Micropropagation of <Emphasis Type="Italic">Penthorum chinense</Emphasis> through axillary bud

This study reports a protocol for successful micropropagation of Penthorum chinense using nodal explants on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA) or kinetin (Kn). The presence of BA promoted a higher rate of shoot multiplication than Kn. Maximum multiple shoot formation was observed in 59.2% of nodal explants cultured on MS medium supplemented with 2.0 mg l⁻¹ BA after 6 wk. After subculture for 4 wk, the maximum number of shoots (6.4) was obtained on a medium with 2.0 mg l⁻¹ BA, but shoots were too short and not suitable for micropropagation. The taller shoots that regenerated in the presence of lower BA concentration (1.0 mg l⁻¹) were selected for root induction study. Most shoots (98.8%) rooted in the presence of 0.5 mg l⁻¹ indole-3-acetic acid after 3 wk, with each shoot forming an average of 10.0 roots. Plantlets were transferred to soil and successfully acclimatized.     

Ubiquinone-10 production using Agrobacterium tumefaciens dps gene in Escherichia coli by coexpression system

Ubiquinone (Coenzyme Q; abbreviation, UQ) acts as a mobile component of the respiratory chain by playing an essential role in the electron transport system, and has been widely used in pharmaceuticals. The biosynthesis of UQ involves 10 sequential reactions brought about by various enzymes. In this study we have cloned, expressed the decaprenyl diphosphate synthase, designated dps gene, from Agrobacterium tumefaciens, and succeeded in detecting UQ-10 in addition to innate UQ-8 in Escherichia coli. Furthermore, the production of UQ-10 was higher than UQ-8. To establish an efficient expression system for UQ-10 production, we used genes, including ubiC, ubiA, and ubiG involved in UQ biosynthesis in E. coli, to construct a better co-expression system. The expression coupled by dps and ubiCA was effective for increasing UQ-10 production by five times than that by expressing single dps gene in the shake flask culture. To study for a large-scale production of UQ-10 in E. coli, fed-batch fermentations were implemented to achieve a high cell density culture. A cell concentration of 85.40 g/L and 94.58 g/L dry cell weight (DCW), and UQ-10 content of 50.29 mg/L and 45.86 mg/L was obtained after 32.5 h and 27.5 h of cultivation, subsequent to isopropyl-β-d-thiogalactopy ranoside and lactose induction, respectively. In addition, plasmid stability was maintained at high level throughout the fermentation.     

Regulation of apoptosis by C. elegans CED-9 in the absence of the C-terminal transmembrane domain

Bcl-2 proteins regulate apoptosis in organisms as diverse as mammals and nematodes. These proteins are often localized at mitochondria by a C-terminal transmembrane domain. Although the transmembrane domain and mitochondrial localization are centrally involved in specific cases of vertebrate Bcl-2 activity, the significance of this localization is not clear for all species. Studying the Caenorhabditis elegans Bcl-2 homolog CED-9, we found that the transmembrane domain was both necessary and sufficient for localization at mitochondrial outer membranes. Furthermore, we found that in our assays, ced-9 transgenes lacking the transmembrane domain, although somewhat less active than equivalent transgenes derived from wild-type ced-9, rescued embryonic lethality of ced-9(lf) animals and responded properly to upstream signals in controlling the fate of Pn.aap neurons. Both of these apoptotic activities were retained in a construct where CED-9 lacking the transmembrane domain was targeted to the cytosolic surface of the endoplasmic reticulum and derived organelles, suggesting that in wild-type animals, accumulation at mitochondria is not essential for CED-9 to either inhibit or promote apoptosis in C. elegans. Taken together, these data are consistent with a multimodal character of CED-9 action, with an ability to regulate apoptosis through interactions in the cytosol coexisting with additional evolutionarily conserved role(s) at the membrane.     

Genetic analysis of a novel plasmid pZMX101 from Halorubrum saccharovorum: determination of the minimal replicon and comparison with the related haloarchaeal plasmid pSCM201

The DNA sequence of a novel haloarchaeal plasmid pZMX101 (3918 bp) from Halorubrum saccharovorum was determined and six ORFs were predicted. The largest ORF encodes a putative replication initiation protein RepA, which shares 40% sequence similarity with the Rep201 of a theta-replication plasmid pSCM201 recently isolated from Haloarcula, suggesting that pZMX101 might replicate via a theta-type mechanism. Using pZMX101 as the only haloarchaeal replicon, a shuttle vector pZMX108 was constructed and successfully transformed into Haloferax volcanii DS70. Based on this in vivo system, the minimal replicon (1978 bp) of pZMX101 was determined. It is composed of the repA gene plus c. 400-bp upstream and 300-bp downstream sequences. Significantly, the putative replication origin of pZMX101 and that of pSCM201 contain different types of sequence motifs, and these two plasmids exhibit distinct host preference for Haloferax and Haloarcula, respectively.     

