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Notes: Reactive oxygen species (ROS) play a major role in recruiting and activating inflammatory cells, inducing angiogenesis, fibroblast proliferation, and collagen synthesis, all processes essential to wound healing. However, the same mediators have been implicated in pulmonary and hepatic fibrosis, vascular occlusion, and necrotic tissue damage. We have previously shown that hyperbaric oxygen caused a prolonged elevation of VEGF and delayed wound healing in a rat model of tissue ischemia. In this study we examine the effect of the ROS scavenger, n-acetylcysteine (NAC), on ischemic wound healing. NAC is a virtually nontoxic free radical scavenger that has been used in many experimental studies to block ROS-mediated signaling.Methods:  Male Sprague-Dawley rats underwent creation of the ischemic flap previously validated by this laboratory. This model provides two ischemic and two nonischemic excisional wounds. Rats were treated daily with HBO for 90 minutes at 2.4 atm, HBO plus 150 mg/kg NAC intraperitoneal, or control (neither HBO nor NAC). Wounds were analyzed for surface area, lactate, and VEGF.Results:  The HBO/NAC-treated animals had markedly impaired healing of their ischemic wounds at day 7 (0.105 ± .005 cm vs. 0.068 ± .002 cm for ctl and 0.064 ± .006 for HBO) and a twofold elevation of VEGF (120 pg/mg protein vs. 60 pg/mg protein). Lactate levels were not altered by NAC treatment and non-ischemic wounds showed no difference in healing, lactate, or VEGF.Conclusion:  Reactive oxygen species are required for wound healing. Our initial hypothesis was that the delay in wound closure with HBO treatment was due to excess ROS and that NAC would improve healing. The finding of elevated VEGF and delayed wound healing in the presence of a potent free radical scavenger suggests that blocking ROS signaling prevents the normal mitogenic response to VEGF. Additional studies to examine the VEGF receptor and tyrosine kinase activation will further elucidate the mechanism of ROS signaling in response to HBO treatment.

Notes: Very few articles have aimed to illuminate the clinical profiles of vitiligo in China. We conducted this retrospective survey involving 4118 outpatients with vitiligo in order to identify the differences among various clinical types of vitiligo and their associated disorders. Completed questionnaires (3742) were validated and analysed. Of this large cohort, 1565 (41.8%) individuals presented vitiligo vulgaris, followed by focal, segmental, acrofacial, and universal, in that order. The mean age of vitiligo onset was 18.88 years. More than 60% of the patients were affected before 20 years of age. Patients with segmental vitiligo were affected earlier than those with other types of vitiligo (15.55 years; (P 〈 0.001). More than 74% of the patients presented with focal vitiligo at onset. After 3–5 years, 99% of active vitiligo was worse and shifted from one clinical type to another. However, there was no transformation between acrofacial vitiligo and segmental vitiligo. Compared with the general population, the patients with vitiligo were more likely to be affected by rheumatoid arthritis (P 〈 0.01), ichthyosis (P 〈 0.01), chronic urticaria (P 〈 0.01), or alopecia areata (P 〈 0.01).

Notes: Significant progress has been made in understanding the structure of high molecular weight (HMW) glutenin subunits and their role in determining the end use quality of wheat grains. However, few reports have dealt with the development and characterization of knock out mutants for HMW glutenin subunit genes. Here, the molecular analysis of MB14, a mutant derived from an elite Chinese wheat variety Xiaoyan 54 through chemical mutagenesis is described. SDS-PAGE and Western blot experiments revealed that, in the seeds of homozygous MB14 plants, the expression of the 1Bx14 subunit was specifically blocked whereas the remaining four subunits (1Ax1, 1By15, 1Dx2, 1Dy12) accumulated to levels comparable to those in the wild type plants. The 5′-flanking region and the open reading frame (ORF) of the mutant 1Bx14 allele were amplified and compared to the corresponding regions of wild type 1Bx14. The nucleotide sequences of the 5′-flanking regions from the mutant and wild type 1Bx14 alleles were identical. However, the ORF of the mutant allele differed from that of the wild type 1Bx14 by three point substitutions, one of which resulted in a premature stop codon in the mutant ORF. Interestingly, the mutant 1Bx14 allele was still transcribed in the developing seeds, but no truncated translation product could be detected by Western blot analysis. Potential application of the 1Bx14 knock out mutant in studying the biological function of 1Bx14 and its contribution to the end use quality control in hexaploid wheat is discussed.

