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Biomedical subjects

S P Lim

Publications and source records attributed to S P Lim.

At least 19 recordsLinked to original sources
PubMed2005

Effects of juvenile hormone analogs on new reproductives and colony growth of Pharaoh ant (Hymenoptera: Formicidae).

Two juvenile hormone analogs (JHAs), pyriproxyfen and S-methoprene, were impregnated into dried tuna fish and fed to colonies of Monomorium pharaonis (L.) at very low concentrations (1.0, 2.0, 3.0, 4.0, and 5.0 microg/ml). Its effects on the production of sexuals and colonial growth were observed. Colonies treated with pyriproxyfen yielded sexuals with physical abnormalities. Both female and male sexuals developed bulbous wings, decreased melanization, and died shortly after emergence. Sexuals emerged from colonies treated with S-methoprene did not possess anomalous characteristics. Both pyriproxyfen and S-methoprene did not have significant effects on colonial growth because of the low concentrations of the baits. A commercial bait containing 0.3% S-methoprene (Bioprene-BM) also was evaluated for its efficacy on Pharaoh's ant colonies. Results showed that Pharaoh's ant colonies succumbed to the lethal effects of S-methoprene. Colony members were reduced significantly. Production of queens also decreased significantly in treated colonies and treated queens were unable to lay eggs. JHAs are slow acting and eliminate ant colonies at a relatively slow rate. At low concentrations, pyriproxyfen recorded baffling results, i.e., bulbous wings and demelanized exoskeleton, and it is vital that further studies are initiated to solidify these findings.

Animals↗
PubMed2001

The hepatitis C virus core protein interacts with NS5A and activates its caspase-mediated proteolytic cleavage.

Viral proteins interact with one another during viral replication, assembly, and maturation. Systematic interaction assays of the hepatitis C virus (HCV) proteins using the yeast two-hybrid method have uncovered a novel interaction between core and NS5A. This interaction was confirmed by in vitro binding assays, and coimmunoprecipitation in mammalian cells. Core and NS5A are also colocalized in COS-7 cells. Interestingly, NS5A is cleaved to give specific-size fragments, when core is coexpressed in mammalian cells. Overexpression of core produced many dying and rounded cells and effects such as DNA laddering and the truncation of poly(ADP-ribose) polymerase 1 (PARP1), both indicators of apoptosis. These observations led us to investigate the link between the induction of apoptosis by core and the cleavage of NS5A. The proteolysis of NS5A and these apoptotic events can be inhibited by caspase inhibitor, Z-VAD, indicating that core induces apoptosis and the cleavage of NS5A by caspases. In cells infected by the HCV, core may provide the intrinsic apoptotic signal, which produces truncated forms of NS5A. The biological function of core-NS5A interaction and the downstream effect of NS5A cleavage are discussed.

Animals↗
PubMed2001

Two cases of large solitary fibrous tumors of the pleura associated with fasting hypoglycemia.

We describe two cases of elderly women who presented hypoglycemic episodes with suppressed levels of insulin and insulin-like growth factor I. They were found to have huge malignant solitary fibrous tumor of the pleura. The tumors had rich vascularization on enhanced CT scans and showed characteristic low signal intensity on T2-weighted MR images. This paraneoplastic hypoglycemia is caused by an incompletely processed precursor of insulin-like growth factor II secreted from the tumor. Hypoglycemia and insulin suppression were resolved after removal of the tumors.

Aged↗
PubMed2001

Identification and molecular characterisation of the complete genome of a Singapore isolate of hepatitis C virus: sequence comparison with other strains and phylogenetic analysis.

The complete genomic sequence of a Singapore isolate of the hepatitis C virus (HCV) was obtained from serum of an individual chronically infected with HCV. Nine overlapping cDNA clones covering the entire viral genome was amplified by reverse-transcription-polymerase chain reaction (RT-PCR), This isolate (HCV-S1) comprised 9,609 nucleotides (nt), including 341 nt of the complete 5' untranslated region (5' UTR), a single open reading frame of 3,011 amino acids (aa) and 235 nt of the complete 3' UTR. Its genotype was identified as type lb from analyses of its sequences in the 5' UTR, NS3 and NS5B regions. When compared against nine reported HCV isolates, the overall aa homology of HCV-SI was closest with an Australian strain, HCV-A (94%) and a Japanese strain, HCV-JT (93.9%). Phylogenetic analysis revealed that it was most closely related to the Taiwan strain, HCV-TW and another Japanese strain, HCV-K1-R1.

