Small Molecular Weight Proteins Research Articles

Helicobacter pylori Outer Membrane Protein 18 (Hp1125) Induces Dendritic Cell Maturation and Function

Dendritic cells (DCs) are potent antigen-presenting cells that initiate T-cell responses. A robust adaptive Th1 immune response is crucial to an adaptive (Th2) immune response necessary for vaccine-induced protective immunity against Helicobacter pylori. It has been shown that several outer membrane proteins (Omps) induce a robust antibody response. However, it is also known that the antibodies generated are not protective. Moreover there is great variation in the recognition of high molecular weight H. pylori proteins by sera from infected patients. In contrast to the high molecular weight proteins, serologic responses to small molecular weight proteins provide assessment of current infection with H. pylori and also of its eradication. The goal of the study was to analyze the activation of the immune response by a specific low molecular weight Omp that is universally expressed by all H. pylori strains. Therefore, we studied interaction of H. pylori Omp18 with DCs. Activation of murine bone marrow-derived DCs and production of cytokines by Omp18 was assessed by fluorescence-activated cell sorter (FACS) for costimulatory markers and ELISA, respectively. The ability of Omp18 stimulated DCs to induce lymphocyte proliferation was measured in a mixed leukocyte reaction. Omp18 induced higher expression of the B7 (CD80 and CD86) costimulatory molecule after 18 hours indicating processing and presentation of the antigen on the surface by bone marrow-derived DCs. The maturing DCs also secreted significant levels of IL-12, but was 4-fold less than that stimulated by whole bacteria. Omp18-primed DCs induced proliferation and release of IFNgamma by syngeneic splenocytes. We concluded that Omp18 is capable of activating DCs initiating a Th1 immune response.

Biochemical and cellular mechanisms of mammalian CDK inhibitors: a few unresolved issues

p21 and p16, first identified as two small molecular weight proteins in CDK and cyclin immunocomplexes, represent two distinct families constituting a total of seven CDK inhibitors in mammalian cells. The physiological functions of these genes are believed to be broadly involved in connecting various cellular pathways to cell cycle control. Extensive studies over the past 10 years have led to a fairly clear understanding of their biochemical and cellular mechanisms and have also left some unresolved and controversial issues.

Disintegrins

The existence of disintegrins, non-enzymatic, small molecular weight proteins from viper venom, has been known for 2 decades, and their impact on cellular research has been substantial and far-reaching. Disintegrins have been the molecular scaffold used in the design of therapeutics for the prevention of thrombosis and cancer. Their sequencing has provided insights into the evolution of proteins over millennia. Production of recombinant disintegrin mutants and fusion proteins has allowed investigations into molecular mechanisms at work in cell-extracellular matrix interactions. Structural homologies with non-snake proteins have shown disintegrin-like molecules in species ranging from slime mold to humans. Intracellular signaling events have been elucidated through the use of venom disintegrins, including events related to programmed cell death, motility, cell proliferation and viral pathogenesis. Disintegrin sequences (protein or genes) have been placed in microbubbles and liposomes and been found to target neovascular endothelium and metastatic tumors in two mouse models. The purpose of this review is to highlight the members of this disintegrin family discovered since 1998 as well as the increased understanding of their usefulness in therapeutics and technical assays.

CDNA Microarray Analysis of Genes Up-Regulated by Treatment with 5-AZA-2′-Deoxycytidine in Combination with Trichostatin A Identifies Aberrant Metallothionein 1H Promoter Methylation at a High Frequency Iin Human AML.

