Spinocerebellar ataxia type 8 larger triplet expansion alters histone modification and induces RNA foci

Background

The POU family genes containing the POU domain are common in vertebrates and invertebrates and play critical roles in cell-type-specific gene expression and cell fate determination.

Results

Har-POU, a new member of the POU gene family, was cloned from the suboesophageal ganglion of Helicoverpa armigera (Har), and its potential functions in the development of the central nervous system (CNS) were analyzed. Southern blot analysis suggests that a single copy of this gene is present in the H. armigera haploid genome. Har-POU mRNA is distributed widely in various tissues and expressed highly in the CNS, salivary gland, and trachea. In vitro-translated Har-POU specifically bound canonical octamer motifs on the promoter of diapause hormone and pheromone biosynthesis activating neuropeptide (DH-PBAN) gene in H. armigera. Expression of the Har-POU gene is markedly higher in the CNS of nondiapause-destined pupae than in diapause-destined pupae. Expression of the Har-POU gene in diapausing pupae was upregulated quickly by injection of ecdysone.

Conclusion

Har-POU may respond to ecdysone and bind to the promoter of DH-PBAN gene to regulate pupal development in H. armigera.     

Genome Sequence of a Cellulose-Producing Bacterium,Gluconacetobacter hansenii ATCC 23769

The Gram-negative bacterium Gluconacetobacter hansenii is considered a model organism for studying cellulose synthesis. We have determined the genome sequence of strain ATCC 23769.Plants produce cellulose, an unbranched chain of β-1,4-linked glucose units, as a structural polysaccharide. It is the most abundant polymer on earth, recently receiving much interest due to its potential use as a feedstock for bioethanol. Bacteria also produce cellulose. Among these, Gluconacetobacter hansenii (previously named Acetobacter xylinus) (4) has been extensively characterized and is a model system for cellulose biosynthesis (1, 2, 7). G. hansenii produces extracellular cellulose that is devoid of lignin or hemicellulose, making it an excellent source for pure cellulose. A lack of a completely sequenced genome for this organism has been a limiting factor in identifying other key proteins involved in cellulose synthesis.The whole-genome sequencing of G. hansenii ATCC 23769 was performed using the 454 FLX-Titanium pyrosequencing technology (5). A combinatorial sequencing approach using 489,201 reads obtained from the shotgun library and 195,088 reads from an 8-kb pair end library (3) produced a total of 221,294,116 bp. These reads were assembled using the Newbler assembler, producing 88 large contigs (>500 bp) and a chromosome-sized scaffold of 3,646,142 bp with an average coverage of ×50.5. This scaffold contained exclusively chromosomal DNA and no plasmid sequences. The gaps in the large scaffold were filled by primer walking and subsequent sequencing of the PCR products. The resulting high-quality draft assembly, consisting of a large scaffold with 71 contigs, was annotated using the Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) service of the National Institute of Biotechnology Information (NCBI).The chromosomal sequence of G. hansenii 23769 contains 3,547,122 bp, with a G+C content of 59%. The genome contains 3,351 genes, of which 3,308 are protein-encoding genes, accounting for 84% of the genome. There are 43 genes for tRNAs and 2 rRNA loci. The genes encoding proteins involved in cellulose synthesis are in an operon consisting of acsAB (GXY_04277), acsC (GXY_04282), and acsD (GXY_04292), as previously shown by Saxena et al. (7). Interestingly, there are two additional copies of acsAB, GXY_08864 and GXY_14452, which share 69% and 72% sequence identity, respectively, with the acsAB genes in the operon; the deduced amino acid sequences are 40% and 46% identical, respectively, with that deduced from acsAB in the operon. There are also two additional copies of acsC, GXY_08869 and GXY_014472, which share 72% and 65% DNA sequence identity, respectively, with the acsC gene in the operon; the deduced amino acid sequences share 28% and 30% amino acid identity, respectively, with that deduced from acsC. acsAB (GXY_08864) and acsC (GXY_08869) are only 17 bp apart, less than the distance (66 bp) between the acsAB and acsC genes in the operon. acsAB (GXY_14452) and acsC (GXY_14472) are separated by 3,299 bp, with three genes in between. However, acsD is present only in the operon, not duplicated elsewhere in the genome. The genome also contains three genes encoding diguanylate cyclase, as previously reported by Tal et al. (8). Diguanylate cyclase catalyzes the formation of cyclic di-GMP, a second messenger in bacteria that functions as an allosteric activator of cellulase synthase AcsAB (6).     

