Effects of hydroxybenzyl alcohols on melanogenesis in melanocyte-keratinocyte co-culture and monolayer culture of melanocytes

In mammalian skin, melanocyte proliferation and melanogenesis can be stimulated by keratinocytes, fibroblasts and other regulatory factors. To determine whether hydroxybenzyl alcohols (HBAs) show more inhibitory in melanocytes cultured alone or in melanocytes co-cultured with keratinocytes, we developed a murine melanocyte-keratinocyte co-culture model to investigate the pigmentation regulators in company with other melanogenic inhibitors and stimulators. It was found that the effects of HBAs and melanogenic factors were more evident in melanocytes co-cultured with keratinocytes. Keratinocytes may play a synergistic role in melanocyte melanogenesis and influence the pigment production. The tests in the co-culture model also imply that the inhibitory effects of HBAs on melanogenesis are due to the direct inhibition of melanosomal tyrosinase activity. HBAs showed a low cytotoxicity. The eventual results proved that HBAs are promising and safe agents for skin whitening in melanocyte alone and in co-culture systems. The co-culture model provides a more physiologically realistic condition to study the interaction between melanocytes and keratinocytes, which enables a reliable screening system for depigmenting compounds.     

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.     

Discovery of a series of aminopiperidines as novel iNOS inhibitors

Nitric oxide (NO), a mediator of various physiological and pathophysiological processes, is synthesized by three isozymes of nitric oxide synthase (NOS). Potential candidate clinical drugs should be devoid of inhibitory activity against endothelial NOS (eNOS), since eNOS plays an important role in maintaining normal blood pressure and flow. A new series of aminopiperidines as potent inhibitors of iNOS were identified from a HTS lead. From this study, we identified compound 33 as a potent iNOS inhibitor, with >25-fold selectivity over eNOS and 16-fold selectivity over nNOS.     

Discovery of amide and heteroaryl isosteres as carbamate replacements in a series of orally active gamma-secretase inhibitors

The design of amide and heteroaryl amide isosteres as replacements for the carbamate substructure in previously disclosed 2,6-disubstituted piperidine N-arylsulfonamides is described. In several cases, amides lessened CYP liabilities in this class of gamma-secretase inhibitors. Selected compounds showed significant reduction of Abeta levels upon oral dosing in a transgenic murine model of Alzheimer's disease.     

Recombination of protein fragments: a promising approach toward engineering proteins with novel nanomechanical properties

Combining single molecule atomic force microscopy (AFM) and protein engineering techniques, here we demonstrate that we can use recombination-based techniques to engineer novel elastomeric proteins by recombining protein fragments from structurally homologous parent proteins. Using I27 and I32 domains from the muscle protein titin as parent template proteins, we systematically shuffled the secondary structural elements of the two parent proteins and engineered 13 hybrid daughter proteins. Although I27 and I32 are highly homologous, and homology modeling predicted that the hybrid daughter proteins fold into structures that are similar to that of parent protein, we found that only eight of the 13 daughter proteins showed beta-sheet dominated structures that are similar to parent proteins, and the other five recombined proteins showed signatures of the formation of significant alpha-helical or random coil-like structure. Single molecule AFM revealed that six recombined daughter proteins are mechanically stable and exhibit mechanical properties that are different from the parent proteins. In contrast, another four of the hybrid proteins were found to be mechanically labile and unfold at forces that are lower than the approximately 20 pN, as we could not detect any unfolding force peaks. The last three hybrid proteins showed interesting duality in their mechanical unfolding behaviors. These results demonstrate the great potential of using recombination-based approaches to engineer novel elastomeric protein domains of diverse mechanical properties. Moreover, our results also revealed the challenges and complexity of developing a recombination-based approach into a laboratory-based directed evolution approach to engineer novel elastomeric proteins.     

Accessory proteins stabilize the acceptor complex for synaptobrevin, the 1:1 syntaxin/SNAP-25 complex

Syntaxin/SNAP-25 interactions precede assembly of the ternary SNARE complex that is essential for neurotransmitter release. This binary complex has been difficult to characterize by bulk methods because of the prevalence of a 2:1 dead-end species. Here, using single-molecule fluorescence, we find the structure of the 1:1 syntaxin/SNAP-25 binary complex is variable, with states changing on the second timescale. One state corresponds to a parallel three-helix bundle, whereas other states show one of the SNAP-25 SNARE domains dissociated. Adding synaptobrevin suppresses the dissociated helix states. Remarkably, upon addition of complexin, Munc13, Munc18, or synaptotagmin, a similar effect is observed. Thus, the 1:1 binary complex is a dynamic acceptor for synaptobrevin binding, and accessory proteins stabilize this acceptor. In the cellular environment the binary complex is actively maintained in a configuration where it can rapidly interact with synaptobrevin, so formation is not likely a limiting step for neurotransmitter release.     

包膜肥料养分控释机理研究进展

利用胶囊技术将易水溶性肥料包被起来是提高肥料利用率和减少施肥环境风险的一个重要手段,在广泛收集国内外有关包膜肥料研究的基础上,探讨了包膜肥料养分控释的原理、过程和特征,概述了影响包膜肥料养分控释的因素和包膜肥料养分控释的数学模拟研究,并提出了我国包膜肥料养分控释机理研究的主要方向。     

Application of mitochondrial control region in population genetic studies of the shrimp Penaeus

This study reports a primer set for amplifying a partial fragment of about 610 bp in the fast mutating mitochondrial control region in shrimps of the genus Penaeus (Decapoda: Penaeidae). The utility of this amplified fragment for studying population differentiation and structuring, compared with more conservative mitochondrial genes (16S rRNA and COI), was explored in P. merguiensis populations over a vast geographical range based on sequence and RFLP analyses. The results indicate that the mitochondrial control region provides more informative sites and reveals more haplotypes, making it most useful for evaluating genetic variations within and between populations of Penaeus species.