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Biomedical subjects

C Khoo

Publications and source records attributed to C Khoo.

16 recordsLinked to original sources
PubMed2007

Molecular characterisation of the gut microflora of healthy and inflammatory bowel disease cats using fluorescence in situ hybridisation with special reference to Desulfovibrio spp.

Inflammatory bowel disease (IBD) is a common cause of chronic large bowel diarrhoea in cats. Although the aetiology of IBD is unknown, an immune-mediated response to a luminal antigen is thought to be involved. As knowledge concerning the colonic microflora of cats is limited and requires further investigation, the purpose of this study was to determine the presence of specific bacterial groups in normal and IBD cats, and the potential role they play in the health of the host. Total bacterial populations, Bacteroides spp., Bifidobacterium spp., Clostridium histolyticum subgp., Lactobacillus-Enterococcus subgp. and Desulfovibrio spp. were enumerated in 34 healthy cats and 11 IBD cats using fluorescence in situ hybridisation. The study is one of the first to show the presence of Desulfovibrio in cats. Total bacteria, Bifidobacterium spp. and Bacteroides spp. counts were all significantly higher in healthy cats when compared with IBD cats, whereas Desulfovibrio spp. (producers of toxic sulphides) numbers were found to be significantly higher in colitic cats. The information obtained from this study suggests that modulation of bacterial flora by increasing bifidobacteria and decreasing Desulfovibrio spp. may be beneficial to cats with IBD. Dietary intervention may be an important aspect of their treatment.

Animals↗
PubMed2002

Inhibition of lipopolysaccharide-induced cyclooxygenase-2, tumor necrosis factor-alpha and [Ca2+]i responses in human microglia by the peripheral benzodiazepine receptor ligand PK11195.

The anti-inflammatory actions of the mitochondrial peripheral benzodiazepine receptor (PBR) agonist PK11195 [1-(2-chloro- phenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline-carboxamide] were investigated in human microglia. Application of the microglial inflammatory stimulus lipopolysaccharide (LPS, at 100 ng/mL for 3 h), induced enhancement of the expressions of the inducible enzyme, cyclooxygenase-2 (COX-2) and the pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). PK11195 (at 50 microm) significantly inhibited the LPS-induced up-regulation of both inflammatory factors; at a lower concentration of PK11195 (2 microm) expression of TNF-alpha, but not COX-2, was reduced. Production of both factors, using immunocytochemistry for COX-2 and ELISA for TNF-alpha, was markedly reduced with 50 microm of PK11195 added to LPS solution. Acute application of LPS induced a transient increase in intracellular Ca2+[Ca2+]i exhibiting both a slow development and recovery in kinetic behavior. This increase in [Ca2+]i consisted primarily of a Ca2+ influx component accompanied by a smaller mobilization from intracellular Ca2+ stores. In the presence of PK11195, the amplitude of the [Ca2+]i response induced by LPS was reduced by 54%. Another mitochondrial agent cyclosporin A (CsA), which also acts at the permeability transition pore (PTP) of mitochondrial membrane but at a site different from the PBR, was ineffective in reducing either the LPS-induced expression of COX-2 and TNF-alpha or the endotoxin increase in [Ca2+]i. These results indicate that the mitochondrial effector PK11195 is a specific and effective agent for inhibiting LPS-induced microglial expressions of COX-2 and TNF-alpha and that modulation of Ca2+-mediated signaling pathways could be involved in the anti-inflammatory actions.

Brain↗
PubMed2001

A comparison of alternative polymer excipients and processing methods for making solid dispersions of a poorly water soluble drug.

Solid dispersions were prepared with the extremely poorly water soluble drug, probucol and the water soluble polymers, polyvinyl pyrrolidone (PVP), polyacrylic acid (PAA) or polyethylene oxide (PEO) and blends of these polymers. The solid dispersions were prepared either by the solvent evaporation method, or by compression moulding into films. The materials were characterised by a combination of thermal analysis and FT-Raman spectroscopy. The physical state of the drug was observed to be dependent on the carrier, thus the PVP solid dispersions contained amorphous probucol, whilst the PAA and PEO systems contained the crystalline polymorph II. The method of production was not found to greatly influence the state of the drug in the solid dispersion. The greatest extent of release into solution was observed for the binary blend of drug and PEO, and the blending of polymers was not found to have any advantageous effects in this study.

