The development of endothermy during pouch life in the eastern barred bandicoot (Perameles gunnii), a marsupial

Marsupials are born ectothermic and gradually become endothermic during pouch occupancy. In order to study the timing of this transition, we measured the metabolic rates of eight pouch-young eastern barred bandicoots (Perameles gunnii) at 25 degrees C (the thermoneutral zone of adults), at 35 degrees C (pouch temperature), and after injection with norepinephrine. From 5 to 7 wk of age, oxygen consumption (VO(2)) was higher at 35 degrees C than at 25 degrees C. At 8 wk, VO(2) was significantly higher at 25 degrees C than at 35 degrees C. Norepinephrine administration at 5 and 6 wk had no effect on metabolic rate, but at 8 wk, near pouch vacation, it resulted in a significant increase in metabolic rate. Our results indicate that signs of thermoregulation for P. gunnii begin at 7 wk, and at 8 wk the species is endothermic, that is, able to increase heat production in response to both cold and norepinephrine. The appearance of fur, first seen at the age of 6 wk and fully developed at 8 wk, coincides with the onset of endothermy.     

From natural products to polymeric derivatives of "eugenol": a new approach for preparation of dental composites and orthopedic bone cements

Polymers with eugenol moieties covalently bonded to the macromolecular chains were synthesized for potential application in orthopedic and dental cements. First, eugenol was functionalized with polymerizable groups. The synthetic methods employed afforded two different methacrylic derivatives, where the acrylic and eugenol moieties were either directly bonded, eugenyl methacrylate (EgMA), or separated through an oxyethylene group, ethoxyeugenyl methacrylate (EEgMA). A typical Fisher esterification reaction was used for the synthesis of EgMA and EEgMA, affording the desired monomers in 80% yields. Polymerization of each of the novel monomers, at low conversion, provided soluble polymers consisting of hydrocarbon macromolecules with pendant eugenol moieties. At high conversions only cross-linked polymers were obtained, attributed to participation of the allylic double bonds in the polymerization reaction. In addition, copolymers of each eugenol derivative with ethyl methacrylate (EMA) were prepared at low conversion, with the copolymerization reaction studied by assuming the terminal model and the reactivity ratios determined according to linear and nonlinear methods. The values obtained were r(EgMA) = 1.48, r(EMA) = 0.55 and r(EEgMA) = 1.22, r(EMA) = 0.42. High molecular weight polymers and copolymers were obtained at low conversion. Analysis of thermal properties revealed a T(g) of 95 degrees C for PEgMA and of 20 degrees C for PEEgMA and an increase in the thermal stability for the eugenol derivatives polymers and copolymers with respect to that of PEMA. Water sorption of the copolymers was found to decrease with the eugenol derivative content. Both monomers EgMA and EEgMA showed antibacterial activity against Streptococcus mutans, producing inhibition halos of 7 and 21 mm, respectively. Finally, cell culture studies revealed that the copolymers did not leach any toxic eluants and showed good cellular proliferation with respect to PEMA. This study thus indicates that the eugenyl methacrylate derivatives are potentially good candidates for dental and orthopedic cements.     

Analysis of ESTs from multiple Gossypium hirsutum tissues and identification of SSRs.

In an effort to expand the Gossypium hirsutum L. (cotton) expressed sequence tag (EST) database, ESTs representing a variety of tissues and treatments were sequenced. Assembly of these sequences with ESTs already in the EST database (dbEST, GenBank) identified 9675 cotton sequences not present in GenBank. Statistical analysis of a subset of these ESTs identified genes likely differentially expressed in stems, cotyledons, and drought-stressed tissues. Annotation of the differentially expressed cDNAs tentatively identified genes involved in lignin metabolism, starch biosynthesis and stress response, consistent with pathways likely to be active in the tissues under investigation. Simple sequence repeats (SSRs) were identified among these ESTs, and an inexpensive method was developed to screen genomic DNA for the presence of these SSRs. At least 69 SSRs potentially useful in mapping were identified. Selected amplified SSRs were isolated and sequenced. The sequences corresponded to the EST containing the SSRs, confirming that these SSRs will potentially map the gene represented by the EST. The ESTs containing SSRs were annotated to help identify the genes that may be mapped using these markers.     

Absence of CuZn superoxide dismutase leads to elevated oxidative stress and acceleration of age-dependent skeletal muscle atrophy

We describe a novel phenotype in mice lacking the major antioxidant enzyme, CuZn-superoxide dismutase (Sod1(-/-) mice), namely a dramatic acceleration of age-related loss of skeletal muscle mass. Sod1(-/-) mice are 17 to 20% smaller and have a significantly lower muscle mass than wild-type mice as early as 3 to 4 months of age. Muscle mass in the Sod1(-/-) mice is further reduced with age and by 20 months, the hind-limb muscle mass in Sod1(-/-) mice is nearly 50% lower than in age-matched wild-type mice. Skeletal muscle tissue from young Sod1(-/-) mice has elevated oxidative damage to proteins, lipids, and DNA compared to muscle from young wild-type mice. The reduction in muscle mass and elevated oxidative damage are accompanied by a 40% decrease in voluntary wheel running by 6 months of age and decreased performance on the Rota-rod test at 13 months of age, but are not associated with a decline in overall spontaneous activity. In some of the old Sod1(-/-) mice, the loss in muscle mass is also associated with the presence of tremors and gait disturbances. Thus, the absence of CuZnSOD imposes elevated oxidative stress, loss of muscle mass, and physiological consequences that resemble an acceleration of normal age-related sarcopenia.     

