有关彩色真菌培养基的应用

彩色真菌培养基具有选择性强、分辨率高、易生长、易观察的特点。在真菌培养方面优于其它培养基,其主要作用机理在于应用了化学生物效应促进真菌生长。     

Liver X receptor activation induces podocyte injury via inhibiting autophagic activity

Podocyte injury plays a key role in the occurrence and development of kidney diseases. Decreased autophagic activity in podocyte is closely related to its injury and the occurrence of proteinuria. Liver X receptors (LXRs), as metabolic nuclear receptors, participate in multiple pathophysiological processes and express in several tissues, including podocytes. Although the functional roles of LXRs in the liver, adipose tissue and intestine are well established; however, the effect of LXRs on podocytes function remains unclear. In this study, we used mouse podocytes cell line to investigate the effects of LXR activation on podocytes autophagy level and related signaling pathway by performing Western blotting, RT-PCR, GFP-mRFP-LC3 transfection, and immunofluorescence staining. Then, we tested this effect in STZ-induced diabetic mice. Transmission electron microscopy and immunohistochemistry were employed to explore the effects of LXR activation on podocytes function and autophagic activity. We found that LXR activation could inhibit autophagic flux through blocking the formation of autophagosome in podocytes in vitro which was possibly achieved by affecting AMPK, mTOR, and SIRT1 signaling pathways. Furthermore, LXR activation in vivo induced autophagy suppression in glomeruli, leading to aggravated podocyte injury. In summary, our findings indicated that activation of LXRs induced autophagy suppression, which in turn contributed to the podocyte injury.

     

Decreased Energy Metabolism Extends Life Span in Caenorhabditis elegans Without Reducing Oxidative Damage

On the basis of the free radical and rate of living theories of aging, it has been proposed that decreased metabolism leads to increased longevity through a decreased production of reactive oxygen species (ROS). In this article, we examine the relationship between mitochondrial energy metabolism and life span by using the Clk mutants in Caenorhabditis elegans. Clk mutants are characterized by slow physiologic rates, delayed development, and increased life span. This phenotype suggests that increased life span may be achieved by decreasing energy expenditure. To test this hypothesis, we identified six novel Clk mutants in a screen for worms that have slow defecation and slow development and that can be maternally rescued. Interestingly, all 11 Clk mutants have increased life span despite the fact that slow physiologic rates were used as the only screening criterion. Although mitochondrial function is decreased in the Clk mutants, ATP levels are normal or increased, suggesting decreased energy utilization. To determine whether the longevity of the Clk mutants results from decreased production of ROS, we examined sensitivity to oxidative stress and oxidative damage. We found no evidence for systematically increased resistance to oxidative stress or decreased oxidative damage in the Clk mutants despite normal or elevated levels of superoxide dismutases. Overall, our findings suggest that decreased energy metabolism can lead to increased life span without decreased production of ROS.MUTATIONS in clk-1 have been shown to increase longevity in both worms and mice, suggesting that these mutations affect an evolutionarily conserved mechanism of life span extension (Lakowski and Hekimi 1996; Liu et al. 2005; Lapointe et al. 2009). The CLK-1 protein encodes a hydroxylase involved in the synthesis of ubiquinone (Ewbank et al. 1997), a multifunctional, lipid-like molecule that transfers electrons in the electron transport chain and may also act as an intracellular antioxidant (Maroz et al. 2009). clk-1 was originally identified in worms in a screen for maternally rescued mutations that result in abnormal development and behavior. In addition to slow development and slow defecation, clk-1 mutants show decreased brood size, a decreased rate of thrashing, and a decreased rate of pharyngeal pumping (Wong et al. 1995). It was a surprise, however, that clk-1 worms also displayed extended longevity, because, at the time that it was discovered, only two other mutants, age-1 and daf-2, with very different phenotypes, had been found to extend longevity (Friedman and Johnson 1988; Kenyon et al. 1993).It is currently uncertain how mutations in clk-1 result in the overall slowing of development and physiologic rates as well as an extended life span. One classic theory of aging, called the rate of living theory, postulates the existence of a link between energy metabolism and aging (Pearl 1922; Speakman 2005). This theory proposes that what determines the life span of an organism is the rate at which it produces and uses energy at the cellular level. Thus, the fact that clk-1 worms exhibit slow physiologic rates and development suggests a decrease in the rate that these worms utilize energy, and, by the rate of living theory, this could account for their long life span.In support of the rate of living theory, the loss of clk-1 has been shown to result in decreased whole-worm oxygen consumption (Felkai et al. 1999; Yang et al. 2007) and decreased electron transfer from complex I to complex III in the electron transport chain (Kayser et al. 2004b), although this has not been observed by all investigators (Miyadera et al. 2001). While some reports have suggested that energy consumption is not reduced in clk-1 worms, at least under liquid culture conditions (Braeckman et al. 2002), the observation that clk-1 worms have higher levels of ATP than wild-type worms (Braeckman et al. 1999) suggests a decreased use of energy in clk-1 worms regardless of whether energy production is normal or decreased. It has also been found that clk-1 double-mutant combinations that exhibit slower development than clk-1 worms live even longer than clk-1 worms (Lakowski and Hekimi 1996). In addition, overexpression of clk-1 prevents the slowing of the defecation rate with age, increases mitochondrial function, and decreases life span (Felkai et al. 1999).Drawing on ideas from the free radical theory of aging (Harman 1956), it has been suggested that a possible mechanism underlying the rate of living theory is that decreased metabolism results in a lower rate of production of reactive oxygen species (ROS). As the free radical theory of aging proposes that aging results from the accumulation of molecular damage caused by ROS, then lower ROS production should result in slower aging. In clk-1 worms, it has not been possible to directly measure levels of ROS in vivo; however, measurement of hydrogen peroxide production from submitochondrial particles has demonstrated increased ROS generation in clk-1 mitochondria compared to wild type (Yang et al. 2009). In addition, the superoxide production potential is increased in clk-1 worms compared to wild-type N2 worms (Braeckman et al. 2002). Despite showing increased levels of ROS production, clk-1 worms have been found to have normal or decreased levels of oxidative damage (Kayser et al. 2004a; Yang et al. 2007, 2009) and decreased accumulation of lipofuscin (Braeckman et al. 2002). The decrease in oxidative damage that occurs in spite of increased ROS production likely results from increased antioxidant defenses. In support of this conclusion, sod-2 and sod-3 mRNA are increased in clk-1 worms compared to wild type (Yang et al. 2007).Clearly, the levels of ROS production and antioxidant defense are altered in clk-1 worms and likely contribute to the physiology and life span of these worms. Evidence supporting a role for altered ROS levels in determining the clk-1 phenotype comes from the demonstration that increasing the levels of ROS through decreasing superoxide dismutase expression has been shown to modulate a variety of phenotypes in clk-1 worms (Shibata et al. 2003; Yang et al. 2007). It is important to note, however, that the decrease in oxidative damage in clk-1 worms appears not to contribute to their long life as it is possible to experimentally increase oxidative damage in clk-1 worms beyond wild-type levels without reducing life span (Yang et al. 2007).In addition to clk-1, four other genes have been identified that yield a clk-1-like phenotype (Clk phenotype), which includes slow development, slow defecation, slow pharyngeal pumping, decreased brood size and long life span coupled with maternal rescue (homozygous mutants from heterozygous mothers are phenotypically normal) (Hekimi et al. 1995; Lemieux et al. 2001). The Clk phenotype has been studied in most detail in clk-1 worms (Wong et al. 1995) and, subsequently, with gro-1 (Lemieux et al. 2001), clk-2 (Benard et al. 2001), and tpk-1 worms (de Jong et al. 2004), while clk-3 worms have not been extensively studied [although clk-3 worm energy metabolism and oxygen consumption have been examined (Braeckman et al. 2002; Shoyama et al. 2009)]. Despite the phenotypic similarity of these mutants, the mutations that have been identified thus far have been shown to occur in genes encoding proteins with a wide range of functions with no obvious relationship to one another. gro-1 encodes a tRNA-modifying enzyme (Lemieux et al. 2001), clk-2 encodes a homolog of yeast Tel2p and a regulator of several PI3K-related protein kinases (Ahmed et al. 2001; Benard et al. 2001; Jiang et al. 2003; Takai et al. 2007), and tpk-1 encodes thiamine pyrophosphokinase, which is necessary for the assimilation of thiamine (vitamin B1) (de Jong et al. 2004).All of the Clk mutants that have been identified exhibit slow physiologic rates and increased life span, suggesting that one may be sufficient for the other. To test this hypothesis, we identified six novel Clk mutants and demonstrate that these strains bear all of the characteristic features of the Clk phenotype, including extended longevity. We further show that mitochondrial function is decreased in the Clk mutants but that this decrease does not result in increased resistance to oxidative stress or decreased oxidative damage. Our results provide a plausible explanation for the extended life span observed in the Clk mutants and support aspects of the rate of living theory of aging while casting further doubt on the free radical theory of aging.     

