Differential mesenteric fat deposition in bovines fed on silage or concentrate is independent of glycerol membrane permeability

In the meat industry, the manipulation of fat deposition in cattle is of pivotal importance to improve production efficiency, carcass composition and ultimately meat quality. There is an increasing interest in the identification of key factors and molecular mechanisms responsible for the development of specific fat depots. This study aimed at elucidating the influence of breed and diet on adipose tissue membrane permeability and fluidity and their interplay on fat deposition in bovines. Two Portuguese autochthonous breeds, Alentejana and Barrosã, recognized as late- and early-maturing breeds, respectively, were chosen to examine the effects of breed and diet on fat deposition and on adipose membrane composition and permeability. Twenty-four male bovines from these breeds were fed on silage-based or concentrate-based diets for 11 months. Animals were slaughtered to determine their live slaughter and hot carcass weights, as well as weights of subcutaneous and visceral adipose depots. Mesenteric fat depots were excised and used to isolate adipocyte membrane vesicles where cholesterol content, fatty acid profile as well as permeability and fluidity were determined. Total accumulation of neither subcutaneous nor visceral fat was influenced by breed. In contrast, mesenteric and omental fat depots weights were higher in concentrate-fed bulls relative to silage-fed animals. Membrane fluidity and permeability to water and glycerol in mesenteric adipose tissue were found to be independent of breed and diet. Moreover, the deposition of cholesterol and unsaturated fatty acids, which may influence membrane properties, were unchanged among experimental groups. Adipose membrane lipids from the mesenteric fat depot of ruminants were rich in saturated fatty acids, and unaffected by polyunsaturated fatty acids dietary levels. Our results provide evidence against the involvement of cellular membrane permeability to glycerol on fat accumulation in mesenteric fat tissue of concentrate-fed bovines, which is consistent with the unchanged membrane lipid profile found among experimental groups.     

The antennal benzoic acid receptor cell of the female silk moth <Emphasis Type="Italic">Bombyx mori</Emphasis> L.: structure–activity relationship studies with halogen substitutes

Studies on structure–activity relationships were carried out to characterize the response specificity of the benzoic acid cell of the female of the moth Bombyx mori by means of single sensillum electrophysiological recordings. We demonstrated that this cell type responds best to a natural key substance (benzoic acid) and has similar response profiles for less effective compounds, including various halogen substitutes of benzoic acid, benzaldehyde and other derivates of the key compound. Using different halogen substitutes (F, Cl, Br, I), we showed that the cellular response decreases with increasing atomic size of the substitute and that halogen substitutes were most effective in the meta-position. Thus, m-fluor benzoic acid was even more effective than benzoic acid. These results indicate that a critical feature of the stimulus molecule is the inductive effect generated by the halogen substitutes. Increasing the atomic size of the halogen substitute impairs the recognition of the molecule by the receptor cell, possibly due to steric effects. Decreasing the electron density in the aromatic ring improves the receptor response. The benzoic acid receptor cell can be considered as specialist despite not being involved in pheromone detection as it responds maximally to a key substance and has similar response profiles for less effective compounds.     

Pushing forces drive the comet-like motility of microtubule arrays in Dictyostelium

Overexpression of dynein fragments in Dictyostelium induces the movement of the entire interphase microtubule array. Centrosomes in these cells circulate through the cytoplasm at rates between 0.4 and 2.5 microm/s and are trailed by a comet-tail like arrangement of the microtubule array. Previous work suggested that these cells use a dynein-mediated pulling mechanism to generate this dramatic movement and that similar forces are at work to maintain the interphase MTOC position in wild-type cells. In the present study, we address the nature of the forces used to produce microtubule movement. We have used a laser microbeam to sever the connection between the motile centrosomes and trailing microtubules, demonstrating that the major force for such motility results from a pushing on the microtubules. We eliminate the possibility that microtubule assembly/disassembly reactions are significant contributors to this motility and suggest that the cell cortex figures prominently in locating force-producing molecules. Our findings indicate that interphase microtubules in Dictyostelium are subject to both dynein- and kinesin-like forces and that these act in concert to maintain centrosome position in the cell and to support the radial character of the microtubule network.     

Adaptation to a high protein, carbohydrate-free diet induces a marked reduction of fatty acid synthesis and lipogenic enzymes in rat adipose tissue that is rapidly reverted by a balanced diet

We have previously shown that in vivo lipogenesis is markedly reduced in liver, carcass, and in 4 different depots of adipose tissue of rats adapted to a high protein, carbohydrate-free (HP) diet. In the present work, we investigate the activity of enzymes involved in lipogenesis in the epididymal adipose tissue (EPI) of rats adapted to an HP diet before and 12 h after a balanced diet was introduced. Rats fed an HP diet for 15 days showed a 60% reduction of EPI fatty acid synthesis in vivo that was accompanied by 45%-55% decreases in the activities of pyruvate dehydrogenase complex, ATP-citrate lyase, acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and malic enzyme. Reversion to a balanced diet for 12 h resulted in a normalization of in vivo EPI lipogenesis, and in a restoration of acetyl-CoA carboxylase activity to levels that did not differ significantly from control values. The activities of ATP-citrate lyase and pyruvate dehydrogenase complex increased to about 75%-86% of control values, but the activities of glucose-6-phosphate dehydrogenase and malic enzyme remained unchanged 12 h after diet reversion. The data indicate that in rats, the adjustment of adipose tissue lipogenic activity is an important component of the metabolic adaptation to different nutritional conditions.     

