The 5'-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle

5'-AMP-activated protein kinase (AMPK) is a metabolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific gamma3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK gamma3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse gamma3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK gamma3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK gamma3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK gamma3 isoform as a therapeutic target for prevention and treatment of insulin resistance.     

Mepyramine, a histamine H1 receptor inverse agonist, binds preferentially to a G protein-coupled form of the receptor and sequesters G protein

Accurate characterization of the molecular mechanisms of the action of ligands is an extremely important issue for their appropriate research, pharmacological, and therapeutic uses. In view of this fact, the aim of the present work was to investigate the mechanisms involved in the actions of mepyramine at the guinea pig H(1) receptor stably expressed in Chinese hamster ovary cells. We found that mepyramine is able to decrease the basal constitutive activity of the guinea pig H(1) receptor, to bind with high affinity to a G(q/11) protein-coupled form of the receptor and to promote a G protein-coupled inactive state of the H(1) receptor that interferes with the G(q/11)-mediated signaling of the endogenously expressed ATP receptor, as predicted by the Cubic Ternary Complex Model of receptor occupancy. The effect of mepyramine on ATP-induced signaling was specifically neutralized by Galpha(11) overexpression, indicating that mepyramine is able to reduce G protein availability for other non-related receptors associated with the same signaling pathway. Finally, we found a loss of mepyramine efficacy in decreasing basal levels of intracellular calcium at high Galpha(11) expression levels, which can be theoretically explained in terms of high H(1) receptor constitutive activity. The whole of the present work sheds new light on H(1) receptor pharmacology and the mechanisms H(1) receptor inverse agonists could use to exert their observed negative efficacy.     

The IL-6 promoter polymorphism is associated with disease activity and disability in systemic sclerosis

Systemic sclerosis (SSc) is a rare, autoimmune disease characterized by cutaneous and visceral fibrosis. Interleukin- 6 (IL-6) is involved in the pathogenesis of many immune-mediated diseases. IL-6 plays an important role in the initiation and promotion of fibrosis. The polymorphism in the position -174 (G/C) of the promoter region of the IL-6 gene (IL-6pr) may alter the expression of the gene. Complete linkage disequilibrium was observed between the -174 and -597 alleles. The aim of this study is to investigate the possible influence of -597 (-174) IL-6pr polymorphism on the susceptibility and/or the clinical course of SSc in Romanian population. Genotyping of -597 variant was performed by an RFLP method on 20 SSc patients and 26 healthy subjects. Patients having the homozygous GG (-597) genotype had higher disease activity and disability scores than heterozygous GA patients: the European Scleroderma Study Group (EScSG) disease activity score was 5.0 +/- 3.3 in homozygous GG subjects vs. 2.4 +/- 3.6 in heterozygous GA patients (p < 0.05), and the Disability Index of the Health Assessment Questionnaire (HAQ-DI) was 1.42 +/- 1.04 in homozygous GG subjects vs. 0.53 +/- 0.55 in heterozygous GA patients (p < 0.05). No difference was observed in the distribution of allele frequencies between SSc patients and healthy controls. Conclusions: The GG homozygosis was found to be associated with a higher degree of illness activity and disability in SSc patients. No statistically significant differences were found between SSc patients and healthy controls with respect to the -597 allele distribution.     

Correlation of protein expression, Gleason score and DNA ploidy in prostate cancer

The prognosis of prostate cancer correlates with tumor differentiation. Gleason score and DNA ploidy are two prognostic factors that correlate with prognosis. We analyzed differences in protein expression in prostate cancer of high and low aggressiveness according to these measures. From 35 prostatectomy specimens, 29 cancer samples and 10 benign samples were harvested by scraping cells from cut surfaces. DNA ploidy was assessed by image cytometry. Protein preparations from cell suspensions were examined by 2-DE. Protein spots that differed quantitatively between sample groups were identified by MS fingerprinting of tryptic fragments and MS/MS sequence analysis. We found 39 protein spots with expression levels that were raised or lowered in correlation with Gleason score and/or DNA ploidy pattern (31 overexpressed in high-malignant cancer, 8 underexpressed). Of these, 30 were identified by MS. Among overexpressed proteins were heat-shock, structural and membrane proteins and enzymes involved in gene silencing, protein synthesis/degradation, mitochondrial protein import (metaxin 2), detoxification (GST-pi) and energy metabolism. Stroma-associated proteins were generally underexpressed. The protein expression of prostate cancer correlates with tumor differentiation. Potential prognostic markers may be found among proteins that are differentially expressed and the clinical value of these should be validated.     

Generation of stable cell clones expressing mixtures of human antibodies

Therapeutic monoclonal antibodies, a highly successful class of biological drugs, are conventionally manufactured in mammalian cell lines. A recent approach to increase the therapeutic effectiveness of monoclonal antibodies has been to combine two or more of them; however this increases the complexity of development and manufacture. To address this issue a method to efficiently express multiple monoclonal antibodies from a single cell has been developed and we describe here the generation of stable cell clones that express high levels of a human monoclonal antibody mixture. PER.C6® cells were transfected with a combination of plasmids containing genes encoding three different antibodies. Clones that express the three corresponding antibody specificities were identified, subcloned, and passaged in the absence of antibiotic selection pressure. At several time points, batch production runs were analyzed for stable growth and IgG production characteristics. The majority (11/12) of subclones analyzed expressed all three antibody specificities in constant ratios with total IgG productivity ranging between 15 and 20 pg/cell/day under suboptimal culture conditions after up to 67 population doublings. The growth and IgG production characteristics of the stable clones reported here resemble those of single monoclonal antibody cell lines from conventional clone generation programs. We conclude that the methodology described here is applicable to the generation of stable PER.C6® clones for industrial scale production of mixtures of antibodies. Biotechnol. Bioeng. 2010;106: 741–750. © 2010 Wiley Periodicals, Inc.     

CXCL10 is the key ligand for CXCR3 on CD8+ effector T cells involved in immune surveillance of the lymphocytic choriomeningitis virus-infected central nervous system

IFN-gamma-inducible protein 10/CXCL10 is a chemokine associated with type 1 T cell responses, regulating the migration of activated T cells through binding to the CXCR3 receptor. Expression of both CXCL10 and CXCR3 are observed during immunopathological diseases of the CNS, and this receptor/ligand pair is thought to play a central role in regulating T cell-mediated inflammation in this organ site. In this report, we investigated the role of CXCL10 in regulating CD8(+) T cell-mediated inflammation in the virus-infected brain. This was done through analysis of CXCL10-deficient mice infected intracerebrally with lymphocytic choriomeningitis virus, which in normal immunocompetent mice induces a fatal CD8(+) T cell-mediated meningoencephalitis. We found that a normal antiviral CD8(+) T cell response was generated in CXCL10-deficient mice, and that lack of CXCL10 had no influence on the accumulation of mononuclear cells in the cerebrospinal fluid. However, analysis of the susceptibility of CXCL10-deficient mice to lymphocytic choriomeningitis virus-induced meningitis revealed that these mice just like CXCR3-deficient mice were partially resistant to this disease, whereas wild-type mice invariably died. Furthermore, despite marked up-regulation of the two remaining CXCR3 ligands: CXCL9 and 11, we found a reduced accumulation of CD8(+) T cells in the brain parenchyma around the time point when wild-type mice succumb as a result of CD8(+) T cell-mediated inflammation. Thus, taken together these results indicate a central role for CXCL10 in regulating the accumulation of effector T cells at sites of CNS inflammation, with no apparent compensatory effect of other CXCR3 ligands.