Multi-tier regulation of the streptomycete morphogenetic peptide SapB

Streptomyces coelicolor is a morphologically complex bacterium requiring the secretion of surface‐active proteins to progress through its life cycle. SapB represents an important class of these biosurfactants, as illustrated by its ability to restore aerial hyphae formation when applied exogenously to developmental mutants. However, such aerial hyphae fail to sporulate, exemplifying the need to co‐ordinate the timing of SapB production with other developmental events. SapB has an unusual lantibiotic structure. Its structural gene, ramS, is only 38 nucleotides downstream of the gene encoding its putative modification enzyme, RamC. Transient, co‐ordinated expression of the operon was thought to be controlled by the response regulator RamR. However, we show that ramS is transcribed throughout the cell cycle with a dual expression profile dissimilar to the tightly controlled ramC expression. Surprisingly, post‐translational modification relies on prior membrane localization of the precursor peptide, RamS, as demonstrated by the absence of RamS modification in S. coelicolor hyphae treated with the Bacillus subtilis lipoprotein surfactin. Our results demonstrate that interspecies interaction can also be mediated by interference of post‐translational events. Further, temporal and spatial regulation of irreversible post‐translational modification of a surface‐active morphogenetic peptide suggests a new model for the control of key developmental events.     

Association of MAOA and COMT gene polymorphisms with palatable food intake in children

Several studies have implicated dopamine (DA) in appetite regulation. The enzymes catechol-o-methyltransferase (COMT) and monoamine oxidase A (MAOA) control DA availability and their genes have well-characterized functional variants. In this study, we examined three polymorphisms in these genes, T941G and MAOAu-VNTR in the MAOA gene and Val158Met in the COMT gene, to investigate how heritable variations in enzymes that determine DA levels might influence food intake and nutritional status. This investigation was a cross-sectional examination of 354 Brazilian children of three to four years old. Polymorphisms were analyzed by PCR-based methods. Means of dietary and anthropometric data were compared among genotypes by one-way analyses of variance or Kruskal Wallis tests. The MAOAu-VNTR and COMT Val158Met polymorphisms were associated with the amount of palatable food intake in boys. Presence of the MAOAu-VNTR*long allele was associated with higher intake of lipid-dense foods (LDF) when compared with the *short allele (P=.009); the amount of sugar-dense foods (SDF) intake was also higher in males carriers of the MAOAu-VNTR *long allele than in carriers of the *short allele (P=.034). In the girls' sample, MAOAu-VNTR polymorphism was not associated with food intake and nutritional status. Carriers of the COMT Val158Met*Val allele presented higher intake of LDF when compared with Met/Met homozygotes (P=.008). This study provides the first indication that genetic variants of enzymes that control DA availability might be involved in determination of the amount of palatable food intake in children.     

Enzymatic synthesis of 8-vinyl- and 8-styryl-2'-deoxyguanosine modified DNA--novel fluorescent molecular probes

Fluorescent analogs of the natural nucleobases are widely used as molecular probes for investigating DNA hybridization and topology. In this study the guanosine analogs 8-vinyl- and 8-styryl-2'-deoxyguanosine were synthesized and converted into the corresponding 5'-triphosphates. These C8 modified nucleotides were processed by various DNA polymerases to create fluorescent DNA. Whereas the 8-styryl modified nucleotide somewhat hampers DNA synthesis 8-vinyl-2'-deoxyguanosine is processed by DNA polymerases emphasizing the broad applicability as a molecular probe for fluorescence spectroscopy.     

The synthesis of 2'-methylseleno adenosine and guanosine 5'-triphosphates

Modified nucleoside triphosphates (NTPs) represent powerful building blocks to generate nucleic acids with novel properties by enzymatic synthesis. We have recently demonstrated the access to 2'-SeCH(3)-uridine and 2'-SeCH(3)-cytidine derivatized RNAs for applications in RNA crystallography, using the corresponding nucleoside triphosphates and distinct mutants of T7 RNA polymerase. In the present note, we introduce the chemical synthesis of the novel 2'-methylseleno-2'-deoxyadenosine and -guanosine 5'-triphosphates (2'-SeCH(3)-ATP and 2'-SeCH(3)-GTP) that represent further candidates for the enzymatic RNA synthesis with engineered RNA polymerases.     

