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.     

The enhancement of Th1 immune response by anti-PD-L1 antibody in cattle infected with Mycobacterium avium subsp. paratuberculosis

Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic enteritis of ruminants. Previous studies have shown that programmed death-ligand 1 (PD-L1) is associated with the disease progression, and PD-L1 blockade activates MAP-specific Th1 responses in vitro. Here, we performed anti-PD-L1 antibody administration using 2 MAP-infected cattle at the late subclinical stage of infection. After administration, bacterial shedding was reduced or maintained at a low level. Additionally, MAP-specific Th1 cytokine production was upregulated, and CD69 expression was increased in T cells. Collectively, the treatment has a potential as a novel control method against Johne’s disease.     

Homo erectus calvarium from the Pleistocene of Java

A Homo erectus calvarium [Sambungmacan 4 (Sm 4)] was recovered from Pleistocene sediments at Sambungmacan in central Java. Micro-computed tomography analysis shows a modern human-like cranial base flexion associated with a low platycephalic vault, implying that the evolution of human cranial globularity was independent of cranial base flexion. The overall morphology of Sm 4 is intermediate between that of earlier and later Javanese Homo erectus; apparent morphological specializations are more strongly expressed in the latter. This supports the hypothesis that later Pleistocene Javanese populations were substantially isolated and made minimal contributions to the ancestry of modern humans.     

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.