Evidence for existence of cAMP-Epac signaling in the heads of mouse epididymal spermatozoa

Cyclic adenosine 3',5'-monophosphate (cAMP) signaling regulates the expression of fertilizing ability in mammalian spermatozoa. Many articles indicate that this signaling is mediated mainly via protein kinase A. Recently, a guanine nucleotide exchange factor for small G protein Rap1 (an exchange protein directly activated by cAMP: Epac) was discovered as a new mediator of cAMP signaling in somatic cells. The aim of this study was to reveal the existence of cAMP-Epac signaling in mouse spermatozoa. Northern blot analysis and in situ hybridization suggested that Epac1 and Epac2 mRNAs were transcribed in the seminiferous epithelia of the testis. This shows that expression of Epac mRNAs is present in mouse testicular germ cells. Indirect immunofluorescence with specific polyclonal antibodies suggested possible co-localization of Epac1 and Rap1 proteins in the heads of epididymal spermatozoa. Moreover, treatment of epididymal spermatozoa with an Epac-specific cAMP analog, 8-pMeOPT-2'-O-Me-cAMP, induced activation of Rap1, as revealed with a commercial kit for pull-down assay. These results indicate the existence of cAMP-Epac signaling in the heads of mouse epididymal spermatozoa.     

Determination of the Chemical Structures of Tandyukisins B–D,Isolated from a Marine Sponge-Derived Fungus

Tandyukisins B–D (1–3), novel decalin derivatives, have been isolated from a strain of Trichoderma harzianum OUPS-111D-4 originally derived from the marine sponge Halichondria okadai, and their structures have been elucidated on the basis of spectroscopic analyses using 1D and 2D NMR techniques. In addition, their chemical structures were established by chemical transformation. They exhibited weak cytotoxicity, but selective growth inhibition on panel screening using 39 human cancer cell lines.     

Disaggregation and reaggregation of gluten proteins by sodium chloride

This study showed that gluten proteins were extracted with distilled water from dough prepared in the presence of NaCl. To elucidate the interrelationship of NaCl and gluten proteins in dough, the extracted proteins were characterized. These proteins were primarily found to be soluble gliadin monomers by N-terminal amino acid sequencing and analytical ultracentrifugation. Extracted proteins were aggregated by the addition of NaCl at concentrations of >10 mM. A decrease in beta-turn structures, which expose tryptophan residues to an aqueous environment in the presence of NaCl, was revealed by Fourier transform infrared analysis and scanning of fluorescence spectra. In addition, cross-linking experiments with disuccinimidyl tartrate showed that a large amount of protein was cross-linked in the dough only in the presence of NaCl. These results suggest that both interactions and distances between proteins were altered by the addition of NaCl.     

Reliable enzyme-linked immunosorbent assay for the determination of walnut proteins in processed foods

Among food allergens of tree nuts, walnuts are a frequent cause of adverse food reactions in allergic patients. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of walnut soluble proteins in processed foods was developed. The sandwich ELISA is highly specific for walnut soluble proteins. The recovery ranged from 83.4 to 123%, whereas the intra- and interassay coefficients of variation were less than 8.8 and 7.2%, respectively. This study showed that the proposed method is a reliable tool for detection in the presence of hidden walnut proteins in processed foods.     

Reliable enzyme-linked immunosorbent assay for the determination of soybean proteins in processed foods

Among allergenic foods, soybean is known as a food causing adverse reactions in allergenic patients. To clarify the validity of labeling, the specific and sensitive detection method for the analysis of the soybean protein would be necessary. The p34 protein, originally characterized to be p34 as an oil-body associated protein in soybean, has been identified as one of the major allergenic proteins and named Gly m Bd 30K. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of the soybean protein in processed foods was developed using polyclonal antibodies raised against p34 as a soybean marker protein and the specific extraction buffer for extract. The developed sandwich ELISA method was highly specific for the soybean protein. The limit of detection (LOD) and the limit of quantification (LOQ) of the developed ELISA were 0.47 ng/mL (equivalent to 0.19 microg/g in foods) and 0.94 ng/mL (equivalent to 0.38 microg/g in foods), respectively. The recovery ranged from 87.7 to 98.7%, whereas the intra- and interassay coefficients of variation were less than 4.2 and 7.5%, respectively. This study showed that the developed ELISA method is a specific, precise, and reliable tool for the quantitative analysis of the soybean protein in processed foods.     

