Modulation of fatty acid biosynthesis by antisense beta-keto reductase expression

Plants with an antisense construct to beta-ketoacyl-acyl carrier protein reductase have reduced seed and leaf fatty acid contents and show distinct phenotypes, including leaf curl, stunted growth and wrinkled seeds. The carbon resource allocation is altered in both of these tissues. In leaves there is an inability to synthesize transient starch and in seeds embryogenesis is foreshortened and storage proteins accumulate prematurely.     

Granule-bound starch synthase: structure, function, and phylogenetic utility

Interest in the use of low-copy nuclear genes for phylogenetic analyses of plants has grown rapidly, because highly repetitive genes such as those commonly used are limited in number. Furthermore, because low- copy genes are subject to different evolutionary processes than are plastid genes or highly repetitive nuclear markers, they provide a valuable source of independent phylogenetic evidence. The gene for granule-bound starch synthase (GBSSI or waxy) exists in a single copy in nearly all plants examined so far. Our study of GBSSI had three parts: (1) Amino acid sequences were compared across a broad taxonomic range, including grasses, four dicotyledons, and the microbial homologs of GBSSI. Inferred structural information was used to aid in the alignment of these very divergent sequences. The informed alignments highlight amino acids that are conserved across all sequences, and demonstrate that structural motifs can be highly conserved in spite of marked divergence in amino acid sequence. (2) Maximum-likelihood (ML) analyses were used to examine exon sequence evolution throughout grasses. Differences in probabilities among substitution types and marked among-site rate variation contributed to the observed pattern of variation. Of the parameters examined in our set of likelihood models, the inclusion of among-site rate variation following a gamma distribution caused the greatest improvement in likelihood score. (3) We performed cladistic parsimony analyses of GBSSI sequences throughout grasses, within tribes, and within genera to examine the phylogenetic utility of the gene. Introns provide useful information among very closely related species, but quickly become difficult to align among more divergent taxa. Exons are variable enough to provide extensive resolution within the family, but with low bootstrap support. The combined results of amino acid sequence comparisons, maximum-likelihood analyses, and phylogenetic studies underscore factors that might affect phylogenetic reconstruction. In this case, accommodation of the variable rate of evolution among sites might be the first step in maximizing the phylogenetic utility of GBSSI.      

Retinal Glia Promote Dorsal Root Ganglion Axon Regeneration

Axon regeneration in the adult central nervous system (CNS) is limited by several factors including a lack of neurotrophic support. Recent studies have shown that glia from the adult rat CNS, specifically retinal astrocytes and Müller glia, can promote regeneration of retinal ganglion cell axons. In the present study we investigated whether retinal glia also exert a growth promoting effect outside the visual system. We found that retinal glial conditioned medium significantly enhanced neurite growth and branching of adult rat dorsal root ganglion neurons (DRG) in culture. Furthermore, transplantation of retinal glia significantly enhanced regeneration of DRG axons past the dorsal root entry zone after root crush in adult rats. To identify the factors that mediate the growth promoting effects of retinal glia, mass spectrometric analysis of retinal glial conditioned medium was performed. Apolipoprotein E and secreted protein acidic and rich in cysteine (SPARC) were found to be present in high abundance, a finding further confirmed by western blotting. Inhibition of Apolipoprotein E and SPARC significantly reduced the neuritogenic effects of retinal glial conditioned medium on DRG in culture, suggesting that Apolipoprotein E and SPARC are the major mediators of this regenerative response.     

The diabetic rat kidney mediates inosituria and selective urinary partitioning of D-chiro-inositol

Diabetic nephropathy is a serious complication of diabetes mellitus with a pressing need for effective metabolic markers to detect renal impairment. Of potential significance are the inositol compounds, myo-inositol (MI), and the less abundant stereoisomer, D-chiro-inositol (DCI), which are excreted at increased levels in the urine in diabetes mellitus, a phenomenon known as inosituria. There is also a selective urinary excretion of DCI compared to MI. As the biological origins of altered inositol metabolism in diabetes mellitus are unknown, the aim of this study was to determine whether the diabetic kidney was directly responsible. Kidneys isolated from four-week streptozotocin-induced diabetic rats were characterized by a 3-fold reduction in glomerular filtration rate (GFR) compared to matched non-diabetic kidneys. When perfused with fixed quantities of MI (50 µM) and DCI (5 µM) under normoglycemic conditions (5 mM glucose), GFR-normalized urinary excretion of MI was increased by 1.7-fold in diabetic vs. non-diabetic kidneys. By comparison, GFR-normalized urinary excretion of DCI was increased by 4-fold. Perfusion conditions replicating hyperglycemia (20 mM glucose) potentiated DCI but not MI urinary excretion in both non-diabetic and diabetic kidneys. Overall, there was a 2.4-fold increase in DCI urinary excretion compared to MI in diabetic kidneys that was independent of glucose ambience. This increased urinary excretion of DCI and MI in diabetic kidneys occurred despite increased renal expression of the inositol transporters, sodium myo-inositol transporter subtype 1 and 2 (SMIT1 and SMIT2). These findings show that the diabetic kidney primarily mediates inosituria and altered urinary partitioning of MI and DCI. Urinary inositol levels might therefore serve as an indicator of impaired renal function in diabetes mellitus with wider implications for monitoring chronic kidney disease.     

