We developed two sets of a multiplex amplification refractory mutation system (M-ARMS) assay to identify specific beta-thalassemia (beta-thal) mutations that are common in Thailand. The first one was for the detection of mutants with codon 17 (A>T), IV S-I-1 (G >T)), codons 41/42 (-TCT T) and codons 71/72 (+A), while the second one was for the -87 (C>A), -28 (A>G) and IVS-II-654 (C>T). Application of the proposed assay to 282 persons with beta-thal trait revealed a positive result in 276 cases (97.8%). There were 258 cases (91.5%) positive for the set 1 M-ARMS assay and 18 cases (6.4%) were positive for set 2. Six cases (2.2%) were negative for both sets 1 and 2, and were further characterized by DNA sequencing. The mutations detected by the set 1 M-ARMS assay were 113 cases (40.1%) of codons 41/42, 95 (33.7%) of codon 17, 41 (14.5%) of IVS-I-1 and nine cases (3.2%) of codons 71/72, while by set 2 there were 12 cases (4.2%) of -28, four cases(1.4%) of -87 and two cases (0.7%) of IVS-II-654. Mutations undetectable by M-ARMS assay were two cases of codons 27/28 (+C), one case of codon 35 (C>A), one of codon 43 (G>T), one of -31 (A>G) and one of IVS-I-5 (C>G). The M-ARMS assay proved to be a valuable tool for the analysis of beta-thal mutations. The method is robust, accurate, simple, speedy and cost-effective. The application of this assay will facilitate genetic counseling and prenatal diagnosis for severe thalassemia in high-risk pregnancies.

We used quantitative real-time polymerase chain reaction (qPCR) and high resolution melting (HRM) analyses for the detection of the common α-thalassemia (α-thal) genotypes in 40 northern Thai Hb H (β4) patients. The α(0)-thal [- -(SEA) (Southeast Asian) deletion] was detected by a multiplex gap real-time PCR. To determine the α(+)-thal, three primer pairs were designed. The A-primer pair was used to amplify the 3' terminal DNA sequences of the HBA2 gene, and the B-primer pair amplified the 5' flanking region of the HBA1 gene. The C-primer pair amplified the 3' terminal DNA sequences of the HBA1 gene and was used as an internal control. The -α(4.2) (leftward) and -α(3.7) (rightward) deletions were determined by monitoring the absence of PCR product(s). The Hb H patients who had a negative PCR result for the A-primer pair but positive for the B- and C-primer pairs carried the -α(4.2) deletion, while the -α(3.7) deletion carriers were negative for the A- and B-primer pairs. In the case of Hb H with Hb Constant Spring (Hb CS, α142, Term→Gln; HBA2: c.427T>C), all primer pairs were positive, HRM analysis of the PCR product of the A-primer pair was introduced to analyze the Hb CS gene. It can distinguish clearly between normal and Hb CS genotypes. The applied methods for the determination of the α(0)- and α(+)-thal genotypes revealed the results to be as accurate as conventional gap-PCR and the direct DNA sequencing methods but resulted in a much simpler and faster procedure.

Sericin-derived oligopeptides obtained from silk cocoons were investigated for the in vivo hypotensive effect and investigated for the underlying mechanism involved in vasodilation in isolated rat thoracic aorta. In normotensive anesthetized rats, oligopeptides induced an immediate and transient hypotensive activity. In rat aortic rings, oligopeptides induced a concentration-dependent vasorelaxation in vessels precontracted with both KCl and phenylephrine (PE) with endothelium-intact or endothelium-denuded rings. In endothelium-intact rings, pretreatment with Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 100 µM), an inhibitor of the NO synthase (NOS) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 µM), a selective inhibitor of the guanylyl cyclase enzyme, significantly reduced the relaxant effect of oligopeptides. However, indomethacin, an inhibitor of the cyclooxygenase, had no effect on oligopeptides-induced relaxation. In addition, pretreatment with tetraethylammonium (TEA, 5 mM) reduced the maximal relaxant effect induced by oligopeptides. By contrast, relaxation was not affected by 4-aminopyridine (4-AP, 1 mM), glibenclamide (10 µM), or barium chloride (BaCl2, 1 mM). In depolarization Ca2+-free solution, oligopeptides inhibited calcium chloride- (CaCl2-) induced contraction in endothelium-denuded rings in a concentration-dependent manner. Nevertheless, oligopeptides attenuated transient contractions in Ca2+-free medium containing EGTA (1 mM) induced by 1 µM PE, but they were not affected by 20 mM caffeine. It is obvious that potent vasodilation effect of oligopeptides is mediated through both the endothelium and the vascular smooth muscle.

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In order to prepare four types of crude extracts, physic nut seed residues (seed cake) were extracted using a Soxhlet apparatus with organic solvents. Hexane, dichloromethane, acetone, and methanol were used to macerate the nut for 72 h to obtain crude extracts, namely, PH, PD, PA, and PM(H), respectively. In the case of cold extraction, physic nut seed residue was macerated with methanol for 72 h at room temperature resulting in the crude extract PM(C). Antibacterial activities of all crude extracts were determined against each of the Gram-negative bacteria, Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi. and against the Gram-positive bacteria, Staphylococcus aureus, Bacillus cereus, and B. megaterium. This was done by using the agar diffusion method compared to ampicillin and streptomycin as positive control. The results showed that E. coli was inhibited by 30 mg/ml of PA and 50 mg/ml of PD. P. aeruginosa could not grow in the medium containing 50 mg/ml of PA. S. aureus, B. cereus, and B. megaterium were inhibited by PM(H) at different concentrations. PM(C) crude extract only inhibited B. megaterium. Salmonella typhi was not inhibited by any of the crude extracts.