Unscrambling the Effect of C-Terminal Tail Deletion on the Stability of a Cold-Adapted,Organic Solvent Stable Lipase from Staphylococcus epidermidis AT2

Terminal moieties of most proteins are long known to be disordered and flexible. To unravel the functional role of these regions on the structural stability and biochemical properties of AT2 lipase, four C-terminal end residues, (Ile–Thr–Arg–Lys) which formed a flexible, short tail-like random-coil segment were targeted for mutation. Swapping of the tail-like region had resulted in an improved crystallizability and anti-aggregation property along with a slight shift of the thermostability profile. The lipolytic activity of mutant (M386) retained by 43 % compared to its wild-type with 18 % of the remaining activity at 45 °C. In silico analysis conducted at 25 and 45 °C was found to be in accordance to the experimental findings in which the RMSD values of M386 were more stable throughout the total trajectory in comparison to its wild-type. Terminal moieties were also observed to exhibit large movement and flexibility as denoted by high RMSF values at both dynamics. Variation in organic solvent stability property was detected in M386 where the lipolytic activity was stimulated in the presence of 25 % (v/v) of DMSO, isopropanol, and diethyl ether. This may be worth due to changes in the surface charge residues at the mutation point which probably involve in protein–solvent interaction.     

A thermoalkaliphilic lipase of <Emphasis Type="Italic">Geobacillus</Emphasis> sp. T1

A thermoalkaliphilic T1 lipase gene of Geobacillus sp. strain T1 was overexpressed in pGEX vector in the prokaryotic system. Removal of the signal peptide improved protein solubility and promoted the binding of GST moiety to the glutathione-Sepharose column. High-yield purification of T1 lipase was achieved through two-step affinity chromatography with a final specific activity and yield of 958.2 U/mg and 51.5%, respectively. The molecular mass of T1 lipase was determined to be approximately 43 kDa by gel filtration chromatography. T1 lipase had an optimum temperature and pH of 70°C and pH 9, respectively. It was stable up to 65°C with a half-life of 5 h 15 min at pH 9. It was stable in the presence of 1 mM metal ions Na⁺, Ca²⁺, Mn²⁺, K⁺ and Mg²⁺ , but inhibited by Cu²⁺, Fe³⁺ and Zn²⁺. Tween 80 significantly enhanced T1 lipase activity. T1 lipase was active towards medium to long chain triacylglycerols (C10–C14) and various natural oils with a marked preference for trilaurin (C12) (triacylglycerol) and sunflower oil (natural oil). Serine and aspartate residues were involved in catalysis, as its activity was strongly inhibited by 5 mM PMSF and 1 mM Pepstatin. The T _m for T1 lipase was around 72.2°C, as revealed by denatured protein analysis of CD spectra.     

Population structure, mitochondrial polyphyly and the repeated loss of human biting ability in anopheline mosquitoes from the southwest Pacific

Australia and New Guinea contain high levels of endemism and biodiversity, yet there have been few evaluations of population‐level genetic diversity in fauna occurring throughout the Australo‐Papuan region. Using extensive geographical sampling, we examined and compared the phylogenetic relationships, phylogeography and population structure of Anopheles farauti, An. hinesorum and An. irenicus throughout their ranges in the southwest Pacific using mitochondrial (mtDNA COI) and nuclear (ribosomal protein S9 and ribosomal DNA ITS2) loci. Phylogenetic analyses suggest that the ability to utilize humans as hosts has been lost repeatedly, coincident with independent colonizations of the Solomon Islands. As some of the species under investigation transmit malaria in the region, this is a medically important finding. Maximum likelihood and Bayesian phylogenetic analyses of nuclear loci also showed that the three species are monophyletic. However, putative introgression of An. hinesorum mtDNA onto a nuclear background of An. farauti was evident in populations from Queensland, Torres Strait and southern New Guinea. Haplotype networks and pairwise F_ST values show that there is significant genetic structure within New Guinea and Australia in both An. farauti and An. hinesorum, consistent with a long‐term history of low gene flow among populations.     

A model of a digital biological ecosystem

In this paper, we propose a model of a digital biological ecosystem of fish. Collections of fish data by scientists in Malaysia have been converted and developed into a digital biological ecosystem using computational technology and methods. We used a combination of Geographical Information System, databases and ontologies for storing, retrieving and visualizing fish species distribution and their abundance as well as their relationship with the environment. We used ArcGIS to build the fish and environmental geodatabase, which is linked to Google Earth for real-time visualization, and Surfer 8 to create contour maps which are superimposed as layers on the ArcGIS interface. Individual fish species on the map are hyperlinked to the FishBase Portal (http://www.fishbase.org/) while the published map is displayed using ArcReader. The three databases that we created contain information about fish species, environment and contours. These are also integrated using a fish ontology which will link all the available information for various fish species. The proposed system can be adopted by marine scientists to better convince economists, fishery managers, coastal developers and government bodies who make important decisions on conservation policies and laws.     

Apelin Attenuates Oxidative Stress in Human Adipocytes

It has been recently recognized that the increased oxidative stress (ROS overproduction) in obese condition is a key contributor to the pathogenesis of obesity-associated metabolic diseases. Apelin is an adipocytokine secreted by adipocytes, and known for its anti-obesity and anti-diabetic properties. In obesity, both oxidative stress and plasma level of apelin are increased. However, the regulatory roles of apelin on oxidative stress in adipocytes remain unknown. In the present study, we provide evidence that apelin, through its interaction with apelin receptor (APJ), suppresses production and release of reactive oxygen species (ROS) in adipocytes. This is further supported by the observations that apelin promotes the expression of anti-oxidant enzymes via MAPK kinase/ERK and AMPK pathways, and suppresses the expression of pro-oxidant enzyme via AMPK pathway. We further demonstrate that apelin is able to relieve oxidative stress-induced dysregulations of the expression of anti- and pro-oxidant enzymes, mitochondrial biogenesis and function, as well as release of pro- and anti-inflammatory adipocytokines. This study, for the first time, reveals the antioxidant properties of apelin in adipocytes, and suggests its potential as a novel therapeutic target for metabolic diseases.     

Locally isolated yeasts from Malaysia: identification, phylogenetic study and characterization

Yeasts are a convenient platform for many applications. They have been widely used as the expression hosts. There is a need to have a new yeast expression system to contribute the molecular cloning demands. Eight yeast isolates were screened from various environment sources and identified through ribosomal DNA (rDNA) Internal Transcribed Spacer (ITS). Full sequence of the rDNA ITS region for each isolate was BLASTed and phylogenetic study was constructed by using MEGA4. Among the isolates, isolate WB from 'ragi' (used to ferment carbohydrates) could be identified as a new species in order Saccharomycetales according to rDNA ITS region, morphology and biochemical tests. Isolate SO (from spoiled orange), RT (rotten tomato) and RG (different type of 'ragi') were identified as Pichia sp. Isolates R1 and R2, S4 and S5 (from the surrounding of a guava tree) were identified as Issatchenkia sp. and Hanseniaspora sp., respectively. Geneticin, 50 μg/mL, was determined to be the antibiotic marker for all isolates excepted for isolates RT and SO which used 500 μg/mL and 100 μg/mL Zeocin, respectively. Intra-extracellular proteins were screened for lipolytic activity at 30°C and 70°C. Thermostable lipase activity was detected in isolates RT and R1 with 0.6 U/mg and 0.1 U/mg, respectively. In conclusion, a new yeast-vector system for isolate WB can be developed by using phleomycin or geneticin as the drugs resistance marker. Moreover, strains RT and R1 can be investigated as a novel source of a thermostable lipase.