Antisense knockdown of PKC-alpha using LNA-oligos

Full-length and 4 nucleotides truncated Locked Nucleic Acid (LNA) modifications of ISIS 3521 were compared for antisense properties in a cellular assay. ISIS 3521 is a 20-mer phosphorothioate designed to hybridise to human protein kinase C-alpha (PKC-alpha) mRNA and is currently submitted to clinical trials against cancer. We report that LNA can potentate this antisense oligo and retain the antisense potential with shorter oligos.     

Monitoring of two-stage anaerobic biodegradation using a BOD biosensor

A previously developed biosensor for fast estimation of short-term biochemical oxygen demand (BOD_st) was used for off-line monitoring of intermediate products from the initial step of an anaerobic process in laboratory scale. Good agreement was generally achieved between the results from the biosensor method and the conventional 5-day test except for samples with high content of organic polymers. During the period of agreement between the measurement principles, good correlation was achieved between the biogas production rate and the organic loading rate. The results from this study demonstrate that BOD_st can be a successful monitoring parameter to achieve a better process control.     

Nitrogen use efficiency by a slow-growing species as affected by CO2 levels, root temperature, N source and availability

This study examines the importance of N source and concentration on plant response to distinct CO₂ concentrations and root temperatures. The experimental design of this work was a factorial combination of: CO₂ concentration, nitrogen concentration, nitrogen source and root temperature. Carob (Ceratonia siliqua L.) was assessed as a potential model of a slow growing Mediterranean species.

The results showed that: 1) biomass increment under high CO₂ varied between 13 and 100 percnt; in relation to plants grown under the same conditions but at ambient CO₂ concentrations, depending on the root temperature and nitrogen source; 2) nitrate-fed plants attained a larger increase in biomass production compared to ammonium-fed ones. This performance seems to be linked to the co-ordinated regulation of the activities of glutamine synthetase and sucrose phosphate synthase. The variations in the magnitude and nature of growth responses to elevated CO₂ observed resulted in substantial changes in the chemical composition of the plant material and consequently in plant nitrogen use efficiency.

Although performed with seedlings and under controlled conditions, this work emphasizes the importance of the nitrogen source used by the plants, a factor rarely taken into consideration when forecasting plant responses to global changes. Particularly, the results presented here, highlight the potential for uncoupling biomass accumulation from increment of air CO₂ concentration and show that more than nitrogen availability N source may offset positive plant growth responses under elevated CO₂ and root temperature.     

Carbon monoxide stimulates insulin release and propagates Ca2+ signals between pancreatic beta-cells

A key question for understanding the mechanisms of pulsatile insulin release is how the underlying beta-cell oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i) are synchronized within and among the islets in the pancreas. Nitric oxide has been proposed to coordinate the activity of the beta-cells by precipitating transients of [Ca2+]i. Comparing ob/ob mice and lean controls, we have now studied the action of carbon monoxide (CO), another neurotransmitter with stimulatory effects on cGMP production. A strong immunoreactivity for the CO-producing constitutive heme oxygenase (HO-2) was found in ganglionic cells located in the periphery of the islets and in almost all islet endocrine cells. Islets from ob/ob mice had sixfold higher generation of CO (1 nmol.min-1.mg protein-1) than the lean controls. This is 100-fold the rate for their constitutive production of NO. Moreover, islets from ob/ob mice showed a threefold increase in HO-2 expression and expressed inducible HO (HO-1). The presence of an excessive islet production of CO in the ob/ob mouse had its counterpart in a pronounced suppression of the glucose-stimulated insulin release from islets exposed to the HO inhibitor Zn-protoporhyrin (10 microM) and in a 16 times higher frequency of [Ca2+]i transients in their beta-cells. Hemin (0.1 and 1.0 microM), the natural substrate for HO, promoted the appearance of [Ca2+]i transients, and 10 microM of the HO inhibitors Zn-protoporphyrin and Cr-mesoporphyrin had a suppressive action both on the firing of transients and their synchronization. It is concluded that the increased islet production of CO contributes to the hyperinsulinemia in ob/ob mice. In addition to serving as a positive modulator of glucose-stimulated insulin release, CO acts as a messenger propagating Ca2+ signals with coordinating effects on the beta-cell rhythmicity.     

PPARalpha /gamma ragaglitazar eliminates fatty liver and enhances insulin action in fat-fed rats in the absence of hepatomegaly

Peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma agonists lower lipid accumulation in muscle and liver by different mechanisms. We investigated whether benefits could be achieved on insulin sensitivity and lipid metabolism by the dual PPARalpha/gamma agonist ragaglitazar in high fat-fed rats. Ragaglitazar completely eliminated high-fat feeding-induced liver triglyceride accumulation and visceral adiposity, like the PPARalpha agonist Wy-14643 but without causing hepatomegaly. In contrast, the PPARgamma agonist rosiglitazone only slightly lessened liver triglyceride without affecting visceral adiposity. Compared with rosiglitazone or Wy-14643, ragaglitazar showed a much greater effect (79%, P < 0.05) to enhance insulin's suppression of hepatic glucose output. Whereas all three PPAR agonists lowered plasma triglyceride levels and lessened muscle long-chain acyl-CoAs, ragaglitazar and rosiglitazone had greater insulin-sensitizing action in muscle than Wy-14643, associated with a threefold increase in plasma adiponectin levels. There was a significant correlation of lipid content and insulin action in liver and particularly muscle with adiponectin levels (P < 0.01). We conclude that the PPARalpha/gamma agonist ragaglitazar has a therapeutic potential for insulin-resistant states as a PPARgamma ligand, with possible involvement of adiponectin. Additionally, it can counteract fatty liver, hepatic insulin resistance, and visceral adiposity generally associated with PPARalpha activation, but without hepatomegaly.     

