Urinary vitamin D-binding protein, a novel biomarker for lupus nephritis, predicts the development of proteinuric flare.

Lupus nephritis (LN) is a major complication of systemic lupus erythematosus (SLE). Conventional biomarkers for assessing renal disease activity are imperfect in predicting clinical outcomes associated with LN. The aim of this study is to identify urinary protein biomarkers that reliably reflect the disease activity or predict clinical outcomes. A quantitative proteomic analysis was performed to identify protein biomarker candidates that can differentiate between SLE patients with and without LN. Selected biomarker candidates were further verified by enzyme-linked immunosorbent assay using urine samples from a larger cohort of SLE patients ( n = 121) to investigate their predictive values for LN activity measure. Furthermore, the association between urinary levels of a selected panel of potential biomarkers and prognosis of LN was assessed with a four-year follow-up study of renal outcomes. Urinary vitamin D-binding protein (VDBP), transthyretin (TTR), retinol binding protein 4 (RBP4), and prostaglandin D synthase (PTGDS) were significantly elevated in SLE patients with LN, especially in patients with active LN ( n = 21). Among them, VDBP well correlated with severity of proteinuria (rho = 0.661, p < 0.001) and renal SLE Disease Activity Index (renal SLEDAI) (rho = 0.520, p < 0.001). In the four-year follow-up, VDBP was a significant risk factor (hazard ratio 9.627, 95% confidence interval 1.698 to 54.571, p = 0.011) for the development of proteinuric flare in SLE patients without proteinuria ( n = 100) after adjustments for multiple confounders. Urinary VDBP correlated with proteinuria and renal SLEDAI, and predicted the development of proteinuria.

Crucial role of calbindin-D28k in the pathogenesis of Alzheimer's disease mouse model.

Calbindin-D28k (CB), one of the major calcium-binding and buffering proteins, has a critical role in preventing a neuronal death as well as maintaining calcium homeostasis. Although marked reductions of CB expression have been observed in the brains of mice and humans with Alzheimer disease (AD), it is unknown whether these changes contribute to AD-related dysfunction. To determine the pathogenic importance of CB depletions in AD models, we crossed 5 familial AD mutations (5XFAD; Tg) mice with CB knock-out (CBKO) mice and generated a novel line CBKO·5XFAD (CBKOTg) mice. We first identified the change of signaling pathways and differentially expressed proteins globally by removing CB in Tg mice using mass spectrometry and antibody microarray. Immunohistochemistry showed that CBKOTg mice had significant neuronal loss in the subiculum area without changing the magnitude (number) of amyloid ß-peptide (Aß) plaques deposition and elicited significant apoptotic features and mitochondrial dysfunction compared with Tg mice. Moreover, CBKOTg mice reduced levels of phosphorylated mitogen-activated protein kinase (extracellular signal-regulated kinase) 1/2 and cAMP response element-binding protein at Ser-133 and synaptic molecules such as N-methyl-D-aspartate receptor 1 (NMDA receptor 1), NMDA receptor 2A, PSD-95 and synaptophysin in the subiculum compared with Tg mice. Importantly, this is the first experimental evidence that removal of CB from amyloid precursor protein/presenilin transgenic mice aggravates AD pathogenesis, suggesting that CB has a critical role in AD pathogenesis.

Intersection of the Kap123p-mediated nuclear import and ribosome export pathways.

Kap123p is a yeast beta-karyopherin that imports ribosomal proteins into the nucleus prior to their assembly into preribosomal particles. Surprisingly, Kap123p is not essential for growth, under normal conditions. To further explore the role of Kap123p in nucleocytoplasmic transport and ribosome biogenesis, we performed a synthetic fitness screen designed to identify genes that interact with KAP123. Through this analysis we have identified three other karyopherins, Pse1p/Kap121p, Sxm1p/Kap108p, and Nmd5p/Kap119p. We propose that, in the absence of Kap123p, these karyopherins are able to supplant Kap123p's role in import. In addition to the karyopherins, we identified Rai1p, a protein previously implicated in rRNA processing. Rai1p is also not essential, but deletion of the RAI1 gene is deleterious to cell growth and causes defects in rRNA processing, which leads to an imbalance of the 60S/40S ratio and the accumulation of halfmers, 40S subunits assembled on polysomes that are unable to form functional ribosomes. Rai1p localizes predominantly to the nucleus, where it physically interacts with Rat1p and pre-60S ribosomal subunits. Analysis of the rai1/kap123 double mutant strain suggests that the observed genetic interaction results from an inability to efficiently export pre-60S subunits from the nucleus, which arises from a combination of compromised Kap123p-mediated nuclear import of the essential 60S ribosomal subunit export factor, Nmd3p, and a DeltaRAI1-induced decrease in the overall biogenesis efficiency.

