Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (bo,+AT) of rBAT.

Cystinuria (MIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids. Mutations in SLC3A1, encoding rBAT, cause cystinuria type I (ref. 1), but not other types of cystinuria (ref. 2). A gene whose mutation causes non-type I cystinuria has been mapped by linkage analysis to 19q12-13.1 (Refs 3,4). We have identified a new transcript, encoding a protein (bo, +AT, for bo,+ amino acid transporter) belonging to a family of light subunits of amino acid transporters, expressed in kidney, liver, small intestine and placenta, and localized its gene (SLC7A9) to the non-type I cystinuria 19q locus. Co-transfection of bo,+AT and rBAT brings the latter to the plasma membrane, and results in the uptake of L-arginine in COS cells. We have found SLC7A9 mutations in Libyan-Jews, North American, Italian and Spanish non-type I cystinuria patients. The Libyan Jewish patients are homozygous for a founder missense mutation (V170M) that abolishes b o,+AT amino-acid uptake activity when co-transfected with rBAT in COS cells. We identified four missense mutations (G105R, A182T, G195R and G295R) and two frameshift (520insT and 596delTG) mutations in other patients. Our data establish that mutations in SLC7A9 cause non-type I cystinuria, and suggest that bo,+AT is the light subunit of rBAT.

The transcription factor PU.1 is involved in macrophage proliferation.

PU.1 is a tissue-specific transcription factor that is expressed in cells of the hematopoietic lineage including macrophages, granulocytes, and B lymphocytes. Bone marrow-derived macrophages transfected with an antisense PU.1 expression construct or treated with antisense oligonucleotides showed a decrease in proliferation compared with controls. In contrast, bone marrow macrophages transfected with a sense PU.1 expression construct displayed enhanced macrophage colony-stimulating factor (M-CSF)-dependent proliferation. Interestingly, there was no effect of sense or antisense constructs of PU.1 on the proliferation of the M-CSF-independent cell line, suggesting that the response was M-CSF dependent. This was further supported by the finding that macrophages transfected with a sense or an antisense PU.1 construct showed, respectively, an increased or a reduced level of surface expression of receptors for M-CSF. The enhancement of proliferation seems to be selective for PU.1, since transfections with several other members of the ets family, including ets-2 and fli-1, had no effect. Various mutants of PU.1 were also tested for their ability to affect macrophage proliferation. A reduction in macrophage proliferation was found when cells were transfected with a construct in which the DNA-binding domain of PU.1 was expressed. The PEST (proline-, glutamic acid-, serine-, and threonine-rich region) sequence of the PU.1 protein, which is an important domain for protein-protein interactions in B cells, was found to have no influence on PU.1-enhanced macrophage proliferation when an expression construct containing PU.1 minus the PEST domain was transfected into bone marrow-derived macrophages. In vivo, PU.1 is phosphorylated on several serine residues. The transfection of plasmids containing PU.1 with mutations at each of five serines showed that only positions 41 and 45 are critical for enhanced macrophage proliferation. We conclude that PU.1 is necessary for the M-CSF-dependent proliferation of macrophages. One of the proliferation-relevant targets of this transcription factor could be the M-CSF receptor.

IFN-gamma-dependent transcription of MHC class II IA is impaired in macrophages from aged mice.

To determine the effect of aging on IFN-gamma-induced MHC class II antigen expression, we produced bone marrow-derived macrophages in vitro. In these conditions, we analyzed the effect of aging on the genomic expression of macrophages without the influence of other cell types that may be affected by aging. Although macrophages from young and aged mice showed an identical degree of differentiation, after incubation with IFN-gamma, the expression at the cell surface of the IA complex and the levels of IAbeta protein and mRNA were lower in aged macrophages. Moreover, the transcription of the IAbeta gene was impaired in aged macrophages. The amount of transcription factors that bound to the W and X, but not to the Y, boxes of the IAbeta promoter gene was lower in aged macrophages. Similar levels of CIITA mRNA were found after IFN-gamma treatment of both young and aged macrophages. This shows that neither the initial cascade that starts after the interaction of IFN-gamma with the receptor nor the second signals involved in the expression of CIITA are impaired in aged macrophages. These data indicate that aging is associated with low levels of MHC class II gene induction by IFN-gamma because of impaired transcription.

