R54C Mutation of NOTCH3 Gene in the First Rungus Family with CADASIL

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a rare hereditary stroke caused by mutations in NOTCH3 gene. We report the first case of CADASIL in an indigenous Rungus (Kadazan-Dusun) family in Kudat, Sabah, Malaysia confirmed by a R54C (c.160C>T, p.Arg54Cys) mutation in the NOTCH3. This mutation was previously reported in a Caucasian and two Korean cases of CADASIL. We recruited two generations of the affected Rungus family (n = 9) and found a missense mutation (c.160C>T) in exon 2 of NOTCH3 in three siblings. Two of the three siblings had severe white matter abnormalities in their brain MRI (Scheltens score 33 and 50 respectively), one of whom had a young stroke at the age of 38. The remaining sibling, however, did not show any clinical features of CADASIL and had only minimal changes in her brain MRI (Scheltens score 17). This further emphasized the phenotype variability among family members with the same mutation in CADASIL. This is the first reported family with CADASIL in Rungus subtribe of Kadazan-Dusun ethnicity with a known mutation at exon 2 of NOTCH3. The penetrance of this mutation was not complete during the course of this study.     

The antimutagenicity of 2-substituted selenazolidine-4-(R)-carboxylic acids

Selenium can have cancer chemopreventive activity, although the mechanism of action has not been well defined. Selenazolidine-4-(R)-carboxylic acids (SCAs) were devised as prodrugs of L-selenocysteine, to provide selenium in a form and at a concentration commensurate with cancer chemopreventive activity. In the present study, a series of selenazolidines has been evaluated in the Salmonella typhimurium TA98 tester strain and all were found to possess antimutagenic activity. There was little difference between the seven selenazolidines in their effectiveness against either benzo[a]pyrene (B[a]P) or 3,6-bis(dimethylamino)acridine (acridine orange), agents which differ in their requirement for mammalian enzyme bioactivation for mutagenicity. Antimutagenic activity against acridine orange was dependent on selenazolidine concentration, and EC50 values were in the 5-10 microM range. At 25 microM, the concentration tested in common for the two mutagens, the selenazolidines were more effective antimutagens against acridine orange than against B[a]P, with reductions in mutant frequency ranging from 54 to 71% for B[a]P and 79 to 93% for acridine orange. Efficacy against B[a]P was not enhanced when the concentration was increased to 50 microM. The similarity in efficacy among the selenazolidines against B[a]P mutagenicity, contrasted with inter-compound differences in their ability to inhibit S9 CYP1A activity. The CYP1A Ki values ranged from a low of 63 microM (2-[2'-hydroxyphenyl]SCA) to a high of 1.1mM (2-cyclohexylSCA), but all were above the concentration required to inhibit mutagenicity by 50%. Thus, all the SCAs possess antimutagenic activity against both B[a]P and acridine orange, the efficacy varies little between the individual selenazolidines, and for B[a]P, the efficacy is not proportional to the inhibitory effect on the mutagen bioactivating enzyme.     

In Vitro Characterization of Novel Protegrin-1 Analogues Against Neoplastic Cells

Even with the great advances in cancer therapies, cancer remains the major cause of death worldwide. The use of high doses of anti-cancer chemotherapeutic drugs eventually causes the inevitable damage in non-neoplastic cells. New, selective, and more effective drugs are therefore urgently required to fight cancer. In this study, the anticancer activity of new peptide analogues (P1 and P2) derived from natural peptide, protegrin-1 (PG-1) were evaluated against human breast carcinoma cell lines (MCF-7) and human non-neoplastic mammary epithelial cell lines (MCF-10A), human hepatocellular carcinoma cells (HepG2) and Vero cells. The CC₅₀ values of cancer cells were significantly lower (P < 0.01) compared to non-neo-plastic cells after treating with P1 and P2 analogues. The analogues of PG-1 showed lower percentage of Lactate Dehydrogenase release (P < 0.001) from non-neoplastic cells compared to cancer cells and low haemolytic potential (P < 0.001) compared to PG-1. The P1 and P2 analogues were shown to be able to induce cancer cell senescence and apoptosis in a p53-dependent pathway which in turn, induced caspase activities and subsequent cell death. Overall, these results suggested that designing shorter peptides, as well as altering the number and position of positive charged residues in P1 and P2 analogues resulted in reduction of their toxicity to non-neoplastic cells and increased selectivity towards cancer cells. Increased selectivity also suggests its potential use to be developed as delivery vectors in the design of chemotherapeutic anticancer drugs.     

