Mapping protein expression in mouse pancreatic islets by immunolabeling and electron energy loss spectrum-imaging

A combination of immuno-electron microscopy and electron energy-loss spectrum-imaging was used to map the distributions of endocrine polypeptide hormones and proteins in mouse pancreatic islet of Langerhans. Tissue was analyzed from control animals and from mice that were heterozygous for the Anx7 gene, which defines a Ca2+/GTP-dependent membrane fusion and ion channel protein. The heterozygous Anx7 (+/-) mouse displays defects in IP3 receptor mediated Ca2+ signaling and insulin secretion. Therefore, information was obtained about the distributions of the hormones insulin and glucagon, as well as the proteins ANX7 and the IP3 receptor. Insulin secretion appears to be defective in the mutants. It was found from immunolabeling experiments that expression of the IP3 receptor is reduced in mutant islets compared to control islets. Subcellular distributions of sulfur and nitrogen obtained by electron energy-loss spectrum-imaging showed that the insulin concentrations of beta granules were essentially the same in control and mutant islets. By contrast, immunogold labeling of mutant islets shows more insulin immunoreactivity in the beta granules. It follows that insulin may be packaged differently in mutant islets, making antigenic determinants more available to the labeling antibody. The increased rate of insulin secretion in the hyperplastic mutant islets can be explained by compensatory increases in islet size, rather than by an increased insulin concentration in the beta cells. The results indicate that reduced ANX7 expression leads to defects in the IP3 receptor expression in the endocrine cells of the mutant mouse. Increased size of the islet or of adrenal medulla may be a compensatory mechanism for secretion defect by individual endocrine cells. Defects in IP3 receptor expression, and documented consequences of a Ca2+ signaling defect, lead to other changes in organelles such as the mitochondrial number in islet beta-cells. The effects and consequences of reduced ANX7 expression on mitochondria are evident in ultrastructural observations.     

Mutasynthesis-derived myxalamids and origin of the isobutyryl-CoA starter unit of myxalamid B

Myxalamids are potent inhibitors of the eukaryotic electron transport chain produced by different myxobacteria. Here, we describe the identification of the myxalamid biosynthesis gene cluster from Myxococcus xanthus. Additionally, new myxalamids (5-13) have been obtained by mutasynthesis from bkd mutants of M. xanthus and Stigmatella aurantiaca. Moreover, as these bkd mutants are still able to produce myxalamid B (2), the origin of the isobutyryl-CoA (IB-CoA) starter unit required for its biosynthesis has been determined. In a M. xanthus bkd mutant, IB-CoA originates from valine, but in S. aurantiaca this starter unit is derived from alpha-oxidation of iso-odd fatty acids, thereby connecting primary and secondary metabolism.     

Methyl-Donors Can Induce Apoptosis and Attenuate Both the Akt and the Erk1/2 Mediated Proliferation Pathways in Breast and Lung Cancer Cell Lines

Dietary methyl-donors play important roles in physiological processes catalyzed by B vitamins as coenzymes, and are used for complementary support in oncotherapy. Our hypothesis was that methyl-donors can not only assist in tolerating cancer treatment but may also directly interfere with tumor growth and proliferation. Therefore, we investigated the proposed cancer inhibitory effects of methyl-donors (in a mixture of L-methionine, choline chloride, folic acid, and vitamin B12) on MCF7 and T47D breast cancer as well as A549 and H1650 lung cancer cell lines. Indeed, methyl-donor treatment significantly reduced the proliferation in all cell lines, possibly through the downregulation of MAPK/ERK and AKT signaling. These were accompanied by the upregulation of the pro-apoptotic Bak and Bax, both in MCF7 and H1650 cells, at reduced anti-apoptotic Mcl-1 and Bcl-2 levels in MCF7 and H1650 cells, respectively. The treatment-induced downregulation of p-p53(Thr55) was likely to contribute to protecting the nuclear localization and apoptosis inducing functions of p53. The presented features are known to improve the sensitivity of cancer therapy. Therefore, these data support the hypothesis, i.e., that methyl-donors may promote apoptotic signaling by protecting p53 functions through downregulating both the MAPK/ERK and the AKT pathways both in breast and lung adenocarcinoma cell lines. Our results can emphasize the importance and benefits of the appropriate dietary supports in cancer treatments. However, further studies are required to confirm these effects without any adverse outcome in clinical settings.     

