A Back Hydrogen Exchange Procedure via the Acid-Unfolded State for a Large Protein

A deuterated protein sample is required for nuclear magnetic resonance (NMR) measurements of a large protein because severe signal broadenings occur because of the high molecular weight. The deuterated sample expressed in (2)H(2)O should subsequently be subjected to a back hydrogen exchange at amide groups. To perform the back exchange, the protein molecule is unfolded or destabilized so that internal residues become accessible to the solvent. However, the refolding yield from the destabilized or unfolded state of a large protein is usually low, leading to a dilemma in NMR measurements of large proteins. In our previous paper [Suzuki, M., et al. (2011) Biochemistry50, 10390-10398], we suggested that an acid-denatured microbial transglutaminase (MTG) consisting of 331 amino acid residues can be recovered effectively under low-salt conditions, escaping from the aggregation-prone intermediate. Here, we demonstrate that proMTG, the pro form of MTG consisting of 376 amino acid residues, can be refolded perfectly from the acid-unfolded state under low-salt conditions, as confirmed by circular dichroism and NMR spectroscopies. By performing the same procedure with a deuterated proMTG expressed in (2)H(2)O, we observed complete back exchanges for internal residues by NMR spectroscopy. Our procedure has potential applications to the back hydrogen exchange of large proteins for NMR measurements.     

Preliminary report of novel technique for cryopreservation--vacuum-assisted cryoprotectant infiltration

To date, cryopreservation of large soft tissues has not been successfully achieved because of limitation of cryoprotective agent (CPA) infiltration into the tissue. This study aimed to investigate the effects of a vacuum on the tissue-infiltration of a CPA. An instant pickle-maker was modified for use as a vacuum apparatus, and glycerol was selected as the CPA. Twenty-six rats were used, and their thighs were divided into three treatment groups. Group 1: fresh control; Group 2: cryopreserved control, i.e., immersed in the CPA for 1h under atmospheric pressure and cryopreserved; Group 3: vacuum-assisted CPA infiltration, i.e., immersed in the CPA under negative pressure (20, 40 and 60 cmHg, for durations of 10, 20 and 30 min at each) and cryopreserved. The Groups 2 and 3 specimens were thawed after 3 weeks of cryopreservation at -80 °C and histologically examined, in comparison with Group 1. Skin: in Groups 2 and 3, the skin was well preserved. Muscle: in Group 2, both extracellular and intracellular ice crystal formation was widely distributed throughout the muscle tissue. In Group 3, under an adequate vacuum, the muscle tissue was well preserved, with no ice crystal formation. However, when the treatment was conducted under excessive vacuum conditions, the muscle tissue showed focal necrosis. Blood vessels: in Group 3, both the arteries and veins were well preserved up to the tunica intima. The method described in this paper may be a useful technique for achieving cryopreservation of large soft tissues.     

Type-I IFN signaling is required for the induction of antigen-specific CD8(+) T cell responses by adenovirus vector vaccine in the gut-mucosa

Microtubules in living cells are very important component for various cellular functions as well as to maintain the cell shape. Mechanical properties of microtubules play a vital role in their functions and structure. To understand the mechanical properties of microtubules in living cells, we developed an orthotropic-Pasternak model and investigated the vibrational behavior when microtubules are embedded in surrounding elastic medium. We considered microtubules as orthotropic elastic shell and its surrounding elastic matrix as Pasternak foundation. We found that due to mechanical coupling of microtubules with elastic medium, the flexural vibration is increased with the stiffening of elastic medium. We noticed that foundation modulus (H) and shear modulus (G) have more effect on radial vibrational mode as compared to longitudinal vibrational mode and torsional vibrational mode.     

Siglec-15 protein regulates formation of functional osteoclasts in concert with DNAX-activating protein of 12 kDa (DAP12)

Osteoclasts are multinucleated giant cells that reside in osseous tissues and resorb bone. Signaling mediated by receptor activator of nuclear factor (NF)-κB (RANK) and its ligand leads to the nuclear factor of activated T cells 2/c1 (NFAT2 or NFATc1) expression, a critical step in the formation of functional osteoclasts. In addition, adaptor proteins harboring immunoreceptor tyrosine-based activation motifs, such as DNAX-activating protein of 12 kDa (DAP12), play essential roles. In this study, we identified the gene encoding the lectin Siglec-15 as NFAT2-inducible, and we found that the protein product links RANK ligand-RANK-NFAT2 and DAP12 signaling in mouse osteoclasts. Both the recognition of sialylated glycans by the Siglec-15 V-set domain and the association with DAP12 through its Lys-272 are essential for its function. When Siglec-15 expression was knocked down, fewer multinucleated cells developed, and those that did were morphologically contracted with disordered actin-ring structures. These changes were accompanied by significantly reduced bone resorption. Siglec-15 formed complexes with Syk through DAP12 in response to vitronectin. Furthermore, chimeric molecules consisting of the extracellular and transmembrane regions of Siglec-15 with a K272A mutation and the cytoplasmic region of DAP12 significantly restored bone resorption in cells with knocked down Siglec-15 expression. Together, these results suggested that the Siglec-15-DAP12-Syk-signaling cascade plays a critical role in functional osteoclast formation.     

