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Biomedical subjects

L Shen

Publications and source records attributed to L Shen.

At least 127 records · Page 7Linked to original sources
PubMed1999

[Ciliary neurotrophic factor in the regeneration of facial nerve].

OBJECTIVE: To study the role of CNTF in regeneration of facial nerve. METHOD: SD Rat facial nerves in temporal bones were transected and then combined end to end. In ciliary neurotrophic factor group (n = 30), lesions were covered by gelatin sponge soaked with CNTF. In control group (n = 18), lesions were covered by gelatin sponge soaked with normal saline. 2, 4, 12 weeks after the operation, both CNTF group and SAL group undergone electrophysiology test and histopathology as well as quantity analysis. RESULT: Two weeks later, both CNTF and SAL group failed in eliciting muscular excitement by stimulating facial nerve; T-test showed significant difference between the two groups(P < 0.05) about the average of myelinated axons 4 weeks later, the latency of facial muscular excitement was 7.650 +/- 2.702 ms in CNTF group and 16.080 +/- 0.162 ms in SAL group. The average number of myelinated axons was 1614 +/- 411 in experimental group and 1094 +/- 343 in control group. T-test showed significant difference between them (P < 0.05). 12 weeks later, latency of facial muscle excitement was 2.016 +/- 0.122 ms in CNTF group and 2.060 +/- 0.162 ms in SAL group. The average number of myelinated axons was 1909 +/- 332 in experimental group and 1847 +/- 422 in control group. T-test showed no significant difference between the two groups (P > 0.05). CONCLUSION: CNTF enhance facial nerve regeneration in adult SD rats.

Administration, Topical↗
PubMed1999

[Determination of alpha-acid in hop by high performance liquid chromatography].

A method for the determination of alpha-acid in hop by reversed-phase high performance liquid chromatgraphy is presented. The alpha-acid in hop and hop exteact was separated in a mu-Bondapak C18 cartridge by using V(CH3OH):V(H2O):V(H3PO4) = 80:20:0.25 as mobile phase. The flow rate was 1.0 mL/min and detection of alpha-acid was performed with UV detector at 314 nm. Quantification of alpha-acid was carried out by measuring peak height/peak area and comparing with external standard. The relative standard deviation of alpha-acid in hop was 2.02% and in hop extract was 1.78%. The method is simple, rapid and satisfactory.

English Abstract↗
PubMed1999

[Cibacron blue F3GA-attached 2 microns non-porous monodisperse silicas for affinity chromatography].

Non-porous monodisperse silica (NPS), 2 microns in diameter, was modified with 3-aminopropyltriethoxysilane for immobilization of Cibacron Blue F3GA (CB), a packing of NPS-ACB for affinity chromatography was obtained. Up to 2 mg of CB could be attached to 1 mL of NPS beads. There was no obvious leakage of dye from NPS-ACB. Oval was not retained by the column, while Lys was specifically adsorbed. The adsorption of Lys varied with pH values and ionic strengths. In addition, alpha-globulin could not be retained by the packing, while beta- and gamma-globulin could be adsorbed on the column. gamma-Globulin was able to be eluted by 20% 1,6-hexanediol and 1 mol/L KCl, while beta-globulin was not able to be eluted by the same eluent. The difference in affinity interaction could be used to separate the three globulins. Furthermore, the column could be used for separation and preparation of Lys from hen egg white. The chromatograms of Lys on non-porous silica diethylamine column (NPS-DEA) showed that retention time of one peak of the crude Lys prepared was in accordance with Lys's, so it could be said that NPS-ACB column can be used for preparation in a small scale.

Alpha-Globulins↗
PubMed1999

[Expression and identification of recombinant 22.6 kDa fusion protein of Schistosoma japonicum].

