Defects in ErbB-Dependent Establishment of Adult Melanocyte Stem Cells Reveal Independent Origins for Embryonic and Regeneration Melanocytes

Adult stem cells are responsible for maintaining and repairing tissues during the life of an organism. Tissue repair in humans, however, is limited compared to the regenerative capabilities of other vertebrates, such as the zebrafish (Danio rerio). An understanding of stem cell mechanisms, such as how they are established, their self-renewal properties, and their recruitment to produce new cells is therefore important for the application of regenerative medicine. We use larval melanocyte regeneration following treatment with the melanocytotoxic drug MoTP to investigate these mechanisms in Melanocyte Stem Cell (MSC) regulation. In this paper, we show that the receptor tyrosine kinase, erbb3b, is required for establishing the adult MSC responsible for regenerating the larval melanocyte population. Both the erbb3b mutant and wild-type fish treated with the ErbB inhibitor, AG1478, develop normal embryonic melanocytes but fail to regenerate melanocytes after MoTP-induced melanocyte ablation. By administering AG1478 at different time points, we show that ErbB signaling is only required for regeneration prior to MoTP treatment and before 48 hours of development, consistent with a role in establishing MSCs. We then show that overexpression of kitla, the Kit ligand, in transgenic larvae leads to recruitment of MSCs, resulting in overproliferation of melanocytes. Furthermore, kitla overexpression can rescue AG1478-blocked regeneration, suggesting that ErbB signaling is required to promote the progression and specification of the MSC from a pre–MSC state. This study provides evidence that ErbB signaling is required for the establishment of adult MSCs during embryonic development. That this requirement is not shared with the embryonic melanocytes suggests that embryonic melanocytes develop directly, without proceeding through the ErbB-dependent MSC. Moreover, the shared requirement of larval melanocyte regeneration and metamorphic melanocytes that develops at the larval-to-adult transition suggests that these post-embryonic melanocytes develop from the same adult MSC population. Lastly, that kitla overexpression can recruit the MSC to develop excess melanocytes raises the possibility that Kit signaling may be involved in MSC recruitment during regeneration.     

α,β-unsaturated 4-oxo acids in heterocyclic synthesis

3-(1,1-Dioxadibenzothien-4-oyl)acrylic acid (1) was condensed with compounds containing active methylene groups under Michael reaction conditions to furnish the Michael adducts (lactones,2a–c, lactams,3a–c, ketones4a, b). The behavior of these adducts toward the action of hydrazine hydrate were investigated. The compounds were tested for their biological properties. 1st part of this series:Egypt. J. Chem. 42, 309 (1999).     

Increasing the thermal stability of the water-soluble pyrroloquinoline quinone glucose dehydrogenase by single amino acid replacement

Based on the characterization of a PCR mutation of water-soluble glucose dehydrogenase possessing pyrroloquinoline quinone (PQQ), PQQGDH-B, Ser231Cys, we have constructed a series of Ser231 variants. The replacement of Ser231 to Cys, Met, Leu, Asp, Asn, His, or Lys resulted in an increase in thermal stability. Among these variants, Ser231Lys showed the highest level of thermal stability and also showed high catalytic activity. Considering that Ser231Lys showed more than an 8-fold increase in its half-life during the thermal inactivation at 55 degrees C compared with the wild-type enzyme, and also retained catalytic activity similar to a wild-type enzyme, the application of this mutant enzyme as a glucose sensor constituent may develop into a stable glucose sensor construction.     

The production of soluble pyrroloquinoline quinone glucose dehydrogenase by Klebsiella pneumoniae, the alternative host of PQQ enzymes

Klebsiella pneumoniae, which produces PQQ and is available for use with a conventional expression vector system, was selected as the host strain for soluble PQQ glucose dehydrogenase (PQQGDH-B) production. The recombinant K. pneumoniaeexpressed PQQGDH-B in its holo-form at about 18000 U l^–1, equal to that achieved in recombinant Escherichia coli. The signal sequence of recombinant PQQGDH-B produced by K. pneumoniaewas correctly processed. K. pneumoniaecan become an alternative host microorganism not only for PQQGDH-B production but also for recombinant PQQ enzymes production.     

Development of novel liver X receptor modulators based on a 1,2,4-triazole scaffold

Liver?X?Receptor (LXR) agonists have been reported as a potential treatment for atherosclerosis, Alzheimer’s disease and hepatitis C virus (HCV) infection. We have designed and synthesized a series of potent compounds based on a 1,2,4-triazole scaffold as novel LXR modulators. In cell-based cotransfection assays these compounds generally functioned as LXR agonists and we observed compounds with selectivity towards LXRα (7-fold) and LXRβ (7-fold) in terms of potency. Assessment of the effects of selected compounds on LXR target gene expression in HepG2 cells revealed that compounds 6a-b and 8a-b behaved as inverse agonists on FASN expression even though they were agonists in the LXRα and LXRβ cotransfection assays. Interestingly, these compounds had no effect on the expression of SREBP-1c confirming a unique LXR modulator pharmacology. Molecular docking studies and evaluation of ADME properties in-silico show that active compounds possess favorable binding modes and ADME profiles. Thus, these compounds may be useful for in vivo characterization of LXR modulators with unique profiles and determination of their potential clinical utility.     

