Interleukin-1 regulates FGF-2 mRNA and localization of FGF-2 protein in human osteoblasts

Interleukin-1 (IL-1) and basic fibroblast growth factor (FGF-2) are potent stimulators of osteoclast formation. However, the role of FGF-2 in the responses to IL-1 in bone has not been reported. We examined the effect of IL-1 on FGF-2 mRNA and protein expression in human osteosarcoma MG-63 osteoblasts, normal human osteoblasts (NHOB), and osteoblasts from osteoarthritic patients (F2 and F13). IL-1 increased FGF-2 mRNA expression in osteoblasts within 1.5 to 3 h. Multiple FGF-2 protein isoforms were expressed in human osteoblasts. Twenty-four hours of treatment of MG-63 and NHOB cells with IL-1 increased the high-molecular-weight(HMW, 22/24 kDa) and low-molecular-weight (LMW, 18 kDa) FGF-2 proteins intracellularly. In contrast, IL-1 preferentially increased the LMW protein signal intracellularly as well as on the cell surface of F2 and F13 osteoblasts. We conclude that IL-1 is a major stimulator of FGF-2 expression in human osteoblasts. Furthermore, selective increases in the exportable LMW protein in osteoblasts from osteoarthritic patients may be of clinical relevance.     

Effect of chlorine treatment on infectivity of hepatitis A virus.

This study examined the effect of chlorine treatment on the infectivity of hepatitis A virus (HAV). Prodromal chimpanzee feces, shown to induce hepatitis in marmosets (Saguinus sp.), was clarified, and the virus was precipitated with 7% polyethylene glycol 6000, harvested, and resuspended. The suspension was layered onto 5 to 30% linear sucrose gradients and centrifuged; the fractions containing HAV were dialyzed, and a 1:500,000 dilution of this preparation induced hepatitis and seroconversion in 2 of 4 marmosets. A 1:50 dilution of this preparation served as inoculum. Untreated inoculum induced overt hepatitis and seroconversion in 100% (5 of 5) of marmosets inoculated intramuscularly. Inoculum treated for various periods (15, 30, or 60 min) with 0.5, 1.0, or 1.5 mg of free residual chlorine per liter induced hepatitis in 14% (2 of 14), 8% (1 of 12), and 10% (1 of 10) of marmosets, respectively, and induced seroconversion in 29, 33, and 10% of the animals. Inoculum treated with 2.0 or 2.5 mg of free residual chlorine per liter was not infectious in marmosets as determined by absence of hepatitis and seroconversion in the 13 animals tested. Thus, treatment levels of 0.5 to 1.5 mg of free residual chlorine per liter inactivated most but not all HAV in the preparation, whereas concentrations of 2.0 and 2.5 mg of free residual chlorine per liter destroyed the infectivity completely. These results suggest that HAV is somewhat more resistant to chlorine than are other enteroviruses.     

Pyrithiamine as a substrate for thiamine pyrophosphokinase

Thiamine pyrophosphokinase transfers a pyrophosphate group from a nucleoside triphosphate, such as ATP, to the hydroxyl group of thiamine to produce thiamine pyrophosphate. Deficiencies in thiamine can result in the development of the neurological disorder Wernicke-Korsakoff Syndrome as well as the potentially fatal cardiovascular disease wet beriberi. Pyrithiamine is an inhibitor of thiamine metabolism that induces neurological symptoms similar to that of Wernicke-Korsakoff Syndrome in animals. However, the mechanism by which pyrithiamine interferes with cellular thiamine phosphoester homeostasis is not entirely clear. We used kinetic assays coupled with mass spectrometry of the reaction products and x-ray crystallography of an equilibrium reaction mixture of thiamine pyrophosphokinase, pyrithiamine, and Mg2+/ATP to elucidate the mechanism by which pyrithiamine inhibits the enzymatic production of thiamine pyrophosphate. Three lines of evidence support the ability of thiamine pyrophosphokinase to form pyrithiamine pyrophosphate. First, a coupled enzyme assay clearly demonstrated the ability of thiamine pyrophosphokinase to produce AMP when pyrithiamine was used as substrate. Second, an analysis of the reaction mixture by mass spectrometry directly identified pyrithiamine pyrophosphate in the reaction mixture. Last, the structure of thiamine pyrophosphokinase crystallized from an equilibrium substrate/product mixture shows clear electron density for pyrithiamine pyrophosphate bound in the enzyme active site. This structure also provides the first clear picture of the binding pocket for the nucleoside triphosphate and permits the first detailed understanding of the catalytic requirements for catalysis in this enzyme.     

Variation in the temperature preference and growth rate of individual fish reconciles differences between two growth models

1. A growth model, originally developed for brown trout (Salmo trutta), has now been fitted to data for Atlantic salmon (S. salar) and stone‐loach (Barbatula barbatula) from English populations, and Arctic charr (Salvelinus alpinus) from Sweden. The model relates growth rate to temperature for a fish of standard size and the functional relationship has a triangular shape with a sharp peak at the optimal temperature for growth and zero growth at the base of the triangle. It was unsuitable for growth data for Norwegian salmon, and a curvilinear Ratkowsky model provided a better fit, though the experimental protocol was different in the Norwegian and English experiments. 2. The Norwegian salmon were kept in groups in each tank, had to compete for food, and had to be divided into slow, moderate and fast growers before the Ratkowsky model could be fitted. Each English salmon was kept in its own tank and fed individually. For replicate experiments, fish of similar size were selected. Variation among fish kept under similar conditions was therefore small, and the triangular model was essentially for individual fish, not groups of fish. 3. The present simulation study tests the hypothesis that individual differences in the growth response could account for the curvilinear growth‐temperature relationship for the Norwegian salmon. The triangular model was used to generate the growth response to temperature for a group of salmon, each fish having a slightly different temperature preference and growth rate. The result was a curvilinear response, well approximated by the Ratkowsky model (adjusted R² = 0.96). When the variability in individual temperature preference was increased, the Ratkowsky model was an even better fit (adjusted R² = 0.98). Therefore, the apparent discrepancy between the two models was reconciled by allowing for individual differences in temperature preference and growth rate within groups of fish.     

