Depressive symptoms are prevalent in childhood-onset systemic lupus erythematosus (cSLE).

BACKGROUND: Depressive symptoms are common in adolescence and young adulthood; however, their prevalence in childhood-onset systemic lupus erythematosus (cSLE) is unknown. OBJECTIVE: The objective of this study was to examine the prevalence of depressive symptoms and their association with disease characteristics in children, adolescents, and young adults with cSLE. METHODS: A cross-sectional sample of patients with cSLE between 10 to 24 years old completed standardized depression inventories. Demographics and disease characteristics were collected. RESULTS: Total depression inventory scores reported were below standard cut-off values for depression. However, 26% (10/38) of children and adolescents, and 44% (seven of 16) of young adults had scores at or above established cut-offs for elevated depression symptoms. Physical symptoms of depression were endorsed most frequently. There were no differences in depressive symptoms by disease characteristics including disease duration, health-related quality of life inventory scores, antiphospholipid antibody status, and a history of renal involvement or neuropsychiatric SLE (NPSLE). However, two patients had a history of depression as an NPSLE manifestation of their SLE. In the children and adolescents, prednisone dose was associated with negative self-esteem (r = 0.37, p = 0.04) and somatic depressive symptoms (r = 0.39, p = 0.02), but we did not observe a significant association in the young adults. CONCLUSION: Depressive symptoms in cSLE are frequent, although similar to the high prevalence rates in the general population. Physical symptoms are most frequently endorsed. Further study will determine if serial evaluations are recommended for early detection in this at-risk population.

The lupus phenotype in B6.NZBc1 congenic mice reflects interactions between multiple susceptibility loci and a suppressor locus.

Lupus susceptibility loci on chromosome 1 have an important role in the development of autoimmunity in the New Zealand Black (NZB) mouse. We have previously shown that C57BL/6 congenic mice with an introgressed homozygous NZB chromosome 1 interval extending from ∼35 to 106 cM develop anti-nuclear antibodies and mild glomerulonephritis. In this study, we produced subcongenic mouse strains to localize the susceptibility loci in this interval and investigate how they promote autoimmunity. Our results indicate at least four susceptibility alleles and a suppressor allele. One allele is located in the 96-100 cM region and is sufficient to breach tolerance to chromatin. Addition of a second locus in the 88-96 cM interval enhances anti-dsDNA antibody production and promotes renal disease, which together with a third susceptibility allele in the 70-88 interval results in significant mortality. We further demonstrate the presence of a suppressor locus in the 35-70 or 100-102 cM interval that abrogates these phenotypes and an additional susceptibility allele in the 102-106 cM interval that restores a milder autoimmune phenotype. Several of these loci alter T-cell function. Thus, there is substantial genetic complexity in the NZB 35-106 cM interval, with disease reflecting a balance between susceptibility and suppressor loci.

Lack of association between the interferon-alpha signature and longitudinal changes in disease activity in systemic lupus erythematosus.

OBJECTIVE: To study the longitudinal expression of interferon (IFN)-inducible genes in systemic lupus erythematosus (SLE) and determine their suitability as disease biomarkers. METHODS: RNA was isolated from the peripheral blood of 94 patients with SLE and 11 controls and reverse transcribed into cDNA. The expression levels of five IFN-responsive genes (LY6E, OAS1, IFIT1, ISG15 and MX1) were determined by quantitative PCR, normalised to GAPDH and summed to generate a global IFN score. Patients were followed longitudinally for a period of 3-12 months, and the association between disease activity, as measured by the SLE disease activity index (SLEDAI-2K), and other clinical and laboratory variables was examined. RESULTS: The expression of all five IFN-responsive genes was significantly higher in patients with SLE than in controls. The expression of LY6E, OAS1, IFIT1 and the global IFN score was associated with high disease activity. The global IFN score was also associated with active renal disease, a decreased C3, and the presence of anti-dsDNA or anti-RNA binding protein antibodies at a single point in time. However, there was a poor correlation between changes in this score and changes in disease activity, C3 or anti-dsDNA antibody levels in patients followed longitudinally. In most patients the levels of IFN-induced gene expression remained relatively stable over 3-12 months despite marked changes in disease activity. Nevertheless, in patients with low/moderate disease activity, those with high IFN scores had a more recent history of sustained high disease activity. CONCLUSION: The findings indicate that IFN-induced gene expression has limited clinical utility as a biomarker of acute changes in disease activity.

Evidence that the NH2 terminus of vph1p, an integral subunit of the V0 sector of the yeast V-ATPase, interacts directly with the Vma1p and Vma13p subunits of the V1 sector.