Anticholesteremic Agents↗
PubMed2001

Telomeric protein Pin2/TRF1 as an important ATM target in response to double strand DNA breaks.

ATM mutations are responsible for the genetic disease ataxia-telangiectasia (A-T). ATM encodes a protein kinase that is activated by ionizing radiation-induced double strand DNA breaks. Cells derived from A-T patients show many abnormalities, including accelerated telomere loss and hypersensitivity to ionizing radiation; they enter into mitosis and apoptosis after DNA damage. Pin2 was originally identified as a protein involved in G(2)/M regulation and is almost identical to TRF1, a telomeric protein that negatively regulates telomere elongation. Pin2 and TRF1, probably encoded by the same gene, PIN2/TRF1, are regulated during the cell cycle. Furthermore, up-regulation of Pin2 or TRF1 induces mitotic entry and apoptosis, a phenotype similar to that of A-T cells after DNA damage. These results suggest that ATM may regulate the function of Pin2/TRF1, but their exact relationship remains unknown. Here we show that Pin2/TRF1 coimmunoprecipitated with ATM, and its phosphorylation was increased in an ATM-dependent manner by ionizing DNA damage. Furthermore, activated ATM directly phosphorylated Pin2/TRF1 preferentially on the conserved Ser(219)-Gln site in vitro and in vivo. The biological significance of this phosphorylation is substantiated by functional analyses of the phosphorylation site mutants. Although expression of Pin2 and its mutants has no detectable effect on telomere length in transient transfection, a Pin2 mutant refractory to ATM phosphorylation on Ser(219) potently induces mitotic entry and apoptosis and increases radiation hypersensitivity of A-T cells. In contrast, Pin2 mutants mimicking ATM phosphorylation on Ser(219) completely fail to induce apoptosis and also reduce radiation hypersensitivity of A-T cells. Interestingly, the phenotype of the phosphorylation-mimicking mutants is the same as that which resulted from inhibition of endogenous Pin2/TRF1 in A-T cells by its dominant-negative mutants. These results demonstrate for the first time that ATM interacts with and phosphorylates Pin2/TRF1 and suggest that Pin2/TRF1 may be involved in the cellular response to double strand DNA breaks.

Amino Acid Sequence↗
PubMed2001

Inhibition of store-operated Ca(2+) influx by acidic extracellular pH in cultured human microglia.

The effects of extracellular acidification on Ca(2+)-dependent signaling pathways in human microglia were investigated using Ca(2+)-sensitive fluorescence microscopy. Adenosine triphosphate (ATP) was used to elicit Ca(2+) responses primarily dependent on the depletion of intracellular endoplasmic reticulum (ER) stores, while platelet-activating factor (PAF) was used to elicit responses primarily dependent on store-operated channel (SOC) influx of Ca(2+). The duration of transient responses induced by ATP was not significantly different in standard physiological pH 7.4 (mean duration 30.2 +/- 2.5 s) or acidified pH 6.2 (mean duration 31.7 +/- 2.8 s) extracellular solutions. However, the time course of the PAF response at pH 7.4 was significantly reduced by 87% with external pH at 6.2. These results suggest that acidification of extracellular solutions inhibits SOC entry of Ca(2+) with little or no effect on depletion of ER stores. Changes of extracellular pH over the range from 8.6 to 6.2 during the development of a sustained SOC influx induced by PAF resulted in instantaneous modulation of SOC amplitude indicating a rapidly reversible effect of pH on this Ca(2+) pathway. Whole-cell patch clamp recordings showed external acidification blocked depolarization-activated outward K(+) current indicating cellular depolarization may be involved in the acid pH inhibition. Since SOC mediated influx of Ca(2+) is strongly modulated by membrane potential, the electrophysiological data suggest that acidification may act to inhibit SOC by cellular depolarization. These results suggest that acidification observed during cerebral ischemia may alter microglial responses and functions.

Acids↗
PubMed2001

Modeling chromosomal instability and epithelial carcinogenesis in the telomerase-deficient mouse.