Differential stable isotope labeling of peptides for quantitation and de novo sequence derivation.

We have demonstrated the use of per-methyl esterification of peptides for relative quantification of proteins between two mixtures of proteins and automated de novo sequence derivation on the same dataset. Protein mixtures for comparison were digested to peptides and resultant peptides methylated using either d0- or d3-methanol. Methyl esterification of peptides converted carboxylic acids, such as are present on the side chains of aspartic and glutamic acid as well as the carboxyl terminus, to their corresponding methyl esters. The separate d0- and d3-methylated peptide mixtures were combined and the mixture subjected to microcapillary high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). Parent proteins of methylated peptides were identified by correlative database searching of peptide tandem mass spectra. Ratios of proteins in the two original mixtures could be calculated by normalization of the area under the curve for identical charge states of d0- to d3-methylated peptides. An algorithm was developed that derived, without intervention, peptide sequence de novo by comparison of tandem mass spectra of d0- and d3-peptide methyl esters.

The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5.

The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) that are expressed on infectious agents, but not on the host. Toll-like receptors (TLRs) recognize PAMPs and mediate the production of cytokines necessary for the development of effective immunity. Flagellin, a principal component of bacterial flagella, is a virulence factor that is recognized by the innate immune system in organisms as diverse as flies, plants and mammals. Here we report that mammalian TLR5 recognizes bacterial flagellin from both Gram-positive and Gram-negative bacteria, and that activation of the receptor mobilizes the nuclear factor NF-kappaB and stimulates tumour necrosis factor-alpha production. TLR5-stimulating activity was purified from Listeria monocytogenes culture supernatants and identified as flagellin by tandem mass spectrometry. Expression of L. monocytogenes flagellin in non-flagellated Escherichia coli conferred on the bacterium the ability to activate TLR5, whereas deletion of the flagellin genes from Salmonella typhimurium abrogated TLR5-stimulating activity. All known TLRs signal through the adaptor protein MyD88. Mice challenged with bacterial flagellin rapidly produced systemic interleukin-6, whereas MyD88-null mice did not respond to flagellin. Our data suggest that TLR5, a member of the evolutionarily conserved Toll-like receptor family, has evolved to permit mammals specifically to detect flagellated bacterial pathogens.

Post-translational modification of the N-terminal His tag interferes with the crystallization of the wild-type and mutant SH3 domains from chicken src tyrosine kinase.

Structural studies of the wild type and mutants of the src SH3 domain were initiated to elucidate the correlation of the native-state topology with protein thermostability and folding kinetics. An extra mass of 178 Da arising from the post-translational modification at the N-terminal His tag was observed. The spontaneous alpha-N-6 gluconoylation at the amino group of the His-tagged SH3 domain contributed to the observed extra mass. The partial modification of the N-terminal His-tag produced heterogeneity, both in size and in charge, in the Escherichia coli expressed SH3 domain. The removal of the His tag from the SH3 domain was essential for the crystallization of both wild-type and mutant src SH3. Both the wild type and the W43I mutant were crystallized by hanging-drop vapor diffusion and are in the hexagonal space group P6(5)22 with one molecule in the asymmetric unit. Data sets were collected to 1.8 and 1.95 A resolution for the the wild type and the W43I mutant, respectively.

A remote flow cell for UV absorbance detection with capillary HPLC based on a single strand fiber optic.

A remote flow cell based on a single strand of fused-silica fiber optic was built for UV absorbance detection with a packed capillary HPLC system, using commercially available pumps, detection electronics (Shimadzu) and fittings. This 'off-column' flow cell design is applicable to both pressure and electro-osmotically driven systems. The goals were to minimize the linearity and light leakage problems that often limit the performance of UV absorbance detection with capillary chromatography. A linear dynamic range of 10(3) (reserpine, lambda = 220 nm), and a concentration detection limit of 5.1 x 10(-8) mol l-1 were observed. Baseline noise was measured at 3.5 x 10(-5) absorbance units (AU), with a standard deviation of 1.7 x 10(-5) AU. The illuminated volume of approximately 3 nl was optimized for capillaries with inner diameters in the range 50-100 microns, and flow rates from 100 nl min-1 to 1 microliter min-1. These modifications of readily available instrumentation have allowed the construction of a practical system for fractionating complex mixtures of peptides in small amounts, prior to mass spectrometry or additional wet chemistry steps.

