Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Umbilical cord-derived mesenchymal stem cells preconditioned with isorhamnetin: potential therapy for burn wounds

BACKGROUNDImpaired wound healing can be associated with different pathological states. Burn wounds are the most common and detrimental injuries and remain a major health issue worldwide. Mesenchymal stem cells (MSCs) possess the ability to regenerate tissues by secreting factors involved in promoting cell migration, proliferation and differentiation, while suppressing immune reactions. Preconditioning of MSCs with small molecules having cytoprotective properties can enhance the potential of these cells for their use in cell-based therapeutics.AIMTo enhance the therapeutic potential of MSCs by preconditioning them with isorhamnetin for second degree burn wounds in rats.METHODSHuman umbilical cord MSCs (hU-MSCs) were isolated and characterized by surface markers, CD105, vimentin and CD90. For preconditioning, hU-MSCs were treated with isorhamnetin after selection of the optimized concentration (5 µmol/L) by cytotoxicity analysis. The migration potential of these MSCs was analyzed by the in vitro scratch assay. The healing potential of normal, and preconditioned hU-MSCs was compared by transplanting these MSCs in a rat model of a second degree burn wound. Normal, and preconditioned MSCs (IH + MSCs) were transplanted after 72 h of burn injury and observed for 2 wk. Histological and gene expression analyses were performed on day 7 and 14 after cell transplantation to determine complete wound healing.RESULTSThe scratch assay analysis showed a significant reduction in the scratch area in the case of IH + MSCs compared to the normal untreated MSCs at 24 h, while complete closure of the scratch area was observed at 48 h. Histological analysis showed reduced inflammation, completely remodeled epidermis and dermis without scar formation and regeneration of hair follicles in the group that received IH + MSCs. Gene expression analysis was time dependent and more pronounced in the case of IH + MSCs. Interleukin (IL)-1β, IL-6 and Bcl-2 associated X genes showed significant downregulation, while transforming growth factor β, vascular endothelial growth factor, Bcl-2 and matrix metallopeptidase 9 showed significant upregulation compared to the burn wound, showing increased angiogenesis and reduced inflammation and apoptosis.CONCLUSIONPreconditioning of hU-MSCs with isorhamnetin decreases wound progression by reducing inflammation, and improving tissue architecture and wound healing. The study outcome is expected to lead to an improved cell-based therapeutic approach for burn wounds.     

Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.     

Genetic Diversity of Ostreopsis ovata (Dinophyceae) from Malaysia

The genus Ostreopsis is an important component of benthic and epiphytic dinoflagellate assemblages in coral reefs and seaweed beds of Malaysia. Members of the species may produce toxins that contribute to ciguatera fish poisoning. In this study, two species have been isolated and cultured, Ostreopsis ovata and Ostreopsis lenticularis. Analyses of the 5.8S subunit and internal transcribed spacer regions ITS1 and ITS2 of the ribosomal RNA gene sequences of these two species showed that they are separate species, consistent with morphological designations. The nucleotide sequences of the 5.8S subunit and ITS1 and ITS2 regions of the rRNA gene were also used to evaluate the interpopulation and intrapopulation genetic diversity of O. ovata found in Malaysian waters. Results showed a low level of sequence divergence within populations. At the interpopulation level, the rRNA gene sequence distinguished two groups of genetically distinct strains, representative of a Malacca Straits group (isolates from Port Dickson) and a South China Sea group (isolates from Pulau Redang and Kota Kinabalu). Part of the sequences in the ITS regions may be useful in the design of oligonucleotide probes specific for each group. Results from this study show that the ITS regions can be used as genetic markers for taxonomic, biogeographic, and fine-scale population studies of this species. Received September 15, 2000; accepted December 15, 2000     

Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.     

Unraveling Comparative Anti-Amyloidogenic Behavior of Pyrazinamide and D-Cycloserine: A Mechanistic Biophysical Insight

Amyloid fibril formation by proteins leads to variety of degenerative disorders called amyloidosis. While these disorders are topic of extensive research, effective treatments are still unavailable. Thus in present study, two anti-tuberculosis drugs, i.e., pyrazinamide (PYZ) and D-cycloserine (DCS), also known for treatment for Alzheimer’s dementia, were checked for the anti-aggregation and anti-amyloidogenic ability on Aβ-42 peptide and hen egg white lysozyme. Results demonstrated that both drugs inhibit the heat induced aggregation; however, PYZ was more potent and decelerated the nucleation phase as observed from various spectroscopic and microscopic techniques. Furthermore, pre-formed amyloid fibrils incubated with these drugs also increased the PC12/SH-SY5Y cell viability as compare to the amyloid fibrils alone; however, the increase was more pronounced for PYZ as confirmed by MTT assay. Additionally, molecular docking study suggested that the greater inhibitory potential of PYZ as compare to DCS may be due to strong binding affinity and more occupancy of hydrophobic patches of HEWL, which is known to form the core of the protein fibrils.     

Effect of coculturing canine notochordal,nucleus pulposus and mesenchymal stromal cells for intervertebral disc regeneration

IntroductionEarly degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs.MethodsCanine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture.ResultsNCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa.ConclusionsNo regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0569-6) contains supplementary material, which is available to authorized users.     

The distribution and evolutionary history of the PRP8 intein

Background  

We recently described a mini-intein in the PRP8 gene of a strain of the basidiomycete Cryptococcus neoformans, an important fungal pathogen of humans. This was the second described intein in the nuclear genome of any eukaryote; the first nuclear encoded intein was found in the VMA gene of several saccharomycete yeasts. The evolution of eukaryote inteins is not well understood. In this report we describe additional PRP8 inteins (bringing the total of these to over 20). We compare and contrast the phylogenetic distribution and evolutionary history of the PRP8 intein and the saccharomycete VMA intein, in order to derive a broader understanding of eukaryote intein evolution. It has been suggested that eukaryote inteins undergo horizontal transfer and the present analysis explores this proposal.     

Phylogenetic relationship of Alexandrium tamiyavanichii (Dinophyceae) to other Alexandrium species based on ribosomal RNA gene sequences

The phylogenetic relationship of the thecate PSP-toxin producing dinoflagellate Alexandrium tamiyavanichii Balech to other species of Alexandrium was studied based on nucleotide sequences of the ITS1, ITS2, 5.8S, 18S and 28S subunits of the ribosomal RNA gene. These are the first such sequences available for A. tamiyavanichii, which is one of the producers of paralytic shellfish poisoning toxins in tropical waters. Based on the nucleotide sequences of the 28S, 18S and 5.8S subunits of the rRNA gene, A. tamiyavanichii grouped together with A. tamarense, A. catenella and A. fundyense. More interestingly, A. tamiyavanichii was most closely affiliated to A. tamarense isolates from Thailand. This result reaffirmed conclusions from previous studies that, for the A. tamarense/fundyense/catenella species complex, geographical origin rather than morphology seems to determine genetic relatedness. Results of this study also suggest that A. tamiyavanichii most probably belongs to the same species complex. Ribosomal RNA gene sequences do not separate the PSP toxin producing from the non-producing species of Alexandrium.