Localization of nitric-oxide synthase in plant peroxisomes

The presence of nitric-oxide synthase (NOS) in peroxisomes from leaves of pea plants (Pisum sativum L.) was studied. Plant organelles were purified by differential and sucrose density gradient centrifugation. In purified intact peroxisomes a Ca(2+)-dependent NOS activity of 5.61 nmol of L-[(3)H]citrulline mg(-1) protein min(-1) was measured while no activity was detected in mitochondria. The peroxisomal NOS activity was clearly inhibited (60-90%) by different well characterized inhibitors of mammalian NO synthases. The immunoblot analysis of peroxisomes with a polyclonal antibody against the C terminus region of murine iNOS revealed an immunoreactive protein of 130 kDa. Electron microscopy immunogold-labeling confirmed the subcellular localization of NOS in the matrix of peroxisomes as well as in chloroplasts. The presence of NOS in peroxisomes suggests that these oxidative organelles are a cellular source of nitric oxide (NO) and implies new roles for peroxisomes in the cellular signal transduction mechanisms.     

Emerging technologies for salivaomics in cancer detection

Salivary diagnostics has great potential to be used in the early detection and prevention of many cancerous diseases. If implemented with rigour and efficiency, it can result in improving patient survival times and achieving earlier diagnosis of disease. Recently, extraordinary efforts have been taken to develop non‐invasive technologies that can be applied without complicated and expensive procedures. Saliva is a biofluid that has demonstrated excellent properties and can be used as a diagnostic fluid, since many of the biomarkers suggested for cancers can also be found in whole saliva, apart from blood or other body fluids. The currently accepted gold standard methods for biomarker development include chromatography, mass spectometry, gel electrophoresis, microarrays and polymerase chain reaction‐based quantification. However, salivary diagnostics is a flourishing field with the rapid development of novel technologies associated with point‐of‐care diagnostics, RNA sequencing, electrochemical detection and liquid biopsy. Those technologies will help introduce population‐based screening programs, thus enabling early detection, prognosis assessment and disease monitoring. The purpose of this review is to give a comprehensive update on the emerging diagnostic technologies and tools for the early detection of cancerous diseases based on saliva.     

Folic acid intestinal absorption in newborn rats at 21 day postpartum: effects of maternal ethanol consumption.

This study was designed to examine the effects of prenatal and postnatal exposure of ethanol in the in vivo absorption of free folic acid in the small intestine in pups rats at the 21st day after birth. The rats were accustomed to increasing amounts of ethanol (5 to 20%, vol/vol) in tap water for 1 month. During pregnancy and suckling period, ethanol-fed dams were assigned again to ethanol 20% in drinking water. Two sets of experiments were performed. In the first set, jejunal free folic acid absorption in control group and litters nursed by dams receiving ethanol showed a gradual increase along with the increase of perfusion time at all the assayed concentrations. In general, in litters of ethanol-fed dams, jejunal free folic acid absorption expressed as nmol/intestinal surface, nmol/g tissue wet weight and nmol/g tissue dry weight were higher than in control animals. In the second set of experiments, in distal ileum loops, free folic acid absorption did not occur in control pups, but appeared in litters exposed to ethanol. Milk folic acid levels are significantly decreased in ethanol-treated dams. However, only a slight decrease in the serum folic acid levels occurs in litters of ethanol-fed dams. In conclusion, the results obtained in the present work suggested a different pattern of free folic acid absorption in distal ileum for the two groups. The exposure of rats to ethanol during the pregnancy and suckling period, can affect postnatal development of intestinal functions and could play a role in the genesis of malnutrition observed in the infant.     

Control of muscle mitochondria by insulin entails activation of Akt2-mtNOS pathway: implications for the metabolic syndrome

Background

In the metabolic syndrome with hyperinsulinemia, mitochondrial inhibition facilitates muscle fat and glycogen accumulation and accelerates its progression. In the last decade, nitric oxide (NO) emerged as a typical mitochondrial modulator by reversibly inhibiting citochrome oxidase and oxygen utilization. We wondered whether insulin-operated signaling pathways modulate mitochondrial respiration via NO, to alternatively release complete glucose oxidation to CO₂ and H₂O or to drive glucose storage to glycogen.