5' Untranslated Regions↗
PubMed2001

Mutations that affect dimer formation and helicase activity of the hepatitis C virus helicase.

Interaction between viral proteins is necessary for viral replication and viral particle assembly. We used the yeast two-hybrid assay to identify interactions among all the mature proteins of the hepatitis C virus. The interaction between NS3 and NS3 was one of the strongest viral protein-protein interactions detected. The minimal region required for this interaction was mapped to a specific subdomain of 174 amino acids in the N terminus of the helicase region. Random mutations in the minimal region were generated by PCR, and mutants that failed to interact with a wild-type minimal fragment were isolated using the yeast two-hybrid assay as a screen. Three of these mutations resulted in a reduction or a loss of interaction between helicases. Analytical gel filtration showed that in the presence of an oligonucleotide, wild-type helicases form dimers whereas the mutants remain mostly monomeric. All three mutants were partially or almost inactive when assayed for helicase activity in vitro. Mixing a mutant helicase (Y267S) with wild-type helicase did not dramatically affect helicase activity. These data indicate that dimerization of the helicase is important for helicase activity. The mutations that reduce self-association of the helicase may define the key residues involved in NS3-NS3 dimerization.

Amino Acid Sequence↗
PubMed2000

The human immunodeficiency virus type 1 Tat protein up-regulates the promoter activity of the beta-chemokine monocyte chemoattractant protein 1 in the human astrocytoma cell line U-87 MG: role of SP-1, AP-1, and NF-kappaB consensus sites.

It has been shown that the human immunodeficiency virus type 1 (HIV-1) Tat protein can specifically enhance expression and release of monocyte chemoattractant protein 1 (MCP-1) from human astrocytes. In this study, we show evidence that Tat-induced MCP-1 expression is mediated at the transcriptional level. Transient transfection of an expression construct encoding the full-length Tat into the human glioblastoma-astrocytoma cell line U-87 MG enhances reporter gene activity from cotransfected deletion constructs of the MCP-1 promoter. HIV-1 Tat exerts its effect through a minimal construct containing 213 nucleotides upstream of the translational start site. Site-directed mutagenesis studies indicate that an SP1 site (located between nucleotides -123 and -115) is critical for both constitutive and Tat-enhanced expression of the human MCP-1 promoter, as mutation of this SP1 site significantly diminished reporter gene expression in both instances. Gel retardation experiments further demonstrate that Tat strongly enhances the binding of SP1 protein to its DNA element on the MCP-1 promoter. Moreover, we also observe an increase in the binding activities of transcriptional factors AP1 and NF-kappaB to the MCP-1 promoter following Tat treatment. Mutagenesis studies show that an upstream AP1 site and an adjacent NF-kappaB site (located at -128 to -122 and -150 to -137, respectively) play a role in Tat-mediated transactivation. In contrast, a further upstream AP1 site (-156 to -150) does not appear to be crucial for promoter activity. We postulate that a Tat-mediated increase in SP1 binding activities augments the binding of AP1 and NF-kappaB, leading to synergistic activation of the MCP-1 promoter.

Astrocytoma↗
PubMed2000

Cloning trap for signal peptide sequences.

Novel secreted and/or type I transmembrane proteins containing N-terminal signal sequences have been successfully cloned using the signal sequence trapping (SST) method. Often this involves random cloning of short 5' cDNA terminal ends into an epitope-tagged expression vector and the detection of expressed recombinant proteins on the cell surfaces of transfected cells with an antibody to the tagged epitope. Here, we report a novel cloning system for the detection of secreted proteins also using SST. In this method, we used the human immunodeficiency virus (HIV-1) p24 as the epitope for tagging. To test the system, two constructs were created. The 5' terminal end of a human beta-chemokine (which was regulated upon activation, expressed by normal T cells and presumably secreted [RANTES]) and the 5' end of a human CD4 receptor were cloned upstream of and in-frame with the p24 cDNA. Secreted p24 was detectable in the culture media two days after transfection of either DNA construct into the human cell lines, HeLa and 293T. When the chimeric p24 expression constructs were transfected at a ratio of 1:100 to the vector pcDNA3.1(+), p24 could still be detected in cell supernatants. The use of a secreted viral antigen like HIV-1 p24 (or of any noncellular protein) as a marker in SST cloning approaches is likely to be advantageous because it reduces the background noise in detection and also renders this system suitable for high-throughput screening.