Rapid isolation method for lipopolysaccharide and lipid A from gram-negative bacteria.

A fast, convenient extraction method for lipopolysaccharide (LPS), using a commercial RNA isolating reagent, allows the isolation of LPS or lipid A from low milligram (dry weight) quantities of bacterial cells. The method avoids the use of specialized equipment and has been used for processing relatively large numbers of samples. The major components of the commercial RNA isolating reagent, Tri-Reagent, are phenol and guanidinium thiocyanate in aqueous solution. The bacterial cell membranes are disrupted with guanidinium thiocyanate, which eliminates the need for mechanical cell disruption (e.g. French press) or heating. LPS and its degradation products, with particular attention paid to its bioactive lipid A portion, were measured and compared with those from the most common conventional extraction method, hot phenol-water. Negative ion quadrupole ion trap and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, fatty acid composition analysis by capillary gas chromatography, total and free phosphate by UV spectrophotometry and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that LPS and lipid A isolated using the Tri-Reagent approach were cleaner and suffered less degradation through loss of phosphate and (or) fatty acyl side chains from lipid A. The Tri-Reagent extraction method generated low free phosphate contamination, 11% of the total phosphate concentration, whereas the hot phenol-water extraction method gave approximately 58% as free, inorganic phosphate. Similar results were observed for the degradation of fatty acyl side chains. The time required by the new method is considerably shorter (two or three days) than that required by conventional hot phenol-water extraction (about two weeks).

A PhoP-regulated outer membrane protease of Salmonella enterica serovar typhimurium promotes resistance to alpha-helical antimicrobial peptides.

The outer membrane protein contents of Salmonella enterica serovar Typhimurium strains with PhoP/PhoQ regulon mutations were compared by two-dimensional gel electrophoresis. At least 26 species of outer membrane proteins (OMPs) were identified as being regulated by PhoP/PhoQ activation. One PhoP/PhoQ-activated OMP was identified by semiautomated tandem mass spectrometry coupled with electronic database searching as PgtE, a member of the Escherichia coli OmpT and Yersinia pestis Pla family of outer membrane proteases. Salmonella PgtE expression promoted resistance to alpha-helical cationic antimicrobial peptides (alpha-CAMPs). Strains expressing PgtE cleaved C18G, an 18-residue alpha-CAMP present in culture medium, indicating that protease activity is likely to be the mechanism of OmpT-mediated resistance to alpha-CAMPs. PhoP/PhoQ did not regulate the transcription or export of PgtE, indicating that another PhoP/PhoQ-dependent mechanism is required for PgtE outer membrane localization. PgtE is a posttranscriptionally regulated component of the PhoP/PhoQ regulon that contributes to Salmonella resistance to innate immunity.

Specific lipopolysaccharide found in cystic fibrosis airway Pseudomonas aeruginosa.

Cystic fibrosis (CF) patients develop chronic airway infections with Pseudomonas aeruginosa (PA). Pseudomonas aeruginosa synthesized lipopolysaccharide (LPS) with a variety of penta- and hexa-acylated lipid A structures under different environmental conditions. CF patient PA synthesized LPS with specific lipid A structures indicating unique recognition of the CF airway environment. CF-specific lipid A forms containing palmitate and aminoarabinose were associated with resistance to cationic antimicrobial peptides and increased inflammatory responses, indicating that they are likely to be involved in airway disease.

Sp1 affinity for GC-rich elements correlates with ornithine decarboxylase promoter activity.

The highly conserved ornithine decarboxylase (E.C.4.1.1.17) promoter contains multiple binding sites for Sp1 within the first 400 bp of the cap site. Therefore the ability of individual Sp1 elements to confer transactivation alone or in combination was tested. We show that different Sp1 sites vary in their affinity for Sp1 and that increasing affinity correlates with enhancer activity. In addition, several adjacent Sp1 sites synergistically enhanced promoter activity. Thus, the strength of promoter transactivation correlated with both the number of GC-rich elements and their affinity for Sp1 protein.