Complexes of hydroxy(thio)pyrone and hydroxy(thio)pyridinone with Zn(II) and Mo(VI). Thermodynamic stability and insulin-mimetic activity

The development of metal-containing pharmaceuticals as insulin-mimetics has been the object of recent worldwide research. We have examined a series of zinc(II) and molybdenum(VI) complexes with model O,S-donor ligands (thiomaltol and 1,2-dimethyl-3-hydroxypyridine-4-thione (DMHTP)) and the corresponding O,O-analogues (maltol and DMHP) for their insulin-mimetic activity. Aimed at getting structure-activity relationships, some physical-chemical properties were also studied, such as metal-complex formation, speciation at different pH conditions and ligand lipophilicity. The Zn-complexes exhibit considerably higher insulin-mimetic activity than the corresponding Mo-analogues. Particularly, the bis(thiomaltolato)zinc(II) complex reveals a very high activity, ascribed to the effect of the thione π character and to the soft nature of the sulfur donor atom enhancing the Zn(II)-ligand affinity and the ligand/complex lipophilicity, two determinant parameters for delivering the metal-drug into the cells. Hence, these preliminary studies indicate that the Zn(thiomaltol)? complex can be considered a potential drug candidate for treatment of diabetes mellitus, upon in vivo evaluations.     

Impacts of Visceral Adipose Tissue and Subcutaneous Adipose Tissue on Metabolic Risk Factors in Middle‐aged Japanese

Regional fat distribution rather than overall fat volume has been considered to be important to understanding the link between obesity and metabolic disorders. We aimed to evaluate the independent associations of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) with metabolic risk factors in apparently healthy middle‐aged Japanese. Participants were 1,119 men and 854 women aged 38–60 years who were not taking medications for diabetes, hypertension, or dyslipidemia. VAT and SAT were measured by use of computed tomography (CT) scanning. VAT and SAT were significantly and positively correlated with each other in men (r = 0.531, P < 0.001) and women (r = 0.589, P < 0.001). In multiple regression analyses, either measure of abdominal adiposity (VAT or SAT) was positively associated with blood pressure, fasting plasma glucose, and log triglyceride (P < 0.001) and inversely with high‐density lipoprotein (HDL)‐cholesterol (P < 0.001). When VAT and SAT were simultaneously included in the model, the association of VAT with triglycerides was maintained (P < 0.001) but that of SAT was lost. The same was true for HDL‐cholesterol in women. For fasting plasma glucose, the association with VAT was strong (P < 0.001) and the borderline association with SAT was maintained (P = 0.060 in men and P = 0.020 in women). Both VAT and SAT were independently associated with blood pressure (P < 0.001). Further adjustment for anthropometric indices resulted in the independent association only with VAT for all risk factors. In conclusion, impacts of VAT and SAT differed among risk factors. VAT showed dominant impacts on triglyceride concentrations in both genders and on HDL‐cholesterol in women, while SAT also had an independent association with blood pressure.     

Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.     

E2 Polyubiquitin-conjugating Enzyme Ubc13 in Keratinocytes Is Essential for Epidermal Integrity

The E2 polyubiquitin-conjugating enzyme Ubc13 is a mediator of innate immune reactions. Ubc13 mediates the conjugation of keratin (K)63-linked polyubiquitin chains onto TNF receptor-associated factor 6 and IKKγ during NF-κB activation. In contrast to K48-linked polyubiquitin chains, K63-linked polyubiquitin chains function in nonproteasomal biological processes. Although Ubc13 has been shown to be critical for Toll-like receptor (TLR) and IL-1 receptor signaling, the function of Ubc13 in the epidermis has not been studied. We generated keratinocyte-specific Ubc13-deficient mice (Ubc13^flox/floxK5-Cre). At birth, the skin of the Ubc13^flox/floxK5-Cre mice was abnormally shiny and smooth; in addition, the mice did not grow and died by postnatal day 2. Histological analysis showed atrophy of the epidermis with keratinocyte apoptosis. Immunohistochemical analyses revealed reduced proliferation, abnormal differentiation, and apoptosis of keratinocytes in the Ubc13^flox/floxK5-Cre mouse epidermis. In culture, Ubc13^flox/floxK5-Cre keratinocyte growth was impaired, and spontaneous cell death occurred. Moreover, the deletion of Ubc13 from cultured Ubc13^flox/flox keratinocytes by means of an adenoviral vector carrying Cre recombinase also resulted in spontaneous cell death. Therefore, Ubc13 is essential for keratinocyte growth, differentiation, and survival. Analyses of intracellular signaling revealed that the IL-1 and TNF-induced activation of JNK, p38, and NF-κB pathways was impaired in Ubc13^flox/floxK5-Cre keratinocytes. In conclusion, Ubc13 appears to be essential for epidermal integrity in mice.