Repression of major histocompatibility complex I-A beta gene expression by dbpA and dbpB (mYB-1) proteins.

The induction of major histocompatibility complex class II gene expression is mediated by three DNA elements in the promoters of these genes (W, X, and Y boxes). The Y box contains an inverted CCAAT box sequence, and the binding activity to the CAAT box is mediated by factor NF-Y, which is composed of subunits NF-YA and NF-YB. We have found that transfection of either dbpA or dbpB (mYB-1) or both inhibits I-A beta gene expression. Although the genes for some members of the Y-box family of binding proteins have been isolated by screening an expression library using the Y-box sequence, under our conditions no binding of dbpA or dbpB to the Y box of the I-A beta or I-E alpha promoter was detected. This suggested that repression of I-A beta gene expression by dbpA and dbpB was not due to competition for binding to the Y-box sequence. The results suggest two other mechanisms by which dbpA and dbpB can inhibit transcription from the I-A beta promoter. When dbpA was added, the binding of NF-YA to DNA increased, which could be explained by interaction between these two proteins whose purpose is to increase the binding affinity of NF-YA for DNA. However, this complex was unable to stimulate transcription from the I-A beta promoter. Thus, dbpA competed for the interaction between NF-YA and NF-YB by binding to NF-YA. When dbpB factor was added together with NF-YA and NF-YB, the binding of the NF-YA--NF-YB complex was reduced. This suggested that dbpB may complete with NF-YB for interaction with NF-YA. These results provide an example of how dbpA and dbpB may regulate transcription of promoters that utilize NF-Y as a transcription factor.

Macrophages and Mitochondria: A Critical Interplay Between Metabolism, Signaling, and the Functional Activity.

Macrophages are phagocytic cells that participate in a broad range of cellular functions and they are key regulators of innate immune responses and inflammation. Mitochondria are highly dynamic endosymbiotic organelles that play key roles in cellular metabolism and apoptosis. Mounting evidence suggests that mitochondria are involved in the interplay between metabolism and innate immune responses. The ability of these organelles to alter the metabolic profile of a cell, thereby allowing an appropriate response to each situation, is crucial for the correct establishment of immune responses. Furthermore, mitochondria act as scaffolds for many proteins involved in immune signaling pathways and as such they are able to modulate the function of these proteins. Finally, mitochondria release molecules, such as reactive oxygen species, which directly regulate the immune response. In summary, mitochondria can be considered as core components in the regulation of innate immune signaling. Here we discuss the intricate relationship between mitochondria, metabolism, intracellular signaling, and innate immune responses in macrophages.

Mapping, cloning and sequencing of a glycoprotein-encoding gene from bovine herpesvirus type 1 homologous to the gE gene from HSV-1.

In order to map and identify the glycoprotein-encoding gene from bovine herpesvirus type 1 (BHV-1), homologous to the gE glycoprotein from herpes simplex virus type 1 (HSV-1), a region of the unique short sequence from the BHV-1 genome has been sequenced. The sequenced region contains an ORF coding for a polypeptide of 575 amino acids (aa). The aa sequence presents substantial similarity to that of the glycoprotein gE from HSV-1 and to homologous proteins of related viruses such as pseudorabies virus, equine herpesvirus type 1 and varicella zoster virus. The aa sequence presents additional characteristics compatible with the structure of a viral glycoprotein: signal peptide, putative glycosylation sites and a long C-terminal transmembrane alpha-helix.

Essential catalytic role of Glu134 in endo-beta-1,3-1,4-D-glucan 4-glucanohydrolase from B. licheniformis as determined by site-directed mutagenesis.

Site-directed mutagenesis experiments designed to identify the active site of Bacillus licheniformis endo-beta-1,3-1,4-D-glucan 4-glucanohydrolase (beta-glucanase) have been performed. Putative catalytic residues were chosen on the basis of sequence similarity analysis to viral and eukaryotic lysozymes. Four mutant enzymes were expressed and purified from recombinant E. coli and their kinetics analysed with barley beta-glucan. Replacement of Glu134 by Gln produced a mutant (E134Q) that retains less than 0.3% of the wild-type activity. The other mutants, D133N, E160Q and D179N, are active but show different kinetic parameters relative to wild-type indicative of their participation in substrate binding and transition-state complex stabilization. Glu134 is essential for activity; it is comprised in a region of high sequence similarity to the active site of T4 lysozyme and matches the position of the general acid catalyst. These results strongly support a lysozyme-like mechanism for this family of Bacillus beta-glucan hydrolases with Glu134 being the essential acid catalyst.

