Influence of specific fermentation conditions on natural microflora of pomace in “Grappa” production

As reported in the European Community regulation, grappa is a spirit beverage made in Italy from marc that has been steam distilled or distilled after the addition of water. Grape marc from red grapes has already undergone alcoholic fermentation with the must and can be distilled immediately. Grape marc from white grapes does not contain ethanol but contains sugars that are fermented by spontaneous anaerobic fermentation during a storage period. The characteristic aroma of grappa consists of a large number of volatile compounds, which arise from various sources, the most important of which is yeast. Very few studies have been undertaken to characterize the natural populations of yeast during the fermentation of grape marc. The goal of this study was to understand how different pHs, temperatures and yeast starter cultures affect the growth and dynamics of yeast species involved in pomace fermentation, which could be the basis for improving the final quality of grappa production. We found that a temperature of 15°C has the greatest effect on improving the quality of the product. Unfortunately, due to the solid state of the grape marc and the impossibility of its mixing, it appears that acidification and the addition of yeast starter cultures during the silage period are not effective.     

Ectopic serotonin production in β-cell specific transgenic mice

Genetically modified mice have been widely used in the field of β-cell research. However, analysis of results gathered using genetically modified organisms should be interpreted carefully as the results may be confounded by several factors. Here, we showed the ectopic serotonin (5-HT) production in β-cells of RIP-Cre^Mgn, MIP-GFP, and MIP-Cre/ERT mice. These mice contained a human growth hormone (hGH) cassette to enhance transgene expression and hGH expression and Stat5 phosphorylation were detected in pancreatic islets of these mice. The expression level of tryptophan hydroxylase 1 (Tph1) was upregulated in pancreatic islets of transgenic mice with an hGH cassette but not in transgenic mice without an hGH cassette. Ectopic 5-HT production was not observed in β-cell-specific prolactin receptor (Prlr) knockout mice or Stat5 knockout mice crossed with RIP-Cre^Mgn. We further confirmed that 5-HT production in β-cells of several transgenic mice was induced by hGH expression followed by the activation of the Prlr-Stat5-Tph1 pathway. These findings indicate that results obtained using transgenic mice containing the hGH cassette should be interpreted with care.     

An application of β-glycosidase to transformation of ginsenosides for the effective production of specific ginsenosides with biological efficacy

Over the past several decades, the pharmacological effects of ginsenosides in Panax ginseng roots have been extensively investigated. Here, we developed a method for producing specific ginsenosides (F1 and F2) with good yields (F1:162 mg/g, F2:305 mg/g) using ??-glycosidase purified from Aspergillus niger. In addition, each ginsenoside (at least 25 species) was separated and purified by high performance liquid chromatography (HPLC) using five different types of solvents and different purification steps. In addition, the Rg3:Rh2 mixture (1:1, w/w) was shown to inhibit a specific lung cancer cell line (NCI-H232) in vivo, displaying an anticancer effect at a dose lower than achieved using treatments with single Rg3 or Rh2. This finding suggests that the combination of ginsenosides for targeting anticancer is more effective than the use of a single ginsenoside from ginseng or red ginseng.     

SUMO1 promotes Aβ production via the modulation of autophagy

Autophagy is one of the main mechanisms in the pathophysiology of neurodegenerative disease. The accumulation of autophagic vacuoles (AVs) in affected neurons is responsible for amyloid-β (Aβ) production. Previously, we reported that SUMO1 (small ubiquitin-like modifier 1) increases Aβ levels. In this study, we explored the mechanisms underlying this. We investigated whether AV formation is necessary for Aβ production by SUMO1. Overexpression of SUMO1 increased autophagic activation, inducing the formation of LC3-II-positive AVs in neuroglioma H4 cells. Consistently, autophagic activation was decreased by the depletion of SUMO1 with small hairpin RNA (shRNA) in H4 cells. The SUMO1-mediated increase in Aβ was reduced by the autophagy inhibitors (3-methyladenine or wortmannin) or genetic inhibitors (siRNA targeting ATG5, ATG7, ATG12, or HIF1A), respectively. Accumulation of SUMO1, ATG12, and LC3 was seen in amyloid precursor protein transgenic mice. Our results suggest that SUMO1 accelerates the accumulation of AVs and promotes Aβ production, which is a key mechanism for understanding the AV-mediated pathophysiology of Alzheimer disease.     