Antigens, Viral↗
PubMed2000

Therapeutic potential of platelet-activating factor antagonism in the management of myocardial infarction.

BACKGROUND: Antagonists of platelet-activating factor (PAF) reduce myocardial postischemia reperfusion injury when given before the onset of ischemia. However, the effects of PAF antagonists when administered at a clinically modelled time (during ischemia but before reperfusion) are controversial. Moreover, the extended survival (eight day) and the characteristics of scar formation after treatment with PAF antagonists have not been investigated. OBJECTIVES: To determine the therapeutic potential of PAF antagonist TCV-309 for the treatment of regional myocardial ischemia-reperfusion injury; and to determine the effects of TCV-309 on cardiovascular recovery, evolution of scar formation and survival eight days after a myocardial infarction treated with reperfusion. ANIMALS AND METHODS: Swine underwent regional myocardial ischemia for 60 mins by ligation of the left anterior descending coronary artery, followed by reperfusion for eight days. The treated group (n=7) received PAF antagonist TCV-309 (0.1 mg/kg) 45 mins after ligation; the untreated group (n=7) received vehicle only. RESULTS: Untreated animals experienced significantly (P<0.001) lower systemic arterial blood pressure during the reperfusion period than animals treated with TCV-309. Furthermore, untreated animals required significantly more (P<0.01) antiarrhythmic and inotropic support. Only two of seven animals in the untreated group survived, which was significantly different (P<0.05) from the six of seven treated animals that survived for eight days. Morphometric analyses did not show differences between groups in the characteristics of scar formation following reperfusion for eight days. CONCLUSIONS: PAF antagonist TCV-309 improves survival and reduces cardiovascular dysfunctions associated with regional myocardial ischemia reperfusion injury when administered at a clinically modelled time.

Animals↗
PubMed1998

A novel extracellular domain variant of the human integrin alpha 7 subunit generated by alternative intron splicing.

The integrin alpha 7 beta 1 laminin receptor, which is expressed on replicating myoblasts, and upregulated during myogenic differentiation, is involved in cell adhesion and communication between muscle cells and the extracellular matrix. It is a major cell-surface substrate in skeletal muscle cells for the cell-surface, argininespecific, ADP-ribosyltransferase. Both the extracellular and cytoplasmic domains of the mouse alpha 7 subunit undergo alternative splicing during development, generating differentially expressed variants with presumably unique ligand-binding and signalling properties. Here human cDNA clones isolated from a fetal heart lambda gt10 cDNA library encoded the complete sequence of the alpha 7 subunit and hybridised to a single major 4.4 kb alpha 7 subunit transcript abundantly expressed in human skeletal muscle, moderately expressed in heart, and weakly expressed in most other tissues. One clone out of four contained a novel 225-nucleotide in-frame deletion corresponding to 75 amino acids in the C-terminal region of the extracellular domain. The variant, whose expression appears to be tissue-specific, is created by alternative splicing at sites flanking an intron in the alpha 7 gene. A related mouse form was identified in P19 embryonal carcinoma cells. Deletion of the spliced region, which either contains or is in very close proximity to the major ADP-ribosylation site of the alpha 7 subunit, may serve to modulate the effects of ADP-ribosylation, or alternatively molecular associations, and receptor-ligand affinity.

Alternative Splicing↗
PubMed1998

The beta7 integrin gene (Itgb-7) promoter is responsive to TGF-beta1: defining control regions.

