Definition of membrane topology and identification of residues important for transport in subunit a of the vacuolar ATPase

Subunit a of the vacuolar H(+)-ATPases plays an important role in proton transport. This membrane-integral 100-kDa subunit is thought to form or contribute to proton-conducting hemichannels that allow protons to gain access to and leave buried carboxyl groups on the proteolipid subunits (c, c', and c″) during proton translocation. We previously demonstrated that subunit a contains a large N-terminal cytoplasmic domain followed by a C-terminal domain containing eight transmembrane (TM) helices. TM7 contains a buried arginine residue (Arg-735) that is essential for proton transport and is located on a helical face that interacts with the proteolipid ring. To further define the topology of the C-terminal domain, the accessibility of 30 unique cysteine residues to the membrane-permeant reagent N-ethylmaleimide and the membrane-impermeant reagent polyethyleneglycol maleimide was determined. The results further define the borders of transmembrane segments in subunit a. To identify additional buried polar and charged residues important in proton transport, 25 sites were individually mutated to hydrophobic amino acids, and the effect on proton transport was determined. These and previous results identify a set of residues important for proton transport located on the cytoplasmic half of TM7 and TM8 and the lumenal half of TM3, TM4, and TM7. Based upon these data, we propose a tentative model in which the cytoplasmic hemichannel is located at the interface of TM7 and TM8 of subunit a and the proteolipid ring, whereas the lumenal hemichannel is located within subunit a at the interface of TM3, TM4, and TM7.     

RACK1 regulates VEGF/Flt1-mediated cell migration via activation of a PI3K/Akt pathway

Vascular endothelial growth factor (VEGF) is vital to physiological as well as pathological angiogenesis, and regulates a variety of cellular functions, largely by activating its 2 receptors, fms-like tyrosine kinase (Flt1) and kinase domain receptor (KDR). KDR plays a critical role in the proliferation of endothelial cells by controlling VEGF-induced phospholipase Cγ-protein kinase C (PLCγ-PKC) signaling. The function of Flt1, however, remains to be clarified. Recent evidence has indicated that Flt1 regulates the VEGF-triggered migration of endothelial cells and macrophages. Here, we show that RACK1, a ubiquitously expressed scaffolding protein, functions as an important regulator of this process. We found that RACK1 (receptor for activated protein kinase C 1) binds to Flt1 in vitro. When the endogenous expression of RACK1 was attenuated by RNA interference, the VEGF-driven migration was remarkably suppressed whereas the proliferation was unaffected in a stable Flt1-expressing cell line, AG1-G1-Flt1. Further, we demonstrated that the VEGF/Flt-mediated migration of AG1-G1-Flt1 cells occurred mainly via the activation of the PI3 kinase (PI3K)/Akt and Rac1 pathways, and that RACK1 plays a crucial regulatory role in promoting PI3K/Akt-Rac1 activation.     

Cellular DBP and E4BP4 proteins are critical for determining the period length of the circadian oscillator

The phenotypes of mice carrying clock gene mutations have been critical to understanding the mammalian clock function. However, behavior does not necessarily reflect cell-autonomous clock phenotypes, because of the hierarchical dominance of the central clock. We performed cell-based siRNA knockdown and cDNA overexpression and monitored rhythm using bioluminescent reporters of clock genes. We found that knockdown of DBP, D-box positive regulator, in our model led to a short-period phenotype, whereas overexpressing of DBP produced a long-period rhythm when compared to controls. Furthermore, knockdown and overexpressing of E4BP4, D-box negative regulator, led to an opposite effect of DBP. Our experiments demonstrated that D-box regulators play a crucial role in determining the period length of Per1 and Per2 promoter-driven circadian rhythms in Rat-1 fibroblasts.     

