Characterization of Defense Signaling Pathways of <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Brassica carinata</Emphasis> in Response to <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> Challenge

Canola (Brassica napus L.) is an agriculturally and economically important crop in Canada, and its growth and yield are frequently influenced by fungal pathogens. Sclerotinia sclerotiorum is among those fungal pathogens and causes stem rot disease in B. napus whereas it has been reported that Brassica carinata is moderately tolerant to S. sclerotiorum. Jasmonic acid/ethylene (JA/ET) and salicylic acid (SA) are phytohormones that are known to be involved in plant disease responses. To investigate the defense signaling cascades involved in the interaction of B. napus and B. carinata with S. sclerotiorum, we examined the expression of five orthologs of B. napus genes involved in JA/ET or SA signaling pathways using quantitative RT-PCR. Our results indicated that there are differences in the timing of JA/ET and SA signaling pathways between B. napus and B. carinata. Our results in these two Brassica species also support previous observations that necrotrophic pathogens trigger JA/ET signaling in response to infection. Finally, we observed that transgenic canola expressing 1-aminocyclopropane-1-carboxylate-deaminase producing low levels of ET was relatively more susceptible to S. sclerotiorum than its wild-type counterpart, suggesting that ET inhibits S. sclerotiorum-induced symptom development.     

Strobilanthes crispus attenuates renal carcinogen, iron nitrilotriacetate (Fe-NTA)-mediated oxidative damage of lipids and DNA

This study was aimed to evaluate the effect of Strobilanthes crispus extract for possible protection against lipid peroxidation and DNA damage induced by iron nitrilotriacetate (Fe-NTA) and hydrogen peroxide (H₂O₂). Fe-NTA is a potent nephrotoxic agent and induces acute and subacute renal proximal tubular necrosis by catalyzing the decomposition of H₂O₂-derived production of hydroxyl radicals, which are known to cause lipid peroxidation and DNA damage. Incubation of postmitochondrial supernatant and/or calf thymus DNA with H₂O₂ (40 mM) in the presence of Fe-NTA (0.1 mM) induces lipid peroxidation and DNA damage to about 2.3-fold and 2.9-fold, respectively, as compared to control (P < 0.05). In lipid peroxidation protection studies, S. crispus treatment showed a dose-dependent inhibition (45–53% inhibition, P < 0.05) of Fe-NTA and H₂O₂ induced lipid peroxidation. Similarly, in DNA damage protection studies, S. crispus treatment also showed a dose-dependent inhibition (18–30% inhibition, P < 0.05) of DNA damage. In addition, the protection was closely related to the content of phenolic compounds as evident by S. crispus extract showing the value of 124.48 mg/g total phenolics expressed as gallic acid equivalent (GAE, mg/g of extract). From these studies, it is concluded that S. crispus inhibits peroxidation of membrane lipids and DNA damage induced by Fe-NTA and H₂O₂ and possesses the potential to be used to treat or prevent degenerative diseases where oxidative stress is implicated.     

Pre and post cloning characterization of a β-1,4-endoglucanase from <Emphasis Type="Italic">Bacillus</Emphasis> sp.

Consistent with its precloning characterization from the cellulolytic Bacillus sp., β-1,4-endoglucanase purified from the recombinant E. coli exhibited maximum activity at 60°C and pH 7.0. It was highly specific for CMC hydrolysis, with stability up to 70°C and over a pH range of 6.0–8.0. The K _m and V _max values for CMCase activity of the enzyme were 4.1 mg/ml and 25 μmole/ml min⁻¹, respectively. The purified enzyme was a monomer of 65 kDa, as determined by SDS-PAGE. The presence of sucrose and IPTG in fermentation media increased the endoglucanase activity of the recombinant enzyme to 5.2-folds as compared with that of the actual one.     

Distribution of the triterpenoid saponins and chemotaxonomy of the genus Clematis L. by high-performance liquid chromatography–mass spectrometry

Selected species of the genus Clematis (Ranunculaceae) have been screened for occurrence of triterpenoid saponins by qualitative HPLC-MSⁿ analysis of root and rhizome materials from 18 Clematis samples as well as the whole plant materials of Clematis puberula var. ganpiniana and Clematis terniflora. The HPLC-MSⁿ analysis allowing the detection of 17 oleanolic acid or hederagenin saponins was carried out in the negative selected ion monitoring (SIM) mode. Triterpenoid saponin profiles of these taxa were used for phylogenetic studies, and results are presented as a dendrogram. Huzhangoside B could be unambiguously identified in all analyzed Clematis taxa, as well as in the investigated Ranunculus taxa. The distribution and chemotaxonomic importance of the triterpenoid saponin profile within this genus are discussed.     

Reproductive performance of repeat breeders in dairy herds

The objectives were to characterize repeat breeding in dairy cows, including reproductive performance and risk factors. Data from 613 Holstein Friesian cows in nine dairy herds across Japan were enrolled. A repeat breeder was defined as a cow that did not become pregnant after three inseminations, despite no clinically detectable reproductive disorders. In contrast, cows that became pregnant within three inseminations were considered to have normal fertility. Of the 613 cows, 87.3% eventually became pregnant after repeated AI (maximum calving to conception interval was 435 d). Mean (±SEM) first AI conception rate, days in milk at first AI, calving to conception interval and service per conception were 38.3%, 82 ± 2 d, 125 ± 3 d, and 2.0 ± 0.1 times, respectively. Normal fertility cows (n = 479) required only 114 ± 3 d to conceive and 1.7 ± 0.1 inseminations per pregnancy, whereas repeat breeders (n = 86) required significantly more days to conceive (211 ± 10) and more inseminations per pregnancy (4.7 ± 0.2). Based on survival analysis, it took 94 d after calving for 50% of normal fertility cows to become pregnant, compared to 155 d for repeat breeders. For repeat breeders, 31.4, 50.0, and 58.1% became pregnant within 210, 300, and 435 d after calving, respectively. The risk factors for repeat breeding were parity (relative risk [RR] = 0.809; P = 0.058), resumption of postpartum ovarian cycles (RR = 1.928; P = 0.009), and days in milk at first AI (RR = 0.991; P = 0.039). In conclusion, repeat breeder dairy cows had very poor reproductive performance. Lower parity, abnormal resumption of postpartum ovarian cycles, and shorter days in milk at first AI were risk factors for repeat breeding.     

