Isolation and differential expression of two isoforms of the ROBO2/Robo2 axon guidance receptor gene in humans and mice

Expression of Robo receptor molecules is important for axon guidance across the midline of the mammalian central nervous system. Here we describe novel isoform a of human ROBO2, which is initially strongly expressed in the fetal human brain but thereafter only weakly expressed in adult brain and a few other tissues. The known isoform b of ROBO2 shows a more or less ubiquitous expression pattern, suggesting diverse functional roles. The genomic structure and distinct expression patterns of Robo2a and Robo2b have been conserved in the mouse, but in contrast to human ROBO2a mouse Robo2a is also abundant in adult brain. Exons 1 and 2 of human ROBO2a lie in an inherently unstable DNA segment at human chromosome 3p12.3 that is associated with segmental duplications, independent chromosome rearrangements during primate evolution, and homozygous deletion and loss of heterozygosity in various human cancers. The 5' end of mouse Robo2a lies in a <150-kb DNA segment of break in synteny between mouse chromosome 16C3.1 and the human genome.     

Ciliary neurotrophic factor fused to a protein transduction domain retains full neuroprotective activity in the absence of cytokine-like side effects

Ciliary neurotrophic factor (CNTF) is a multifunctional cytokine that can regulate the survival and differentiation of many types of developing and adult neurons. CNTF prevents the degeneration of motor neurons after axotomy and in mouse mutant progressive motor neuronopathy, which has encouraged trials of CNTF for human motor neuron disease. Given systemically, however, CNTF causes severe side effects, including cachexia and a marked immune response, which has limited its clinical application. The present work describes a novel approach for administering recombinant human CNTF (rhCNTF) while conserving neurotrophic activity and avoiding deleterious side effects. rhCNTF was fused to a protein transduction domain derived from the human immunodeficiency virus-1 TAT (transactivator) protein. The resulting fusion protein (TAT-CNTF) crosses the plasma membrane within minutes and displays a nuclear localization. TAT-CNTF was equipotent to rhCNTF in supporting the survival of cultured chicken embryo dorsal root ganglion neurons. Local or subcutaneous administration of TAT-CNTF, like rhCNTF rescued motor neurons from death in neonatal rats subjected to sciatic nerve transection. In contrast to subcutaneous rhCNTF, which caused a 20–30% decrease in body weight in neonatal rats between postnatal days 2 and 7 together with a considerable fat mobilization in brown adipose tissue, TAT-CNTF lacked such side effects. Together, these results indicate that rhCNTF fused with the protein transduction domain/TAT retains neurotrophic activity in the absence of CNTFs cytokine-like side effects and may be a promising candidate for the treatment of motor neuron and other neurodegenerative diseases.     

Effects of Agave salmiana Otto Ex Salm-Dyck silage as forage on ruminal fermentation and growth in goats

Ensiling of Agave salmiana Otto Ex Salm-Dyck, a widespread plant in Mexico, as a viable preservation method to create a potential animal feed resource for ruminants was investigated. Fresh A. salmiana with 205 g dry matter (DM)/kg and wilted alfalfa with 602 g DM/kg were ensiled in combinations (DM:DM) of 1000:0, 500:500 and 350:650, to evaluate feeding value of agave:alfalfa silages on ruminal fermentation and growth of goats. Chemical composition and in situ ruminal disappearance of three total mixed rations (TMRs), which included 240 g/kg DM of each silage (1000:0, 500:500 and 350:650) were determined. The TMR were used to assess ruminal fermentation and growth of 15 goats (20 ± 2.2 kg body weight (BW)). Silage pH (≤4), lactate (>25 g/kg DM) and ammonia (<50 g/kg total N) concentrations indicate that silage quality was good. Lactic acid was the main acid in all silages, acetic acid concentrations were relatively low, and butyrate was only detected in only the 1000:0 agave:alfalfa silage. Potential DM disappearance of the TMR increased quadratically as the amount of alfalfa included in the silage mixture increased. The BW gain and feed efficiency were not changed by treatment, even though DM intake decreased and aNDF intake increased linearly as the amount of alfalfa included in the silage mixture increased. Ruminal pH and butyrate increased, and ammonia N, lactate and propionate decreased linearly as alfalfa proportion of alfalfa in the silage mixture was increased. The TMR ingredient selectivity by the goats may have limited goat performance when alfalfa was included in agave silage mixtures. Because the agave:alfalfa blend improved nutritional quality, ruminal digestibility and intake of agave silage, alfalfa inclusion may improve nutritional characteristics of agave plants silages for ruminants.     