The importance of species pool size for community composition

We investigated if an increase in species pool size leads to more pronounced turnover in local communities and assessed if this increase relates to stronger competition for environmental niches or to more random placement of species. We compared compositional turnover of pteridophytes (ferns and lycophytes) at 15 sites in mountain ecosystems on 13 islands in southeast Asia and Melanesia that mainly differed in the size of their species pool. Each site was sampled with 16 plots of 20 × 20 m². Using multiple regression on distance matrices, we investigated the relationship between environmental distance and compositional turnover at different spatial extents within sites with different species pool sizes. Additionally, we tested the hypothesis that the intensity of competition increases with increasing species pool size. This was done by assessing how realized niche overlap and unevenness of communities relate to environmental distance and species pool size. With increasing species pool size, there was an increase in: a) proportional turnover in community composition, b) the importance of environmental distance for explaining turnover in community composition and c) a decrease in environmental niche overlap between species indicating an increasing importance of competition for community composition. Our results support the idea that increasing species pool size increases the competition for available environmental niches, and thereby leads to a tighter connection between environmental factors and community composition.     

Enolpyruvate transferase MurAAA149E,identified during adaptation of Enterococcus faecium to daptomycin,increases stability of MurAA–MurG interaction

Daptomycin (DAP) is an antibiotic frequently used as a drug of last resort against vancomycin-resistant enterococci. One of the major challenges when using DAP against vancomycin-resistant enterococci is the emergence of resistance, which is mediated by the cell-envelope stress system LiaFSR. Indeed, inhibition of LiaFSR signaling has been suggested as a strategy to “resensitize” enterococci to DAP. In the absence of LiaFSR, alternative pathways mediating DAP resistance have been identified, including adaptive mutations in the enolpyruvate transferase MurAA (MurAA^A149E), which catalyzes the first committed step in peptidoglycan biosynthesis; however, how these mutations confer resistance is unclear. Here, we investigated the biochemical basis for MurAA^A149E-mediated adaptation to DAP to determine whether such an alternative pathway would undermine the potential efficacy of therapies that target the LiaFSR pathway. We found cells expressing MurAA^A149E had increased susceptibility to glycoside hydrolases, consistent with decreased cell wall integrity. Furthermore, structure–function studies of MurAA and MurAA^A149E using X-ray crystallography and biochemical analyses indicated only a modest decrease in MurAA^A149E activity, but a 16-fold increase in affinity for MurG, which performs the last intracellular step of peptidoglycan synthesis. Exposure to DAP leads to mislocalization of cell division proteins including MurG. In Bacillus subtilis, MurAA and MurG colocalize at division septa and, thus, we propose MurAA^A149E may contribute to DAP nonsusceptibility by increasing the stability of MurAA–MurG interactions to reduce DAP-induced mislocalization of these essential protein complexes.     

Surface-sterilization of Glomus mosseae sporocarps for studying endomycorrhization in vitro

 The effects of sterilization time, sterilizing agents (ethanol, Chloramine T, calcium hypochlorite) and antibiotics (streptomycin and gentamycin) on Glomus mosseae (BEG 12) sporocarp germination and contamination were evaluated. Incubation for 10 s in 96 % ethanol, followed by 10 min in a solution of 2% Chloramine T, 0.02% streptomycin, 0.01% gentamycin and Tween 20, and then 6 min in 6% calcium hypochlorite greatly reduced fungal and bacterial contamination from sporocarps and caused little change in germination rate in water agar medium. Accepted: 4 March 1999     

The Application of Biotechnology to Orchids

This review provides an informative and broad overview of orchid biotechnology, addressing several important aspects such as molecular systematics, modern breeding, in vitro morphogenesis, protoplast culture, flowering control, flower color, somaclonal variation, orchid mycorrhiza, pathogen resistance, virus diagnosis and production of virus-free plants, functional genomics, genetic transformation, conservation biotechnology and pharmaceutical biotechnology. This resource will provide valuable insight to researchers who are involved in orchid biology and floriculture, using biotechnology to advance research objectives. Producing an improved orchid through biotechnology for industrial purposes or to serve as a model plant for pure and applied sciences is well within reach and many of the current techniques and systems are already employed at the commercial production level.