Screening and identification of extracellular lipase-producing thermophilic bacteria from a Malaysian hot spring

Seven lipase-producing thermophilic bacteria (ST 1, ST 4, ST 6, ST 7, ST 8, ST 9 and ST 10) were isolated from the Setapak hot spring in Malaysia. The crude extracellular lipases recovered by ultrafiltration of cell-free culture supernatant were reacted in an olive oil mixture and their lipolytic activities were compared. Identification of the bacteria was carried out using the Biolog system and biochemical tests. Strain ST 7 that exhibited the highest lipolytic activity of 4.58 U/ml was identified as belonging to the Bacillus genus. Strain ST 6 with an activity of 3.51 U/ml, was identified as Ralstonia paucula. The lipolytic activities of strains ST 1, ST 4, ST 8, ST 9 and ST 10 were 2.39, 1.84, 2.38, 1.80 and 2.62 U/ml respectively. Strains ST 1, ST 4, and ST 10 were identified as Ralstonia paucula while strains ST 8 and ST 9 were Bacillus spp. Strains ST 7 and ST 9 were tentatively identified as Bacillus thermoglucosidasius, Bacillus stearothermophilus or Bacillus coagulans, whereas strain ST 8 was tentatively identified as Bacillus subtilis.     

Trypsin cleaves exclusively C-terminal to arginine and lysine residues

Almost all large-scale projects in mass spectrometry-based proteomics use trypsin to convert protein mixtures into more readily analyzable peptide populations. When searching peptide fragmentation spectra against sequence databases, potentially matching peptide sequences can be required to conform to tryptic specificity, namely, cleavage exclusively C-terminal to arginine or lysine. In many published reports, however, significant numbers of proteins are identified by non-tryptic peptides. Here we use the sub-parts per million mass accuracy of a new ion trap Fourier transform mass spectrometer to achieve more than a 100-fold increased confidence in peptide identification compared with typical ion trap experiments and show that trypsin cleaves solely C-terminal to arginine and lysine. We find that non-tryptic peptides occur only as the C-terminal peptides of proteins and as breakup products of fully tryptic peptides N-terminal to an internal proline. Simulating lower mass accuracy led to a large number of proteins erroneously identified with non-tryptic peptide hits. Our results indicate that such peptide hits in previous studies should be re-examined and that peptide identification should be based on strict trypsin specificity.     

Subcellular localization of microsomal triglyceride transfer protein

Microsomal triglyceride transfer protein (MTP) is essential for the assembly of apolipoprotein B-containing lipoproteins. Within the endoplasmic reticulum, it transfers lipid from the membrane to the forming lipoprotein. Recent evidence suggests that it may also function within the Golgi apparatus. To address this hypothesis, we developed a polyclonal antibody to MTP and used it in a series of studies on mouse liver and McArdle-RH7777 (McA) cells. Western blot analysis demonstrated the presence of MTP within mouse hepatic-Golgi apparatus-rich fractions. In addition, in vitro lipid transfer assays demonstrated the presence of triglyceride transfer activity within the Golgi fractions. Immunohistochemical studies with mouse liver demonstrated the presence of MTP within all hepatocytes, but not in nonparenchymal cells. The subcellular location of MTP in McA cells was investigated using confocal microscopy. MTP colocalized with the trans-Golgi network (TGN) 38 and Golgi SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptor) of 28 kDa (GS28), markers for the trans- and cis-Golgi apparatus, respectively. Morphometric analyses indicated that approximately 17% of the MTP signal colocalized with the TGN38, while 33% of the trans-Golgi marker colocalized with the MTP. Approximately 17% of the MTP signal colocalized with the GS28, whereas 53% of the cis-Golgi marker colocalized with the MTP. The results provide unequivocal evidence for the location of MTP within the Golgi apparatus, and further highlight the importance of this organelle in the assembly of lipoproteins.     

