Cycloheximide resistance in carrot culture: a differentiated function

Cultured carrot cells grow as unorganized callus tissue in medium containing auxin. Upon removal of the auxin from the medium, they grow in an organized manner and differentiate into embryos. In the normal cell line, W001C, the callus growth can be inhibited by cycloheximide, but the embryonic growth cannot. A variant cell line, WCH105, whose callus growth is resistant to cycloheximide, was isolated. The mechanism of cycloheximide resistance in embryos of both lines and in WCH105 callus was found to be cycloheximide inactivation. In addition to auxin, bromodeoxyuridine can also promote callus growth in carrot culture. Callus cultures maintained by bromodeoxyuridine behave the same as do those maintained by auxin. WCH105 callus is resistant, whereas W001C callus is sensitive to cycloheximide inhibition. Except for the onset of embryogenesis, cycloheximide inactivation is expressed throughout the embryo developmental stages up to the plantlets. These results suggest that cycloheximide inactivation is a function expressed in the differentiated, but not in the undifferentiated, tissues.     

Cytological Studies on the Embryogenesis of Hybrids between Oryza sativa L.(Pennisetum sp.

(1) The pollen grains of Pennisetum can germinate normally on the stigma of rice and the pollen tubes can grow into the style and enter the embryo sacs. However, the process of double fertilization is slow and more or less abnormal and phenomenon of simple fertilization often occurs. (2) It has been found that in the majority of cases the development of the embryos is slow and stays long in the stage of globular embryos, thus, the differentiation of the embryos is very difficult and degeneration of the embryos appears many times. Simple differentiation was observed only in some embryos during 16–24 days after pollination. Normal differenting and developing embryos were not observed. The cause of the degeneration of the embryos is related to the state of endosperm development and also to the non-coordination of the genomes of both parents. (3) The development of the endosperm is abnormal. The change from the free nuclei into the cells in the endosperm is delayed as late as the 8th day after pollination. The whole endosperm tissue is composed of the cell masses which are quite different both in shape and function, a part of these endospemn cells lacks the ability to synthesize starch. The disintegration of the endosperm could be frequently observed during their development. (4) A lots of starch are accumulated in the nucellar cells near the antipodals, It is shown that there was some metabolic confusion resulted from the crossing in the embryo sacs. Based on the above mentioued results the authers consider that the failure of producing seeds by crossing is at least related to the nutrient condition which are essential for the development of embryos. If embryo culture technique is employed at the early stage of the embryo development the hybrid seeds could be obtained.     

Enantioselective hydrolysis of racemic styrene oxide by epoxide hydrolase of<Emphasis Type="Italic">Rhodosporidium kratochvilovae</Emphasis> SYU-08

Enantioselective hydrolysis for the production of chiral styrene oxide was investigated using the epoxide hydrolase activity of a newly isolatedRhodosporidium kratochvilovae SYU-08. The effects of reaction prameters—buffer type, pH, temperature, initial substrate concentrations, phenyl-1,2-ethanediol concentrations on hydrolysis rate, and enantioselectivity—were analyzed. Optically active (S)-styrene oxide with an enantiomeric excess higher than 99 % was obtained from its racemate with a yield of 38 % (theoretically 50% maximum yield) from an initial concentration of 80 mM.     

Immunization with attenuated Salmonella typhimurium producing catalase in protection against gastric Helicobacter pylori infection in mice

Aim. To evaluate the protective effect of live attenuated Salmonella typhimurium expressing catalase against gastric Helicobacter pylori infection in mice, and to explore the underlying mechanisms of the protective immune reaction. Materials and Methods The H. pylori catalase gene was introduced into attenuated S. typhimurium strain SL3261. C₅₇BL/6 mice were orally immunized with the SL3261 vaccine strain expressing catalase or with SL3261 alone or phosphate‐buffered saline (PBS). Mice were sacrificed 4 weeks after immunization and 5 weeks after H. pylori challenge, respectively. Results. All PBS control mice were infected. Eight of 13 (61.5%) mice immunized with the SL3261 vaccine strain and three of 14 (21%) mice immunized with SL3261 alone showed protection against H. pylori infection. Serum anti‐H. pylori IgG2a levels of S. typhimurium‐immunized mice were higher than those of PBS controls, both before and after H. pylori challenge, while there were no differences for IgG1 and IgA. Similarly, mRNA expression of interleukin (IL)‐2, IL‐12 and interferon‐γ in the gastric mucosa of S. typhimurium‐immunized mice was significantly higher than that of PBS controls both before and after challenge. Moreover, S. typhimurium‐immunized mice were characterized by marked infiltration of lymphocyte and mononuclear cells in the gastric mucosa after challenge. IL‐4 and IL‐10 were not detected in any of the three groups. IL‐6 expression was increased in the PBS group compared with the S. typhimurium‐immunized groups after challenge. Conclusions. This study demonstrates that oral immunization of mice with catalase delivered by an attenuated S. typhimurium strain offers protection against H. pylori infection. This protective immunity was mediated through a predominantly Th1‐type response and was associated with post‐immunization gastritis.     

