Identification of a UPTG inhibitor protein from maize endosperm: high homology with sucrose synthase protein

A thermolabile UPTG inhibitor protein (IP) was isolated and purified from a developing maize endosperm preparation. High homology of two internal peptides of IP with known plant sucrose synthase (SS) sequences suggested that IP might be related somehow with SS. IP and SS activities were found in the same preparation and showed thermolability between 60-65 degrees C. IP and SS activities presented the same ionic charge and molecular mass in native conditions (Mono Q and Superose-12 columns chromatographies). Western blot experiments with an anti-SS antibody as well as with an anti-IP antibody showed a single 80 kDa polypeptide band where IP and SS activities were present. Anti-SS antibody can neutralize SS as well as IP activities in a neutralization assay. It was found that in the maize mutant shrunken-1, lacking SS1 protein, the UPTG activity was not inhibited. Furthermore, the solubilized preparation of the sh1 endosperm is unable of inhibiting UPTG activity from potato tuber. The high correlation between IP and SS properties suggests that IP might be in fact a form of SS. Moreover, the relation between IP and the SS1 isoform is discussed. So, a new biological activity of SS is suggested.     

Molecular cloning in Arabidopsis thaliana of a new protein phosphatase 2C (PP2C) with homology to ABI1 and ABI2

Trapeziectomy, ligament reconstruction and tendon interposition arthroplasty is one of the most commonly performed procedures to address pain and instability due to osteoarthritis at the basal joint of the thumb. To determine the effect of stress on first metacarpal subsidence, 15 ligament reconstruction and tendon interposition basal joint arthroplasties were evaluated after a mean follow-up of 32 months. Radiographs were obtained of the arthroplasty at rest and then with maximal effort key pinch stress, which is known to subject the first carpometacarpal joint to considerable axial compression stress. Compared with the preoperative x-rays, the first metacarpal had subsided 21% of the arthroplasty space at rest. Under stress, the first metacarpal was found to subside another 10.5% in height. No subluxation of the metacarpal base could be detected. Key pinch strength improved 17% from the preoperative strength. Tip-to-tip pinch strength improved 17% from the preoperative measurement. Grip strength improved 17% from the preoperative measurement. Grip strength was 9% greater than the preoperative grip strength. There was no statistical association between the amount of first metacarpal subsidence and follow-up key pinch, tip pinch, or grip strength. With axial compressive loading of the arthroplasty, such as in lateral pinch, there is some further proximal migration of the first metacarpal, but this is minimal and does not correlate with functional outcome.     

Prediction of protein side-chain rotamers from a backbone-dependent rotamer library: a new homology modeling tool

Modeling by homology is the most accurate computational method for translating an amino acid sequence into a protein structure. Homology modeling can be divided into two sub-problems, placing the polypeptide backbone and adding side-chains. We present a method for rapidly predicting the conformations of protein side-chains, starting from main-chain coordinates alone. The method involves using fewer than ten rotamers per residue from a backbone-dependent rotamer library and a search to remove steric conflicts. The method is initially tested on 299 high resolution crystal structures by rebuilding side-chains onto the experimentally determined backbone structures. A total of 77% of chi1 and 66% of chi(1 + 2) dihedral angles are predicted within 40 degrees of their crystal structure values. We then tested the method on the entire database of known structures in the Protein Data Bank. The predictive accuracy of the algorithm was strongly correlated with the resolution of the structures. In an effort to simulate a realistic homology modeling problem, 9424 homology models were created using three different modeling strategies. For prediction purposes, pairs of structures were identified which shared between 30% and 90% sequence identity. One strategy results in 82% of chi1 and 72% chi(1 + 2) dihedral angles predicted within 40 degrees of the target crystal structure values, suggesting that movements of the backbone associated with this degree of sequence identity are not large enough to disrupt the predictive ability of our method for non-native backbones. These results compared favorably with existing methods over a comprehensive data set.     

