Pancreatic β cell identity is maintained by DNA methylation-mediated repression of Arx

Adult pancreatic β cells can replicate during growth and after injury to maintain glucose homeostasis. Here, we report that β cells deficient in Dnmt1, an enzyme that propagates DNA methylation patterns during cell division, were converted to α cells. We identified the lineage determination gene aristaless-related homeobox (Arx), as methylated and repressed in β cells, and hypomethylated and expressed in α cells and Dnmt1-deficient β cells. We show that the methylated region of the Arx locus in β cells was bound by methyl-binding protein MeCP2, which recruited PRMT6, an enzyme that methylates histone H3R2 resulting in repression of Arx. This suggests that propagation of DNA methylation during cell division also ensures recruitment of enzymatic machinery capable of modifying and transmitting histone marks. Our results reveal that propagation of DNA methylation during cell division is essential for repression of α cell lineage determination genes to maintain pancreatic β cell identity.     

Activation of anti-tumor immune response and reduction of regulatory T cells with Mycobacterium indicus pranii (MIP) therapy in tumor bearing mice

Background

Role of immune system in protecting the host from cancer is well established. Growing cancer however subverts immune response towards Th2 type and escape from antitumor mechanism of the host. Activation of both innate and Th1 type response is crucial for host antitumor activity. In our previous study it was found, that Mycobacterium indicus pranii (MIP) also known as M. w induces Th1 type response and activates macrophages in animal model of tuberculosis. Hence, we studied the immunotherapeutic potential of MIP in mouse tumor model and the underlying mechanisms for its antitumor activity.

Methodology and Principal Findings

Tumors were implanted by injecting B16F10 melanoma cells subcutaneously into C57BL/6 mice. Using the optimized dose and treatment regimes, anti-tumor efficacy of heat killed MIP was evaluated. In MIP treated group, tumor appeared in only 50–60% of mice, tumor growth was delayed and tumor volume was less as compared to control. MIP mediated immune activation was analysed in the tumor microenvironment, tumor draining lymph node and spleen. Induction of Th1 response and higher infiltration of immune cells in the tumor microenvironment was observed in MIP treated mice. A large fraction of these immune cells were in activated state as confirmed by phenotypic and functional analysis. Interestingly, percentage of Treg cells in the tumor milieu of treated mice was less. We also evaluated efficacy of MIP along with chemotherapy and found a better response as compared to chemotherapy alone.

Conclusion

MIP therapy is effective in protecting mice from tumor. It activates the immune cells, increases their infiltration in tumor, and abrogates tumor mediated immune suppression.     

Environmental and auxin regulation of wood formation involves members of the Aux/IAA gene family in hybrid aspen

Indole acetic acid (IAA/auxin) profoundly affects wood formation but the molecular mechanism of auxin action in this process remains poorly understood. We have cloned cDNAs for eight members of the Aux/IAA gene family from hybrid aspen (Populus tremula L. x Populus tremuloides Michx.) that encode potential mediators of the auxin signal transduction pathway. These genes designated as PttIAA1-PttIAA8 are auxin inducible but differ in their requirement of de novo protein synthesis for auxin induction. The auxin induction of the PttIAA genes is also developmentally controlled as evidenced by the loss of their auxin inducibility during leaf maturation. The PttIAA genes are differentially expressed in the cell types of a developmental gradient comprising the wood-forming tissues. Interestingly, the expression of the PttIAA genes is downregulated during transition of the active cambium into dormancy, a process in which meristematic cells of the cambium lose their sensitivity to auxin. Auxin-regulated developmental reprogramming of wood formation during the induction of tension wood is accompanied by changes in the expression of PttIAA genes. The distinct tissue-specific expression patterns of the auxin inducible PttIAA genes in the cambial region together with the change in expression during dormancy transition and tension wood formation suggest a role for these genes in mediating cambial responses to auxin and xylem development.     

Clinical Utility of Waist Circumference in Predicting All‐cause Mortality in a Preventive Cardiology Clinic Population: A PreCIS Database Study

Although obesity is a risk factor for mortality, it is unclear whether waist circumference (WC) is a better predictor of mortality than BMI in a clinical setting of patients at high risk for coronary artery disease (CAD). Thus, we compared the association between WC and BMI with all‐cause mortality in relation to traditional CAD risk factors in a high‐risk cohort. Study population included 5,453 consecutive new patients seen between 1996 and 2005 for management of CAD risk factors in a preventive cardiology clinic. Mortality was determined from the Social Security Death Index. There were 359 deaths over a median follow‐up of 5.2 years. Mortality was greater in high (>102 cm in men and >88 cm in women) vs. normal WC in both genders (P < 0.01). The unadjusted Cox proportional hazard ratio (HR) for continuous WC (per cm) was 1.02 (P < 0.001) in both genders and remained significant after adjustment for CAD risk factors (HR = 1.01 in men, HR = 1.03 in women, both P < 0.05). BMI did not associate statistically with mortality. WC associated with diabetes mellitus (DM) and CAD prevalence (P < 0.001). BMI associated only with DM (P < 0.001) and this association disappeared when WC was added to the model. We conclude that WC is an independent predictor of all‐cause mortality in a preventive cardiology population. These data affirm the clinical importance of WC measurements for mortality, DM, and CAD risk prediction and suggest that obesity‐specific interventions targeting WC in addition to traditional risk factor management may favorably impact these outcomes.     