Neural induction: Historical views and application to pluripotent stem cells

Embryonic stem (ES) cells are a useful experimental material to recapitulate the differentiation steps of early embryos, which are usually invisible and inaccessible from outside of the body, especially in mammals. ES cells have greatly facilitated the analyses of gene expression profiles and cell characteristics. In addition, understanding the mechanisms during neural differentiation is important for clinical purposes, such as developing new therapeutic methods or regenerative medicine. As neurons have very limited regenerative ability, neurodegenerative diseases are usually intractable, and patients suffer from the disease throughout their lifetimes. The functional cells generated from ES cells in vitro could replace degenerative areas by transplantation. In this review, we will first demonstrate the historical views and widely accepted concepts regarding the molecular mechanisms of neural induction and positional information to produce the specific types of neurons in model animals. Next, we will describe how these concepts have recently been applied to the research in the establishment of the methodology of neural differentiation from mammalian ES cells. Finally, we will focus on examples of the applications of differentiation systems to clinical purposes. Overall, the discussion will focus on how historical developmental studies are applied to state‐of‐the‐art stem cell research.     

Protein Composition and Biomedical Potential of the Skin Secretion of Hylarana erythraea (Schlegel, 1837) (Anura: Ranidae) from Langkawi Archipelago,Kedah, Peninsular Malaysia

International Journal of Peptide Research and Therapeutics - The skin secretion of amphibians is known for its high content of bioactive compounds. These bioactive compounds are essential for the...     

Effect of spatial inlet velocity profiles on the vortex formation pattern in a dilated left ventricle

Despite the advancement of cardiac imaging technologies, these have traditionally been limited to global geometrical measurements. Computational fluid dynamics (CFD) has emerged as a reliable tool that provides flow ?eld information and other variables essential for the assessment of the cardiac function. Extensive studies have shown that vortex formation and propagation during the filling phase acts as a promising indicator for the diagnosis of the cardiac health condition. Proper setting of the boundary conditions is crucial in a CFD study as they are important determinants, that affect the simulation results. In this article, the effect of different transmitral velocity profiles (parabolic and uniform profile) on the vortex formation patterns during diastole was studied in a ventricle with dilated cardiomyopathy (DCM). The resulting vortex evolution pattern using the uniform inlet velocity profile agreed with that reported in the literature, which revealed an increase in thrombus risk in a ventricle with DCM. However the application of a parabolic velocity profile at the inlet yields a deviated vortical flow pattern and overestimates the propagation velocity of the vortex ring towards the apex of the ventricle. This study highlighted that uniform inlet velocity profile should be applied in the study of the filling dynamics in a left ventricle because it produces results closer to that observed experimentally.     

Properties and Expression of Na+/K+-ATPase α-Subunit Isoforms in the Brain of the Swamp Eel,Monopterus albus,Which Has Unusually High Brain Ammonia Tolerance

The swamp eel, Monopterus albus, can survive in high concentrations of ammonia (>75 mmol l⁻¹) and accumulate ammonia to high concentrations in its brain (∼4.5 µmol g⁻¹). Na⁺/K⁺-ATPase (Nka) is an essential transporter in brain cells, and since NH₄ ⁺ can substitute for K⁺ to activate Nka, we hypothesized that the brain of M. albus expressed multiple forms of Nka α-subunits, some of which might have high K⁺ specificity. Thus, this study aimed to clone and sequence the nka α-subunits from the brain of M. albus, and to determine the effects of ammonia exposure on their mRNA expression and overall protein abundance. The effectiveness of NH₄ ⁺ to activate brain Nka from M. albus and Mus musculus was also examined by comparing their Na⁺/K⁺-ATPase and Na⁺/NH₄ ⁺-ATPase activities over a range of K⁺/NH₄ ⁺ concentrations. The full length cDNA coding sequences of three nkaα (nkaα1, nkaα3a and nkaα3b) were identified in the brain of M. albus, but nkaα2 expression was undetectable. Exposure to 50 mmol l⁻¹ NH₄Cl for 1 day or 6 days resulted in significant decreases in the mRNA expression of nkaα1, nkaα3a and nkaα3b. The overall Nka protein abundance also decreased significantly after 6 days of ammonia exposure. For M. albus, brain Na⁺/NH₄ ⁺-ATPase activities were significantly lower than the Na⁺/K⁺-ATPase activities assayed at various NH₄ ⁺/K⁺ concentrations. Furthermore, the effectiveness of NH₄ ⁺ to activate Nka from the brain of M. albus was significantly lower than that from the brain of M. musculus, which is ammonia-sensitive. Hence, the (1) lack of nkaα2 expression, (2) high K⁺ specificity of K⁺ binding sites of Nkaα1, Nkaα3a and Nkaα3b, and (3) down-regulation of mRNA expression of all three nkaα isoforms and the overall Nka protein abundance in response to ammonia exposure might be some of the contributing factors to the high brain ammonia tolerance in M. albus.     