Some human immunodeficiency virus type 1 Vpu proteins are able to antagonize macaque BST-2 in vitro and in vivo: Vpu-negative simian-human immunodeficiency viruses are attenuated in vivo

Human immunodeficiency virus type 1 (HIV-1) Vpu enhances the release of viral particles from infected cells by targeting BST-2/tetherin, a cellular protein inhibiting virus release. The widely used HIV-1(NL4-3) Vpu functionally inactivates human BST-2 but not murine or monkey BST-2, leading to the notion that Vpu antagonism is species specific. Here we investigated the properties of the CXCR4-tropic simian-human immunodeficiency virus DH12 (SHIV(DH12)) and the CCR5-tropic SHIV(AD8), each of which carries vpu genes derived from different primary HIV-1 isolates. We found that virion release from infected rhesus peripheral blood mononuclear cells was enhanced to various degrees by the Vpu present in both SHIVs. Transfer of the SHIV(DH12) Vpu transmembrane domain to the HIV-1(NL4-3) Vpu conferred antagonizing activity against macaque BST-2. Inactivation of the SHIV(DH12) and SHIV(AD8) vpu genes impaired virus replication in 6 of 8 inoculated rhesus macaques, resulting in lower plasma viral RNA loads, slower losses of CD4(+) T cells, and delayed disease progression. The expanded host range of the SHIV(DH12) Vpu was not due to adaptation during passage in macaques but was an intrinsic property of the parental HIV-1(DH12) Vpu protein. These results demonstrate that the species-specific inhibition of BST-2 by HIV-1(NL4-3) Vpu is not characteristic of all HIV-1 Vpu proteins; some HIV-1 isolates encode a Vpu with a broader host range.     

Clinical characteristics of primary hyperaldosteronism due to adrenal microadenoma

An increasing number of patients are being diagnosed with primary aldosteronism (PA) due to aldosterone-producing macroadenoma (APA). However, there are only limited data available on the clinical characteristics of PA that are associated with adrenal microadenoma. Of the 55 patients that were diagnosed with PA in our study, 22 patients showed a unilateral adrenal over-production of aldosterone. The histopathology of the surgically removed adrenal tissues led to six patients being diagnosed with microadenoma, and the clinical features of microadenoma, macroadenoma and idiopathic hyperaldosteronism (IHA) were studied. The expression levels of CYP11B2, CYP17, CYP21 and 3β-hydroxysteroid dehydrogenase 2 (HSD3B2) mRNA in the adrenal cortices (n = 5 and 6, respectively) that remained attached to the adrenal microadenomas or macroadenomas were examined by real time-PCR and then compared to the expression levels in the adrenal cortices (n = 5) of non-functioning adrenal adenomas (NF). The patients with microadenoma (n = 6) had significantly higher diastolic blood pressure than the patients with macroadenoma (n = 16) or IHA (n = 33) (p < 0.05). The systolic blood pressure, plasma aldosterone concentration, serum potassium level and renal function did not differ between the PA sub-groups. The levels of CYP11B2 and CYP17 mRNA were significantly increased in the adjacent tissues of microadenomas, as compared with macroadenomas or NF (p < 0.05), whereas no significant differences in the CYP21 and HSD3B2 mRNA levels were found between the PA sub-groups. The tumor size did not influence the clinical characteristics of APA. The non-tumor portions of the microadenomas showed marked and sustained CYP11B2 mRNA expression under the suppressed renin-angiotensin system. We suggest that an increased number of microadenomas should be sampled, and the immunohistochemistry for steoridogenic enzymes should be investigated to clarify the etiology of microadenoma.     

Genome-wide association study on and the clinical application to chronic hepatitis C

Based on the data and technology generated in previous international projects, such as the Human Genome Project and the HapMap, for the building of the common patterns of genetic variation in humans, a genome-wide association study (GWAS) to HCV infection was conducted to reveal genetic effects against treatment response or the induction of side effects. Single nucleotide polymorphisms (SNPs) associated with response to pegylated-interferon (PEG-IFN) and ribavirin (RBV) therapy were determined around IL-28B in chromosome 19, and the strong association was also observed in spontaneous viral clearance regardless of population. These data imply that an important interaction between HCV infection and IL-28B is critical for viral persistence or clearance. PEG-IFN and RBV therapy is associated with a range of treatment-limiting adverse effects. One of the frequent side effects induced by the combination therapy is haemolytic anaemia. The severe anaemia requires the reduction of the RBV dose, which could lead to treatment failure. Genetic variants around inosine triphosphatase gene (ITPA) were associated with heamolytic anaemia. Interestingly, the significant SNPs observed in Europe and the United States were not strongly associated with Japanese population although all significant SNPs were located around ITPA gene, suggesting that SNPs typing using individual population are required for the collection of precise data. These significant SNPs would be useful for prediction prior to treatment for individualized medicine.     