The vacuolar-type H(+)-ATPase (V-ATPase) is composed of a peripherally bound (V(1)) and a membrane-associated (V(0)) complex. V(1) ATP hydrolysis is thought to rotate a central stalk, which in turn, is hypothesized to drive V(0) proton translocation. Transduction of torque exerted by the rotating stalk on V(0) requires a fixed structural link (stator) between the complexes to prevent energy loss through futile rotation of V(1) relative to V(0); this work sought to identify stator components. The 95-kDa V-ATPase subunit, Vph1p, has a cytosolic NH(2) terminus (Nt-Vph1p) and a membrane-associated COOH terminus. Two-hybrid assays demonstrated that Nt-Vph1p interacts with the catalytic V(1) subunit, Vma1p. Co-immunoprecipitation of Vma1p with Nt-Vph1p confirmed the interaction. Expression of Nt-Vph1p in a Deltavph1 mutant was necessary to recruit Vma13p to V(1). Vma13p bound to Nt-Vph1p in vitro demonstrating direct interaction. Limited trypsin digests cleaves both Nt-Vph1p and Vma13p. The same tryptic treatment results in a loss of proton translocation while not reducing bafilomycin A(1)-sensitive ATP hydrolysis. Trypsin cleaved Vph1p at arginine 53. Elimination of the tryptic cleavage site by substitution of arginine 53 to serine partially protected vacuolar acidification from trypsin digestion. These results suggest that Vph1p may function as a component of a fixed structural link, or stator, coupling V(1) ATP hydrolysis to V(0) proton translocation.

Substrate- and inhibitor-induced conformational changes in the yeast V-ATPase provide evidence for communication between the catalytic and proton-translocating sectors.

The vacuolar-type H(+)-ATPases (V-ATPases) are composed of two distinct sectors, a catalytic complex (V(1)) involved in ATP hydrolysis and a membrane-associated complex (V(0)) mediating proton translocation across a lipid bilayer. To date, little is known about the mechanism by which these two functions are coupled. We sought to examine the impact of nucleotide and cation binding on the structure of the core components of the catalytic complex and to determine whether conformational changes within the catalytic complex impact subunits of the membrane-associated complex. Nucleotide- and cation- induced changes in the catalytic core of the V-ATPase were investigated by monitoring changes in the rate and pattern of tryptic digests. ATP.Mg-induced changes were detected in both the catalytic (Vma1p or 69 kDa) and the regulatory subunits (Vma2p or 60 kDa) of the V(1) sector. ATP alone increased the rate of trypsinization of the regulatory subunit, but did not have any effect on Vma1p. Surprisingly, ATP also had an impact on the 95-kDa subunit, a component of the V(0) sector of the V-ATPase. Although the presence of divalent cations had no impact on the V(1) sector, the rate of trypsinization of the 95-kDa subunit was greatly enhanced. The effect of divalent cations on the structure of the 95-kDa subunit was abrogated when trypsinization was performed in the absence of the catalytic sector. Addition of bafilomycin A(1), a V-ATPase inhibitor that putatively binds to the 95-kDa subunit, increased the rate of trypsinization of the catalytic subunit. These data suggest that structural alterations within the V(1) sector result in alterations within the V(0) sector and vice versa. Clearly, a structural link must exist to couple the two sectors. The 95-kDa subunit is ideally suited to fulfill this role. Hydropathy analysis suggests a bipartite structure, with the NH(2)-terminal portion predicted to lie in an aqueous environment and the C-terminal portion predicted to contain 6 transmembrane segments. Tryptic digests of sealed vacuolar vesicles and immunofluorescence studies revealed that the large hydrophilic NH(2)-terminal domain of the 95-kDa subunit is localized toward the cytosol. This region therefore is ideally positioned to interact with components of the V(1) complex, potentially functioning as the elusive link between the two sectors of the V-ATPase.

Two glycoprotein populations of band 3 dimers are present in human erythrocytes.

The human erythrocyte Band 3 anion exchanger contains a single site of N-glycosylation that contains either a short complex oligosaccharide or an extended polylactosaminyl oligosaccharide. Approximately equal amounts of the different glycosylated forms of Band 3 are found in human red cells. As Band 3 exists predominantly as dimers, they may be uniform and consist of a subunit containing an extended oligosaccharide paired with a subunit containing a short oligosaccharide chain. Alternatively, Band 3 dimers may be comprised of subunits that either contain polylactosaminyl or short oligosaccharide chains. To distinguish between these two extremes, the ability of Band 3 membrane domain dimers to bind to immobilized tomato lectin, which specifically binds polylactosaminyl oligosaccharide, was tested. The dimeric membrane domain of Band 3 could be resolved into two fractions by tomato lectin chromatography. This shows that Band 3 dimers are not homogeneous and that two pools exist in red cells, some with a long polylactosaminyl oligosaccharide and the other with a short complex type. The amount of short chain form recovered in the unbound fraction was higher than expected for a random distribution of oligosaccharide chains on Band 3 dimers. Detergent extraction experiments showed that Band 3 glycoforms did not display a differential interaction with the cytoskeleton. The ability to separate Band 3 dimers into two glycoform populations suggests that subunit exchange between dimers does not occur in the membrane or in detergent solution. Furthermore, the results show that while one population of Band 3 dimers is processed to contain polylactosaminyl oligosaccharide, the other largely escapes this processing step.