Cloning of a human multispanning membrane protein cDNA: evidence for a new protein family

We report the cloning of a human cDNA encoding a protein of calculated 68.8 kDa molecular mass, named hMP70. The deduced protein sequence shows a large N-terminal hydrophilic part and a C-terminal part with nine putative hydrophobic regions characteristic of integral transmembrane domains. Computer searches with sequence databases revealed homologies with three complete yeast proteins and with at least 19 human, 10 plant and one nematode short unidentified protein sequences translated from Expressed Sequence Tags (ESTs). Remarkably, this hMP70 protein retains between 27 and 31% overall sequence identity with the yeast proteins. We propose that hMP70 and related genes have evolved from a common ancestral gene and form a new multispanning membrane protein family which we call the MP70 protein family. Gene expression of hMP70 appears to be ubiquitous, as the mRNA is detectable in all human tissues analysed so far, as shown by Northern blot analysis. Furthermore, a protein of about 70 kDa is detectable in different mammalian cell lines, as shown by immunoblot analysis. From its widespread expression and conservation from yeast, plants to mammals, it is likely that hMP70 has a fundamental biological function in the cell.     

The intrinsic factor-vitamin B12 receptor and target of teratogenic antibodies is a megalin-binding peripheral membrane protein with homology to developmental proteins

The present report shows the molecular characterization of the rat 460-kDa epithelial glycoprotein that functions as the receptor facilitating uptake of intrinsic factor-vitamin B12 complexes in the intestine and kidney. The same receptor represents also the yolk sac target for teratogenic antibodies causing fetal malformations in rats. Determination of its primary structure by cDNA cloning identified a novel type of peripheral membrane receptor characterized by a cluster of eight epidermal growth factor type domains followed by a cluster of 27 CUB domains. In accordance with the absence of a hydrophobic segment, the receptor could be released from renal cortex membranes by nonenzymatic and nonsolubilizing procedures. The primary structure has no similarity to known endocytic receptors but displays homology to epidermal growth factor and CUB domain proteins involved in fetal development, e.g. the bone morphogenic proteins. Electron microscopic immunogold double labeling of rat yolk sac and renal proximal tubules demonstrated subcellular colocalization with the endocytic receptor megalin, which is expressed in the same epithelia as the 460-kDa receptor. Furthermore, megalin affinity chromatography and surface plasmon resonance analysis revealed a calcium-dependent high affinity binding of the 460-kDa receptor to megalin, which thereby may mediate its vesicular trafficking. Due to the high number of CUB domains, accounting for 88% of the protein mass, we propose the name cubilin for the novel receptor.     

Isolation of a granulocyte inhibitory protein from uraemic patients with homology of beta 2-microglobulin

Increased incidence of infection in uraemic patients is mainly caused by granulocyte dysfunction. Recently we discovered a granulocyte inhibitory protein (GIP I) in the ultrafiltrate of haemodialysis patients, that inhibits four fundamental functions of polymorphonuclear leukocytes (PMNLs). We now report on the isolation of a further polypeptide in end-stage renal disease patient ultrafiltrate using a polyamide filter with biological activity inhibiting healthy PMNL function in vitro. This protein (GIP II) has a molecular weight of about 9500 Da. In-vitro nanomolar concentrations inhibit PMNL O2- production and glucose uptake stimulated by phorbol-myristate-acetate (PMA), but not by formyl-methionyl-leucyl-phenylalanine (FMLP). In-vitro studies were performed to compare the effects of GIP I and GIP II on several PMNL functions. In contrast to GIP II, GIP I inhibits only FMLP-, but not PMA-stimulated PMNL glucose uptake. The NH2 terminal amino acid sequence (21 amino acids) of GIP II shows homology to beta 2-microglobulin. Commercially available intact beta 2-microglobulin had no effect on PMNL glucose uptake and O2- production. The beta 2-microglobulin homologue protein isolated from plasma ultrafiltrates of uraemic patients cross-reacts with three different commercially available assays for intact beta 2-microglobulin. Therefore, beta 2-microglobulin levels measured in the plasma ultrafiltrates of regular haemodialysis patients are overestimated with contribution of an uncertain amount of the beta 2-microglobulin homologue protein (GIP II).     

