Profiling of morphine and codeine in urine after the ingestion of curry containing poppy seed as an evidence for opiates defence in Malaysia.

Consumption of curry containing poppy seeds has raised an issue concerning the opiate content in the urine that might exceed the cut-off value (300ng/mL). The main objective of this study was to examine the morphine and codeine contents in the urine of the consumers after partaking poppy seed-enriched curry in. The volunteers were asked to partake: (a) a single meal and their urines were collected within 24h, or (b) Two meals a day for three consecutive days and their urines were collected within 72h. Two different dosages were also tested in this study: (a) low dosage: 1g/100ml curry (containing 138µg of morphine and 66µg of codeine) and (b) high dosage: 5g/100ml curry (containing 690µg of morphine and 330µg of codeine). The subjects were randomised into the groups using the method of stratified randomization with age and gender groups as covariates. A total of 6 subjects was allocated for each group and placebos were used as control. Results showed that all subjects who consumed low dosage of poppy seeds either in single meal or multiple meals experiment were found negative. However, 1 out of 6high dosage subjects was confirmed positive at a period of 3-6h after the consumption of curry in the single meal study. This outlier maybe due to the lack of water consumption after consuming the curry, thus the low volume of urine was collected and the opiate was concentrated in the urine. On the other hand, 5 out of 6high dosage subjects in the multiple meals experiment were found positive. Majority of these subjects were found positive on the second and third day of the experiment after the second curry meal was consumed. The outlier (negative) in this group might be due to the high consumption of water throughout the experiment and the subject's urine volumes and frequency of urine collection were much higher compared to other subjects. From the result of this study, it can be concluded that partaking high dosages of poppy seed in curry could give a positive response (>300ng/ml+uncertainty of measurement) in the urine, and the water consumption after partaking curry has significant influence for the opiate contents in the urine.

(+)-Spectaline and Iso-6-Spectaline Induce a Possible Cross-Talk between Autophagy and Apoptosis in Trypanosoma brucei rhodesiense.

In our previous study, two known piperidine alkaloids (+)-spectaline (1) and iso-6-spectaline (2) were isolated from the leaves of Senna spectabilis and showed no toxic effect on L6 cells. In view of the potential use of piperidine alkaloids in S. spectabilis for the treatment of sleeping sickness, further investigation on the cell death actions of the parasite after treatment with compound 1 and 2 suggested that the treated parasites died by a process of autophagy based on the characteristic morphological alterations observed in intracellular T. b. rhodesiense. In search for apoptosis, interestingly, trypanosomes treated with high concentration of compound 1 and 2 after 72 h significantly induced an early apoptosis-like programmed cell death (PCD) such as phosphatidylserine (PS) exposure, loss of mitochondrial membrane potential and caspases activation. No DNA laddering discriminated late apoptosis event. Taken together, these findings demonstrated the potential of compound 1 and 2 as a natural chemotherapeutic capable of inducing a possible cross-talk between autophagy and apoptosis in T. b. rhodesiense.

Inhibitory effects of (+)-spectaline and iso-6-spectaline from Senna spectabilis on the growth and ultrastructure of human-infective species Trypanosoma brucei rhodesiense bloodstream form.

In our ongoing work searching for new trypanocidal lead compounds from Malaysian plants, two known piperidine alkaloids (+)-spectaline (1) and iso-6-spectaline (2) were isolated from the leaves of Senna spectabilis (sin. Cassia spectabilis). Analysis of the 1H and 13C NMR spectra showed that 1 and 2 presented analytical and spectroscopic data in full agreement with those published in the literature. All compounds were screened in vitro against Trypanosoma brucei rhodesiense in comparison to the standard drug pentamidine. Compound 1 and 2 inhibited growth of T. b. rhodesiense with an IC50 value of 0.41 ± 0.01 µM and 0.71 ± 0.01 µM, without toxic effect on L6 cells with associated a selectivity index of 134.92 and 123.74, respectively. These data show that piperidine alkaloids constitute a class of natural products that feature a broad spectrum of biological activities, and are potential templates for the development of new trypanocidal drugs. To our knowledge, the compounds are being reported for the first time to have inhibitory effects on T. b. rhodesiense. The ultrastructural alterations in the trypanosome induced by 1 and 2, leading to programmed cell death were characterized using electron microscopy. These alterations include wrinkling of the trypanosome surface, formation of autophagic vacuoles, disorganization of kinetoplast, and swelling of the mitochondria. These findings evidence a possible autophagic cell death.

Development of resazurin-based assay in 384-well format for high throughput whole cell screening of Trypanosoma brucei rhodesiense strain STIB 900 for the identification of potential anti-trypanosomal agents.

To accelerate the discovery of novel leads for the treatment of Human African Trypanosomiasis (HAT), it is necessary to have a simple, robust and cost-effective assay to identify positive hits by high throughput whole cell screening. Most of the fluorescence assay was made in black plate however in this study the HTS assay developed in 384-well format using clear plate and black plate, for comparison. The HTS assay developed is simple, sensitive, reliable and reproducible in both types of plates. Assay robustness and reproducibility were determined under the optimized conditions in 384-well plate was well tolerated in the HTS assay, including percentage of coefficient of variation (% CV) of 4.68% and 4.74% in clear and black 384-well plate, signal-to-background ratio (S/B) of 12.75 in clear 384-well plate and 12.07 in black 384-well plate, Z' factor of 0.79 and 0.82 in clear 384-well plate and black 384-well plate, respectively and final concentration of 0.30% dimethylsulfoxide (DMSO) in both types of plate. Drug sensitivity was found to be comparable to the reported anti-trypanosomal assay in 96-well format. The reproducibility and sensitivity of this assay make it compliant to automated liquid handler use in HTS applications.