The beta7 integrins LPAM-1 (alpha4beta7) and M290 (alphaEbeta7) mediate the homing of lymphocytes to gut-associated lymphoid tissue, and the proposed retention of intraepithelial lymphocytes (IEL), respectively. Here we show that the gut mucosal cytokine TGF- beta1 increases the expression of beta7 and alphaE subunit mRNA transcripts and the cell-surface expression of M290 on T cells, and that it decreases the level of alpha4 integrin transcripts. Induced beta7 integrin gene expression was inhibited by the protein tyrosine kinase inhibitor genistein, implicating a role for tyrosine phosphorylation. An analysis of the beta7 integrin gene promoter revealed three DNAse I hypersensitivity sites, two of which mapped to the 5' and 3' ends of a promoter fragment (nucleotides +690 to +63) that directed both the basal and the TGF-beta1-induced expression of a heterologous reporter gene. Deletion analysis identified two TGF-beta1 response regions encompassing nucleotides -509 to -398 (TGFBRR1), and -122 to +32 (TGFBRR2). TGFBRR1 interacted with at least five protein complexes, whose binding could be induced with TGF-beta1 stimulation and could be antagonized by TGFBRR2 which harbored both similar and distinctive cis-elements. TGFBRR2 interacted specifically with at least two major nuclear protein complexes, whose binding was phosphorylation dependent. These data provide new insights into the mechanism by which TGF-beta may switch LPAM-1(+ve) migrating T cells to express M290, facilitating their retention in the gut.

Animals↗
PubMed1998

The relationship of ischemia-reperfusion injury of transplanted lung and the up-regulation of major histocompatibility complex II on host peripheral lymphocytes.

OBJECTIVES: This study was designed to examine the relationship between ex vivo preservation time of the transplanted lung and the extent of injury and to relate this to the severity of rejection with and without allogenicity. METHODS: Single lung transplantation was performed on two groups of domestic swine. Group A (n = 7) and group B (n = 6) had ex vivo preservation times of 4 and 15 hours, respectively, at 4 degrees C hypothermia. Group C (n = 6) underwent 2 hours of warm ischemia via dissection and isolation of the left lung with ligation of its bronchial artery and crossclamping of the left pulmonary artery, vein, and bronchus without explantation. Assessment measures included lung function, antioxidant enzyme activities in the plasma and lung tissue, levels of inflammatory mediators in the recipient plasma, and quantification of major histocompatibility complex II HLA-DR-beta on host peripheral lymphocytes. RESULTS: All groups demonstrated increases in interleukin-10, lung weight, and HLA-DR-1beta expression and decreases in lung-tissue antioxidant enzyme activities, gas exchange, and lung compliance. There was a strong positive correlation between ex vivo preservation time and the expression of HLA-DR-beta and a negative correlation between ischemic time and lung-tissue superoxide dismutase. CONCLUSIONS: These results suggest that the intensity of the host immunogenic response is related to the severity of ischemia-reperfusion injury and is independent of tissue incompatibility and/or the type of ischemic insult. We conclude that the extension of ex vivo preservation time may predispose the transplanted lung to more severe rejection.

Animals↗
PubMed1995

Acetaminophen-induced microvascular injury in the rat liver: protection with misoprostol.

Studies into the mechanism of acetaminophen (APAP)-induced hepatotoxicity have focused mainly at the hepatocellular level. This study aimed to investigate the effect of acetaminophen on the hepatic microvasculature using a vascular casting technique. Acetaminophen was administered at a dose of 650 mg/kg body weight (intraperitoneally) to fasted male Long Evans rats. Microvascular casting was performed at various points after drug administration. Liver casts from control rats showed good patency with normal hepatic microvasculature. Thirty-six hours after overdose with acetaminophen, liver casts showed rounded centrilobular cavities of various sizes, representing regions in which cast-filled sinusoids were absent with relatively normal microvasculature within periportal regions. Evidence of microvascular injury occurred as early as 5 hours after acetaminophen overdose. This injury consisted of changes to centrilobular sinusoids including areas of incomplete filling and dilated centrilobular sinusoids. Misoprostol (a prostaglandin E1 analog) treatment (6 x 25 micrograms/kg) given before and after acetaminophen administration markedly reduced the extent of microvascular injury with only small focal unfilled areas in the casts and a generally intact microvasculature. In conclusion, this study shows that overdosage with APAP resulted in an extensive, characteristic pattern of hepatic microvascular injury in the centrilobular region. The results also suggest that microvascular injury is an early event in the pathogenesis of acetaminophen hepatotoxicity. Misoprostol was found to protect against injury occurring at the microvascular level.

