Effect of DNA modifications on DNA processing by HIV-1 integrase and inhibitor binding: role of DNA backbone flexibility and an open catalytic site

Integration of the viral cDNA into host chromosomes is required for viral replication. Human immunodeficiency virus integrase catalyzes two sequential reactions, 3'-processing (3'-P) and strand transfer (ST). The first integrase inhibitors are undergoing clinical trial, but interactions of inhibitors with integrase and DNA are not well understood in the absence of a co-crystal structure. To increase our understanding of integrase interactions with DNA, we examined integrase catalysis with oligonucleotides containing DNA backbone, base, and groove modifications placed at unique positions surrounding the 3'-processing site. 3'-Processing was blocked with substrates containing constrained sugars and alpha-anomeric residues, suggesting that integrase requires flexibility of the phosphodiester backbone at the 3'-P site. Of several benzo[a]pyrene 7,8-diol 9,10-epoxide (BaP DE) adducts tested, only the adduct in the minor groove at the 3'-P site inhibited 3'-P, suggesting the importance of the minor groove contacts for 3'-P. ST occurred in the presence of bulky BaP DE DNA adducts attached to the end of the viral DNA suggesting opening of the active site for ST. Position-specific effects of these BaP DE DNA adducts were found for inhibition of integrase by diketo acids. Together, these results demonstrate the importance of DNA structure and specific contacts with the viral DNA processing site for inhibition by integrase inhibitors.     

Cytopathic effects of the cytomegalovirus-encoded apoptosis inhibitory protein vMIA

Replication of human cytomegalovirus (CMV) requires the expression of the viral mitochondria-localized inhibitor of apoptosis (vMIA). vMIA inhibits apoptosis by recruiting Bax to mitochondria, resulting in its neutralization. We show that vMIA decreases cell size, reduces actin polymerization, and induces cell rounding. As compared with vMIA-expressing CMV, vMIA-deficient CMV, which replicates in fibroblasts expressing the adenoviral apoptosis suppressor E1B19K, induces less cytopathic effects. These vMIA effects can be separated from its cell death-inhibitory function because vMIA modulates cellular morphology in Bax-deficient cells. Expression of vMIA coincided with a reduction in the cellular adenosine triphosphate (ATP) level. vMIA selectively inhibited one component of the ATP synthasome, namely, the mitochondrial phosphate carrier. Exposure of cells to inhibitors of oxidative phosphorylation produced similar effects, such as an ATP level reduced by 30%, smaller cell size, and deficient actin polymerization. Similarly, knockdown of the phosphate carrier reduced cell size. Our data suggest that the cytopathic effect of CMV can be explained by vMIA effects on mitochondrial bioenergetics.     

Focal adhesion size controls tension-dependent recruitment of alpha-smooth muscle actin to stress fibers

Expression of alpha-smooth muscle actin (alpha-SMA) renders fibroblasts highly contractile and hallmarks myofibroblast differentiation. We identify alpha-SMA as a mechanosensitive protein that is recruited to stress fibers under high tension. Generation of this threshold tension requires the anchoring of stress fibers at sites of 8-30-microm-long "supermature" focal adhesions (suFAs), which exert a stress approximately fourfold higher (approximately 12 nN/microm2) on micropatterned deformable substrates than 2-6-microm-long classical FAs. Inhibition of suFA formation by growing myofibroblasts on substrates with a compliance of < or = 11 kPa and on rigid micropatterns of 6-microm-long classical FA islets confines alpha-SMA to the cytosol. Reincorporation of alpha-SMA into stress fibers is established by stretching 6-microm-long classical FAs to 8.1-microm-long suFA islets on extendable membranes; the same stretch producing 5.4-microm-long classical FAs from initially 4-microm-long islets is without effect. We propose that the different molecular composition and higher phosphorylation of FAs on supermature islets, compared with FAs on classical islets, accounts for higher stress resistance.     

