelial outgrowth from segments of five such pannus specimens were analyzed by Western blot and reverse transcription-polymerase chain reaction, respectively. RESULTS: Cross sections of all eight specimens showed a stratified epithelium with goblet cells expressing mucin (MUC)-5AC, and a stroma showing blood vessels and inflammatory cell infiltrates. Immunostaining showed full-thickness expression of K19 in the entire pannus of all eight specimens. Expression of K3 was negative in seven patients, but was sporadically positive in a patient with Stevens-Johnson syndrome. In culture, all five pannus specimens generated a compact, small epithelial cell outgrowth, and except for one, reached confluence in 2 to 3 weeks. The K3/K12 pair was expressed by extracts of cell outgrowth from the control limbal epithelial explant, but not in all five pannus specimens. A 60-kDa band of DeltaNp63 was expressed in the control specimen and in all five pannus specimens. Cell outgrowth expressed K3 transcript in three, but none showed K12 transcript. CONCLUSIONS: The resultant epithelial phenotype of the pannus tissue was not corneal, as evidenced by the negative staining to cornea-specific K12 mRNA and protein, but was conjunctival, as evidenced by the presence of goblet cells, the weak expression of K3, and the strong expression of K19. The abundant expression of DeltaNp63 in such conjunctiva-derived epithelium in eyes with total limbal SC deficiency raises doubts as to its validity as a limbal SC marker.

Adult↗
PubMed2004

Calcium-induced abnormal epidermal-like differentiation in cultures of mouse corneal-limbal epithelial cells.

PURPOSE: To develop a reproducible method for expanding mouse corneal-limbal epithelial cells and determine the role of extracellular Ca(2+) concentration and serum in modulating their growth and differentiation. METHODS: Intact and viable corneal epithelial sheets were isolated from CD-1 albino mouse eyeballs by incubating for 18 hours at 4 degrees C in 15 mg/mL dispase II with sorbitol in defined keratinocyte serum-free medium (KSFM) or supplementary hormonal epithelial medium (SHEM). These sheets were trypsinized into single cells and cultured on plastic in KSFM or SHEM. Cultures in KSFM were further manipulated by increasing Ca(2+) concentration to 0.9 mM, with or without 5% FBS. Epithelial growth was compared in KSFM/KSFM (digestion medium/culture medium) and SHEM/SHEM by continuous passaging at a 1:3 split and by crystal violet staining of confluent dishes. Epithelial differentiation was assessed by immunostaining and/or immunoblotting to ZO-1, cytokeratin K12 (K12), connexin 43 (Cx43), cytokeratin K10 (K10), and involucrin. RESULTS: Intact and viable corneal-limbal epithelial sheets were consistently isolated from more than 200 mouse eyes. Gradual increases in cell sizes and expression of ZO-1, K12, and Cx43 were noted from KSFM/KSFM to SHEM/KSFM, KSFM/SHEM, and SHEM/SHEM at passage 0. Epithelial growth ended at passage 1 in SHEM/SHEM but continued until passage 3 in KSFM/KSFM. Immunoblot analysis revealed that K12 expression was the highest in SHEM/SHEM, decreased from passages 0 to 1, and disappeared in passage 2 in KSFM/KSFM, with complete replacement of K10 and increasing expression of involucrin. Appearance of K10 was facilitated by 0.9 mM Ca(2+) but suppressed by 5% FBS in KSFM at passage 0. CONCLUSIONS: Mouse corneal-limbal epithelial sheets can be used for initiating primary cultures, and their differentiation is promoted, whereas growth is suppressed, by a high Ca(2+) concentration, even during enzymatic digestion. In serum-free medium, abnormal epidermal-like differentiation is promoted by increasing Ca(2+) concentrations but prevented by serum. These results provide the ability to devise a medium to promote growth while maintaining normal differentiation.

Animals↗
PubMed2004

[The efficacy of two type of membrane plasma separator on the treatment of patients with chronic severe hepatitis B].

