Rapid ethanol fermentation of cellulose hydrolysate. I. Batch versus continuous systems

Rapid fermentation of bagasse hydrolysate to ethanol under anaerobic conditions by a strain of Saccharomyces cerevisiae has been studied in batch and continuous cultures at pH 4.0 and 30°C temperature with cell recycle. By using a 23.6 g/liter cell concentration, a concentation of 9.7% (w/v)ethanol was developed in a period of 6 hr. The rate of fermentation was found to increase with supplementation of yeast vitamins in the hydrolysate. In continuous culture employing cell recycle and a 0.127 v/v/m air flow rate, a cell mass concentration of 48.5 g/liter has been achieved. The maximum fermentor productivity of ethanol obtained under these conditions was 32.0 g/liter/hr, which is nearly 7.5 times higher than the normal continuous process without cell recycle and air sparging. The ethanol productivity was found to decrease linearly with ethanol concentration. Conversion of glucose in the hydrolysate to ethanol was achieved with a yield of 95 to 97% of theoretical.     

Cytotoxic activity of nucleoside diphosphate kinase secreted from Mycobacterium tuberculosis.

Pathogenicity of Mycobacterium tuberculosis is closely related to its ability to survive and replicate in the hostile environment of macrophages. For some pathogenic bacteria, secretion of ATP-utilizing enzymes into the extracellular environment aids in pathogen survival via P2Z receptor-mediated, ATP-induced death of infected macrophages. A component of these enzymes is nucleoside diphosphate kinase (Ndk). The ndk gene was cloned from M. tuberculosis H37Rv and expressed in Escherichia coli. Ndk was secreted into the culture medium by M. tuberculosis, as determined by enzymatic activity and Western blotting. Purified Ndk enhanced ATP-induced macrophage cell death, as assayed by the release of [14C]adenine. A catalytic mutant of Ndk failed to enhance ATP-induced macrophage cell death, and periodate-oxidized ATP (oATP), an irreversible inhibitor of P2Z receptor, blocked ATP/Ndk-induced cell death. Purified Ndk was also found to be autophosphorylated with broad specificity for all nucleotides. Conversion of His117-->Gln, which is part of the nucleotide-binding site, abolished autophosphorylation. Purified Ndk also showed GTPase activity. Collectively, these results indicate that secreted Ndk of M. tuberculosis acts as a cytotoxic factor for macrophages, which may help in dissemination of the bacilli and evasion of the immune system.     

Diacylglycerol generation and phosphoinositide turnover in human neutrophils: effects of particulate versus soluble stimuli

Serum-treated, or "opsonized" zymosan (OZ), a particulate material which can be phagocytized by polymorphonuclear leukocytes, activates the superoxide-generating respiratory burst in these cells. The use of dual wavelength spectroscopy in the present studies has allowed accurate continuous monitoring of superoxide generation (cytochrome c reduction) upon cellular activation by this turbid material; activation occurs after a short lag period (about 20 s) which is similar to the lag seen after activation with the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP). Unlike the fMLP response which terminates after about 90 s, superoxide generation in response to OZ continues beyond 10 min, and is similar in this regard to the response seen with the protein kinase C activator phorbol myristate acetate (PMA). OZ and fMLP, but not PMA, also activate receptor-linked phospholipase C mechanisms as judged by the appearance of inositol trisphosphate (IP3) (as well as other inositol phosphates) and diacylglycerol (DAG), with the latter measured by a mass assay. The appearance of these potential mediators corresponded to the loss of phosphoinositides, in particular phosphatidylinositol 4,5-bisphosphate (PIP2). The magnitude of DAG and inositol sugar generation as well as the breakdown of PIP2 was considerably greater using OZ than with fMLP. In addition, while fMLP resulted in a transient increase in IP3 and DAG, OZ resulted in a sustained elevation of these molecules. With both agonists, the onset and duration of generation of putative mediators corresponded to the period of generation of O2-, consistent with a role for DAG and/or IP3 in the activation of the respiratory burst.     

Thrombospondin-1/HIV-1 tat protein interaction: modulation of the biological activity of extracellular Tat.

