Is there a core set of organisms that structure macroinvertebrate assemblages in freshwater wetlands?

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Peripheral profiling analysis for bipolar disorder reveals markers associated with reduced cell survival

Little is known about the molecular factors that are altered in remitting bipolar disorder (BD) patients. We carried out proteome profiling of peripheral blood mononuclear cells (PBMCs) and serum from BD patients who were not experiencing mania or major depression (euthymia) compared to matched healthy controls using liquid chromatography–mass spectrometry (LC‐MS^E) and Multi‐Analyte Profiling (Human Map^®) platforms. This resulted in the identification of approximately 60 differentially expressed molecules involved predominantly in cell death/survival pathways. In PBMCs, this was manifested in cytoskeletal and stress response‐associated proteins, whereas most serum analytes were associated with the inflammatory response. The predicted effect of serum analytes on physiological systems was tested by treating PBMCs with serum obtained from the same patients, resulting in reduced cellular survival. These preliminary results suggest that BD patients carry a peripheral fingerprint that has detrimental effects on cell function and that could be used to distinguish BD patients from healthy controls despite being in a remission phase. It is hoped that additional studies of BD patients in the manic and depressed stages could lead to the identification of a molecular fingerprint that could be used for predicting episodic switching and for guiding treatment strategies.     

Interaction of the antiestrogen [3H]H1285 with the two forms of the molybdate-stabilized calf uterine estrogen receptor

The high affinity antiestrogen [3H]H1285 bound to the cytosol calf uterine estrogen receptor dissociated very slowly (t 1/2 approx 30 h at 20 degrees C) and did not demonstrate a change in dissociation rate in the presence of molybdate, which is characteristic of [3H]estradiol-receptor complexes. [3H]H1285-Receptor complexes sediment at approx 6S on 5-20% sucrose density gradients containing 0.3M KCl with or without 10 mM molybdate. This is in contrast to [3H]estradiol-receptor complexes which sedimented at approx 4.5S without molybdate and at approx 6S with molybdate. These results suggest a physicochemical difference in the estrogen receptor when occupied by antiestrogens versus estrogens. We recently reported that the cytoplasmic uterine estrogen receptor, when bound by estradiol and prepared in 10 mM molybdate, eluted from DEAE-Sephadex columns as Peak I (0.21 M KCl) & Peak II (0.25 M KCl). However, [3H]H1285 bound to the estrogen receptor eluted only as one peak at 0.21 M KCl, also suggesting that the initial interaction of antiestrogens with the estrogen receptor is different. We have extended these studies and report that H1285 can compete with [3H]estradiol for binding to both forms of the estrogen receptor and [3H]H1285 can bind to both forms if the unoccupied receptor is first separated by DEAE-Sephadex chromatography. However, if the receptor is first bound by unlabeled H1285, eluted from the column and post-labeled by exchange with [3H]estradiol, only one peak is measured. Thus, it appears that H1285 binding alters the properties of the receptor such that all receptor components seem to elute as one form. These partially purified [3H]H1285-receptor complexes obtained from DEAE-Sephadex columns sedimented as 5.5S in sucrose density gradients in contrast to the sedimentation values for the [3H]estradiol-receptor components eluting as Peak I (4.5S) and Peak II (6.3S). These differences in the physicochemical characteristics of the estrogen receptor when bound by estrogen versus antiestrogens may be related to some of the biological response differences induced by these ligands.     

