RNA interference-mediated repression of sucrose-phosphatase in transgenic potato tubers (Solanum tuberosum) strongly affects the hexose-to-sucrose ratio upon cold storage with only minor effects on total soluble carbohydrate accumulation

Storage of potato tubers at low temperatures leads to the accumulation of glucose and fructose in a process called 'cold sweetening'. The aim of this work was to investigate the role of sucrose-phosphatase (SPP) in potato tuber carbohydrate metabolism at low temperature (4 degrees C). To this end, RNA interference (RNAi) was used to reduce SPP expression in transgenic potato tubers. Analysis of SPP specific small interfering RNAs (siRNAs), SPP protein accumulation and enzyme activity indicated that SPP silencing in transgenic tubers was stable during the cold treatment. Analysis of soluble carbohydrates showed that in transgenic tubers, cold-induced hexogenesis was inhibited while, despite strongly reduced SPP activity, sucrose levels exceeded wild-type (WT) values four- to fivefold after 34 d of cold treatment. This led to a drastic change in the hexose-to-sucrose ratio from 1.9 in WT tubers to 0.15 to 0.11 in transgenic tubers, while the total amount of soluble sugars was largely unchanged in both genotypes. Sucrose-6(F)-phosphate (Suc6P), the substrate of SPP, accumulated in transgenic tubers in the cold which most likely enables the residual enzyme to operate with maximal catalytic activity in vivo and thus, in the long term, counterbalances reduced SPP activity in the transformants. Northern analysis revealed that cold-induced expression of vacuolar invertase (VI) was blocked in SPP-silenced tubers explaining a reduced sucrose-to-hexose conversion. Suc6P levels were found to negatively correlate with VI expression. A possible role of Suc6P in regulating VI expression is discussed.     

In vivo induction of necrosis in mice fibrosarcoma via intravenous injection of type B staphylococcal enterotoxin

The bacterial superantigen staphylococcal enterotoxin B (SEB) is a potent inducer of cytotoxic T-cell activity and cytokine production in vivo. We investigated the possibility of the therapeutic application of SEB in patients with fibrosarcoma. The anti-tumor effect of SEB in mice with inoculated fibrosarcoma (WEHI-164) was examined by intravenous (IV) and intratumoral (IT) injection and the sizes of the inoculated tumors, IFN-γ production, and CD4+/CD8+ T cell infiltration were determined. The inoculated tumors were also examined histologically. In the mice in the IV-injected group, a significant reduction (P < 0.02) of tumor size was observed in comparison with mice in the IT-injected and control groups. Furthermore, the mice in the IV-injected group showed significantly higher levels of IFN-γ (P < 0.009) and CD4+/CD8+ T cell infiltration when compared with the other groups (P < 0.02). A significantly higher frequency of necrosis in tumor tissues was also observed in mice in the IV-injected group (P < 0.05). Our present findings suggest that tumor cell death is caused by increased cytotoxic T-cell activity and cytokine levels in response to the IV injection of SEB and that SEB may be a good option for use as a novel therapy in patients with fibrosarcoma. An erratum to this article can be found at     

Sex hormones affect the production of recombinant Factor IX in CHO and HEK-293 cell lines

Recombinant human Factor IX (rFIX) was cloned in a mammalian expression vector and transfected into CHO and HEK-293. Treatment with 10⁻⁹ M methyl testosterone increased rFIX production by 30–50% in CHO and HEK clones. However, 10⁻⁹ M 17β-oestradiol increased production of rFIX by ~50% in CHO-F7 clone and decreased production by 48% and 37% in CHO-F8 and HEK-F2-6, respectively. Progesterone treatment inhibited rFIX production in both cell lines. Production of rFIX can thus be increased by sex hormone treatment and therefore used to enhance biotechnological production in mammalian cells.     

Molecular detection of nematicidal crystalliferous Bacillus thuringiensis strains of Iran and evaluation of their toxicity on free-living and plant-parasitic nematodes

