X-ray crystal structure of leukocyte type core 2 beta1,6-N-acetylglucosaminyltransferase. Evidence for a convergence of metal ion-independent glycosyltransferase mechanism

Leukocyte type core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT-L) is a key enzyme in the biosynthesis of branched O-glycans. It is an inverting, metal ion-independent family 14 glycosyltransferase that catalyzes the formation of the core 2 O-glycan (Galbeta1-3[GlcNAcbeta1-6]GalNAc-O-Ser/Thr) from its donor and acceptor substrates, UDP-GlcNAc and the core 1 O-glycan (Galbeta1-3GalNAc-O-Ser/Thr), respectively. Reported here are the x-ray crystal structures of murine C2GnT-L in the absence and presence of the acceptor substrate Galbeta1-3GalNAc at 2.0 and 2.7A resolution, respectively. C2GnT-L was found to possess the GT-A fold; however, it lacks the characteristic metal ion binding DXD motif. The Galbeta1-3GalNAc complex defines the determinants of acceptor substrate binding and shows that Glu-320 corresponds to the structurally conserved catalytic base found in other inverting GT-A fold glycosyltransferases. Comparison of the C2GnT-L structure with that of other GT-A fold glycosyltransferases further suggests that Arg-378 and Lys-401 serve to electrostatically stabilize the nucleoside diphosphate leaving group, a role normally played by metal ion in GT-A structures. The use of basic amino acid side chains in this way is strikingly similar to that seen in a number of metal ion-independent GT-B fold glycosyltransferases and suggests a convergence of catalytic mechanism shared by both GT-A and GT-B fold glycosyltransferases.     

Neutralizing epitopes of the SARS-CoV S-protein cluster independent of repertoire,antigen structure or mAb technology

Neutralizing antibody responses to the surface glycoproteins of enveloped viruses play an important role in immunity. Many of these glycoproteins, including the severe acute respiratory syndrome-coronavirus (SARS-CoV) spike (S) protein form trimeric units in the membrane of the native virion. There is substantial experimental and pre-clinical evidence showing that the S protein is a promising lead for vaccines and therapeutics. Previously we generated a panel of monoclonal antibodies (mAbs) to whole inactivated SARS-CoV which neutralize the virus in vitro.^1,2 Here, we define their specificity and affinity, map several of their epitopes and lastly characterise chimeric versions of them. Our data show that the neutralizing mAbs bind to the angiotensin-converting enzyme 2 (ACE2) receptor-binding domain (RBD) of the SARS S protein. Three of the chimeric mAbs retain their binding specificity while one conformational mAb, F26G19, lost its ability to bind the S protein despite high level expression. The affinity for recombinant S is maintained in all of the functional chimeric versions of the parental mAbs. Both parental mAb F26G18 and the chimeric version neutralize the TOR2 strain of SARS-CoV with essentially identical titres (2.07 and 2.47 nM, respectively). Lastly, a comparison with other neutralizing mAbs to SARS-CoV clearly shows that the dominance of a 33 amino acid residue loop of the SARS-CoV RBD is independent of repertoire, species, quaternary structure, and importantly, the technology used to derive the mAbs. In cases like this, the dominance of a compact RBD antigenic domain and the central role of the S protein in pathogenesis may inherently create immunoselection pressure on viruses to evolve more complex evasion strategies or die out of a host species. The apparent simplicity of the mechanism of SARS-CoV neutralization is in stark contrast to the complexity shown by other enveloped viruses.Key words: SARS coronavirus, monoclonal antibody, neutralizing, epitope, immunochemistry     

Establishment of a plant tissue culture system and genetic transformation for agronomic improvement of Indonesian black rice (Oryza sativa L.)

Plant Cell, Tissue and Organ Culture (PCTOC) - Indonesian black rice has the potential to be developed as a functional food due to their high anthocyanin content and other nutritional benefits such...     

