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Existence of two emm-like "mrp" and "emm" genes in the mga regulon of the Streptococcus pyogenes strain ST4547

Eshaghi M, Ali AM, Jamal F, Yusoff K

J. Biochem. Mol. Biol. Biophys., 2002 Feb;6(1):23-8.
PMID: 12186779

Streptococcus pyogenes ST4547 is an opacity factor negative strain, which has been recently reported as a new emm type from Malaysia. Nucleotide sequencing of the mga regulon of this strain showed the existence of two emm-like genes. The emm gene located upstream of the scpA gene comprises 1305 nucleotides encoding the putative precursor M protein of 435 amino acids in length with an M(r) of 49 kDa. or a predicted mature protein of 394 amino acids with an M(r) of 44.8 kDa. Another gene mrpST4547 was located upstream of the emm gene and downstream of the mga gene. The sequence of this mrp gene comprises 1167 nucleotides encoding a predicted protein of 388 amino acids in length with an M(r) of 42.2 kDa. or a predicted mature protein of 347 amino acids with an M(r) of 37.9 kDa. The mga regulon of strain ST4547 has a mosaic structure comprising segments, which originated from different OF positive and OF negative strains. The sequences flanking the hyper-variable and C repeats of the emmST4547 gene showed high similarity to corresponding regions in the mga regulon of OF positive strains notably M15, M4, M22 and M50. In contrast, the sequence within the hyper-variable and C repeat regions of the emmST4547 gene revealed high similarity to equivalent regions in the OF negative strains. These data indicates that horizontal transfer of emm-like gene could have occurred between OF positive and OF negative strains resulting in architectural divergence in the mga regulon.

Matched MeSH terms: DNA, Bacterial/genetics; DNA, Bacterial/isolation & purification
Combine-ARMS: a rapid and cost-effective protocol for molecular characterization of beta-thalassemia in Malaysia

Tan KL, Tan JA, Wong YC, Wee YC, Thong MK, Yap SF

Genet. Test., 2001;5(1):17-22.
PMID: 11336396 DOI: 10.1089/109065701750168626

Beta-thalassemia major patients have chronic anemia and are dependent on blood transfusions to sustain life. Molecular characterization and prenatal diagnosis of beta3-thalassemia is essential in Malaysia because about 4.5% of the population are heterozygous carriers for beta-thalassemia. The high percentage of compound heterozygosity (47.62%) found in beta-thalassemia major patients in the Thalassaemia Registry, University of Malaya Medical Centre (UMMC), Malaysia, also supports a need for rapid, economical, and sensitive protocols for the detection of beta-thalassemia mutations. Molecular characterization of beta-thalassemia mutations in Malaysia is currently carried out using ARMS, which detects a single beta-thalassemia mutation per PCR reaction. We developed and evaluated Combine amplification refractory mutation system (C-ARMS) techniques for efficient molecular detection of two to three beta-thalassemia mutations in a single PCR reaction. Three C-ARMS protocols were evaluated and established for molecular characterization of common beta-thalassemia mutations in the Malay and Chinese ethnic groups in Malaysia. Two C-ARMS protocols (cd 41-42/IVSII #654 and -29/cd 71-72) detected the beta-thalassemia mutations in 74.98% of the Chinese patients studied. The CARMS for cd 41-42/IVSII #654 detected beta-thalassemia mutations in 72% of the Chinese families. C-ARMS for cd 41-42/IVSI #5/cd 17 allowed detection of beta-thalassemia mutations in 36.53% of beta-thalassemia in the Malay patients. C-ARMS for cd 41-42/IVSI #5/cd 17 detected beta-thalassemia in 45.54% of the Chinese patients. We conclude that C-ARMS with the ability to detect two to three mutations in a single reaction provides more rapid and cost-effective protocols for beta-thalassemia prenatal diagnosis and molecular analysis programs in Malaysia.

