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Biomedical subjects

L Guo

Publications and source records attributed to L Guo.

At least 145 records · Page 8Linked to original sources
PubMed2000

The human polycystic kidney disease 2-like (PKDL) gene: exon/intron structure and evidence for a novel splicing mechanism.

Polycystin-L is a member of the expanding family of polycystins. Mutations in polycystin-1 or -2 cause human autosomal dominant polycystic kidney disease (ADPKD). The mouse ortholog of PKDL, Pkdl, is deleted in a mouse line with renal and retinal defects. We recently have shown that polycystin-L has calcium channel properties. In the current study, we determined the exon/intron organization of the PKDL gene and its alternative splicing. We show that PKDL has 16 exons. All splice acceptor/donor sites for these exons conform to the GT-AG rule. The positions of introns and the sizes of exons in the PKDL gene are very similar to those of PKD2, except for the last two 3' end exons. RT-PCR demonstrates the existence of at least three polycystin-L splice variants: PKDL(Delta5), PKDL(Delta456), and PKDL(Delta15) that are expressed in a tissue-specific manner. In addition, we have localized polymorphic marker D10S603 to intron 4 and exon 5 of PKDL. Elucidation of the gene structure, exact location, and alternative splicing patterns of PKDL will facilitate its evaluation as a candidate gene in cystic or other genetic disorders.

Alternative Splicing↗
PubMed2000

Structure and function of a cap-independent translation element that functions in either the 3' or the 5' untranslated region.

Barley yellow dwarf virus RNA lacks both a 5' cap and a poly(A) tail, yet it is translated efficiently. It contains a cap-independent translation element (TE), located in the 3' UTR, that confers efficient translation initiation at the AUG closest to the 5' end of the mRNA. We propose that the TE must both recruit ribosomes and facilitate 3'-5' communication. To dissect its function, we determined the secondary structure of the TE and roles of domains within it. Nuclease probing and structure-directed mutagenesis revealed that the 105-nt TE (TE105) forms a cruciform secondary structure containing four helices connected by single-stranded regions. TE105 can function in either UTR in wheat germ translation extracts. A longer viral sequence (at most 869 nt) is required for full cap-independent translation in plant cells. However, substantial translation of uncapped mRNAs can be obtained in plant cells with TE105 combined with a poly(A) tail. All secondary structural elements and most primary sequences that were mutated are required for cap-independent translation in the 3' and 5' UTR contexts. A seven-base loop sequence was needed only in the 3' UTR context. Thus, this loop sequence may be involved only in communication between the UTRs and not directly in recruiting translational machinery. This structural and functional analysis provides a framework for understanding an emerging class of cap-independent translation elements distinguished by their location in the 3' UTR.

3' Untranslated Regions↗
PubMed2000

Minocycline inhibits caspase-1 and caspase-3 expression and delays mortality in a transgenic mouse model of Huntington disease.

Huntington disease is an autosomal dominant neurodegenerative disease with no effective treatment. Minocycline is a tetracycline derivative with proven safety. After ischemia, minocycline inhibits caspase-1 and inducible nitric oxide synthetase upregulation, and reduces infarction. As caspase-1 and nitric oxide seem to play a role in Huntington disease, we evaluated the therapeutic efficacy of minocycline in the R6/2 mouse model of Huntington disease. We report that minocycline delays disease progression, inhibits caspase-1 and caspase-3 mRNA upregulation, and decreases inducible nitric oxide synthetase activity. In addition, effective pharmacotherapy in R6/2 mice requires caspase-1 and caspase-3 inhibition. This is the first demonstration of caspase-1 and caspase-3 transcriptional regulation in a Huntington disease model.

Animals↗
PubMed2000

Selecting and expressing protective single-chain Fv fragment to stabilize L-asparaginase against inactivation by trypsin.