Gene promoter methylation is a major mechanism of transcriptional silencing in cancer cells that is mediated by CpG methylation and histone deacetylase activity, with methylation being dominant. Importantly, pharmacological treatment of cancer cells with inhibitors of DNA methylation and histone deacetylation have been shown to synergistically reactivate transcription of previously silenced genes. In an effort to identify novel genes silenced by DNA methylation in human acute myeloid leukemia (AML), we used cDNA microarray technology to screen for genes upregulated in the human cell line AML193 following treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, Trichostatin A (TSA). Analysis of 1,700 gene microarrays in triplicate revealed seven genes consistently upregulated by combined 5-Aza-dC and TSA treatment, all of which were confirmed by semi-quantitative RT-PCR. Interestingly, four of these genes (MT1H, MT1E, MT2A, MT1G) are members of the metallothionein family of cysteine-rich small molecular weight proteins. These proteins are considered to be important mediators of cellular detoxification and are induced by toxic heavy metals, UV irradiation and reactive oxygen species. As well, MT1G promoter methylation has been implicated in the pathogenesis of sporadic papillary thyroid carcinoma. The methylation status of candidate metallothionein genes reactivated by 5-Aza-dC and TSA treatment in AML193 cells was assessed by methylation specific PCR (MSP) in seven human AML cell lines. Methylated promoter alleles were detected in MT1H, MT1E and MT1G in 71%, 29% and 29% of cell lines, respectively, and there was no methylation of MT2A in any of the cell lines tested. To investigate for the occurrence of MT1H, MT1E and MT1G methylation in human AML in vivo, we analyzed AML patient blasts (n=39) and non-leukemic controls (n=13) by MSP. MT1H, MT1E and MT1G methylation was detected in 51%, 0% and 3% of AML patient samples, respectively, and there was no methylation of any of these genes in control samples. Our findings implicate MT1H promoter methylation in the pathogenesis of human AML and suggest that the use of cDNA microarray technology following pharmacological manipulation is a useful approach for identifying novel epigenetically silenced genes in this disease.

NTAL phosphorylation is a common event in the signaling pathways initiated by antigen and stem cell factor (SCF) in mast cells

Background SCF potentiates antigen mediated secretory responses in mast cells. We therefore explored whether the signaling pathways regulated by their respective receptors Kit and FcϵRI may share a common early event to account for these synergistic responses. Methods Human mast cells (HuMCs) were obtained by culturing CD34 + peripheral blood cells in media containing IL-3 (one week only), SCF and IL-6 for 6-8 weeks. Mouse bone marrow (BM) MCs were grown in media containing IL-3. Results As with antigen stimulation, SCF induced the rapid tyrosine phosphorylation of a small MW protein of 25-30 kD. Immunodepletion and immunoprecipitation studies identified this protein as Non T Cell Activation Linker (NTAL), also termed LAB (Linker for Activation of B cells), a small MW transmembrane adaptor molecule similar to LAT. Unlike the antigen response, SCF induced tyrosine phosphorylation of NTAL in the absence of detectable LAT. We explored the signaling pathways regulating NTAL phosphorylation in BMMCs derived from wild type (WT), lyn −/− , btk −/− mice, and lyn/btk double knock out mice. FcϵRI-mediated NTAL phosphorylation, was markedly ablated in the lyn −/− , and lyn −/−btk −/− and to a lesser extent in the btk −/− BMMCs, however Kit-mediated NTAL phosphorylation appeared little changed in these cells compared to the WT response. Conclusions Taken together, these data suggest that NTAL phosphorylation may be a common early signal for both antigen and SCF mediated degranulation and/or cytokine release in mast cells however, whereas the antigen induced response may be mediated by both Lyn- and Btk-dependent pathways, Kit may directly phosphorylate NTAL independently of both Lyn and Btk.

Control of nutrient-sensitive transcription programs by the unconventional prefoldin URI.

Prefoldins (PFDs) are members of a recently identified, small-molecular weight protein family able to assemble into molecular chaperone complexes. Here we describe an unusually large member of this family, termed URI, that forms complexes with other small-molecular weight PFDs and with RPB5, a shared subunit of all three RNA polymerases. Functional analysis of the yeast and human orthologs of URI revealed that both are targets of nutrient signaling and participate in gene expression controlled by the TOR kinase. Thus, URI is a component of a signaling pathway that coordinates nutrient availability with gene expression.