Human carcinomas are intimately linked to advancing age. These cancers have complex cytogenetic profiles, including aneuploidy and chromosomal structural aberrations. While aged humans sustain a high rate of carcinomas, mice bearing common tumor suppressor gene mutations typically develop soft tissue sarcomas and lymphomas. One marked species distinction between human and mouse that bears on the predisposition to carcinogenesis lies in the radical differences in length and regulation of the telomere, nucleoprotein complexes that cap the ends of eukaryotic chromosomes. Recent cancer modeling studies in the telomerase knockout p53 mutant mice revealed that telomere dynamics might be relevant to carcinogenesis. In these mice, there is a shift in the tumor spectrum towards epithelial carcinomas, and these cancers emerge with complex cytogenetic profiles classical for human carcinomas. In this review, we suggest that the mechanism of fusion-bridge-breakage-translocation, triggered by critically short telomeres, may be one of the generators of genomic instability commonly seen in human carcinomas.

Animals↗
PubMed2001

Tropane alkaloid production by shoot culture of Duboisia myoporoides R. Br.

This work demonstrates the presence of hyoscyamine and scopolamine at different stages of shoot regeneration from non-organogenic and organogenic calli. The 11-week-old non-organogenic calli contained 0.41+/-0.03 and 0.23+/-0.02 microg g(-1) dry wt hyoscyamine and scopolamine respectively. However, no root meristem was found in the calli. The alkaloids were absent in 2-week-old organogenic calli. The shoot-buds induced on the non-organogenic and organogenic calli did not contain these alkaloids. Hyoscyamine and scopolamine contents of the 6-week-old non-rooted shoots regenerated from non-organogenic calli were 7.8+/-0.1 and 6.5+/-0.4 microg g(-1) dry wt respectively and those in the 9-week-old non-rooted shoot regenerated from organogenic calli were 38.5+/-0.4 and 3.6+/-0.1 microg g(-1) dry wt respectively. Hyoscyamine and scopolamine contents of the 4-week-old roots regenerated from non-organogenic and organogenic calli were higher than those in the non-rooted shoots. Since the presence of hyoscyamine and scopolamine in the non-rooted shoot depends on the stage of differentiation, manipulation of culture environment may improve hyoscyamine and scopolamine contents of the non-rooted shoots.

Alkaloids↗
PubMed2001

Distinct patterns of lipoproteins with apoB defined by presence of apoE or apoC-III in hypercholesterolemia and hypertriglyceridemia.

Apolipoprotein (apo) E and apoC-III concentrations in VLDL and LDL are associated with coronary heart disease. We studied the relationship between apoE and apoC-III and the abnormal concentrations and distribution of apoB lipoproteins in 10 hypercholesterolemic and 13 hypertriglyceridemic patients compared with 12 normolipidemic subjects (mean age, 45 years). Sixteen distinct types of apoB lipoprotein particles were separated by first using anti-apoE and anti-apoC-III immunoaffinity chromatography in sequence and then ultracentrifugation [light VLDL, dense VLDL, IDL, and LDL, with apoE with or without apoC-III (E(+)C-III(+), E(+)C-III(-)) or without apoE with or without apoC-III (E(-)C-III(+), E(-)C-III(-))]. The concentrations of VLDL particles with apoC-III (E(+)C-III(+), E(-)C-III(+)) were increased in the hypertriglyceridemic group compared with the hypercholesterolemic and normolipidemic groups. These particles were the most triglyceride rich of the particle types, and their triglyceride content was twice as high in hypertriglyceridemics compared with the other two groups. Hypertriglyceridemics had a similar concentration of total E(-)C-III(-) particles compared with normolipidemics, but the E(-)C-III(-) particles were distributed more to VLDL and IDL than to LDL. Hypercholesterolemics, in contrast, were distinguished from the normolipidemic group by 2-fold higher concentrations of apoB lipoproteins without apoE or apoC-III (E(-)C-III(-)), mainly LDL, which had high cholesterol content. Nonetheless, both normolipidemics and hypercholesterolemics had apoC-III-containing VLDL, which comprised 68% and 43% of their total VLDL particles. E(+)C-III(-) particles were a minor type, comprising <10% of particles in all lipoproteins and patient groups. Therefore, VLDL particles with apoC-III may play a central role in identifying the high risk of coronary heart disease in hypertriglyceridemia, but their substantial prevalence in normolipidemics may be of clinical significance as well.