Lamina lucida of basement membrane: an artefact

In tissues prepared with chemical fixation followed by conventional dehydration, basement membranes have been observed to be laminated structures composed of a lamina lucida and lamina densa as well as a poorly limited transitional zone referred to as the pars fibroreticularis. Scattered attempts in the application of new techniques of tissue preparation such as cryofixation or freeze substitution for the study of the basement membrane structure have been made in recent years. From these studies, the possibility has arisen in which basement membranes are composed of only the lamina densa without a lamina lucida. In recent studies in this laboratory, the attempt was made to determine whether or not this lamina lucida is an artefact, and if so, which step in the conventional method of tissue preparation is responsible for its formation. Basement membranes from diverse sources in the mouse and rat including the testis, ductus epididymis, eye, thyroid, kidney, and skin, were observed after either cryofixation by slam freezing followed by freeze substitution, or aldehyde fixation followed by freeze substitution. The basement membranes after preservation with either of these two methods were composed of only the lamina densa with no lamina lucida. It indicates that an artefactual formation of the lamina lucida occurs during dehydration in conventional tissue preparation rather than during chemical fixation. In view of the well known superiority of freeze substitution over conventional dehydration, the lamina lucida of the basement membrane is likely to be an artefact. Therefore, it is concluded that the lamina lucida is an artefact formed during conventional tissue preparation, and in its original condition in the living state, the basement membrane is composed of a single layer made up of lamina densa material.     

Immunopurification and characterization of a collagenase/gelatinase domain issued from basement membrane fibronectin

The proteolytic potential of cellular fibronectin fragments issued from a basement membrane hydrolysate was investigated. Three different gelatinase activities (47, 43 and 37 kDa), located by gelatin zymography, were isolated using successively heparin-agarose, gelatin-agarose and immunopurification with polyclonal antibodies directed against bovine plasma fibronectin. These fragments were also characterized using a monoclonal antibody directed against the extra-domain EDA of cellular fibronectin as a probe. A collagenase activity, reliably indicated by the gelatin zymography pattern, was also found using MCA-Pro-Leu-Gly-Leu-DPA-Ala-Arg-NH2, the intramolecularly quenched fluorogenic substrate of collagenases. From these results, cellular fibronectin was found to be able to exhibit a proteolytic function after limited proteolysis. This MMP-like function could be associated with tissue remodeling in both normal and pathological states, such as metastasis, angiogenesis and tissue repair.     

Characteristics and outcome of anti-glomerular basement membrane disease: a single-center experience

The relative virulence and avirulence of Mycobacterium tuberculosis strains H37Rv and H37Ra were previously defined using animal infection models. To investigate host species' specificity of mycobacterial virulence, growth of the 2 M. tuberculosis strains in human monocyte-derived macrophages in vitro was studied. Mycobacterial growth was evaluated by acid-fast staining, electron microscopy, and colony-forming units (cfu) assay. As expected, the 2 strains demonstrated significantly different growth rates in mouse macrophages in vitro (53 h for H37Rv, 370 h for H37Ra). In marked contrast, in human macrophages the average division times of the strains were nearly equal (80 h for H37Rv and 76 h for H37Ra by cfu measurement, and 96 h for H37Rv and 104 h for H37Ra by acid-fast staining). These findings indicate that observations of mycobacterial virulence in murine systems may not necessarily translate to the human system, in which different mechanisms to control mycobacterial growth may be expressed.     

A mouse Fas-associated protein with homology to the human Mort1/FADD protein is essential for Fas-induced apoptosis

The Fas cell surface receptor belongs to the tumor necrosis factor receptor family and can initiate apoptosis in a variety of cell types. Using the Fas cytoplasmic domain as bait in a yeast two-hybrid screening, we isolated a mouse cDNA encoding a 205-amino-acid protein. Its predicted protein sequence shows 68% identity and 80% similarity with the sequence of recently described human Mort/FADD. This protein, most likely the mouse homolog of human FADD, associates with Fas in vivo only upon the induction of cell death. A fraction of this protein is highly phosphorylated at serine/threonine residues, with both phosphorylated and unphosphorylated forms being capable of binding to FAS. Stable expression of a truncated form of the Mort/FADD protein protects cells from Fas-mediated apoptosis by interfering with the wild-type protein-Fas interaction. Thus, mouse Mort/FADD is an essential downstream component that mediates Fas-induced apoptosis.     

Membranoproliferative glomerulonephritis with disruption of the glomerular basement membrane

Seven patients with a form of membranoproliferative glomerulonephritis distinct in its glomerular ultrastructure from other forms are described. The use of silver impregnated electron micrographs revealed contiguous subepithelial and subendothelial deposits associated with basement membrane disruption, replication and layering of lamina densalike material. By light and fluorescence microscopy the appearance was distinctive but not diagnostic. Immunohistology consistently showed abundant C3 and properdin in a granular pattern while immunoglobulins and Clq were variably present. Low serum C3 concentrations were observed at some time in each patient, often accompanied by low levels of properdin, whereas the concentrations of Clq and C4 were normal. The patients were indistinguishable in their clinical course from those with other types of MPGN.     

