Growth and hepatic acetyl coenzyme-A carboxylase activity are affected by dietary protein level in European seabass (Dicentrarchus labrax)

Two trials were undertaken with European seabass (Dicentrarchus labrax) to estimate the protein requirements for maintenance and growth as well as the effect of dietary protein level on the activity of hepatic acetyl coenzyme-A carboxylase (ACoAC). Six diets were formulated to contain graded levels of protein (from 5 to 55% crude protein (CP)) at a constant (12%) lipid level. Three other diets were also formulated to contain 35, 45 and 55% CP, but with a higher lipid level (19%). Groups of 10 individually marked fish (IBW: 100 g) and groups of 8 fish (IBW: 160 g) were used in trial I and II, respectively. Fish were fed to visual satiety and intake was recorded. At the end of both studies, whole body, liver and plasma samples were withdrawn for analyses. Growth rate was improved with increasing dietary CP level. Despite not being the object of a statistical analysis, feed efficiency tended to be enhanced at higher dietary CP level and protein efficiency ratio tended to decrease with increased protein intake. The reduction of the dietary protein/energy ratio, due to the increase of dietary lipids further improved growth and feed utilisation. Data from both experiments indicate 4.5+/-0.5 g kg(-1) d(-1) as the daily protein intake for maximum N gain and 520+/-50 mg kg(-1) d(-1) as the maintenance needs for nitrogen balance. An increase of dietary CP level, up to 25%, increased ACoAC activity. A further increase in dietary CP level (35 to 55%) did not affect liver ACoAC activity. The increase in dietary lipid level depressed significantly liver ACoAC specific activity.     

The J-helix of Escherichia coli DNA polymerase I (Klenow fragment) regulates polymerase and 3'- 5'-exonuclease functions

To assess the functional importance of the J-helix region of Escherichia coli DNA polymerase I, we performed site-directed mutagenesis of the following five residues: Asn-675, Gln-677, Asn-678, Ile-679, and Pro-680. Of these, the Q677A mutant is polymerase-defective with no change in its exonuclease activity. In contrast, the N678A mutant has unchanged polymerase activity but shows increased mismatch-directed exonuclease activity. Interestingly, mutation of Pro-680 has a Q677A-like effect on polymerase activity and an N678A-like effect on the exonuclease activity. Mutation of Pro-680 to Gly or Gln results in a 10-30-fold reduction in k(cat) on homo- and heteropolymeric template-primers, with no significant change in relative DNA binding affinity or K(m)((dNTP)). The mutants P680G and P680Q also showed a nearly complete loss in the processive mode of DNA synthesis. Since the side chain of proline is generally non-reactive, mutation of Pro-680 may be expected to alter the physical form of the J-helix itself. The biochemical properties of P680G/P680Q together with the structural observation that J-helix assumes helical or coiled secondary structure in the polymerase or exonuclease mode-bound DNA complexes suggest that the structural alteration in the J-helix region may be responsible for the controlled shuttling of DNA between the polymerase and the exonuclease sites.     

Activation of malonyl-CoA decarboxylase in rat skeletal muscle by contraction and the AMP-activated protein kinase activator 5-aminoimidazole-4-carboxamide-1-beta -D-ribofuranoside

Alterations in the concentration of malonyl-CoA, an inhibitor of carnitine palmitoyltransferase I, have been linked to the regulation of fatty acid oxidation in skeletal muscle. During contraction decreases in muscle malonyl-CoA concentration have been related to activation of AMP-activated protein kinase (AMPK), which phosphorylates and inhibits acetyl-CoA carboxylase (ACC), the rate-limiting enzyme in malonyl-CoA formation. We report here that the activity of malonyl-CoA decarboxylase (MCD) is increased in contracting muscle. Using either immunopurified enzyme or enzyme partially purified by (NH(4))(2)SO(4) precipitation, 2-3-fold increases in the V(max) of MCD and a 40% decrease in its K(m) for malonyl-CoA (190 versus 119 micrometer) were observed in rat gastrocnemius muscle after 5 min of contraction, induced by electrical stimulation of the sciatic nerve. The increase in MCD activity was markedly diminished when immunopurified enzyme was treated with protein phosphatase 2A or when phosphatase inhibitors were omitted from the homogenizing solution and assay mixture. Incubation of extensor digitorum longus muscle for 1 h with 2 mm 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside, a cell-permeable activator of AMPK, increased MCD activity 2-fold. Here, too, addition of protein phosphatase 2A to the immunopellets reversed the increase of MCD activity. The results strongly suggest that activation of AMPK during muscle contraction leads to phosphorylation of MCD and an increase in its activity. They also suggest a dual control of malonyl-CoA concentration by ACC and MCD, via AMPK, during exercise.     

