Hepatic triglyceride contents are genetically determined in mice: results of a strain survey

To assess whether genetic factor(s) determine liver triglyceride (TG) levels, a 10-mouse strain survey of liver TG contents was performed. Hepatic TG contents were highest in BALB/cByJ, medium in C57BL/6J, and lowest in SWR/J in both genders. Ninety and seventy-six percent of variance in hepatic TG in males and females, respectively, was due to strain (genetic) effects. To understand the physiological/biochemical basis for differences in hepatic TG among the three strains, studies were performed in males of the BALB/cByJ, C57BL/6J, and SWR/J strains. In vivo hepatic fatty acid (FA) synthesis rates and hepatic TG secretion rates ranked BALB/cByJ approximately C57BL/6J > SWR/J. Hepatic 1-(14)C-labeled palmitate oxidation rates and plasma beta-hydroxybutyrate concentrations ranked in reverse order: SWR/J > BALB/cByJ approximately C57BL/6J. After 14 h of fasting, plasma-free FA and hepatic TG contents rose most in BALB/cByJ and least in SWR/J. beta-Hydroxybutyrate concentrations rose least in BALB/cByJ and most in SWR/J. Adaptation to fasting was most effective in SWR/J and least in BALB/cByJ, perhaps because BALB/cByJ are known to be deficient in SCAD, a short-chain FA oxidizing enzyme. To assess the role of insulin action, glucose tolerance test (GTT) was performed. GTT-glucose levels ranked C57BL/6J > BALB/cByJ approximately SWR/J. Thus strain-dependent (genetic) factors play a major role in setting hepatic TG levels in mice. Processes such as FA production and hepatic export in VLDL on the one hand and FA oxidation on the other, explain some of the strain-related differences in hepatic TG contents. Additional factor(s) in the development of fatty liver in BALB/cByJ remain to be demonstrated.     

Design of a data model for developing laboratory information management and analysis systems for protein production

Data management has emerged as one of the central issues in the high-throughput processes of taking a protein target sequence through to a protein sample. To simplify this task, and following extensive consultation with the international structural genomics community, we describe here a model of the data related to protein production. The model is suitable for both large and small facilities for use in tracking samples, experiments, and results through the many procedures involved. The model is described in Unified Modeling Language (UML). In addition, we present relational database schemas derived from the UML. These relational schemas are already in use in a number of data management projects.     

Novel insulin-elicited phosphoproteins in adipocytes

Akt is a key insulin-activated protein kinase. We searched for Akt substrates in 3T3-L1 adipocytes by means of immunoprecipitation with an Akt phosphomotif-specific antibody (PAS antibody). Four insulin-elicited phosphoproteins were isolated and identified by mass spectrometry. The identity of each protein was established by isolating the protein from lysates of untreated and insulin-treated adipocytes with an antibody specific for the protein and showing that the PAS antibody reacted only with the protein in the immunoprecipitate from insulin-treated cells. These proteins have sizes of 47, 75, 105, and 250 kDa on SDS PAGE, and have been designated pp47, 75, 105, and 250. The effect of inhibitors on the phosphorylation of the proteins, the identified sites of phosphorylation, and in vitro phosphorylation by recombinant Akt further indicated that pp47, 105, and 250 are likely to be Akt substrates, whereas pp75 may not be. pp47 and 105 are novel proteins with no known or predicted function. pp75 was previously found as a protein that associated with the colony-stimulating factor receptor, designated as Fms-interacting protein. pp250 is a novel protein with a predicted GTPase activating protein (GAP) domain for Rheb and/or Rap at its carboxy terminus. The subcellular and tissue distributions of the four proteins were determined.     