Glycoprotein E of bovine herpesvirus type 1 is involved in virus transmission by direct cell-to-cell spread.

In order to identify the role of the bovine herpesvirus type 1 (BHV-1) glycoprotein E (gE) in the viral infection cycle, we have constructed a BHV-1 gE deletion mutant strain (BHV-1 gE-). This strain was assayed in vitro by comparing its growth kinetics with the wild type strain used as a host of the deletion. Our results indicate that those conditions which prevent the infection by direct adsorption to the cells (presence of a semi-solid medium or presence of neutralizing antibodies in the medium) selectively inhibit the growth of the gE- strain, suggesting that gE plays a central role in the BHV-1 spread by direct cell-to-cell transmission, a major mechanism of the BHV-1 in vivo virulence.

Repression mechanisms of the I-A beta gene of the major histocompatibility complex.

The mechanisms of regulation of I-A beta gene expression in the murine major histocompatibility complex by transcriptional repression are reviewed. Active and passive repression mechanisms are presented. The transcription factor PU.1 actively inhibits the expression of I-A beta through the binding to a DNA sequence near the Y box, a cis-element in the promoter necessary for transcription. This interaction probably interferes with the preinitiation complex assembly. NF-Y is a transcription factor that binds to the Y box and has two constituents: NF-YA (that binds weakly to DNA) and NF-YB (that increases the binding of NF-YA to DNA). The dbpA protein represses the expression of I-A beta by a quenching mechanism, forming a complex with NF-YA and the dbpB protein by sequestering the NF-YB protein. A similar mechanism is observed with the glucocorticoid receptor that binds to the X-box binding proteins and inhibits their interaction with the X box. These results are examples of cross-talk between proteins, which may help us to understand the regulation of I-A beta gene expression.

Molecular mechanisms involved in macrophage survival, proliferation, activation or apoptosis.

Macrophages play a critical role during the immune response. Like other cells of the immune system, macrophages are produced in large amounts and most of them die through apoptosis. Macrophages survive in the presence of soluble factors, such as IFN-gamma, or extracellular matrix proteins like decorin. The mechanism toward survival requires the blocking of proliferation at the G1/S boundary of the cell cycle that is mediated by the cyclin-dependent kinase (cdk) inhibitor, p27kip and the induction of a cdk inhibitor, p21waf1. At the inflammatory loci, macrophages need to proliferate or become activated in order to perform their specialized activities. Although the stimuli inducing proliferation and activation follow different intracellular pathways, both require the activation of extracellular signal-regulated kinases (ERKs) 1 and 2. However, the kinetics of ERK-1/2 activation is different and is determined by the induction of the MAP-kinase phosphatase-1 (MKP-1) that dephosphorilates ERK-1/2. This phosphatase plays a critical role in the process of proliferation versus activation of the macrophages.

Effect of cycloheximide on different stages of Drosophila melanogaster.

Cycloheximide, an antibiotic inhibiting protein synthesis, exerted a toxic effect on different developmental stages egg, larva and adult of Drosophila melanogaster. At the egg stage the early embryos were most sensitive. With larvae, a strong decrease in viability was found, with no sex difference. In adults, there was a dose-effect relationship, mortality increasing with concentration. At 10 and 15 mM, males were more sensitive than females. There were consistent differences between the control and cycloheximide-fed females in respect of the average number of eggs deposited and offspring produced.

An ORF from Bacillus licheniformis encodes a putative DNA repressor.

The complete sequence of a reading frame adjacent to the endo-beta-1,3-1,4-D-glucanase gene from Bacillus licheniformis is reported. It encodes a putative 171 amino acid residues protein with either, low significant sequence similarity in data banks or the corresponding orthologue in the recently sequenced Bacillus subtilis genome. Computer analyses predict a canonical Helix-Turn-Helix motif characteristic of bacterial repressors/DNA binding proteins. A maxicells assay shows that the encoded polypeptide is expressed. A DNA-protein binding, assay performed by gel electrophoresis shows that the expressed protein specifically binds to Bacillus licheniformis DNA.

