Structure-based design of a leucine zipper protein with new DNA contacting region

We have employed a structure-based design to construct a small folding domain from the F-actin bundling protein villin that contains the amino acids necessary for the DNA binding of the basic leucine zipper protein GCN4 and have compared its DNA binding with GCN4. The monomeric motif folds into a stable domain and binds DNA in a rigid-body mechanism, while its affinity is not higher than that of the basic region peptide. The addition of the leucine zipper region to the folded domain restored its sequence-specific DNA binding comparable to that of GCN4. Unlike the monomeric folded domain, its leucine zipper derivative undergoes a conformational change upon DNA binding. CD spectral and thermodynamic studies indicate that the DNA-contacting region is folded in the presence or absence of DNA and suggest that the junction between the DNA-contacting and the leucine zipper regions transits to a helix in the presence of DNA. These results demonstrate that the structural transition outside the direct-contacting region, which adjusts the precise location of the DNA-contacting region, plays a critical role in the specific complex formation of basic leucine zipper proteins.     

Structure and host recognition of serotype 13 glycopeptidolipid from Mycobacterium intracellulare

The Mycobacterium avium-M. intracellulare complex (MAIC) is divided into 28 serotypes by a species-specific glycopeptidolipid (GPL). Previously, we clarified the structures of serotype 7 GPL and two methyltransferase genes (orfA and orfB) in serotype 12 GPL. This study elucidated the chemical structure, biosynthesis gene, and host innate immune response of serotype 13 GPL. The oligosaccharide (OSE) structure of serotype 13 GPL was determined to be 4-2'-hydroxypropanoyl-amido-4,6-dideoxy-β-hexose-(1 → 3)-4-O-methyl-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 3)-α-L-rhamnose-(1 → 2)-α-L-6-deoxy-talose by using chromatography, mass spectrometry, and nuclear magnetic resonance (NMR) analyses. The structure of the serotype 13 GPL was different from those of serotype 7 and 12 GPLs only in O-methylations. We found a relationship between the structure and biosynthesis gene cluster. M. intracellulare serotypes 12 and 13 have a 1.95-kb orfA-orfB gene responsible for 3-O-methylation at the terminal hexose, orfB, and 4-O-methylation at the rhamnose next to the terminal hexose, orfA. The serotype 13 orfB had a nonfunctional one-base missense mutation that modifies serotype 12 GPL to serotype 13 GPL. Moreover, the native serotype 13 GPL was multiacetylated and recognized via Toll-like receptor 2. The findings presented here imply that serotypes 7, 12, and 13 are phylogenetically related and confirm that acetylation of the GPL is necessary for host recognition. This study will promote better understanding of the structure-function relationships of GPLs and may open a new avenue for the prevention of MAIC infections.     

Human aquaporin adipose (AQPap) gene. Genomic structure, promoter analysis and functional mutation.

Aquaporin adipose (AQPap), which we identified from human adipose tissue, is a glycerol channel in adipocyte [Kishida et al. (2000) J. Biol. Chem. 275, 20896-20902]. In the current study, we determined the genomic structure of the human AQPap gene, and identified three AQPap-like genes that resembled (approximately 95%) AQPap, with little expression in human tissues. The AQPap promoter contained a putative peroxisome proliferator response element (PPRE) at -46 to -62, and a putative insulin response element (IRE) at -542/-536. Deletion of the PPRE abolished the pioglitazone-mediated induction of AQPap promoter activity in 3T3-L1 adipocytes. Deletion and single base pair substitution analysis of the IRE abolished the insulin-mediated suppression of the human AQPap gene. Analysis of AQPap sequence in human subjects revealed three missense mutations (R12C, V59L and G264V), and two silent mutations (A103A and G250G). The cRNA injection of the missense mutants into Xenopus oocytes revealed the absence of the activity to transport glycerol and water in the AQPap-G264V protein. In the subject homozygous for AQPap-G264V, exercise-induced increase in plasma glycerol was not observed in spite of the increased plasma noradrenaline. We suggest that AQPap is responsible for the increase of plasma glycerol during exercise in humans.     