Does age, sex, or ACE genotype affect glucose and insulin responses to strength training?

The purpose of the present study was to determine whether age, sex, or angiotensin I-converting enzyme (ACE) genotype influences the effects of strength training (ST) on glucose homeostasis. Nineteen sedentary young (age = 20-30 yr) men (n = 10) and women (n = 9) were studied and compared with 21 sedentary older (age = 65-75 yr) men (n = 12) and women (n = 9) before and after a 6-mo total body ST program. Fasting insulin concentrations were reduced in young men and in older men with ST (P < 0.05 in both). In addition, total insulin area under the curve decreased by 21% in young men (P < 0.05), and there was a trend for a decrease (11%) in older men (P = 0.06). No improvements in insulin responses were observed in young or older women. The ACE deletion/deletion genotype group had the lowest fasting insulin and insulin areas under the oral glucose tolerance test (OGTT) curve before training (all P < 0.05), but those with at least one insertion allele had a trend for a greater reduction in total insulin area than deletion homozygotes (P = 0.07). These results indicate that ST has a more favorable effect on insulin response to an OGTT in men than in women and offer some support for the hypothesis that ACE genotype may influence insulin responses to ST.     

Prostaglandin F2α: A bone remodeling mediator

Prostaglandin F2α (PGF2α) plays multiple roles on bone metabolism by regulating a wide range of signaling pathways. PGF2α, via activation of PKC, stimulates Na‐dependent inorganic phosphate (Pi) transport system in osteoblasts; up‐regulates interleukin (IL)‐6 synthesis; increases vascular endothelial growth factor (VEGF). In addition, PGF2α acts as a strong mitogenic and survival agent on osteoblasts, and these effects are, at least in part, mediated by the binding of fibroblast growth factor‐2 (FGF‐2) to the specific receptor FGFR1. The understanding of PGF2α intracellular network, albeit complex to clarify, provides molecular bases useful to identify the players of osteoblast proliferation, apoptosis, and the associated angiogenic processes. Indeed, the molecular mechanism that underline PGF2α‐regulated bone metabolism may be a promising platform for the development of novel targeted therapies in the treatment of bone disorders and disease. J. Cell. Physiol. 228: 25–29, 2013. © 2012 Wiley Periodicals, Inc.     

Disentangling Ethnic and Contextual Influences Among Parents Raising Youth in High-Risk Communities

This article reports on analyses examining contextual influences on parenting with an ethnically and geographically diverse sample of parents (predominantly mothers) raising 387 children (49% ethnic minority; 51% male) in high-risk communities. Parents and children were followed longitudinally from first through tenth grades. Contextual influences included geographical location, neighborhood risk, SES, and family stress. The cultural variable was racial socialization. Parenting constructs created through the consensus decision-making of the Parenting Subgroup of the Study Group on Race, Culture, and Ethnicity (see Le et al., 2008) included Monitoring, Communication, Warmth, Behavioral Control and Parenting Efficacy. Hierarchical regressions on each parenting construct were conducted for each grade for which data were available. Analyses tested for initial ethnic differences and then for remaining ethnic differences once contextual influences were controlled. For each construct, some ethnic differences did remain (Monitoring, ninth grade; Warmth, third grade; Communication, kindergarten; Behavioral Control, eighth grade; and Parenting Efficacy, kindergarten through fifth grade). Ethnic differences were explained by contextual differences in the remaining years. Analyses examining the impact of cultural influences revealed a negative relation between racial socialization messages and Communication or Monitoring.     

Binding of G-Quadruplex-interactive Agents to Distinct G-Quadruplexes Induces Different Biological Effects in MiaPaCa Cells

Our previous studies have demonstrated the preference of telomestatin for intramolecular, rather than the intermolecular, G-quadruplex structures, while TMPyP4 has selectivity for intermolecular over intramolecular G-quadruplex structures. However, it was not clear whether the difference in the selectivity between two different G-quadruplex-interactive agents could determine the corresponding biological effects in cultured human tumor cells. Here we evaluated the biological effects of both TMPyP4 and telomestatin in the human pancreatic carcinoma cell line (MiaPaCa) using subtoxic and cytotoxic concentrations. The cytotoxicity of these agents against MiaPaCa cells is quite different, and the IC ₅₀ of telomestatin (0.5 μM) is about 100 times less than that of TMPyP4 (50 μM). At IC ₅₀ concentrations, TMPyP4 induced anaphase bridge formation in MiaPaCa cells, while telomestatin failed to induce anaphase bridge formation. At subtoxic concentrations, TMPyP4 induced MiaPaCa cell growth arrest, senescence, apoptosis, and telomere length shortening within 5 weeks, while similar biological effects were evident after 12 weeks following treatment with telomestatin. Our data suggest that binding of G-quadruplex-interactive agents to distinct G-quadruplexes could induce different biological effects in human cancer cells.     

Analysis and Optimization of the Cationic Lipid Component of a Lipid/Peptide Vector Formulation for Enhanced Transfection In Vitro and In Vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K₁₆GACRRETAWACG) and Lipofectin^®, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.