Methodology/Principal Findings

We illustrate here that NO produced by translocated nNOS (mtNOS) is the insulin-signaling molecule that controls mitochondrial oxygen utilization. We evoke a hyperinsulinemic-normoglycemic non-invasive clamp by subcutaneously injecting adult male rats with long-lasting human insulin glargine that remains stable in plasma by several hours. At a precise concentration, insulin increased phospho-Akt2 that translocates to mitochondria and determines in situ phosphorylation and substantial cooperative mtNOS activation (+4–8 fold, P<.05), high NO, and a lowering of mitochondrial oxygen uptake and resting metabolic rate (−25 to −60%, P<.05). Comparing in vivo insulin metabolic effects on gastrocnemius muscles by direct electroporation of siRNA nNOS or empty vector in the two legs of the same animal, confirmed that in the silenced muscles disrupted mtNOS allows higher oxygen uptake and complete (U-¹⁴C)-glucose utilization respect to normal mtNOS in the vector-treated ones (respectively 37±3 vs 10±1 µmolO₂/h.g tissue and 13±1 vs 7.2±1 µmol ³H₂O/h.g tissue, P<.05), which reciprocally restricted glycogen-synthesis by a half.

Conclusions/Significance

These evidences show that after energy replenishment, insulin depresses mitochondrial respiration in skeletal muscle via NO which permits substrates to be deposited as macromolecules; at discrete hyperinsulinemia, persistent mtNOS activation could contribute to mitochondrial dysfunction with insulin resistance and obesity and therefore, to the progression of the metabolic syndrome.     

牛生长激素基因的人工合成,重组克隆及高效表达

本文通过设计选择大肠杆菌高频利用密码子代替天然密码子,人工全合成３２个寡聚核苷酸片段;连接并克隆获得Ａ（２７８ｂｐ）、Ｂ（８８ｂｐ）、Ｃ（２２４ｂｐ）３个较大ＤＮＡ片段;经重组克隆、定位突变等获得阳性克隆;双向ＤＮＡ序列测定分析表明获得了两种人工全合成牛生长激素（ｂＧＨ）编码基因,即编码Ｎ－Ｍｅｔ－Ａｌａ－ｂＧＨ和Ｎ－Ｍｅｔ－Ｐｈｅ－ｂＧＨ的两个基因,而至今尚未见有人工全合成ｂＧＨ基因的报道。构建了在ＰＬｐｒｏｍｏｔｅｒ控制下含上述合成基因的表达质位ｐＢＬｂＧＨＥ７Ａ１和ｐＢＬｂＧＨＥ８,并在大肠杆菌中进行了温敏诱导表达。经ＳＤＳ－ＰＡＧＥ分析,表达产物占全菌蛋白的３７％以上。Ｗｅｓｔｅｒｎ－ｂｌｏｔ分析和纯化产物的Ｎ端氨基酸序列测定结果都表明上述两种人工全合成ｂＧＨ基因在大肠杆菌中得到了高效表达,表达量为现今国际文献报道的高限水平。     

Row orientation effect on UV-B,UV-A and PAR solar irradiation components in vineyards at Tuscany,Italy