Rapid tyrosine phosphorylation of a major small MW protein following FcεRI aggregation in human mast cells (HuMCs): Identification as a novel transmembrane adaptor protein similar to LAT

Rapid tyrosine phosphorylation of a major small MW protein following FcεRI aggregation in human mast cells (HuMCs): Identification as a novel transmembrane adaptor protein similar to LAT

Abstracts from Other Presentations at the International Symposium on Toxins and Natural Products in Honor of Professor Anthony T. Tu

Abstracts from Other Presentations at the International Symposium on Toxins and Natural Products in Honor of Professor Anthony T. Tu

Manganese Distribution Across the Blood–Brain Barrier: I. Evidence for Carrier-Mediated Influx of Manganese Citrate as Well as Manganese and Manganese Transferrin

Manganese (Mn) is an essential element and a neurotoxicant. Regulation of Mn movement across the blood–brain barrier (BBB) contributes to whether the brain Mn concentration is functional or toxic. In plasma, Mn associates with water, small molecular weight ligands and proteins. Mn speciation may influence the kinetics of its movement through the BBB. In the present work, the brain influx rates of 54Mn 2+, 54Mn citrate and 54Mn transferrin ( 54Mn Tf) were determined using the in situ brain perfusion technique. The influx rates were compared to their predicted diffusion rates, which were determined from their octanol/aqueous partitioning coefficients and molecular weights. The in situ brain perfusion fluid contained 54Mn 2+, 54Mn citrate or 54Mn Tf and a vascular volume/extracellular space marker, 14C -sucrose, which did not appreciably cross the BBB during these short experiments (15–180 s). The influx transfer coefficient ( K in) was determined from four perfusion durations for each Mn species in nine brain regions and the lateral ventricular choroid plexus. The brain K in was (5–13)×10 −5, (3–51)×10 −5, and (2–13)×10 −5 ml/s/g for 54Mn 2+, 54Mn citrate, and 54Mn Tf, respectively. Brain K in values for any one of the three Mn species generally did not significantly differ among the nine brain regions and the choroid plexus. However, the brain K in for Mn citrate was greater than Mn 2+ and Mn Tf K in values in a number of brain regions. When compared to calculated diffusion rates, brain K in values suggest carrier-mediated brain influx of 54Mn 2+, 54Mn citrate and 54Mn Tf. 55Mn citrate inhibited 54Mn citrate uptake, and 55Mn 2+ inhibited 54Mn 2+ uptake, supporting the conclusion of carrier-mediated brain Mn influx. The greater K in values for Mn citrate than Mn 2+ and its presence as a major non-protein-bound Mn species in blood plasma suggest Mn citrate may be a major Mn species entering the brain.

Chemokine Expression in Myocardial Ischemia: MIP-2 Dependent MCP-1 Expression Protects Cardiomyocytes from Cell Death

Chemokines are small molecular weight proteins that play important roles in inflammation. Originally described as chemotactic cytokines, chemokines stimulate the influx of leukocytes into specific tissue compartments. These molecules also modulate gene expression in both infiltrating and resident cells to mediate a vast array of cellular functions, and their importance in disease processes has been well documented. This study examined the expression of chemokines during myocardial ischemia and established a pathway by which two, MIP-2 and JE/MCP-1, modulate cardiac myocyte viability during this process. To focus on the direct effects of chemokines on these cells, a mouse model of ischemia without reperfusion was used. The expression of chemokines and chemokine receptors was induced in the left ventricular free wall as early as 1 h post-ischemia, with the most significant increases in MIP-2 (CXCL2) and JE/MCP-1 (CCL2). Expression of their respective receptors, CXCR2 and CCR2, was also induced. Similar changes in gene expression occurred at the mRNA and protein levels in isolated neonatal mouse cardiac myocytes stimulated by hypoxia. Antibody to MIP-2 inhibited hypoxia-induced JE/MCP-1 expression, demonstrating that MIP-2 is critical for this event. Moreover, in vivo intramyocardial injection of either an adenovirus expressing MIP-2 or the recombinant protein itself was sufficient to upregulate JE/MCP-1 production even in the absence of ischemia. Thus, MIP-2 regulates JE/MCP-1 expression both in cell culture and in vivo. Furthermore, JE/MCP-1 markedly decreased hypoxia-induced cell death in cultured cardiac myocytes. Thus, JE/MCP-1 appears to mediate an unanticipated survival pathway in target cardiac myocytes themselves. These findings indicate an important role for MIP-2 and JE/MCP-1 in regulating the response of cardiac myocytes to myocardial ischemia.