Notes: Tetraploid cotton is one of the most extensively cultivated species. Two tetraploid species, Gossypium hirsutum L. and G. barbadense L., dominate the world's cotton production. To better understand the genetic basis of cotton fibre traits for the improvement of fibre quality, a genetic linkage map of tetraploid cotton was constructed using sequence-related amplified polymorphisms (SRAPs), simple sequence repeats (SSRs) and random amplified polymorphic DNAs (RAPDs). A total of 238 SRAP primer combinations, 368 SSR primer pairs and 600 RAPD primers were used to screen polymorphisms between G. hirsutum cv. Handan208 and G. barbadense cv. Pima90 which revealed 749 polymorphic loci in total (205 SSRs, 107 RAPDs and 437 SRAPs). Sixty-nine F2 progeny from the interspecific cross of ‘Handan208’בPima90’ were genotyped with the 749 polymorphic markers. A total of 566 loci were assembled into 41 linkage groups with at least three loci in each group. Twenty-eight linkage groups were assigned to corresponding chromosomes by SSR markers with known chromosome locations. The map covered 5141.8 cM with a mean interlocus space of 9.08 cM. A × test for significance of deviations from the expected ratio (1: 2: 1 or 3: 1) identified 135 loci (18.0%) with skewed segregation, most of which had an excess of maternal parental alleles. In total, 13 QTL associated with fibre traits were detected, among which two QTL were for fibre strength, four for fibre length and seven for micronaire value. These QTL were on nine linkage groups explaining 16.18-28.92% of the trait variation. Six QTL were located in the A subgenome, six QTL in the D subgenome and one QTL in an unassigned linkage group. There were three QTL for micronaire value clustered on LG1, which would be very useful for improving this trait by molecular marker-assisted selection.

Notes: A simple protocol of transformation of cotton (Gossypium hirsutum L.) at a high frequency has been developed via Agrobacterium mediation, coupled with the use of embryogenic calli as explants. Embryogenic calli derived from only one to two somatic embryogenic calli lines of two Chinese cotton cultivars, the cvs. Ekang 9 and Jihe 321 which have low embryogenic potency were first inoculated with the A. tumefaciens strain LBA4404 harbouring binary vector pBin438 carrying a synthetic Bacillus thuringiensis-active Cry1Ac and API-B chimeric gene. Infected embryogenic calli were co-cultivated for 48 h and were then moved on to the selection medium with kanamycin (100 mg/l) for 7-8 weeks. Then, the kanamycin-resistant calli (Km1) subcultured in proliferation medium would re-differentiate to form somatic embryos in 30 days. Cotyledon embryos were transferred to 100-ml Erlenmeyer flasks for germination and regeneration. Putative transformants were confirmed by polymerase chain reaction and Southern blot analysis. Forty-five regenerated plants were successfully transferred to soil, of which 12 proved to have the active Cry1Ac and API-B chimeric gene. Insect resistance was tested by bioassay. The transgenic plants were highly resistant to cotton bollworm (Heliothis armigera) larvae, with mortality (insect resistance) ranging from 95.8 to 100%. In comparison with the methods used in Agrobacterium-mediated transformation of cotton hypocotyls or cotyledons, about 6 months are saved by using the method presented in this paper to obtain a large number of transgenic plants.

Notes: The nature of antigens and functional state of dendritic cells (DC) are important in antigen presentation. The ability of DC for the induction of T-cell responses is promoted by maturation. It has been confirmed that mannose receptors mediate highly efficient endocytosis and presentation of mannosylated proteins. In the present study, L2 domain of ErbB2 ectodomain was expressed in Escherichia coli, purified and mannosylated. The maturation and functional capacity of DC induced by mannosylated L2 (mL2) protein were investigated. The results showed that L2 protein could induce DC maturation, which was accompanied by elevated expression of MHC and co-stimulatory molecules. The effect of mL2 protein on DC maturation was more remarkable than that of non-mL2 proteins. Uptake of mL2 antigens by DC was more efficient. Furthermore, the T cells can be stimulated to proliferate in vitro and secrete Th1 and Th2 cytokines. Higher levels of both IFN-γ and IL-10 were detected from the T cells stimulated by mL2-pulsed DC, suggesting a concurrent activation of CD4+ and CD8+ T cells. The results demonstrated that L2 domain of ErbB2 receptor is an immunodominant molecule. The mL2 domain of ErbB2 can induce an enhanced maturation and functional capacity of DC. It may become an effective strategy to induce anti-ErbB2 response.

