Source and role of intestinally derived lysophosphatidic acid in dyslipidemia and atherosclerosis

We previously reported that i) a Western diet increased levels of unsaturated lysophosphatidic acid (LPA) in small intestine and plasma of LDL receptor null (LDLR^−/−) mice, and ii) supplementing standard mouse chow with unsaturated (but not saturated) LPA produced dyslipidemia and inflammation. Here we report that supplementing chow with unsaturated (but not saturated) LPA resulted in aortic atherosclerosis, which was ameliorated by adding transgenic 6F tomatoes. Supplementing chow with lysophosphatidylcholine (LysoPC) 18:1 (but not LysoPC 18:0) resulted in dyslipidemia similar to that seen on adding LPA 18:1 to chow. PF8380 (a specific inhibitor of autotaxin) significantly ameliorated the LysoPC 18:1-induced dyslipidemia. Supplementing chow with LysoPC 18:1 dramatically increased the levels of unsaturated LPA species in small intestine, liver, and plasma, and the increase was significantly ameliorated by PF8380 indicating that the conversion of LysoPC 18:1 to LPA 18:1 was autotaxin dependent. Adding LysoPC 18:0 to chow increased levels of LPA 18:0 in small intestine, liver, and plasma but was not altered by PF8380 indicating that conversion of LysoPC 18:0 to LPA 18:0 was autotaxin independent. We conclude that i) intestinally derived unsaturated (but not saturated) LPA can cause atherosclerosis in LDLR^−/− mice, and ii) autotaxin mediates the conversion of unsaturated (but not saturated) LysoPC to LPA.     

PTOV1 is associated with UCH-L1 and in response to estrogen stimuli during the mouse oocyte development

To investigate the biological significance of ubiquitin carboxyl-terminal hydrolase L1 (UCH-L1) involvement in oocyte maturation, we screened for proteins that bound to UCH-L1 in mouse ovaries, and we found that the prostate tumor overexpressed-1 (PTOV1) protein was able to bind to UCH-L1. PTOV1 is highly expressed in prostate cancers and considered as a potential marker for carcinogenesis and the progress of prostate cancer. It was reported that PTOV1 plays an important role in cell cycle regulation, but its role in mammalian oocyte development and meiosis is still unclear. In this paper, it was found that the expression levels of PTOV1 in mouse ovaries progressively increased from prepubescence to adulthood. And we found by immunohistochemistry that PTOV1 spreaded in both the cytoplasm and nuclei of oocytes during prepuberty, but in normal adult mouse oocytes, it concentrated not only in nuclei but also on the plasma membrane, though in some oocytes with abnormal shapes, PTOV1 did not display the typical distribution patterns. In granulosa cells, however, it was found to locate in the cytoplasm at all the selected ages. In postnatal mouse ovaries (28 days), estradiol treatment induced the adult-specific distribution pattern of PTOV1 in oocytes. In addition, UCH-L1 was shown to be associated with CDK1, which participated in the regulation of cell cycle and oocyte maturation. Therefore, we propose that the distribution changes of PTOV1 are age-dependent, and significant for mouse oocyte development and maturation. Moreover, the discovery that PTOV1 is associated with UCH-L1 in mouse oocytes supports the explanations for that UCH-L1 is involved in oocyte development and maturation, especially under the regulation of estrogen.     

Strand 6B deformation and residues exposure towards N-terminal end of helix B during proteinase inhibition by Serpins

Serine Protease inhibitors (Serpins) like antithrombin, antitrypsin, neuroserpin, antichymotrypsin, protein C-inhibitor and plasminogen activator inhibitor is involved in important biological functions like blood coagulation, fibrinolysis, inflammation, cell migration and complement activation. Serpins native state is metastable, which undergoes transformation to a more stable state during the process of protease inhibition. Serpins are prone to conformation defects, however little is known about the factors and mechanisms which promote its conformational change and misfolding. Helix B region in serpins is with several point mutations which result in pathological conditions due to polymerization. Helix B analysis for residue burial and cavity was undertaken to understand its role in serpin structure function. A structural overlap and an accessible surface area analysis showed the deformation of strand 6B and exposure of helix B at N-terminal end in cleaved conformation but not in the native and latent conformation of various inhibitory serpins. A cleaved polymer like conformation of antitrypsin also showed deformation of s6B and helix B exposure. Cavity analysis showed that helix B residues were part of the largest cavity in most of the serpins in the native state which increase in size during the transformation to cleaved and latent states. These data for the first time show the importance of strand 6B deformation and exposure of helix B in smooth insertion of the reactive center loop during serpin inhibition and indicate that helix B exposure due to variants may increase its polymer propensity. ABBREVIATIONS: serpin -serine protease inhibitors RCL -reactive center loop ASA -accessible surface area.     