A computational approach toward label-free protein quantification using predicted peptide detectability

We propose here a new concept of peptide detectability which could be an important factor in explaining the relationship between a protein's quantity and the peptides identified from it in a high-throughput proteomics experiment. We define peptide detectability as the probability of observing a peptide in a standard sample analyzed by a standard proteomics routine and argue that it is an intrinsic property of the peptide sequence and neighboring regions in the parent protein. To test this hypothesis we first used publicly available data and data from our own synthetic samples in which quantities of model proteins were controlled. We then applied machine learning approaches to demonstrate that peptide detectability can be predicted from its sequence and the neighboring regions in the parent protein with satisfactory accuracy. The utility of this approach for protein quantification is demonstrated by peptides with higher detectability generally being identified at lower concentrations over those with lower detectability in the synthetic protein mixtures. These results establish a direct link between protein concentration and peptide detectability. We show that for each protein there exists a level of peptide detectability above which peptides are detected and below which peptides are not detected in an experiment. We call this level the minimum acceptable detectability for identified peptides (MDIP) which can be calibrated to predict protein concentration. Triplicate analysis of a biological sample showed that these MDIP values are consistent among the three data sets.     

Glucocorticoid-induced TNF receptor family related gene activation overcomes tolerance/ignorance to melanoma differentiation antigens and enhances antitumor immunity

Glucocorticoid-induced TNF receptor family related protein (GITR) is present on many different cell types. Previous studies have shown that in vivo administration of an anti-GITR agonist mAb (DTA-1) inhibits regulatory T cells (Treg)-dependent suppression and enhances T cell responses. In this study, we show that administration of DTA-1 induces >85% tumor rejection in mice challenged with B16 melanoma. Rejection requires CD4+, CD8+, and NK1.1+ cells and is dependent on IFN-gamma and Fas ligand and independent of perforin. Depletion of Treg via anti-CD25 treatment does not induce B16 rejection, whereas 100% of the mice depleted of CD25+ cells and treated with DTA-1 reject tumors, indicating a predominant role of GITR on effector T cell costimulation rather than on Treg modulation. T cells isolated from DTA-1-treated mice challenged with B16 are specific against B16 and several melanoma differentiation Ags. These mice develop memory against B16, and a small proportion of them develop mild hypopigmentation. Consistent with previous studies showing that GITR stimulation increases Treg proliferation in vitro, we found in our model that GITR stimulation expanded the absolute number of FoxP3+ cells in vivo. Thus, we conclude that overall, GITR stimulation overcomes self-tolerance/ignorance and enhances T cell-mediated antitumor activity with minimal autoimmunity.     

FGF21 N- and C-termini play different roles in receptor interaction and activation

Fibroblast growth factor-21 (FGF21) signaling requires the presence of β-Klotho, a co-receptor with a very short cytoplasmic domain. Here we show that FGF21 binds directly to β-Klotho through its C-terminus. Serial C-terminal truncations of FGF21 weakened or even abrogated its interaction with β-Klotho in a Biacore assay, and led to gradual loss of potency in a luciferase reporter assay but with little effect on maximal response. In contrast, serial N-terminal truncations of FGF21 had no impact on β-Klotho binding. Interestingly, several of them exhibited characteristics of partial agonists with minimal effects on potency. These data demonstrate that the C-terminus of FGF21 is critical for binding to β-Klotho and the N-terminus is critical for fibroblast growth factor receptor (FGFR) activation.

Structured summary

MINT-6799939: FGFR1c (uniprotkb:P11362) binds (MI:0407) to β-Klotho (uniprotkb: Q86Z14) by surface plasmon resonance (MI:0107)MINT-6799907, MINT-6799922: FGF21 (uniprotkb: Q9NSA1) binds (MI:0407) to β-Klotho (uniprotkb: Q86Z14) by surface plasmon resonance (MI:0107)     

Short-time glucose exposure of embryonic carcinoma cells impairs their function as terminally differentiated cardiomyocytes

The fetal and postnatal phenotype is influenced by developmental conditions experienced prenatally. Among prenatal development metabolic factors are of particular importance as they are supposed to predispose for pathophysiological alterations later in life and to pioneer functional impairment in senescence (metabolic programming). Till now the mechanisms of metabolic programming are not well understood. We have investigated various concentrations of glucose during differentiation of pluripotent P19 embryonic carcinoma cells (ECC) into cardiomyocytes. Undifferentiated P19 cells were exposed to 5mM (low), 25 mM (control), 40 mM or 100mM (high) glucose for 48 h during embryoid body (EB) formation, followed by plating and differentiation into cardiomyocytes in vitro with standard glucose supplementation (25 mM) for 10-15 days. The amount of cardiac clusters, the frequency of spontaneous beatings as well as the expression of metabolic and cardiac marker genes and their promoter methylation were measured. We observed a metabolic programming effect of glucose during cardiac differentiation. Whereas the number of beating clusters and the expression of the cardiac marker alpha myosin heavy chain (α-MHC) were comparable in all groups, the frequencies of beating clusters were significantly higher in the high glucose group compared to low glucose. However, neither the insulin receptor (IR) or insulin like growth factor 1 receptor (IGF1R) nor the metabolic gene glucose transporter 4 (GLUT4) were influenced in RNA expression or in promoter methylation. Our data indicate that a short time glucose stress during embryonic cell determination leads to lasting effects in terminally differentiated cell function.