AIM: To obtain a large amount of purified 22.6 kDa antigen of Schistosoma japonicum (Sj22.6) in large quantity. METHODS: The sequence of the gene fragment encoding Sj22.6 was reformed by PCR and subcloned into plasmid vector pGEX-1 lambda T that coded for the 26 kDa GST antigen of Schistosoma japonicum (Sj26 GST). The recombinant plasmid was transformed into E. coli TG2 and then the positive recombinant clone was expressed by induction with IPTG. RESULTS: The recombinant Sj22.6/Sj26 GST fusion protein was expressed in 5.1% of total bacterial protein and was easy to be purified with glutathione sepharose 4B. Moreover, the purified recombinant Sj22.6 antigen could be cut off easily from the fusion protein with thrombin and had high immunogenicity. CONCLUSION: The purified recombinant Sj22.6 protein and Sj22.6/Sj26 GST fusion protein had the same immunological activity as the native Sj22.6 kDa protein.

Animals↗
PubMed1999

[Studies on immunoprotection in mice after immunization with Schistosoma japonicum 22.6 kDa recombinant protein].

AIM: To evaluate the immunoprotective effect of Schistosoma japonicum recombinant 22.6 kDa (rSj22.6) and Sj22.6/Sj26 GST fusion protein. METHODS: The Sj22.6/Sj26 GST fusion protein was prepared by affinity chromatography using glutathione Sepharose 4B. The purified rSj22.6 could be cleaved easily from the fusion protein with Thrombin. 17 and 12 mice immunized with rSj22.6 and Sj22.6/Sj26 GST separately were each challenged with 40 +/- 1 S. japonicum cercariae. RESULTS: In BALB/c mice, the rSj22.6 and Sj22.6/Sj26 GST could induce 32.1 (P < 0.005) and 34.9% (P < 0.02) worm reduction, respectively, as well as 28.4% (P < 0.02) and 45.1% (P < 0.005) total egg reduction, respectively. CONCLUSION: Bpth rSj22.6 and Sj22.6/Sj26 GST fusion protein are partially effective against S. japonicum.

Animals↗
PubMed1999

[Development and clinical application of dental spot weld & heat-treat tri-uses device].

A spot weld & heating treatment tri-uses device based on modern mechanical-electric technique, was developed for dental spot weld, stainless steel orthodontic wire heating-reversion and Ni-Ti orthodontic wire heating-overload metamorphism. The device will be very helpful instrument in orthodontic clinic. The paper not only described the operating methods of the device, but also discussed the Performance-improved principles of stainless steel orthodontic wire and the principles of heating overload metamorphism of Ni-Ti orthodontic wire.

Dental Soldering↗
PubMed1998

Enhancement of human protein C function by site-directed mutagenesis of the gamma-carboxyglutamic acid domain.

This study reports properties of site-directed mutants of human protein C that display enhanced calcium and/or membrane binding properties. Mutants containing the S11G modification all showed increased affinity for membranes at saturating calcium concentration. Ser-11 is unique to human protein C, whereas all other vitamin K-dependent proteins contain glycine. This site is located in a compact region of the protein, close to a suggested membrane contact site. Additional changes of H10Q or S12N resulted in proteins with lower calcium requirement for membrane contact but without further increase in membrane affinity at saturating calcium. Mutations Q32E and N33D did not, by themselves, alter membrane affinity to a significant degree. These mutations were included in other mutant proteins and may contribute somewhat to higher function in these mutants. This family of mutants helped discriminate events that are necessary for protein-membrane binding. These include calcium binding to the free protein and subsequent protein-membrane contact. Depending on conditions of the assay used, the mutants displayed increased activity of the corresponding activated protein C (APC) derivatives. The degree of enhanced activity (up to 10-fold) was dependent on the concentration of phospholipid and quality of phospholipid (+/- phosphatidylethanolamine) used in the assay. This was expected, because APC is active in its membrane-associated form, which can be regulated by changes in either the protein or phospholipid. As expected, the largest impact of the mutants occurred at low phospholipid concentration and in the absence of phosphatidylethanolamine. The anticoagulant activity of all proteins was stimulated by protein S, with the greatest impact on the enhanced mutants. Whereas plasma containing Factor V:R506Q was partially resistant to all forms of APC, the enhanced variants were more active than normal APC. Protein C variants with enhanced function present new reagents for study of coagulation and may offer improved materials for biomedical applications.