OBJECTIVE: In order to compare the efficacy of two kinds of membrane plasma separator on the treatment of patients with severe hepatitis B. METHODS: 63 cases suffering from chronic severe hepatitis B were divided into two groups, 25 cases were treated with plasma exchange using Evacure-4A membrane plasma separator (A group) or 38 cases were using PS-06 membrane plasma separator (B group). Both of them also were treated with similar basic medical treatment. The level of serum total bilirubin, non-conjugated bilirubin, prothrombin time and albumin were tested at baseline and the end of the treatment with PE. RESULTS: Evacure-4A and PS-06 membrane plasma separators can effientively remove bilirubin, the levels of serum total bilirubin, non-conjugated bilirubin of all patients were significantly decreased after treated with PE. In A group, the level of serum total bilirubin, non-conjugated bilirubin decreased from (464.2+/-193.8)micromol/L to (279.4+/-158.7)micromol/L, (293.5+/-129.1)micromol/L to (175.5+/-106.7)micromol/L (t=5.45, 10.36, P<0.01) respectively. In B group, the level of serum total bilirubin, non-conjugated bilirubin decreased from (493.2+/-126.9)micromol/L to (299.7+/-96.5)micromol/L, (300.2+/-74.3)micromol/L to (171.5+/-53.1)micromol/L (t=5.17, 12.04, P<0.01) respectively. The level of serum albumin increased after treated with PE in A and B groups, to contrast with PS-06, the increasing percentage of albumin was higher when the patients were treated with PE using Evacure-4A membrane plasma separator [(8.3+/-0.7) % vs. (3.4+/-9.3) %, t = 2.76, P<0.01]. CONCLUSION: Evacure-4A membrane plasma separator may be better than PS-06 membrane plasma separator on the treatment of patients with chronic severe hepatitis B.

Adult↗
PubMed2004

[The difference between the positive rate of anti-HCV in the patients with severe viral hepatitis detected by the first generation or second generation enzyme linked-immunosorbent assay].

OBJECTIVE: To compare the positive rate of antibody to hepatitis C virus (anti-HCV) in sera of patients with severe viral hepatitis between 1984-1990 year and 1997-2003 year. METHODS: Serum anti-HCV was detected by enzyme linked-immunosorbent assay (ELISA). It was detected by the first generation (1st) ELISA (Ortho Co. USA) in 79 cases of severe viral hepatitis during 1984-1990 year, and it was detected by the second generation (2nd) ELISA (Xiamen Xingchuang Co. China) in 251 cases of severe viral hepatitis during 1997-2003 year. RESULTS: The positive rate of serum anti-HCV was 51.9% detected by the 1st ELISA in 79 cases of severe viral hepatitis during 1984-1990 year, and it was 1.2% detected by the 2nd ELISA in 251 cases of severe hepatitis during 1997-2003 year (chi2 = 133.68, P </= 0.001). CONCLUSION: To compare with the positive rate of serum antibody to hepatitis C virus in severe viral hepatitis detected by the 1st ELISA, it was lower that detected by the 2nd ELISA

Adult↗
PubMed2003

Post-translational modifications of three members of the human MAP1LC3 family and detection of a novel type of modification for MAP1LC3B.

The molecular machinery required for autophagy is highly conserved in all eukaryotes as seen by the high degree of conservation of proteins involved in the formation of the autophagosome membranes. Recently, both yeast Apg8p and its rat homologue Map1lc3 were identified as essential constituents of autophagosome membrane as a processed form. In addition, both the yeast and human proteins exist in two modified forms produced by a series of post-translational modifications including a critical C-terminal cleavage after a conserved Gly residue, and the smaller processed form is associated with the autophagosome membranes. Herein, we report the identification and characterization of three human orthologs of the rat Map1LC3, named MAP1LC3A, MAP1LC3B, and MAP1LC3C. We show that the three isoforms of human MAP1LC3 exhibit distinct expression patterns in different human tissues. Importantly, we found that the three isoforms of MAP1LC3 differ in their post-translation modifications. Although MAP1LC3A and MAP1LC3C are produced by the proteolytic cleavage after the conserved C-terminal Gly residue, like their rat counterpart, MAP1LC3B does not undergo C-terminal cleavage and exists in a single modified form. The essential site for the distinct post-translation modification of MAP1LC3B is Lys-122 rather than the conserved Gly-120. Subcellular localization by cell fractionation and immunofluorescence revealed that three human isoforms are associated with membranes involved in the autophagic pathway. These results revealed different regulation of the three human isoforms of MAP1LC3 and implicate that the three isoforms may have different physiological functions.

