Effect of Three Training Systems on Grapes in a Wet Region of China: Yield, Incidence of Disease and Anthocyanin Compositions of Vitis vinifera cv. Cabernet Sauvignon.

Grapevine training systems determine the suitability for grape varieties in a specific growing region. We evaluated the influence of three training systems, Single Guyot (SG), Spur-pruned Vertical Shoot-Positioned (VSP), and Four-Arm Kniffin (4AK), on the performance of grapes and vines of Vitis vinifera L. cv. Cabernet Sauvignon in the 2012 and 2013 growing seasons in a wet region of central China. 4AK was the most productive system in comparison to SG and VSP. SG and VSP had lower disease infections of leaves and berries, especially in the mid- and final stage of berry ripening. Three training systems had no impact on berry maturity. PLS-DA (Partial Least Squares-Discriminant) analysis showed that the relatively dry vintage could well discriminate three training systems, but the wet vintage was not. A wet vintage of 2013 had more accumulation of 3'5'-substituted and acylated anthocyanins, including malvidin-3-O-(6-O-acetyl)-glucoside, malvidin-3-O-glucoside, and petunidin-3-O-(cis-6-O-coumaryl)-glucoside, etc. With regard to the effect of training systems, 4AK grapes had the lowest concentrations of total anthocyanins and individual anthocyanins, SG and VSP differed according to the different vintages, and showed highest concentration of total individual anthocyanins in 2012 and 2013, respectively. Generally, VSP benefited the most, contributing to significantly highest levels of total individual anthocyanins, and major anthocyanin, including malvidin-3-O-glucoside and malvidin-3-O-(6-O-acetyl)-glucoside, and the grapes obtained from VSP presented significantly highest proportion of 3'5'-substituted anthocyanins. With regard to the ratios of 3'5'/3'-substituted, methoxylated/non-methoxylated and acylated/non-acylated anthocyanins, the significantly higher levels were also shown in VSP system. In summary, VSP was the best training system for Cabernet Sauvignon to accumulate relatively stable individual anthocyanins in this wet region of China and potentially in other rainy regions.

Promoting effect of foliage sprayed zinc sulfate on accumulation of sugar and phenolics in berries of Vitis vinifera cv. Merlot growing on zinc deficient soil.

The effect of foliage sprayed zinc sulfate on berry development of Vitis vinifera cv. Merlot growing on arid zone Zn-deficient soils was investigated over two consecutive seasons, 2013 and 2014. Initial zinc concentration in soil and vines, photosynthesis at three berry developmental stages, berry weight, content of total soluble solids, titratable acidity, phenolics and expression of phenolics biosynthetic pathway genes throughout the stages were measured. Foliage sprayed zinc sulfate showed promoting effects on photosynthesis and berry development of vines and the promotion mainly occurred from veraison to maturation. Zn treatments enhanced the accumulation of total soluble solids, total phenols, flavonoids, flavanols, tannins and anthocyanins in berry skin, decreasing the concentration of titratable acidity. Furthermore, foliage sprayed zinc sulfate could significantly influence the expression of phenolics biosynthetic pathway genes throughout berry development, and the results of expression analysis supported the promotion of Zn treatments on phenolics accumulation. This research is the first comprehensive and detailed study about the effect of foliage sprayed Zn fertilizer on grape berry development, phenolics accumulation and gene expression in berry skin, providing a basis for improving the quality of grape and wine in Zn-deficient areas.

[Azoospermia factor microdeletions in idiopathic azoospermia and severe oligozoospermia].