A PCR-directed cell-free approach to optimize protein expression using diverse fusion tags

N-terminal fusion tags that enhance translation initiation or protein solubility are often used to facilitate protein overexpression. As the optimal tag for a given target protein cannot be predicted a priori, valuable time can be lost in cloning and manipulating the corresponding gene to generate different fusion constructs for expression analysis. We have developed a cell-free strategy that consolidates these steps, enabling the utility of a panel of nine fusion-tags to be determined within one to two days. This approach exploits the fact that PCR-amplified DNA can be used as a template for cell-free protein synthesis. Overlap/extension PCR using the TEV protease site as the overlap region allows the fusion of different T7 promoter (T7p)-tag-TEV DNA fragments with a TEV-gene-T7 terminator (T7ter) fragment. For tag sequences where the TEV site is not compatible, a short C?G? repeat (CGr) sequence can be used as the overlap region. The resulting T7p-tag-TEV-gene-T7ter constructs are then used as templates for PCR-directed cell-free protein synthesis to identify which tag-TEV-gene fusion protein produces the highest amount of soluble protein. We have successfully applied this approach to the overexpression of the Adiponectin hypervariable domain (AHD). Five of the nine N-terminal fusion tags tested enabled the synthesis of soluble recombinant protein. The best of these was the Peptidyl-prolylcis-trans isomerise B (PpiB) fusion tag which produces 1mg/ml amounts of soluble fusion protein. PpiB is an example of a new class of fusion tag known as the "stress-responsive proteins". Our results suggest that this cell-free fusion-tag expression screen facilitates the rapid identification of suitable fusion-tags that overcome issues such as poor expression and insolubility, often encountered using conventional approaches.     

<Emphasis Type="Italic">Lactococcus lactis</Emphasis> M4, a potential host for the expression of heterologous proteins

Background  

Many plasmid-harbouring strains of Lactococcus lactis have been isolated from milk and other sources. Plasmids of Lactococcus have been shown to harbour antibiotic resistance genes and those that express some important proteins. The generally regarded as safe (GRAS) status of L. lactis also makes it an attractive host for the production of proteins that are beneficial in numerous applications such as the production of biopharmaceutical and nutraceutical. In the present work, strains of L. lactis were isolated from cow's milk, plasmids were isolated and characterised and one of the strains was identified as a potential new lactococcal host for the expression of heterologous proteins.     

Isolated Diaphorase From Bovine Erythrocyte Cannot Reduce Oxidized Cytoglobin (Metcygb)

Background:Cytoglobin (Cygb) is a relatively newly identified globin protein that acts as an oxygen transporter in tissues like hemoglobin (Hb) in erythrocytes and myoglobin (Mb) in muscles. The natural oxidation of the Fe²⁺ ion in its heme group into metglobin (globin-Fe³⁺) made the loses of oxygen binding functions. It is known metHb and metMb can be reduced enzymatically using diaphorase or cyb5r3. However, metCygb reductase had not been previously identified. This study aims to analyze the reducing activity of bovine diaphorase on metCygb.Methods:Diaphorase was isolated from bovine erythrocyte and purified using gel filtration and cationic-exchanger chromatography. Its purity was verified by SDS-PAGE and western blot (WB). The metCygb was obtained from Cygb oxidation with potassium ferrocyanide and its reducing activity was determined by spectroscopy.Results:The diaphorase (MW=30.09 kDa) was purified 10.77-fold from crude enzyme with specific activity against metHb 8.479 U/mg. The purity was confirmed by WB using primary antibody anti-cyb5r3. The purified enzyme reduced metCygb at 0.785 µgmin^-1, which was 13.7 times less than the Vmax of metHb.DiscussionIn conclusion, the purified diaphorase from bovine erythrocytes did not significantly reduce metCygb rather than metHb, a natural substrate in cells.Key Words: Bovine Erythrocyte, Cytochrome B5 Reductase, Diaphorase, Metcytoglobin, Reduction     

Plasma Protein Profile of Lactating Women from Two Primary Health Centers in Jakarta,Indonesia

Background:Plasma protein profile test is a potential laboratory method to assess nutritional status especially albumin and globulins levels which reflects protein adequacy. The purpose of this study is to evaluate plasma protein profile of lactating women from two primary health centers in Jakarta.Methods:A cross-sectional study was conducted involving lactating women attending routine maternal examinations in two public primary health centers in Jakarta, Indonesia. The mother’s plasma total protein, albumin, globulin, and immunoglobulin levels were measured.Results:Sixty lactating women were recruited, mostly were 28 years old, slightly overweight, bearing two children, and their recent children were 2 months old. The mean total protein level was 8.13 g/dl, albumin 5.00 g/dl, globulin 3.18 g/dl, albumin: globulin ratio 1.558, mean total IgG level of 1255.98 and mean total IgM level of 135.819. All the measured plasma protein parameters were shown to be not correlated with maternal age, maternal BMI, or maternal parity.DiscussionThe plasma total protein, albumin, globulin, as well as total IgG and IgM level of lactating women in Jakarta were within normal range. These biochemical parameters were shown to be not correlated with anthropometrical data such as maternal age and BMI. The small and relatively homogenous samples were supposed to be the cause of such findings.Conclusion:The plasma protein profile of lactating women in Jakarta was adequate. Further studies are required to evaluate the eligibility of plasma protein profile as biochemical parameter of nutritional status in lactating women.Key Words: Blood protein, lactation, protein, albumin/globulin, immunoglobulin M/G