Integrins regulate centrosome integrity and astrocyte polarization following a wound

In response to a wound, astrocytes in culture extend microtubule‐rich processes and polarize, orienting their centrosomes and Golgi apparatus woundside. β1 Integrin null astrocytes fail to extend processes toward the wound, and are disoriented, and often migrate away orthogonal, to the wound. The centrosome is unusually fragmented in β1 integrin null astrocytes. Expression of a β1 integrin cDNA in the null background yields cells with intact centrosomes that polarize and extend processes normally. Fragmented centrosomes rapidly assemble following integrin ligation and cell attachment. However, several experiments indicated that cell adhesion is not necessary. For example, astrocytes in suspension expressing a chimeric β1 subunit that can be activated by an antibody assemble centrosomes suggesting that β1 activation is sufficient to cause centrosome assembly in the absence of cell adhesion. siRNA knockdown of PCM1, a major centrosomal protein, inhibits cell polarization, consistent with the notion that centrosomes are necessary for polarity and that integrins regulate polarity via centrosome integrity. Screening inhibitors of molecules downstream of integrins indicate that neither FAK nor ILK is involved in regulation of centrosome integrity. In contrast, blebbistatin, a specific inhibitor of non‐muscle myosin II (NMII), mimics the response of β1 integrin null astrocytes by disrupting centrosome integrity and cell polarization. Blebbistatin also inhibits integrin‐mediated centrosome assembly in astrocytes attaching to fibronectin, consistent with the hypothesis that NMII functions downstream of integrins in regulating centrosome integrity. © 2012 Wiley Periodicals, Inc. Develop Neurobiol 73: 333–353, 2013.     

Serum Metabolome and Lipidome Changes in Adult Patients with Primary Dengue Infection

Background

Dengue virus (DENV) is the most widespread arbovirus with an estimated 100 million infections occurring every year. Endemic in the tropical and subtropical areas of the world, dengue fever/dengue hemorrhagic fever (DF/DHF) is emerging as a major public health concern. The complex array of concurrent host physiologic changes has hampered a complete understanding of underlying molecular mechanisms of dengue pathogenesis.

Methodology/Principle Findings

Systems level characterization of serum metabolome and lipidome of adult DF patients at early febrile, defervescence, and convalescent stages of DENV infection was performed using liquid chromatography- and gas chromatography-mass spectrometry. The tractability of following metabolite and lipid changes in a relatively large sample size (n = 44) across three prominent infection stages allowed the identification of critical physiologic changes that coincided with the different stages. Sixty differential metabolites were identified in our metabolomics analysis and the main metabolite classes were free fatty acids, acylcarnitines, phospholipids, and amino acids. Major perturbed metabolic pathways included fatty acid biosynthesis and β-oxidation, phospholipid catabolism, steroid hormone pathway, etc., suggesting the multifactorial nature of human host responses. Analysis of phospholipids and sphingolipids verified the temporal trends and revealed association with lymphocytes and platelets numbers. These metabolites were significantly perturbed during the early stages, and normalized to control levels at convalescent stage, suggesting their potential utility as prognostic markers.

Conclusions/Significance

DENV infection causes temporally distinct serum metabolome and lipidome changes, and many of the differential metabolites are involved in acute inflammatory responses. Our global analyses revealed early anti-inflammatory responses working in concert to modulate early pro-inflammatory processes, thus preventing the host from development of pathologies by excessive or prolonged inflammation. This study is the first example of how an omic- approach can divulge the extensive, concurrent, and dynamic host responses elicited by DENV and offers plausible physiological insights to why DF is self limiting.