Ethanol production from paper material using a simultaneous saccharification and fermentation system in a fed-batch basis

In this work, a recycled paper-derived feedstock was used to produce ethanol by the simultaneous saccharification and fermentation (SSF) process using the thermotolerant yeast Kluyveromyces marxianus CECT 10875. At standard SSF conditions, the highest yield (about 80% of theoretical) was obtained at low substrate concentration and high enzyme loading. With increasing substrate concentration, mixing difficulties appeared which prevented an adequate SSF process performance and limited ethanol production. An SSF fed-batch procedure was then used which permitted an increase in substrate concentrations while maintaining SSF yields similar to that obtained at standard SSF, thus allowing an increased final ethanol production (about 18 g/l).     

Lactic acid production from submerged fermentation of broken rice using undefined mixed culture

The present work aimed to characterize and optimize the submerged fermentation of broken rice for lactic acid (LA) production using undefined mixed culture from dewatered activated sludge. A microorganism with amylolytic activity, which also produces LA, Lactobacillus amylovorus, was used as a control to assess the extent of mixed culture on LA yield. Three level full factorial designs were performed to optimize and define the influence of fermentation temperature (20–50?°C), gelatinization time (30–60 min) and broken rice concentration in culture medium (40–80 g L^?1) on LA production in pure and undefined mixed culture. LA production in mixed culture (9.76 g L^?1) increased in sixfold respect to pure culture in optimal assessed experimental conditions. The optimal conditions for maximizing LA yield in mixed culture bioprocess were 31?°C temperature, 45 min gelatinization time and 79 g L^?1 broken rice concentration in culture medium. This study demonstrated the positive effect of undefined mixed culture from dewatered activated sludge to produce LA from culture medium formulated with broken rice. In addition, this work establishes the basis for an efficient and low-cost bioprocess to manufacture LA from this booming agro-industrial by-product.     

Insularinase A, a prothrombin activator from Bothrops insularis venom, is a metalloprotease derived from a gene encoding protease and disintegrin domains

The first low-molecular-mass metalloprotease presenting prothrombin activating activity was purified from Bothrops insularis venom and named insularinase A. It is a single-chain protease with a molecular mass of 22 639 Da. cDNA sequence analysis revealed that the disintegrin domain of the precursor protein is post-translationally processed, producing the mature insularinase A. Analysis of its deduced amino acid sequence showed a high similarity with several fibrin(ogen)olytic metalloproteases and only a moderate similarity with prothrombin activators. However, SDS-PAGE of prothrombin after activation by insularinase A showed fragment patterns similar to those generated by group A prothrombin activators, which convert prothrombin into meizothrombin independently of the prothrombinase complex. In addition, insularinase A activates factor X and hydrolyses fibrinogen and fibrin. Chelating agents fully inhibit all insularinase A activities. Insularinase A induced neither detachment nor apoptosis of human endothelial cells and was also not able to trigger an endothelial proinflammatory cell response. Nitric oxide and prostacyclin levels released by endothelial cells were significantly increased after treatment with insularinase A. Our results show that, although its primary structure is related to class P-I fibrin(ogen)olytic metalloproteases, insularinase A is functionally similar to group A prothrombin activators.     

Kunitz-type Bauhinia bauhinioides inhibitors devoid of disulfide bridges: isolation of the cDNAs, heterologous expression and structural studies

Bauhinia bauhinoides cruzipain inhibitor (BbCI) and Bauhinia bauhinioides kallikrein inhibitor (BbKI) are cysteine and serine proteinase inhibitors structurally homologous to plant Kunitz-type inhibitors, but are devoid of disulfide bridges. Based on cDNA sequences, we found that BbKI and BbCI are initially synthesized as a prepropeptide comprising an N-terminal signal peptide (19 residues), the mature protein (164 residues) and a C-terminal targeting peptide (10 residues). Partial cDNAs encoding the mature enzymes plus N-terminal His-tags and thrombin cleavage sites were expressed in E. coli and the soluble proteins were purified by one-step nickel affinity chromatography. After thrombin cleavage, both proteins exhibited potent inhibitory activities toward their cognate proteinases like the wild-type proteins. BbCI inhibits human neutrophil elastase ( K i(app) 5.3 nM), porcine pancreatic elastase ( K i(app) 40 nM), cathepsin G ( K i(app) 160 nM) and the cysteine proteinases cruzipain ( K i(app) 1.2 nM), cruzain ( K i(app) 0.3 nM) and cathepsin L ( K i(app) 2.2 nM), while BbKI strongly inhibits plasma kallikrein ( K i(app) 2.4 nM) and plasmin ( K i(app) 33 nM). Circular dichroism spectra of BbCI and BbKI were in agreement with the beta-trefoil fold described for Kunitz inhibitors. The inhibitory potency of both BbCI- and BbKI-type inhibitors suggests that other, non-covalent interactions may compensate for the lack of disulfide bridges.     