Follistatin-derived peptide expression in muscle decreases adipose tissue mass and prevents hepatic steatosis

Myostatin, a member of the transforming growth factor (TGF)-β superfamily, plays a potent inhibitory role in regulating skeletal muscle mass. Inhibition of myostatin by gene disruption, transgenic (Tg) expression of myostatin propeptide, or injection of propeptide or myostatin antibodies causes a widespread increase in skeletal muscle mass. Several peptides, in addition to myostatin propeptide and myostatin antibodies, can bind directly to and neutralize the activity of myostatin. These include follistatin and follistatin-related gene. Overexpression of follistatin or follistatin-related gene in mice increased the muscle mass as in myostatin knockout mice. Follistatin binds to myostatin but also binds to and inhibits other members of the TGF-β superfamily, notably activins. Therefore, follistatin regulates both myostatin and activins in vivo. We previously reported the development and characterization of several follistatin-derived peptides, including FS I-I (Nakatani M, Takehara Y, Sugino H, Matsumoto M, Hashimoto O, Hasegawa Y, Murakami T, Uezumi A, Takeda S, Noji S, Sunada Y, Tsuchida K. FASEB J 22: 477-487, 2008). FS I-I retained myostatin-inhibitory activity without affecting the bioactivity of activins. Here, we found that inhibition of myostatin increases skeletal muscle mass and decreases fat accumulation in FS I-I Tg mice. FS I-I Tg mice also showed decreased fat accumulation even on a control diet. Interestingly, the adipocytes in FS I-I Tg mice were much smaller than those of wild-type mice. Furthermore, FS I-I Tg mice were resistant to high-fat diet-induced obesity and hepatic steatosis and had lower hepatic fatty acid levels and altered fatty acid composition compared with control mice. FS I-I Tg mice have improved glucose tolerance when placed on a high-fat diet. These data indicate that inhibiting myostatin with a follistatin-derived peptide provides a novel therapeutic option to decrease adipocyte size, prevent obesity and hepatic steatosis, and improve glucose tolerance.     

Colocalization of NO and VIP in neurons of the submucous plexus in the rat intestine

Since very few previous studies have carried out the quantitative analysis for the colocalization of nitric oxide (NO) and vasoactive intestinal peptide (VIP) in the submucous neurons in the rat digestive tract, we applied in vivo treatment of colchicine to enhance the immunoreactivity and examined the colocalization of NO synthase (nNOS) and VIP in neurons of the submucous plexus throughout the rat digestive tract. The density of nNOS-containing neurons in the submucous plexus in the stomach corpus (103±25 cells/cm², n=3) and that in the antrum (157±9 cells/cm², n=3) were significantly lower than those in small and large intestine. However no difference was detected in the cell density among duodenum (1967±188 cells/cm², n=3), jejunum (2640±140 cells/cm², n=3), ileum (2070±42 cells/cm², n=3), proximal colon (2243±138 cells/cm², n=3) and distal colon (2633±376 cells/cm², n=3). The proportion of nNOS-immunoreactive (IR), nNOS/VIP-IR and VIP-IR neurons to the total number of submucous neurons was examined. nNOS/VIP-IR neurons comprised 45–55% of total number of submucous neurons from the duodenum to the proximal colon, however those comprised 66.4±5.1% in the distal colon. The results showed that the dense distribution of nNOS-containing neurons was found in the submucous plexus throughout the small and large intestine, and large population of submucous neurons co-stored nNOS and VIP.     

A long Y chromosome in man

Summary An unusually long Y chromosome was described in the phenotypically normal father and paternal grandfather of a girl with Down's syndrome, and likewise in a male infant with multiple malformations and his father, normal in phenotype. Measurements revealed that the long Y chromosome corresponded in length to autosomes of group 16–18.Information was obtained to show that the increased length of the Y chromosome was an inheritable character, and that a long Y chromosome was not always associated with an abnormal phenotype (or phenotypes).Contribution No. 585 from the Zoological Institute, Hokkaido University.     

