Identification of a novel Na+-independent acidic amino acid transporter with structural similarity to the member of a heterodimeric amino acid transporter family associated with unknown heavy chains

We identified a novel Na(+)-independent acidic amino acid transporter designated AGT1 (aspartate/glutamate transporter 1). AGT1 exhibits the highest sequence similarity (48% identity) to the Na(+)-independent small neutral amino acid transporter Asc (asc-type amino acid transporter)-2 a member of the heterodimeric amino acid transporter family presumed to be associated with unknown heavy chains (Chairoungdua, A., Kanai, Y., Matsuo, H., Inatomi, J., Kim, D. K., and Endou, H. (2001) J. Biol. Chem. 276, 49390-49399). The cysteine residue responsible for the disulfide bond formation between transporters (light chains) and heavy chain subunits of the heterodimeric amino acid transporter family is conserved for AGT1. Because AGT1 solely expressed or coexpressed with already known heavy chain 4F2hc (4F2 heavy chain) or rBAT (related to b(0,+)-amino acid transporter) did not induce functional activity, we generated fusion proteins in which AGT1 was connected with 4F2hc or rBAT. The fusion proteins were sorted to the plasma membrane and expressed the Na(+)-independent transport activity for acidic amino acids. Distinct from the Na(+)-independent cystine/glutamate transporter xCT structurally related to AGT1, AGT1 did not accept cystine, homocysteate, and l-alpha-aminoadipate and exhibited high affinity to aspartate as well as glutamate, suggesting that the negative charge recognition site in the side chain-binding site of AGT1 would be closer to the alpha-carbon binding site compared with that of xCT. The AGT1 message was predominantly expressed in kidney. In mouse kidney, AGT1 protein was present in the basolateral membrane of the proximal straight tubules and distal convoluted tubules. In the Western blot analysis, AGT1 was detected as a high molecular mass band in the nonreducing condition, whereas the band shifted to a 40-kDa band corresponding to the AGT1 monomer in the reducing condition, suggesting the association of AGT1 with other protein via a disulfide bond. The finding of AGT1 and Asc-2 has established a new subgroup of the heterodimeric amino acid transporter family whose members associate not with 4F2hc or rBAT but with other unknown heavy chains.     

Ligation of HLA class I molecules on endothelial cells induces phosphorylation of Src,paxillin, and focal adhesion kinase in an actin-dependent manner

The development of chronic rejection is the major limitation to long-term allograft survival. HLA class I Ags have been implicated to play a role in this process because ligation of class I molecules by anti-HLA Abs stimulates smooth muscle cell and endothelial cell proliferation. In this study, we show that ligation of HLA class I molecules on the surface of human aortic endothelial cells stimulates phosphorylation of Src, focal adhesion kinase, and paxillin. Signaling through class I stimulated Src phosphorylation and mediated fibroblast growth factor receptor (FGFR) translocation to the nucleus. In contrast, Src kinase activity was not involved in class I-mediated transfer of FGFR from cytoplasmic stores to the cell surface. Inhibition of Src protein kinase activity blocked HLA class I-stimulated tyrosine phosphorylation of paxillin and focal adhesion kinase. Furthermore, HLA class I-mediated phosphorylation of the focal adhesion proteins and FGFR expression was inhibited by cytochalasin D and latrunculin A, suggesting a role for the actin cytoskeleton in the signaling process. These findings indicate that anti-HLA Abs have the capacity to transduce activation signals in endothelial cells that may promote the development of chronic rejection.     

Identification of a type 1 peroxisomal targeting signal in a viral protein and demonstration of its targeting to the organelle

Peroxisomes are unimembrane, respiratory organelles of the cell. Transport of cellular proteins to the peroxisomal matrix requires a type 1 peroxisomal targeting signal (PTS1) which essentially constitutes a tripeptide from the consensus sequence S/T/A/G/C/N-K/R/H-L/I/V/M/A/F/Y. Although PTS-containing proteins have been identified in eukaryotes, prokaryotes, and parasites, viral proteins with such signals have not been identified so far. We report here the first instance of a virus, the rotavirus, which causes infantile diarrhea worldwide, containing a functional C-terminal PTS1 in one of its proteins (VP4). Analysis of 153 rotavirus VP4-deduced amino acid sequences identified five groups of conserved C-terminal PTS1 tripeptide sequences (SKL, CKL, GKL, CRL, and CRI), of which CRL is represented in approximately 62% of the sequences. Infection of cells by a CRL-containing representative rotavirus (SA11 strain) and confocal immunofluorescence analysis revealed colocalization of VP4 with peroxisomal markers and morphological changes of peroxisomes. Further, transient cellular expression of green fluorescent protein (GFP)-fused VP4CRL resulted in transport of VP4 to peroxisomes, whereas the chimera lacking the PTS1 signal, GFP-VP4DeltaCRL, resulted in diffuse cytoplasmic staining, suggesting a CRL-dependent targeting of the protein. The present study therefore demonstrates hitherto unreported organelle involvement, specifically of the peroxisomes, in rotaviral infections as demonstrated by using the SA11 strain of rotavirus and opens a new line of investigation toward understanding viral pathogenesis and disease mechanisms.     