Sensitivity and specificity of troponin t in diagnosis of acute myocardial infarction

Lately, in the diagnosis of acute myocardial infarction (MI), special attention is paid to the study of small molecular weight proteins, representing the strucrutal components of cardial muscle controctile proteins, such as cardial troponin T (cTnT). Therefore, the aim of this study was to determine and compare the specificity and sensitivity of cTnT myoglobine and the activity of standard tested enzymes such as: creatine kinase (CK), CK MB isoenzymes and lactic delytrogenase (LDH) in early diagnosis of acute MI. The study concerned the four groups of patients. The first group consisted of healty blood donors (n = 105), the second of patients with verified acute MI (n = 30), the third of a patients with polytrauma (n = 30), and the fourth of patients with cardiovascular diseases with no proved MI (n = 30). For the determination of cTnT level, the commercial Eleccys Troponin T STAT test was used (third generation), Boehringer Mannheim company, based on ECLIA (tehnique of electrochemical luminiscent analysis), designated for the work with automated analysis Elecsys 2010. The concentration of myoglobine (Mi) was determined by immunoturbiometric method, while the activity of other enzymes was determined by the standard IFCC methods with biochemical analyser HITACHI 911, Boehringer Manheim, and with original test reagents. The reference values of blood donor individuals found in the blood ranged from 0.01 to 0.028 ng/mL. In the group of patients with acute MI, biochemical markers (cTnT, CK, CK MB, Mi and LDH) were determined as soon as the material was received by the 24 h service and 4, 8 16, 24, 48, 72, 96, 120, 144, 168, 192 and 216 hours later. In patients with polytrauma the markers were tested 8 hours after surgery and in patients with other cardiovascular deseases with non documented acute MI immediattely after admission, and 4, 8, 16 and 24 hours later. The level of cTnT in patients with proved acute MI was increased at the first testing after admission. The highest values were found at hour 16, gradually declining thereafter, but maintaining above the higher control level in the next 216 hours after admission. In patients with cardiovascular diseases and non documented acute MI the level of cTnT was unchanged. Comparison of results of these two groups of patients showed statistically significant differences in cTnT levels in patients with acute MI during all tested time intervals. In the group of patients with polytrauma only the value of cTnT was within the normal level. Diagnostic precision of tested cardiac markers was also tested by ROC analysis. According to the data obtained, only cTnT exhibited statistically significiant diagnostic precision immediately after the admission of patients with the following calculated ROC AUC: 0.855 for cTnT, 0.716 for CK, 0.503 for CK MB and 0.552 for LDH, respectively. According to the presented data, it can be concluded that cTnT was the most specific and the most sensitive marker in the diagnosis of acute MI.

Increase in milk metalloproteinase activity and vascular permeability in bovine endotoxin-induced and naturally occurring Escherichia coli mastitis