Structural and functional consequences of an N-glycosylation mutation (HEMPAS) affecting human erythrocyte membrane glycoproteins.

Band 3, the human erythrocyte anion exchanger (AE1), and the glucose transporter (GLUT1) proteins each contain a single site of N-glycosylation that is heterogeneously glycosylated. Lectin binding and enzymatic deglycosylation assays showed that the polylactosaminyl oligosaccharide structure of these glycoproteins was altered to a high mannose or hybrid glycan form in three patients with hereditary erythroblastic multinuclearity, with a positive acidified-serum lysis test (HEMPAS). Offspring from one of the HEMPAS patients had intermediate levels of polylactosaminyl oligosaccharide associated with AE1 and GLUT1, suggesting they may have been heterozygous for the genetic defect. The array of polylactosaminyl-containing glycoproteins present in EBV-transformed lymphoblasts derived from fresh blood of HEMPAS patients was similar to control lymphoblasts. HEMPAS lymphoblasts do not therefore express the defect in polylactosamine synthesis found in erythroid cells, indicating that lymphoid cells are not deficient in the processing enzymes or contain an alternative oligosaccharide processing pathway. Purified HEMPAS band 3 had an unaltered oligomeric structure but dimers aggregated more rapidly in detergent solution than normal band 3. The altered oligosaccharide structure did not affect the sensitivity of band 3 to proteolytic digestion in intact red cells but a greater amount of HEMPAS band 3 was associated with the cytoskeleton. The transport activities of AE1 and GLUT1 in HEMPAS erythrocytes were similar to those in normal controls. This shows that the HEMPAS glycosylation defect does not impair the functional accumulation of these two important erythrocyte membrane transporters even though it produces subtle structural changes in band 3 that result in its increased cytoskeletal interaction and self association in detergent solution.

Glycosylation of multiple extracytosolic loops in Band 3, a model polytopic membrane protein.

N-glycosylated sites in polytopic membrane proteins are usually localized to single extracytosolic (EC) loops containing more than 30 residues [Landolt-Marticorena and Reithmeier (1994) Biochem. J. 302, 253-260]. This may be due to a biosynthetic restriction whereby only a single loop of nascent polypeptide is available to the oligosaccharyl transferase in the lumen of the endoplasmic reticulum. To test this hypothesis, two types of N-glycosylation mutants were constructed using Band 3, a polytopic membrane protein that contains up to 14 transmembrane segments and a single endogenous site of N-glycosylation at Asn-642 in EC loop 4. In the first set of mutants, an additional N-glycosylation acceptor site (Asn-Xaa-Ser/Thr) was constructed by site-directed mutagenesis in EC loop 3, with or without retention of the endogenous site. In the second set of mutants, EC loop 4 was duplicated and inserted into EC loop 2, again with or without retention of the endogenous site. Cell-free translation experiments using reticulocyte lysates showed that microsomes were able to N-glycosylate multiple EC loops in these Band 3 mutants. The acceptor site in EC loop 3 was poorly N-glycosylated, probably due to the suboptimal size (25 residues) of this EC loop. The localization of N-glycosylation sites to single EC loops in multi-span membrane proteins is probably due to the absence of suitably positioned acceptor sites on multiple loops.

Transmembrane helix-helix interactions and accessibility of H2DIDS on labelled band 3, the erythrocyte anion exchange protein.