The characterization of nematode-effective strains and cry genes in the Iranian Bacillus thuringiensis (Bt) collection (70 isolates) is presented. Characterization was based on PCR analysis using 12 specific primers for cry5, cry6, cry12, cry13, cry14, and cry21 genes encoding proteins active against nematodes, crystal morphology, and protein band patterns as well as their nematicidal activity on root-knot nematode (Meloidogyne incognita) and two free-living nematodes (Chiloplacus tenuis and Acrobeloides enoplus). PCR results with primers for these genes showed that 22 isolates (31.5%) contain a minimum of one nematode-active cry gene. Strains containing the cry6 gene were the most abundant and represent 22.8% of the isolates. Bt strains harboring cry14 genes were also abundant (14.2%). cry21 and cry5 genes were less abundant, found in 4.2% and 2.8% of the strains, respectively. In total, six different nematode-active cry gene profiles were detected in this collection. Four isolates did not show the expected PCR product size for cry5, cry6, and cry21 genes; they might contain potentially novel insecticidal crystal protein genes. Twenty-two Bt isolates containing nematode-active cry genes were selected for preliminary bioassays on M. incognita. Based on these bioassays, four isolates were selected for detailed bioassays. Isolates YD5 and KON4 at 2 x 10(8) CFU/mL concentrations showed 77% and 81% toxicity on M. incognita, respectively. The free-living nematodes C. tenuis and A. enoplus were more susceptible and the highest mortality was observed within 48 h of incubation at all of the concentrations tested. Maximum mortality was recorded for isolates SN1 and KON4 at 2 x 10(8) CFU/mL concentrations and resulted in 68% and 77% adults deaths of C. tenuis and 68% and 72% for A. enoplus, respectively. Our results showed that PCR is a useful technique for toxicity prediction of nematicidal Bt isolates.     

Quercus infectoria galls possess antioxidant activity and abrogates oxidative stress-induced functional alterations in murine macrophages

The present study reports the antioxidant activity of ethanolic extract of Quercus infectoria galls. The antioxidant potency of galls was investigated employing several established in vitro model systems. Their protective efficacy on oxidative modulation of murine macrophages was also explored. Gall extract was found to contain a large amount of polyphenols and possess a potent reducing power. HPTLC analysis of the extract suggested it to contain 19.925% tannic acid (TA) and 8.75% gallic acid (GA). The extract potently scavenged free radicals including DPPH (IC(50)~0.5 microg/ml), ABTS (IC(50)~1 microg/ml), hydrogen peroxide (H(2)O(2)) (IC(50)~2.6 microg/ml) and hydroxyl (*OH) radicals (IC(50)~6 microg/ml). Gall extract also chelated metal ions and inhibited Fe(3+) -ascorbate-induced oxidation of protein and peroxidation of lipids. Exposure of rat peritoneal macrophages to tertiary butyl hydroperoxide (tBOOH) induced oxidative stress in them and altered their phagocytic functions. These macrophages showed elevated secretion of lysosomal hydrolases, and attenuated phagocytosis and respiratory burst. Activity of macrophage mannose receptor (MR) also diminished following oxidant exposure. Pretreatment of macrophages with gall extract preserved antioxidant armory near to control values and significantly protected against all the investigated functional mutilations. MTT assay revealed gall extract to enhance percent survival of tBOOH exposed macrophages. These results indicate that Q. infectoria galls possess potent antioxidant activity, when tested both in chemical as well as biological models.     

The inhibition of tube formation in a collagen-fibrinogen, three-dimensional gel by cleaved kininogen (HKa) and HK domain 5 (D5) is dependent on Src family kinases

Cleaved high molecular weight kininogen (HKa), as well as its domain 5 (D5), inhibits migration and proliferation induced by angiogenic factors and induces apoptosis in vitro. To study its effect on tube formation we utilized a collagen-fibrinogen, three-dimensional gel, an in vitro model of angiogenesis. HKa, GST-D5 and D5 had a similar inhibitory effect of tube length by 90+/-4.5%, 86+/-5.5% and 77+/-12.9%, respectively. D5-derived synthetic peptides: G440-H455 H475-H485 and G486-K502 inhibited tube length by 51+/-3.7%, 54+/-3.8% and 77+/-1.7%, respectively. By a comparison of its inhibitory potency and its sequences, a functional sequence of HKa was defined to G486-G496. PP2, a Src family kinase inhibitor, prevented tube formation in a dose-dependent manner (100-400 nM), but PP3 at 5 microM, an inactive analogue of PP2, did not. HKa and D5 inhibited Src 416 phosphorylation by 62+/-12.3% and 83+/-6.1%, respectively. The C-terminal Src kinase (Csk) inhibits Src kinase activity. Using a siRNA to Csk, expression of Csk was down-regulated by 86+/-7.0%, which significantly increased tube length by 27+/-5.8%. The addition of HKa and D5 completely blocked this effect. We further showed that HKa inhibited Src family kinase activity by disrupting the complex of uPAR, alphavbeta3 integrin and Src. Our results indicate that the anti-angiogenic effect of HKa and D5 is mediated at least in part through Src family kinases and identify a potential novel target for therapeutic inhibition of neovascularization in cancer and inflammatory arthritis.     

