A repressor element in the 5'-untranslated region of human Pax5 exon 1A

Five members of the RecQ helicase family, RECQL, WRN, BLM, RTS and RECQL5, have been found in human and three of them (WRN, BLM and RTS) were disclosed to be the genes responsible for Werner, Bloom and Rothmund–Thomson syndromes, respectively. RECQL5 (RecQ helicase protein-like 5) was isolated as the fifth member of the family in humans through a search of homologous expressed sequence tags. The gene is expressed with at least three alternative splicing products, , β and γ. Here, we isolated mouse RECQL5β and determined the DNA sequence of full-length cDNA as well as the genome organization and chromosome locus. The mouse RECQL5β gene consists of 2949 bp coding 982 amino acid residues. Comparison of amino acid sequence among human (Homo sapiens), mouse (Mus musculus), Drosophila melanogaster and Caenorhabditis elegans RECQL5β homologs revealed three portions of highly conserved regions in addition to the helicase domain. Nineteen exons are dispersed over 40 kbp in the genome and all of the acceptor and donor sites for the splicing of each exon conform to the GT/AG rule. The gene is localized to the mouse chromosome 11E2, which has a syntenic relation to human 17q25.2-q25.3 where human RECQL5β exists. Our genetic characterizations of the mouse RECQL5β gene will contribute to functional studies on the RECQL5β products.     

Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 μM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.     

A Clinical,Neuropathological and Genetic Study of Homozygous A467T POLG-Related Mitochondrial Disease

Mutations in the nuclear gene POLG (encoding the catalytic subunit of DNA polymerase gamma) are an important cause of mitochondrial disease. The most common POLG mutation, A467T, appears to exhibit considerable phenotypic heterogeneity. The mechanism by which this single genetic defect results in such clinical diversity remains unclear. In this study we evaluate the clinical, neuropathological and mitochondrial genetic features of four unrelated patients with homozygous A467T mutations. One patient presented with the severe and lethal Alpers-Huttenlocher syndrome, which was confirmed on neuropathology, and was found to have a depletion of mitochondrial DNA (mtDNA). Of the remaining three patients, one presented with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), one with a phenotype in the Myoclonic Epilepsy, Myopathy and Sensory Ataxia (MEMSA) spectrum and one with Sensory Ataxic Neuropathy, Dysarthria and Ophthalmoplegia (SANDO). All three had secondary accumulation of multiple mtDNA deletions. Complete sequence analysis of muscle mtDNA using the MitoChip resequencing chip in all four cases demonstrated significant variation in mtDNA, including a pathogenic MT-ND5 mutation in one patient. These data highlight the variable and overlapping clinical and neuropathological phenotypes and downstream molecular defects caused by the A467T mutation, which may result from factors such as the mtDNA genetic background, nuclear genetic modifiers and environmental stressors.     

Seasonality and vertical structure of light-attracted insect communities in a dipterocarp forest in Sarawak

Nocturnal flying insects were collected monthly for 13 months using ultra violet light-traps set at various vertical levels in a weakly-seasonal, tropical lowland dipterocarp forest in Sarawak, Malaysia. Abundance, faunal composition, size distribution and guild structure of these samples were analyzed with respect to temperal and vertical distributions. The nocturnal flying insect community in the canopy level was highly dominated by fig wasps (84%) in individual number, and by scarabaeid beetles (28%) in weight. A principal component analysis on monthly catches detected non-random, seasonal trends of insect abundance. The first two principal trends were an alternation of wetter (September to January) and less wet seasons (February to August) and an alternation between the least wet (January to March) and the other seasons. Many insect groups were less abundant in the least wet season than the other seasons, whilst inverse patterns were found in Scarabaeidae and Tenebrionidae. Significantly positive and negative correlations between monthly catch and rainfall were detected only in ovule-feeders and in phloem-feeders, respectively. Delayed, significant negative correlations between monthly catch and 1–3 month preceding rainfall were more frequently detected in phytophages, phloem-feeders, seed-feeders, wood-borers and scavengers. The peak in abundance along vertical levels were found at the canopy level (35 m) for phloem-, ovule-, seed-, root-, fungal-feeders and nectar collectors, at an upper subcanopy level (25 m) for scavengers and aquatic predators, and at a middle subcanopy level (17 m) for ants. Catches at the emergent level (45 m) did not exceed those at the canopy level.     

In vitro propagation ofMitragyna parvifolia Korth.

Multiple shoot formation and their elongation from excised apical vegetative shoots of a 40-year old-tree ofMitragyna parvifolia Korth. was achieved in Murashige and Skoog's medium supplemented with 4.44 M benzyl adenine. The in vitro regenerated shoots rooted when cultured on modified Murashige and Skoog's medium containing low inorganic salts and the three auxins. Regeneration by this method was suitable for mass propagation of the plant.     

