Purification and partial amino acid sequence analysis of two distinct tumor necrosis factor receptors from HL60 cells

Two distinct tumor necrosis factor (TNF) receptors of 55- and 75-kDa apparent molecular masses previously identified on the cell surface by monoclonal antibodies have been solubilized with Triton X-100 from HL60 cells. A filter-based dot blot assay was developed to monitor specific 125I-TNF alpha binding during fractionation of the cell extract. By a combination of immuno- and ligand affinity chromatography and reverse phase high performance liquid chromatography both receptor proteins were purified to apparent homogeneity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two bands at 55 and 51 kDa for the 55-kDa TNF receptor and a major 75-kDa and a minor 65-kDa band for the 75-kDa TNF receptor. All these bands specifically bound TNF alpha and TNF beta in ligand blot experiments. The exclusive specificity of monoclonal antibodies of the utr series for the 75.65-kDa bands and of the htr series for the 55.51-kDa bands was demonstrated with the purified antigens on Western blots. Both TNF receptor types were found to contain N-linked carbohydrates. N-terminal amino acid sequence analysis of the 55- and 51-kDa bands of the 55-kDa TNF receptor revealed identical sequences suggesting a possible truncation at the C-terminal end. Two different N-terminal sequences were determined for the 65-kDa band. One corresponded to the published sequence of ubiquitin; the other was therefore assumed to be a unique sequence of the 75-kDa TNF receptor. Additional internal sequences of this receptor were determined after proteolytic cleavage.     

Partial Deficiency of Sphingosine-1-Phosphate Lyase Confers Protection in Experimental Autoimmune Encephalomyelitis

Background

Sphingosine-1-phosphate (S1P) regulates the egress of T cells from lymphoid organs; levels of S1P in the tissues are controlled by S1P lyase (Sgpl1). Hence, Sgpl1 offers a target to block T cell-dependent inflammatory processes. However, the involvement of Sgpl1 in models of disease has not been fully elucidated yet, since Sgpl1 KO mice have a short life-span.

Methodology

We generated inducible Sgpl1 KO mice featuring partial reduction of Sgpl1 activity and analyzed them with respect to sphingolipid levels, T-cell distribution, and response in models of inflammation.

Principal Findings

The partially Sgpl1 deficient mice are viable but feature profound reduction of peripheral T cells, similar to the constitutive KO mice. While thymic T cell development in these mice appears normal, mature T cells are retained in thymus and lymph nodes, leading to reduced T cell numbers in spleen and blood, with a skewing towards increased proportions of memory T cells and T regulatory cells. The therapeutic relevance of Sgpl1 is demonstrated by the fact that the inducible KO mice are protected in experimental autoimmune encephalomyelitis (EAE). T cell immigration into the CNS was found to be profoundly reduced. Since S1P levels in the brain of the animals are unchanged, we conclude that protection in EAE is due to the peripheral effect on T cells, leading to reduced CNS immigration, rather than on local effects in the CNS.

Significance

The data suggest Sgpl1 as a novel therapeutic target for the treatment of multiple sclerosis.     

A new highly selective physicochemical assay to measure NAD+ in intact cells

A simple, fast, and highly specific chromatographic method for measuring the content of NAD+ in intact cells has been developed. This procedure involves the separation of NAD+ from the bulk of acid-soluble nucleosides, nucleotides, and other pyridine containing molecules by affinity chromatography on dihydroxyboronyl-Bio-Rex. The boronate purified preparations were utilized for the quantification of NAD+ by strong anion exchange high-pressure liquid chromatography under isocratic conditions using a low salt buffer system. The overall recovery of the method exceeded 80%. This new method was applied to determine the extent of NAD+ consumption in intact hepatocytes following treatment with two different DNA damaging agents. A major advantage of this method is that it allows for the simultaneous determination of poly(ADP-ribose) in the acid-insoluble fraction of the same sample.     