4,4'-Diisothiocyanodihydrostilbene-2,2'-disulphonate (H2DIDS), a bifunctional inhibitor of anion exchange in erythrocytes, reacts with Lys-539 in band 3 at neutral pH and crosslinks to Lys-851 at alkaline pH. The accessibility of H2DIDS-labelled band 3 was determined using an anti-H2DIDS antibody and proteolysis. Competitive enzyme-linked immunosorbent assays (ELISAs) showed that a polyclonal antibody raised against H2DIDS-labelled keyhole limpet hemocyanin bound a variety of stilbene disulphonates in the following order of affinities, H2DIDS having the highest affinity: H2DIDS > 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS) > 4-acetamido-4'-isothiocyanostilbene-2,2'disulphonate (SITS) > 4,4'-dinitrostilbene-2,2'-disulphonate (DNDS) > 4,4'-diaminostilbene-2,2'-disulphonate (DADS). The antibody readily detected mono- or bifunctionally H2DIDS-labelled band 3 and proteolytic fragments on immunoblots. H2DIDS attached to Lys-539 is retained in a 7.5 kDa membrane-associated peptide after papain treatment of ghost membranes while the sequence around Lys-851 is more accessible. The band 3 proteolytic fragments protected by the membrane from proteolysis remained associated as a specific complex with a Stokes radius slightly smaller than the dimeric membrane domain after solubilization in detergent solution and retained 82% of the amino acid content of the membrane domain. Circular dichroism (CD) measurements of this H2DIDS-labelled complex showed that it had a very high helical content (86%). The loops connecting the transmembrane segments in H2DIDS-labelled band 3 are therefore not required to maintain transmembrane helix-helix interactions. Denatured band 3 prelabelled with H2DIDS was more readily immunoprecipitated with the anti-H2DIDS antibody than was native band 3 in detergent solution. Deglycosylation of band 3 or proteolytic cleavage of the extramembranous loops did not enhance immunoprecipitation of H2DIDS-labelled band 3. The stilbene disulphonate inhibitor site is therefore relatively inaccessible and is bound by a bundle of helices in the native band 3 protein.

Asparagine-linked oligosaccharides are localized to single extracytosolic segments in multi-span membrane glycoproteins.

A comprehensive survey of mammalian multi-span (polytopic) membrane proteins showed that asparagine(N)-linked oligosaccharides are localized to single extracytosolic segments. In most membrane proteins this is because potential consensus sites for N-glycosylation (Asn-Xaa-Ser/Thr, X not equal to Pro) are not found in multiple extracytosolic segments. In functional proteins where consensus N-glycosylation sites are contained within more than one extracytosolic segment, only the first segment contains N-linked carbohydrate. An exception is the alpha-subunit of the Na+ channel, which consists of a duplicated structure containing two glycosylated segments. The average size of established N-glycosylated loops connecting two transmembrane segments is 62 residues, with the smallest glycosylated loop being 33 residues in size. N-glycosylated sites are more highly conserved than non-glycosylated (primarily cytosolic) sites and are more common toward the N-terminus of the membrane domain of multi-span membrane proteins. The optimal conditions for glycosylation of consensus sites within an extracytosolic domain of a multi-span membrane protein are (i) the acceptor site is well-spaced (greater than 10 residues) from the transmembrane domain, (ii) the loop is greater than 30 residues in size and (iii) the segment is the first in the protein to contain a suitable extracytosolic consensus site. The localization of N-linked oligosaccharide chains to a single protein segment suggests either glycosylation of multiple loops may compromise protein folding or function, or only a single polypeptide domain can be optimally glycosylated during biosynthesis in vivo.

Non-random distribution of amino acids in the transmembrane segments of human type I single span membrane proteins.

The distribution of amino acids in the transmembrane segments and flanking regions of 115 human type I single span (amino terminus extracellular and carboxyl terminus cytosolic) plasma membrane proteins was found to be non-random. In this sample, Ile was preferentially localized to the amino-terminal region of the hydrophobic transmembrane segments, followed by Val, while Leu predominated in the carboxyl-terminal half of the segment. Although Gly residues were preferentially located in the transmembrane segment, this residue was excluded from the carboxyl-terminal and adjacent boundary regions. Aromatic residues (Tyr, Trp and Phe) occurred preferentially at the cytoplasmic boundary, with Trp also favored at the extracellular boundary. The extracellular flanking sequence amino-terminal to the transmembrane segment was enriched in residues predicted to initiate helix formation (Pro, Asn and Ser), while Arg and Lys were enriched in the cytoplasmic flank where they may function as topological determinants. The positional preferences of these particular amino acids within the transmembrane segment and flanking regions suggests that, in addition to lipid-protein interactions, these residues may participate in specific protein-protein interactions. A consensus sequence motif for type I membrane proteins is proposed and its role in the biosynthesis, folding, assembly and function of these segments is discussed.

Antibiotic properties of ectomycorrhizae and saprophytic fungi growing on Pinus radiata D. Don I.

Extracts of thirty six Agaricales fungi were assayed for their antibacterial activity against Staphylococcus aureus ATCC6538, Bacillus subtilis ATCC6633 and Escherichia coli UCCL51. Approximately thirty species showed activity against one or more of the tested organisms.

Screening of Chilean plants for anticancer activity. I.

Extracts prepared from 519 samples of plant material were evaluated for anticancer activity against the usual systems. The results of the antitumor testing showed that 156 of these extracts gave initial indications of anticancer activity in one or more tumor systems. The activity was confirmed in 14 samples.