Leishmania major: effects of proteophosphoglycan on reactive oxygen species, IL-12, IFN-gamma and IL-10 production in healthy individuals

Protozoan parasites of the genus Leishmania secrete a range of proteophosphoglycans (PPG) known to be important for successful colonization of Leishmania in the sandfly and for virulence in the mammalian host. PPGs are a large family of extensively glycosylated proteins with some unusual and unique features. In this study we purified PPG from culture supernatant of Leishmania major metacyclic promastigotes. In discontinuous SDS-PAGE, PPG could not enter the resolving gel but after mild acid hydrolysis several bands resolved. Agarose gel electrophoresis and immunoblot analysis using monoclonal antibody (WIC 79.3) indicated that the PPG preparation consisted of heterogeneous molecules. Compositional analysis showed that the PPG preparation contained 67% glycan, 28% protein and 5% phosphate. Additionally, the effect of PPG on reactive oxygen species (ROS) production and induction of IL-10, IL-12 and IFN-γ secretion by human peripheral blood mononuclear cells (PBMCs) isolated from healthy individuals was investigated. The water-soluble secreted form of PPG at a concentration of 1 μg glycan/ml seems to be a potent inducer of ROS and IL-10 and to a lesser extent of IFN-γ and IL-12. Cytokines and ROS production was decreased in a dose-dependent manner as the concentration of PPG was increased to 100 μg glycan/ml.     

Docosahexaenoic acid sensitizes Ramos cells to Gamma-irradiation-induced apoptosis through involvement of PPAR-γ activation and NF-κB suppression

The aim of the present study was to characterize the effects of chronic nitric oxide synthase (NOS) inhibition on the alterations of regulatory myocardial proteins of intracellular signaling pathways (mitogen-activated protein kinase (MAPK) and Akt kinase cascades) and matrix metalloproteinases (MMP). Chronic NO deficiency (NOD) was induced by N^G-nitro-L-arginine methyl ester (L-NAME, 40mg/kg/day, 4 weeks). Protein levels and activation of protein kinases were determined using specific antibodies, activities of MMP were analyzed by zymography in gels containing gelatin as a substrate. The development of NOD was associated with decreased activation of endothelial NOS (eNOS) and down-regulation of protein level of inducible NOS (iNOS). Investigation of kinase pathways revealed that the activation of extracellular signal-regulated kinases (ERK) and the levels of upstream activators of ERK (aFGF, H-Ras) were decreased after L-NAME treatment. Western blot analysis revealed that chronic application of L-NAME also decreased the activation of Akt kinase as compared with control hearts. Study of MMPs showed that in L-NAME-treated rat hearts activities of tissue MMP-2 were decreased. It is concluded that development of NOD resulted in inhibition of ERK and Akt kinase pathways and these changes suggest the involvement of these cascades in responses of myocardium to NOD. The results also point to the possible relationship between ERK and Akt kinase pathways and activation of eNOS and/or MMP-2. Anna Špániková and Petra Šimončíková have contributed equally to the study.     

Estimates of economic values for traits of Arabic sheep in village system

A deterministic bio-economic model was used to estimate the economic values of different traits in Arabic sheep native to the Khuzestan province of Iran. In the studied system, variable costs accounted for about 98.5% of the total costs and among variable costs, feed costs had the highest proportion with 70.7%. Revenue sources included meat, wool, and manure, where meat was the most important one and formed 95.5% of total revenues. Economic value for a trait was estimated as the amount of change in the profit of system as its mean increased by one unit, while the means of other traits were constant. The most important trait in this system was litter size, followed by ewe survival, dressing percentage, and wool weight, respectively. Birth weight had a negative economic value but weight at older ages especially weaning weight and 12-month weight had positive economic values. The sensitivity of economic values of traits was investigated by changing feed and non-feed costs, meat and wool prices by ±10%. Results showed that economic values for dressing percentage and wool weight are not sensitive to change in costs. In addition, changes in marketing and management costs had no effect on the economic value for traits related to body weight in different ages. In general, the economic value for traits which showed sensitivity to the changes of costs, except ewe survival, decreased due to an increase in costs. The economic value for all traits, except birth and wool weight, changed because of a change in meat price. Increasing meat price meant a higher economic importance. Among different factors, meat price fluctuations had the most effect on the economic value of traits.