Tat protein, a trans-activating factor of the human immunodeficiency virus type 1, acts also as an extracellular molecule modulating gene expression, cell survival, growth, transformation, and angiogenesis. Here we demonstrate that human thrombospondin-1 (TSP), a plasma glycoprotein and constituent of the extracellular matrix, binds to glutathione-S-transferase (GST)-Tat protein but not to GST. Scatchard plot analysis of the binding of free GST-Tat to immobilized TSP reveals a high-affinity interaction (Kd equal to 25 nM). Accordingly, TSP inhibits cell internalization and HIV-1 LTR trans-activating activity of extracellular Tat in HL3T1 cells with ID50 equal to 10-30 nM. Also, TSP inhibits cell interaction and mitogenic activity of extracellular Tat in T53 Tat-less cells. TSP is instead ineffective when administered after the interaction of Tat with cell surface heparan-sulfate proteoglycans has occurred, in keeping with its ability to prevent but not disrupt Tat/heparin interaction in vitro. Finally, TSP inhibits the autocrine loop of stimulation exerted by endogenous Tat in parental T53 cells. Accordingly, TSP overexpression inhibits cell proliferation, angiogenic activity, and tumorigenic capacity of stable T53 transfectants. Our data demonstrate the ability of TSP to bind to Tat protein and to affect its LTR trans-activating, mitogenic, angiogenic, and tumorigenic activity. These findings suggest that TSP may be implicated in the progression of AIDS and in AIDS-associated pathologies by modulating the bioavailability and biological activity of extracellular Tat.     

Increased endogenous H2S generation by CBS, CSE, and 3MST gene therapy improves ex vivo renovascular relaxation in hyperhomocysteinemia

Hydrogen sulfide (H(2)S) has recently been identified as a regulator of various physiological events, including vasodilation, angiogenesis, antiapoptotic, and cellular signaling. Endogenously, H(2)S is produced as a metabolite of homocysteine (Hcy) by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). Although Hcy is recognized as vascular risk factor at an elevated level [hyperhomocysteinemia (HHcy)] and contributes to vascular injury leading to renovascular dysfunction, the exact mechanism is unclear. The goal of the current study was to investigate whether conversion of Hcy to H(2)S improves renovascular function. Ex vivo renal artery culture with CBS, CSE, and 3MST triple gene therapy generated more H(2)S in the presence of Hcy, and these arteries were more responsive to endothelial-dependent vasodilation compared with nontransfected arteries treated with high Hcy. Cross section of triple gene-delivered renal arteries immunostaining suggested increased expression of CD31 and VEGF and diminished expression of the antiangiogenic factor endostatin. In vitro endothelial cell culture demonstrated increased mitophagy during high levels of Hcy and was mitigated by triple gene delivery. Also, dephosphorylated Akt and phosphorylated FoxO3 in HHcy were reversed by H(2)S or triple gene delivery. Upregulated matrix metalloproteinases-13 and downregulated tissue inhibitor of metalloproteinase-1 in HHcy were normalized by overexpression of triple genes. Together, these results suggest that H(2)S plays a key role in renovasculopathy during HHcy and is mediated through Akt/FoxO3 pathways. We conclude that conversion of Hcy to H(2)S by CBS, CSE, or 3MST triple gene therapy improves renovascular function in HHcy.     

Molecular cloning and expression profile of snow trout GPDH gene in response to abiotic stress

Glycerol-3-phosphate dehydrogenase (GPDH) gene possibly plays a key role for cold acclimation process in snow trout during winter months when water temperature goes down to 4–5?°C. In this study, 1,012?bp nucleotide fragment of GPDH gene was obtained from two snow trout species (Schizothorax richardsonii and S. niger; family: Cyprinidae), distributed in several Himalayan rivers. The gene encoded a protein of 334 amino acids. The encoded protein sequence was very similar to GPDH of Danio rerio (94.36?%) using BLASTx searches. In S. richardsonii the qRT-PCR showed highest expression in muscle tissue followed by liver and also revealed 19 fold gene expression in liver tissue under cold (5?°C) in comparison with warm (15?°C) condition. The elevated expression levels of GPDH cDNA on cold treatment furthermore suggest that GPDH plays a role in stress related responses in S. richardsonii. The phylogenetic analysis showed that the two snow trout species GPDH share the same clade with characterized GPDHs from other teleost fishes suggesting a common evolutionary origin and a similar catalytic function. In addition, the Ka/Ks ratios of these sequences suggested that they are under purifying selection. Moreover, the expression profile of GPDH gene among co generic species of genus Schizothorax showed that GPDH cDNA expression was highest in S. richardsonii and lowest in S. esocinus which gives an indication of species specific adaptation in relation to different geographical areas.     