Histidine 352 (His352) and Tryptophan 355 (Trp355) Are Essential for Flax UGT74S1 Glucosylation Activity toward Secoisolariciresinol

Flax secoisolariciresinol diglucoside (SDG) lignan is a natural phytoestrogen for which a positive role in metabolic diseases is emerging. Until recently however, much less was known about SDG and its monoglucoside (SMG) biosynthesis. Lately, flax UGT74S1 was identified and characterized as an enzyme sequentially glucosylating secoisolariciresinol (SECO) into SMG and SDG when expressed in yeast. However, the amino acids critical for UGT74S1 glucosyltransferase activity were unknown. A 3D structural modeling and docking, site-directed mutagenesis of five amino acids in the plant secondary product glycosyltransferase (PSPG) motif, and enzyme assays were conducted. UGT74S1 appeared to be structurally similar to the Arabidopsis thaliana UGT72B1 model. The ligand docking predicted Ser³⁵⁷ and Trp³⁵⁵ as binding to the phosphate and hydroxyl groups of UDP-glucose, whereas Cys³³⁵, Gln³³⁷ and Trp³⁵⁵ were predicted to bind the 7-OH, 2-OCH₃ and 17-OCH₃ of SECO. Site-directed mutagenesis of Cys³³⁵, Gln³³⁷, His³⁵², Trp³⁵⁵ and Ser³⁵⁷_, and enzyme assays revealed an alteration of these binding sites and a significant reduction of UGT74S1 glucosyltransferase catalytic activity towards SECO and UDP-glucose in all mutants. A complete abolition of UGT74S1 activity was observed when Trp³⁵⁵ was substituted to Ala³⁵⁵ and Gly³⁵⁵ or when changing His³⁵² to Asp³⁵²_, and an altered metabolite profile was observed in Cys335Ala, Gln337Ala, and Ser357Ala mutants. This study provided for the first time evidence that Trp³⁵⁵ and His³⁵² are critical for UGT74S1’s glucosylation activity toward SECO and suggested the possibility for SMG production in vitro.     

Retinal Photoreceptor Expresses Toll-Like Receptors (TLRs) and Elicits Innate Responses Following TLR Ligand and Bacterial Challenge

Toll-like receptors (TLRs) play an important role in host defense against microbial pathogens. Our previous studies have shown that TLRs are expressed on various retinal cells (Microglia and Müller glia) and orchestrate retinal innate responses in bacterial endophthalmitis. In this study, we used a well-characterized mouse cone photoreceptor cell line (661W); and demonstrated that these cells express all known TLRs. Although the stimulation of 661W cells with TLR ligands (Pam3Cys, PolyI:C, LPS, Flagellin, Poly DT, and ODN) did not alter TLR expression, downstream TLR-signaling pathways (NF-κB, p38, and ERK) are activated. Moreover, TLR-activated 661W cells secreted significant amounts of inflammatory mediators (IL-6, IL-1β, MIP-2, and KC) in their culture supernatant, as assessed by ELISA. A similar trend was observed in 661W cells challenged with live bacteria (Staphylococcus aureus). Interestingly, the neutralization of TLR2, a major receptor for S. aureus recognition, did not significantly attenuate bacterial-induced inflammatory mediators, suggesting the existence of TLR2-independent mechanisms in photoreceptor cells. Together, these results indicate that photoreceptors constitutively express functional TLRs and possess the ability to initiate innate responses following pathogen challenge, implicating their role in retinal innate immunity.     

