Biomedical subjects

L H Guo

Publications and source records attributed to L H Guo.

At least 19 recordsLinked to original sources

PubMed2002

Induction of acetylcholinesterase expression during apoptosis in various cell types.

Acetylcholinesterase (AChE) plays a key role in terminating neurotransmission at cholinergic synapses. AChE is also found in tissues devoid of cholinergic responses, indicating potential functions beyond neurotransmission. It has been suggested that AChE may participate in development, differentiation, and pathogenic processes such as Alzheimer's disease and tumorigenesis. We examined AChE expression in a number of cell lines upon induction of apoptosis by various stimuli. AChE is induced in all apoptotic cells examined as determined by cytochemical staining, immunological analysis, affinity chromatography purification, and molecular cloning. The AChE protein was found in the cytoplasm at the initiation of apoptosis and then in the nucleus or apoptotic bodies upon commitment to cell death. Sequence analysis revealed that AChE expressed in apoptotic cells is identical to the synapse type AChE. Pharmacological inhibitors of AChE prevented apoptosis. Furthermore, blocking the expression of AChE with antisense inhibited apoptosis. Therefore, our studies demonstrate that AChE is potentially a marker and a regulator of apoptosis.

Acetylcholine↗

PubMed2001

Overexpression of gamma-aminobutyric acid transporter subtype I leads to susceptibility to kainic acid-induced seizure in transgenic mice.

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter, and the GABAergic synaptic transmission is normally terminated by the rapid uptake through GABA transporters. With transgenic mice ubiquitously overexpressing GABA transporter subtype I (GAT1), the present study explored the pathophysiological role of GAT1 in epileptogenesis. Though displaying no spontaneous seizure activity, these mice exhibit altered electroencephalographic patterns and increased susceptibility to seizure induced by kainic acid. In addition, the GABA(A) receptor and glutamate transporters are up-regulated in transgenic mice, which perhaps reflects a compensatory or corrective change to the elevated level of GAT1. These preliminary findings support the hypothesis that excitatory and inhibitory neurotransmission, and seizure susceptibility can be altered by neurotransmitter transporters.

ATP-Binding Cassette Transporters↗

PubMed2001

Adenovirus-mediated expression of pig alpha(1, 3) galactosyltransferase reconstructs Gal alpha(1, 3) gal epitope on the surface of human tumor cells.

Gal alpha(1, 3) Gal (gal epitope) is a carbohydrate epitope and synthesized in large amount by alpha(1, 3) galactosyltransferase [alpha(1, 3) GT] enzyme on the cells of lower mammalian animals such as pigs and mice. Human has no gal epitope due to the inactivation of alpha(1, 3) GT gene but produces a large amount of antibodies (anti-Gal) which recognize Gal alpha(1, 3) Gal structures specifically. In this study, a replication-deficient recombinant adenoviral vector Ad5sGT containing pig alpha(1, 3) GT cDNA was constructed and characterized. Adenoviral vector-mediated transfer of pig alpha(1, 3) GT gene into human tumor cells such as malignant melanoma A375, stomach cancer SGC-7901, and lung cancer SPC-A-1 was reported for the first time. Results showed that Gal epitope did not increase the sensitivity of human tumor cells to human complement-mediated lysis, although human complement activation and the binding of human IgG and IgM natural antibodies to human tumor cells were enhanced significantly after Ad5sGT transduction. Appearance of gal epitope on the human tumor cells changed the expression of cell surface carbohydrates reacting with Ulex europaeus I (UEA I) lectins, Vicia villosa agglutinin (VVA), Arachis hypogaea agglutinin (PNA), and Glycine max agglutinin (SBA) to different degrees. In addition, no effect of gal epitope on the growth in vitro of human tumor cells was observed in MTT assay.

Adenoviridae↗

PubMed2001

GABA transporter 1 transcriptional starting site exhibiting tissue specific difference.

GABA transporter 1(GAT1) takes important roles in multiple physiological processes through the uptake and release of GABA, but the regulation of GAT1 gene expression in different tissues is rarely known. To address the question, first, 5' Rapid amplification of cDNA end (RACE) was used to determine GAT1 transcriptional starting sites in neonatal mouse cerebral cortex and intestine, adult mouse brain and adult rat testis. The products of 5'RACE were confirmed by DNA sequencing. We found that the transcript of GAT1 in neonatal mouse cerebral cortex and adult mouse brain starts at the same site (inside of exon 1), while in mouse intestine, GAT1 starts transcription in intron 1, and in rat testis, the transcript of GAT1 has an additional untranslation exon to the 5' direction.