1-Carboxyglutamic Acid↗
PubMed1998

Four ubiquitously expressed genes, RD (D6S45)-SKI2W (SKIV2L)-DOM3Z-RP1 (D6S60E), are present between complement component genes factor B and C4 in the class III region of the HLA.

The association of the HLA class III region with many diseases motivates the investigation of unidentified genes in the 30-kb segment between complement component genes Bf and C4. RD, which codes for a putative RNA binding protein, is 205 bp downstream of Bf. SKI2W (HGMW-approved symbol SKIV2L), a DEVH-box gene probably involved in RNA turnover, is 171 bp downstream of RD (HGMW-approved symbol D6S45). RP1 (HGMW-approved symbol D6S60E) is located 611 bp upstream of C4. The DNA sequence between human RD and RP1 was determined and the exon-intron structure of SKI2W elucidated. SKI2W consists of 28 exons. The putative RNA helicase domain of Ski2w is encoded by 9 exons. Further analysis of the 2.5-kb intergenic sequence between SKI2W and RP1 led to the discovery of DOM3Z. The full-length cDNA sequence of DOM3Z encodes 396 amino acids with a leucine zipper motif. Dom3z-related proteins are present in simple and complex eukaryotes. In Caenorhabditis elegans, Dom3z-related protein could be involved in the development of germ cells. Human RD-SKI2W and DOM3Z-RP1 are arranged as two head-to-head oriented gene pairs with unmethylated CpG sequences at the common 5' regulatory region of each gene pair. The ubiquitous expression pattern suggests that these four genes are probably housekeeping genes.

Amino Acid Sequence↗
PubMed1998

Amino acid residues in thrombin-sensitive region and first epidermal growth factor domain of vitamin K-dependent protein S determining specificity of the activated protein C cofactor function.

Human protein S (PS) potentiates the anticoagulant activity of human but not bovine activated protein C (APC), whereas bovine PS is a cofactor to APC from both species. The structural requirements for the specificity of the APC cofactor function of human PS are located in its thrombin-sensitive region (TSR) and the first epidermal growth factor (EGF1)-like module. To elucidate which residues in these two modules determine the specificity of the APC cofactor activity, 41 human PS mutants were expressed. All mutants were cofactors to human APC and some also to bovine APC. Residues in TSR (positions 49 and 52) and EGF1 (residues 97 and 106) together determined the specificity of the APC cofactor function, whereas substitution of individual residues did not change specificity. Bovine PS, and mutants expressing cofactor activity to bovine APC, stimulated phospholipid binding of bovine APC. In contrast, human PS and mutants lacking cofactor activity to bovine APC failed to support binding of bovine APC to phospholipids. These data indicate that residues in TSR and EGF1 cause the specificity of the APC cofactor activity and support the concept that key residues in these two modules interact with APC on the phospholipid surface.

Amino Acid Sequence↗
PubMed1998

The human DEVH-box protein Ski2w from the HLA is localized in nucleoli and ribosomes.

The human helicase gene SKI2W is located between RD and RP1 in the class III region of the major histocompatibility complex. Transcripts of SKI2W are detectable in RNA samples isolated from multiple tissues. The protein product Ski2w shares striking amino acid sequence similarities to the yeast antiviral protein Ski2p that controls the translation of mRNAs, probably based on the mRNA structural integrity. Whether this translational regulation mechanism for cellular and viral RNAs exists in mammals is under investigation. Antisera against human Ski2w were generated using fusion proteins produced in bacteria or insect cells. Western blot analysis showed that the endogenous Ski2w protein is approximately 140 kDa in size and is enriched in polysomal fractions of cytoplasmic extracts from HeLa cells. Ribosomal profile studies revealed that Ski2w distributed throughout the entire sucrose gradient in the presence of Mg2+, but co-sedimented with the 18S rRNA-containing 40S subunit and the small ribosomal subunit protein S27a in the presence of EDTA. The co-sedimentation of Ski2w with the 40S subunit is not affected by RNase A treatment of the cell extract, or the addition of KCl to 0.5 M, suggesting that Ski2w is associated with the 40S ribosomal subunit. Indirect immunofluorescence experiments showed that human Ski2w is localized in the nucleoli and in the cytoplasm. In essence, human Ski2w is present at the sites of ribosome biogenesis and protein synthesis.