Four non-inhibitory specific single-chain Fv (sc Fv) fragments directed against L-asparaginase (ASNase) of Escherichia coli were selected from a synthetic phage-display scFv library. The scFv46 fragment could enhance the resistance of ASNase to trypsin proteolysis, with 70% of the initial ASNase activity present after the ASNase-scFv46 complex had been treated with trypsin for 30 min at 37 degrees C, whereas little residual activity was detected without the scFv46 fragment. The scFv46 gene was cloned to an expression vector pET-21a and expressed at high levels (about 45% of total cell protein) in E. coli BL21 (DE3) as inclusion bodies. The refolded and purified scFv46 fragment was proved to protect ASNase, and the protective effect was further confirmed by SDS/PAGE. It was found that under optimum conditions of molar ratio of scFv to ASNase, incubation time and temperature, the residual activity of the ASNase-scFv46 complex could reach about 78% after treatment with trypsin for 30 min at 37 degrees C. The results demonstrated that scFv fragments prepared by phage-antibody library technology could be used to protect target proteins.

Asparaginase↗
PubMed2000

The influence of prednisolone on the secretion of MUC5AC from TH29-MTX cell culture

INTRODUCTION: Glucocorticoids have been used in the treatment of otitis media with effusion with promising but inconsistent resluts. The HT29-MTX cell line is a completely differentiated and almost exclusively mucous secreting cell line, which therefore has the potential to act as an ideal model for the study of mucus-secreting epithelia. To assess this potential of steroids in suppressing mucin secretion we have studied the response of the cell culture to prednisolone. METHODS: Confluent cell cultures were trypsinized, subcultured in six-well plates and incubated with four doses of prednisolone from 10-5 M to 10-11 M and over varying time courses from 12 to 36 h. ELISA was performed using a monoclonal mouse antibody to human gastric mucin by dot-blot ELISA. RESULTS: Prednisolone caused a reduction in mucin production from the cell line consistently. Media control values ranged from 3495 to 3559 compared with untreated controls of 18 998-27 176 and steroid treatment values of 12 244-27 792. Increasing concentrations of prednisolone result in increasing suppression of MUC5AC (P = 0.005). There was no independent time-related effect. CONCLUSION: There does appear to be a genuine suppression of mucus production by prednisolone and this appears to be dose-dependent but not time-dependent at the times studied. The effect size was small.

Journal Article↗
PubMed2000

Mutant DnaA proteins defective in duplex opening of oriC, the origin of chromosomal DNA replication in Escherichia coli.

We characterized three mutant DnaA proteins with an amino acid substitution of R334H, R342H and E361G that renders chromosomal replication cold (20 degrees C) sensitive. Each mutant DnaA protein was highly purified from overproducers, and replication activities were assayed in in vitro oriC replication systems. At 30 degrees C, all three mutant proteins exhibited specific activity similar to that seen with the wild-type protein, whereas at 20 degrees C, there was much less activity in a replication system using a crude replicative extract. Regarding the affinity for ATP, the dissociation rate of bound ATP and binding to oriC DNA, the three mutant DnaA proteins showed a capacity indistinguishable from that of the wild-type DnaA protein. Activity for oriC DNA unwinding of the two mutant DnaA proteins, R334H and R342H, was more sensitive to low temperature than that of the wild-type DnaA protein. We propose that R334H and R342H have a defect in their potential to unwind oriC DNA at low temperatures, the result being the cold-sensitive phenotype in oriC DNA replication. The two amino acid residues of DnaA protein, located in a motif homologous to that of NtrC protein, may play a role in the formation of the open complex. The E361 residue may be related to interaction with another protein present in a crude cell extract.

Adenosine Triphosphate↗
PubMed2000

Selection of peptide ligands that bind to acid fibroblast growth factor.

Acid fibroblast growth factor (aFGF) binds to its cell-surface receptors in a heparin-dependent manner. In an attempt to define the aFGF recognition site on fibroblast growth factor receptor 1 (FGFR1), we developed a screening strategy for identifying FGF ligands that bind to the receptor-binding region of FGF. To retain the natural conformation of aFGF during screening, we used biotinylated heparin to immobilize aFGF on a streptavidin-coated dish. A 15-mer phage display peptide library was then screened in the dish and a group of related peptide sequences was identified. These peptide sequences contain two conserved motifs, SSG and VPS, corresponding to two protein sequences of the immunoglobulin-like (Ig-like) domain II of FGFR1 at amino acids 180-182 and 221-223 (CPSSG-VPSDKGNYTC). Further experiments demonstrate that the phage displaying these sequences can specifically bind to aFGF and that the synthesized peptide corresponding in sequence can inhibit mitogenic activity of aFGF. These sequences may thus constitute part of the aFGF-binding region on FGFR1, and the synthesized peptide has the potential to become a therapeutic agent as an aFGF antagonist.