Aging↗

PubMed2001

[lambda N gene expression regulated by the leader sequence].

It was reported recently that changing the TIR (translational initiation region) of lambda N gene resulted in the increasing expression of lambda N gene and it was regulated at translational level. According to the alignment, the leader sequence of lambda N gene had three parts: a code region for ORF lambda N, the upstream sequence of ORF lambda N and 17 bp of spacer between ORF lambda N and downstream of lambda N gene. ORF lambda N is an open reading frame located at upstream of lambda N gene coding a peptide of 19 amino acids. To study the mechanism of regulation of lambda N gene expression, three serials of plasmids with mutant leader region of lambda N gene were constructed. (1) pMC1403-XT, in which the start codon or the partial code of ORF lambda N was connected with lacZ to obtain the ORF lambda N-lacZ fusion gene and in which the ORF lambda N-lacZ expression was under the control of a strong trp/lac promoter; (2) The ORF lambda N mutants in which the termination codon TAA was introduced into ORF lambda N at different positions by site-directed mutagenesis to preterminate the ORF lambda N on plasmid pMC1403N; (3) Mutants in which a deletion was located at upstream ORF lambda N in the ORF lambda N mutants above. The results obtained from determination of the beta-galactosidase activity in the transformants harboring the different plasmids showed that the ORF lambda N-lacZ expression was suppressed by the ORF lambda N code region and the lambda N expression was increased in both the second and third serials of mutants. At the same time the results from RNA-DNA dot hybridization showed that the lambda N gene expression was regulated at translational level. Therefore it was predicted that the reason of relatively low expression for lambda N gene in pMC1403N was due to the low efficiency of ORF lambda N translation. There are two ways to increase the expression of lambda N gene. One is to preterminate the translation of ORF lambda N at a suitable position to decrease the ORF lambda N effects on downstream lambda N gene translation; the other is to change the upstream sequence of ORF lambda N to improve its translation efficiency.

5' Untranslated Regions↗

PubMed2001

Human xenoreactivity is reduced in mice bearing porcine antisense alpha(1,3) galactosyltransferase cDNA.

AIM: To explore the effect of antisense alpha(1,3) galactosyltransferase alpha(1,3) GT cDNA on production of Gal alpha(1,3) Gal (Gal epitope) xenoantigen in vivo. METHODS: Transgenic mice bearing the porcine antisense alpha(1,3) GT cDNA (nt 1alpha-640) were generated by pronuclei microinjection method. The integration of transgene was identified by PCR and Southern-blot analysis. The expression of murine alpha(1,3) GT was characterized by RT-PCR. Morphology of the spleen was examined by histological technique. Gal epitope was detected by immunofluorescent analysis. Binding of human natural xenoantibodies (IgM and IgG) and complement (C3c) to cells from mice was determined by flow cytometric assay. RESULTS: Transgenic mice bearing the porcine antisense alpha(1,3) GT cDNA were born healthy and developed normally. However, necrosis occurred in the spleen of some mice heterozygous for transgene. Cell surface Gal epitope in transgenic heterozygotes was evidently reduced. Substantially less (30 % - 60 %) xenoantibodies in human serum bound to cells from a variety of tissues of transgenic heterozygotes compared with wild-type controls. Consequentially, human complement activation on cells from these mice was reduced by 40 % - 50 %. CONCLUSION: Human xenoreactivity could be effectively reduced by inhibiting the expression of alpha(1,3) galactosyltransferase with an antisense gene.

Animals↗

PubMed2001

Transgenic mice ubiquitously expressing human Fas ligand develop a slight form of graft-versus-host-like disease.