OBJECTIVE: To observe the relationship between microdeletions of AZF( azoospermia factor) on Y chromosome in male with idiopathic azoospermia and severe oligozoospermia. METHODS: Only patients with an apparently normal 46,XY karyotype and normal FSH, LH and T were included in this study. Multiplex PCR was used to detect the sequence-tagged sites( STS) as follows :sY84, sY86, sY127, sY134, sY152, sY153, sY254, sY255, and ZFX/Y was used as internal control gene. RESULTS: No microdeletion was detected in the control whereas 8 microdeletion cases existed in 67 idiopathic azoospermia and severe oligozoospermia, including 4 in AZFc, 2 in AZFa + AZFc, 1 in AZFc + AZFb, and 1 in AZFb. The prevalence rate of microdeletion was 11.94%, which was statistically different from the control. CONCLUSION: Microdeletions in the AZF regions on the long arm of the Y-chromosome are associated with idiopathic azoospermic and severely oligozoospermic men. Multiplex PCR was a rapid and reliable method for screening microdeletions of AZF.

Effect of NS398 on metastasis-associated gene expression in a human colon cancer cell line.

AIM: To investigate the effect of NS398 on the metastasis-associated gene expression in LoVo colorectal cancer cells. METHODS: LoVo cells were treated with NS398 at the concentration of 100 micromol/L for 24 and 48 h respectively. Total RNA was extracted with TRIzol reagents and reverse transcribed with Superscript II and hybridized with cDNA microarray (containing oncogenes, tumor suppressor genes, signal transduction molecules, adhesive molecules, growth factors, and ESTs) fabricated in our laboratory. After normalization, the ratio of gene expression of NS398 treated to untreated LoVo cells was either 2-fold up or 0.5-fold down was defined as the differentially expressed genes. Semi-quantitative RT-PCR was used to validate the microarray results. RESULTS: Among the 447 metastasis-associated genes, 9 genes were upregulated and 8 genes were downregulated in LoVo cells treated with NS398 for 24 h compared to untreated cells. While 31 genes were upregulated and 14 genes were downregulated in LoVo cells treated with NS398 for 48 h. IGFBP-5, PAI-2, JUN, REL, BRCA1, and BRCA2 might be the new targets of NS398 in treatment of colorectal cancer. CONCLUSION: NS398 might exert its anti-metastasis effect on colorectal cancer by affecting several metastasis-associated gene expression.

Melatonin treatment of pre-veraison grape berries to increase size and synchronicity of berries and modify wine aroma components.

A comprehensive investigation was carried out to determine the effect of exogenous melatonin treatment of pre-veraison grapes on grape berries and its wines. Two melatonin treatments of pre-veraison grape berries increased the weight of the berries by approximately 6.6%. Meanwhile, this melatonin treatment could be beneficial in the reduction of underripe and overripe fruits and in enhancing the synchronicity of the berries. In addition, there were significant differences in the volatile compound composition between the wine produced from the melatonin-treated berries and the wines made from untreated berries. The wine from melatonin-treated pre-veraison grape berries had stronger fruity, spicy, and sweet sensory properties, compared to the wines made from untreated berries. Prolonging the treatment through repeated applications can enhance these effects and under different seasonal conditions, more pronounced effects on the grape quality and wine properties can be observed.

Viral replication modulated by synthetic peptide derived from hepatitis B virus X protein.

AIM: A strategy for viral vaccine design is the use of conserved peptides to overcome the problem of sequence diversity. At present it is still unclear whether conserved peptide is safe as a candidate vaccine. We reported it here for the first time not only to highlight the biohazard issue and safety importance for viral peptide vaccine, but also to explore the effect of a fully conserved peptide on HBV replication within the carboxyl terminus of HBx. METHODS: We synthesized the fully conserved peptide (CP) with nine residues, FVLGGCRHK. HBV-producing 2.2.15 cells were treated with or without 3.5 microM CP for 36 hours. Quantitative detection of viral DNA was performed by real-time PCR. HBV antigens were determined by enzyme-linked immunoadsorbent assay (ELISA). Quantitative analyses of p53 and Bax proteins were based on immunofluorescence. Flow cytometry was performed to detect cell cycle and apoptosis. RESULTS: Both extracellular and intracellular copies of HBV DNA per ml were significantly increased after incubation with 3.5 microM of CP. HBsAg and HBeAg in the cultured medium of CP-treatment cells were as abundant as untreated control cells. CP influenced negatively the extracellular viral gene products, and 3.5 microM CP could significantly inhibit intracellular HBsAg expression. In response to CP, intracellular HBeAg displayed an opposite pattern to that of HBsAg, and 3.5 microM CP could efficiently increase the level of intracellular HBeAg. Flow cytometric analyses exhibited no significant changes on cell cycle, apoptosis, p53 and Bax proteins in 2.2.15 cells with or without CP. CONCLUSION: Together with the results generated from the synthetic peptide, we address that the conserved region, a domain of HBx, may be responsible for modulating HBV replication. As conserved peptides from infectious microbes are used as immunogens to elicit immune responses, their latent biological hazard for human beings should be evaluated.