Dexamethasone enhances macrophage colony stimulating factor- and granulocyte macrophage colony stimulating factor-stimulated proliferation of bone marrow-derived macrophages.

Glucocorticoids are effective repressors of the immune system. We have examined the effect of glucocorticoids on the proliferation of murine macrophages. Dexamethasone by itself did not affect proliferation of differentiated or undifferentiated bone marrow-derived macrophages (BMM) and elicited peritoneal macrophages. However, dexamethasone enhanced the proliferation induced by macrophage colony stimulating factor (M-CSF) of these cells. The effect of dexamethasone was not restricted to M-CSF-dependent proliferation. Similarly, dexamethasone enhanced granulocyte macrophage colony stimulating factor (GM-CSF)-dependent proliferation of BMM. In agreement, macrophages transfected with the glucocorticoid receptor showed an enhancement of M-CSF-dependent proliferation. The enhancement of proliferation by dexamethasone or the glucocorticoid receptor was abolished by RU 486, an antagonist of the glucocorticoid receptor. Moreover, the addition of antibodies against M-CSF inhibits the effect of dexamethasone, suggesting that dexamethasone increases the autocrine production of M-CSF. This only occurs when M-CSF or GM-CSF, which induce M-CSF, are present in the media. In tissues, dexamethasone may enhance macrophage proliferation and contribute to the resolution of the inflammatory states.

Molecular cloning, expression and nucleotide sequence of the endo-beta-1,3-1,4-D-glucanase gene from Bacillus licheniformis. Predictive structural analyses of the encoded polypeptide.

A Bacillus licheniformis gene coding for an endo-beta-1,3-1,4-D-glucanase have been cloned in Escherichia coli and sequenced. The open reading frame contains a sequence of 731 bp, encoding a polypeptide of 243 amino acid residues, with a molecular mass of 27404 Da (24418 Da without the putative signal peptide), which corresponds to the enzyme we had previously isolated and characterized. The signal peptide is functional in E. coli. More than 60% of the endo-beta-1,3-1,4-D-glucanase activity is extracellular or periplasmic. The polypeptide is highly similar to other reported Bacillus beta-glucanases. Several structural predictive analyses (secondary structure, hydropathic plots, similarity with other related enzymes, etc.) have been performed. From these analyses we assign a tentative three-functional-domain structure for the enzyme (signal peptide, substrate binding and catalytic domains) and a putative lysozyme-like active site.

Regulation of murine Tap1 and Lmp2 genes in macrophages by interferon gamma is mediated by STAT1 and IRF-1.

The genes of the transporter associated with antigen processing (Tap)-1, and the low molecular weight peptide (Lmp)-2, are crucial for class I major histocompatibility complex function and share a common bidirectional promoter. In murine bone marrow-derived macrophages, interferon gamma (IFN-gamma) induced Tap-1 and upregulated Lmp-2, which is constitutively expressed at low levels. The IFN-gamma-induction was independent of early gene synthesis. The mRNA induced by IFN-gamma was very stable. In macrophages from STAT1 knockout mice, IFN-gamma did not induce the expression of Tap-1 or Lmp-2. Several areas in the promoter can be controlled by IFN-gamma, such as proximal and distal GAS boxes in the direction of the Tap-1 gene, NFgammaB and IRF-1 boxes. By making deletions of the promoter, we found that only the proximal GAS and IRF-1 boxes are required for IFN-gamma induction of Tap-1 and Lmp-2. Experiments using nuclear extracts from macrophages treated for 30 min with IFN-gamma and gel shift analysis indicated that STAT1 binds to the GAS box. The nuclear extracts from macrophages treated for at least 2 h with IFN-gamma bound to the IRF-1 box. These results indicate that both STAT1 and IRF-1 are required for the IFN-gamma induction of Tap-1 and Lmp-2 genes.

Interferon gamma induces the expression of p21waf-1 and arrests macrophage cell cycle, preventing induction of apoptosis.