Individual detection of genetically modified maize varieties in non-identity-preserved maize samples

In many countries, the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved GM varieties. The GMO content in a maize sample containing the combined-trait (stacked) GM maize as determined by the currently available methodology is likely to be overestimated. However, there has been little information in the literature on the mixing level and varieties of stacked GM maize in real sample grains. For the first time, the GMO content of non-identity-preserved (non-IP) maize samples imported from the United States has been successfully determined by using a previously developed individual kernel detection system coupled to a multiplex qualitative PCR method followed by multichannel capillary gel electrophoresis system analysis. To clarify the GMO content in the maize samples imported from the United States, determine how many stacked GM traits are contained therein, and which GM trait varieties frequently appeared in 2005, the GMO content (percent) on a kernel basis and the varieties of the GM kernels in the non-IP maize samples imported from the United States were investigated using the individual kernel analysis system. The average (+/-standard deviation) of the GMO contents on a kernel basis in five non-IP sample lots was determined to be 51.0+/-21.6%, the percentage of a single GM trait grains was 39%, and the percentage of the stacked GM trait grains was 12%. The MON810 grains and NK603 grains were the most frequent varieties in the single GM traits. The most frequent stacked GM traits were the MON810xNK603 grains. In addition, the present study would provide the answer and impact for the quantification of GM maize content in the GM maize kernels on labeling regulation.     

A mixed apocrine gland tumor with metastases to the bone and bone marrow in a miniature poodle

A 10-year-old female miniature poodle had a mass in its carpal joint of the left forelimb. The tumor was divided into small multiple lobules by delicate connective tissues, and necroses were found in some of the central lobules. In some connective stromal areas, chondroid and osteoid tissues were formed. The tumor cells were similar to the structure of apocrine gland epithelial cells with apical blebs resembling apocrine secretion and eosinophilic secretary materials within the luminal space, and spindle cells were sometimes found in the basal area of the glandular structure. In some areas, tumor cells invaded in the blood vessels, bone and bone marrow. Immunohistochemically, the tumor cells forming tubulo-acinar to solid structures were intensely positive for cytokeratin and keratin K8/K18, and the spindle cells were positive for vimentin and alpha-smooth muscle actin. This case was diagnosed as a malignant mixed apocrine gland tumor with metastases to the bone and bone marrow.     

A potential live vector, foamy virus, directed intra-cellular expression of ovine interferon-tau exhibited the resistance to HIV infection

Interferon-tau (IFN-tau), produced by the embryonic trophectoderm, is a member of type I IFNs required for the establishment of pregnancy in the ruminant ungulates. Although this IFN possesses antiviral activity similar to other type I IFNs, the effectiveness of IFN-tau as an antiviral agent has not been well characterized. To investigate possible antiviral effects of ovine IFN-tau (oIFN-tau), oIFN-tau-GST fusion protein was expressed in E. coli BL21, from which the purified protein isolated possessed anti-viral activity. An apathogenic human foamy virus (hFV) was then used to establish a potential recombinant live vector consisting of oIFN-tau cDNA sense (+) or antisense (-) sequence, oIFN-tau(+)/hFV or oIFN-tau(-)/hFV, respectively. Human hematopoietic and other mammalian cell lines that had been transduced with hFV vector consisting of no oIFN-tau, oIFN-tau(+)/hFV or oIFN-tau(-)/hFV construct were cultured initially for 12 days, and three of cell lines were then maintained for up to 90 days. These cells with oIFN-tau expression directed by hFV exhibited the in vitro cytopathic effect minimally. Transduced cell lines that had been cultured for 90 days were subjected to studies on human immunodeficiency virus type-1 (HIV-1) infection, which was measured with infectivity of viral particles resulted from the GFP inserted T-cell tropic HIV SF2 or macrophage tropic HIV SF162: the number of HIV-1 positive cells was reduced by the hFV driven-intra-cellular oIFN-tau expression. Since oIFN-tau/hFV transduced cells exhibited the resistance to HIV-1 infection and/or replication, oIFN-tau could be considered as one of effective antiviral agents against HIV-1. These results suggest that the hFV genome could be an effective recombinant live vector for the expression of a targeted gene in various cell types.