Amino Acid Sequence↗
PubMed1998

Induction of apoptosis in glioblastoma cells by inhibition of protein kinase C and its association with the rapid accumulation of p53 and induction of the insulin-like growth factor-1-binding protein-3.

Increased protein kinase C(alpha) (PKC(alpha)) expression in glioblastoma cells is associated with proliferation and resistance to drug-induced apoptosis by an undefined anti-apoptotic pathway. To clarify the role of PKC in apoptosis, we have investigated the effect of the selective PKC inhibitor Ro 31-8220 (3-[1-[3-(amidinothio)propyl]-3-indolyl]-4-(1-methyl-3-indolyl)-1H -pyrrole-2,5-dione methanesulfonate) in two glioblastoma cell lines whose proliferation is dependent on high levels of PKC(alpha). U-87 and A172 cells treated with an IC50 of Ro 31-8220 exhibited nucleosomal DNA fragmentation that coincided with an increase in the number of apoptotic cells. This effect was preceded by the rapid nuclear accumulation of wild-type p53 within 2 hr, and an increased level of the pro-apoptotic protein, insulin-like growth factor-1-binding protein-3, (IGFBP3) but not other p53-regulated proteins such as p21WAF1 or Bax. Accumulation of p53 was also associated with the hypophosphorylated and activated form of the retinoblastoma tumor suppressor protein (RB) at later times after treatment. These results suggest that PKC(alpha) suppresses apoptosis in glioblastoma cells primarily by restricting the accumulation of p53 and the expression of insulin-like growth factor-1-binding protein, as well as by maintaining RB in an inactive hyperphosphorylated state.

Apoptosis↗
PubMed1998

In vivo effects of a bacterial superantigen on macaque TCR repertoires.

A macaque model was employed to explore staphylococcal enterotoxin B (SEB) superantigen-driven T lymphocyte responses. The SEB-reactive Vbeta+ cell subpopulations demonstrated a striking tri-phase response in rhesus monkeys following an SEB challenge in vivo. The hyperacute down-regulation, seen as early as 2 h through 2 days after SEB injection, was characterized by a disappearance of the reactive Vbeta-restricted PBL subpopulations from the circulation and decreased expression of these cell subpopulations in lymphoid tissues. Following this, a dominant expansion of reactive Vbeta-expressing CD4+ cell subpopulations occurred in lymph nodes and spleens, whereas in the peripheral blood a preferential expansion of reactive Vbeta-expressing CD8+ cell subpopulations was seen. An exhaustion of this response was then seen, with a prolonged decrease in the number of the reactive Vbeta+ CD4+ lymphocyte subpopulations. Interestingly, monoclonal or oligoclonal dominance was seen in the reactive Vbeta+ cell subpopulations in the period of the transition from the polyclonal cellular expansion to the exhaustion of the response, suggesting that some Vbeta+ cell clones may be more resistant than others to superantigen-mediated depletion. These results indicate that in vivo SEB superantigen-mediated effect on lymphocyte subpopulations in macaques is complex, suggesting that profound dynamics in the TCR repertoires may in part account for the susceptibility of higher primates to SEB-induced diseases.

Animals↗
PubMed1998

Dependence of germinal center B cells on expression of CD21/CD35 for survival.

Affinity-driven selection of B lymphocytes within germinal centers is critical for the development of high-affinity memory cells and host protection. To investigate the role of the CD21/CD35 coreceptor in B cell competition for follicular retention and survival within the germinal center, either Cr2+ or Cr2null lysozyme-specific transgenic B cells were adoptively transferred into normal mice immunized with duck (DEL) or turkey (TEL) lysozyme, which bind with different affinities. In mice injected with high-affinity turkey lysozyme, Cr2null B cells responded by follicular retention; however, they could not survive within germinal centers. This suggests that CD21 provides a signal independent of antigen that is required for survival of B cells in the germinal center.

Adoptive Transfer↗
PubMed1998

Expression and regulation of GFRalpha3, a glial cell line-derived neurotrophic factor family receptor.

