The intracellular signal transduction of AngII (angiotensin II) has been implicated in cardiovascular diseases, such as hypertension, atherosclerosis and restenosis after injury. AT(1) receptor (AngII type-1 receptor), a G-protein-coupled receptor, mediates most of the physiological and pathophysiological actions of AngII, and this receptor is predominantly expressed in cardiovascular cells, such as VSMCs (vascular smooth muscle cells). AngII activates various signalling molecules, including G-protein-derived second messengers, protein kinases and small G-proteins (Ras, Rho, Rac etc), through the AT(1) receptor leading to vascular remodelling. Growth factor receptors, such as EGFR (epidermal growth factor receptor), have been demonstrated to be 'trans'-activated by the AT(1) receptor in VSMCs to mediate growth and migration. Rho and its effector Rho-kinase/ROCK are also implicated in the pathological cellular actions of AngII in VSMCs. Less is known about the endothelial AngII signalling; however, recent studies suggest the endothelial AngII signalling positively, as well as negatively, regulates the NO (nitric oxide) signalling pathway and, thereby, modulates endothelial dysfunction. Moreover, selective AT(1)-receptor-interacting proteins have recently been identified that potentially regulate AngII signal transduction and their pathogenic functions in the target organs. In this review, we focus our discussion on the recent findings and concepts that suggest the existence of the above-mentioned novel signalling mechanisms whereby AngII mediates the formation of cardiovascular diseases.

The complete genomic sequence of the archaeon Thermoplasma volcanium, possessing optimum growth temperature (OGT) of 60°C, is reported. By systematically comparing this genomic sequence with the other known genomic sequences of archaea, all possessing higher OGT, a number of strong correlations have been identified between characteristics of genomic organization and the OGT. With increasing OGT, in the genomic DNA, frequency of clustering purines and pyrimidines into separate dinucleotides rises (e.g., by often forming AA and TT, whereas avoiding TA and AT). Proteins coded in a genome are divided into two distinct subpopulations possessing isoelectric points in different ranges (i.e., acidic and basic), and with increasing OGT the size of the basic subpopulation becomes larger. At the metabolic level, genes coding for enzymes mediating pathways for synthesizing some coenzymes, such as heme, start missing. These findings provide insights into the design of individual genomic components, as well as principles for coordinating changes in these designs for the adaptation to new environments.

Here we describe the large-scale domain movements and hydration structure changes in the active-site cleft of unligated glutamate dehydrogenase. Glutamate dehydrogenase from Thermococcus profundus is composed of six identical subunits of M(r) 46K, each with two distinct domains of roughly equal size separated by a large active-site cleft. The enzyme in the unligated state was crystallized so that one hexamer occupied a crystallographic asymmetric unit, and the crystal structure of the hexamer was solved and refined at a resolution of 2.25 A with a crystallographic R-factor of 0.190. In that structure, the six subunits displayed significant conformational variations with respect to the orientations of the two domains. The variation was most likely explained as a hinge-bending motion caused by small changes in the main chain torsion angle of the residue composing a loop connecting the two domains. Small-angle X-ray scattering profiles both at 293 and 338 K suggested that the apparent molecular size of the hexamer was slightly larger in solution than in the crystalline state. These results led us to the conclusion that (i) the spontaneous domain motion was the property of the enzyme in solution, (ii) the domain motion was trapped in the crystallization process through different modes of crystal contacts, and (iii) the magnitude of the motion in solution was greater than that observed in the crystal structure. The present cryogenic diffraction experiment enabled us to identify 1931 hydration water molecules around the hexamer. The hydration structures around the subunits exhibited significant changes in accord with the degree of the domain movement. In particular, the hydration water molecules in the active-site cleft were rearranged markedly through migrations between specific hydration sites in coupling strongly with the domain movement. We discussed the cooperative dynamics between the domain motion and the hydration structure changes in the active site of the enzyme. The present study provides the first example of a visualized hydration structure varying transiently with the dynamic movements of enzymes and may form a new concept of the dynamics of multidomain enzymes in solution.