Structure of the chromo barrel domain from the MOF acetyltransferase

We report here the structure of the putative chromo domain from MOF, a member of the MYST family of histone acetyltransferases that acetylates histone H4 at Lys-16 and is part of the dosage compensation complex in Drosophila. We found that the structure of this domain is a beta-barrel that is distinct from the alpha + beta fold of the canonical chromo domain. Despite the differences, there are similarities that support an evolutionary relationship between the two domains, and we propose the name "chromo barrel." The chromo barrel domains may be divided into two groups, MSL3-like and MOF-like, on the basis of whether a group of conserved aromatic residues is present or not. The structure suggests that, although the MOF-like domains may have a role in RNA binding, the MSL3-like domains could instead bind methylated residues. The MOF chromo barrel shares a common fold with other chromatin-associated modules, including the MBT-like repeat, Tudor, and PWWP domains. This structural similarity suggests a probable evolutionary pathway from these other modules to the canonical chromo domains (or vice versa) with the chromo barrel domain representing an intermediate structure.     

Valence and anion binding of bovine ribonuclease A between pH 6 and 8

Several studies have shown that divalent anion binding to ribonuclease A (RNase A) contributes to RNase A folding and stability. However, there are conflicting reports about whether chloride binds to or stabilizes RNase A. Two broad-zone experimental approaches, membrane-confined electrophoresis and analytical ultracentrifugation, were used to examine the electrostatic and electrohydrodynamic characteristics of aqueous solutions of bovine RNase A in the presence of 100 mM KCl and 10 mM Bis-Tris propane over a pH range of 6.00-8.00. The results of data analysis using a Debye-Huckel-Henry model, compared with expectations based on pK(A) values, are consistent with the binding of two chlorides by RNase A. The decreased protein valence resulting from anion binding contributes 2-3 kJ/mol to protein stabilization. This work demonstrates the utility of first-principle valence determinations to detect protein solution properties that might otherwise remain undetected.     

Fatty liver in familial hypobetalipoproteinemia: roles of the APOB defects, intra-abdominal adipose tissue, and insulin sensitivity

Fatty liver is frequent in the apolipoprotein B (apoB)-defective genetic form of familial hypobetalipoproteinemia (FHBL), but interindividual variability in liver fat is large. To explain this, we assessed the roles of metabolic factors in 32 affected family members with apoB-defective FHBL and 33 related and unrelated normolipidemic controls matched for age, sex, and indices of adiposity. Two hour, 75 g oral glucose tests, with measurements of plasma glucose and insulin levels, body mass index, and waist-hip ratios were obtained. Abdominal subcutaneous, intraperitoneal (IPAT), and retroperitoneal adipose tissue masses were quantified by MR imaging, and hepatic fat was quantified by MR spectroscopy. Mean +/- SD liver fat percentage values of FHBL and controls were 14.8 +/- 12.0 and 5.2 +/- 5.9, respectively (P = 0.001). Means for these measures of obesity and insulin action were similar in the two groups. Important determinants of liver fat percentage were FHBL-affected status, IPAT, and area under the curve (AUC) insulin in both groups, but the strongest predictors were IPAT in FHBL (partial R(2) = 0.55, P < 0.0002) and AUC insulin in controls (partial R(2) = 0.59, P = 0.0001). Regression of liver fat percentage on IPAT fat was significantly greater for FHBL than for controls (P < 0.001). In summary, because apoB-defective FHBL imparts heightened susceptibility to liver triglyceride accumulation, increasing IPAT and insulin resistance exert greater liver fat-increasing effects in FHBL.     

Mechanical integrin stress and magnetic forces induce biological responses in mesenchymal stem cells which depend on environmental factors

The control of mesenchymal stem cells (MSC) by physical cues is of great interest in regenerative medicine. Because integrin receptors function as mechanotransducers, we applied drag forces to β1 integrins on the apical surface of adherent human MSC. In addition to mechanical forces, the technique we used involved also the exposure of the cells to an inhomogeneous magnetic field. In order to assess the influence of the substrate on cell adhesion, cells were cultured on plain tissue culture polystyrene (TCP) or on coated well plates, which allowed only adhesion to embedded fibronectin or RGD peptides. We found that the expression of collagen I, which is involved in osteogenesis, and VEGF, a factor which stimulates angiogenesis, increased as a result of short-term mechanical integrin stress. Whereas, collagen I expression was stimulated by mechanical forces when the cells were cultured on fibronectin and RGD peptides but not on TCP, VEGF expression was enhanced by physical stimulation on TCP. The study further revealed that magnetic forces enhanced Sox 9 expression, a marker of chondrogenesis, and reduced the expression of ALP. Concerning the intracellular mechanisms involved, we found that the expression of VEGF induced by physical forces depended on Akt activation. Together, the results implicate that biological functions of MSC can be stimulated by integrin-mediated mechanical forces and a magnetic field. However, the responses of cells depend strongly on the substrate to which they adhere and on the cross-talk between integrin-mediated signals and soluble factors.     