Importance of the basement membrane protein SPARC for viability and fertility in Caenorhabditis elegans

The basement membrane is a specialized extracellular matrix located at epithelial-mesenchymal boundaries that supports cell adhesion, migration, and proliferation; it is highly conserved between invertebrates and vertebrates [1,2]. One of its component proteins, SPARC (osteonectin/BM-40), binds calcium and collagens, and can modulate cell-matrix interactions, so altering cell shape, growth, and differentiation [3,5]. The tissue distribution of a secreted fusion protein containing SPARC and green fluorescent protein (GFP) was analyzed in Caenorhabditis elegans. The protein localized to most basement membranes along body wall and sex muscles, and was also deposited around the pharynx and the gonad, in the spermatheca and at the distal tip cells. The contributions of SPARC to C. elegans development were determined using RNA interference, which accurately phenocopies loss-of-function defects [6-8]. A reduction in the amount of SPARC protein resulted in embryonic or larval lethality in a significant proportion of progeny. Those that survived developed a 'clear' phenotype characterized by a lack of gut granules, which made the animals appear transparent, plus small size, and sterility or reduced fecundity. No significant morphological abnormalities were observed, indicating that SPARC plays a regulatory rather than structural role in modulating cell-matrix interactions during normal development and reproduction.     

Grb2 forms an inducible protein complex with CD28 through a Src homology 3 domain-proline interaction

CD28 provides a costimulatory signal that results in optimal activation of T cells. The signal transduction pathways necessary for CD28-mediated costimulation are presently unknown. Engagement of CD28 leads to its tyrosine phosphorylation and subsequent binding to Src homology 2 (SH2)-containing proteins including the p85 subunit of phosphatidylinositol 3'-kinase (PI3K); however, the contribution of PI3K to CD28-dependent costimulation remains controversial. Here we show that CD28 is capable of binding the Src homology 3 (SH3) domains of several proteins, including Grb2. The interaction between Grb2 and CD28 is mediated by the binding of Grb2-SH3 domains to the C-terminal diproline motif present in the cytoplasmic domain of CD28. While the affinity of the C-terminal SH3 domain of Grb2 for CD28 is greater than that of the N-terminal SH3 domain, optimal binding requires both SH3 domains. Ligation of CD28, but not tyrosine-phosphorylation, is required for the SH3-mediated binding of Grb2 to CD28. We propose a model whereby the association of Grb2 with CD28 occurs via an inducible SH3-mediated interaction and leads to the recruitment of tyrosine-phosphorylated proteins such as p52(shc) bound to the SH2 domain of Grb2. The inducible interaction of Grb2 to the C-terminal region of CD28 may form the basis for PI3K-independent signaling through CD28.     

Cloning and characterization of a new silver-stainable protein SSP29, a member of the LRR family

Silver-stainable proteins (SSPs) are aspartic acid-rich nuclear proteins which are silver stained under very specific conditions. Using a degenerate oligodeoxyncleotide probe which codes for acidic amino acid residues, a cDNA for a new SSP, referred to as SSP29, has been isolated. The cDNA-derived amino acid sequence shows SSP29 has a molecular mass of 29 kDa, leucine-rich repeats (LRR) near the NH2-terminal region and acidic clusters at the COOH-terminal portion, indicating that SSP29 is also a member of the LRR subfamily of acidic proteins which have been shown to be involved in antigen-mediated cellular responses, leukemogenesis and differentiation. SSP29 can be stained by Ag-NOR staining. SSP29 is expressed in all human tissues and cell lines tested, localized to nucleoplasm and translocated partially to the nucleoli after heat shock. Its interaction with RNA polymerase I suggests that SSP29 may participate in signal transduction that directs nucleolar activities by regulating ribosomal RNA biosynthesis.     