Association of rs780094 in GCKR with Metabolic Traits and Incident Diabetes and Cardiovascular Disease: The ARIC Study

Objective

The minor T-allele of rs780094 in the glucokinase regulator gene (GCKR) associates with a number of metabolic traits including higher triglyceride levels and improved glycemic regulation in study populations of mostly European ancestry. Using data from the Atherosclerosis Risk in Communities (ARIC) Study, we sought to replicate these findings, examine them in a large population-based sample of African American study participants, and to investigate independent associations with other metabolic traits in order to determine if variation in GKCR contributes to their observed clustering. In addition, we examined the association of rs780094 with incident diabetes, coronary heart disease (CHD), and stroke over up mean follow-up times of 8, 15, and 15 years, respectively.

Research Design and Methods

Race-stratified analyses were conducted among 10,929 white and 3,960 black participants aged 45–64 at baseline assuming an additive genetic model and using linear and logistic regression and Cox proportional hazards models.

Results

Previous findings replicated among white participants in multivariable adjusted models: the T-allele of rs780094 was associated with lower fasting glucose (p = 10⁻⁷) and insulin levels (p = 10⁻⁶), lower insulin resistance (HOMA-IR, p = 10⁻⁹), less prevalent diabetes (p = 10⁻⁶), and higher CRP (p = 10⁻⁸), 2-h postprandial glucose (OGTT, p = 10⁻⁶), and triglyceride levels (p = 10⁻³¹). Moreover, the T-allele was independently associated with higher HDL cholesterol levels (p = 0.022), metabolic syndrome prevalence (p = 0.043), and lower beta-cell function measured as HOMA-B (p = 0.011). Among black participants, the T-allele was associated only with higher triglyceride levels (p = 0.004) and lower insulin levels (p = 0.002) and HOMA-IR (p = 0.013). Prospectively, the T-allele was associated with reduced incidence of diabetes (p = 10⁻⁴) among white participants, but not with incidence of CHD or stroke.

Conclusions

Our findings indicate rs780094 has independent associations with multiple metabolic traits as well as incident diabetes, but not incident CHD or stroke. The magnitude of association between the SNP and most traits was of lower magnitude among African American compared to white participants.     

Novel Escherichia coli hybrids with enhanced butanol tolerance

Hybrids between Escherichia coli and Lactobacillus brevis were generated via protoplast fusion. Growth kinetics of five hybrid strains and E. coli were used to evaluate the butanol tolerance of the novel strains under different conditions. The hybrid strains tolerated up to 2% (v/v) butanol compared to the 1% (v/v) maximum for E. coli. The growth inhibitory effects of butanol were also significantly less in several of the hybrids compared to E. coli. These results demonstrate the potential use of protoplast fusion to generate butanol-tolerant strains.     

Neuroprotection by tetramethylpyrazine against ischemic brain injury in rats

In traditional Chinese medicine, Ligusticum wallichii Franchat (Chuan Xiong) and its active ingredient tetramethylpyrazine (TMP) have been used to treat cardiovascular diseases and to relieve various neurological symptoms such as ischemic deficits. However, scientific evidence related to their effectiveness or precise modes of neuroprotective action is largely unclear. In the current study, we elicited the neuroprotective mechanisms of TMP after focal cerebral ischemic/reperfusion (I/R) by common carotid arteries and middle cerebral artery occlusion model in rats. TMP was administrated 60 min before occlusion via intraperitoneal injection. TMP concentration-dependently exhibited significant neuroprotective effect against ischemic deficits by reduction of behavioral disturbance. Neuronal loss and brain infarction in the ischemic side of rats were markedly lowered by treatment with TMP. Cerebral I/R-induced internucleosomal DNA fragmentation, caspase-8, caspase-9, and caspase-3 activation, and cytochrome c release were reduced by TMP treatment. Western blot analysis revealed the down-regulation of Bcl-2 and Bcl-xL and the up-regulation of Bax and Bad by cerebral I/R insult. Among them, only the alteration in Bcl-xL expression was reversed by TMP treatment. Moreover, the activation of microglia and/or recruitment of inflammatory cells within the ischemic side and the consequent production of monocyte chemoattractant protein 1 (MCP-1) were suppressed by TMP pre-treatment. Our findings suggest that TMP might provide neuroprotection against ischemic brain injury, in part, through suppression of inflammatory reaction, reduction of neuronal apoptosis, and prevention of neuronal loss.     

Novel trihydroxamate-containing peptides: design, synthesis, and metal coordination

