Effect of sedation with detomidine and butorphanol on pulmonary gas exchange in the horse

Background  

Sedation with α₂-agonists in the horse is reported to be accompanied by impairment of arterial oxygenation. The present study was undertaken to investigate pulmonary gas exchange using the Multiple Inert Gas Elimination Technique (MIGET), during sedation with the α₂-agonist detomidine alone and in combination with the opioid butorphanol.     

YMR313c/TGL3 encodes a novel triacylglycerol lipase located in lipid particles of Saccharomyces cerevisiae

Previous work from our laboratory (Athenstaedt, K., Zweytick, D., Jandrositz, A., Kohlwein, S. D., and Daum, G. (1999) J. Bacteriol. 181, 6441-6448) showed that the gene product of YMR313c (named Tgl3p) is a component of yeast lipid particles, and deletion of this gene led to an increase in the cellular level of triacylglycerols (TAG). These observations suggested that TGL3 may encode a TAG lipase of Saccharomyces cerevisiae. Here we demonstrate by cell fractionation and by microscopic inspection of a strain bearing a Tgl3p-GFP hybrid that this polypeptide is highly enriched in the lipid particle fraction but virtually absent from other organelles. The entire TAG lipase activity of lipid particles is attributed to Tgl3p, because the activity in this organelle is completely absent in a Deltatgl3 deletion mutant, whereas it is significantly enhanced in a strain overexpressing Tgl3p. A His6-tagged Tgl3p hybrid purified close to homogeneity from a yeast strain overexpressing this fusion protein exhibited high TAG lipase activity. Most importantly, experiments in vivo using the fatty acid synthesis inhibitor cerulenin demonstrated that deletion of TGL3 resulted in a decreased mobilization of TAG from lipid particles. The amino acid sequence deduced from the open reading frame YMR313c contains the consensus sequence motif GXSXG typical for lipolytic enzymes. Otherwise, Tgl3p has no significant sequence homology to other lipases identified so far. In summary, our data identified Tgl3p as a novel yeast TAG lipase at the molecular level and by function in vivo and in vitro.     

Alterations in the muscle-to-capillary interface in patients with different degrees of chronic obstructive pulmonary disease

Background

It is hypothesized that decreased capillarization of limb skeletal muscle is implicated in the decreased exercise tolerance in COPD patients. We have recently demonstrated decreased number of capillaries per muscle fibre (CAF) but no changes in CAF in relation to fibre area (CAFA), which is based on the diffusion distance between the capillary and muscle fibre. The aim of the current study is to investigate the muscle-to-capillary interface which is an important factor involved in oxygen supply to the muscle that has previously been suggested to be a more sensitive marker for changes in the capillary bed compared to CAF and CAFA.

Methods

23 COPD patients and 12 age-matched healthy subjects participated in the study. Muscle-to-capillary interface was assessed in muscle biopsies from the tibialis anterior muscle using the following parameters:1) The capillary-to-fibre ratio (C:F_i) which is defined as the sum of the fractional contributions of all capillary contacts around the fibre2) The ratio between C:F_i and the fibre perimeter (CFPE-index)3) The ratio between length of capillary and fibre perimeter (LC/PF) which is also referred to as the index of tortuosity.Exercise capacity was determined using the 6-min walking test.

Results

A positive correlation was found between CFPE-index and ascending disease severity with CFPE-index for type I fibres being significantly lower in patients with moderate and severe COPD. Furthermore, a positive correlation was observed between exercise capacity and CFPE-index for both type I and type IIa fibres.

Conclusion

It can be concluded that the muscle-to-capillary interface is disturbed in the tibialis anterior muscle in patients with COPD and that interface is strongly correlated to increased disease severity and to decreased exercise capacity in this patient group.     

Glycodelin protein and mRNA is downregulated in human first trimester abortion and partially upregulated in mole pregnancy.

Glycodelin (Gd) is a major reproductive glycoprotein and a mediator for immunomodulatory effects directed to cellular, humoral, and innate immunity. Human pregnancy depends on a diversity of physiological processes including modulation of the maternal immunosystem. We evaluated the expression of Gd protein and mRNA in first trimester decidual tissue of normal pregnancies and spontaneous abortion and hydatidiform moles. Furthermore, in vitro experiments on endometrial cancer cells to analyze the effect of human chorionic gonadotropin (hCG) on Gd regulation were performed. In decidual tissue of abortion patients, Gd expression was significantly decreased compared with normal gestation, which was confirmed by in situ hybridization. In mole pregnancy, an upregulation of Gd in the first 8 weeks of pregnancy was present. Gd is a main product of decidual tissue in the first trimester of human pregnancy. Reduced Gd expression in abortive pregnancy could lead to an increased activation of the maternal immunosystem, thus causing rejection of the developing fetus. Moreover, Gd expression in endometrial cancer cells in vitro could be stimulated by addition of hCG. Therefore, we speculate that hCG could be one of the factors regulating Gd expression because hCG is downregulated in women with abortion and upregulated in mole pregnancy. In addition, we found a positive feedback loop in Gd and hCG expression in human pregnancy.     

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

Deciphering proteins and their functions in the regenerating retina

Neurons of the mammalian CNS, including retinal ganglion cells, lack, in contrast to the PNS, the ability to regenerate axons spontaneously after injury. Regeneration of the CNS is extremely complex and involves various molecular factors and cells. Therewith the regenerative process remains an enormous scientific and clinical challenge. This article provides an overview of proteins that play a crucial role in axon regeneration of retinal ganglion cells and their underlying signaling pathways. In this context, we elucidate the role of 2D gel electrophoresis and highlight some additional proteins, altered upon regeneration by using this highly sensitive method.     

Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites

O-GalNAc-glycosylation is one of the main types of glycosylation in mammalian cells. No consensus recognition sequence for the O-glycosyltransferases is known, making prediction methods necessary to bridge the gap between the large number of known protein sequences and the small number of proteins experimentally investigated with regard to glycosylation status. From O-GLYCBASE a total of 86 mammalian proteins experimentally investigated for in vivo O-GalNAc sites were extracted. Mammalian protein homolog comparisons showed that a glycosylated serine or threonine is less likely to be precisely conserved than a nonglycosylated one. The Protein Data Bank was analyzed for structural information, and 12 glycosylated structures were obtained. All positive sites were found in coil or turn regions. A method for predicting the location for mucin-type glycosylation sites was trained using a neural network approach. The best overall network used as input amino acid composition, averaged surface accessibility predictions together with substitution matrix profile encoding of the sequence. To improve prediction on isolated (single) sites, networks were trained on isolated sites only. The final method combines predictions from the best overall network and the best isolated site network; this prediction method correctly predicted 76% of the glycosylated residues and 93% of the nonglycosylated residues. NetOGlyc 3.1 can predict sites for completely new proteins without losing its performance. The fact that the sites could be predicted from averaged properties together with the fact that glycosylation sites are not precisely conserved indicates that mucin-type glycosylation in most cases is a bulk property and not a very site-specific one. NetOGlyc 3.1 is made available at www.cbs.dtu.dk/services/netoglyc.