From membrane to molecule to the third amino acid from the left with a membrane transport protein

The lac permease of E. coli is a paradigm for secondary active transporter proteins that transduce the free energy stored in electrochemical ion gradients into work in the form of a concentration gradient. This hydrophobic, polytopic, cytoplasmic membrane protein catalyses the coupled, stoichiometric translocation of beta-galactosides and H+, and it has been solubilized, purified, reconstituted into artificial phospholipid vesicles and shown to be solely responsible responsible for beta-galactoside transport as a monomer. The lacY gene which encodes the permease has been cloned and sequenced, and all available evidence indicates that the protein has 12 transmembrane domains in alpha-helical configuration that traverse the membrane in zigzag fashion connected by hydrophilic loops with the N and C termini on the cytoplasmic face of the membrane. Extensive use of site-directed and Cys-scanning mutagenesis indicates that very few residues in the permease are directly involved in the transport mechanism, but the permease appears to be a highly flexible protein that undergoes widespread conformational changes during turnover. Based on a variety of site-directed approaches which include second-site suppressor analysis and site-directed mutagenesis, excimer fluorescence, engineered divalent metal binding sites, chemical cleavage, EPR, thiol crosslinking and identification of discontinuous mAb epitopes, a helix packing model has been formulated.A mechanism for the coupled translocate ion of substrate and H+ by the lac permease of E. coli is proposed. Four residues are irreplaceable with respect to coupling, and the residues are paired in the tertiary structure--Arg-302 (helix IX) with Glu-325 (helix 10) and His-322 (helix 10) with Glu-269 (helix VIII). In an adjacent region of the molecule at the interface between helices VIII and V is the substrate translocation pathway in which Glu-126 and Arg-144 appear to play key roles. Because of this arrangement, interfacial changes between helices VIII and V are transmitted to the interface between helices IX and X and vice versa. Upon ligand binding, a structural change at the interface between helices V and VIII disrupts the interaction between Glu-269 and His-322, Glu-269 displaces Glu-325 from Ag-302 and Glu-325 is protonated.Simultaneously, protonated Glu-325 becomes inaccessible to water which drastically increases its pKa. In this configuration, the permease undergoes a freely reversible conformational change that corresponds to translocation of the ternary complex. In order to return to ground state after release of substrate, the Arg-302-Glu-325 interaction must be reestablished which necessitates loss of H+ from Glu-325. The H+ is released into a water-filled crevice between helices IX and X which becomes transiently accessible to both sides of the membrane due to a change in helix tilt, where it is acted upon equally by either the membrane potential or the pH gradient across the membrane. Remarkably few amino-acid residues appear to be critically involved in the transport mechanism of lac permease, suggesting that relatively simple chemistry drives the mechanism. On the other hand, widespread, cooperative conformational changes appear to be involved in turnover. As a whole the data suggest that the 12 helices which comprise the permease are loosely packed with a considerable amount of water in the interstices and that surface contours are important for sliding or tilting motions that occur during turnover. This surmise coupled with the indication that few residues are essential to the mechanism is encouraging in that it suggest that the possibility that a relatively low resolution structure (i.e. helix packing) plus localization of the critical residues and the translocation pathway can provide important insights into the mechanism. (ABSTRACT TRUNCATED)     

IFI60/ISG60/IFIT4, a new member of the human IFI54/IFIT2 family of interferon-stimulated genes

1. Disturbances in cortisol secretory patterns and excessive secretion of cortisol after a variety of neuroendocrine stimulation tests indicate excessive activity of the hypothalamic-pituitary-adrenocortical axis in depression. 2. Peripheral indicators of hypercortisolemia have also been observed (e.g. enlarged adrenal glands, glucocorticoid insensitivity and insulin intolerance). 3. Excessive cortisol production may also result in altered bone metabolism and bone architecture, and a recent study by Michelson et al. (1996) found slightly lower bone density in depressed women with hypercortisoluria versus healthy controls. 4. In this study, the authors examined bone mineral density (BMD) using dual energy radiographic absorptometry (DEXA) technique in 6 depressed patients (3 with and 3 without hypercortisoluria) with a mean (+/- SD) age of 41 +/- 13 years, and in 5 healthy, controls with mean age 38 +/- 4 years). 5. DEXA images of the lumbar vertebrae (L1 to L4) for BMD were acquired over a 5-minute interval. 6. Overall, the authors observed no difference in mean BMD values between patients and controls, nor were differences observed between patients with and without hypercortisoluria.