Molecular cloning and expression of the Fabs of human autoantibodies in Escherichia coli. Determination of the heavy or light chain contribution to the anti-DNA/-cardiolipin activity of the Fab

The Fabs of three human autoantibodies (B3/33H11, anti-DNA; UK4, anti-phospholipid) and six related hybrids have been cloned, expressed in Escherichia coli, and purified to homogeneity. SDS-polyacrylamide gel electrophoresis and Western blot analysis of the recombinant Fab demonstrated the purified Fab to be of correct size and in assembled form. Protein expression levels of up to 5-9 mg per liter of culture were achievable. A sensitive and reliable comparative anti-DNA enzyme-linked immunosorbent assay, involving a defined biotinylated 35-mer oligonucleotide in its single- or double-stranded form, is also described. Crithidia assay and anti-DNA or anti-cardiolipin antibody enzyme-linked immunosorbent assay analyses demonstrated convincing binding of the recombinant Fab proteins to DNA/cardiolipin, confirming the expression of functional molecule. The comparative DNA/cardiolipin binding analyses of the nine Fabs revealed that the anti-DNA (light, B3/33H11) or anti-cardiolipin (heavy, UK4) activity lies predominantly on one of the two chains. However, a compatible partner chain is necessary for optimum antigen binding activity of the antibody.     

Sequence analysis of glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 and comparison of the enzymatic characteristics of native and recombinant GDHs

The gdhA gene encoding glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned and sequenced. Phylogenetic analysis was performed on an alignment of 25 GDH sequences including KOD1-GDH, and two protein families were distinguished, as previously reported. KOD1-GDH was classified as new member of the hexameric GDH Family II. The gdhA gene was expressed in Escherichia coli, and recombinant KOD1-GDH was purified. Its enzymatic characteristics were compared with those of the native KOD1-GDH. Both enzymes had a molecular mass of 47 300 Da and were shown to be functional in a hexameric form (284 kDa). The N-terminal amino acid sequences of native KOD1-GDH and the recombinant GDH were VEIDPFEMAV and MVEIDPFEMA, respectively, indicating that native KOD1-GDH does not retain the initial methionine at the N-terminus. The recombinant GDH displayed enzyme characteristics similar to those of the native GDH, except for a lower level of thermostability, with a half-life of 2 h at 100° C, compared to 4 h for the native enzyme purified from KOD1. Kinetic studies suggested that the reaction is biased towards glutamate production. KOD1-GDH utilized both coenzymes NADH and NADPH, as do most eukaryal GDHs. Received: 6 May 1997 / Accepted: 23 September 1997     

MPDB 1.0: a medicinal plant database of Bangladesh

The term of medicinal plants include a various types of plants used in herbalism with medicinal activities. These plants are considered as rich resources of ingredients which can be used as complementary and alternative medicines and, also in drug developments and synthesis. In addition, some plants regarded as valuable origin of nutrition. Thus, all these plants are recommended as therapeutic agents. Information related to medicinal plants and herbal drugs accumulated over the ages are scattered and unstructured which make it prudent to develop a curated database for medicinal plants. MPDB 1.0 database is dedicated to provide the first window to find the plants around Bangladesh claimed to have medicinal and/or nutritive values by accumulating data from the published literatures. This database contains 406 medicinal plants with their corresponding scientific, family and local names as well as utilized parts for treatment from different districts of Bangladesh. Information regarding ailments is available for 353 plants. In addition, we have found active compounds for 78 plants with their corresponding PubMed ID.

Availability

www.medicinalplantbd.net     

Identification of quantitative trait loci (QTL) for oil and protein contents and their relationships with other seed quality traits in Brassica juncea

A detailed RFLP-genomic map was used to study the genetics of oil, seed and meal protein and sum of oil and seed/meal protein contents in a recombinant doubled-haploid population developed by crossing black- and yellow-seeded Brassica juncea lines. Two yellow seed color genes (SC-B4, SC-A6) and one QTL for erucic acid content (E_1b) showed pleiotropic effect for oil, protein and sum of oil and seed/meal protein contents. Six (O-A1, O-A6, O-A9, O-B3, O-B4, O-B5) and five (SP-A1, SP-A9, SP-B4, SP-B6, SP-C) QTLs were significant for oil and seed protein contents, respectively. Tight linkage of three of these QTLs (SP-A1, SP-A9, SP-B4, O-A1, O-A9, O-B4), with opposite effects, poses challenge to the plant breeders for simultaneous improvement of negatively correlated (r = −0.7**) oil and seed protein contents. However, one QTL for oil content (O-B3) and two for seed protein content (SP-B6, SP-C) were found to be unlinked, which offer the possibility for simultaneous improvement of these two traits. QTLs significant for meal protein (MP-A1, MP-A6, MP-A9, MP-B5, MP-B6) were significant at least for oil, seed protein or sum of oil and seed/meal protein contents (T-A6, T-A7, T-B4, T-B5). Sum of oil and seed protein contents and sum of oil and meal protein contents had a perfect correlation, as well as same epistatic interactions and QTLs with similar additive effect. This indicates that protein in seed or meal has practically the same meaning for breeding purposes. Epistatic interactions were significant for the quality traits, and their linkage reflected association among the traits.