AIM: To construct transgenic mice bearing human Fas ligand (FasL/CD95L) cDNA, and further explore the physiological effects of ubiquitous expression of FasL on such animals. METHODS: Transgenic mice were produced by pronuclei microinjection method. Integration and transmission of transgene were identified by nest-PCR and Southern-blot analysis. Level of FasL mRNA was evaluated by semi-quantitative RT-PCR analysis. FasL protein was detected by immunofluorescence analysis. Morphological alterations in tissues were analyzed by histological examination. The percentage of alphabetaT cells in the spleen was determined by flow cytometry analysis. RESULTS: Two independent founder mice bearing human FasL cDNA under the control of CMV promoter were generated healthily. Human FasL was moderately expressed in the majority of tissues examined in F1 heterozygotic mice. Although developing normally, adult transgenic mice exhibited a slight form of graft-versus-host (GVH)-like disease characterized by many morphological abnormalities occurring locally in the spleen, testis, lung and liver. In addition, the percentage of alphabetaT cells in the spleen was respectively decreased approximately by 32 % and 24 % in two independent transgenic lines, relative to wild-type mice. CONCLUSION: Ubiquitous expression of Fas ligand can lead to slight GVH-like disease

Animals↗

PubMed2001

Overexpression of gamma-aminobutyric acid transporter subtype I leads to cognitive deterioration in transgenic mice.

AIM: To explore the physiological role of gamma-aminobutyric acid transporter subtype I (GAT1) in cognition. METHODS: Transgenic mice were produced by pronuclei microinjection method. Integration of transgene was identified by Southern-blot and PCR analysis in various generations. Level of GAT1 mRNA in a variety of tissues was evaluated by semi-quantitative RT-PCR analysis. GAT1 protein was detected by immunofluorescence and histochemistry analysis. Associative learning capacity was analyzed by conditioned avoidance task. Memory retention was assessed by novel object recognition test. Morphology of synaptosomes was examined by electron microscope. RESULTS: Four independent founder mice bearing various copies of transgene were generated. GAT1 was evidently overexpressed at both mRNA and protein level in a variety of tissues from transgenic mice. In comparison with wild-type mice, transgenic mice exhibited significantly declined associative learning capacity (P < 0.01) and decreased memory retention (P < 0.01 in 1-h-retention, and P < 0.05 in 1-d-retention). In addition, the amount of asymmetric synapses in the brain of transgenic mice was reduced approximately by 24 %, relative to wild-type mice. CONCLUSION: Overexpression of GAT1 in mice results in cognitive deterioration, indicating that the alteration in the expression of gamma-aminobutyric acid transporters is involved in the pathophysiological mechanism underlying some cognitive deficiencies.

Animals↗

PubMed2000

Functional significance of N- and C-terminus of the amino acid transporters EAAC1 and ASCT1: characterization of chimeric transporters.

To localize functionally significant domains in the amino acid transporters of mouse brain mEAAC1 and mASCT1, cRNA encoding for wild-type and chimeric transporters was injected into Xenopus oocytes. Activity of expressed transporters was investigated by measurements of uptake of 3H-labeled glutamate and serine and of glutamate- and serine-induced currents under voltage clamp. Though all transporters accept glutamate and serine as substrate, the central part of the protein (Ala94-Met418 of mEAAC1 and Ala119-Ile393 of mASCT1) determines substrate selectivity. The C-terminus rectifies the interaction with the respective substrate. A channel mode of the glutamate transporter can be activated by glutamate and serine, and the N- and C-termini of the mEAAC1 seem to be essential for the channel formation.

Amino Acid Sequence↗

PubMed2000

Gamma-aminobutyric acid transporter (GAT1) overexpression in mouse affects the testicular morphology.

Gamma-aminobutyric acid and GABAergic receptors were previously reported to be distributed in reproductive systems besides CNS and predicted to participate in the modulation of testicular function. Gamma-aminobutyric acid transporter was implicated to be involved in this process. However, the potential role of gamma-aminobutyric transporter in testis has not been explored. In this study, we investigated the existence of mouse gamma-aminobutyric acid transporter subtype I (mGAT1) in testis. Wild-type and transgenic mice, which overexpressing mGAT1 in a variety of tissues, especially in testis, were primarily studied to approach the profile of mGAT1 in testis. Mice with overexpressed mGAT1 develop normally but with reduced mass and size of testis as compared with wild-type. Testicular morphology of transgenic mice exhibited overt abnormalities including focal damage of the spermatogenic epithelium accompanied by capillaries proliferation and increased diameter of seminiferous tubules lumen. Reduced number of spermatids was also found in some seminiferous tubules. Our results clearly demonstrate the presence of GAT1 in mouse testis and imply that GAT1 is possibly involved in testicular function.