Simultaneous analysis of isomers of escin saponins in human plasma by liquid chromatography–tandem mass spectrometry: Application to a pharmacokinetic study after oral administration

A rapid and sensitive bioassay based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) for the simultaneous determination of four isomeric escin saponins (escin Ia, escin Ib, isoescin Ia and isoescin Ib) in human plasma has been developed and validated. Sample preparation of plasma after addition of telmisartan as internal standard (I.S.) involved solid-phase extraction (SPE) on C18 cartridges. Separation was based on reversed phase chromatography using gradient elution with methanol–acetonitrile (50:50, v/v) and 10 mM ammonium acetate solution (pH 6.8). MS/MS detection in the positive ion mode used multiple reaction monitoring of the transition at m/z 1113.8 → 807.6. Stability issues with the four saponins required the addition of formic acid to plasma samples prior to storage at ?80 °C and analysis within 30 days. The method was linear at concentrations up to 10 ng/mL with correlation coefficients > 0.996 for all analytes. The lower limit of quantitation (LLOQ) for all four saponins was 33 pg/mL. Intra- and inter-day precisions (as relative standard deviation) were all <15% and accuracies (as relative error) in the range ?5.3% to 6.1%. The method was successfully applied to a pharmacokinetic study of escins in healthy volunteers after oral administration of sodium aescinate tablets containing 60 mg escin saponins.     

Retinoblastoma protein co-purifies with proteasomal insulin-degrading enzyme: Implications for cell proliferation control

Previous investigations on proteasomal preparations containing insulin-degrading enzyme (IDE; EC 3.4.24.56) have invariably yielded a co-purifying protein with a molecular weight of about 110 kDa. We have now found both in MCF-7 breast cancer and HepG2 hepatoma cells that this associated molecule is the retinoblastoma tumor suppressor protein (RB). Interestingly, the amount of RB in this protein complex seemed to be lower in HepG2 vs. MCF-7 cells, indicating a higher (cytoplasmic) protein turnover in the former vs. the latter cells. Moreover, immunofluorescence showed increased nuclear localization of RB in HepG2 vs. MCF-7 cells. Beyond these subtle differences between these distinct tumor cell types, our present study more generally suggests an interplay between RB and IDE within the proteasome that may have important growth-regulatory consequences.     

NCK is critical for the development of deleted in colorectal cancer (DCC) sensitive spinal circuits

As our understanding of motor circuit function increases, our need to understand how circuits form to ensure proper function becomes increasingly important. Recently, deleted in colorectal cancer (DCC) has been shown to be important in the development of spinal circuits necessary for gait. Importantly, humans with mutation in DCC show mirror movement disorders pointing to the significance of DCC in the development of spinal circuits for coordinated movement. Although DCC binds a number of ligands, the intracellular signaling cascade leading to the aberrant spinal circuits remains unknown. Here, we show that the non‐catalytic region of tyrosine kinase adaptor (NCK) proteins 1 and 2 are distributed in the developing spinal cord. Using dissociated dorsal spinal neuron cultures we show that NCK proteins are necessary for the outgrowth and growth cone architecture of DCC^+ve dorsal spinal neurons. Consistent with a role for NCK in DCC signaling, we show that loss of NCK proteins leads to a reduction in the thickness of TAG1^+ve commissural bundles in the floor plate and loss of DCC mRNA in vivo. We suggest that DCC signaling functions through NCK1 and NCK2 and that both proteins are necessary for the establishment of normal spinal circuits necessary for gait.

     

RT-qPCR reveals opsin gene upregulation associated with age and sex in guppies (Poecilia reticulata) - a species with color-based sexual selection and 11 visual-opsin genes

Background  

PCR-based surveys have shown that guppies (Poecilia reticulata) have an unusually large visual-opsin gene repertoire. This has led to speculation that opsin duplication and divergence has enhanced the evolution of elaborate male coloration because it improves spectral sensitivity and/or discrimination in females. However, this conjecture on evolutionary connections between opsin repertoire, vision, mate choice, and male coloration was generated with little data on gene expression. Here, we used RT-qPCR to survey visual-opsin gene expression in the eyes of males, females, and juveniles in order to further understand color-based sexual selection from the perspective of the visual system.