An endotoxin-induced mastitis model was used to study permeability changes associated with increased milk matrix metalloproteinase (MMP) activity in early inflammation. One quarter of two cows was inoculated with endotoxin ( Escherichia coli 055:B5). Blood, milk, and whey were collected before and repeatedly after inoculation for 48 h. The profile and amounts of gelatinolytic MMPs were determined by zymography; gelatinase A (72 kD MMP-2) and gelatinase B (92 kD MMP-9) were identified by Western immunoblotting. Bovine serum albumin (BSA) and trypsin inhibitor capacity (TIC) were used as markers of capillary permeability with parallel examination of neutrophil penetration from blood to milk. Five clinical E. coli mastitis milk samples and five milk samples from cows with healthy udders were analyzed to detect whether increased levels of gelatinolytic MMP-2 and MMP-9 have a role in naturally occurring mastitis with endotoxin involvement. Milk MMP levels increased 2 h after the endotoxin challenge. Both MMP-2 and MMP-9 were involved in this early proteolytic event. These increased MMP levels are associated with increased capillary permeability, evidenced first by the penetration of small molecular weight proteins, such as BSA and TIC. Later, 6–12 h post endotoxin inoculation, neutrophilic leucocytes also entered the site, as they require larger tissue damage in basal membrane and interstitial tissue structures than BSA and TIC to extravasate. In naturally occurring disease, increased MMP-2 and MMP-9 levels were detected in milk. Thus, gelatinases, especially MMP-2, are involved in the early degradation of the blood–milk barrier, which precedes the penetration of blood-derived cellular components into milk in endotoxin-induced mastitis. In the future, measuring MMP in milk/whey might be a useful tool for diagnosing early mastitis.

Crosstalk between ARF6 and protein kinase Cα in FcγRI-mediated activation of phospholipase D1

Fc receptors play a pivotal role linking the cellular and humoral arms of the immune system [1–3]. Our previous studies have shown that the human high-affinity immunoglobulin G receptor FcγRI couples to a novel intracellular signaling pathway requiring phospholipase D activation [4]. The mechanisms that regulate receptor coupling to phospholipase D in intact cells are poorly understood but involve small molecular weight GTPases and protein kinase C [5–7]. Here, we show that immune complex aggregation of FcγRI stimulates the association of phospholipase D1 with ARF6 and protein kinase Cα. Surprisingly, PKCα activity per se is not required. Rather, all of the FcγRI-mediated increase in PKC activity requires phospholipase D1, as treatment of cells with butan-1-ol (0.3%) or specific downregulation of phospholipase D1 using antisense oligonucleotides inhibits FcγRI-coupled PKC activation. Moreover, treatment of cells with butan-1-ol or phospholipase D1 antisense oligonucleotides inhibits translocation of PKCδ, -ϵ, and -ζ but had no effect on the association of PKCα or ARF6 with phospholipase D1. These data indicate that association with ARF6 and PKCα plays a role in coupling FcγRI to phospholipase D1 activation and that PLD1 lies upstream of all FcγRI-mediated PKC activity.

Posttranslational modifications of microfibril associated glycoprotein-1 (MAGP-1).

Microfibril-associated glycoprotein-1 (MAGP-1) is a small molecular weight protein associated with extracellular matrix microfibrils. Biochemical studies have shown that MAGP-1 undergoes several posttranslational modifications that may influence its associations with other microfibrillar components. To identify the sites in the molecule where posttranslational modifications occur, we expressed MAGP-1 constructs containing various point mutations as well as front and back half truncations in CHO cells. Characterization of transiently expressed protein showed that MAGP-1 undergoes O-linked glycosylation and tyrosine sulfation at sites in its amino-terminal half. This region of the protein also served as a major amine acceptor site for transglutaminase and mediated self-assembly into high molecular weight multimers through a glutamine-rich sequence. Fine mapping of the modification sites through mutational analysis demonstrated that Gln20 is a major amine acceptor site for the transglutaminase reaction and confirmed that a canonical tyrosine sulfation consensus sequence is the site of MAGP-1 sulfation. Our results also show that O-glycosylation occurs at more than one site in the molecule.

Selective extraction of small proteins from biological samples using a novel restricted access column with cation exchange properties

The determination of proteins utilising a polymer-based restricted access suppor material with ion exchange properties (IERAM) is outlined. Solid phase extraction coupled on-line with a mincrobore reversed phase HPLC system for the quantitation of small marker proteins is demonstrated. The cation-exchange restricted access packings were characterised with respect to their adsorption and desorption kinetics. The IERAM material was also investigated by capacity, selectivity, and biocompatibility determinations when applied to the quantification of small molecular weight proteins such as cytochrome C, Lysozyme, Ribonuclease A, Myoglobin, Insulin, human serum albumin, and a Tryptic inhibitor.