Notes: It has been shown that many antihistamines may have anti-inflammatory activity in addition to being H1 antagonists. Mizolastine (MIZ), a novel antihistamine, might also have anti-angiogenesis properties. In this study, we investigated the influence of MIZ on proangiogenesis factors, vascular endothelial cell growth factor (VEGF), tumour necrosis factor (TNF)-α and keratinocyte-derived chemokine (KC) in murine mast cells by using ELISA and RT–PCR, as compared with dexamethasone (DEX) and loratadine (LOR). Our results show that MIZ is effective in the inhibition of KC, VEGF and TNF-α release induced by an IgE-dependent mechanism, in a time- and dose-dependent manner. The differences between the inhibitory effects of the three drugs on these proangiogenic factors were rather subtle. Semiquantitative analysis using RT–PCR showed that the three drugs significantly reduced VEGF165, VEGF120, TNF-α and KC mRNA expression. Statistical results revealed that the effect of DEX on VEGF165 mRNA was different from that of MIZ or LOR (P 〈 0.01) and the differences between the three drugs on VEGF120, TNF-α and KC mRNA were not statistically significant (P 〉 0.05). These findings raise the possibility that MIZ can mediate anti-angiogenesis activity and that the effect may depend not only on the inhibition on the levels of cytokine proteins but also at the mRNA level.

Notes: Thermal shock strengths of a plate of a functionally graded material (FGM) are analyzed when the plate is suddenly exposed to an environmental medium of different temperature. A finite element/mode superposition method is proposed to solve the time-dependent temperature field. The admissible temperature jump that the material can sustain is studied using the stress-based and fracture mechanics-based criteria. The critical parameters governing the level of the transient thermal stress in the medium are identified. The strength of FGMs under transient thermal stresses is analyzed using both maximum local tensile stress and maximum stress intensity factor criteria.

Notes: Background  Vascular endothelial growth factor (VEGF) is overexpressed in malignant melanoma (MM).Objectives  To develop an RNA interference approach that specifically targets VEGF by constructing a eukaryotic expression plasmid containing short interfering RNA (siRNA), and to evaluate the effects of this vector on the proliferation and apoptosis of MM in vitro and in vivo.Methods  pU-VEGF-siRNA plasmid was transfected into MM cell line A375 and colorectal carcinoma cell line Lovo by electroporation. Expression of VEGF mRNA and protein in A375 and Lovo cells after gene transfer was detected by reverse transcription–polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Proliferation of pU-VEGF-siRNA-transfected A375 and Lovo cells and control cells was observed by cell counting through the microscope. The proliferation of human umbilical vein endothelial cells (ECV-304) cultured in medium containing supernatants of transfected and control A375 cells was measured by the cell counting method. Flow cytometry (FCM) was used to analyse the apoptosis of transfected and control groups. In a mouse model, tumorigenicity and tumour growth of transfected cells were studied in vivo. VEGF expression and microvessel density (MVD) in tumour tissue were measured by immunohistochemistry. Apoptosis in tumours was detected by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling.Results  Expression of VEGF mRNA and protein in pU-VEGF-siRNA-transfected A375 and Lovo cells was significantly decreased on days 3, 10, 17 and 24 post-transfection, compared with controls. The greatest suppression occurred on days 3 and 10 post-transfection. The proliferation of transfected A375 cells and ECV-304 cocultured with supernatants of transfected A375 cells was inhibited. FCM analysis showed that a hypodiploidy peak was found only in A375 cells transfected by pU-VEGF-siRNA. After subcutaneous inoculation with pU-VEGF-siRNA-transfected A375 cells, tumour growth in mice was inhibited, VEGF expression and MVD were decreased, and tumour apoptosis was increased significantly, in comparison with mice inoculated with untransfected A375 cells.Conclusions  The delivery of siRNA directed against VEGF was shown not only to give efficient and specific downregulation of the expression of VEGF, inhibit proliferation of A375 and ECV-304 cells and induce apoptosis of A375 cells in vitro, but also to suppress growth of MM in vivo. These results suggest that a strategy based on siRNA targeting of VEGF may build the foundation to the clinical management of MM.