Reversible silencing of CFTR chloride channels by glutathionylation

The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation- and ATP-dependent chloride channel that modulates salt and water transport across lung and gut epithelia. The relationship between CFTR and oxidized forms of glutathione is of potential interest because reactive glutathione species are produced in inflamed epithelia where they may be modulators or substrates of CFTR. Here we show that CFTR channel activity in excised membrane patches is markedly inhibited by several oxidized forms of glutathione (i.e., GSSG, GSNO, and glutathione treated with diamide, a strong thiol oxidizer). Three lines of evidence indicate that the likely mechanism for this inhibitory effect is glutathionylation of a CFTR cysteine (i.e., formation of a mixed disulfide with glutathione): (a) channels could be protected from inhibition by pretreating the patch with NEM (a thiol alkylating agent) or by lowering the bath pH; (b) inhibited channels could be rescued by reducing agents (e.g., DTT) or by purified glutaredoxins (Grxs; thiol disulfide oxidoreductases) including a mutant Grx that specifically reduces mixed disulfides between glutathione and cysteines within proteins; and (c) reversible glutathionylation of CFTR polypeptides in microsomes could be detected biochemically under the same conditions. At the single channel level, the primary effect of reactive glutathione species was to markedly inhibit the opening rates of individual CFTR channels. CFTR channel inhibition was not obviously dependent on phosphorylation state but was markedly slowed when channels were first "locked open" by a poorly hydrolyzable ATP analogue (AMP-PNP). Consistent with the latter finding, we show that the major site of inhibition is cys-1344, a poorly conserved cysteine that lies proximal to the signature sequence in the second nucleotide binding domain (NBD2) of human CFTR. This region is predicted to participate in ATP-dependent channel opening and to be occluded in the nucleotide-bound state of the channel based on structural comparisons to related ATP binding cassette transporters. Our results demonstrate that human CFTR channels are reversibly inhibited by reactive glutathione species, and support an important role of the region proximal to the NBD2 signature sequence in ATP-dependent channel opening.     

Synthesis, characterisation and cytotoxicity of chloro derivatives of prenylnaphthohydroquinone

From the Diels-Alder adduct between alpha-myrcene and 2-chloro-1,4-benzoquinone, a family of chloro derivatives of prenylnaphthohydroquinone have been synthesised and evaluated for their cytotoxicity against 14 neoplastic cell lines.     

Effects of fetal ovine adrenalectomy on sympathetic and baroreflex responses at birth

Studies were performed to test the hypothesis that the absence of adrenal glucocorticoids late in gestation alters sympathetic and baroreflex responses before and immediately after birth. Fetal sheep at 130-131 days gestation (term 145 days) were subjected to bilateral adrenalectomy before the normal prepartum increase in plasma cortisol levels. One group of fetuses (n = 5) received physiological cortisol replacement with a continuous infusion of hydrocortisone (2 mg x day(-1) x kg(-1) for 10 days), whereas the other group received 0.9% NaCl vehicle (n = 5). All animals underwent a second surgery 48 h before the study for placement of a renal nerve recording electrode. Heart rate (HR), mean arterial blood pressure (MABP), renal sympathetic nerve activity (RSNA), and baroreflex control of HR and RSNA were studied before and after cesarean section delivery. At the time of study (140-141 days gestation), fetal plasma cortisol concentration was undetectable in adrenalectomized (ADX) fetuses and 58 +/- 9 ng/ml in animals receiving cortisol replacement (ADX + F). Fetal and newborn MABP was significantly greater in ADX + F relative to ADX animals. One hour after delivery, MABP increased 13 +/- 3 mmHg and RSNA increased 91 +/- 12% above fetal values in ADX + F (both P < 0.05) but remained unchanged in ADX lambs. The midpoint pressures of the fetal HR and RSNA baroreflex function curves were significantly greater in ADX + F (54 +/- 3 and 56 +/- 3 mmHg for HR and RSNA curves, respectively) than ADX fetuses (45 +/- 2 and 46 +/- 3 mmHg). After delivery, the baroreflex curves reset toward higher pressure in ADX + F but not ADX lambs. These results suggest that adrenal glucocorticoids contribute to cardiovascular regulation in the late-gestation fetus and newborn by modulating arterial baroreflex function and sympathetic activity.     

Mating types in yeast,vomeronasal organ in rodents,homosexuality in humans: does a guiding thread exist?

Pheromones and their receptors are the molecules used by very different organisms in order to join two haploid cells. It happens evidently in yeast, since the two blending haploid cells are also the two mating organisms, whereas in rodents pheromone receptors are the triggers of the vomeronasal system which, supervising sexual behaviors, is responsible for copulation and therefore for fertilization. The debate is still open about the real significance of pheromones in humans but a working vomeronasal organ, able to recognize pheromones of the same sex, could be the simplest biological explanation of homosexuality. This hypothesis is discussed and connected with some well known experimental data.