A protocol for expression of foreign genes in chloroplasts

Several major costs associated with the production of biopharmaceuticals or vaccines in fermentation-based systems could be minimized by using plant chloroplasts as bioreactors, which facilitates rapid scale-up. Oral delivery of chloroplast-derived therapeutic proteins through plant cells eliminates expensive purification steps, low temperature storage, transportation and sterile injections for their delivery. Chloroplast transformation technology (CTT) has also been successfully used to engineer valuable agronomic traits and for the production of industrial enzymes and biomaterials. Here, we provide a detailed protocol for the construction of chloroplast expression and integration vectors, selection and regeneration of transformants, evaluation of transgene integration and inheritance, confirmation of transgene expression and extraction, and quantitation and purification of foreign proteins. Integration of appropriate transgenes into chloroplast genomes and the resulting high levels of functional protein expression can be achieved in approximately 6 months in lettuce and tobacco. CTT is eco-friendly because transgenes are maternally inherited in most crop plants.     

Histochemical analysis of sperm storage in Helicolenus dactylopterus dactylopterus (Teleostei: Scorpaenidae).

The bluemouth rockfish, Helicolenus dactylopterus dactylopterus (De la Roche, 1809), is a zygoparous species with internal fertilization. The male urogenital papilla acts as the copulating organ, and the females retain the spermatozoa in their ovaries for up to 10 months. The objective of this study is to extend our knowledge of the mechanisms that allow the sperm to be retained in the ovaries for prolonged periods. To this end, we analyze the histochemical properties of: 1) the epithelium of the testicular sperm duct, 2) the sperm of the males, 3) the internal epithelium of the ovary wall, 4) the ovarian fluid, and 5) the spermatozoa storage crypts of females. The PAS (Periodic acid-Schiff) and bright Coomassie blue positive reactions of the epithelium of the spermatic duct point to the secretion of polysaccharides and proteins that could promote the bundling of the spermatozoa. The internal epithelium of the ovarian wall secretes polysaccharides, protein, and lipid compounds throughout the storage and spawning period. The acid nature of the ovarian fluid during the storage period may maintain the bundling of spermatozoa when they enter the ovary and may also inhibit sperm motility until the moment of fertilization. The polysaccharide granules that come from the cryptal epithelium into the cavity where spermatozoa are maintained may supply them with nutrients for the storage period. The presence of glucosaminoglycans on the surface of the sperm is probably related to the inhibition of spermatic motility produced by the acidic environment. They are absent in the spermatozoa located in the testicular ducts, relatively scarce in those of the duct of the copulating organ, and abundant in those within the intraovarian cryptal structures.     

Association Study of a Functional Variant on ABCG2 Gene with Sunitinib-Induced Severe Adverse Drug Reaction

Sunitinib is a tyrosine kinase inhibitor and used as the first-line treatment for advanced renal cell carcinoma (RCC). Nevertheless, inter-individual variability of drug’s toxicity was often observed among patients who received sunitinib treatment. This study is to investigate the association of a functional germline variant on ABCG2 that affects the pharmacokinetics of sunitinib with sunitinib-induced toxicity of RCC patients in the Japanese population. A total of 219 RCC patients were recruited to this pharmacogenetic study. ABCG2 421C>A (Q141K) was genotyped by using PCR-Invader assay. The associations of both clinical and genetic variables were evaluated with logistic regression analysis and subsequently receiver operating characteristic (ROC) curve was plotted. About 43% (92/216) of RCC patients that received sunitinib treatment developed severe grade 3 or grade 4 thrombocytopenia according to the National Cancer Institute-Common Terminology Criteria for Adverse Events version 3.0, the most common sunitinib-induced adverse reaction in this study. In the univariate analysis, both age (P = 7.77x10^-3, odds ratio (OR) = 1.04, 95%CI = 1.01–1.07) and ABCG2 421C>A (P = 1.87x10^-2, OR = 1.71, 95%CI = 1.09–2.68) showed association with sunitinib-induced severe thrombocytopenia. Multivariate analysis indicated that the variant ABCG2 421C>A is suggestively associated with severe thrombocytopenia (P = 8.41x10^-3, OR = 1.86, 95% CI = 1.17–2.94) after adjustment of age as a confounding factor. The area under curve (AUC) of the risk prediction model that utilized age and ABCG2 421C>A was 0.648 with sensitivity of 0.859 and specificity of 0.415. Severe thrombocytopenia is the most common adverse reaction of sunitinib treatment in Japanese RCC patients. ABCG2 421C>A could explain part of the inter-individual variability of sunitinib-induced severe thrombocytopenia.     