Generation and Characterization of Thymidine/d-Alanine Auxotrophic Recombinant Lactococcus lactis subsp. lactis IL1403 Expressing BmpB

Genetic engineering of Lactococcus lactis to produce a heterologous protein may cause potential risks to the environment despite the industrial usefulness of engineered strains. To reduce the risks, we generated three auxotrophic recombinant L. lactis subsp. lactis IL1403 strains expressing a heterologous protein, BmpB, using thyA- and alr-targeting integration vectors: ITD (thyA ⁻ alr ⁺ bmpB ⁺), IAD (thyA ⁺ alr ⁻ bmpB ⁺), and ITDAD (thyA ⁻ alr ⁻ bmpB ⁺). After construction of integration vectors, each vector was introduced into IL1403 genome. Integration of BmpB expression cassette, deletion of thyA, and inactivation of alr were verified by using PCR reaction. All heterologous DNA fragments except bmpB were eliminated from those recombinants during double crossover events. By using five selective agar plates, we also showed thymidine auxotrophy of ITD and ITDAD and d-alanine auxotrophy of IAD and ITDAD. In M17G and skim milk (SYG) media, the growth of the three recombinants was limited. In MRS media, the growth of IAD and ITDAD was limited, but ITD showed a normal growth pattern as compared with the wild-type strain (WT). All the recombinants showed maximal BmpB expression at an early stationary phase when they were cultivated in M17G supplemented with thymidine and d-alanine. These results suggest that auxotrophic recombinant L. lactis expressing a heterologous protein could be generated to reduce the ecological risks of a recombinant L. lactis.     

Syncyte formation in the microsporangium of <Emphasis Type="Italic">Chrysanthemum</Emphasis> (Asteraceae): a pathway to infraspecific polyploidy

Polyploidy, which is thought to have played an important role in plant evolution and speciation, is prevalent in Chrysanthemum (x = 9). In fact, polyploid series are known in C. zawadskii (2x, 4x, 6x, 8x, and 10x) and C. indicum (2x, 4x, and 6x), but the mechanism by which polyploidization occurs is unknown. Here we show that in diploid individuals of both C. zawadskii and C. indicum, the fusion between two adjacent pollen mother cells (PMCs) occurs at a frequency of 1.1–1.3% early in the first meiotic division. While possessing the chromosomes of both PMCs, the fused cell or syncyte undertakes subsequent meiotic division processes as a single large PMC, producing four 2n pollen grains that are able to germinate. Despite their low frequency, syncyte formation may have played a major role in the production of infraspecific polyploids in Chrysanthemum.     

Pre-treatment neutrophil to lymphocyte ratio is elevated in epithelial ovarian cancer and predicts survival after treatment

Purpose  Inflammatory cells can both suppress and stimulate tumor growth, and the influence of inflammatory cells on clinical outcome has been the focus of many studies. The purpose of this study was to evaluate the effectiveness of the neutrophil to lymphocyte ratio (NLR), a measure of the systemic inflammatory response, as an additional discriminative biomarker in epithelial ovarian cancer and to determine whether it predicts survival and recurrence. Methods  We studied 192 patients with epithelial ovarian cancer, 173 with benign ovarian tumors, 229 with benign gynecologic disease, and 405 healthy controls. Serum CA125 levels and leukocyte counts according to subtypes were recorded prior to treatment in all study subjects. In epithelial ovarian cancer, the diagnostic usefulness of NLR, in combination with CA125, was evaluated. The correlation between NLR and overall and disease-free survival was analyzed using both univariate and multivariate analyses adjusting for the known prognostic factors (age, stage, cell type, and grade). Results  Preoperative NLR in ovarian cancer subjects (mean 6.02) was significantly higher than that in benign ovarian tumor subjects (mean 2.57), benign gynecologic disease subjects (mean 2.55), and healthy controls (mean 1.98) (P < 0.001). The sensitivity and specificity of NLR in detecting ovarian cancer was 66.1% (95% CI, 59.52–72.68%) and 82.7% (95% CI, 79.02–86.38%), respectively (cutoff value: 2.60). In early stage ovarian cancer, CA125 was not elevated in 19 out of 49 patients. Seven (36.8%) of these 19 patients were NLR positive. On Cox multivariate analysis, NLR positive, stage III/IV, and older age were independent poor prognostic factors, and being NLR positive was the most powerful predictive variable (Hazard Ratio = 8.42 [95% CI: 1.09–64.84], P = 0.041). Conclusions  Our findings provide evidence for the association between NLR and epithelial ovarian cancer. Preoperative NLR, in combination with CA125, may represent a simple and cost-effective method of identifying ovarian cancers, and an elevated NLR may predict an adverse outcome in ovarian cancer.     

Tauroursodeoxycholate (TUDCA), chemical chaperone, enhances function of islets by reducing ER stress

The exposure to acute or chronic endoplasmic reticulum (ER) stress has been known to induce dysfunction of islets, leading to apoptosis. The reduction of ER stress in islet isolation for transplantation is critical for islet protection. In this study, we investigated whether tauroursodeoxycholate (TUDCA) could inhibit ER stress induced by thapsigargin, and restore the decreased glucose stimulation index of islets. In pig islets, thapsigargin decreased the insulin secretion by high glucose stimulation in a time-dependent manner (1 h, 1.35 ± 0.16; 2 h, 1.21 ± 0.13; 4 h, 1.17 ± 0.16 vs. 0 h, 1.81 ± 0.15, n = 4, p < 0.05, respectively). However, the treatment of TUDCA restored the decreased insulin secretion index induced by thapsigargin (thapsigargin, 1.25 ± 0.12 vs. thapsigargin + TUDCA, 2.13 ± 0.19, n = 5, p < 0.05). Furthermore, the culture of isolated islets for 24 h with TUDCA significantly reduced the rate of islet regression (37.4 ± 5.8% vs. 14.5 ± 6.4%, n = 12, p < 0.05). The treatment of TUDCA enhanced ATP contents in islets (27.2 ± 3.2 pmol/20IEQs vs. 21.7 ± 2.8 pmol/20IEQs, n = 9, p < 0.05). The insulin secretion index by high glucose stimulation is also increased by treatment of TUDCA (2.42 ± 0.15 vs. 1.92 ± 0.12, n = 12, p < 0.05). Taken together, we suggest that TUDCA could be a useful agent for islet protection in islet isolation for transplantation.