3T3 Cells↗
PubMed2000

Caries patterns and their relationship to infant feeding and socio-economic status in 2-4-year-old Chinese children.

AIMS: To describe the prevalence, severity and patterns of caries in 2-4-year-old children and to evaluate the association between caries experience of the children and their feeding patterns and socio-economic background in terms of mothers' education and family income. DESIGN: Cross-sectional survey. SETTING: Suburban area of Hanchuan in Hubei province, China. PARTICIPANTS: A sample of 426 children (250 boys and 176 girls). METHODS: Dental-examinations were undertaken in kindergartens using World Health Organization diagnostic criteria for dental caries. Mothers completed a short questionnaire. OUTCOME MEASURES: Prevalence of caries; rampant caries; caries in incisors; caries in incisors and/or canines and molars; mean number of decayed, missing and filled teeth/surfaces (dmft/s). RESULTS: 36% of the children had caries, 7% had rampant caries. The more extensive pattern of caries involving primary molars as well as incisors and/or canines was seen in 12% of children. Children who had been wholly bottle-fed had five times the risk of having rampant caries compared to children who were breast-fed. CONCLUSIONS: The results indicated that infant feeding practice might be a key risk factor for the development of caries at an early age in this country as elsewhere.

Bottle Feeding↗
PubMed2000

All-trans retinoic acid modulates fas expression and enhances chemosensitivity of human medulloblastoma cells.

Retinoic acid (RA) can promote human medulloblastoma cells Med-3 toward differentiation but is not sufficient to induce cell death, suggesting its limited effect on medulloblastomas. On the other hand, the differentiated tumour cells have been supposed to be more sensitive to chemotherapeutic drugs. To elucidate this possibility for medulloblastoma cells, 10 microM/l RA, 1.0 microg/ml cisplatin (CP) and their half-dosage combinations were utilized in this study to treat Med-3 cells and their influences in cell proliferation, morphology and death patterns were evaluated. In parallel, the expressions of Fas and its ligand (FasL) were analyzed by immunocytochemical staining and Western blot hybridization. Anti-Fas antibody was used to incubate the Med-3 cells pretreated by 10 microM/l RA or 1.0 microg/ml CP. It was revealed that RA and CP could inhibit cell growth but rarely induce apoptosis. Combination of half doses each of RA and CP effectively caused most of tumour cells to die of apoptosis within 6 days. FasL molecules in 29 kDa and 37 kDa were detected in Med-3 cells with and without the treatments. The Fas molecule around 30 kDa and located in the cytoplasm was found in the normally cultured cells and the cells treated by CP. An additional 45 kDa Fas band with the appearance of its cell surface labeling was detected in the cells treated by 10 microM/l RA and by 5 microM/l RA + 0.5 microg/ml CP. The anti-Fas antibody could efficiently induce apoptosis only in the cell populations pretreated by RA. Our data thus suggest that RA can enhance the chemosensitivity of human medulloblastoma Med-3 cells presumably via modulating the Fas expression pattern. The RA/CP combined regimen would be a potential therapeutic approach for medulloblastomas.

Antineoplastic Agents↗
PubMed2000

Frequent expression of soluble Fas and Fas ligand in Chinese stomach cancer and its preneoplastic lesions.

To investigate the frequency and pattern of Fas and FasL expression in the gastric mucosa at different stages of gastrocarcinogenesis, the combined examinations of pathology, immunocytochemistry and Western blot hybridisation were performed on the cancer specimens as well as their preneoplastic and non-cancerous counterparts. The frequencies of Fas and FasL expression were found to be 6.3% (1/16) and 62.5% (10/16) in non-cancerous mucosa, 60% (6/10) and 80% (8/10) in atrophic gastritis, 75% (9/12) and 83% (10/12) in intestinal metaplasia, 100% in both dysplasia Grades II (20/20) and Grade III (15/15) and 4 types of gastric carcinomas (74/74). Two forms of FasL protein in 37 kDa and 26 kDa were detected in all FasL+ cases. Soluble Fas (30 kDa) but not the membrane-type (43 kDa) is predominantly expressed in the Fas+ cases. Our data thus suggest a close correlation of soluble Fas with stomach tumour progression P<0.01. The sFas protein, together with the tumor-derived soluble and membrane FasL, may confer on the transforming and transformed gastric epithelial cells an immune advantage enabling escape from endogenous and exogenous suicide signal(s).