Aggregate formation of hepatitis B virus X protein affects cell cycle and apoptosis.

AIM: To investigate whether the formation of aggregated HBx has a potential linking with its cellular responses. METHODS: Recombinant HBx was expressed in Escherichia coli and purified by Ni-NTA metal-affinity chromatography. Anti-HBx monoclonal antibody was developed for immunocytochemical detection. Bicistronic expression vector harboring full-length DNA of HBx was employed for transfection of human HepG2 cells. Immunocytochemical staining was used to examine the intracellular HBx aggregates in cells. The effects of HBx aggregation on cell cycle and apoptosis were assessed by flow cytometry. RESULTS: Immunocytochemical staining revealed most of the HBx was formed intracellular aggregate in cytoplasm and frequently accumulated in large granules. Flow cytometry analysis showed that HepG2 cells transfected with vector harboring HBx significantly increased apoptosis and largely accumulated in the G0-G1 phase by maintenance in serum medium for 36 hours. Control cells without HBx aggregates in the presence of serum entered S phase and proliferated more rapidly at the same time. EGFP fluorescence in HBx expression cells was significantly decreased. CONCLUSION: Our observations show that cells with HBx aggregate undergo growth arrest and apoptosis, whereas control cells without HBx remain in growth and progression into S phase. Our data may provide helpful information to understand the biological effects of HBx aggregates on cells.

[rpsL gene analysis associated with Streptomycin resistance in Mycobacterium tuberculosis].

The mutation of the rpsL gene in Mycobacterium tuberculosis (M.TB) is related to Streptomycin resistance. In this experiment, 77 TB strains obtained in hospital were selected, the routine drug susceptibility test was done by using BACIEC460 system, meanwhile the TB-rspL gene was amplicated by PCR, and analyzed by SSCP, RFLP and sequence analysis. There are 20 strains sensitive to SM and 57 strains resistant to it in routine drug susceptibility test. The SSCP result displayed that 34 strains were rspL gene wild type and 43 strains were mutant type. Compared with routine drug susceptibility test, the specificity was 95% and the positive anticipate value was 98%. RFLP cut by MobII displayed that 81.4% of mutation happened in codon 43 of TB-rspL. Sequence analysis displayed that there was single base mutation in codon 43(AAG-->AGG). In conclusion, Drug resistance of TB to SM is related to rspL gene mutation and the mutation in codon 43 is the most common cause.

Erythrocyte-based analgesic peptides.

Human erythrocyte discards the major organelles in a bid to maximize cellular hemoglobin. Hemoglobin, approximately 98% of the intraerythrocytic protein, serves as the principle transport medium of gaseous conveyance. The accumulated data speaks in favor of erythrocyte not merely engaging in gas exchange, but building molecular signaling as a side job during its 4-month sojourn in blood circulation. The production mechanism of erythrocyte-based bioactive peptides is not clear. Recent studies indicate that proteasome and its subunits persist in mature erythrocyte. The intraerythrocytic proteasome is involved in the formation of hemoglobin-derived analgesic peptides and enables erythrocyte to exert the erythrocrine function. Erythrocrine describes erythrocyte for generation and excretion of signaling molecules and has the potential of shedding light on our understanding of novel actions of erythrocyte. Different types of erythrocrine analgesic peptides are originated from the intraerythrocytic degradation of hemoglobin and manifest the systemic influence in physiology and pathophysiology along its travel through the body via the bloodstream. Translational research from bench to bedside will expand our knowledge of erythrocrine concept and facilitate the development of therapeutic strategies for clinical pain.