Carotenoid inhibitors reduce strigolactone production and Striga hermonthica infection in rice

The strigolactones are internal and rhizosphere signalling molecules in plants that are biosynthesised through carotenoid cleavage. They are secreted by host roots into the rhizosphere where they signal host-presence to the symbiotic arbuscular mycrorrhizal (AM) fungi and the parasitic plants of the Orobanche, Phelipanche and Striga genera. The seeds of these parasitic plants germinate after perceiving these signalling molecules. After attachment to the host root, the parasite negatively affects the host plant by withdrawing water, nutrients and assimilates through a direct connection with the host xylem. In many areas of the world these parasites are a threat to agriculture but so far very limited success has been achieved to minimize losses due to these parasitic weeds. Considering the carotenoid origin of the strigolactones, in the present study we investigated the possibilities to reduce strigolactone production in the roots of plants by blocking carotenoid biosynthesis using carotenoid inhibitors. Hereto the carotenoid inhibitors fluridone, norflurazon, clomazone and amitrole were applied to rice either through irrigation or through foliar spray. Irrigation application of all carotenoid inhibitors and spray application of amitrole significantly decreased strigolactone production, Striga hermonthica germination and Striga infection, also in concentrations too low to affect growth and development of the host plant. Hence, we demonstrate that the application of carotenoid inhibitors to plants can affect S. hermonthica germination and attachment indirectly by reducing the strigolactone concentration in the rhizosphere. This finding is useful for further studies on the relevance of the strigolactones in rhizosphere signalling. Since these inhibitors are available and accessible, they may represent an efficient technology for farmers, including poor subsistence farmers in the African continent, to control these harmful parasitic weeds.     

Clustered diffusion of integrins

We discuss the diffusion of clusters of integrins (and other similar membrane proteins) on a cell membrane with a cortical cytoskeleton. We argue that protein clusters—in contrast with normal oligomers, which are forced to pass through cytoskeletal barriers all at once—should be treated essentially as many-legged random walkers that can pass through a cytoskeletal barrier by putting one leg at a time through the fence. We present the mathematics that should describe the phenomenon, which result in a two-parameter model of diffusion that should apply to any cluster size. We also perform and discuss numerical simulations of the effect in the erythrocyte model system.     

Cancer cell stiffness: integrated roles of three-dimensional matrix stiffness and transforming potential

While significant advances have been made toward revealing the molecular mechanisms that influence breast cancer progression, much less is known about the associated cellular mechanical properties. To this end, we use particle-tracking microrheology to investigate the interplay among intracellular mechanics, three-dimensional matrix stiffness, and transforming potential in a mammary epithelial cell (MEC) cancer progression series. We use a well-characterized model system where human-derived MCF10A MECs overexpress either ErbB2, 14-3-3ζ, or both ErbB2 and 14-3-3ζ, with empty vector as a control. Our results show that MECs possessing ErbB2 transforming potential stiffen in response to elevated matrix stiffness, whereas non-transformed MECs or those overexpressing only 14-3-3ζ do no exhibit this response. We further observe that overexpression of ErbB2 alone is associated with the highest degree of intracellular sensitivity to matrix stiffness, and that the effect of transforming potential on intracellular stiffness is matrix-stiffness-dependent. Moreover, our intracellular stiffness measurements parallel cell migration behavior that has been previously reported for these MEC sublines. Given the current knowledge base of breast cancer mechanobiology, these findings suggest that there may be a positive relationship among intracellular stiffness sensitivity, cell motility, and perturbed mechanotransduction in breast cancer.     

Expression of <Emphasis Type="Italic">rol</Emphasis> genes in transgenic soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) leads to changes in plant phenotype,leaf morphology,and flowering time

Soybean (Glycine max L.) cultivar NARC-4 was transformed with constructs carrying rolA, rolB, or rolC genes, each under the control of the Cauliflower Mosaic Virus 70S promoter. Cotyledonary nodes of soybean seeds were infected with Agrobacterium tumefaciens strain LBA4404 carrying one of the three rol genes, along with nptII in the plasmid pLBR. The efficiency of the transformation varied slightly among the three constructs, with frequencies of 6, 7, and 5% for the rolA, rolB, and rolC genes, respectively, being observed. Southern blot analysis confirmed the integration of rol genes in the soybean genome with varying numbers of copies of the transgene. All transformed plants showed enhanced rooting, but the number of adventitious roots was higher for transformants carrying the rolB transgene. rolA and rolC transformants showed dwarf phenotypes, clustered branching, and variations in leaf morphology. Furthermore, these plants flowered within a short period of time and produced lower numbers of flowers. rolB transformants showed variations in phenotype, including dwarf to semi-dwarf and shrubby growth, abnormal stem growth, short internodes, variations in leaf morphology, and greenish to yellowish-green colored leaves. These plants also flowered early, but dwarf plants produced low numbers of flowers, while shrubby plants produced high numbers of flowers, but these were mostly infertile.