Incubation of bone marrow macrophages with lipopolysaccharide (LPS) or interferon gamma (IFN gamma) blocks macrophage proliferation. LPS treatment or M-CSF withdrawal arrests the cell cycle at early G1 and induces apoptosis. Treatment of macrophages with IFN gamma stops the cell cycle later, at the G1/S boundary, induces p21Waf1, and does not induce apoptosis. Moreover, pretreatment of macrophages with IFN gamma protects from apoptosis induced by several stimuli. Inhibition of p21Waf1 with antisense oligonucleotides or using KO mice shows that the induction of p21Waf1 by IFN gamma mediates this protection. Thus, IFN gamma makes macrophages unresponsive to apoptotic stimuli by inducing p21Waf1 and arresting the cell cycle at the G1/S boundary. Therefore, the cells of the innate immune system could only survive while they were functionally active.

IFN-gamma up-regulates the A2B adenosine receptor expression in macrophages: a mechanism of macrophage deactivation.

Adenosine is a potent endogenous anti-inflammatory agent released by cells in metabolically unfavorable conditions, such as hypoxia or ischemia. Adenosine modulates different functional activities in macrophages. Some of these activities are believed to be induced through the uptake of adenosine into the macrophages, while others are due to the interaction with specific cell surface receptors. In murine bone marrow-derived macrophages, the use of different radioligands for adenosine receptors suggests the presence of A2B and A3 adenosine receptor subtypes. The presence of A2B receptors was confirmed by flow cytometry using specific Abs. The A2B receptor is functional in murine macrophages, as indicated by the fact that agonists of A2B receptors, but not agonists for A1, A2A, or A3, lead to an increase in cAMP levels. IFN-gamma up-regulates the surface protein and gene expression of the A2B adenosine receptor by induction of de novo synthesis. The up-regulation of A2B receptors correlates with an increase in cAMP production in macrophages treated with adenosine receptor agonist. The stimulation of A2B receptors by adenosine or its analogues inhibits the IFN-gamma-induced expression of MHC class II genes and also the IFN-gamma-induced expression of nitric oxide synthase and of proinflammatory cytokines. Therefore, the up-regulation of the A2B adenosine receptor expression induced by IFN-gamma could be a feedback mechanism for macrophage deactivation.

A beta-glucosidase gene (bgl3) from Streptomyces sp. strain QM-B814. Molecular cloning, nucleotide sequence, purification and characterization of the encoded enzyme, a new member of family 1 glycosyl hydrolases.

A beta-glucosidase gene (bgl3) from Streptomyces sp. QM-B814 (American Type Culture Collection 11238) has been cloned by functional complementation of a beta-glucosidase-negative mutant of Streptomyces lividans. An open-reading frame of 1440 nucleotides encoding a polypeptide of 479 amino acids was found by sequencing. The encoded protein (Bgl3) shows extensive similarity (over 45% identity) with beta-glycosidases from family-1 glycosyl hydrolases. The cloned enzyme, purified following ammonium sulphate precipitation and two chromatographic steps, is monomeric with molecular mass 52.6 kDa, as determined by mass spectrometry, and an isoelectric point of pI 4.4. The enzyme appears to be a beta-glucosidase with broad substrate specificity, is active on cellooligomers, and performs transglycosylation reactions. The estimated apparent Km values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose are 0.27 mM and 7.9 mM, respectively. The Ki values for glucose and delta-gluconolactone, using p-nitrophenyl-beta-D-glucopyranoside as a substrate, are 65 mM and 0.08 mM, respectively. The purified enzyme has a pH optimum of pH 6.5 and the temperature optimum for activity is 50 degrees C.

Repression of I-A beta gene expression by the transcription factor PU.1.

The PU.1 protein is an ets-related transcription factor that is expressed in macrophages and B lymphocytes. We present evidence that PU.1 binds to the promoter of the I-A beta gene, i.e. a PU box located next to the Y box. Transfection of PU.1 in B lymphocytes or in interferon-gamma-treated macrophages represses I-A beta gene expression. The inhibitory effect of PU.1 was obtained with the DNA binding domain of the protein, but not with the activation domain. Using the gel shift retardation assay we found that in vitro transcribed/translated NF-YA and NF-YB bind to the Y box of the I-A beta promoter. When PU.1 was added to the assay, a supershifted DNA band was found, indicating that PU.1 and NFY proteins bind to the same DNA molecule. We conclude that I-A beta gene expression is repressed by PU.1 binding to the PU box domain.