Fas Ligand Protein↗
PubMed2000

BU48: a novel buprenorphine analog that exhibits delta-opioid-mediated convulsions but not delta-opioid-mediated antinociception in mice.

N-Cyclopropylmethyl-[7alpha,8alpha,2', 3']-cyclohexano-1'[S]-hydroxy-6,14-endo-ethenotetrahydronororip avine (BU48) is a novel, ring-constrained analog of buprenorphine. In vivo, BU48 (0.1-10 mg/kg s.c.) produced brief, nonlethal convulsions in mice followed by brief Straub tail and a short period of catalepsy characteristic of BW373U86 and other nonpeptidic delta-receptor agonists. BU48-induced convulsions were sensitive to antagonism by naltrindole (10 mg/kg s.c.) and were also prevented by administration of the putative delta(1) antagonist 7-benzylidenenaltrexone and the putative delta(2) antagonist naltriben, with the latter being more potent. In the abdominal stretch assay in the mouse, only low-efficacy antinociceptive activity of BU48 (0.1-10 mg/kg) was seen. This was reversed by the kappa-opioid antagonist norbinaltorphimine (32 mg/kg s.c.) but not by the delta-opioid antagonist naltrindole (10 mg/kg s.c.). BU48 (10 mg/kg s.c.) acted as a delta-antagonist in this assay. In mouse brain homogenates, BU48 had high (nanomolar) binding affinity for all three opioid receptors in the order mu > delta = kappa. In vitro, the compound acted as a potent (EC(50) = 1.4 nM) kappa-opioid agonist in the guinea pig ileum and a potent (EC(50) = 0.2 nM) delta-opioid agonist in the mouse vas deferens but showed partial agonist activity at the rat cloned delta-opioid (40%) and human cloned kappa-opioid (59%) receptors with very low efficacy at the rat cloned mu-opioid receptor (10%); findings consistent with its in vivo profile. BU48 is the first described compound that produces delta-opioid-mediated convulsions without any evidence of delta-opioid-mediated antinociception and will be a useful tool in investigations of the delta-opioid receptor.

Analgesics, Opioid↗
PubMed2000

Alternative splicing of RHAMM gene in chinese gastric cancers and its in vitro regulation.

OBJECTIVE: To identify the receptor of hyaluronic acid mediated motility (RHAMM), expression pattern(s) in the gastric mucosa with various lesions and to elucidate the effect of hyaluronic acid(HA) on in vitro RHAMM expression. METHODS: Immunocytochemical staining and Western blot hybridization were performed to check the expression patterns and locations of RHAMM molecules in the noncancerous, preneoplastic and malignant gastric specimens. RHAMM expression of the two stomach cancer cell lines cultured conventionally or under the conditions with gelatin or HA supplementation was evaluated as well. RESULTS: Mr 93000-95000 intracellular RHAMM, so-called IHABP, proteins were detected in 17% noncancerous mucosa. The multiple spectrums and increased frequencies of RHAMM expression could be observed in the majority of the cancer cases with both intracellular and cell surface labeling. HA but not gelatin could promote membrane type RHAMM (mRHAMM) expression of stomach cancer cells in a dose associated pattern. CONCLUSION: The expression of mRHAMM variants is closely associated with tumor progression, which may be promoted and modulated by HA.

Alternative Splicing↗
PubMed2000

Expression of hyaluronan receptors CD44 and RHAMM in stomach cancers: relevance with tumor progression.