We report the identification of an additional member of the glial cell line-derived neurotrophic factor (GDNF) family receptor, termed GFRalpha3, that is homologous to the previously identified GDNF and neurturin ligand binding receptors GFRalpha1 and GFRalpha2. GFRalpha3 is 32% and 37% identical to GFRalpha1 and GFRalpha2, respectively. RNase protection assays show that whereas gfralpha1 and gfralpha2 are abundant in both developing and adult brain, gfralpha3 is exclusively expressed during development. All receptors are widely present in both the developing and adult peripheral nervous system and in peripheral organs. For instance, in situ hybridization shows that the developing liver, stomach, intestine, kidney, and sympathetic chain, which all contain ret-expressing cells, transcribe unique complementary and overlapping patterns of most or all of the GDNF family receptors and ligands. In sensory neurons of the trigeminal ganglion gfralpha2 and gfralpha3 are expressed in different subpopulations of neurons, whereas gfralpha1 is coexpressed in some gfralpha2 and gfralpha3-positive neurons. We find that the gfralpha1 population of trigeminal neurons is absent in GDNF null mutant mice, suggesting that GDNF signals in vivo by interacting with GFRalpha1. Thus, our results show that there are at least three members in the GDNF family of ligand binding receptors and that these receptors may be crucial in conferring ligand specificity in vivo. The unique complementary and overlapping expression of gfralpha3 implies distinct functions in the developing and adult mouse from that of GFRalpha1 and GFRalpha2.

Amino Acid Sequence↗
PubMed1998

Tumor necrosis factor alpha levels in plasma and whole-blood culture in dengue-infected patients: relationship between virus detection and pre-existing specific antibodies.

The pathogenesis of dengue hemorrhagic fever (DHF) is not well known, but the role of host factors has been suggested. The level of immunoreactive circulating and cell-generated tumor necrosis factor alpha (TNF alpha) was studied in 35 patients with DHF; its relationship with virus isolation and/or genome detection by reverse transcription polymerase chain reaction (RT-PCR) and specific antibodies were detected by hemagglutination inhibition (HI). Large variation of TNF alpha plasma levels was obtained in dengue-infected patients at the same stage of the disease and at the same day after infection. Most of the patients (14 out of 17 patients) who displayed augmented spontaneous in vitro production of TNF alpha by heparinized whole-blood culture compared with controls also had elevated levels of TNF alpha in the plasma. The TNF alpha values in lipopolysaccharide and phytohemagglutinin heparinized whole-blood cultures were not higher in patients than in controls, but low TNF alpha levels were obtained in three out of 30 patients. An inverse correlation was observed between spontaneous in vitro TNF alpha production and viral replication, which raises the issue of the antiviral effect of TNF alpha in dengue infection. The results do not support the hypothesis of the role of antibody-dependent enhancement giving rise to increased viremic titers and production of TNF alpha in patients. The present study demonstrates the activation of the TNF alpha-producing cells in dengue-infected patients and suggests further investigation to define the mechanism and the role of TNF alpha in the pathogenesis of dengue virus infection.

Adolescent↗
PubMed1998

Alkylation of 2'-deoxynucleosides and DNA by the Premarin metabolite 4-hydroxyequilenin semiquinone radical.