Calcium channel blocker use and serum ferritin in adults with hypertension

Iron overload cardiomyopathy is becoming more prevalent, and early recognition and intervention may alter outcomes. Calcium channels are key transporters of iron under iron-overloaded conditions, and potentially represent a new therapeutic target for iron overload. The purpose of this study was to examine the relationship between Calcium channel blocker (CCB) use and serum ferritin among adults with diagnosed hypertension. We analyzed the nationally representative NHANES (National Health and Nutrition Examination Survey) 1999–2002 for adults ≥40?years with diagnosed hypertension. The association between CCBs and serum ferritin was assessed using a t-test and adjusted multiple regressions.The study population included 2143 individuals (representing 37.4 million individuals, 42.0?% males). 12.6?% of the population reported taking CCBs in the last month. Individuals taking CCBs had lower mean serum ferritin (129.3?ng/mL versus 154.5?ng/mL, p?=?0.02). After adjusting for age, sex, menopause and hysterectomy status for women, race/ethnicity, and C-reactive protein, mean serum ferritin for individuals taking CCBs was 26.3?ng/mL lower than for those not taking CCBs (p?=?0.01). In an adjusted regression, individuals who took CCBs and had a daily vitamin C intake of ≥500?mg had a mean serum ferritin that was 60.1?ng/mL lower than people not taking CCBs and with daily vitamin C?<?500?mg (p?<?0.001). In conclusion, this study found an association between use of CCBs and lower serum ferritin levels in individuals with hypertension. Further studies are needed to assess the possible use of CCBs as non-traditional chelating agents for treatment of iron overload cardiomyopathy.     

Benzimidazole inhibitors of the protein kinase CHK2: Clarification of the binding mode by flexible side chain docking and protein–ligand crystallography

Two closely related binding modes have previously been proposed for the ATP-competitive benzimidazole class of checkpoint kinase 2 (CHK2) inhibitors; however, neither binding mode is entirely consistent with the reported SAR. Unconstrained rigid docking of benzimidazole ligands into representative CHK2 protein crystal structures reveals an alternative binding mode involving a water-mediated interaction with the hinge region; docking which incorporates protein side chain flexibility for selected residues in the ATP binding site resulted in a refinement of the water-mediated hinge binding mode that is consistent with observed SAR. The flexible docking results are in good agreement with the crystal structures of four exemplar benzimidazole ligands bound to CHK2 which unambiguously confirmed the binding mode of these inhibitors, including the water-mediated interaction with the hinge region, and which is significantly different from binding modes previously postulated in the literature.     

Triazole pyrimidine nucleosides as inhibitors of Ribonuclease A. Synthesis,biochemical, and structural evaluation

Five ribofuranosyl pyrimidine nucleosides and their corresponding 1,2,3-triazole derivatives have been synthesized and characterized. Their inhibitory action to Ribonuclease A has been studied by biochemical analysis and X-ray crystallography. These compounds are potent competitive inhibitors of RNase A with low μM inhibition constant (K_i) values with the ones having a triazolo linker being more potent than the ones without. The most potent of these is 1-[(β-d-ribofuranosyl)-1,2,3-triazol-4-yl]uracil being with K_i = 1.6 μM. The high resolution X-ray crystal structures of the RNase A in complex with three most potent inhibitors of these inhibitors have shown that they bind at the enzyme catalytic cleft with the pyrimidine nucleobase at the B₁ subsite while the triazole moiety binds at the main subsite P₁, where P-O5′ bond cleavage occurs, and the ribose at the interface between subsites P₁ and P₀ exploiting interactions with residues from both subsites. The effect of a susbsituent group at the 5-pyrimidine position at the inhibitory potency has been also examined and results show that any addition at this position leads to a less efficient inhibitor. Comparative structural analysis of these RNase A complexes with other similar RNase A—ligand complexes reveals that the triazole moiety interactions with the protein form the structural basis of their increased potency. The insertion of a triazole linker between the pyrimidine base and the ribose forms the starting point for further improvement of these inhibitors in the quest for potent ribonucleolytic inhibitors with pharmaceutical potential.     

Degradation of mutant huntingtin via the ubiquitin/proteasome system is modulated by FE65

An unstable expansion of the polyglutamine repeat within exon 1 of the protein Htt (huntingtin) causes HD (Huntington's disease). Mounting evidence shows that accumulation of N-terminal mutant Htt fragments is the source of disruption of normal cellular processes which ultimately leads to neuronal cell death. Understanding the degradation mechanism of mutant Htt and improving its clearance has emerged as a new direction in developing therapeutic approaches to treat HD. In the present study we show that the brain-enriched adaptor protein FE65 is a novel interacting partner of Htt. The binding is mediated through WW-polyproline interaction and is dependent on the length of the polyglutamine tract. Interestingly, a reduction in mutant Htt protein level was observed in FE65-knockdown cells, and the process requires the UPS (ubiquitin/proteasome system). Moreover, the ubiquitination level of mutant Htt was found to be enhanced when FE65 is knocked down. Immunofluroescence staining revealed that FE65 associates with mutant Htt aggregates. Additionally, we demonstrated that overexpression of FE65 increases mutant Htt-induced cell death both in vitro and in vivo. These results suggest that FE65 facilitates the accumulation of mutant Htt in cells by preventing its degradation via the UPS, and thereby enhances the toxicity of mutant Htt.