Leukotriene B4 receptor-1 is essential for allergen-mediated recruitment of CD8+ T cells and airway hyperresponsiveness

Recent studies in both human and rodents have indicated that in addition to CD4+ T cells, CD8+ T cells play an important role in allergic inflammation. We previously demonstrated that allergen-sensitized and -challenged CD8-deficient (CD8-/-) mice develop significantly lower airway hyperresponsiveness (AHR), eosinophilic inflammation, and IL-13 levels in bronchoalveolar lavage fluid compared with wild-type mice, and that all these responses were restored by adoptive transfer of in vivo-primed CD8+ T cells or in vitro-generated effector CD8+ T cells (T(EFF)). Recently, leukotriene B4 and its high affinity receptor, BLT1, have been shown to mediate in vitro-generated T(EFF) recruitment into inflamed tissues. In this study we investigated whether BLT1 is essential for the development of CD8+ T cell-mediated allergic AHR and inflammation. Adoptive transfer of in vivo-primed BLT1+/+, but not BLT1-/-, CD8+ T cells into sensitized and challenged CD8-/- mice restored AHR, eosinophilic inflammation, and IL-13 levels. Moreover, when adoptively transferred into sensitized CD8-/- mice, in vitro-generated BLT1+/+, but not BLT1-/-, T(EFF) accumulated in the lung and mediated these altered airway responses to allergen challenge. These data are the first to show both a functional and an essential role for BLT1 in allergen-mediated CD8+ T(EFF) recruitment into the lung and development of AHR and airway inflammation.     

Gonadal sex differentiation and effect of rearing temperature on sex ratio in black rockfish (Sebastes schlegeli)

Temperature-dependent sex determination has been demonstrated in some species of fish, and a high temperature during the period of sex differentiation typically produces a male-dominant population. This research investigated the gonadal sex differentiation and effect of rearing temperature on the sex ratio in larval black rockfish Sebastes schlegeli, which is a viviparous species. Two types of gonads were histologically distinguishable in fish 20 mm in total length (TL). The putative ovary started forming an ovarian cavity, while the putative testis was not clearly differentiated until 51 mm TL. In a temperature-controlled experiment, the proportions of females were 45% at 10°C, 46% at 14°C, 50% at 18°C, 63% at 22°C, and 83% (significantly different from 1:1 sex ratio) at 24°C. These results suggest that morphological sex differentiation in black rockfish occurs at approximately 20 mm in TL, and it is possible that high temperatures (24°C) induce not a male- but a female-dominant population in this species.     

Inhibition of mTOR signaling with rapamycin attenuates renal hypertrophy in the early diabetic mice

Early diabetic nephropathy is characterized by renal hypertrophy that is mainly due to proximal tubular hypertrophy. Mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase, and its signaling has been reported to regulate protein synthesis and cellular growth, specifically, hypertrophy. Therefore, we examined the effect of mTOR signaling on diabetic renal hypertrophy by using the specific inhibitor for mTOR, rapamycin. Ten days after streptozotocin-induced diabetes, mice showed kidney hypertrophy with increases in the phosphorylation of p70S6kinase and the expression of cyclin kinase inhibitors, p21(Cip1) and p27(Kip1), in the kidneys. The intraperitoneal injection of rapamycin (2 mg/kg/day) markedly attenuated the enhanced phosphorylation of p70S6kinase, the increment of cyclin-dependent kinase inhibitors, and renal enlargement without any changes of clinical parameters, including blood glucose, blood pressure, and food intake. Overexpression of a constitutive active form of p70S6kinase resulted in increased cell size of cultured mouse proximal tubule cells; thus, activation of p70S6kinase causes hypertrophy of proximal tubular cells. Our findings suggest that activation of mTOR signaling causes renal hypertrophy at the early stage of diabetes.     

Conjugated indole-imidazole derivatives displaying cytotoxicity against multidrug resistant cancer cell lines

We report herein the SAR studies of a series of indole-imidazole compounds. that demonstrate substantial in vitro anti-proliferative activities against cancer cell lines, including multidrug resistance (MDR) phenotypes. The in vitro cytotoxic effects have been demonstrated across a wide array of tumor types, including hematologic and solid tumor cell lines of various origins (e.g., leukemia, breast, colon, and uterine).