Apolipoprotein C-III↗
PubMed1999

Effects of estrogenic oral contraceptives on the lipoprotein B particle system defined by apolipoproteins E and C-III content.

Apolipoproteins E and C-III are modulators of lipoprotein metabolism that could affect development of atherosclerosis. The prevalence in plasma of apoB-containing particles (LpB) that contain either apoE or apoC-III, both or neither, and the effect of estrogen on these lipoproteins are unknown. The LpB particle system, defined by the presence or absence of apoE or C-III, was studied in 13 normolipidemic women, 7 nonusers and 6 users of oral contraceptives. Fasting plasma was separated by anti-apoE and C-III affinity chromatography and ultracentrifugation into four types of VLDL, IDL, and LDL particles: with apoE but not apoC-III (E+C-), apoC-III but not apoE (E-C+), both (E+C+) or neither (E-C-). The predominant VLDL particles were E-C- (42% in nonusers, 56% in users) and E+C+ (39% in nonusers, 24% in users), suggesting that apoE and apoC-III mainly exist together in VLDL. In IDL, E-C- was the major fraction (74% nonusers, 81% users), and in LDL, it was 99% in both groups. The triglycerides in VLDL and IDL were mainly contained in C+ particles (79% and 66% of the total VLDL and IDL triglycerides, respectively). Within VLDL, IDL, and LDL, E-C- particles had the smallest size and E+C+ or E-C+ the largest. Users had higher concentrations of VLDL E-C- (280%) and IDL E-C- (90%) particles than nonusers. They also had higher free cholesterol and cholesteryl ester concentrations associated with these fractions and with VLDL E-C+. The triglyceride contents of VLDL E-C- particles were lower in users of oral contraceptives than in nonusers. This study demonstrates that the elevated VLDL TG concentrations in users of estrogen-dominant oral contraceptives is mainly caused by an increased concentration of small VLDL particles that have reduced TG content, and that do not have apoE and C-III. These particles may have lower atherogenicity than particles enriched with apoE and C-III.-Khoo, C., H. Campos, H. Judge, and F. M. Sacks. Effects of estrogenic oral contraceptives on the lipoprotein B particle system defined by apolipoproteins E and C-III content.

Adult↗
PubMed1997

Human cysteine-rich intestinal protein: cDNA cloning and expression of recombinant protein and identification in human peripheral blood mononuclear cells.

Cysteine-rich intestinal protein (CRIP) is a small, 8.5-kDa protein with one double zinc-finger motif called a LIM domain. It is very abundant in intestine and some immune cells in rodents, and expression is influenced by development and the immune response. We have cloned a human CRIP cDNA from human small intestine poly(A)+ RNA by RT-PCR. Through sequencing, we found that the human intestinal CRIP protein (hCRIP) differed from the previously cloned rat CRIP by two amino acids (residues 8 and 58). hCRIP was expressed with the pET vector/bacterial system and isolated by gel filtration and ion-exchange chromatography. The protein was purified to homogeneity as confirmed by PAGE, Western blotting, and immunodetection. Recombinant hCRIP has a molecular mass of 8390 Da based on mass spectrum analysis. Southern analysis suggests that there are three copies of the CRIP gene in the human genome. hCRIP mRNA was detected by RT-PCR in human monocytes purified from peripheral blood and THP-1 cells, a human monocytic cell line. Incubation of THP-1 cells with 65Zn and chromatography of the cytosol show that a significant amount of the radioactivity is associated with CRIP as was shown previously for rat intestine. The results are consistent with a functional role for CRIP in proliferation/differentiation of specific cell types, particularly those associated with host defense.

Amino Acid Sequence↗
PubMed1997

Necrotising fasciitis--an old enemy or a new foe.