A targeted apoB38.9 mutation in mice is associated with reduced hepatic cholesterol synthesis and enhanced lipid peroxidation

Familial hypobetalipoproteinemia (FHBL) due to truncation-specifying mutations of apolipoprotein B (apoB), which impair hepatic lipid export in very low-density lipoprotein (VLDL) particles, is associated with fatty liver. In an FHBL-like mouse with the apoB38.9 mutation, fatty liver develops despite reduced hepatic fatty acid synthesis. However, hepatic cholesterol contents in apoB38.9 mice are normal. We found that cholesterogenic enzymes (3-hydroxy-3-methylglutaryl-coenzyme A reductase, sterol-C5-desaturase, and 7-dehydrocholesterol reductase) were consistently downregulated in two separate expression-profiling experiments using a total of 19 mice (n = 7 each for apob(+/+) and apob(+/38.9), and n = 5 for apob(38.9/38.9)) and Affymetrix Mu74Av2 GeneChip microarrays. Results were confirmed by real-time PCR. Cholesterol synthesis rates in cultured hepatocytes were reduced by 35% and 25% in apob(38.9/38.9) and apob(+/38.9), respectively, vs. apob(+/+). Hepatic triglycerides and lipid peroxides, the latter measured by thiobarbituric acid-reactive substances (TBARS) assay, were significantly elevated in apob(+/38.9) (117%) and apob(38.9/38.9) (132%) vs. apob(+/+) (100%), as were mRNA expression of the microsomal lipid peroxidizing enzymes Cyp4A10 and Cyp4A14. Hepatic lipid peroxide levels were positively correlated with triglyceride contents (r = 0.601, P = 0.0065). Thus the fatty liver due to a VLDL secretion defect is associated with insufficient adaptation to triglyceride accumulation and with increased lipid peroxidation. In contrast, apoB38.9 mice effectively maintain cholesterol homeostasis in the liver, at least in part, by reducing hepatic cholesterol synthesis.     

Immuno-gene therapy of cancer with tumour-mRNA transfected dendritic cells

We have developed immuno-gene therapy for malignant melanoma and prostate cancer. The therapy is based on monocyte-derived dendritic cells (DCs) that are transfected with autologous melanoma-mRNA or mRNA from three prostate cancer cell lines (DU-145, LN-CaP and PC-3). A broad spectrum of tumour-associated antigens will be included in both DC-vaccines. The use of autologous melanoma-mRNA moreover allows targeting of individual tumour antigens that are specific to each patient. Effective protocols have been established for mRNA-transfection by square wave electroporation and for the generation of clinical grade DCs. A full scale preclinical evaluation demonstrated in vitro T cell responses in 6/6 advanced melanoma patients. The responses were specific to antigens encoded by the transfected tumour-mRNA. Recently, we have conducted two phase I/II trials, in advanced malignant melanoma and androgen-resistant prostate cancer. Successful vaccine preparations were obtained for all 41 patients elected. No serious adverse effects were observed. Specific T cell responses (T cell proliferation and/or IFNγ ELISPOT) were demonstrated in 9/19 evaluable melanoma patients and in 12/19 prostate cancer patients. The response rates were higher for patients receiving intradermal vaccination, compared to intranodal injection. Thirteen prostate cancer patients developed a decrease in log-slope PSA. The PSA-response was significantly related to the T cell response (P=0.002). We conclude that the DC-vaccine is feasible and safe, and that T cell responses are elicited in about 50% of patients.This article is a symposium paper from the Annual Meeting of the “International Society for Cell and Gene Therapy of Cancer”, held in Shenzhen, China, on 9–11 December 2005.