Multi-imaging approach to analyze bioactive compounds in Abelmoschus moschatus and Cinnamomum zeylanicum via planar chromatography hyphenated with effect-directed assays and high-resolution mass spectrometry

Citizens in developing countries rely on indigenous knowledge and practices and use locally available medicinal plants for different treatments. Due to limited instrumentation for chemical and biological characterization, most studies investigating the bioactive properties of Sri Lankan medicinal plants rarely progress to the molecular level. While for some plants, the whole plant extracts have been tested, most of the individual active compounds and their effect mechanisms have still not been identified. The separation of bioactive compounds from natural sources is a challenging task. Multi-imaging high-performance thin-layer chromatography (HPTLC-UV/Vis/FLD) combined with biological/biochemical assays (effect- directed analysis, EDA) and high-resolution mass spectrometry (HRMS) provide the straightforward identification of natural products without prior tedious fractionation and compound isolation. Separating complex plant extracts into individual compounds, and still on the same adsorbent surface, studying their effects highlighted their potential. These findings can be used to substantiate current traditional medicinal knowledge and to evaluate their benefits, risks, and limitations. The developed hyphenated HPTLC-UV/Vis/FLD-EDA-HESI- HRMS methods for identification of single compound effects in Sri Lankan Abelmoschus moschatus and Cinnamomum zeylanicum included (1) non-target screening, (2) assignment of prominent individual bioactive compounds, and (3) comparison of product profiles to check the quality of commercial products. Both studies revealed not only the phytochemical profiles but also prioritized bioactive constituents. Diverse antimicrobials, antioxidants, and inhibitors of glucosidase, tyrosinase, and cholinesterase were detected. Running reference standards in parallel to identified compounds, confirmed the assignments and bioactivities. The sustainable and environmentally friendly technique can be used in developing countries for profiling and valorization of plant-based preparations.     

WOOLY — an expert system for the wool dyer

The knowledge acquired by many generations of dyers and by today's wool dyeing specialists can now be gathered and stored in databases. This has allowed the development of a computerised expert system capable of solving different standard tasks of the wool dyer. This is demonstrated by simulated screen displays.     

Practical Synthesis of Allylic Silanes from Allylic Esters and Carbamates by Stereoselective Copper‐Catalyzed Allylic Substitution Reactions

The first copper‐catalyzed allylic substitution reactions of allylic acetates and carbamates employing a bis(triorganosilyl)zinc reagent are reported. This novel procedure avoids the use of stoichiometric amounts of copper salts which are usually mandatory with this chemistry. Application of this methodology to standard model substrates substantiates a high diastereoselectivity for the double bond geometry (E : Z) as well as the relative configuration (syn : anti).     

A second mutant allele (V199I) at the PRKAG3 (RN) locus-II. Effect on colour characteristics of pork loin

Three alleles at the PRKAG3 (RN) locus that influence the glycogen content of pork were found to be segregating in Hampshire×Landrace crossbred pigs, RN(-), rn(+) as well as second mutant allele V 199I (here denoted rn*). The effect of these three alleles on ultimate pH, pigment content, internal reflectance (FOP), surface colour measured by tristimulus colorimetry (L*, a*, b*) and fractions of deoxymyoglobin (Mb), oxymyoglobin (MbO(2)) and metmyoglobin (MetMb) of pork loin was studied. Moreover, the effect of sex, entire male versus female pigs, on these traits was also analysed. The three PRKAG3 alleles affected ultimate pH, internal reflectance, colour and distribution of myoglobin derivatives of pork loin, while the pigment content was not influenced. Ultimate pH values of loins from the three genotypes were found to be in the order RN(-)/- genotypes rn(+)/rn(+) genotype=rn(+)/rn* genotype=rn*/rn* genotype. The RN(-) allele was dominant resulting in higher redness (a* value) and yellowness (b* value), while the rn* allele tended to result in lower redness and yellowness compared with the rn* allele. The RN(-) allele was dominant over the rn* allele in lightness (L* value) giving a lighter colour. Surface colour differences were mainly explained by differences in the distribution of the myoglobin derivatives. Finally, surface lightness was higher and pigment content, redness and fraction of MbO(2) lower in loin from entire males compared with females.