Interactions of hyaluronic acid (HA) with its binding proteins CD44 and RHAMM (receptor for HA-mediating motility) have been proposed to be important in promoting tumor progression and dissemination. However, a comparative study of their expression patterns in stomach cancer and its associated lesions is not yet available. To address this issue, the combined examinations of pathology, immunocytochemistry and Western blot hybridization were performed on advanced gastric cancer specimens as well as their preneoplastic and non-cancerous counterparts. Alternative CD44 expression was observed in the gastric mucosa with different lesions. CD44 proteins harboring variant exon 6 (CD44 v6) was detected only in cancer tissues with a total positive rate of 14% (10/74). Intracellular RHAMM molecules in Mr 93000 to 95000 were expressed in 3/31 non-cancerous mucosa. RHAMM detection rates increased along with tumor progression. Irrespective of the differences of gross and morphological pattern, majority (54/74) of cancer cases expressed multiple RHAMM isoforms in Mr 40000-45000, 64000, 70000-73000, 85000 and 93000-95000 with the appearance of cell surface immunocytochemical labeling. Among CD44 variant isoforms, v6 is more relevant with malignant transformation of gastric epithelium. Expression of RHAMM, especially the cell surface variants, is closely correlated with tumor progression (P<0.01). Expression of CD44 and RHAMM may benefit the invasion and metastasis of gastric cancer cells presumably in a reciprocal manner.

Blotting, Western↗
PubMed2000

[Modification of L-asparaginase with colominic acid and the new characteristics of the modified enzyme].

The colominic acid was covalently coupled to L-asparaginase molecule by reductive amination. Depending on the molar ratios of colominic acid-asparaginase (30:1, 50:1 and 100:1), a modified enzyme molecule contained 4.7, 7.2 and 12 colominic acid molecule, they retained 58%, 56% and 33.2% of the initial asparaginase activity, respectively. In comparison with the native enzyme, modified enzyme had lower immunogenicity and antigenicity, longer half-life time (in vitro), more resistance ability to trypsin proteolysis, and similar Km value for L-asparagine.

Asparaginase↗
PubMed2000

Screening for mt-DNA mutations in optic neuritis of unknown cause.

OBJECTIVE: To investigate mitochondrial DNA (mt-DNA) mutations in optic neuritis of unknown cause (ONUC) and to assess the practical value of mt-DNA mutation detection in etiologically and differentially diagnosing ONUC. METHODS: Thirty patients with ONUC were screened for mt-DNA mutations of nt11778, nt3460 and nt15257 by using SSCP, mutation-specific primer PCR and sequencing. RESULTS: mt-DNA mutations were found in twelve of thirty ONUC patients. All of the mutations were at nt11778 position, but no one at nt3460 and nt15257. CONCLUSIONS: Forty percent (12/30) of ONUC patients were caused by an mt-DNA mutation. Combined with other routine measures, screening for mt-DNA mutations in ONUC patients is of great significance in diagnosing ONUC etiologically and differentially.

Adolescent↗
PubMed2000

Comparison of computer-photoscreening with non-cycloplegic retinoscopy for amblyopiogenic risk factors in children.

OBJECTIVE: To determine the sensitivity and specificity of using a computer-photoscreener and non-cycloplegic retinoscopy in the detection of amblyopiogenic factors in nine to fifty months old infants and children. METHODS: Three hundred children, nine to fifty months old, were screened with the computer-photoscreener and non-cycloplegic retinoscopy. With a blinded standardized clinical assessment as the standard, an overall comparison of the sensitivity of and specificity results obtained with the two techniques was made. Photoscreen images on the computer monitor screen were reviewed and analyzed immediately by two independent observers for indicators of amblyopiogenic risk factors. Simultaneously, the results were compared to the findings of a full ophthalmologic examination. RESULTS: The computer-photoscreener revealed a sensitivity of 94.6% and specificity of 90.1%, and the non-cycloplegic retinocopy revealed a sensitivity of 85.7% and specificity of 81.0% for the detection of amblyopiogenic risk factors, including hyperopia (+2.75 D or more), myopia (-1.50 D or more), astigmatism (1.75 D or more), anisometropia (2.00 D or more), ocular misalignment (5 degrees or more), and media opacity (1.5 mm or more). CONCLUSIONS: The computer-photoscreener offers an opportunity to identify problems that limit vision, and could provide a feasible and sufficiently reliable screening technique in infants and preschool children to be screened successfully for amblyopiogenic risk factors.

Amblyopia↗