New strepsirrhine primate from the late Eocene of Peninsular Thailand (Krabi Basin)

In this paper, we describe the newly discovered lower jaw of a primate from the late Eocene Krabi coal mine (Bang Mark pit) of Peninsular Thailand. We performed microtomographic examinations at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) to analyze different morphological aspects of the jaw and teeth. Although partially preserved, this fossil mandible reveals a set of distinctive dental traits (e.g., double-rooted P(2) and molarized P(4)) that allow us to describe a new stepsirrhine adapiform: Muangthanhinius siami, new genus and species. This taxon is somewhat atypical among Paleogene adapiforms, and more specialized than the sivaladapid adapiforms (hoanghoniines) that existed in Asia in the same epoch. In fact, Muangthanhinius shows a degree of dental specialization approximating that of some modern strepsirrhine lemuriforms, although it lacks the highly specialized anterior dentition characterizing this living primate group (canine + incisors forming a very procumbent toothcomb). In contrast, Muangthanhinius exhibits a large canine deeply anchored within the dentary that probably protruded high above the toothrow. Finally, despite the development of a molarized P(4) as in Miocene sivaladapid sivaladapines, Muangthanhinius differs in molar morphology from this group, and the position of this new taxon within the Adapiformes remains indeterminate. Clarification of its phylogenetic position will require more morphological evidence than is currently available.     

Khoratpithecus piriyai, a Late Miocene hominoid of Thailand

A Khoratpithecus piriyai lower jaw corresponds to a well-preserved Late Miocene hominoid fossil from northeastern Thailand. Its morphology and internal structure, using a microcomputed tomography scan, are described and compared to those of other known Miocene hominoids. It originated from fluviatile sand and gravel deposits of a large river, and was associated with many fossil tree trunks, wood fragments, and large vertebrate remains. A biochronological analysis by using associated mammal fauna gives an estimated geological age between 9-6 Ma. The flora indicates the occurrence of a riverine tropical forest and wide areas of grassland. K. piriyai displays many original characters, such as the great breadth of its anterior dentition, suggesting large incisors, large lower M3, a canine with a flat lingual wall, and symphysis structure. Several of its morphological derived characters are shared with the orangutan, indicating sister-group relationship with that extant ape. This relationship is additionally strongly supported by the absence of anterior digastric muscle scars. These shared derived characters are not present in Sivapithecus, Ankarapithecus, and Lufengpithecus, which are therefore considered more distant relatives to the orangutan than Khoratpithecus. The Middle Miocene K. chiangmuanensis is older, displays more primitive dental characters, and shares several dental characters with the Late Miocene form. It is therefore interpreted as its probable ancestor. But its less enlarged M3 and more wrinkled enamel may suggest an even closer phylogenetic position to orangutan ancestors, which cannot yet be supported because of the incomplete fossil record. Thus Khoratpithecus represents a new lineage of Southeast Asian hominoids, closely related to extant great ape ancestors.     

Evidence of a functional role for interaction between ICAM-1 and nonmuscle alpha-actinins in leukocyte diapedesis

ICAM-1 is involved in both adhesion and extravasation of leukocytes to endothelium during inflammation. It has been shown that the ICAM-1 cytoplasmic domain is important for transendothelial migration of leukocytes but the precise molecular mechanisms involving the intracytoplasmic portion of ICAM-1 is not known. To characterize precisely the molecular scaffolding associated with ICAM-1, we have used the yeast two-hybrid system, and we have identified six different proteins interacting with the ICAM-1 cytoplasmic domain. In this study, we report that the two forms of nonmuscle alpha-actinin (i.e., alpha-actinin 1 and alpha-actinin 4) associate with ICAM-1, and that these interactions are essential for leukocyte extravasation. These interactions were further confirmed by coimmunoprecipitation and immunofluorescence in endothelial cells and in ICAM-1-transfected Chinese hamster ovary cells. The function of these interactions was analyzed by point mutation of charged amino acids located on ICAM-1 cytoplasmic domain. We have identified three charged amino acids (arginine 480, lysine 481, and arginine 486) which are essential in the binding of alpha-actinins to the ICAM-1 cytoplasmic tail. Mutation of these amino acids completely inhibited ICAM-1-mediated diapedesis. Experiments with siRNA inhibiting specifically alpha-actinin 1 or alpha-actinin 4 on endothelial cells indicated that alpha-actinin 4 had a major role in this phenomenon. Thus, our data demonstrate that ICAM-1 directly interacts with cytoplasmic alpha-actinin 1 and 4 and that this interaction is required for leukocyte extravasation.     