Necrotising fasciitis when described by Meleney was caused predominantly by Streptococcus pyogenes. Since then, there were several reports which confirmed streptococcus as the main organism identified in this disease entity. However, recently there were reports of necrotising fasciitis caused by organisms other than Streptococcus. We analysed 19 cases of necrotising fasciitis seen in the Department of Orthopaedic Surgery over a period of 24 months to see if this disease entity has changed significantly. The patients in our series were between the ages of 19 to 85 years with an average age of 57.2 years. Males were more often affected (16 out of 19). The majority of the patients have some form of underlying disease (16 out of 19 cases). There was a trend towards polymicrobial infection and many were resistant to standard antimicrobial therapy. The mortality rate was 21.1%. Our results are comparable to many earlier series.

Adult↗
PubMed1996

Lipopolysaccharide regulates cysteine-rich intestinal protein, a zinc-finger protein, in immune cells and plasma.

Cysteine-rich intestinal protein (CRIP), a double zinc-finger LIM protein, is expressed in great abundance in the intestine. We have found comparable levels of CRIP mRNA in peritoneal macrophages, peripheral blood mononuclear cells (PBMC), and lesser amounts in thymus and spleen. Because CRIP expression was high in immune cells, rats were challenged with lipopolysaccharide (LPS) to determine whether expression was altered during the acute-phase immune response. Immunocytochemistry showed that, in adherent mononuclear cells, CRIP protein was localized in the cytoplasm. CRIP mRNA levels increased over time after LPS injection in peritoneal macrophages, PBMC, spleen, and intestine. No changes in CRIP mRNA level were seen in either liver or thymus. In PBMC, the level of CRIP mRNA decreased before increasing later in the acute-phase immune response. CRIP protein was found in the plasma. Shortly after LPS administration plasma CRIP decreased, suggesting that CRIP was either passively diffused out of capillaries or was actively shunted into tissues to execute its function. Increased CRIP expression seen in response to LPS suggests that CRIP may play a role in immune cell activation or differentiation or in processes associated with cellular repair.

Acute-Phase Reaction↗
PubMed1996

Differential expression of cysteine-rich intestinal protein in liver and intestine in CCl4-induced inflammation.

Cysteine-rich intestinal protein (CRIP) is a double zinc finger (LIM domain) protein that is developmentally regulated but has an unknown function. CRIP is highly expressed in the intestine, but expression is low in liver. To determine if CRIP expression is regulated under altered physiological status, we used CCl4-induced injury as a model to produce hepatic injury and systemic effects associated with inflammation. Since CRIP is a zinc finger protein and zinc decreases the hepatic response to CCl4, the effect of supplemental dietary zinc (300 mg/kg diet) was also examined. Our results show that this supplemental level of dietary zinc did not affect the index of hepatic injury (plasma alanine aminotransferase), indicating zinc did not have a protective effect. Liver CRIP mRNA increased with CCl4 and CRIP protein was shown by immunohistochemistry to be localized in hepatocytes near the vascular supply. In the intestine, CCl4 caused a transient decrease in CRIP mRNA, but supplemental dietary zinc treatment prevented this decrease. These current results show that CRIP expression changes in response to cellular damage due to acute hepatic injury and are consistent with a functional role for CRIP in proliferation, differentiation, or turnover.

Animals↗
PubMed1994

Purification and properties of rat cysteine-rich intestinal protein.

Cysteine-rich intestinal protein (CRIP) is a zinc-binding protein where the binding domain is in the so-called LIM double zinc finger motif. Methods are described for the preparation of CRIP from rat small intestine. Gel-filtration and ion-exchange chromatography and preparative PAGE gave homogeneous CRIP, based upon analytical PAGE, mass spectrometry and microsequencing. Initial localization of CRIP during chromatography was based on binding of 65Zn radioisotope introduced into the intestine. The stoichiometry of binding by CRIP is less than 2 atoms of zinc per molecule. The metal-binding affinity in vitro is zinc > cadmium > copper > iron, at low metal concentrations. Zinc is the predominant metal bound when these metals are taken up from the intestinal lumen. Zinc binding was not influenced by pH between values of 4.5 to 7.5. Metallothionein has a much greater zinc-binding affinity than CRIP. The tissue concentration of CRIP is of the order of 15-20 micrograms/g of mucosal tissue, suggesting that the protein is more abundant than zinc-finger-containing transcription factors. The metal-binding properties of CRIP are consistent with proposed zinc-related functions for this cytoplasmic protein, which is expressed in the small intestine during the postnatal period.

Amino Acid Sequence↗