Matched MeSH terms: DNA Mutational Analysis/economics*; DNA Mutational Analysis/methods*
p53 codon 72 polymorphisms and random amplified polymorphic DNA analysis of non-melanoma skin cancer through archival formalin-fixed paraffin-embedded tissue

Yoke-Kqueen C, Ab Mutalib NS, Sidik SM, Learn-Han L, Geok-Chin T

Oncol Rep, 2012 Mar;27(3):753-63.
PMID: 22159872 DOI: 10.3892/or.2011.1581

Non-melanoma skin cancer (NMSC) is classified among the ten most frequent cancers in Malaysia. A common polymorphism at codon 72 of the p53 tumor suppressor gene and its influence on cancer risk has been studied for different types of cancer with mixed and inconsistent results with limited published data on the Malaysian population so far. In the present study, the frequency of p53 codon 72 polymorphism in 60 patients with NMSC was investigated from archival formalin-fixed paraffin-embedded (FFPE) tissue obtained from Hospital Universiti Kebangsaan Malaysia (HUKM). Additionally, random amplified polymorhic DNA -polymorphic chain reaction (RAPD-PCR) was employed for preliminary biomarker development. NMSC FFPE samples (70%) possess Arg/Arg, 20% with Pro/Pro and 10% with Arg/Pro. In total, there was no significant difference in the p53 codon 72 genotypes between histological types of NMSC, gender, race, tumor location and age group. However, there was an apparent age-associated increase in the Arg/Arg genotype but did not reach statistical significance (P=0.235). NMSC types and demographic characteristics did not influence genotype distribution. On the other hand, BCC and SCC distributions are influenced by age group, race and tumor location.

Matched MeSH terms: DNA Mutational Analysis; Random Amplified Polymorphic DNA Technique/methods
Low-density lipoprotein receptor gene mutations in a Southeast Asian population with familial hypercholesterolemia

Khoo KL, van Acker P, Defesche JC, Tan H, van de Kerkhof L, Heijnen-van Eijk SJ, et al.

Clin Genet, 2000 Aug;58(2):98-105.
PMID: 11005141 DOI: 10.1034/j.1399-0004.2000.580202.x

The aim of this study was to detect mutations in the genes coding for the low-density lipoprotein receptor and apolipoprotein B in patients of Southeast Asian origin with clinically diagnosed familial hypercholesterolemia (FH) and to relate these findings with the observed lower incidence of coronary heart disease in this part of the world. A total of 86 unrelated patients with FH were selected on clinical grounds, and complete DNA analysis of the low-density lipoprotein (LDL)-receptor and apolipoprotein B (apoB) genes by DGGE and DNA-sequencing was performed. In the majority (73%) of the cohort studied, no mutations could be detected, even after extensive analysis of the LDL-receptor and apoB genes. However, the 22 patients with a mutation had significantly more xanthomas and a higher incidence of coronary heart disease and levels of low-density lipoproteins were also significantly different. There was no correlation between the type of the mutation and lipoprotein levels or clinical signs of atherosclerosis. The fact that the majority of the FH patients studied had no detectable mutation and that this group had a significant milder phenotype, suggests the presence of a third gene in the Southeast Asian population, predominantly leading to a disorder resembling a milder form of FH. A similar, but less frequent, trait has recently been described in a number of European families.

Matched MeSH terms: Sequence Analysis, DNA; DNA Primers/chemistry
Fulltext Agarolytic bacterium Persicobacter sp. CCB-QB2 exhibited a diauxic growth involving galactose utilization pathway

Furusawa G, Lau NS, Suganthi A, Amirul AA

Microbiologyopen, 2017 02;6(1).
PMID: 27987272 DOI: 10.1002/mbo3.405

The agarolytic bacterium Persicobacter sp. CCB-QB2 was isolated from seaweed (genus Ulva) collected from a coastal area of Malaysia. Here, we report a high-quality draft genome sequence for QB2. The Rapid Annotation using Subsystem Technology (RAST) annotation server identified four β-agarases (PdAgaA, PdAgaB, PdAgaC, and PdAgaD) as well as galK, galE, and phosphoglucomutase, which are related to the Leloir pathway. Interestingly, QB2 exhibited a diauxic growth in the presence of two kinds of nutrients, such as tryptone and agar. In cells grown with agar, the profiles of agarase activity and growth rate were very similar. galK, galE, and phosphoglucomutase genes were highly expressed in the second growth phase of diauxic growth, indicating that QB2 cells use galactose hydrolyzed from agar by its agarases and exhibit nutrient prioritization. This is the first report describing diauxic growth for agarolytic bacteria. QB2 is a potential novel model organism for studying diauxic growth in environmental bacteria.

