Screening for eukaryotic signal transduction and Mycobacterium isocitrate lyase inhibitor from actinomycetes and fungi of dipterocarp rain forests at Imabak Valey, Sabah, Malaysia

A diversity of actinomycetes and fungi was isolated from various sites during the Imbak Valley Scientific Expedition 2000. A total of 144 soil samples were collected under trees that have been identified to species or genus level. Imbak Valley is a lowland dipterocarp forest, which is interestingly dominated by Dryobalanops beccarii. Isolation of Streptomyces and non-Streptomyces actinomycetes on HV medium and other specific isolation media for non-Streptomyces yielded 203 isolates from 89 soil samples. Morphological characterisation of the isolated actinomycetes was carried out based on aerial mycelium colour, substrate mycelium colour and diffusible pigment production on oatmeal medium. Nine strains of fungi were isolated from the six soil samples plated on PDA medium. All actinomycetes isolates were grown under aerobic condition in liquid culture and extracted with acetone, and used for screening against proteins involved eukaryotic signal transduction. Yeast MAPK kinase and MAP kinase phosphatase were some of the targeted proteins used in this research. MKK1P386 and MKK1P386-MSG5 mutant yeasts were used to screen for these inhibitors, as these yeast kinase and phosphatase have homologous proteins in the MAP kinase signal transduction pathway in human. No inhibitors in the extracts were found in these screenings. Type 1 protein serine/ threonine phosphatase (GLC7) in yeast was used to screen inhibitors against PP1 inhibitors and no inhibitor was found. None of the fungal extracts showed any inhibitory activities in all the screening systems. No Ras/Raf inhibitor was found in the in vivo Ras/Raf interaction with the yeast two-hybrid screening system, which used to screen for inhibitor against Ras/ Raf protein interaction inhibitor. There were 11 actinomycetes extracts that showed toxicity against yeast strain LZ (transformant of Ras/ Raf). H7667, a Streptomycete toxic to yeast is further screened for inhibitors of the GSK3-beta pathway. H7763, a Streptomyces species that showed positive in the primary screen for inhibitor of isocitrate lyase (ICL) which is not itaconic acid (known ICL inhibitor). H7240 showed the strongest susceptibility towards the resin in which the concentration of 5g/l of resin is sufficient to produce growth inhibition of the bacteria

Comparison of clinical profiles between rs12084215 genotypes among FH patients.

<p>Data are presented in mean ± SD.</p><p>p-values were obtained by comparing the phenotypes among the genotypes using Analysis of Variance (ANOVA).</p

Minor allele frequency of significant SNPs.

<p>rs no, NCBI Reference SNP (rs) Number, an identification tag assigned by NCBI to SNPs.</p><p>Chr, Chromosome.</p><p>p-value obtained by comparing frequencies using Chi-Square Test.</p><p>MAF (Total), minor allele frequency obtained from total sum of case and control subjects in this study.</p><p>MAF (PD), minor allele frequency information from public database, NCBI dbSNP Build 137.</p

Genetic Polymorphisms in <i>LDLR</i>, <i>APOB</i>, <i>PCSK9</i> and Other Lipid Related Genes Associated with Familial Hypercholesterolemia in Malaysia

<div><p>Familial hypercholesterolemia (FH) is an autosomal dominant disorder characterized by elevations in total cholesterol (TC) and low density lipoprotein cholesterol (LDLc). Development of FH can result in the increase of risk for premature cardiovascular diseases (CVD). FH is primarily caused by genetic variations in <i>Low Density Lipoprotein Receptor</i> (<i>LDLR</i>), <i>Apolipoprotein B</i> (<i>APOB</i>) or <i>Proprotein Convertase Subtilisin/Kexin type 9</i> (<i>PCSK9</i>) genes. Although FH has been extensively studied in the Caucasian population, there are limited reports of FH mutations in the Asian population. We investigated the association of previously reported genetic variants that are involved in lipid regulation in our study cohort. A total of 1536 polymorphisms previously implicated in FH were evaluated in 141 consecutive patients with clinical FH (defined by the Dutch Lipid Clinic Network criteria) and 111 unrelated control subjects without FH using high throughput microarray genotyping platform. Fourteen Single Nucleotide Polymorphisms (SNPs) were found to be significantly associated with FH, eleven with increased FH risk and three with decreased FH risk. Of the eleven SNPs associated with an increased risk of FH, only one SNP was found in the <i>LDLR</i> gene, seven in the <i>APOB</i> gene and three in the <i>PCSK9</i> gene. SNP rs12720762 in <i>APOB</i> gene is associated with the highest risk of FH (odds ratio 14.78, p<0.001). Amongst the FH cases, 108 out of 141 (76.60%) have had at least one significant risk-associated SNP. Our study adds new information and knowledge on the genetic polymorphisms amongst Asians with FH, which may serve as potential markers in risk prediction and disease management.</p> </div

Comparison of clinical profiles between rs12084215 genotypes among Control subjects.

<p>Data are presented in mean ± SD.</p><p>p-values were obtained by comparing the phenotypes among the genotypes using Analysis of Variance (ANOVA).</p

Demographics and clinical profiles of the subjects.

<p>BMI, Body Mass Index.</p><p>WC, Waist circumference.</p><p>TG, Triglyceride.</p><p>TC, Total Cholesterol.</p><p>HDLc, High Density Lipoprotein Cholesterol.</p><p>LDLc, Low Density Lipoprotein Cholesterol.</p><p>The data are expressed as mean (±SD).</p><p>p-values were obtained by comparing the phenotypes between the two groups using Student’s t-test.</p

Comparison of clinical profiles between rs57825321 genotypes among FH patients.

<p>Data are presented in mean ± SD.</p><p>p-values were obtained by comparing the phenotypes among the genotypes using Analysis of Variance (ANOVA).</p

Comparison of clinical profiles between rs13306194 genotypes among FH patients.

<p>Data are presented in mean ± SD.</p><p>p-values were obtained by comparing the phenotypes among the genotypes using Analysis of Variance (ANOVA).</p

FH associated SNPs, (p<0.05).

<p>rs number, NCBI Reference SNP (rs) Number, an identification tag assigned by NCBI to SNPs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060729#pone.0060729-Sherry1" target="_blank">[30]</a>.</p><p>CI, Confidence interval.</p><p>Odds ratio (OR) between groups was determined by logistic regression.</p

Comparison of clinical profiles between rs12720772 genotypes among FH patients.

<p>Data are presented in mean ± SD.</p><p>p-values were obtained by comparing the phenotypes among the genotypes using Analysis of Variance (ANOVA).</p