Novel trihydroxamate-containing peptides were designed to mimic desferrioxamine (Desferal(R), DFO, a naturally occurring siderophore) but possess distinct conformational restrictions and varied lipophilicity to probe structure vs. metal coordination. The synthesis was performed via fragment condensation of hydroxamate-containing oligopeptides such as Fmoc-Leu- Psi[CON(OBz)]-Phe-Ala-Pro-OH and H-Leu-Psi[CON(OBz)]-Phe-Ala-Pro-OBu(t) (Fmoc: 9-fluor enylmethoxycarbonyl; OBz: benzyl; OBu(t): tert-butyl) either in solution or on a solid support. The metal-binding properties were studied by electrospray ionization-mass spectroscopy (ESI-MS), ultraviolet (UV)-visible spectroscopy, and (1)H nuclear magnetic resonance (NMR). Similar to the dihydroxamate analogs previously explored [Biopolymers (Peptide Science), 2003, Vol. 71, pp. 489-515], the compounds with three hydroxamates arrayed at 10-atom intervals, i.e., H-[Leu-Psi[CON(OH)]-Phe-Ala-Pro](3)-OH (P1), cyclo[Leu-Psi[CON(OH)]-Phe-Ala-Pro](3) (P2), and H-[Leu-Psi(CONOH)-Phe-Ala-Pro](2)-Leu-NHOH (P7), exhibited high affinities for intramolecular coordination with Fe(III) and Ga(III). As expected, both P1 and P2 showed higher relative Fe(III)-binding affinities than the corresponding dihydroxamate-containing peptide analogs (P11 and P12). Even though both P1 and P2 did not compete with DFO in the relative metal-binding affinity in both solution and gas phases, P1, P2, and DFO exhibited similar relative binding selectivities to 11 different metal ions including Fe(III), Fe(II), Al(III), Ga(III), In(III), Zn(II), Cu(II), Co(II), Ni(II), Gd(III), and Mn(II). Compared to the other metal ions, they had higher relative binding affinities with Fe(III), Fe(II), Al(III), Ga(III), and In(III). The decreased metal-binding affinities of P1 and P2 in comparison with DFO suggested the conformational restrictions of their backbones perturb their three hydroxamate groups from optimal hexadentate orientations for metal coordination. As detected by ESI-MS, P2 was distinguished from both P1 and DFO by solvation of its Ga(III) and Fe(III) complexes (such as acetonitrile or water), thereby stabilizing the resulting complexes in the gas phase. Noteworthy, P2 led to 69% death rate in Hela cells at a concentration of 50 microM, exhibiting higher cytotoxicity than DFO in vitro despite its much lower affinity for iron. This enhanced toxicity may simply reflect the increased lipophilicity of the cyclic trihydroxamate (P2) together with the improvements in its cell penetration, and/or subsequent intracellular molecular recognition of both side chains and hydroxamate groups. The cytotoxicity was significantly suppressed by precoordination with Ga(III) or Fe(III), suggesting a mechanism of toxicity via sequestration of essential metal ions as well as the importance of curbing the metal coordination before targeting. The potential of such siderophore-mimicking peptides in oncology needs further exploration.     

Myeloperoxidase-derived 2-chlorohexadecanal forms Schiff bases with primary amines of ethanolamine glycerophospholipids and lysine

Numerous studies have suggested relationships between myeloperoxidase, inflammation, and atherosclerosis. MPO-derived reactive chlorinating species (RCS) attack membrane plasmalogens releasing alpha-chloro-fatty aldehydes (alpha-Cl-FALDs) including 2-chlorohexadecanal (2-ClHDA). The molecular targets of alpha-Cl-FALDs are not known. The current study demonstrates 2-ClHDA adducts with ethanolamine glycerophospholipids and Fmoc-lysine. Utilizing electrospray ionization mass spectrometry, chlorinated adducts were observed that are apparent Schiff base adducts. Reduction of these Schiff base adducts with sodium cyanoborohydride resulted in a novel, stable adduct produced by the elimination of HCl. NMR further confirmed this structure. 2-ClHDA adducts with ethanolamine glycerophospholipids were also substrates for phospholipase D (PLD). The hydrolysis products were derivatized to pentafluorobenzoyl esters, and further structurally confirmed by GC-MS. Multiple molecular species of 2-ClHDA-N-modified ethanolamine glycerophospholipids were observed in endothelial cells treated with 2-ClHDA. These results show novel Schiff base adducts of alpha-Cl-FALDs with primary amines, which may represent an important fate of alpha-Cl-FALDs.     

The role of the laminin beta subunit in laminin heterotrimer assembly and basement membrane function and development in C. elegans

Laminins are components of basement membranes that are required for morphogenesis, organizing cell adhesions and cell signaling. Studies have suggested that laminins function as alpha(x) beta(y) gamma(z) heterotrimers in vivo. In C. elegans, there is only one laminin beta gene, suggesting that it is required for all laminin functions. Our analysis is consistent with the role of the laminin beta as a subunit of laminin heterotrimers; the same cells express the laminin alpha, beta, and gamma subunits, the laminin beta subunit localizes to all basement membranes throughout development, and secretion of the beta subunit requires an alpha subunit. RNAi inhibition of the beta subunit gene or of the other subunit genes causes an embryonic lethality phenotype. Furthermore, a distinctive set of phenotypes is caused by both viable laminin alpha and beta partial loss-of-function mutations. These results show developmental roles for the laminin beta subunit, and they provide further genetic evidence for the importance of heterotrimer assembly in vivo.