A novel β-loop scaffold of phage-displayed peptides for highly specific affinities

Loop peptides stabilized by two β-strands were used as a scaffold for a phage displayed peptide library. Affinity-based screening for insulin provided peptides, which showed affinity constants of 10(5) M(-1) order for insulin over 100 times greater than their affinity for the structurally similar insulin-like growth factor 1. The results suggested that the scaffold offers a powerful tool for generating and screening peptides as ligands for drugs and biologics.     

SUMO1 promotes Aβ production via the modulation of autophagy

Autophagy is one of the main mechanisms in the pathophysiology of neurodegenerative disease. The accumulation of autophagic vacuoles (AVs) in affected neurons is responsible for amyloid-β (Aβ) production. Previously, we reported that SUMO1 (small ubiquitin-like modifier 1) increases Aβ levels. In this study, we explored the mechanisms underlying this. We investigated whether AV formation is necessary for Aβ production by SUMO1. Overexpression of SUMO1 increased autophagic activation, inducing the formation of LC3-II-positive AVs in neuroglioma H4 cells. Consistently, autophagic activation was decreased by the depletion of SUMO1 with small hairpin RNA (shRNA) in H4 cells. The SUMO1-mediated increase in Aβ was reduced by the autophagy inhibitors (3-methyladenine or wortmannin) or genetic inhibitors (siRNA targeting ATG5, ATG7, ATG12, or HIF1A), respectively. Accumulation of SUMO1, ATG12, and LC3 was seen in amyloid precursor protein transgenic mice. Our results suggest that SUMO1 accelerates the accumulation of AVs and promotes Aβ production, which is a key mechanism for understanding the AV-mediated pathophysiology of Alzheimer disease.     

A high-throughput screen for the engineered production of β-lactam antibiotics

High-throughput screens and selections have had profound impact on our ability to engineer proteins possessing new, desired properties. These methods are especially useful when applied to the modification of existing enzymes to create natural and unnatural products. In an advance upon existing methods we developed a high-throughput, genetically regulated screen for the in vivo production of β-lactam antibiotics using a green fluorescent protein (gfp) reporter. This assay proved reliable and sensitive and presents a dynamic range under which a wide array of β-lactam architectural subclasses can be detected. Moreover, the graded response elicited in this assay can be used to rank mutant activity. The utility of this development was demonstrated in vivo and then applied to the first experimental investigation of a putative catalytic residue in carbapenem synthase (CarC). Information gained about the mutability of this residue defines one parameter for enzymatic activity and sets boundaries for future mechanistic and engineering efforts.     

Overexpression of SNX7 reduces Aβ production by enhancing lysosomal degradation of APP

Abnormal production of amyloid-β peptides (Aβ) by proteolytic processing of amyloid precursor protein (APP) is thought to be central to the pathogenesis of Alzheimer's disease (AD). Although many efforts have been made to investigate mechanisms that regulate APP processing, many details remain incompletely understood. Sorting nexins (SNXs) are a family of proteins which are involved in many intracellular trafficking events. Several SNXs have been implicated in APP processing and Aβ production. In this study, we extended the investigation to SNX7. We found that overexpression of SNX7 in HEK293T cells reduces the levels of secreted Aβ and β-cleaved N-terminal APP fragments (sAPPβ). Moreover, SNX7 overexpression caused a significant reduction of the steady-state levels of APP as well as of the cell surface APP levels. By using NH₄Cl and Bafilomycin A1 to inhibit the lysosomal degradative pathway, we found that the reduction of APP induced by SNX7 overexpression was prevented by such inhibition. No change in the cell surface distribution or steady-state levels of BACE1 was detected after overexpression of SNX7. Taken together, these results suggest that SNX7 regulates Aβ production by directing APP for degradation.     

Tumour necrosis factor-α production in fibrosing alveolitis is macrophage subset specific

Background  

Previous studies have revealed that tumour necrosis factor (TNF)-α is upregulated in fibrosing alveolitis (FA) in humans. The aim of this study was to compare the TNF-α secretory profile of alveolar macrophages (AMs) and peripheral blood monocytes (Mos) of patients with cryptogenic FA and systemic sclerosis (SSc), a rheumatological disorder in which lung fibrosis can occur. In particular, we wished to assess whether TNF-α levels differ between SSc patients with FA (FASSc) and a nonfibrotic group.     