Matched MeSH terms: DNA, Bacterial/genetics; Sequence Analysis, DNA
Fulltext Aluminosilicate Nanocomposite on Genosensor: A Prospective Voltammetry Platform for Epidermal Growth Factor Receptor Mutant Analysis in Non-small Cell Lung Cancer

Ramanathan S, Gopinath SCB, Arshad MKM, Poopalan P, Anbu P, Lakshmipriya T, et al.

Sci Rep, 2019 11 19;9(1):17013.
PMID: 31745155 DOI: 10.1038/s41598-019-53573-9

Lung cancer is one of the most serious threats to human where 85% of lethal death caused by non-small cell lung cancer (NSCLC) induced by epidermal growth factor receptor (EGFR) mutation. The present research focuses in the development of efficient and effortless EGFR mutant detection strategy through high-performance and sensitive genosensor. The current amplified through 250 µm sized fingers between 100 µm aluminium electrodes indicates the voltammetry signal generated by means of the mutant DNA sequence hybridization. To enhance the DNA immobilization and hybridization, ∼25 nm sized aluminosilicate nanocomposite synthesized from the disposed joss fly ash was deposited on the gaps between aluminium electrodes. The probe, mutant (complementary), and wild (single-base pair mismatch) targets were designed precisely from the genomic sequences denote the detection of EGFR mutation. Fourier-transform Infrared Spectroscopy analysis was performed at every step of surface functionalization evidences the relevant chemical bonding of biomolecules on the genosensor as duplex DNA with peak response at 1150 cm-1 to 1650 cm-1. Genosensor depicts a sensitive EGFR mutation as it is able to detect apparently at 100 aM mutant against 1 µM DNA probe. The insignificant voltammetry signal generated with wild type strand emphasizes the specificity of genosensor in the detection of single base pair mismatch. The inefficiency of genosensor in detecting EGFR mutation in the absence of aluminosilicate nanocomposite implies the insensitivity of genosensing DNA hybridization and accentuates the significance of aluminosilicate. Based on the slope of the calibration curve, the attained sensitivity of aluminosilicate modified genosensor was 3.02E-4 A M-1. The detection limit of genosensor computed based on 3σ calculation, relative to the change of current proportional to the logarithm of mutant concentration is at 100 aM.

Matched MeSH terms: DNA/genetics; DNA/chemistry
Detection of Hepatocystis sp. in southeast Asian flying foxes (Pteropodidae) using microscopic and molecular methods

Olival KJ, Stiner EO, Perkins SL

J Parasitol, 2007 Dec;93(6):1538-40.
PMID: 18314711 DOI: 10.1645/GE-1208.1

Three species of flying fox (Pteropus hypomelanus, P. vampyrus, and P. lylei) from Malaysia and Vietnam were screened for apicomplexan parasites by thin blood smears and polymerase chain reaction. Only 1 of 16 bats sampled from 3 localities in southeast Asia was found to be infected (P. hypomelanus from Pulau Pangkor, Malaysia). We observed micro- and macrogametocytes, with morphology consistent with Hepatocystis sp. parasites, using light microscopy. Phylogenetic analysis of the cytochrome b gene showed that the parasite from P. hypomelanus groups with 2 published sequences from Hepatocystis spp., including one from Cynopterus brachyotis, another fruit bat in the Pteropodidae.

Matched MeSH terms: DNA, Protozoan/blood; DNA, Protozoan/chemistry
Molecular basis of beta thalassemia in the south of Thailand

Laosombat V, Fucharoen SP, Panich V, Fucharoen G, Wongchanchailert M, Sriroongrueng W, et al.

Am J Hematol, 1992 Nov;41(3):194-8.
PMID: 1415194

A total of 103 beta thalassemia genes from 78 children (45 with Hb E/beta thalassemia, 8 with beta thalassemia heterozygotes, and 25 with homozygous beta thalassemia) were analyzed using dot-blot hybridization of the polymerase chain reaction-amplified DNA and direct DNA sequencing. Nine mutations were characterized in 98/103 (95%) of beta thalassemia alleles, of which six (a 4 bp deletion in codons 41-42, a G-C transition at position 5 of IVS-1, A-G transition at codon 19, an A-T transition at codon 17, an A-G transition at position -28 upstream of the beta globin gene, a G-T transition at position 1 of IVS-1), accounted for 92%. The spectrum of beta thalassemia mutations in Chinese Thai is similar to that reported among the Chinese from other parts of the world. The distribution of beta thalassemia mutations in Muslim Thai is similar to that reported among Malaysians. The most common beta thalassemia mutation in Thai and Chinese Thai patients is the frameshift mutation at codons 41-42, in comparison with the Muslim Thai in whom the G-C transition at position 5 of the IVS-1 mutation predominates. The heterogeneity of molecular defects causing beta thalassemia should aid in the planning of a prenatal diagnosis program for beta thalassemia in the South of Thailand.