Acetaminophen↗
PubMed1994

Acquisition of immunogenicity by AKR leukemic cells following DNA-mediated gene transfer is associated with the reduction of constitutive reactive superoxide radicals.

We have employed the DNA-mediated gene transfer method to introduce the allogenic major-histocompatibility-complex(MHC)-class-I gene H-2Kb into the K36.16 tumor cells, H-2k, in order to generate tumor-specific immunity. The acquisition of immunogenicity by the H-2Kb-transformed clones following gene transfer is associated with the reduction of constitutive reactive superoxide radicals. When the levels of cellular superoxide for the H-2Kb-positive immunogenic clones were determined, they were significantly lower (30 to 60%) than that of the parental K36.16 tumor cells. This reduction of superoxide in the H-2Kb-transformed cells was associated with a significant increase in the level of Cu-Zn superoxide dismutase (SOD) and GPX I, together with a reduction in the DNA-binding form of the NF-kappa B transcription factor. The K36.16 parental tumor cells were also found to be relatively more resistant to the cytotoxic effects of hydrogen peroxide in vitro. To further support the role of superoxide anion radicals in tumorigenesis, in vivo depletion of glutathione promoted the tumorigenicity of the H-2Kb-transformed clones in (AKR/J x C57BL/6/J) F1 mice, whereas SOD was able to reduce their tumorigenicity. In addition, the presence of R-sulfoxine (BSO) in spleen-cell cultures in vitro abolished the ability of the immune lymphocytes to develop into tumor-specific cytotoxic T lymphocytes (CTL). These observations support the concept that oxidative processes in tumor cells may have a strong influence on the host response against tumors.

AKR murine leukemia virus↗
PubMed1994

Characterization of a novel IRF-1-deficient mutant cell line.

The transcriptional activation of the major histocompatibility complex (MHC) class I genes by both type I (alpha/beta) and II (gamma) interferons (IFNs) has been extensively studied, and it has been shown that the upregulation of several DNA-binding proteins is critical for this process. In our laboratory, we introduced the mouse H-2Kb gene into the AKR mouse leukaemia cell line K36.16 to effect the generation of tumor-specific immunity. Individual clones were selected and studied. Whereas the MHC class I genes in most of the clones obtained could be stimulated by interferons, one of the clones obtained, clone Kb-S27, failed to be induced, or was at best poorly induced by IFN-alpha/beta and -gamma. Both the exogenous H-2Kb and the endogenous H-2Dk genes behaved in the same manner and were not stimulated by IFNs. The lack of response to IFNs by clone Kb-S27 also resulted in its resistance to the antiproliferative effects of IFNs. This lack of IFN-induction by clone Kb-S27 was not simply due to a change in its surface interferon receptors. Gel-retardation assay and northern blot analysis both demonstrated the lack of induction of the IRF-1 DNA-binding factor in clone Kb-S27. In addition, northern blot analysis showed that the IRF-2 gene expression in clone Kb-S27 was upregulated when compared with the other IFN-inducible clones.

Animals↗
PubMed1994

Microvascular changes in liver after ischemia-reperfusion injury. Protection with misoprostol.

Morphological changes in the hepatic microvasculature were studied in experimentally induced ischemia-reperfusion injury in the rat using a vascular casting technique. Partial hepatic ischemia was induced for 90 min followed by 24 hr reperfusion. Microvascular casting was performed after 24 hr reperfusion by either intraarterial or intravenous infusion of acrylic resin (Mercox). After corrosion of the tissue, the cast was examined by scanning electron microscopy. Casts of normal livers showed good patency with no evidence of unfilled areas. The mean diameter of sinusoids was 14 +/- 3 microns with those in zone 1 slightly smaller than those in zone 3. Liver casts from rats subjected to ischemia and reperfusion resulted in gross disruption of normal architecture. The common characteristics seen in both prograde and retrograde casts were clusters of closed sinusoids around zones 2 and 3 of the liver acini, which resulted in cavities of various sizes. Varicosities were observed in some areas. The mean diameter of sinusoids in areas of patent microvascular structure (10 +/- 2 microns) was significantly smaller compared to those in normal livers (P < 0.001). Misoprostol given at 1 min before reperfusion markedly reduced the microvascular injury. The hepatic microvascular was generally intact with mild focal unfilled areas. The majority of the sinusoids were of normal size and no clusters of blind ending sinusoids were detected. The present study shows that hepatic ischemia-reperfusion results in extensive microvascular injury in the liver. The protective effects of misoprostol against this injury may occur at the vascular level.