Amino Acid Sequence↗
PubMed2003

Human keratocytes cultured on amniotic membrane stroma preserve morphology and express keratocan.

PURPOSE: To develop a new method of expanding human corneal keratocytes in serum while maintaining their characteristic morphology and keratocan expression. METHODS: Human keratocytes were isolated from central corneal buttons by digestion in 1 mg/mL of collagenase A in DMEM and seeded on plastic or the stromal matrix of human amniotic membrane (AM) in DMEM with different concentrations of FBS. On confluence, cells on AM were continuously subcultured for six passages on AM or plastic. In parallel, cells cultured on plastic at passages 3 and 11 were reseeded on AM. Cellular morphology and cell-cell networks were assessed by phase-contrast microscopy and a cell viability assay, respectively. Expression of keratocan was determined by RT-PCR and Western blot analysis. RESULTS: Trephined stroma yielded 91,600 +/- 26,300 cells (ranging from 67,000 to 128,000 cells per corneal button). Twenty-four hours after seeding, cells appeared dendritic on AM, even in 10% FBS but fibroblastic on plastic. Such a difference in morphology correlated with expression of keratocan assessed by RT-PCR and Western blot, which was high and continued at least to passage 6 on AM, even in 10% FBS, but was rapidly lost each time when cells on AM were passaged on plastic. Fibroblasts continuously cultured on plastic to passages 3 and 11 did not reverse their morphology or synthesize keratocan when reseeded on plastic in 1% FBS or on AM. CONCLUSIONS: Human keratocytes maintain their characteristic morphology and keratocan expression when subcultured on AM stromal matrix even in the presence of high serum concentrations. This method can be used to engineer a new corneal stroma.

Amnion↗
PubMed2002

Deficiency in ClC-3 chloride channels prevents rat aortic smooth muscle cell proliferation.

Recent growing evidence suggests that chloride (Cl-) channels are critical to the cell cycle. In cultured rat aortic vascular smooth muscle cells (VSMCs), we have previously found that Cl- channel blockers inhibit endothelin-1 (ET-1)-induced cell proliferation. The present study was designed to further identify the specific Cl- channels responsible for VSMC proliferation. Due to the lack of a specific blocker or opener of any known Cl- channels, we used the antisense strategy to investigate the potential role of ClC-3, a member of the voltage-gated Cl- channel gene family, in cell proliferation of cultured rat aortic VSMCs. With [3H]-thymidine incorporation and immunoblots, we found that ET-1-induced cell proliferation was parallel to a significant increase in the endogenous expression of ClC-3 protein. Transient transfection of rat aortic VSMCs with antisense oligonucleotide specific to ClC-3 caused an inhibition in ET-1-induced expression of ClC-3 protein and cell proliferation of VSMCs in the same concentration- and time-dependent pattern, whereas sense and missense oligonucleotides resulted in no effects on ClC-3 protein expression and cell proliferation. These results strongly suggest that ClC-3 may be the Cl- channel involved in VSMC proliferation and thus provide compelling molecular evidence linking a specific Cl- channel to cell proliferation. The full text of this article is available at http://www.circresaha.org.

Animals↗
PubMed2002

Human serum and glucocorticoid-inducible kinase-like kinase (SGKL) phosphorylates glycogen syntheses kinase 3 beta (GSK-3beta) at serine-9 through direct interaction.