Effect of water stress on proline content and transcript levels in Lathyrus sativus

Response of Lathyrus sativus plants to water stress showed that ABA responsive genes such as PLE 25, TAS 14 and RAB 17 are synthesized constitutively, the levels of which decline gradually with increase in water stress or ABA levels. Proline accumulation was highest in leaves (65-fold) followed by stem (56-fold), root (38-fold) and marginal increase in etiolated seedlings. Proline increase was also observed in plant parts not exposed to light.     

Agro-industrial waste materials and wastewater sludge for rhizobial inoculant production: a review

Inoculating legumes with commercial rhizobial inoculants is a common agriculture practice. Generally, inoculants are sold in liquid or in solid forms (mixed with carrier). The production of inoculants involves a step in which a high number of cells are produced, followed by the product formulation. This process is largely governed by the cost related to the medium used for rhizobial growth and by the availability of a carrier source (peat) for production of solid inoculant. Some industrial and agricultural by-products (e.g. cheese whey, malt sprouts) contain growth factors such as nitrogen and carbon, which can support growth of rhizobia. Other agro-industrial wastes (e.g. plant compost, filtermud, fly-ash) can be used as a carrier for rhizobial inoculant. More recently, wastewater sludge, a worldwide recyclable waste, has shown good potential for inoculant production as a growth medium and as a carrier (dehydrated sludge). Sludge usually contains nutrient elements at concentrations sufficient to sustain rhizobial growth and heavy metals are usually below the recommended level. In some cases, growth conditions can be optimized by a sludge pre-treatment or by the addition of nutrients. Inoculants produced in wastewater sludge are efficient for nodulation and nitrogen fixation with legumes as compared to standard inoculants. This new approach described in this review offers a safe environmental alternative for both waste treatment/disposal and inoculant production.     

Cystathionine-beta-synthase gene transfer and 3-deazaadenosine ameliorate inflammatory response in endothelial cells

Although elevated levels of homocysteine (Hcy) known as hyperhomocysteinemia (HHcy) are associated with increased inflammation and vascular remodeling, the mechanism of Hcy-mediated inflammation and vascular remodeling is unclear. The matrix metalloproteinases (MMPs) and adhesion molecules play an important role in vascular remodeling. We hypothesized that HHcy induces inflammation by increasing adhesion molecules and matrix protein expression. Endothelial cells were supplemented with high methionine, and Hcy accumulation was measured by HPLC. Nitric oxide (NO) bioavailability was detected by a NO probe. The protein expression was measured by Western blot analysis. MMP-9 activity was detected by gelatin-gel zymography. We demonstrated that methionine supplement promoted upregulation of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) through increased Hcy accumulation. In addition, increased synthesis of collagen type-1 was also observed. MMP-9 gene expression and protein activity were increased in methionine supplement groups. 3-Deazaadenosine (DZA), an adenosine analogue, prevented high methionine-induced ICAM-1 and VCAM-1 expression and collagen type-1 synthesis. Transfection of endothelial cells with cystathionine-beta-synthase (CBS) gene construct, which converts Hcy to cystathionine, reduced Hcy accumulation in high methionine-fed cells. CBS gene transfection reduced the inflammatory response, as evident by attenuated ICAM-1 and VCAM-1 expression. Furthermore, collagen type-1 expression and MMP-9 activity were dramatically attenuated with CBS gene transfection. These results suggested that methionine supplement increased Hcy accumulation, which was associated with inflammatory response and matrix remodeling such as collagen type-1 synthesis and MMP-9 activity. However, in vitro DZA and CBS gene therapy successfully treated the HHcy-induced inflammatory reaction in the methionine metabolism pathway.     

Large-scale polymorphism of heterochromatic repeats in the DNA of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

The composition of the individual eukaryote's genome and its variation within a species remain poorly defined. Even for a sequenced genome such as that of the model plant Arabidopsis thaliana accession Col-0, the large arrays of heterochromatic repeats are incompletely sequenced, with gaps of uncertain size persisting in them.