Animals↗
PubMed1994

Misoprostol protection against acetaminophen-induced hepatotoxicity in the rat.

The hepatoprotective effects of misoprostol on acetaminophen (APAP)-induced toxicity were studied in the rat. Liver injury was evaluated at 36 hr after APAP administration by measuring serum ornithine carbamoyltransferase (OCT) and alanine aminotransferase (ALT) levels, by using tetranitroblue tetrazolium (TNBT) staining and by histological analysis. After APAP administration, peak serum levels of the drug were detected at 15 min. Liver GSH was depleted from control levels of 448 +/- 48 micrograms/g to 82 +/- 2 micrograms/g (P < 0.01) within 3 hr. Serum ALT levels increased significantly after 16 hr and H&E staining revealed significant hepatic necrosis after 12 hr. Rats treated with misoprostol before and after APAP administration showed reduced OCT and ALT levels at 36 hr of overdose (454 +/- 446 IU/liter and 2571 +/- 2944 IU/liter, respectively) compared to those without misoprostol treatment (1348 +/- 480 IU/liter and 6077 +/- 3025 IU/liter, respectively, P < 0.01). TNBT staining showed a reduced area of damage from 28.6 +/- 22.3% to 7.3 +/- 8.9% (P < 0.01), and H&E staining also showed less extensive hepatic necrosis in rats treated with misoprostol before and after the overdose. In a time sequence study, misoprostol treatment starting within 10 hr of overdose showed the same protective effect as when it was given before and after APAP ingestion. No protection was detected when the treatment was started during the development of hepatic injury. However, misoprostol given when injury was established seemed to be protective. Our results show that misoprostol protects the liver against APAP-induced injury if given within 10 hr of overdose.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaminophen↗
PubMed1992

Misoprostol hepatoprotection against ischemia-reperfusion-induced liver injury in the rat.

The hepatoprotective effects of misoprostol, a PGE1 analog, against ischemia-reperfusion liver injury were studied using a rat partial liver ischemia model. Serum ornithine carbamoyltransferase (OCT) and alanine aminotransferase (ALT) levels were determined as biochemical indices of injury. Hepatic cell necrosis was assessed histologically using tetranitroblue tetrazolium (TNBT) and hematoxylin and eosin (H&E) staining. With placebo treatment, 90 min of partial hepatic ischemia followed by 24 hr of reperfusion resulted in increased levels of serum OCT (760 +/- 521 IU/liter) and ALT (4327 +/- 1982 IU/liter), while extensive hepatic necrosis was evident by TNBT and H&E staining. Treatment with two doses of 25 micrograms misoprostol/kg body weight at 1 min before ischemia and 1 min before reperfusion significantly reduced the serum levels of OCT and ALT (207 +/- 189 IU/liter, P less than 0.01 and 2075 +/- 1217 IU/liter, P less than 0.01, respectively) and hepatic necrosis. When a single dose of misoprostol was administered 1 min before reperfusion, similar protective effects were observed. However, when the treatment of misoprostol was delayed to 1 min after reperfusion, significantly less hepatoprotection was seen. Misoprostol exerted no hepatoprotection at all when it was administered at 5 min or later after reperfusion. These results demonstrate that misoprostol partially protects the liver against ischemia-reperfusion injury in the rat. The observation that the protective effect of misoprostol occurs only within the first minute of reperfusion suggests that its mechanism of action involves an early event in reperfusion injury, such as modifying the effects of reactive oxygen metabolites.

Animals↗