Inhibition of intraerythrocytic proteasome retards the generation of hemorphins.

Hemorphins are a set of hemoglobin-derived opioid peptides. The production mechanism of these structural overlap peptides remains unclear. Based on the sequences of hemorphins, it could be inferred that hemorphins are probably generated by cleavage of hemoglobin ß chain at sites favored by the chymotrypsin-like protease. 20S proteasome possesses the chymotrypsin-like activity and still persists in mature erythrocytes. This study attempts to clarify whether the intraerythrocytic proteasome involves in the formation of hemorphins. Hemorphins containing hemorphin-7 and V-hemorphin-7 are isolated by immunoprecipitation from culture supernatant of human erythrocytes. Bortezomib inhibits the chymotrypsin-like activity of intraerythrocytic proteasome and prevents the yield of hemorphins in a dose-dependent manner. The present study suggests that intraerythrocytic proteasome contributes to the generation of hemorphins.

[The structure and virus-like particle vaccine of the HIV-1 capsid protein].

The HIV-1 capsid protein (CA) plays an essential role in viral core assembly and maturation. Proteolytic cleavage at the MA-CA junction of the retroviral gag polyprotein refolds the amino-terminal end of capsid into a beta-helix structure that is stabilized by a salt bridge between the protein's processed amino-terminus and a conserved acidic residue. The refolded capsid aminoterminus then creates a new CA-CA interface, allowing assembly of the mature capsid core. Recently, researches focus on assembly of CA in vitro and development of CA vaccine. CA vaccine will provide widely immune protection because CA is comparatively conserved. Experiments demonstrate that fusing as few as four matrix residues onto the amino-terminus of capsid redirects protein assembly from cylinder to spheres in vitro. Evaluation of immunogenicity showed that immunization with virus-like particles induced both cellular and neutralizing antibody responses. Furthermore, mucosal administration of virus-like particles effectively induced both mucosal and systemic immune responses. These results indicate that virus-like particles consisting of HIV structural proteins are an attractive vaccine platform for eliciting anti-viral immune responses, especially neutralizing antibody responses. The production of antigens for vaccines in plants indicates that plant-based transgenic expression represents a viable means of producing CA vaccine for the development of HIV vaccine and for use in HIV diagnostic procedures and it has the potential as a safe and cost-effective alternative to traditional production systems.

Effects of aspirin on metastasis-associated gene expression detected by cDNA microarray.

AIM: To investigate the effect of aspirin on the metastasis-associated gene expression in 3AO ovarian cancer cells. METHODS: 3AO cells were treated with aspirin at the concentration of 1.2 mmol/L for 16 and 48 h, respectively. The total RNA was extracted with Trizol reagents and reverse transcribed with Superscript II and hybridized with cDNA microarray (containing oncogenes, tumor suppressor genes, signal transduction pathway molecules, adhesive molecules, growth factors and ESTs) fabricated in our lab. After normalization, the ratio of gene expression of aspirin treated to untreated 3AO cells being either 2 fold up higher or 0.5 fold down (lower) were defined as differential expression. Semi-quantitative RT-PCR was used to validate the microarray results. RESULTS: Among the 447 metastasis-associated genes, 4 genes were up-regulated and 14 genes were down-regulated in 3AO cells treated with aspirin for 16 h compared with untreated cells. While 24 genes were up-regulated and 10 genes were down-regulated in cells treated with aspirin for 48 h. Several up or down-regulated gene expression changes continued from 16 h to 48 h. CONCLUSION: Aspirin might exert its anti-metastasis effects on ovarian cancer by affecting metastasis-associated gene expression.