转基因动物整合位点的研究进展

转基因动物整合位点克隆及相关序列特征研究是探索外源基因整合机制和整合稳定性的重要手段。转基因整合位点序列的克隆方法主要有：个体基因组文库筛选法、反向PCR法、热不对称交互式PCR法和质粒回收法。4种方法各有优缺点,可根据不同条件选用。克隆到的整合位点序列表明,整合位点可能存在一定的共同特征。 Integration Sites of Transgenes in Transgenic Animals WU Bo,ZHU Zuo-Yan State Key Laboratory of Freshwater Ecology and Biotechnology,Institute of Hydrobiology, Chinese Academy of Science,Wuhan 430072,China Abstract:To study the mechanism of transgene ' s integration in the host genome,first step is to clone the integration-site sequence.The method employed for this purpose includes selection from genomic pool,IPCR,TAIL-PCR and plasmid rescued.Different method fits different cases depending on how the transgenics have been produced.The data of integration-site sequences showed some similar features existed. Key words：transgene; integration site; clone method     

Disruption of the endocytic protein HIP1 results in neurological deficits and decreased AMPA receptor trafficking

Huntingtin interacting protein 1 (HIP1) is a recently identified component of clathrin-coated vesicles that plays a role in clathrin-mediated endocytosis. To explore the normal function of HIP1 in vivo, we created mice with targeted mutation in the HIP1 gene (HIP1(-/-)). HIP1(-/-) mice develop a neurological phenotype by 3 months of age manifest with a failure to thrive, tremor and a gait ataxia secondary to a rigid thoracolumbar kyphosis accompanied by decreased assembly of endocytic protein complexes on liposomal membranes. In primary hippocampal neurons, HIP1 colocalizes with GluR1-containing AMPA receptors and becomes concentrated in cell bodies following AMPA stimulation. Moreover, a profound dose-dependent defect in clathrin-mediated internalization of GluR1-containing AMPA receptors was observed in neurons from HIP1(-/-) mice. Together, these data provide strong evidence that HIP1 regulates AMPA receptor trafficking in the central nervous system through its function in clathrin-mediated endocytosis.     

GADD45γ, down-regulated in 65% Hepatocellular Carcinoma (HCC) from 23 Chinese patients,inhibits cell growth and induces cell cycle G2/M arrest for Hepatoma Hep-G2 cell lines

Growth-arrest and DNA-damage inducible (GADD) genes and Myeloid differentiation primary response (MyD) genes represent a family of genes that play a key role in negative control of cell growth. In the present study, following clone and location of human GADD45 (MyDL) gene, we have found that its mRNA expression level was down-regulated in 15/23 cases of clinic hepatocellular carcinoma (HCC) by comparing the northern hybridization results between the tumor tissues and adjacent normal tissues. Transient transfection of GADD45 cDNA with intact open reading frame sequence into the human hepatoma cells Hep-G2 resulted in dramatic growth suppression in colony formation assays. Furthermore, flow cytometry analysis indicated that GADD45 caused cell cycle arrest at G2/M transition when transfected into Hep-G2 cells. Therefore, the possible role of GADD45 in cell growth control was further confirmed in this paper.     

Systemic poly(ADP-ribose) polymerase-1 activation,chronic inflammation,and oxidative stress in COPD patients

Oxidative stress and systemic inflammation in chronic obstructive pulmonary disease (COPD) strongly suggest a role for the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1, E.C.2.4.2.30) in the disease pathophysiology. PARP-1 is highly activated by reactive oxygen species-induced DNA strand breaks, upon which it forms extensive poly(ADP-ribose) (PAR) polymers from its substrate NAD(+). We hypothesized that in COPD, chronic inflammation and oxidative stress would lead to systemic PARP-1 activation and to a reduced NAD(+) status. In a patient-control study, systemic PARP-1 activation was assessed by immunofluorescent detection of PAR polymers in peripheral blood lymphocytes. The percentage of PAR polymer-positive lymphocytes appeared to be higher in COPD patients (27 +/- 3%) than in healthy age-matched controls (17 +/- 2%, p <.05). Trolox equivalent antioxidant capacity (TEAC) of deproteinized plasma (p <.001), plasma uric acid (p <.05), as well as blood NAD(+) (p <.01) of stable COPD patients were significantly reduced when compared to controls. In addition, levels of proinflammatory cytokines IL-6, IL-8, and sICAM-1 were increased (p <.005) in COPD patients. In this study, evidence was found for the presence of systemic inflammation, chronic oxidative stress, and systemic PARP-1 activation in stable COPD patients. These data support a contribution of oxidative stress-induced PARP-1 activation to the pathophysiology of COPD.