Besides playing an essential role in plant photosynthesis, solar radiation is also involved in many other important biological processes. In particular, it has been demonstrated that ultraviolet (UV) solar radiation plays a relevant role in grapevines (Vitis vinifera) in the production of certain important chemical compounds directly responsible for yield and wine quality. Moreover, the exposure to UV-B radiation (280–320 nm) can affect plant–disease interaction by influencing the behaviour of both pathogen and host. The main objective of this research was to characterise the solar radiative regime of a vineyard, in terms of photosynthetically active radiation (PAR) and UV components. In this analysis, solar spectral UV irradiance components, broadband UV (280–400 nm), spectral UV-B and UV-A (320–400 nm), the biological effective UVBE, as well as the PAR (400–700 nm) component, were all considered. The diurnal patterns of these quantities and the UV-B/PAR and UV-B/UV-A ratios were analysed to investigate the effect of row orientation of the vineyard in combination with solar azimuth and elevation angles. The distribution of PAR and UV irradiance at various heights of the vertical sides of the rows was also studied. The results showed that the highest portion of plants received higher levels of daily radiation, especially the UV-B component. Row orientation of the vines had a pronounced effect on the global PAR received by the two sides of the rows and, to a lesser extent, UV-A and UV-B. When only the diffused component was considered, this geometrical effect was greatly attenuated. UV-B/PAR and UV-A/PAR ratios were also affected, with potential consequences on physiological processes. Because of the high diffusive capacity of the UV-B radiation, the UV-B/PAR ratio was significantly lower on the plant portions exposed to full sunlight than on those in the shade.     

The dehydrogenase-mediated recycling of NADPH is a key antioxidant system against salt-induced oxidative stress in olive plants

NADPH is an important molecule in the redox balance of the cell. In this paper, using olive tissue cultures as a model of the function of the NADPH-generating dehydrogenases in the mechanism of oxidative stress induced by severe salinity conditions was studied. When olive (Olea europaea) plants were grown with 200 mM NaCl, a 40% reduction in leaf fresh weight was produced. The content of non-enzymatic antioxidants such as ascorbate and glutathione was diminished between 20% to 39%, whereas the H2O2 content was increased threefold. In contrast, the analysis of the activity and protein contents of the main antioxidative enzymes showed a significant increase of catalase, superoxide dismutase and glutathione reductase. Overall, these changes strongly suggests that NaCl induces oxidative stress in olive plants. On the other hand, while the content of glucose-6-phosphate was increased almost eightfold in leaves of plants grown under salt stress, the content of NAD(P)H (reduced and oxided forms) did not show significant variations. Under salt stress conditions, the activity and protein contents of the main NADPH-recycling enzymes, glucose-6-phosphate dehydrogenase (G6PDH), isocitrate dehydrogenase (ICDH), malic enzyme (ME) and ferrodoxin-NADP reductase (FNR) showed an enhancement of 30-50%. In leaves of olive plants grown with 200 mM NaCl, analysis of G6PDH by immunocytochemistry and confocal laser scanning microscopy showed a general increase of this protein in epidermis, palisade and spongy mesophyll cells. These results indicate that in olive plants, salinity causes reactive oxygen species (ROS)-mediated oxidative stress, and plants respond to this situation by inducing different antioxidative enzymes, especially the NADPH-producing dehydrogenases in order to recycle NADPH necessary for the protection against oxidative damages. These NADP-dehydrogenases appear to be key antioxidative enzymes in olive plants under salt stress conditions.     

Functional characterization of the enzymes with 2,3-bisphosphoglycerate phosphatase activity from pig skeletal muscle

In pig skeletal muscle exist four enzymes with 2,3-bisphosphoglycerate phosphatase activity. Two of them (forms I-A and I-C) are multi-functional enzymes which, in addition to the phosphatase activity, possess 2,3-bisphosphoglycerate synthase and phosphoglycerate mutase activities. The other two enzyme forms (II-A and II-B) only show the phosphatase activity. The four enzymes differ in substrate specificity. Form I-C is highly specific for glycerate 2,3-P2; form I-A also hydrolyzes the monophosphoglycerates and forms II-A and II-B are specific for phosphoester bonds adjacent to a C-1 carboxylic group. The enzymes possess similar Km, Kcat and optimum pH value, but they are differently inhibited by the reaction products. They are also differently affected by glycolate-2-P (their main activator) and by other modifiers. Probably form I-A, which corresponds to M-type phosphoglycerate mutase, is the main enzyme implicated in the breakdown of glycerate 2,3-P2 in pig muscle.