Animals↗

PubMed2000

Transgenic mice overexpressing gamma-aminobutyric acid transporter subtype I develop obesity.

Transgenic mice ubiquitously overexpressing murine gamma-aminobutyric acid transporter subtype I were created. Unexpectedly, these mice markedly exhibited heritable obesity, which features significantly increased body weight and fat deposition. Behavioral examination revealed that transgenic mice have slightly reduced spontaneous locomotive capacity and altered feeding pattern. This preliminary finding indicates that the inappropriate level of gamma-aminobutyric acid transporters may be directly or indirectly involved in the pathogenic mechanism underlying certain types of obesity.

Animals↗

PubMed2000

[Study on gallic acid preparation by using immobilized tannase from Aspergillus niger].

Immobilized tannase which had higher ability of transforming tannin from Chinese gallotannin into GA was prepared by embedding tannase with calcium alginate carrier. The immobilization conditions and some properties of the immobilized tannase were studied. The results showed that: The optimal immobilized parametres were 90 mg sodium alginate, 546 u(182 u/mL) tannase and 1%-2% CaCl2. The optimum temperature and pH value of immobilized tannase were 45 degrees C and 6.5, respectively. It was stable in the temperature range of 10-50 degrees C and between pH5-7. Based on the optimum program, the laboratory gramme-grade preparation experiments of GA were done. The average yield of GA product in three preparations was upto 61%, and was about equivalent to the yield of acidic hydrolytic method which is used to produce GA industrially now. Thus, the research results possessed potential applied value.

Aspergillus niger↗

PubMed2000

Adenovirus-mediated expression of antisense RNA transcripts complementary to pig alpha(1,3) galactosyltransferase mRNA inhibits expression of Gal alpha(1,3) Gal epitope.

AIM: To examine the effects of the expression of antisense RNA transcripts complementary to the pig alpha(1,3) galactosyltransferase [alpha(1,3)GT]mRNA on the expression of Gal alpha(1,3) Gal structure (gal epitope) in cultured cell lines. METHODS: Human adenoviral vectors were used to mediate the expression of antisense RNA. The expression levels of H blood group antigens and gal epitopes were analyzed by flow cytometry using FITC-UEA-I and FITC-GS-IB4 lectins, respectively. RESULTS: Recombinant adenoviruses, Ad5anti-sGT600 and Ad5-anti-sGT1100, which express antisense RNA complementary to different regions of the pig alpha(1,3) GT mRNA, were constructed and used to infect cell line of NIH3T3. The results showed about 30% reduction in the expression level of gal epitopes on the surface of NIH3T3 cells. In addition, co-expression of human secretor type alpha(1,2) fucosyltransferase [alpha(1,2)FT]cDNA and antisense RNA complementary to the pig alpha(1,3) GT mRNA led to a further reduction in the gal epitope level. CONCLUSION: Recombinant adenoviruses, Ad5anti-sGT600 and Ad5anti-sGT1100, are effective to down-regulate the gal epitope expression.

3T3 Cells↗

PubMed2000

FUT2 gene involved in expression of H blood group antigen on surface of human tumor cell lines BEL-7404, SGC-7901, and SPC-A-1.

AIM: To investigate the expression of blood group H antigen on the surface of several human tumor cell lines such as BEL-7404, SPC-A-1, and SGC-7901. METHODS: In vitro and in vivo expression of the blood group H antigen was analyzed with flow cytometry and immunohistochemistry. Expression of FUT2 gene was determined by RT-PCR, Southern blot, and restriction digestion. RESULTS: Flow cytometric analysis showed that SGC-7901 cells, BEL-7404 cells, and SPC-A-1 cells had mean fluorescence intensity (MFI) values of 162 +/- 43, 81 +/- 25, and 28 +/- 17, respectively. DNA fragments of about 1.0 kb in length were obtained by RT-PCR from RNA isolated from these cells and were detected by FUT2-specific [alpha-32P]dATP labeled DNA probes. CONCLUSION: Human tumor cell lines BEL-7404, SGC-7901, and SPC-A-1 all expressed blood group H antigen on their cell membranes in vitro and in vivo, but their expression levels varied significantly between different cell lines. FUT2 gene expression resulted in the production of these antigens on the cell membrane.