Matched MeSH terms: DNA/analysis; DNA/genetics
Gene mapping of Malaysian alpha thalassemias with alpha and zeta globin gene probes

Lie-Injo LE, Herrera AR, Lebo RV, Hassan K, Lopez CG

Am J Hematol, 1985 Mar;18(3):289-96.
PMID: 2983536

Restriction enzyme analysis of the alpha and zeta globin genes was carried out in four cases of Hb Bart's hydrops fetalis, in three patients with Hb H disease without Hb CoSp, in three patients with Hb H disease with Hb CoSp, in 47 individuals with alpha thalassemia trait, and in 47 normal individuals. All four cases of Hb Bart's hydrops fetalis resulted from deletions of alpha 1 and alpha 2 globin genes which did not extend to the psi zeta 1 and zeta 2 globin genes. The same type of deletion was observed in alpha thal1 carriers, but two newborns (one Malay and one of Chinese extraction) had a nondeletion type of alpha thal1 which was confirmed by quantitative alpha globin gene analysis. In addition, two other newborns diagnosed as alpha thal1 trait carriers (one Malay, one Chinese) were shown to have a deletion of both alpha globin genes by quantitative alpha globin gene analysis, but further testing with zeta globin gene probe failed to reveal an abnormal fragment length characteristic of an alpha globin gene deletion. We believe that this last condition is due to a large deletion which includes all alpha globin genes and all zeta globin genes on the same chromosome. On another front, Bgl II restriction analysis of all four Hb Bart's hydrops fetalis cases and the alpha thal1 trait carriers showed a 10.5-kb Bgl II restriction fragment, in the hydrops fetalis as a single band, while in the carriers this 10.5-kb fragment was accompanied by the usual normal 12.5-kb and 11.3-kb fragments. We report that this 10.5-kb fragment, previously thought to be specific for the Southeast Asian alpha thal1 gene deletion, is also common in normal individuals. Nevertheless, digestion with other enzymes can clearly differentiate the alpha thal1 and normal genotypes. We distinguish the findings in the alpha thalassemias from the extensive DNA polymorphism in the region of the alpha and zeta globin genes.

Matched MeSH terms: DNA/analysis*; DNA Restriction Enzymes
Molecular methods for diagnosis and epidemiological studies of parasitic infections

Singh B

Int J Parasitol, 1997 Oct;27(10):1135-45.
PMID: 9394184

Direct microscopy is widely used for the diagnosis of parasitic infections although it often requires an experienced microscopist for accurate diagnosis, is labour intensive and not very sensitive. In order to overcome some of these shortcomings, molecular or nucleic acid-based diagnostic methods for parasitic infections have been developed over the past 12 years. The parasites which have been studied with these techniques include the human Plasmodia, Leishmania, the trypanosomes, Toxoplasma gondii, Entamoeba histolytica, Giardia, Trichomonas vaginalis, Cryptosporidium parvum, Taenia, Echinococcus, Brugia malayi, Wuchereria bancrofti, Loa loa and Onchocerca volvulus. Early methods, which involved hybridisation of specific probes (radiolabelled and non-radiolabelled) to target deoxyribonucleic acid (DNA), have been replaced by more sensitive polymerase chain reaction (PCR)-based assays. Other methods, such as PCR-hybridisation assays, PCR-restriction fragment length polymorphism (PCR-RFLP) assays and random amplified polymorphic DNA (RAPD) analysis have also proved valuable for epidemiological studies of parasites. The general principles and development of DNA-based methods for diagnosis and epidemiological studies will be described, with particular reference to malaria. These methods will probably not replace current methods for routine diagnosis of parasitic infections in developing countries where parasitic diseases are endemic, due to high costs. However, they will be extremely useful for genotyping parasite strains and vectors, and for accurate parasite detection in both humans and vectors during epidemiological studies.