The angiotensin II (AngII) type 1 receptor (AT(1)) plays a critical role in hypertrophy of vascular smooth muscle cells (VSMCs). Although it is well known that G(q) is the major G protein activated by the AT(1) receptor, the requirement of G(q) for AngII-induced VSMC hypertrophy remains unclear. By using cultured VSMCs, this study examined the requirement of G(q) for the epidermal growth factor receptor (EGFR) pathway, the Rho-kinase (ROCK) pathway, and subsequent hypertrophy. AngII-induced intracellular Ca(2+) elevation was completely inhibited by a pharmacological G(q) inhibitor as well as by adenovirus encoding a G(q) inhibitory minigene. AngII (100nm)-induced EGFR transactivation was almost completely inhibited by these inhibitors, whereas these inhibitors only partially inhibited AngII (100nm)-induced phosphorylation of a ROCK substrate, myosin phosphatase target subunit-1. Stimulation of VSMCs with AngII resulted in an increase of cellular protein and cell volume but not in cell number. The G(q) inhibitors completely blocked these hypertrophic responses, whereas a G protein-independent AT(1) agonist did not stimulate these hypertrophic responses. In conclusion, G(q) appears to play a major role in the EGFR pathway, leading to vascular hypertrophy induced by AngII. Vascular G(q) seems to be a critical target of intervention against cardiovascular diseases associated with the enhanced renin-angiotensin system.

Bonobos (Pan paniscus) inhabit regions south of the Congo River including all areas between its southerly tributaries. To investigate the genetic diversity and evolutionary relationship among bonobo populations, we sequenced mitochondrial DNA from 376 fecal samples collected in seven study populations located within the eastern and western limits of the species’ range. In 136 effective samples from different individuals (range: 7–37 per population), we distinguished 54 haplotypes in six clades (A1, A2, B1, B2, C, D), which included a newly identified clade (D). MtDNA haplotypes were regionally clustered; 83 percent of haplotypes were locality-specific. The distribution of haplotypes across populations and the genetic diversity within populations thus showed highly geographical patterns. Using population distance measures, seven populations were categorized in three clusters: the east, central, and west cohorts. Although further elucidation of historical changes in the geological setting is required, the geographical patterns of genetic diversity seem to be shaped by paleoenvironmental changes during the Pleistocene. The present day riverine barriers appeared to have a weak effect on gene flow among populations, except for the Lomami River, which separates the TL2 population from the others. The central cohort preserves a high genetic diversity, and two unique clades of haplotypes were found in the Wamba/Iyondji populations in the central cohort and in the TL2 population in the eastern cohort respectively. This knowledge may contribute to the planning of bonobo conservation.

Thermococcus profundus, a hyperthermophilic archaeon, did not exhibit detectable glutamine synthetase activity, although the organism possessed an extraordinarily high level of glutamate dehydrogenase (GDH), the content of which reached over 10% of total soluble proteins. This GDH was purified to homogeneity. The enzyme had a molecular weight of 263,000 and was composed of six homogeneous subunits of molecular weight 43,000. The enzyme was extremely thermostable with a half life of 1 h at 90 degrees C. Circular dichroism (CD) spectra of the enzyme revealed gradual unfolding of alpha-helices upon exposure to increasing temperature. The enzyme reaction was strongly biased toward glutamate formation. T. profundus excreted L-alanine into the medium, and the concentration reached mM. High activity of alanine aminotransferase (AAT) was present in the cells, while no alanine dehydrogenase activity was detected. The alanine formation may be initiated by ammonia uptake by GDH followed by aminotransfer from glutamate to pyruvate by AAT.

Background-Angiotensin II (Ang II) promotes growth of vascular smooth muscle cells (VSMCs) via epidermal growth factor (EGF) receptor (EGFR) transactivation mediated through a metalloprotease-dependent shedding of heparinbinding EGF-like growth factor (HB-EGF). However, the identity of the metalloprotease responsible for this process remains unknown. Methods and Results-To identify the metalloprotease required for Ang II-induced EGFR transactivation, primary cultured aortic VSMCs were infected with retrovirus encoding dominant negative (dn) mutant of ADAM10 or ADAM17. EGFR transactivation induced by Ang II was inhibited in VSMCs infected with dnADAM17 retrovirus but not with dnADAM10 retrovirus. However, Ang II comparably stimulated intracellular Ca 2ϩ elevation and JAK2 tyrosine phosphorylation in these VSMCs. In addition, dnADAM17 inhibited HB-EGF shedding induced by Ang II in A10 VSMCs expressing the AT 1 receptor. Moreover, Ang II enhanced protein synthesis and cell volume in VSMCs infected with control retrovirus, but not in VSMCs infected with dnADAM17 retrovirus.

Vector engineering and gene disruption in host cells were attempted for the enhancement of alpha-agglutinin-based display of proteins on the cell surface in yeast. To evaluate the display efficiency by flow cytometric analysis, DsRed-monomer fused with FLAG-tag was displayed and immunostained as a model protein. The use of leu2-d in the expression vector resulted in the enhanced efficiency and ratio of the accessible display of proteins. Moreover, the amount of displayed proteins in SED1-disrupted cells increased particularly during the stationary growth phase. The combination of these improvements resulted in the quantitatively enhanced accessible display of DsRed-monomer on the yeast cell surface. The improved yeast display system would be useful in a wider range of its applications in biotechnology.