Bone marrow mesenchymal stem cells suppress lymphocyte proliferation in vitro but fail to prevent graft-versus-host disease in mice

Several reports have suggested that mesenchymal stem cells (MSCs) could exert a potent immunosuppressive effect in vitro, and thus may have a therapeutic potential for T cell-dependent pathologies. We aimed to establish whether MSCs could be used to control graft-vs-host disease (GVHD), a major cause of morbidity and mortality after allogeneic hemopoietic stem cell transplantation. From C57BL/6 and BALB/c mouse bone marrow cells, we purified and expanded MSCs characterized by the lack of expression of CD45 and CD11b molecules, their typical spindle-shaped morphology, together with their ability to differentiate into osteogenic, chondrogenic, and adipogenic cells. These MSCs suppressed alloantigen-induced T cell proliferation in vitro in a dose-dependent manner, independently of their MHC haplotype. However, when MSCs were added to a bone marrow transplant at a MSCs:T cells ratio that provided a strong inhibition of the allogeneic responses in vitro, they yielded no clinical benefit on the incidence or severity of GVHD. The absence of clinical effect was not due to MSC rejection because they still could be detected in grafted animals, but rather to an absence of suppressive effect on donor T cell division in vivo. Thus, in these murine models, experimental data do not support a significant immunosuppressive effect of MSCs in vivo for the treatment of GVHD.     

Molecular mechanisms of neonatal hyperinsulinism

Congenital hyperinsulinism (CHI), characterized by profound hypoglycaemia related to inappropriate insulin secretion, may be associated histologically with either diffuse insulin hypersecretion or focal adenomatous hyperplasia, which share a similar clinical presentation, but result from different molecular mechanisms. Whereas diffuse CHI is of autosomal recessive, or less frequently of autosomal dominant, inheritance, focal CHI is sporadic. The most common mechanism underlying CHI is dysfunction of the pancreatic ATP-sensitive potassium channel (K(+)(ATP)). The two subunits of the K(+)(ATP) channel are encoded by the sulfonylurea receptor gene (SUR1 or ABCC8) and the inward-rectifying potassium channel gene (KIR6.2 or KCNJ11), both located in the 11p15.1 region. Germ-line, paternally inherited, mutations of the SUR1 or KIR6.2 genes, together with somatic maternal haplo-insufficiency for 11p15.5, were shown to result in focal CHI. Diffuse CHI results from germ-line mutations in the SUR1 or KIR6.2 genes, but also from mutations in several other genes, namely glutamate dehydrogenase (with associated hyperammonaemia), glucokinase, short-chain L-3-hydroxyacyl-CoA dehydrogenase, and insulin receptor gene. Hyperinsulinaemic hypoglycaemia may be observed in several overlapping syndromes, such as Beckwith-Wiedemann syndrome (BWS), Perlman syndrome, and, more rarely, Sotos syndrome. Mosaic genome-wide paternal isodisomy has recently been reported in patients with clinical signs of BWS and CHI. The primary causes of CHI are genetically heterogeneous and have not yet been completely unveiled. However, secondary causes of hyperinsulinism have to be considered such as fatty acid oxidation deficiency, congenital disorders of glycosylation and factitious hypoglycaemia secondary to Munchausen by proxy syndrome.     

Stoichiometry of ATP and metal cofactor interaction with the sarcoplasmic reticulum Ca(2+)-ATPase: a binding model accounting for radioisotopic and fluorescence results

Sarcoplasmic reticulum Ca-ATPase belongs to the P-type ATPases family and transports calcium at the expense of ATP hydrolysis. For years, a complex pattern of activity has been observed as a function of ATP and metal cofactor concentrations, leaving the stoichiometry of both metal and ATP in the active site as an open question. In agreement with recent structural studies we present here-using Mn as analogue of Mg-radioisotopic and fluorescence results showing that two metal ions bind to the Ca-ATPase favoring ATP binding. We further show that low ATP concentration favors the binding of these ions, whereas high ATP concentration is inhibitory. We propose a binding model for ATP and metal ions, which permits simulation of our data. Finally, we suggest that (i) the contribution of two metal ions as cofactors of ATP is essential to get maximal activity; (ii) the contribution of two ATP molecules can activate or inhibit the Ca-ATPase depending on metal concentration.