Influence of ethanol on the release of growth factors in human blood-derived platelet gels

Platelet gels (PG) are new topical single-donor blood products which are attracting great interest in regenerative medicine. They are obtained by mixing a platelet-rich plasma fraction with thrombin to generate a fibrin gel enriched in platelet growth factors (GF). The type of thrombin preparation may affect PG reproducibility. We have determined the impact of 14.6% (v/v) ethanol-stabilized thrombin (EHT) on the release of GF by platelets. Various ratios of EHT and platelet concentrates were mixed to obtain from 2.43 to 7.96% ethanol concentration. Platelet-derived growth factor-AB (PDGF-AB), transforming growth factor-ß1 (TGF-ß1), vascular endothelium growth factor (VEGF), epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) were assessed at 5, 120, and 300 min after PG formation. Protein profiles of thrombin and PG releasates were analyzed by SDS-PAGE. The amount of PDGF-AB, TGF-ß1, and VEGF released per platelet decreased significantly (p < 0.05) with increasing ethanol concentrations but, however, not that of EGF. IGF-1 content was stable, consistent with its presence mostly in plasma. SDS-PAGE indicated that ethanol did not affect fibrin formation. In conclusion, ethanol has a significant impact on the amount of GF released by platelets and should be strictly controlled to standardize PG and optimize clinical benefits.     

Direct Isolation of High-Quality Low Molecular Weight RNA of Pear Peel from the Extraction Mixture Containing Nucleic Acid

Low molecular weight RNA (LMW RNA) is generally obtained either from the total RNA or from total nucleic acids solution. Many steps and chemical reagents are involved in traditional methods for LMW RNA isolation where degradation of LMW RNA often occurs, especially for plant materials with high levels of secondary catabolites. In this study, an efficient method was developed to directly isolate pure LMW RNA from pear peel, a material rich in polyphenolics that is covered with a layer of wax. The method was based on polyethylene glycol (PEG) precipitation combining CTAB buffer which is often used to isolate RNA from polysaccharide-rich and polyphenolics-rich materials. The entire procedure could be completed within 6 h and many samples could be processed at the same time. Few and common chemicals are used with this method. Hence, it could be used as an ordinary method in the laboratory. The developed method was further tested by isolating LMW RNA from Arabidopsis. Using the isolated LMW RNA samples, microRNAs were successfully detected and characterized.     

Functional analysis of the inhibitor of apoptosis genes in <Emphasis Type="Italic">Antheraea pernyi</Emphasis> nucleopolyhedrovirus

The inhibitor of apoptosis proteins (IAP) plays an important role in cell apoptosis. We cloned two novel IAP family members, Ap-iap1 and Ap-iap2, from Antheraea pernyi nucleopolyhedrovirus (ApNPV) genome. Ap-IAP1 contains two baculoviral IAP repeat (BIR) domains followed by a RING domain, but Ap-IAP2 has only one BIR domain and RING. The result of transient expression in Spodoptera frugiperda (Sf21) showed that Ap-iap1 blocked cell apoptosis induced by actinomycin D treatment and also rescued the p35 deficient Autographa californica nucleopolyhedrovirus (AcNPV) to replicate in Sf9 cells, while Ap-iap2 does not have this function. Several Ap-IAP1 truncations were constructed to test the activity of BIRs or RING motif to inhibit cell apoptosis. The results indicated that BIRs or RING of Ap-IAP1 had equally function to inhibit cell apoptosis. Therefore deletion of above both of the above domains could not block apoptosis induced by actinomycin D or rescue the replication of AcMNPVΔp35. We also screened two phage-display peptides that might interact with Ap-IAP1.     

Genetic Analysis and Molecular Mapping of a Novel Gene Conferring Resistance to Rice Stripe Virus

Rice stripe virus (RSV) is one of the most damaging diseases affecting rice in East Asia. Rice variety 502 is highly resistant to RSV, while variety 5112 is extremely susceptible. Field statistical data revealed that all “502 × 5112” F₁ individuals were resistant to RSV and the ratio of resistant to susceptible plants was 3:1 in the F₂ population and 1:1 in the BC₁F₁ population. These results indicated that a dominant gene, designated RSV1, controlled the resistance. Simple sequence repeat (SSR) analysis was subsequently carried out in an F₂ population. Sixty SSR markers evenly distributed on the 12 rice chromosomes were screened and tested. Two markers, RM229 and RM206, showed linkage with RSV1. Based on this result, six SSR markers flanking RM229 and RM206 were further selected and tested. Results indicated that SSR markers RM457 and RM473E were linked to RSV1 with a genetic distance of 4.5 and 5.0 cM, respectively. All of the four SSR markers (RM229, RM473E, RM457 and RM206) linked to RSV1 were all located on chromosome 11, therefore RSV1 should be located on chromosome 11 also. In order to find some new markers more closely linked to the RSV1 gene, sequence-related amplified polymorphism (SRAP) analysis was performed. A total of 30 SRAP primer-pairs were analyzed, and one marker SR1 showed linkage with RSV1 at a genetic distance of 2.9 cM. Finally, RSV1 gene was mapped on chromosome 11 between SSR markers RM457 and SRAP marker SR1 with a genetic distance of 4.5 cM and 2.9 cM, respectively.     