Are all species of Pseudorhabdosynochus strictly host specific? — A molecular study

Species of the diplectanid monogenean genus Pseudorhabdosynochus are strictly host-specific (specialist), with the exception of P. cyanopodus, which was reported in New Caledonia, South Pacific, from two host species, Epinephelus cyanopodus and E. chlorostigma. We sequenced the COI gene of both host fish species and of their monogeneans. Morphological identification and pairwise distances showed that the two fish species were distinct (difference 6.1-6.6%), but that their monogeneans were not (difference 0-1.5%). A morphological study of sclerotised parts showed that specimens of P. cyanopodus are similar in both fish. Most species of groupers and their associated Pseudorhabdosynochus species are from warm surface waters, but the two groupers E. cyanopodus and E. chlorostigma are usually caught in deep-sea on the outer slope of the coral reef. This suggests that acquisition of a less strict host specificity is an adaptation of P. cyanopodus to deep-sea hosts.     

Biodeterioration problems of North Sea oil and gas production—A review

The North Sea is both a highly corrosive and highly biologically productive environment. Oil and gas production structures in this environment not only suffer from the physical effects, corrosion, wave and storm action, of being immersed in this environment, but also the effects of biological activity. Such biodeterioration problems occur both on the external and internal surfaces of the structure and the internal surfaces of any plant or pipework which contains, or has contained, water. The platform jacket may experience biological problems due to external macro- and microfouling or to internal microfouling. Pipelines, risers and topside systems have usually all been hydrostatically tested leaving residual water and possible sites for bacterial activity. Waterflooding with seawater and the separation of produced water from oil also provide sites suitable for biological activity and associated corrosion, slime and blockade problems. This paper reviews these areas in the light of recent advances in investigation and uderstanding of the problems.     

A whole-cell process for the production of ε-caprolactone in aqueous media

ε-Caprolactone is an industrially important intermediate produced in multi-10,000 ton scale annually with broad applications. We report on a whole-cell biocatalytic conversion of cyclohexanol to ε-caprolactone using the combination of alcohol dehydrogenase (ADH) with two stability-improved variants (QM and M15) of the Baeyer-Villiger monooxygenase CHMO with a special focus on process development at the 200 mM scale. Influence of parameters such as volumetric mass transfer co-efficient, stirrer speed and catalytic loading (amount of E. coli whole-cells expressing ADH and CHMO) on the process efficiency were studied and optimised. This resulted in over 98% conversion, a product titer of 20 g L^–1 and an isolated product amount of 9.1 g (80%). This corresponds to a space-time yield of 1.1 g L^–1 h⁻¹ and a reaction yield (mole of product per mole substrate) of 0.9. Comparing the two CHMO variants a significant difference in catalytic yield (weight of product to weight of catalyst; 0.6 vs 0.3) was observed without any inherent changes in the process. Hence, the reported process can accommodate in the future improved variants of the CHMO.     

Continuous production of α-peptide using immobilized recombinant yeast cells: A model for continuous production of foreign peptide by recombinant yeast

α-Peptide, a portion of Escherichia coli β-galactosidase, was cloned downstream of the yeast α-factor promoter and the signal peptide by one of the authors. In this study, we utilized recombinant yeast cells, transformed the α-peptide secretion vector and attempted continuous production of α-peptide as a model of foreign peptide production. The continuous production of α-peptide was performed by using immobilized recombinant yeast cells on a column reactor, after characterizing the secretion, using minimal and complex medium. Utilizing minimal medium, with a productivity of 100 000 U h⁻¹ l⁻¹, α-peptide was continuously produced for more than 200 h. We then attempted to improve the productivity of α-peptide by alternating minimal and complex medium. Utilizing this medium changing method, 1.4 times higher α-peptide was produced during 150 h of operation compared with that achieved only by feeding minimal medium.     

A specific effect of deoxycytidine 5′-triphosphate on DNA chain initiation in vitro

Increased levels of dCTP increase the frequency of initiation of discontinuous DNA intermediates in a cellophane disc in vitro system. Increased levels of other ribo- and deoxyribonucleoside triphosphates show no effect.     