Interactive effects of α-NAA and UV-B radiation on the endogenous hormone contents and growth of Trichosanthes kirilowii Maxim seedlings

The impact of UV-B radiation on endogenous hormones in plants has recently drawn attention from researchers. The mechanism for reduced stem elongation by UV-B might be due to changes in the phytohormone levels, especially IAA, which plays a role in stem elongation. In this study, effects of UV-B radiation on Trichosanthes kirilowii Maxim (T. kirilowii) seedlings in greenhouse-grown plants were investigated. The results indicated that: (1) In comparison to controls, exposure to 0.029 Jm^?2 s^?1. UV-B radiation led to accumulation of endogenous abscisic acid (ABA) and zeatinriboside (ZR) in the plant contents, and decreased contents of endogenous indole-3-acetic acid (IAA) and gibberellic acid (GA_1/3). Exposure to UV-B radiation reduced the height and leaf area of plants. As a result, total biomass (plant dry weight) was lower. (2) In comparison to controls, addition of 2 mg l^?1 α-naphthaleneacetic acid (α-NAA) slightly increased the contents of IAA, GA_1/3 and ZR, and decreased the content of ABA in leaves. This addition of α-NAA significantly increased plant height and leaf area, but only slightly increased total biomass. (3) Addition of α-NAA to UV-B-exposed plants: increased the content of endogenous IAA, GA_1/3 and ZR; decreased accumulation of endogenous ABA; and increased plant height and leaf area in comparison to plants that only were exposed to UV-B. Moreover, total biomass increased slightly. This suggests that addition of α-NAA may compensate to a certain extent for the lack of IAA resulting from UV-B radiation; it also increases the content of GA_1/3 and ZR, decreases the accumulation of ABA, and promotes the growth of plants.     

Effects of indole-3-acetic acid on <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> survival and on symbiotic nitrogen fixation and stem dry weight production

We evaluated the effects of the main auxin phytohormone, indole-3-acetic acid (IAA), on the central metabolism of Sinorhizobium meliloti 1021. We either treated S. meliloti 1021 wild-type cells with 0.5 mM IAA, 1021+, or use a derivative, RD64, of the same strain harboring an additional pathway for IAA biosynthesis (converting tryptophan into IAA via indoleacetamide). We assayed the activity of tricarboxylic acid cycle (TCA) key enzymes and found that activity of citrate synthase and α-ketoglutarate dehydrogenase were increased in both 1021+ and RD64 as compared to the wild-type strain. We also showed that the intracellular acetyl-CoA content was enhanced in both RD64 and 1021+ strains when compared to the control strain. The activity of key enzymes, utilizing acetyl-CoA for poly-β-hydroxybutyrate (PHB) biosynthesis, was also induced. The PHB level measured in these cells were significantly higher than that found in control cells. Moreover, 4-week-long survival experiments showed that 80% of 1021 cells died, whereas 50% of RD64 cells were viable. Medicago truncatula plants nodulated by RD64 (Mt-RD64) showed an induction of both acetylene reduction activity and stem dry weight production.     

牛POMC基因多态性及其与南阳牛生长性状的相关分析

为研究阿片黑皮质素前体(POMC)在动物采食和能量平衡调控中发挥重要作用, 文章采用PCR-SSCP结合DNA测序方法, 对秦川牛、南阳牛、郏县红牛、晋南牛、鲁西牛、安格斯牛和荷斯坦奶牛共计480头个体POMC基因的多态性进行研究, 并分析了多态位点与南阳牛生长性状的相关性。结果表明, 牛POMC基因3个位点中, 在3′侧翼区P3位点新发现3个连锁存在的SNP(811845 C>T、811821 T>C和811797 A>G, 与NW_928357对照)。POMC基因3′侧翼区多态位点与南阳牛6月龄体重和0~6月龄平均日增重显著相关, BB型个体显著大于AA型(P<0.05)。     

Transgenic expression of n‐3 fatty acid desaturase (fat‐1) in C57/BL6 mice: Effects on glucose homeostasis and body weight