3T3 Cells↗

PubMed2000

Adenovirus-mediated expression of human secretor type alpha(1,2) fucosyltransferase reduces level of Gal alpha(1,3)Gal epitope.

AIM: To test the potential of human secretor type alpha(1, 2) fucosyltransferase [Se alpha(1,2)FT] to downregulate the expression of Gal alpha(1,3)Gal epitope (gal epitope) in cultured cell lines. METHODS: Expression of Se alpha(1,2) FT was mediated by human adenoviral vector. Flow cytometric analysis was used to compare the expression level of H blood group antigen or gal epitope. MTT was employed to assess the susceptibility of mouse NIH3T3 cells to human natural antibody and complement mediated lysis. RESULTS: A recombinant replication-deficient adenovirus (rAdv) containing human Se alpha(1,2)FT cDNA (Ad5hSeFT) was designed and successfully constructed. Flow cytometric analysis showed that after mock infection, Ad5null infection, and Ad5hSeFT infection, the mean fluorescence intensity (MFI) values for the binding of Ulex europaeus I (UEA-I) lectin to NIH3T3 cells were 2.3 +/- 0.6, 2.1 +/- 1.0, and 36.5 +/- 5.9, respectively; MFI values for the binding of Griffonia simplicifolia isolectin B4 (GS-IB4) lectin to NIH3T3 cells were 167 +/- 23, 170 +/- 19, and 100 +/- 14, respectively; MFI values for the binding of human natural IgG and IgM antibodies to NIH3T3 cells were 31 +/- 3, 32 +/- 4, and 22 +/- 4, respectively. CONCLUSION: H blood group antigen was detected on NIH3T3 cells after Ad5hSeFT infection and resulted in more than 40% reduction in the level of gal epitope on the cell surface. This reduction increased the resistance of NIH3T3 cells to lysis by normal human serum.

3T3 Cells↗

PubMed1999

[Modulation of the secretion of active renin in human decidual cells by progesterone].

Uterine decidua is a major source of renin, both active and inactive. Active renin cleaves angiotensinogen to produce angiotensin I and exerts its effect on the expression of angiotensin II in the local tissue. Our experiments showed that (1) in the decidua of early pregnancy the contents of active renin increased gradually with advancing pregnancy, and at 8th week of gestation the level of active renin reached to a maximum (63.37 +/- 12.84) AI ng/gww.h(-1); (2) active renin accounted for about 1/4 of the total renin; and (3) the synthesis and secretion of active renin were regulated by progesterone in incubated decidual cells. These data indicate that a high level of active renin in decidual tissue at early pregnancy plays an important role in the uterine renin-angiotensin system during pregnancy.

Cells, Cultured↗

PubMed1998

Nuclear proteins from liver and kidney bind a 37 bp sequence in the 5' upstream region of the mGAT1 gene.

The mouse GABA transporter (mGAT1) gene has been shown to be exclusively expressed in brain by Northern and Western blot analyses. The interactions between the 5' flanking region of the mGAT1 gene and nuclear proteins from different mouse tissues were studied by means of gel-shift assay. Our results show that nuclear protein factors from non-nervous tissues can specifically recognize a 37 bp sequence that is conserved in the 5' flanking region between the human and mouse GAT1 genes. Similar nuclear protein factors were also found to exist in rat, rabbit and pig.

Animals↗

PubMed1998

Analysis of the 5' flanking sequence of the human norepinephrine transporter gene.

The human norepinephrine transporter (NET) gene was cloned and structurally analyzed. The far 5' fragment containing exon 1 (a non-coding exon) and exon 2 was sequenced. The transcription start site of the gene in human brain stem tissue was determined by primer extension analysis. It was found that the gene could be transcribed from multiple starting points. The 5' flanking sequence contains a proximal G-C rich region, one possible GSG element and several SP1 sites. However it does not contain TATA box and CAAT box motifs. Gel shift analysis with nuclear extracts from different tissues of mouse shows that the G-C rich region may be involved in tissue specific expression of the gene.

Animals↗