N,N-bis(2-mercaptoethyl)methylamine: a new coligand for Tc-99m labeling of hydrazinonicotinamide peptides

Hydrazinonicotinamide (HYNIC) forms stable coordination complexes with Tc-99m when reacted with Tc(V)oxo species such as Tc-mannitol or other Tc-polyhydric complexes. However, radio-HPLC of [Tc-For-MLFK-HYNIC] labeled via Tc-polyhydric ligands demonstrated multiple radiochemical species each with unique biodistribution patterns. This is likely due to the fact that Tc can bind to the hydrazino moiety, as well as polyhydric ligands, in a variety of coordination geometries. Tridentate ligands, such as bis(mercaptoethyl)methylamine (NS2), may constrain the possible coordination geometries and improve overall stability. To investigate this, we synthesized NS2, converted the [Tc-mannitol-For-MLFK-HYNIC] to the corresponding NS2-containing complex [Tc-NS2-For-MLFK-HYNIC], and compared its infection imaging and biodistribution properties with [Tc-mannitol-For-MLFK-HYNIC]. Conversion to the NS2 complex was confirmed by HPLC which showed a single unique hydrophobic species with retention time greater than the [Tc-mannitol-For-MLFK-HYNIC] complex. Imaging experiments with both preparations were performed in rabbits with E. coli infections in the left thigh. Tissue radioactivity measurements demonstrated that compared to Tc-mannitol-peptide, accumulation of Tc-NS2-peptide was lower in blood, heart, and normal muscle and higher in spleen, infected muscle, and pus (p < 0.01). These results indicate that the Tc-NS2-peptide complex is chemically more homogeneous and exhibits improved infection localization and biodistribution properties. In an effort to model the interactions of the metal-HYNIC core with NS2 and related ligand types, the reactions of [ReCl3(NNC5H4NH)(NHNC5H4N)] and [99TcCl3(NNC5H4NH)(NHNC5H4N)], effective structural analogues for the [M(NNC5H4NH(x))2] core, with NS2, C5H3N-2,6-(CH2SH)2, O(CH2CH2SH)2, and S(CH2CH2SH)2 were investigated and the compounds [M[CH3N(CH2CH2S)2](NNC5H4N)(NHNC5H4N] (M = 99Tc (5a), Re (5b)), [Re[C5H3N-2,6-(CH2S)2](NNC5H4N)(NHNC5H4N)].CH2Cl2.0.5MeOH (7), [Re[SCH2CH2)2O] (NNC5H4N)(NHNC5H4N)] (8), and [Re[(SCH2CH2)2S](NNC5H4NH)(NHNC5H4N)]Cl (9) were isolated. Similarly, the reaction of [ReCl3(NNC5H4NH)(NHNC5H4N)] with the bidentate ligands pyridine-2-methanethiol and 3-(trimethlysilyl)pyridine-2-thiol led to the isolation of [ReCl(C5H4N-2-CH2S) (NNC5H4N)(NHNC5H4N)] (10) and [Re(2-SC5H3N-3-SiMe3)2 (NNC5H4N)(NHNC5H4N)] (11), respectively, while reaction with N-methylimidazole-2-thiol yielded the binuclear complex [Re(OH)Cl(SC3H2N2CH3)2(NNC5H4N)2 (NHNC5H4N)2] (12). The analogous metal-(HYNIC-OH) precursor, [ReCl3[NNC5H3NH(CO2R)] [NHNC5H3N(CO2R)]] (R = H, 13a; R = CH3, 13b) has been prepared and coupled to lysine to provide [RCl3[NNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)] [NHNC5H3NH(CONHCH2CH2CH2CH2CH(NH2)CO2H)]].2HCl (14.2HCl), while the reaction of the methyl ester 13b with 2-mercaptopyridine yields [Re(2-SC5H4N)2[NNC5H3N(CO2Me)][NHNC5H3N(CO2Me)]] (15). While the chemical studies confirm the robustness of the M-HYNIC core (M = Tc, Re) and its persistence in ligand substitution reactions at adjacent coordination sites of the metal, the isolation of oligomeric structures and the insolubility of the peptide conjugates of 13, 14, and 15 underscore the difficulty of characterizing these materials on the macroscopic scale, an observation relevant to the persistent concerns with reagent purity and identity on the tracer level.     