Premarin (Wyeth-Ayerst) is the estrogen replacement treatment of choice and continues to be one of the most widely dispensed prescriptions in the United States. In addition to endogenous estrogens, Premarin contains unsaturated estrogens including equilenin. We synthesized the catechol metabolite of equilenin, 4-hydroxyequilenin (4-OHEN), and found that the semiquinone radical of 4-OHEN reacted with 2'-deoxynucleosides generating very unusual adducts. 2'-Deoxyguanosine (dG), 2'-deoxyadenosine (dA), or 2'-deoxycytosine (dC) all gave four isomers, but no product was observed for thymidine under similar physiological conditions. The structures of these adducts were determined by electrospray mass spectrometry and NMR experiments including 1H, 13C, DQF-COSY, ROESY, HOHAHA, HMQC, and HMBC. The spectral data show that dG forms a cyclic adduct with the 4-OHEN producing 2-N1,3-N2-deoxyguanosyl-1,3-dihydroxy-5,7,9(10)-estratriene-4,17-d ione. Similarly, reaction with dA produced 1-N6,3-C2-deoxyadenosyl-2,3-dihydroxy-5,7,9(10)-estratriene-4,17-d ione, and incubations with dC resulted in 1-N3,3-N4-deoxycytosyl-2,3-dihydroxy-5,7,9(10)-estratriene-4,17-di one. We found that care needed to be taken during the isolation of the dA adducts in particular, as any exposure to acidic environments caused hydrolysis of the sugar moiety leaving alkylated adenine. In mixtures of the deoxynucleosides treated with 4-OHEN, reaction occurred primarily with dG followed by dC and dA. With DNA significant apurinic sites were produced as 4-OHEN-adenine adducts were detected in the ethanol wash prior to hydrolysis. When the DNA was hydrolyzed to deoxynucleosides and analyzed by electrospray mass spectrometry, only one isomer of 4-OHEN-dG and one isomer of 4-OHEN-dC were observed. Our data suggest that several different types of DNA lesions could be expected from 4-OHEN including apurinic sites and bulky stable adducts, in addition to the published oxidized damage to DNA caused by 4-OHEN. The production of these semiquinone radical-derived DNA adducts could play a role in the carcinogenic effects of Premarin estrogens.

Alkylation↗
PubMed1998

Inhibition of glutathione S-transferase activity by the quinoid metabolites of equine estrogens.

The risk factors for women developing breast and endometrium cancers are all associated with a lifetime of estrogen exposure. Estrogen replacement therapy (ERT) in particular has been correlated with a slight increased cancer risk, although the numerous benefits of ERT may negate this harmful side effect. Equilenin and equilin are equine estrogens which make up between 30% and 45% of the most widely prescribed estrogen replacement formulation, Premarin (Wyeth-Ayerst). In this study we have synthesized the catechol metabolites of equilenin [4-hydroxyequilenin (4-OHEN)] and equilin [4-hydroxyequilin (4-OHEQ)] and examined how changing unsaturation in the B ring affects the formation of o-quinone GSH conjugates and the ability of the o-quinones and/or GSH conjugates to inhibit glutathione S-transferase (GST). Interestingly, both 4-OHEN and 4-OHEQ autoxidized to o-quinones without the need of oxidative enzyme catalysis. 4-OHEN-o-quinone reacts with GSH to give two mono-GSH conjugates and one diadduct. The behavior of 4-OHEQ was found to be more complex than 4-OHEN as conjugates resulting from 4-OHEN were detected in addition to the 4-OHEQ GSH adducts. Both 4-OHEN and 4-OHEQ were found to be potent inhibitors of GST-catalyzed conjugation of GSH with 1-chloro-2,4-dinitrobenzene. In contrast, the endogenous catechol estrogens, 4-hydroxyestrone (4-OHE) and 2-hydroxyestrone (2-OHE), were without effect unless tyrosinase was present to convert the catechols to o-quinones. Scavengers of reactive oxygen species and metal chelators had no effect on GST inhibition by catechol estrogens with the exception of the catalase which protected GST activity. Kinetic studies showed that 4-OHEN was a potent irreversible inactivator of GST. Preincubation of the enzyme with 4-OHEN showed a time-dependent increase in inhibitory effect, and gel filtration did not restore GST activity confirming the irreversible nature of the enzyme inactivation. Analysis of the Kitz-Wilson plot gave a dissociation constant of the reversible enzyme-inhibitor complex (Ki = 620 microM) and a rate constant of conversion of the reversible enzyme-inhibitor complex to the irreversibly inhibited enzyme (k2 = 7.3 x 10(-)3 s-1). These data suggest that 4-OHEN is an irreversible inactivator with relatively low affinity for GST; however, once formed the 4-OHEN enzyme complex is rapidly converted to the irreversibly inhibited enzyme. The inhibition mechanism likely involves oxidation of the catechol estrogens to o-quinones and covalent modification and/or oxidation of critical amino acid residues on GST. In addition, hydrogen peroxide generated through redox cycling of the o-quinone and/or semiquinone radical and GSH could cause oxidative damage to GST.

Animals↗