2,3,7,8 Tetrachlorodibenzo-p-dioxin induction of cytochrome P4501A in cultured rat and human hepatocytes

We report here a novel observation that 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) induced predominantly cytochrome P4501A1 (CYP1A1) in rat hepatocytes and predominantly CYP1A2 in human hepatocytes. As part of our research program to evaluate species-differences in response to CYP inducers, we studied the effects of TCDD on CYP1A activity, protein, and gene expression in primary cultures of rat and human hepatocytes. TCDD was found to induce CYP1A activity, measured as ethoxyresorufin-O-deethylase (EROD) activity, in both rat and human hepatocytes. TCDD induction of EROD activity in human hepatocytes (2-5 fold of concurrent solvent control), was significantly lower than that found in rat hepatocytes ( 20-fold of concurrent solvent control). Two structural analogs of TCDD, 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 6-nitro-1,3,8-trichlorodibenzofuran (6-NCDF), were also evaluated. As observed for TCDD, human hepatocytes consistently showed a lower response than rat hepatocytes. As most TCDD-related effects are believed to be mediated via binding of the TCDD-Ah receptor (AhR) complex to DNA, nuclear AhR levels were measured in rat and human hepatocytes after TCDD treatment. We found that the nuclear AhR levels in TCDD-treated rat hepatocytes were approximately 4 times higher than found in TCDD-treated human hepatocytes. However, the estimated binding affinity of [3H]TCDD to nuclear AhR from rat hepatocytes was similar. The species difference in response to TCDD was further evaluated by analysis of CYP1A1 and CYP1A2 mRNA levels using Northern analysis, and P4501A1 and 1A2 protein levels using Western immunoblotting. Results showed that, at both gene expression and protein levels, TCDD induced predominantly CYP1A1 in rat hepatocytes and CYP1A2 in human hepatocytes.     

Proteomic analysis of human osteoarthritis synovial fluid

Background

Osteoarthritis is a chronic musculoskeletal disorder characterized mainly by progressive degradation of the hyaline cartilage. Patients with osteoarthritis often postpone seeking medical help, which results in the diagnosis being made at an advanced stage of cartilage destruction. Sustained efforts are needed to identify specific markers that might help in early diagnosis, monitoring disease progression and in improving therapeutic outcomes. We employed a multipronged proteomic approach, which included multiple fractionation strategies followed by high resolution mass spectrometry analysis to explore the proteome of synovial fluid obtained from osteoarthritis patients. In addition to the total proteome, we also enriched glycoproteins from synovial fluid using lectin affinity chromatography.

Results

We identified 677 proteins from synovial fluid of patients with osteoarthritis of which 545 proteins have not been previously reported. These novel proteins included ADAM-like decysin 1 (ADAMDEC1), alanyl (membrane) aminopeptidase (ANPEP), CD84, fibulin 1 (FBLN1), matrix remodelling associated 5 (MXRA5), secreted phosphoprotein 2 (SPP2) and spondin 2 (SPON2). We identified 300 proteins using lectin affinity chromatography, including the glycoproteins afamin (AFM), attractin (ATRN), fibrillin 1 (FBN1), transferrin (TF), tissue inhibitor of metalloproteinase 1 (TIMP1) and vasorin (VSN). Gene ontology analysis confirmed that a majority of the identified proteins were extracellular and are mostly involved in cell communication and signaling. We also confirmed the expression of ANPEP, dickkopf WNT signaling pathway inhibitor 3 (DKK3) and osteoglycin (OGN) by multiple reaction monitoring (MRM) analysis of osteoarthritis synovial fluid samples.

Conclusions

We present an in-depth analysis of the synovial fluid proteome from patients with osteoarthritis. We believe that the catalog of proteins generated in this study will further enhance our knowledge regarding the pathophysiology of osteoarthritis and should assist in identifying better biomarkers for early diagnosis.     

MALDI imaging MS reveals candidate lipid markers of polycystic kidney disease

Autosomal recessive polycystic kidney disease (ARPKD) is a severe, monogenetically inherited kidney and liver disease. PCK rats carrying the orthologous mutant gene serve as a model of human disease, and alterations in lipid profiles in PCK rats suggest that defined subsets of lipids may be useful as molecular disease markers. Whereas MALDI protein imaging mass spectrometry (IMS) has become a promising tool for disease classification, widely applicable workflows that link MALDI lipid imaging and identification as well as structural characterization of candidate disease-classifying marker lipids are lacking. Here, we combine selective MALDI imaging of sulfated kidney lipids and Fisher discriminant analysis (FDA) of imaging data sets for identification of candidate markers of progressive disease in PCK rats. Our study highlights strong increases in lower mass lipids as main classifiers of cystic disease. Structure determination by high-resolution mass spectrometry identifies these altered lipids as taurine-conjugated bile acids. These sulfated lipids are selectively elevated in the PCK rat model but not in models of related hepatorenal fibrocystic diseases, suggesting that they be molecular markers of the disease and that a combination of MALDI imaging with high-resolution MS methods and Fisher discriminant data analysis may be applicable for lipid marker discovery.     