Identification of NBS-Type Resistance Gene Homologs in Tobacco Genome

Tobacco (Nicotiana tabacum) is an important cash crop and an ideal experimental system for studies on plant–pathogen interaction. The sequenced tobacco genome provides an opportunity for examining resistance gene homologs (RGHs) in the tobacco genome. Thirty nucleotide-binding site-type RGHs were annotated from genomic data, and another 281 putative RGHs were identified via PCR amplification from wild and cultivated tobacco. The newly identified RGHs are similar to other known RGHs, and some were categorized into new groups or branches that are different from known Nicotiana R genes or RGHs. Of the 281RGHs, 146 were identified from a single tobacco genome. We did not find any polymorphism at the RGHs in cultivated accessions, implying that strong domestication selection and/or demographic effects might have caused a sharp reduction in nucleotide diversity. Three positive selection sites were found in several RGH groups, while purifying selection is pervasive in the RGH family. Our results provide a primary RGH pool and several positively selected sites for the further functional validation of resistance genes in tobacco.     

Carnosine protects against permanent cerebral ischemia in histidine decarboxylase knockout mice by reducing glutamate excitotoxicity

Recently, we showed that carnosine protects against NMDA-induced excitotoxicity in differentiated PC12 cells through a histaminergic pathway. However, whether the protective effect of the carnosine metabolic pathway also occurs in ischemic brain is unknown. Utilizing the model of permanent middle cerebral artery occlusion (pMCAO) in mice, we found that carnosine significantly improved neurological function and decreased infarct size in both histidine decarboxylase knockout and the corresponding wild-type mice to the same extent. Carnosine decreased the glutamate levels and preserved the expression of glutamate transporter-1 (GLT-1) but not the glutamate/aspartate transporter in astrocytes exposed to ischemia in vivo and in vitro. It suppressed the dissipation of ΔΨ_m and generation of mitochondrial reactive oxygen species (ROS) induced by oxygen–glucose deprivation in astrocytes. Furthermore, carnosine also decreased the mitochondrial ROS and reversed the decrease in GLT-1 induced by rotenone. These findings are the first to demonstrate that the mechanism of carnosine action in pMCAO may not be mediated by the histaminergic pathway, but by reducing glutamate excitotoxicity through the effective regulation of the expression of GLT-1 in astrocytes due to improved mitochondrial function. Thus, our study reveals a novel antiexcitotoxic agent in ischemic injury.     

Genetic introgression between an introduced babbler, the Chinese hwamei Leucodioptron c. canorum, and the endemic Taiwan hwamei L. taewanus: a multiple marker systems analysis

Identification of interspecific hybrids is often a subject of primary concern in the development of conservation strategies. Here we performed a genetic analysis combining mitochondrial DNA (mtDNA), microsatellites and single nucleotide polymorphic sites (SNPs) to assay the level of hybridization and introgression between an introduced babbler, Chinese hwamei Leucodioptron canorus, and its close relative, the endemic Taiwan hwamei L. taewanus in Taiwan. Fifty‐five Chinese hwameis from the Asian mainland and 69 Taiwan hwameis, including nine morphological hybrids, were sampled and analyzed. Evidences of mitochondrial introgression were found in three hybrids and one Taiwan hwamei. Five unlinked interspecific SNPs were identified at nine anonymous nuclear loci with interspecific differentiation (total Fst=0.77) that was much higher than that at seven highly polymorphic microsatellite loci combined (total Fst=0.1). Bayesian cluster analysis based on five interspecific SNP loci and two highly differentiated microsatellite loci (Fst>0.08) suggested that twelve individuals sampled in Taiwan were likely F2 or backcross hybrids, among which eight were morphological intermediates. A total of 20.3% (14/69) individuals sampled in Taiwan were suggested to be hybrids, suggesting that fitness reduction in hybrids might be negligible. These results imply that without an effective management strategy, the entire Taiwan hwamei population could easily become an admixed with Chinese hwamei and loose its evolutionary integrity. To reduce introgressive hybridization, illegal trade of Chinese hwamei should be strictly regulated and only the expensive male Chinese hwameis should be legally imported to minimize the chance for Chinese hwameis being released into the field. In our study we also found interspecific SNP markers to outperform microsatellite loci in detecting hybridization and introgression between two closely related species, which may be ascribed to the lower level of homoplasy of SNP loci.