-
[show abstract]
[hide abstract]
ABSTRACT: Type 2 diabetes is the most common form of diabetes worldwide. Some of its complications, such as retinopathy and neuropathy, are long-term and protracted, with an unclear etiology. Given this problem, genetic model systems, such as in flies where type 2 diabetes can be modeled and studied, offer distinct advantages.
We used individual flies in experiments: control and mutant individuals with partial loss-of-function insulin pathway genes. We measured wing size and tested body weight for growth phenotypes, the latter by means of a microbalance. We studied total lipid and carbohydrate content, lipids by a reaction in single fly homogenates with vanillin-phosphoric acid, and carbohydrates with an anthrone-sulfuric acid reaction. Cholinesterase activity was measured using the Ellman method in head homogenates from pooled fly heads, and electroretinograms with glass capillary microelectrodes to assess performance of central brain activity and retinal function.
Flies with partial loss-of-function of insulin pathway genes have significantly reduced body weight, higher total lipid content, and sometimes elevated carbohydrate levels. Brain function is impaired, as is retinal function, but no clear correlation can be drawn from nervous system function and metabolic state.
These studies show that flies can be models of type 2 diabetes. They weigh less but have significant lipid gains (obese); some also have carbohydrate gains and compromised brain and retinal functions. This is significant because flies have an open circulatory system without microvasculature and can be studied without the complications of vascular defects.
Diabetes 04/2011; 60(5):1632-6. · 8.29 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Cellulomonas flavigena CDBB-531 was found to secrete a bifunctional cellulase/xylanase with a molecular mass of 49 kDa and pI 4.3. This enzyme was active on Remazol brilliant blue-carboxymethylcellulose (RBB-CMC) and Remazol brilliant blue-xylan (RBB-X). Based on thin-layer chromatographic analysis of the degradation products, the cellulase activity produced glucose, cellobiose, cellotriose, and cellotetraose from CMC as the substrate. When xylan from birchwood was used, end products were xylose, arabinose, and xylobiose. The bifunctional enzyme showed a pH optimum of 6 for cellulase activity and 9 for xylanase activity, which pointed out that this enzyme had separate sites for each activity. In both cases, the apparent optimum temperature was 50 degrees C. The predicted amino acid sequence of purified protein showed similarity with the catalytic domain of several glycosyl hydrolases of family 10.
Current Microbiology 08/2008; 57(1):39-44. · 1.82 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The diversity of cellulases and xylanases secreted by Cellulomonas flavigena cultured on sugar cane bagasse, Solka-floc, xylan, or glucose was explored by two-dimensional gel electrophoresis. C. flavigena produced the largest variety of cellulases and xylanases on sugar cane bagasse. Multiple extracellular proteins were expressed with these growth substrates, and a limited set of them coincided in all substrates. Thirteen proteins with carboxymethyl cellulase or xylanase activity were liquid chromatography/mass spectrometry sequenced. Proteins SP4 and SP18 were identified as products of celA and celB genes, respectively, while SP20 and SP33 were isoforms of the bifunctional cellulase/xylanase Cxo recently sequenced and characterized in C. flavigena. The rest of the detected proteins were unknown enzymes with either carboxymethyl cellulase or xylanase activities. All proteins aligned with glycosyl hydrolases listed in National Center for Biotechnology Information database, mainly with cellulase and xylanase enzymes. One of these unknown enzymes, protein SP6, was cross-induced by sugar cane bagasse, Solka-floc, and xylan. The differences in the expression maps of the presently induced cultures revealed that C. flavigena produces and secretes multiple enzymes to use a wide range of lignocellulosic substrates as carbon sources. The expression of these proteins depends on the nature of the cellulosic substrate.