The chromosomes of Dall's porpoise,Phocoenoides dallii (True), with remarks on the phylogenetic relation of the Cetacea

Summary The chromosome complex of Dall's porpoise Phocoenoides dallii (True), a species of the Delphinidae (Cetacea), was investigated in male germ cells during the course of spermatogenesis. The diploid number of chromosomes in this species was 44 in the spermatogonia and the haploid number was 22 in both primary and secondary spermatocytes. Sex chromosomes of the typical XY-type were found to occur in this species. The X element is represented by one of the medium-sized chromosomes of rod-type characterized by a globular body located at its inner extremity, while the Y is very minute, attaining a size approximately one third that of the smallest autosome.Morphological analysis of the chromosomes shows the chromosome complement of this species to be strikingly characterized by the prevalence of medium-sized elements having subterminal fibre attachments. Comparison of the chromosomes with those of related forms of mammals shows that the chromosome constitution of this species approximates closely that of the pig. The question of the phylogenetical affinity of the Cetacea was discussed on the basis of the karyological evidence here reported.Contribution No. 213 form the Zoological Institute, Faculty of Science, Hokkaido University, Sapporo, Japan.     

A cytological study of the yoshida sarcoma. An ascites tumor op white rats

Summary In the Yoshida sarcoma a strain of tumor cells is present which have their own characteristic chromosome constitution and multiply by regular mitosis. The well-balanced complement of ±40 chromosomes consists of two distinct groups: one is represented by 22 to 24 rodshaped elements which probably come directly and without change from the original normal cell, the other group comprises 16 to 18 Vand J-shaped chromosomes which are specific for the tumor cells. Their exact origin is unknown, but must be mutational in character. Because of this morphological peculiarity, the chromosomes of the tumor cells are markedly different from those of the host cells for which 42 rodshaped elements are typical. No transitional types bridging the gap between ordinary and tumor cells occur. The individuality of the chromosomes in the strain cells remains unchanged during successive transplant generations from rat to rat. The growth of the tumor is primarily caused by the proliferation of these strain cells. In the course of multiplication part of the proliferating cells become abnormal and undergo aberrant mitotic processes owing probably to an alteration of the spindle mechanism, structural changes of the chromosomes, and some other causes. The frequently occurring tumor cells showing mitotic abnormalities are, therefore, derivatives of the sub-diploid strain cells. Destruction of the derivative cells by chemical treatment (podophyllin, CaCl₂) is followed by multiplication of the resistant strain cells.Comparable evidence has been found in two new strains of ascites tumor similar to the Yoshida sarcoma. Their strain cells have the same total chromosome number as the Yoshida strain cells and, within the set, the same two groups of rod-shaped and of Vor J-shaped chromosomes, but differ from each other as well as from the Yoshida sarcoma in the number of Vand J-shaped chromosomes.Contribution No. 263 from the Zoological Institute, Faculty of Science, Hokkaido University, Sapporo, Japan.     

Oxygenation-linked changes in the ultraviolet absorption and circular dichroism spectra of spiny lobster hemocyanin

As an approach to elucidate the mechanism of the protein structure change in the cooperative ligand binding, the UV difference and CD spectra of aromatic residues in Panulirus japonicus (spiny lobster) hemocyanin were examined. The native hemocyanin showed an O2-induced narrow-banded change in the absorption spectrum around 290 nm, which was not affected by pH in the range of 7.5 to 9.5. When the native hexameric protein was stripped of divalent cations with EDTA (at pH 7.5), the magnitude of the narrow-banded difference was reduced to about half, whereas it was almost completely abolished on dissociation into subunits (stripped at pH 9.5). The magnitude of the absorption change was found to be proportional to the degree of O2 saturation in the native and stripped hemocyanins. It was inferred that the spectral difference reflects a tertiary structure change directly linked to the oxygenation, though it depends greatly on the subunit association. Panulirus hemocyanin showed negative CD bands in the region of 260 to 300 nm, the intensities of which were considerably reduced by oxygenation and also by dissociation into subunits.