Predicting stable functional peptides from the intergenic space of E. coli

Expression of synthetic proteins from intergenic regions of E. coli and their functional association was recently demonstrated (Dhar et al. in J Biol Eng 3:2, 2009. doi:10.1186/1754-1611-3-2). This gave birth to the question: if one can make ‘user-defined’ genes from non-coding genome—how big is the artificially translatable genome? (Dinger et al. in PLoS Comput Biol 4, 2008; Frith et al. in RNA Biol 3(1):40–48, 2006a; Frith et al. in PLoS Genet 2(4):e52, 2006b). To answer this question, we performed a bioinformatics study of all reported E. coli intergenic sequences, in search of novel peptides and proteins, unexpressed by nature. Overall, 2500 E. coli intergenic sequences were computationally translated into ‘protein sequence equivalents’ and matched against all known proteins. Sequences that did not show any resemblance were used for building a comprehensive profile in terms of their structure, function, localization, interactions, stability so on. A total of 362 protein sequences showed evidence of stable tertiary conformations encoded by the intergenic sequences of E. coli genome. Experimental studies are underway to confirm some of the key predictions. This study points to a vast untapped repository of functional molecules lying undiscovered in the non-expressed genome of various organisms.     

In Vitro Culture of Functionally Active Buffalo Hepatocytes Isolated by Using a Simplified Manual Perfusion Method

Background

In farm animals, there is no suitable cell line available to understand liver-specific functions. This has limited our understanding of liver function and metabolism in farm animals. Culturing and maintenance of functionally active hepatocytes is difficult, since they survive no more than few days. Establishing primary culture of hepatocytes can help in studying cellular metabolism, drug toxicity, hepatocyte specific gene function and regulation. Here we provide a simple in vitro method for isolation and short-term culture of functionally active buffalo hepatocytes.

Results

Buffalo hepatocytes were isolated from caudate lobes by using manual enzymatic perfusion and mechanical disruption of liver tissue. Hepatocyte yield was (5.3±0.66)×10⁷ cells per gram of liver tissue with a viability of 82.3±3.5%. Freshly isolated hepatocytes were spherical with well contrasted border. After 24 hours of seeding onto fibroblast feeder layer and different extracellular matrices like dry collagen, matrigel and sandwich collagen coated plates, hepatocytes formed confluent monolayer with frequent clusters. Cultured hepatocytes exhibited typical cuboidal and polygonal shape with restored cellular polarity. Cells expressed hepatocyte-specific marker genes or proteins like albumin, hepatocyte nuclear factor 4α, glucose-6-phosphatase, tyrosine aminotransferase, cytochromes, cytokeratin and α1-antitrypsin. Hepatocytes could be immunostained with anti-cytokeratins, anti-albumin and anti α1-antitrypsin antibodies. Abundant lipid droplets were detected in the cytosol of hepatocytes using oil red stain. In vitro cultured hepatocytes could be grown for five days and maintained for up to nine days on buffalo skin fibroblast feeder layer. Cultured hepatocytes were viable for functional studies.

Conclusion

We developed a convenient and cost effective technique for hepatocytes isolation for short-term culture that exhibited morphological and functional characteristics of active hepatocytes for studying gene expression, regulation, hepatic genomics and proteomics in farm animals.     

Evaluation of Hs-CRP Levels and Interleukin 18 (-137G/C) Promoter Polymorphism in Risk Prediction of Coronary Artery Disease in First Degree Relatives

Background

Coronary Artery Disease (CAD) is clearly a multifactorial disease that develops from childhood and ultimately leads to death. Several reports revealed having a First Degree Relatives (FDRS) with premature CAD is a significant autonomous risk factor for CAD development. C - reactive protein (CRP) is a member of the pentraxin family and is the most widely studied proinflammatory biomarker. IL-18 is a pleiotrophic and proinflammatory cytokine which is produced mainly by macrophages and plays an important role in the inflammatory cascade.

Methods and Results

Hs-CRP levels were estimated by ELISA and Genotyping of IL-18 gene variant located on promoter -137 (G/C) by Allele specific PCR in blood samples of 300 CAD patients and 300 controls and 100 FDRS. Promoter Binding sites and Protein interacting partners were identified by Alibaba 2.1 and Genemania online tools respectively. Hs-CRP levels were significantly high in CAD patients followed by FDRS when compared to controls. In IL-18 -137 (G/C) polymorphism homozygous GG is significantly associated with occurrence of CAD and Hs-CRP levels were significantly higher in GG genotype subjects when compared to GC and CC. IL-18 was found to be interacting with 100 protein interactants.

Conclusion

Our results indicate that Hs-CRP levels and IL-18-137(G/C) polymorphism may help to identify risk of future events of CAD in asymptomatic healthy FDRS.