Conformational Flexibility Determines Selectivity and Antibacterial, Antiplasmodial, and Anticancer Potency of Cationic ��-Helical Peptides

We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.     

Quantitative analysis of sugar constituents of glycoproteins by capillary electrophoresis

A method for quantitative analysis of monosaccharides including N- acetylneuraminic acid derived from sialic acid-containing oligosaccharides and glycoproteins is presented. The analysis is based on the combination of chemical and enzymatic methods coupled with capillary electrophoretic (CE) separation and laser-induced fluorescence (LIF) detection. The present method utilizes a simplified acid hydrolysis procedure consisting of mild hydrolysis (0.1 M TFA) to release sialic acid and strong acid hydrolysis (2.0 N TFA) to produce amino and neutral sugars. Amino sugars released from strong acid hydrolysis of oligosaccharides and glycoproteins were reacetylated and derivatized with 8-aminopyrene-1,3,6-trisulfonate (APTS) along with neutral sugars in the presence of sodium cyanoborohydride to yield quantitatively the highly stable fluorescent APTS adducts. N- acetylneuraminic acid (Neu5Ac), a major component of most mammalian glycoproteins, was converted in a fast specific reaction by the action of neuraminic acid aldolase (N-acylneuraminate pyruvate-lyase EC 4.1.3.3) to N-acetylmannosamine (ManNAc) and pyruvate. ManNAc was then derivatized with APTS in the same manner as the other monosaccharides. This method was demonstrated for the quantitation of pure Neu5Ac and the species derived from mild acid hydrolysis of 6'-sialyl-N- acetyllactosamine and bovine fetuin glycan. Quantitative recovery of the N-acetylmannosamine was obtained from a known amount of Neu5Ac in a mixture of seven other monosaccharides or from the sialylated oligosaccharides occurring in glycoproteins. The sequence of procedures consists of acid hydrolysis, enzymatic conversion and APTS derivatization which produced quantitative recovery of APTS- monosaccharide adducts. The detection limits for sugars derivatized with APTS and detected by CE-LIF are 100 pmol for Neu5Ac and 50 pmol for the other sugars.      

Examining the fish microbiome: vertebrate-derived bacteria as an environmental niche for the discovery of unique marine natural products

Historically, marine invertebrates have been a prolific source of unique natural products, with a diverse array of biological activities. Recent studies of invertebrate-associated microbial communities are revealing microorganisms as the true producers of many of these compounds. Inspired by the human microbiome project, which has highlighted the human intestine as a unique microenvironment in terms of microbial diversity, we elected to examine the bacterial communities of fish intestines (which we have termed the fish microbiome) as a new source of microbial and biosynthetic diversity for natural products discovery. To test the hypothesis that the fish microbiome contains microorganisms with unique capacity for biosynthesizing natural products, we examined six species of fish through a combination of dissection and culture-dependent evaluation of intestinal microbial communities. Using isolation media designed to enrich for marine Actinobacteria, we have found three main clades that show taxonomic divergence from known strains, several of which are previously uncultured. Extracts from these strains exhibit a wide range of activities against both gram-positive and gram-negative human pathogens, as well as several fish pathogens. Exploration of one of these extracts has identified the novel bioactive lipid sebastenoic acid as an anti-microbial agent, with activity against Staphylococcus aureus, Bacillus subtilis, Enterococcus faecium, and Vibrio mimicus.