Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system

Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed “HiP4” (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this system, we developed a tandem affinity purification (TAP) and mass spectrometry (TAP-MS) system for comprehensive protein interactome analysis. The integrated use of nickel bead purification followed by HiP4 tag immunoprecipitation made it possible to reduce nonspecific binding and improve selectivity, leading to the recovery of previously unrecognized proteins that interact with hepatitis B virus X (HBx) protein or TAR DNA-binding protein 43 (TARDBP or TDP-43). Our results indicate that this system may be viable as a simple and powerful tool for TAP-MS that can achieve low background and high selectivity in comprehensive protein–protein interaction analyses.     

Spermatogenesis in Animals as Revealed by Electron Microscopy : VI. Researches on the Spermatozoon-Dimorphism in a Pond Snail, Cipangopaludina malleata

This paper reports an electron microscope study of typical and atypical spermatogenesis in the pond snail, Cipangopaludina malteata. In the typical spermatid the nucleus undergoes profound changes as development proceeds, affecting both its form and internal fine structure. A large number of roughly parallel, dense filaments, arranged along the long axis of the nucleus, fuse with each other to form in the end the homogeneous helical body characteristic of the head of the adult spermatozoa. The nebenkern is apparently mitochondrial in nature and, in its early development, is similar to that of insects except that it appears as a double structure from the beginning. As differentiation proceeds, the mitochondria lose their membranes, and the residual, now denuded cristae, reorganize to give a parallel radial arrangement. In the last stages of development, the nebenkern derivations become applied to the sheath of the middle piece in a compact helical fashion. In the development of the atypical spermatozoa, the nucleus fails to differentiate and simply shrinks in volume until only a remnant, devoid of DNA, is left. The cytoplasm shows numerous vesicles containing small Feulgen-positive bodies, 80 to 130 mµ in diameter. These vesicles plus contents increase in number as spermatogenesis proceeds. The "head" structure of the atypical spermatozoa consists of a bundle (7 to 17) of tail flagella, each with a centriole at its anterior end. The end-piece of the atypical form appears brush-like and is made up of the free ends of the several flagella.     

Microcalorimetric measurements of heat production in isolated rat brown adipocytes.

Heat production, oxygen consumption, and lipolysis in isolated interscapular brown adipocytes from the rat were investigated. Epinephrine, norepinephrine, and isoproterenol increased heat production in a concentration-dependent manner, showing, about 6-, 4-, and 5-fold higher effects than controls, respectively. The concentration of isoproterenol for threshold heat production and glycerol release were 10(-10) M and 10(-9) M, respectively. The fact that 10(-9) M isoproterenol increased heat production by about 3-fold while glycerol release had no effect at all indicates that calorimetry is more appropriate for investigation of brown adipocytes. At least the method is more sensitive than that of measuring glycerol release.     

Structural characterization of the dihydroflavonol 4-reductase B (DFR-B) gene in the sweet potato.

A genomic fragment containing the dihydroflavonol 4-reductase B (DFR-B) gene was cloned from the sweet potato (Ipomoea batatas) and its nucleotide sequence was analyzed. The exons and flanking regions were highly homologous to those of previously reported DFR-B genes of the Japanese morning glory, whereas the introns and the intergenic region were less conserved. In addition to the sequences of three miniature inverted-repeat transposable elements (MITEs) and one direct repeat previously reported in the DFR-B gene of Japanese morning glory, two mobile element-like sequences were newly identified in the sweet potato DFR-B gene. At least four allelic sequences were found to exist by amplification of the DFR-B gene from various sweet potato cultivars. One of these allelic sequences had a 2-kb deletion in the intergenic region and was observed in the cultivars with high anthocyanin content in their storage roots.     

Development of a method for immobilization of non-flocculating cells in loofa (Luffa cylindrica) sponge

Both the matrix structure of loofa sponge and the flocculating property of cells were necessary for efficient immobilization. The addition of chitosan to a reactor containing a bed of loofa sponge and a Candida brassicae cell suspension induced cell flocculation and the cells were efficiently immobilized. During ethanol production by the immobilized cells, the free cell concentration in the broth was controlled at the desired level by intermittent addition of chitosan to the reactor. The immobilized cell concentration increased but their specific ethanol productivity decreased with an increase in the chitosan concentration. The maximum ethanol productivity was obtained at a low chitosan concentration of 0·03 g/litre. With this optimal concentration, the cell concentration, ethanol yield and productivity were, respectively, 2, 1·3 and 3 times higher than those of the suspension culture.