Association of mitochondrial DNA 10398 polymorphism in invasive breast cancer in malay population of peninsular malaysia.

BACKGROUND: The mitochondrial DNA (mtDNA) 10398 polymorphism is hypothesised to alter a mitochondrial subunit of the electron transfer chain and is associated with several neurodegenerative disorders and cancers. METHODS: In this study, an mtDNA polymorphism at nucleotide position 10398 was screened in 101 Malay female patients with invasive breast cancer and 90 age-matched healthy female controls using minisequencing analysis. RESULTS: The Malay women with the 10398G variant showed a significantly increased risk of invasive breast cancer (OR = 2.29, 95% CI 1.25-4.20, P = 0.007). Immunohistochemistry analysis was conducted to investigate the effect of this polymorphism on the levels of apoptosis in breast cancer cells. The level of Bax (a pro-apoptotic protein) expression was significantly higher than that of Bcl-2 (an anti-apoptotic protein) in patients carrying the G allele (P = 0.016) but not in those carrying the A allele (P = 0.48). CONCLUSION: Based on these findings, we propose that the mtDNA 10398 polymorphism may be a potential risk marker for breast cancer susceptibility in the Malay population.

Mutation detection in GJB2 gene among Malays with non-syndromic hearing loss.

OBJECTIVES: To identify the mutations in the GJB2 gene and to determine its association with non-syndromic hearing loss in Malays. METHODS: A comparative cross sectional study was conducted on a group of children from the deaf schools and the normal schools. A total of 91 buccal cell samples of non-syndromic hearing loss and 91 normal hearing children were taken. Polymerase chain reaction was used to amplify the coding region of GJB2 gene. The PCR product of GJB2 coding region was preceded with screening for mutations using denaturing high performance liquid chromatography (dHPLC) and mutations detected were confirmed by DNA sequencing. RESULTS: Twelve sequence variations including mutations and polymorphisms were found in 32 patients and 37 control subjects. The variations were G4D, V27I, E114G, T123N, V37I and R127H in both groups, W24X, R32H, 257_259 del CGC and M34L in patients only and I203T and V153I in control subjects only. There were no association between homozygous (P=0.368) or heterozygous (P=0.164) GJB2 gene and non-syndromic hearing loss. CONCLUSIONS: The types of GJB2 gene mutation were different and vary in Malay non-syndromic hearing loss as compared to the other races. Furthermore, the mutation did not associate with hearing loss in the population. Other related genes are believed to be involved and need to be sought in this group of patients.

Association of Mitochondrial DNA 10398 Polymorphism in Invasive Breast Cancer in Malay Population of Peninsular Malaysia

Background: The mitochondrial DNA (mtDNA) 10398 polymorphism is hypothesised to alter a mitochondrial subunit of the electron transfer chain and is associated with several neurodegenerative disorders and cancers. Methods: In this study, an mtDNA polymorphism at nucleotide position 10398 was screened in 101 Malay female patients with invasive breast cancer and 90 age-matched healthy female controls using minisequencing analysis. Results: The Malay women with the 10398G variant showed a significantly increased risk of invasive breast cancer (OR = 2.29, 95% CI 1.25–4.20, P = 0.007). Immunohistochemistry analysis was conducted to investigate the effect of this polymorphism on the levels of apoptosis in breast cancer cells. The level of Bax (a pro-apoptotic protein) expression was significantly higher than that of Bcl-2 (an anti-apoptotic protein) in patients carrying the G allele (P = 0.016) but not in those carrying the A allele (P = 0.48). Conclusion: Based on these findings, we propose that the mtDNA 10398 polymorphism may be a potential risk marker for breast cancer susceptibility in the Malay population.

Analysis of hair samples using microscopical and molecular techniques to ascertain claims of rare animal species

Background: An unidentified animal species named the Jenglot and claimed to be a rare living animal species was recently found in the deep jungle of Irian Jaya, Indonesia; brought to Kuala Lumpur, Malaysia by a businessman; and exhibited in a local museum. The owner of the Jenglot carcasses had made a request to perform DNA analysis on the Jenglot to ascertain its species. Methods: Because the muscle appeared very dry and recovery of DNA was extremely difficult, we therefore used the animals’ hair for further analysis. Hair samples were collected from three different Jenglots that were different in colour and physical appearance. The samples were labelled as A, B, C and D, respectively. Results: Microscopic characteristics indicated that all four hair samples were of human origin, with a medullary index less than 1/3 and pigment distribution towards the periphery. The scale pattern on the hair samples was of the imbricate type, adding certainty to the hypothesis of human origin. A dried root sheath was found in samples B and C, which was contrary to expectations since the sample collection method left a few cm of hair on the body of the Jenglots. Sample D had black dye granules over the cuticular surface. Sequencing of the mitochondrial DNA (mtDNA) hypervariable segment I (HVS-I) region showed polymorphisms at positions 16140, 16182C, 16183C, 16189, 16217 and 16274 and heteroplasmy at positions 16112, 16232 and 16251, a human-specific mtDNA haplotype that was consistent across all the samples. Conclusions: Based on these findings, it was concluded that it is unlikely that the samples of Jenglot hair originated from an animal species.

Single, rapid coastal settlement of Asia revealed by analysis of complete mitochondrial genomes.

A recent dispersal of modern humans out of Africa is now widely accepted, but the routes taken across Eurasia are still disputed. We show that mitochondrial DNA variation in isolated "relict" populations in southeast Asia supports the view that there was only a single dispersal from Africa, most likely via a southern coastal route, through India and onward into southeast Asia and Australasia. There was an early offshoot, leading ultimately to the settlement of the Near East and Europe, but the main dispersal from India to Australia approximately 65,000 years ago was rapid, most likely taking only a few thousand years.