Matched MeSH terms: DNA, Protozoan/genetics; DNA, Protozoan/isolation & purification
Microtetraspora malaysiensis sp. nov., isolated from Malaysian primary dipterocarp forest soil

Nakajima Y, Ho CC, Kudo T

J Gen Appl Microbiol, 2003 Jun;49(3):181-9.
PMID: 12949699

The taxonomic position of three actinomycete strains isolated from Malaysian soil was established by using a polyphasic approach. The isolates formed chains composed of four spores on the tip of sporophores branching from the aerial mycelium, and their chemotaxonomic properties were common to those of members of the family Streptosporangiaceae. These phenotypic properties as well as a phylogenetic analysis based on 16S rRNA gene sequences indicated that they should be classified in the genus Microtetraspora. The three isolates showed a unique pattern of cultural, physiological and biochemical properties that distinguished them from previously described species of the genus Microtetraspora. The isolates showed more than 72% DNA relatedness to each other, but only 58% or less relatedness to any previously described species. On the basis of the data presented, a new species of the genus Microtetraspora, Microtetraspora malaysiensis, is proposed. The type strain of the new species is strain H47-7(T) (=JCM 11278(T)=DSM 44579(T)).

Matched MeSH terms: DNA, Bacterial/genetics; DNA, Bacterial/chemistry
Fulltext Molecular characterization of multi-drug resistant Escherichia coli isolates from tropical environments in Southeast Asia

Hara H, Yusaimi YA, Zulkeflle SNM, Sugiura N, Iwamoto K, Goto M, et al.

J Gen Appl Microbiol, 2019 Jan 24;64(6):284-292.
PMID: 29877296 DOI: 10.2323/jgam.2018.02.003

The emergence of antibiotic resistance among multidrug-resistant (MDR) microbes is of growing concern, and threatens public health globally. A total of 129 Escherichia coli isolates were recovered from lowland aqueous environments near hospitals and medical service centers in the vicinity of Kuala Lumpur, Malaysia. Among the eleven antibacterial agents tested, the isolates were highly resistant to trimethoprim-sulfamethoxazole (83.7%) and nalidixic acid (71.3%) and moderately resistant to ampicillin and chloramphenicol (66.7%), tetracycline (65.1%), fosfomycin (57.4%), cefotaxime (57.4%), and ciprofloxacin (57.4%), while low resistance levels were found with aminoglycosides (kanamycin, 22.5%; gentamicin, 21.7%). The presence of relevant resistance determinants was evaluated, and the genotypic resistance determinants were as follows: sulfonamides (sulI, sulII, and sulIII), trimethoprim (dfrA1 and dfrA5), quinolones (qnrS), β-lactams (ampC and blaCTX-M), chloramphenicol (cmlA1 and cat2), tetracycline (tetA and tetM), fosfomycin (fosA and fosA3), and aminoglycosides (aphA1 and aacC2). Our data suggest that multidrug-resistant E. coli strains are ubiquitous in the aquatic systems of tropical countries and indicate that hospital wastewater may contribute to this phenomenon.

Matched MeSH terms: DNA, Bacterial/genetics; Sequence Analysis, DNA
Fulltext AP1S3 Mutations Cause Skin Autoinflammation by Disrupting Keratinocyte Autophagy and Up-Regulating IL-36 Production

Mahil SK, Twelves S, Farkas K, Setta-Kaffetzi N, Burden AD, Gach JE, et al.

J Invest Dermatol, 2016 11;136(11):2251-2259.
PMID: 27388993 DOI: 10.1016/j.jid.2016.06.618

Prominent skin involvement is a defining characteristic of autoinflammatory disorders caused by abnormal IL-1 signaling. However, the pathways and cell types that drive cutaneous autoinflammatory features remain poorly understood. We sought to address this issue by investigating the pathogenesis of pustular psoriasis, a model of autoinflammatory disorders with predominant cutaneous manifestations. We specifically characterized the impact of mutations affecting AP1S3, a disease gene previously identified by our group and validated here in a newly ascertained patient resource. We first showed that AP1S3 expression is distinctively elevated in keratinocytes. Because AP1S3 encodes a protein implicated in autophagosome formation, we next investigated the effects of gene silencing on this pathway. We found that AP1S3 knockout disrupts keratinocyte autophagy, causing abnormal accumulation of p62, an adaptor protein mediating NF-κB activation. We showed that as a consequence, AP1S3-deficient cells up-regulate IL-1 signaling and overexpress IL-36α, a cytokine that is emerging as an important mediator of skin inflammation. These abnormal immune profiles were recapitulated by pharmacological inhibition of autophagy and verified in patient keratinocytes, where they were reversed by IL-36 blockade. These findings show that keratinocytes play a key role in skin autoinflammation and identify autophagy modulation of IL-36 signaling as a therapeutic target.