Serum and glucocorticoid-inducible kinase-like kinase (SGKL) has been identified as a new integrator that decodes lipid signals produced by the activation of phosphoinositide 3-kinase (PI3K). SGKL is activated via its lipid-binding domain (phox homology domain) in response to PI3K signaling. However, downstream targets of SGKL as well as the role of SGKL as a mediator in PI3K signaling in human tissues remain to be established. In this study, we identified human glycogen synthase kinase 3 beta (GSK-3beta) as a specific interacting partner with SGKL in a yeast two-hybrid screening of human brain cDNA library. The association between these two proteins is confirmed independently in human embryonic kidney (HEK293) cells by co-immunoprecipitation. Furthermore, the kinase activity of wild-type SGKL was required for the in vitro phosphorylation of a GSK-3 crosstide fusion protein at serine-21/9 as demonstrated with a Phospho-GSK-3alpha/beta (Ser21/9) specific antibody. The present results provide strong evidences that SGKL could utilize GSK-3beta as a direct downstream target by phosphorylating GSK-3beta at serine-9.

Brain↗
PubMed2002

Effects of Cl- channel blockers on endothelin-1-induced proliferation of rat vascular smooth muscle cells.

The effects of Cl- channel blockers on endothelin-1 (ET-1)-induced proliferation of rat aortic vascular smooth muscle cells (VSMC) were examined. We found ET-1 concentration-dependently increased cell count and [3H]-thymidine incorporation into VSMC, with EC50 values of 24.8 and 11.4 nM, respectively. Both nifedipine and SK&F96365 inhibited 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC with the maximal inhibitory concentrations of 1 and 10 microM, respectively. DIDS inhibited 10 nM ET-1-induced increase in cell count and [3H]-thymidine incorporation into VSMC in a concentration-dependent manner, whereas other Cl- channel blockers including IAA-94, NPPB, DPC, SITS and furosemide did not produce these effects. 3 microM DIDS reduced 10 nM ET-1-induced sustained increase in cytoplasmic Ca2+ concentration ([Ca2+]) by 52%. Pretreatment of VSMC with 1 microM nifedipine completely inhibited the DIDS effect on 10 nM ET-1-induced [3H]-thymidine incorporation into VSMC and sustained increase in [Ca2+]i, whereas pretreatment with 10 microM SK&F96365 did not completely block these effects of DIDS. DIDS did not affect ET-1-induced Ca2+ release and 30 mM KCl-induced increase in [Ca2+]i. Our data suggest that DIDS-sensitive Cl- channels mediate VSMC proliferation induced by ET-1 by mechanisms related to membrane depolarization and Ca2+ influx through voltage-dependent Ca2+ channels.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2002

Identification and characterization of nine novel human small GTPases showing variable expressions in liver cancer tissues.

Digestion and detoxification are the two most important functions of the liver, and liver cells always keep a high metabolism level and active vesicular traffic. The malfunction of the vesicular traffic system might be a cause of the abnormal biological behavior of cancerous liver cells. The Ras superfamily is known to regulate various steps of vesicular traffic in eukaryotic cells. It would be significant to determine the change of vesicular transport molecules such as the members of Ras superfamily in carcinogenesis of liver cells. In the present study, we have cloned nine novel genes encoding human small GTPases: RAB1B, RAB4B, RAB10, RAB22A, RAB24, RAB25 ARL5, SARA1, and SARA2, among which the former six belong to the RAB family and the latter three belong to the ARF/SAR1 family. The identification of these new genes has greatly enlarged the pool of the Ras superfamily. It is interesting to find that they are upregulated in most of the 11 hepatocellular carcinoma and 1 cholangiohepatoma cases. Furthermore, the expression in 16 normal human adult tissues, the chromosome loci, and the gene structures of the nine genes are also described. The above findings could be valuable for understanding the vesicular transport system and elucidating the molecular basis of liver cancer carcinogenesis.

Amino Acid Sequence↗