The fat‐1 gene, derived from Caenorhabditis elegans, encodes for a fatty acid n‐3 desaturase. In order to study the potential metabolic benefits of n‐3 fatty acids, independent of dietary fatty acids, we developed seven lines of fat‐1 transgenic mice (C57/BL6) controlled by the regulatory sequences of the adipocyte protein‐2 (aP2) gene for adipocyte‐specific expression (AP‐lines). We were unable to obtain homozygous fat‐1 transgenic offspring from the two highest expressing lines, suggesting that excessive expression of this enzyme may be lethal during gestation. Serum fatty acid analysis of fat‐1 transgenic mice (AP‐3) fed a high n‐6 unsaturated fat (HUSF) diet had an n‐6/n‐3 fatty acid ratio reduced by 23% (P < 0.025) and the n‐3 fatty acid eicosapentaenoic acid (EPA) concentration increased by 61% (P < 0.020). Docosahexaenoic acid (DHA) was increased by 19% (P < 0.015) in white adipose tissue. Male AP‐3‐fat‐1 line of mice had improved glucose tolerance and reduced body weight with no change in insulin sensitivity when challenged with a high‐carbohydrate (HC) diet. In contrast, the female AP‐3 mice had reduced glucose tolerance and no change in insulin sensitivity or body weight. These findings indicate that male transgenic fat‐1 mice have improved glucose tolerance likely due to increased insulin secretion while female fat‐1 mice have reduced glucose tolerance compared to wild‐type mice. Finally the inability of fat‐1 transgenic mice to generate homozygous offspring suggests that prolonged exposure to increased concentrations of n‐3 fatty acids may be detrimental to reproduction. J. Cell. Biochem. 107: 809–817, 2009. © 2009 Wiley‐Liss, Inc.     

Enzymic synthesis of levan and fructo-oligosaccharides by <Emphasis Type="Italic">Bacillus circulans</Emphasis> and improvement of levansucrase stability by carbohydrate coupling

Bacillus circulans was able to produce extracellular levansucrase using sucrose as carbon source optimally at 35°C. The enzymic synthesis of levan and fructo-oligosaccharides was studied using a 50% ethanol fraction of crude extract. The molecular weight of the synthesized levan was markedly affected by sucrose concentration, the molecular weight of levan decreased with increased sucrose concentration up to 32% whereby fructo-oligosaccharides were isolated. Temperature and the reaction time clearly affected the conversion of fructose to levan with molecular weight values ranging from 10 to 38 kDa. Identification of levan indicated that fructose was the building unit of the levan obtained. Thermal and pH stabilities of B. circulans levansucrase could be improved by enzyme glycosylation using sodium metaperiodate treatment. Chemical modification provides additional points of attachment of the enzyme to the support which offered the modified enzyme greater stabilization than did the free enzyme. The modified enzyme exhibited thermal tolerance up to 50°C, where it retained 88.25% of its activity, while the free enzyme only retained 64.55% of its original activity. The half-life significantly increased from 130 min for the free enzyme to 347 min for the modified enzyme at 50°C, however, it increased from 103 min for the free enzyme to 210 min for the modified enzyme at 60°C. Other properties i.e., the response to some metal ions as well as the ability to convert higher substrate levels and tolerance to an extension of the reaction periods were also improved upon modification. Obviously, the results obtained outlined the conditions leading to the formation of important high or low molecular weight or levan and fructo-oligosaccharides suitable for different industrial applications.     

RELATIVE CONTRIBUTION OF ADDITIVE, DOMINANCE, AND IMPRINTING EFFECTS TO PHENOTYPIC VARIATION IN BODY SIZE AND GROWTH BETWEEN DIVERGENT SELECTION LINES OF MICE

Epigenetic effects attributed to genomic imprinting are increasingly recognized as an important source of variation in quantitative traits. However, little is known about their relative contribution to phenotypic variation compared to those of additive and dominance effects, and almost nothing about their role in phenotypic evolution. Here we address these questions by investigating the relative contribution of additive, dominance, and imprinting effects of quantitative trait loci (QTL) to variation in "early" and "late" body weight in an intercross of mice selected for divergent adult body weight. We identified 18 loci on 13 chromosomes; additive effects accounted for most of the phenotypic variation throughout development, and imprinting effects were always small. Genetic effects on early weight showed more dominance, less additive, and, surprisingly, less imprinting variation than that of late weight. The predominance of additivity of QTL effects on body weight follows the expectation that additive effects account for the evolutionary divergence between selection lines. We hypothesize that the appearance of more imprinting effects on late body weight may be a consequence of divergent selection on adult body weight, which may have indirectly selected for alleles showing partial imprinting effects due to their associated additive effects, highlighting a potential role of genomic imprinting in the response to selection.