Matched MeSH terms: DNA/genetics*; DNA Mutational Analysis
Fulltext Haplotype divergence and multiple candidate genes at Rphq2, a partial resistance QTL of barley to Puccinia hordei

Yeo FK, Wang Y, Vozabova T, Huneau C, Leroy P, Chalhoub B, et al.

Theor Appl Genet, 2016 Feb;129(2):289-304.
PMID: 26542283 DOI: 10.1007/s00122-015-2627-5

Rphq2, a minor gene for partial resistance to Puccinia hordei , was physically mapped in a 188 kbp introgression with suppressed recombination between haplotypes of rphq2 and Rphq2 barley cultivars.

Matched MeSH terms: Sequence Analysis, DNA; DNA, Plant/genetics
Varroa jacobsoni (Acari: Varroidae) is more than one species

Anderson DL, Trueman JW

Exp Appl Acarol, 2000 Mar;24(3):165-89.
PMID: 11108385

Varroa jacobsoni was first described as a natural ectoparasitic mite of the Eastern honeybee (Apis cerana) throughout Asia. It later switched host to the Western honeybee (A. mellifera) and has now become a serious pest of that bee worldwide. The studies reported here on genotypic, phenotypic and reproductive variation among V. jacobsoni infesting A. cerana throughout Asia demonstrate that V. jacobsoni is a complex of at least two different species. In a new classification V. jacobsoni is here redefined as encompassing nine haplotypes (mites with distinct mtDNA CO-I gene sequences) that infest A. cerana in the Malaysia Indonesia region. Included is a Java haplotype, specimens of which were used to first describe V. jacobsoni at the beginning of this century. A new name, V. destructor n. sp., is given to six haplotypes that infest A. cerana on mainland Asia. Adult females of V. destructor are significantly larger and less spherical in shape than females of V. jacobsoni and they are also reproductively isolated from females of V. jacobsoni. The taxonomic positions of a further three unique haplotypes that infest A. cerana in the Philippines is uncertain and requires further study. Other studies reported here also show that only two of the 18 different haplotypes concealed within the complex of mites infesting A. cerana have become pests of A. mellifera worldwide. Both belong to V. destructor, and they are not V. jacobsoni. The most common is a Korea haplotype, so-called because it was also found parasitizing A. cerana in South Korea. It was identified on A. mellifera in Europe, the Middle East, Africa, Asia, and the Americas. Less common is a Japan/Thailand haplotype, so-called because it was also found parasitizing A. cerana in Japan and Thailand. It was identified on A. mellifera in Japan, Thailand and the Americas. Our results imply that the findings of past research on V. jacobsoni are applicable mostly to V. destructor. Our results will also influence quarantine protocols for bee mites, and may present new strategies for mite control.

Matched MeSH terms: DNA, Mitochondrial/genetics*; Sequence Analysis, DNA
The requirements for sterol regulatory element-binding protein (Srebp) and stimulatory protein 1 (Sp1)-binding elements in the transcriptional activation of two freshwater fish Channa striata and Danio rerio elovl5 elongase

Goh PT, Kuah MK, Chew YS, Teh HY, Shu-Chien AC

Fish Physiol Biochem, 2020 Aug;46(4):1349-1359.
PMID: 32239337 DOI: 10.1007/s10695-020-00793-w

Fish are a major source of beneficial n-3 LC-PUFA in human diet, and there is considerable interest to elucidate the mechanism and regulatory aspects of LC-PUFA biosynthesis in farmed species. Long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis involves the activities of two groups of enzymes, the fatty acyl desaturase (Fads) and elongase of very long-chain fatty acid (Elovl). The promoters of elovl5 elongase, which catalyses the rate-limiting reaction of elongating polyunsaturated fatty acid (PUFA), have been previously described and characterized from several marine and diadromous teleost species. We report here the cloning and characterization of elovl5 promoter from two freshwater fish species, the carnivorous snakehead fish (Channa striata) and zebrafish. Results show the presence of sterol-responsive elements (SRE) in the core regulatory region of both promoters, suggesting the importance of sterol regulatory element-binding protein (Srebp) in the regulation of elovl5 for both species. Mutagenesis luciferase and electrophoretic mobility shift assays further validate the role of SRE for basal transcriptional activation. In addition, several Sp1-binding sites located in close proximity with SRE were present in the snakehead promoter, with one having a potential synergy with SRE in the regulation of elovl5 expression. The core zebrafish elovl5 promoter fragment also directed in vivo expression in the yolk syncytial layer of developing zebrafish embryos.