Microfilariae in association with other diseases. A report of six cases

BACKGROUND: Lymphatic filariasis is a major public health problem in tropical countries. Earlier reports have reported microfilariae as an incidental finding in body fluids and fine needle aspiration smears from various sites. CASES: The findings of body fluid cytology and fine needle aspiration smears from six patients with microfilariae in association with other conditions--tubercular pleural effusion/lymphadenitis, pregnancy and non-Hodgkin's lymphoma--are presented. Three patients demonstrated an associated eosinophilic cellular exudate. Adherence of inflammatory cells to microfilariae was seen in two patients. CONCLUSION: Although microfilariae in cytologic smears are considered incidental findings, the association of microfilariae with debilitating conditions suggests that it is an opportunistic infection and needs further study.     

Spectrophotometric determination of serum nitrite and nitrate by copper-cadmium alloy

A macro and micro assay for the spectrophotometric determination of serum nitrite and nitrate was developed. Nitrite/nitrate in biological samples can be estimated in a single step by this method. The principle of the assay is the reduction of nitrate by copper-cadmium alloy, followed by color development with Griess reagent (sulfanilamide and N-naphthylethylenediamine) in acidic medium. This assay is sensitive to 1 microM nitrate and is suitable for different biological fluids, including sera with a high lipid concentration. The copper-cadmium alloy used in the present method is easy to prepare and can completely reduce nitrate to nitrite in an hour. The present method provides a simple, cost-effective assay for the estimation of stable oxidation products of nitric oxide in biological samples.     

Synthesis of alkyl and aryl esters of N-protected beta-homoamino acids from N-protected alpha-aminodiazoketones

A simple and concomitant esterification method for the synthesis of methyl, ethyl, t-butyl, benzyl, and 9-fluorenylmethyl esters of Fmoc-/Boc-/Z-beta-homoamino acids employing Fmoc-/Boc-/Z-alpha-aminodiazoketones by Wolff rearrangement is described. The method offers good yield with purity.     

Novel indolo[2,1-b]quinazoline analogues as cytostatic agents: synthesis,biological evaluation and structure-activity relationship

In our endeavor to design and synthesize novel anticancer agents, a new series of indoloquinazoline compounds were prepared and tested initially for anticancer activity in vitro against a panel of human cancer cell lines. Most of these compounds exhibited cytotoxic activity in in vitro screens. Compounds were selected and further evaluated using a modified Hollow Fiber Assay for their preliminary in vivo activity against 12 cell lines implanted in the subcutaneous and intraperitoneal compartments in mice. The results indicate that these compounds may constitute a new class of anticancer agents.     

The localization of the integral membrane protein Cps1p to the cell division site is dependent on the actomyosin ring and the septation-inducing network in Schizosaccharomyces pombe

Schizosaccharomyces pombe cells divide by medial fission through the use of an actomyosin-based contractile ring. Constriction of the actomyosin ring is accompanied by the centripetal addition of new membranes and cell wall material. In this article, we characterize the mechanism responsible for the localization of Cps1p, a septum-synthesizing 1,3-beta-glucan synthase, to the division site during cytokinesis. We show that Cps1p is an integral membrane protein that localizes to the cell division site late in anaphase. Neither F-actin nor microtubules are essential for the initial assembly of Cps1p to the medial division site. F-actin, but not microtubules, is however important for the eventual incorporation of Cps1p into the actomyosin ring. Assembly of Cps1p into the cell division ring is also dependent on the septation-inducing network (SIN) proteins that regulate division septum formation after assembly of the actomyosin ring. Fluorescence-recovery after-photobleaching experiments reveal that Cps1p does not diffuse appreciably within the plasma membrane and is retained at the division site by a mechanism that does not depend on an intact F-actin cytoskeleton. We conclude that the actomyosin ring serves as a spatial cue for Cps1p localization, whereas the maintenance of Cps1p at the division site occurs by a novel F-actin- and microtubule-independent mechanism. Furthermore, we propose that the SIN proteins ensure localization of Cps1p at the appropriate point in the cell cycle.     

Detection of root mucilage using an anti-fucose antibody

Plant root mucilage is known to enhance soil quality by contributing towards the soil carbon pool, soil aggregation, detoxification of heavy metal ions and interactions with rhizospheric microflora. Mucilage consists of many monosaccharide units, including fucose which can be used as an indicator for plant root based polysaccharides. This is the first report of an immunological technique developed to use anti-fucose antibodies as markers for probing and localizing fucosyl residues in mucilage polysaccharide and, in turn, for localization of plant root mucilage. Fucose was complexed with bovine serum albumin to raise antibodies against fucose. A fucose-directed antibody was shown to cross-react with root cap mucilages from grasses. This antibody was used to localize root mucilage polysaccharide in maize and wheat root caps using immunogold electron microscopy. Abundant labelling could be localized on the cell wall, and in the intercellular matrix and vesicles of the peripheral root cap cells. Labelling was less intense in cells towards the centre of the root cap tissue. Control experiments confirmed that immunogold localization of fucose was specific and reliable.