Calreticulin transacetylase mediated upregulation of thioredoxin by 7,8-diacetoxy-4-methylcoumarin enhances the antioxidant potential and the expression of vascular endothelial growth factor in peripheral blood mononuclear cells

Extensive research carried out in our group on polyphenolic acetates (PAs) substantiated the potential role of PAs in causing diverse biological and pharmacological actions. Our earlier investigations firmly established the calreticulin transacetylase (CRTAase) catalyzed activation of nitric oxide synthase (NOS) by PAs. In this report, we have studied the effect of 7,8-diacetoxy-4-methylcoumarin (DAMC, a model PA) and other acetoxy coumarins on the thioredoxin and VEGF expression in human peripheral blood mononuclear cells (PBMCs), with a view to substantiate our earlier observation that DAMC was a superb inducer of angiogenesis. Real time RT-PCR analysis revealed the enhanced expression of thioredoxin reductase (TRXR) and diminished expression of thioredoxin interacting protein (TRXIP) leading to the increased expression and activity of thioredoxin (TRX) in PBMCs due to the the action of DAMC. The fact that TRX activity of PBMCs was enhanced by various acetoxy coumarins in tune with their affinity to CRTAase as substrate, suggested the possible activation of TRX due to acetylation. The overexpression of thioredoxin was found to correlate with that of VEGF as proved by real time RT-PCR and VEGF -ELISA results, apart from the DAMC-caused enhanced production of NO acting as an inducer of VEGF. Moreover, the intracellular ROS levels were also found to be reduced drastically, by DAMC thus reducing the oxidative stress in cells. These observations strongly evidenced the crucial role of TRX in DAMC-induced tissue angiogenesis with the involvement of VEGF.     

State of the science: an update on renal cell carcinoma

Renal cell carcinomas (RCC) are emerging as a complex set of diseases that are having a major socioeconomic impact and showing a continued rise in incidence throughout the world. As the field of urologic oncology faces these trends, several major genomic and mechanistic discoveries are altering our core understanding of this multitude of cancers, including several new rare subtypes of renal cancers. In this review, these new findings are examined and placed in the context of the well-established association of clear cell RCC (ccRCC) with mutations in the von Hippel-Lindau (VHL) gene and resultant aberrant hypoxia inducible factor (HIF) signaling. The impact of novel ccRCC-associated genetic lesions on chromatin remodeling and epigenetic regulation is explored. The effects of VHL mutation on primary ciliary function, extracellular matrix homeostasis, and tumor metabolism are discussed. Studies of VHL proteostasis, with the goal of harnessing the proteostatic machinery to refunctionalize mutant VHL, are reviewed. Translational efforts using molecular tools to elucidate discriminating features of ccRCC tumors and develop improved prognostic and predictive algorithms are presented, and new therapeutics arising from the earliest molecular discoveries in ccRCC are summarized. By creating an integrated review of the key genomic and molecular biological disease characteristics of ccRCC and placing these data in the context of the evolving therapeutic landscape, we intend to facilitate interaction among basic, translational, and clinical researchers involved in the treatment of this devastating disease, and accelerate progress toward its ultimate eradication.     

Elevated expression of phospholipid transfer protein in bone marrow derived cells causes atherosclerosis

Background

Phospholipid transfer protein (PLTP) is expressed by various cell types. In plasma, it is associated with high density lipoproteins (HDL). Elevated levels of PLTP in transgenic mice result in decreased HDL and increased atherosclerosis. PLTP is present in human atherosclerotic lesions, where it seems to be macrophage derived. The aim of the present study is to evaluate the atherogenic potential of macrophage derived PLTP.