Matched MeSH terms: DNA/genetics; DNA/isolation & purification
Fulltext Potential use of Pichia pastoris strain SMD1168H expressing DNA topoisomerase I in the screening of potential anti‑breast cancer agents

Xin J, Wan Mahtar WNA, Siah PC, Miswan N, Khoo BY

Mol Med Rep, 2019 Jun;19(6):5368-5376.
PMID: 31059050 DOI: 10.3892/mmr.2019.10201

Cancer chemotherapy possesses high toxicity, particularly when a higher concentration of drugs is administered to patients. Therefore, searching for more effective compounds to reduce the toxicity of treatments, while still producing similar effects as current chemotherapy regimens, is required. Currently, the search for potential anticancer agents involves a random, inaccurate process with strategic deficits and a lack of specific targets. For this reason, the initial in vitro high‑throughput steps in the screening process should be reviewed for rapid identification of the compounds that may serve as anticancer agents. The present study aimed to investigate the potential use of the Pichia pastoris strain SMD1168H expressing DNA topoisomerase I (SMD1168H‑TOPOI) in a yeast‑based assay for screening potential anticancer agents. The cell density that indicated the growth of the recombinant yeast without treatment was first measured by spectrophotometry. Subsequently, the effects of glutamate (agonist) and camptothecin (antagonist) on the recombinant yeast cell density were investigated using the same approach, and finally, the effect of camptothecin on various cell lines was determined and compared with its effect on recombinant yeast. The current study demonstrated that growth was enhanced in SMD1168H‑TOPOI as compared with that in SMD1168H. Glutamate also enhanced the growth of the SMD1168H; however, the growth effect was not enhanced in SMD1168H‑TOPOI treated with glutamate. By contrast, camptothecin caused only lower cell density and growth throughout the treatment of SMD1168H‑TOPOI. The findings of the current study indicated that SMD1168H‑TOPOI has similar characteristics to MDA‑MB‑231 cells; therefore, it can be used in a yeast‑based assay to screen for more effective compounds that may inhibit the growth of highly metastatic breast cancer cells.

Matched MeSH terms: DNA Topoisomerases, Type I/genetics; DNA Topoisomerases, Type I/metabolism*
Genome characterization of a novel vibriophage VpKK5 (Siphoviridae) specific to fish pathogenic strain of Vibrio parahaemolyticus

Lal TM, Sano M, Ransangan J

J Basic Microbiol, 2016 Aug;56(8):872-88.
PMID: 26960780 DOI: 10.1002/jobm.201500611

Vibrio parahaemolyticus has long been known pathogenic to shrimp but only recently it is also reported pathogenic to tropical cultured marine finfish. Traditionally, bacterial diseases in aquaculture are often treated using synthetic antibiotics but concern due to side effects of these chemicals is elevating hence, new control strategies which are both environmental and consumer friendly, are urgently needed. One promising control strategy is the bacteriophage therapy. In this study, we report the isolation and characterization of a novel vibriophage (VpKK5), belonging to the family Siphoviridae that was specific and capable of complete lysing the fish pathogenic strain of V. parahaemolyticus. The VpKK5 exhibited short eclipse and latent periods of 24 and 36 min, respectively, but with a large burst size of 180 pfu/cell. The genome analysis revealed that the VpKK5 is a novel bacteriophage with the estimated genome size of 56,637 bp and has 53.1% G + C content. The vibriophage has about 80 predicted open reading frames consisted of 37 complete coding sequences which did not match to any protein databases. The analysis also found no lysogeny and virulence genes in the genome of VpKK5. With such genome features, we suspected the vibriophage is novel and could be explored for phage therapy against fish pathogenic strains of V. parahaemolyticus in the near future.