红火蚁病原真菌AUGM47早期发育超微结构研究

在前期筛选已获得对红火蚁Solenopsis invicta Buren高效致病真菌罗伯茨绿僵菌Metarhizium robertsii AUGM47的基础上,为进一步明确病原真菌对寄主昆虫的侵染机制。本试验在室内条件下,以红火蚁工蚁为侵染对象,利用荧光显微镜和透射电镜观察了罗伯茨绿僵菌AUGM47侵染单元分生孢子在体表附着萌发、穿透和体内增殖的早期发育过程。结果表明,菌株AUGM47分生孢子在红火蚁体表可萌发并形成附着胞侵入,接种后12 h观察到萌发,在36 h内普遍出现穿透结构穿透体壁。接种后48 h为菌体在血腔内的增殖阶段。菌丝体在穿透表皮和体腔内增殖过程中伴随着机械压力和酶的活动。接种后96 h,观察到自噬现象,菌体通过自噬降解并回收细胞器,为从体内穿出的晚期发育过程提供物质基础。本研究对罗伯茨绿僵菌AUGM47分生孢子在红火蚁体外至体内的发育进程研究证实了菌株的高致病性,为红火蚁生防真菌菌种改良和后续开发利用奠定理论基础。     

Bioactivity studies and adhesion of human osteoblast (hFOB) on silicon-biphasic calcium phosphate material

Surface reactivity of bioactive ceramics contributes in accelerating bone healing by anchoring osteoblast cells and the connection of the surrounding bone tissues. The presence of silicon (Si) in many biocompatible and bioactive materials has been shown to improve osteoblast cell adhesion, proliferation and bone regeneration due to its role in the mineralisation process around implants. In this study, the effects of Si-biphasic calcium phosphate (Si-BCP) on bioactivity and adhesion of human osteoblast (hFOB) as an in vitro model have been investigated. Si-BCP was synthesised using calcium hydroxide (Ca(OH)₂) and phosphoric acid (H₃PO₄) via wet synthesis technique at Ca/P ratio 1.60 of material precursors. SiO₂ at 3 wt% based on total precursors was added into apatite slurry before proceeding with the spray drying process. Apatite powder derived from the spray drying process was pressed into discs with Ø 10 mm. Finally, the discs were sintered at atmospheric condition to obtain biphasic hydroxyapatite (HA) and tricalcium phosphate (TCP) peaks simultaneously and examined by XRD, AFM and SEM for its bioactivity evaluation. In vitro cell viability of L929 fibroblast and adhesion of hFOB cell were investigated via AlamarBlue® (AB) assay and SEM respectively. All results were compared with BCP without Si substitution. Results showed that the presence of Si affected the material’s surface and morphology, cell proliferation and cell adhesion. AFM and SEM of Si-BCP revealed a rougher surface compared to BCP. Bioactivity in simulated body fluid (SBF) was characterised by pH, weight gain and apatite mineralisation on the sample surface whereby the changes in surface morphology were evaluated using SEM. Immersion in SBF up to 21 days indicated significant changes in pH, weight gain and apatite formation. Cell viability has demonstrated no cytotoxic effect and denoted that Si-BCP promoted good initial cell adhesion and proliferation. These results suggest that Si-BCP’s surface roughness (164 nm) was significantly higher than BCP (88 nm), thus enhancing the adhesion and proliferation of the osteoblast.     

Comparison of Different Protein Extraction Methods for Gel-Based Proteomic Analysis of <Emphasis Type="Italic">Ganoderma</Emphasis> spp.

Ganoderma species are a group of fungi that have the ability to degrade lignin polymers and cause severe diseases such as stem and root rot and can infect economically important plants and perennial crops such as oil palm, especially in tropical countries such as Malaysia. Unfortunately, very little is known about the complex interplay between oil palm and Ganoderma in the pathogenesis of the diseases. Proteomic technologies are simple yet powerful tools in comparing protein profile and have been widely used to study plant–fungus interaction. A critical step to perform a good proteome research is to establish a method that gives the best quality and a wide coverage of total proteins. Despite the availability of various protein extraction protocols from pathogenic fungi in the literature, no single extraction method was found suitable for all types of pathogenic fungi. To develop an optimized protein extraction protocol for 2-DE gel analysis of Ganoderma spp., three previously reported protein extraction protocols were compared: trichloroacetic acid, sucrose and phenol/ammonium acetate in methanol. The third method was found to give the most reproducible gels and highest protein concentration. Using the later method, a total of 10 protein spots (5 from each species) were successfully identified. Hence, the results from this study propose phenol/ammonium acetate in methanol as the most effective protein extraction method for 2-DE proteomic studies of Ganoderma spp.     

The ameliorative effects of Eurycoma longifolia Jack on testosterone-induced reproductive disorders in female rats

The objective of this research was to study the ameliorative effects of a standardized quassinoid-rich extract (TAF 273) of Eurycoma longifolia root on some reproductive disorders in female rats. An irregular estrous cycle and ovarian cystic follicles were induced in 21-day-old females by the daily administration of testosterone (10 mg/kg, sc) for three weeks. The hormone-treated rats exhibited persistent diestrous as well as ovaries containing cystic follicles. Upon treatment with TAF 273, fewer animals showed irregular estrous cycles and there was less follicular morphological damage. The reversal effect may be derived from the anti-estrogenic properties of the plant quassinoids.