Yeast population dynamics of industrial fuel-ethanol fermentation process assessed by PCR-fingerprinting

Yeast population used in industrial production of fuel-ethanol may vary according to the plant process condition and to the environmental stresses imposed to yeast cells. Therefore, yeast strains isolated from a particular industrial process may be adapted to such conditions and should be used as starter strain instead of less adapted commercial strains. This work reports the use of PCR-fingerprinting method based on microsatellite primer (GTG)₅ to characterize the yeast population dynamics along the fermentation period in six distilleries. The results show that indigenous fermenting strains present in the crude substrate can be more adapted to the industrial process than commercial strains. We also identified new strains that dominate the yeast population and were more present either in molasses or sugar cane fermenting distilleries. Those strains were proposed to be used as starters in those industrial processes. This is the first report on the use of molecular markers to discriminate Saccharomyces cerevisiae strains from fuel-ethanol producing process.     

Insights into the molecular determinants of substrate specificity in glycoside hydrolase family 5 revealed by the crystal structure and kinetics of Cellvibrio mixtus mannosidase 5A

The enzymatic hydrolysis of the glycosidic bond is central to numerous biological processes. Glycoside hydrolases, which catalyze these reactions, are grouped into families based on primary sequence similarities. One of the largest glycoside hydrolase families is glycoside hydrolase family 5 (GH5), which contains primarily endo-acting enzymes that hydrolyze beta-mannans and beta-glucans. Here we report the cloning, characterization, and three-dimensional structure of the Cellvibrio mixtus GH5 beta-mannosidase (CmMan5A). This enzyme releases mannose from the nonreducing end of mannooligosaccharides and polysaccharides, an activity not previously observed in this enzyme family. CmMan5A contains a single glycone (-1) and two aglycone (+1 and +2) sugar-binding subsites. The -1 subsite displays absolute specificity for mannose, whereas the +1 subsite does not accommodate galactosyl side chains but will bind weakly to glucose. The +2 subsite is able to bind to decorated mannose residues. CmMan5A displays similar activity against crystalline and amorphous mannans, a property rarely attributed to glycoside hydrolases. The 1.5 A crystal structure reveals that CmMan5A adopts a (beta/alpha)(8) barrel fold, and superimposition with GH5 endo-mannanases shows that dramatic differences in the length of three loops modify the active center accessibility and thus modulate the specificity from endo to exo. The most striking and significant difference is the extended loop between strand beta8 and helix alpha8 comprising residues 378-412. This insertion forms a "double" steric barrier, formed by two short beta-strands that function to "block" the substrate binding cleft at the edge of the -1 subsite forming the "exo" active center topology of CmMan5A.     

Bismuth citrate in the quantification of inorganic phosphate and its utility in the determination of membrane-bound phosphatases

This paper describes a rapid and sensitive method to determine inorganic phosphate, even in the presence of labile organic phosphate compounds and large quantities of proteins. The method eliminates the use of sodium arsenite, a highly toxic compound, substituting bismuth citrate for it to stabilize the phosphomolybdic acid complex formed during the interaction of inorganic phosphate and molybdate reduced by ascorbic acid. This method has also been adapted to microplates and has been used to determine the activities of Na/K ATPase and alkaline phosphatase of intestinal basolateral and luminal plasma membranes.     

Gas chromatography/mass spectrometry characterization of urinary metabolites of danazol after oral administration in human

Danazol (17alpha-pregna-2,4-dien-20-yno [2,3-d]-isoxazol-17beta-ol), is a synthetic derivative of ethisterone, structurally related to stanozolol. For this reason its use as doping agent has been investigated. Danazol (Runch) (200 mg) were orally administered to two healthy male volunteers. Urine samples were collected up to 1-week post-dose. Four new metabolites have been identified in addition to the five previously reported. We propose the monitorization of 6beta-hydroxy-2-hydroxymethyl-1,2-dehydroethisterone and 6beta,16epsilon-dihydroxy-2epsilon-hydroxymethyl-ethisterone by free fraction analysis. In a same way, we proposed to detect the principal isomer of a mono-hydroxylated metabolite of 6beta-hydroxy-2epsilon-hydroxymethylethisterone in the conjugated fraction. We conclude that new metabolites can be included for the detection of danazol abuse since the main metabolite ethisterone is excreted relatively fast in urine.