Molecular and expression characterization of a nanos1 homologue in Chinese sturgeon, Acipenser sinensis

The nanos gene family was essential for germ line development in diverse organisms. In the present study, the full-length cDNA of a nanos1 homologue in A. sinensis, Asnanos1, was isolated and characterized. The cDNA sequence of Asnanos1 was 1489 base pairs (bp) in length and encoded a peptide of 228 amino acid residues. Multiple sequence alignment showed that the zinc-finger motifs of Nanos1 were highly conserved in vertebrates. By RT-PCR analysis, Asnanos1 mRNAs were ubiquitously detected in all tissues examined except for the fat, including liver, spleen, heart, ovary, kidney, muscle, intestines, pituitary, hypothalamus, telencephalon, midbrain, cerebellum, and medulla oblongata. Moreover, a specific polyclonal antibody was prepared from the in vitro expressed partial AsNanos1 protein. Western blot analysis revealed that the tissue expression pattern of AsNanos1 was not completely coincided with that of its mRNAs, which was not found in fat, muscle and intestines. Additionally, by immunofluoresence localization, it was observed that AsNanos1 protein was in the cytoplasm of primary oocytes and spermatocytes. The presented results indicated that the expression pattern of Asnanos1 was differential conservation and divergence among diverse species.     

Biotransformation of menadione to its prenylated derivative MK-3 using recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Prenylated quinones, especially menaquinones, have significant physiological activities, but are arduous to synthesize efficiently. Due to the relaxed aromatic substrate specificity and prenylation regiospecificity at the ortho- site of the phenolic hydroxyl group, the aromatic prenyltransferase NovQ from Streptomyces may be useful in menaquinone synthesis from menadione. In this study, NovQ was overexpressed in Pichia pastoris. After fermentation optimization, NovQ production increased by 1617%. Then the different effects of metal ions, detergents and pH on the activity of purified NovQ were investigated to optimize the prenylation reaction. Finally, purified NovQ and cells containing NovQ were used for menadione prenylation in vitro and in vivo, respectively. Menaquinone-1 (MK-1) was detected as the only product in vitro with γ,γ-dimethylallyl pyrophosphate and menadione hydroquinol substrates. MK-3 at a concentration of 90.53 mg/L was detected as the major product of whole cell catalysis with 3-methyl-2-buten-1-ol and menadione hydroquinol substrates. This study realized whole cell catalysis converting menadione to menaquinones.