Increased Aβ production prompts the onset of glucose intolerance and insulin resistance

Type 2 diabetes (T2D) mellitus and Alzheimer's disease (AD) are two prevalent diseases with comparable pathophysiological features and genetic predisposition. Patients with AD are more susceptible to develop T2D. However, the molecular mechanism linking AD and T2D remains elusive. In this study, we have generated a new mouse model to test the hypothesis that AD would prompt the onset of T2D in mice. To test our hypothesis, we crossed Alzheimer APPswe/PS1dE9 (APP/PS1) transgenic mice with mice partially deficient in leptin signaling (db/+). Body weight, plasma glucose, and insulin levels were monitored. Phenotypic characterization of glucose metabolism was performed using glucose and insulin tolerance tests. β-Cell mass, islet volume, and islet number were analyzed by histomorphometry. APP/PS1 coexpression in mice with intact leptin receptor signaling did not show any metabolic perturbations in glucose metabolism or insulin sensitivity. In contrast, APP/PS1 coexpression in db/+ mice resulted in nonfasting hyperglycemia, hyperinsulinemia, and hypercholesterolemia without changes in body weight. Conversely, fasting blood glucose and cholesterol levels remained unchanged. Coinciding with altered glucose metabolism, APP/PS1 coexpression in db/+ mice resulted in glucose intolerance, insulin resistance, and impaired insulin signaling. In addition, histomorphometric analysis of pancreata revealed augmented β-cell mass. Taken together, these findings provide experimental evidence to support the notion that aberrant Aβ production might be a mechanistic link underlying the pathology of insulin resistance and T2D in AD.     

Are mechanisms of exocytosis neurotransmitter specific?

In their review, Langley and Grant (1997) investigate the question whether mechanisms of exocytosis are neurotransmitter specific. There is now much evidence that the mechanisms governing the exocytosis of the two principal storage organelles—granules (large dense core vesicles) and electron-lucent vesicles—differ. But much less is known concerning potential differences in the release mechanisms of electron-lucent vesicles that store different types of fast neurotransmitters or of granules in different types of neurons. It is an open question whether there is a unifying control mechanism for the exocytosis of, for example, a peptide-containing granule of a glutamatergic neuron, a chromaffin granule, a noradrenergic granule or a granule from a neurosecretory neuron in the pituitary. The small electron-lucent synaptic vesicles of various kind apparently share common molecular components of regulated release. They carry the calcium sensor synaptotagmin, small GTP-binding proteins of the rab3 group or the v-SNARE synaptobrevin. Nevertheless, there may be differences in the regulatory mechanisms. This concerns the type of calcium channel involved or the absence of some of the presynaptic molecules such as rab3a, synapsin I or the t-SNAREs SNAP-25 or syntaxin from distinct types of neurons or sensory cells.     

Coumarin derivatives protection against ROS production in cellular models of Aβ toxicities

The role of oxidative stress and free radicals in the development of Alzheimer's disease (AD) has been the focus of many recent studies. The role of hydrogen peroxide (H₂O₂) in AD is thought to be associated with Aβ (amyloid – β) damage in cells. A number of coumarin derivatives were previously found to be potent anti-inflammatory and antioxidant agents. Herein, these coumarin derivatives were tested as H₂O₂ scavengers with the DCF assay using two types of neuronal cells: (a) wild type (N2a) neuroblastoma cells and (b) APP/PS1 transgenic cell line expressing Aβ. Their scavenging activity was varied between the types of cell cultures and it was found to be concentration and time dependent in the mutant cells. Their protective role against cell death further supports this notion. These results suggest that these compounds could be used as a template in the design of new molecules with a possible role in AD.     

O2 sensitivity and H2 production activity of hydrogenases—A review

Hydrogenases are metalloproteins capable of catalyzing the interconversion between molecular hydrogen and protons and electrons. The iron–sulfur clusters within the enzyme enable rapid relay of electrons which are either consumed or generated at the active site. Their unparalleled catalytic efficiency has attracted attention, especially for potential use in H₂ production and/or fuel cell technologies. However, there are limitations to using hydrogenases, especially due to their high O₂ sensitivity. The subclass, called [FeFe] hydrogenases, are particularly more vulnerable to O₂ but proficient in H₂ production. In this review, we provide an overview of mechanistic and protein engineering studies focused on understanding and enhancing O₂ tolerance of the enzyme. The emphasis is on ongoing studies that attempt to overcome O₂ sensitivity of the enzyme while it catalyzes H₂ production in an aerobic environment. We also discuss pioneering attempts to utilize the enzyme in biological H₂ production and other industrial processes, as well as our own perspective on future applications.