GFP chimeric models exhibited a biphasic pattern of mesenchymal cell invasion in tendon healing

The healing of an injured musculoskeletal system requires an influx of mesenchymal cells that can differentiate into osteoblasts, fibroblasts, chondroblasts, and skeletal myoblasts. However, whether these mesenchymal cells arise from the circulation (bone marrow) or the injured tissues themselves has been controversial. To reveal the spatiotemporal characteristics of the reparative mesenchymal cells, we investigated the healing process after patellar tendon injury using two types of green fluorescent protein (GFP) chimeric rats; one expressing GFP in the circulating cells, and the other expressing it in the patellar tendon. We analyzed the behavior of GFP-positive cells after experimental tendon injury in both chimeric rats to clarify the origin of reparative cells. At 24 h after the injury, the wound contained circulation-derived cells but not tendon-derived cells. Tendon-derived cells first appeared in the wounded area at 3 days after the injury, and had significantly increased in number with time and had maintained a high level of proliferative activity until 7 days after the injury, whereas the circulation-derived cells had decreased in number and had been replaced by the tendon-derived cells. These findings suggest that circulation-derived and tendon-derived cells contribute to the healing of tendons in different periods as part of a biphasic process.     

Structure of a hyperthermophilic archaeal homing endonuclease, I-Tsp061I: contribution of cross-domain polar networks to thermostability

A novel LAGLIDADG-type homing endonuclease (HEase), I-Tsp061I, from the hyperthermophilic archaeon Thermoproteus sp. IC-061 16 S rRNA gene (rDNA) intron was characterized with respect to its structure, catalytic properties and thermostability. It was found that I-Tsp061I is a HEase isoschizomer of the previously described I-PogI and exhibits the highest thermostability among the known LAGLIDADG-type HEases. Determination of the crystal structure of I-Tsp061I at 2.1 A resolution using the multiple isomorphous replacement and anomalous scattering method revealed that the overall fold is similar to that of other known LAGLIDADG-type HEases, despite little sequence similarity between I-Tsp061I and those HEases. However, I-Tsp061I contains important cross-domain polar networks, unlike its mesophilic counterparts. Notably, the polar network Tyr6-Asp104-His180-107O-HOH12-104O-Asn177 exists across the two packed alpha-helices containing both the LAGLIDADG catalytic motif and the GxxxG hydrophobic helix bundle motif. Another important structural feature is the salt-bridge network Asp29-Arg31-Glu182 across N and C-terminal domain interface, which appears to contribute to the stability of the domain/domain packing. On the basis of these structural analyses and extensive mutational studies, we conclude that such cross-domain polar networks play key roles in stabilizing the catalytic center and domain packing, and underlie the hyperthermostability of I-Tsp061I.     

Asexual growth of Babesia bovis is inhibited by specific sulfated glycoconjugates

In the present study, inhibitory effects of several sulfated and nonsulfated glycoconjugates were evaluated on the in vitro asexual growth of Babesia bovis. Among the selected sulfated glycoconjugates, dextran sulfate, heparin, heparan sulfate, fucoidan, and chondroitin sulfate B strongly inhibited the parasitic growth, and all but chondroitin sulfate B induced a significant accumulation of extracellular merozoites in culture. In contrast, chondroitin sulfate A, keratan sulfate, and protamine sulfate, as well as nonsulfated dextran and hyaluronic acid, did not influence the growth. These findings indicate that the asexual growth of B. bovis merozoites is inhibited by specific sulfated glycoconjugates, possibly providing us with an important insight into the molecular interaction(or interactions) during the process of the erythrocyte invasion by B. bovis merozoites.