Methods and Findings

Here we show that macrophages from human PLTP transgenic mice secrete active PLTP. Subsequently, we performed bone marrow transplantations using either wild type mice (PLTPwt/wt), hemizygous PLTP transgenic mice (huPLTPtg/wt) or homozygous PLTP transgenic mice (huPLTPtg/tg) as donors and low density lipoprotein receptor deficient mice (LDLR−/−) as acceptors, in order to establish the role of PLTP expressed by bone marrow derived cells in diet-induced atherogenesis. Atherosclerosis was increased in the huPLTPtg/wt→LDLR−/− mice (2.3-fold) and even further in the huPLTPtg/tg→LDLR−/− mice (4.5-fold) compared with the control PLTPwt/wt→LDLR−/− mice (both P<0.001). Plasma PLTP activity levels and non-HDL cholesterol were increased and HDL cholesterol decreased compared with controls (all P<0.01). PLTP was present in atherosclerotic plaques in the mice as demonstrated by immunohistochemistry and appears to co-localize with macrophages. Isolated macrophages from PLTP transgenic mice do not show differences in cholesterol efflux or in cytokine production. Lipopolysaccharide activation of macrophages results in increased production of PLTP. This effect was strongly amplified in PLTP transgenic macrophages.

Conclusions

We conclude that PLTP expression by bone marrow derived cells results in atherogenic effects on plasma lipids, increased PLTP activity, high local PLTP protein levels in the atherosclerotic lesions and increased atherosclerotic lesion size.     

ATP synthase motor components: proposal and animation of two dynamic models for stator function

Recent research indicates that ATP synthases (F(0)F(1)) contain two distinct nanomotors, one an electrochemically driven proton motor contained within F(0) that drives an ATP hydrolysis-driven motor (F(1)) in reverse during ATP synthesis. This is depicted in recent models as involving a series of events in which each of the three alphabeta pairs comprising F(1) is induced via a centrally rotating subunit (gamma) to undergo the sequential binding changes necessary to synthesize ATP (binding change mechanism). Stabilization of this rotary process (i.e., to minimize "wobble" of F(1)) is provided in current models by a peripheral stalk or "stator" that has recently been shown to extend from near the bottom of the ATP synthase molecule to the very top of F(1). Although quite elegant, these models envision the stator as fixed during ATP synthesis, i.e., bound to only a single alphabeta pair. This is despite the fact that the binding change mechanism views each alphabeta pair as going through the same sequential order of conformational changes which demonstrate a chemical equivalency among them. For this reason, we propose here two different dynamic models for stator function during ATP synthesis. Both models have been designed to maintain chemical equivalency among the three alphabeta pairs during ATP synthesis and both have been animated.     

Rickettsial disease: classical and modern aspects

Rickettsial diseases have been reassessed in recent years since they represent an important field in today's medicine. New agents have been described: some are non-pathogenic agents and the others are associated with well-defined or peculiar clinical patterns. In addition, different species of rickettsiosis are found in relation to the geographic areas of the world. Some agents may be defined as variants of older diseases whereas most of the newly described forms of rickettsiosis represent distinct entities with unique epidemiologial and clinical features. Probably the main news regards the group of the spotted fevers. An additional new aspect is linked to the medicine of travellers and tourists. However, this aspect may not be significant for the rickettsial diseases in relation to other human illnesses, such as malaria. Therefore, an investigation into the geographical origin of patients has to enter our routine medical work.     

Monokariotic fruiting body and clamp cell formation in Mycoleptodonoides aitchisonii (Bunaharitake)

The ability to produce monokaryotic fruiting bodies and clamp cells in culture was examined in monokaryotic strain isolated from several dikaryotic parental strains of the edible mushroom, Mycoleptodonoides aitchisonii (Bunaharitake). We describe a single dikaryotic M. aitchisonii strain, TUFC50005, and 20 monokaryons derived from it, which exhibited a wide spectrum of monokaryotic fruiting types. Most strains formed primordia, or young fruiting body-like structures, but only one of the monokaryons, strain TUFC50005-4, formed a fruiting body, even though it had only one nucleus and produced only two spores after meiosis. We demonstrated that dikariotization was not required for clamp cell formation, fruiting body formation, or meiosis, in this mushroom.