Matched MeSH terms: DNA, Viral/genetics; Sequence Analysis, DNA
HbA2 levels in β-thalassaemia carriers with the Filipino β0-deletion: are the levels higher than what is found with non-deletional forms of β0-thalassaemia?

George E, Teh LK, Tan J, Lai MI, Wong L

Pathology, 2013 01;45(1):62-5.
PMID: 23222244 DOI: 10.1097/PAT.0b013e32835af7c1

AIMS: Classical carriers of β-thalassaemia are identified by a raised HbA2 level. Earlier studies indicated that the Filipino β-deletion has high raised HbA2 levels. The introduction of automated high performance liquid chromatography (HPLC) for thalassaemia screening is an important advance in technology for haematology laboratories. The BioRad Variant II Hb analyser is a common instrument used to quantify HbA2 levels in thalassaemia screening. This study aimed to determine HbA2 levels in carriers of Filipino β-mutation using the BioRad Variant II Hb analyser.
METHODS: The Filipino β-deletion was identified using gap-polymerase chain reaction (PCR) in the parents of transfusion dependent β-thalassaemia patients who were homozygous for the Filipino β-deletion in the indigenous population of Sabah, Malaysia. Hb subtypes were quantified on the BioRad Variant II Hb analyser. Concurrent α-thalassaemia was identified by multiplex gap-PCR for deletions and amplification refractory mutation system (ARMS)-PCR for non-deletional mutations.
RESULTS: The mean HbA2 level for Filipino β-thalassaemia trait was 5.9 ± 0.47 and with coinheritance of α-thalassaemia was 6.3 ± 0.44 (-α heterozygous) and 6.7 ± 0.36 (-α homozygous). The HbA2 levels were all >4% in keeping with the findings of classical β-thalassaemia trait and significantly higher than levels seen in non-deletional forms of β-thalassaemia.
CONCLUSION: The HbA2 level measured on the BioRad Variant II Hb analyser was lower than the level in the first description of the Filipino β-thalassaemia. β-thalassaemia trait with coinheritance of α-thalassaemia (-α) is associated with significantly higher HbA2 level.

Matched MeSH terms: DNA/blood; DNA/chemistry
No difference in the occurrence of mismatch repair defects and APC and CTNNB1 genes mutation in a multi-racial colorectal carcinoma patient cohort

Tan LP, Ng BK, Balraj P, Lim PK, Peh SC

Pathology, 2007 Apr;39(2):228-34.
PMID: 17454753

BACKGROUND AND AIMS: Colorectal cancers of different subtypes involve different pathogenic pathways like the Wnt and the mutator pathways. In this study, we screened 73 colorectal cancer cases from a multi-racial group for genetic and expression profile defects with the aim of correlating these with patients' clinicopathological characteristics.
METHODS: Mutation screening of the entire coding region of APC and exon 3 of CTNNB1, loss of heterozygosity (LOH) of APC, and microsatellite instability (MSI) status were assessed for 44 patients with available paired frozen normal and tumour tissues. In addition, 29 cases with available paraffin embedded tumour blocks were screened for mutation in exon 3 of CTNNB1, the APC mutation cluster region (codon 1286-1513), and hMLH1, hMSH2, hMSH6 protein expressions by immunohistochemistry method.
RESULTS: In our study, 15/73 cases showed APC mutations (20.5%), 1/73 cases had CTNNB1 mutation (1.4%), 5/32 cases had APC LOH (15.6%), and 16/70 (22.9%) cases revealed at least some form of mismatch repair (MMR) defect. Tumour grade (poor differentiation) was found to correlate significantly with right-sided tumour and mucinous histology (p = 0.01879 and 0.00320, respectively). Patients of younger age (below 45 years) more often had tumours of mucinous histology (p = 0.00014), while patients of older age (above 75 years) more often had tumours on the right side of the colon (p = 0.02448). Tumours of the mucinous histology subtype often had MMR defects (p = 0.02686). There was no difference in the occurrence of APC and CTNNB1 mutations and MMR defects found within our multi-racial colorectal cancer patient cohort.
CONCLUSION: Our findings support the notion that racial factor may not be related to the occurrence of MMR defects and APC and CTNNB1 mutations in our multi-racial patient cohort.

Matched MeSH terms: DNA Mutational Analysis; DNA Mismatch Repair*

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