Applied Microbiology and Biotechnology 01/2008; 77(3):589-95. · 3.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The mutant strain PN-120 of Cellulomonas flavigena produces a ss-glucosidase that is 10-fold more active than the corresponding enzyme isolated from the parental strain. These enzymes were partially purified through Q Sepharose and Bio-Gel filtration. A single protein band was detected on polyacrylamide-gel electrophoresis/zymogram using 4-methylumbelliferyl-beta-D-glucoside. On sodium dodecyl sulfate-PAGE, the enzyme displayed three protein bands, suggesting that in C. flavigena the enzyme is oligomeric with a molecular mass of 210 kDa. On purification, the specific activity of ss-glucosidase isolated from PN-120 was increased 16-fold and showed three times more affinity for cellobiose than the enzyme of the parental strain; nevertheless, the optimum pH and temperature were similar for both enzymes. The kinetic parameters suggested that the increase in the activity of the enzyme, from the mutant strain, was caused by a mutation that affects the catalytic site of the enzyme. The partial amino-acid sequence of the isolated enzyme confirmed that it is a beta-glucosidase because of its homology with other beta-glucosidases produced by cellulolytic bacteria and fungi.
Current Microbiology 05/2007; 54(4):266-70. · 1.82 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: An endoglucanase gene called celcflB was isolated from a genomic library of C. flavigena. Its nucleotide sequence showed an ORF of 1725 bp with a GTG start codon. A promoter sequence was found inside the adjacent gene upstream from the start point of translation of celcflB gene. The gene celcflB was induced by sugarcane bagasse and repressed by glucose when C. flavigena was grown on these carbon sources, suggesting that celcflB gene is regulated at transcriptional level. The predicted amino acid sequence of the CelcflB protein presented a catalytic domain with a high homology to family 5 of glycosil hydrolases, and a cellulose binding domain identical to cellulose binding domains type II. The coding region of the celcflB gene was cloned into the expression vector pQE30 and the recombinant protein of 58 kDa presented endoglucanase activity towards carboxymethyl cellulose (CMC).
Current Microbiology 12/2003; 47(5):359-63. · 1.82 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Insulin resistance is defined as the decrease in the glucose disposal in response to insulin by the target tissues. High concentrations of nonesterified fatty acids (NEFA) in plasma have been implicated with many insulin resistance states. We evaluated several aspects of the insulin resistance induced by palmitic acid in rats and found that after treatment with 0.09 g/kg of palmitic acid there is a delay in the curve of tolerance to glucose. We measured the changes in protein phosphorylation in samples from abdominus rectus muscle and there was a decrease of 64 and 75% in the levels of phosphorylation in tyrosine of the insulin receptor and insulin receptor substrate-1, respectively. This diminution in the tyrosine phosphorylation is consistent with a decrease in the main pathway known to be activated after insulin treatment, the mitogen activated protein kinases (MAPKs). If the animals were treated with inhibitors of PKC, like sphingosine, there was a prevention of the effect of palmitic acid determined at the level of tyrosine phosphorylation. According with this result, we found an increase in the phosphorylations in serine of the insulin receptor after the treatment with palmitate. These results suggest that PKC has a role as negative regulator (by phosphorylation in serine) of the insulin receptors activation in the insulin resistance induced by palmitic acid.
Molecular and Cellular Biochemistry 05/2003; 246(1-2):155-62. · 2.06 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Xylanases, an important group of enzymes for biomass degradation in the industry, are commonly found forming complex multienzyme systems. As a preliminary step to the construction of efficient xylanase producers using genetic engineering, we have characterized a gene encoding an endo-beta-1,4 xylanase (xyncflA) from Cellulomonas flavigena. The xylanase activity and the xyncflA synthesis were higher when C. flavigena was grown on sugarcane bagasse. In this substrate, both activity and transcript increased with approximately the same rate during the culture period. When C. flavigena grew on glucose, low signal of mRNA was observed, suggesting that the xyncflA gene is regulated at the transcriptional level.
FEMS Microbiology Letters 10/2002; 214(2):205-9. · 2.04 Impact Factor