Quality of Animal Experiments in Anti-Angiogenic Cancer Drug Development – A Systematic Review

Translation from preclinical animal research to clinical bedside has proven to be difficult to impossible in many fields of research (e.g. acute stroke, ALS and HIV vaccination development) with oncology showing particularly low translation rates (5% vs. 20% for cardiovascular diseases). Several investigations on published preclinical animal research have revealed that apart from plain species differences, translational problems can arise from low study quality (e.g. study design) or non-representative experimental conditions (e.g. treatment schedule).This review assessed the published experimental circumstances and quality of anti-angiogenic cancer drug development in 232 in vivo studies. The quality of study design was often insufficient; at least the information published about the experiments was not satisfactory in most cases. There was no quality improvement over time, with the exception of conflict of interest statements. This increase presumably arose mainly because journal guidelines request such statements more often recently.Visual inspection of data and a cluster analysis confirmed a trend described in literature that low study quality tends to overestimate study outcome. It was also found that experimental outcome was more favorable when a potential drug was investigated as the main focus of a study, compared to drugs that were used as comparison interventions. We assume that this effect arises from the frequent neglect of blinding investigators towards treatment arms and refer to it as hypothesis bias.In conclusion, the reporting and presumably also the experimental performance of animal studies in drug development for oncology suffer from similar shortcomings as other fields of research (such as stroke or ALS). We consider it necessary to enforce experimental quality and reporting that corresponds to the level of clinical studies. It seems that only clear journal guidelines or guidelines from licensing authorities, where failure to fulfill prevents publication or experimental license, can help to improve this situation.     

Differential regulation of chemokine production in human peritoneal mesothelial cells: IFN-gamma controls neutrophil migration across the mesothelium in vitro and in vivo.

Leukocyte recruitment into the infected peritoneal cavity consists of an early, predominant polymorphonuclear leukocyte (PMN) influx and subsequent, prolonged mononuclear cell migration phase. Although chemokine secretion by resident peritoneal cells plays a primary role in mediating this migration, the mechanisms involved in controlling the switch in phenotype of cell infiltrate remain unclear. The present study investigates a potential role for the Th1-type cytokine IFN-gamma in the process of leukocyte recruitment into the peritoneal cavity. Stimulation of cultured human peritoneal mesothelial cells with IFN-gamma (1-100 U/ml) alone or in combination with IL-1beta (100 pg/ml) or TNF-alpha (1000 pg/ml) resulted in significant up-regulation of monocyte chemoattractant protein-1 and RANTES protein secretion. In contrast, IFN-gamma inhibited basal and IL-1beta-, and TNF-alpha-induced production of IL-8. The modulating effects of IFN-gamma on chemokine production occurred at the level of gene expression, and the degree of regulation observed was dependent on the doses of IL-1beta and TNF-alpha used. Analysis of the functional effects of IFN-gamma on IL-1beta-induced transmesothelial PMN migration with an in vitro human transmigration system and an in vivo murine model of peritoneal inflammation demonstrated that IFN-gamma was able to down-regulate PMN migration induced by optimal doses of IL-1beta. These effects were mediated in vivo via down-regulation of CXC chemokine synthesis. These findings suggest that IFN-gamma may play a role in controlling the phenotype of infiltrating leukocyte during the course of an inflammatory response, in part via regulation of resident cell chemokine synthesis.     

Structural Insights into the Lipid A Transport Pathway in MsbA

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High resolution crystal structures of the trans-enamine intermediates formed by sulbactam and clavulanic acid and E166A SHV-1 {beta}-lactamase

Antibiotic resistance mediated by constantly evolving beta-lactamases is a serious threat to human health. The mechanism of inhibition of these enzymes by therapeutic beta-lactamase inhibitors is probed using a novel approach involving Raman microscopy and x-ray crystallography. We have presented here the high resolution crystal structures of the beta-lactamase inhibitors sulbactam and clavulanic acid bound to the deacylation-deficient E166A variant of SHV-1 beta-lactamase. Our previous Raman measurements have identified the trans-enamine species for both inhibitors and were used to guide the soaking time and concentration to achieve full occupancy of the active sites. The two inhibitor-bound x-ray structures revealed a linear trans-enamine intermediate covalently attached to the active site Ser-70 residue. This intermediate was thought to play a key role in the transient inhibition of class A beta-lactamases. Both the Raman and x-ray data indicated that the clavulanic acid intermediate is decarboxylated. When compared with our previously determined tazobactam-bound inhibitor structure, our new inhibitor-bound structures revealed an increased disorder in the tail region of the inhibitors as well as in the enamine skeleton. The x-ray crystallographic observations correlated with the broadening of the O-C=C-N (enamine) symmetric stretch Raman band near 1595 cm(-1). Band broadening in the sulbactam and clavulanic acid inter-mediates reflected a heterogeneous conformational population that results from variations of torsional angles in the O-(C=O)-C=C=NH-C skeleton. These observations led us to conclude that the conformational stability of the trans-enamine form is critical for their transient inhibitory efficacy.