Inactivation of FGF8 in early mesoderm reveals an essential role in kidney development

To bypass the essential gastrulation function of Fgf8 and study its role in lineages of the primitive streak, we have used a new mouse line, T-Cre, to generate mouse embryos with pan-mesodermal loss of Fgf8 expression. Surprisingly, despite previous models in which Fgf8 has been assigned a pivotal role in segmentation/somite differentiation, Fgf8 is not required for these processes. However, mutant neonates display severe renal hypoplasia with deficient nephron formation. In mutant kidneys, aberrant cell death occurs within the metanephric mesenchyme (MM), particularly in the cortical nephrogenic zone, which provides the progenitors for recurring rounds of nephron formation. Prior to mutant morphological changes, Wnt4 and Lim1 expression, which is essential for nephrogenesis, is absent in MM. Furthermore, comparative analysis of Wnt4-null homozygotes reveals concomitant downregulation of Lim1 and diminished tubule formation. Our data support a model whereby FGF8 and WNT4 function in concert to induce the expression of Lim1 for MM survival and tubulogenesis.     

Mutational study of the bacterial hemoglobin distal heme pocket

Ligand binding experiments on three mutants in the distal heme pocket of Vitreoscilla hemoglobin (GlnE7His, ProE8Ala, and GlnE7His,ProE8Ala) were used to probe the role of GlnE7 and ProE8 in the pocket's unusual structure. The oxygen dissociation constants for the wild type, E8Ala mutant, and E7His mutant proteins were 4.5, 4.7, and 1.7microM, respectively; the K(d) for the double mutant was not determinable by our technique. Visible-Soret spectra of the carbonyl and cyanyl forms and FT-IR of the carbonyl form of the E8 mutant were similar to those of the wild type; the opposite was true for the GlnE7His and GlnE7His,ProE8Ala mutants, which also differed from wild type in the visible-Soret spectra of their oxidized forms. Models of the effects of the mutations on distal pocket structure were consistent with the experimental findings, particularly the larger effects of the GlnE7His change.     

Anti-diabetic effect of ginsenoside Re in ob/ob mice

We evaluated the anti-diabetic effects of ginsenoside Re in adult male C57BL/6J ob/ob mice. Diabetic ob/ob mice with fasting blood glucose levels of approximately 230 mg/dl received daily intraperitoneal injections of 7, 20 and 60 mg/kg ginsenoside Re for 12 consecutive days. Dose-related effects of ginsenoside Re on fasting blood glucose levels were observed. After the 20 mg/kg treatment, fasting blood glucose levels were reduced to 188+/-9.2 and 180+/-10.8 mg/dl on Day 5 and Day 12, respectively (both P<0.01 compared to vehicle group, 229+/-9.5 and 235+/-13.4 mg/dl, respectively). The EC(70) of ginsenoside Re was calculated to be 10.3 mg/kg and was used for subsequent studies. Consistent with the reduction in blood glucose, there were significant decreases in both fed and fasting serum insulin levels in mice treated with ginsenoside Re. With 12 days of ginsenoside treatment, glucose tolerance of ob/ob mice increased significantly, and the area under the curve for glucose decreased by 17.8% (P<0.05 compared to vehicle treatment). The hypoglycemic effect of the ginsenoside persisted even at 3 days of treatment cessation (blood glucose levels: 198+/-13.1 with ginsenoside treatment vs. 253+/-20.3 mg/dl with vehicle, P<0.01). There were no significant changes in body weight or body temperature. Preliminary microarray analysis revealed differential expression of skeletal muscle genes associated with lipid metabolism and muscle function. The results suggest that ginsenoside Re may prove to be useful in treating type 2 diabetes.     

Effect of propolis extract on acute carbon tetrachloride induced hepatotoxicity

Ethanolic extract of propolis was administered to rats intoxicated by carbon tetrachloride. Administration of bolus dose of CCl4 (1.5 ml/kg, ip) resulted in elevation of serum transaminases and serum alkaline phosphatase activities. Levels of hepatic lipid peroxidation were significantly increased. On the contrary, there was significant decrease in hepatic reduced glutathione level. The propolis extract (100 and 200 mg/kg, po) exhibited recoupment in both pre- and post-treatment (prophylactic and curative studies) of biochemical changes induced by CCl4. The post treatment of 200 mg/kg, po extract showed most significant hepatoprotective effect. Histopathological studies showed damage in hepatocytes and disturbed chord arrangement after toxicant administration. Propolis extract (200 mg/kg, po) was found to be more effective in restoring CCl4 induced histopathological alterations.