Effects of L-malate on physical stamina and activities of enzymes related to the malate-aspartate shuttle in liver of mice

L-malate, a tricarboxylic acid cycle (TCA) intermediate, plays an important role in transporting NADH from cytosol to mitochondria for energy production and may be involved in the beneficial effects of improving physical stamina. In the present study, we investigated the effects of L-malate on the performance of forced swimming time and blood biochemical parameters related to fatigue - blood urea nitrogen (BUN), glucose (Glc), creatine kinase (CK),total protein (TP) and lactic acid (LA). To investigate the effects of L-malate on the malate-aspartate shuttle and energy metabolism in mice, the activities of enzymes related to the malate-aspartate shuttle were measured. L-malate was orally administered to mice continuously for 30 days using a feeding atraumatic needle. The swimming time was increased by 26.1 % and 28.5 %, respectively, in the 0.210 g/kg and 0.630 g/kg L-malate-treated group compared with the control group. There were no differences in the concentrations of Glc, BUN and TP between the L-malate-treated groups and the control groups. However, the levels of CK were significantly decreased in the L-malate-treated groups. The results predict a potential benefit of L-malate for improving physical stamina and minimizing muscle damage during swimming exercise. The activities of cytosolic and mitochondrial malate dehydrogenase were significantly elevated in the L-malate-treated group compared with the control group. These enzymatic activities may be useful indicators for evaluating changes affecting the malate-aspartate shuttle and energy metabolism in the liver of mice.     

Fish oil induced increase in walking distance, but not ankle brachial pressure index, in peripheral arterial disease is dependent on both body mass index and inflammatory genotype

Peripheral arterial disease (PAD) is an atherosclerotic disease. Evidence suggests that atherosclerosis is an inflammatory condition and long chain n-3 fatty acids, found in oily fish and fish oils, have been shown to reduce inflammation. Genetic and lifestyle factors such as body mass index (BMI) also influence inflammation. In this study we have examined the effect of fish oil in patients with claudication secondary to PAD. Fish oil supplementation, providing 1g EPA and 0.7 g DHA per day for 12 weeks, increased walking distance on a treadmill set at 3.2 km/h with a 7% incline. Walking distance to first pain increased from 76.2+/-8.5 m before fish oil to 140.6+/-25.5 m after fish oil (mean+/-SEM, p=0.004) and total distance walked increased from 160.0+/-21.5 m before fish oil to 242.1+/-34.5 m after fish oil (p=0.002). Fish oil supplementation also improved ankle brachial pressure index (ABPI) from 0.599+/-0.017 before fish oil to 0.776+/-0.030 after fish oil (p<0.001). The increase in walking distance was dependent on both BMI and genotype for single nucleotide polymorphisms in the genes encoding the pro-inflammatory cytokines tumour necrosis factor-alpha and interleukin (IL)-1beta and the anti-inflammatory cytokine IL-10 (detected using amplification refractory mutation system polymerase chain reaction). Neither BMI nor any of the genotypes examined affected the ability of fish oil to increase ABPI. The mechanisms by which fish oil affects walking distance and ABPI do not appear to be the same.     

Characterization of AFLP Markers Associated with Growth in the Kuruma Prawn, <Emphasis Type="Italic">Marsupenaeus japonicus</Emphasis>, and Identification of a Candidate Gene

Growth rate of the Kuruma prawn, Marsupenaeus japonicus is an important economic trait, with larger animals commanding higher market prices. To identify gene markers associated with growth, a genetic map of a full-sib F₂ intercross family of M. japonicus has previously been generated and quantitative trait loci (QTL) influencing weight, total length, and carapace length were identified. In this study, amplified fragment length polymorphism (AFLP) markers associated with the major QTL region, contributing 16% to phenotypic variation, were characterized. Flanking sequence has been obtained and allelic variants responsible for segregation patterns of these markers have been identified. The genomic sequence surrounding the AFLP band 7.21a, residing under the QTL peak, contains a gene sequence homologous to the elongation of very long chain fatty acids-like (ELOVL) protein family. A full-length mRNA (ELOVL-MJ) encoding this protein was isolated from M. japonicus, representing both the first ELOVL gene in crustacea and the first candidate gene identified via QTL studies in crustacea.