Activation of cytokines as a mechanism of disease progression in heart failure

The scientific quest for the basic mechanism(s) responsible for the development and progression of congestive heart failure in humans has been practically exhaustive; none the less, the mechanisms responsible for...

Chemokine and chemokine receptor expression by liver-derived dendritic cells: MIP-1α production is induced by bacterial lipopolysaccharide and interaction with allogeneic T cells

Dendritic cells (DC) are highly-specialized antigen-presenting cells (APC), that initiate and modulate immune responses. Their specialized migratory and tissue-homing properties are regulated by small molecular weight proteins (chemokines) that govern leukocyte migration and activation. Little is known about the capacity of liver DC to produce or respond to chemokines. Here we examined chemokine and chemokine receptor (CR) gene expression in both immature DC progenitors (DCp) and comparatively mature DC generated from mouse liver. Factors affecting production of the chemokine macrophage inflammatory protein (MIP)-1α, and the influence of MIP-1α on liver DC migration were also investigated. Dendritic cells were propagated in response to granulocyte–macrophage colony stimulating factor (GM-CSF)±interleukin (IL)-4 from bone marrow (BM) cells or liver non-parenchymal cells (NPC) isolated from normal mice, or from mice treated with the hematopoietic growth factor Flt3 ligand (FL). Their phenotype and allostimulatory function were assessed by monoclonal antibody (mAb) staining and flow cytometry, and by the capacity to induce mixed leukocyte reactions, respectively. Specific chemokine and CR gene expression was studied using the RNase protection assay (RPA). Production of MIP-1α was determined by enzyme-linked immunoabsorbent assay (ELISA), and the migratory activity of liver DC induced by MIP-1α quantitated using microchemotaxis chambers. Like DC generated simultaneously from BM, liver-derived DC expressed mRNA for a variety of CC and CXC chemokines. RANTES (regulated upon activation, normal T cell expressed and secreted) transcripts were the most strongly expressed. Gene transcripts for the receptor CCR1, that binds RANTES and MIP-1α were also readily detected, as was CCR2, the receptor for the monocyte chemotactic proteins (MCP)1–4. No major differences in chemokine or CR mRNA expression were detected between immature and more mature liver DC. MIP-1α production by liver-derived DC was stimulated by bacterial lipopolysaccharide (LPS), and high levels were also detected in co-cultures of hepatic DC and allogeneic T cells. Chemotactic migration of liver-derived DC was stimulated by MIP-1α. Thus, liver-derived DC express mRNA for several CC and CXC chemokines and their receptors that may play key roles in the regulation of hepatic inflammatory responses. Production of MIP-1α by liver DC, and their migratory responses to this chemokine, suggest that MIP-1α and other chemokines may play significant roles in the regulation of liver DC function and in interactions of liver DC with other leukocytes, under normal and inflammatory conditions.

Initiating Ocular Proteomics for Cataloging Bovine Retinal Proteins: Microanalytical Techniques Permit the Identification of Proteins Derived from a Novel Photoreceptor Preparation

Though some mechanisms of photoreception have been well characterized, others remain obscure. Presumably, most, if not all, of the major players in photoreceptor-specific functions are present in large amounts in the photoreceptor layer, and a catalog of these proteins will prove a useful tool for vision researchers. As a first step toward a complete catalog of photoreceptor cells, we have developed a novel method for isolating the photoreceptor cell monolayer from bovine retina. Electron microscopic studies of both the photoreceptor layer and the residual retina from which the photoreceptor layer had been removed, indicate that the preparation contains the photoreceptor outer segments and the majority of the inner segments. Proteins were extracted from the isolated photoreceptor cell layer as well as the rest of the retina with isoelectric focusing lysis buffer, and the protein components were separated by two-dimensional gel electrophoresis. The obtained protein maps reveal several classes of proteins that appear to be expressed more abundantly or specifically in the photoreceptor layer than in the rest of the retina. Four of these protein spots were excised and in-gel digested with trypsin, and the digests were extracted with solvent. The mixture of peptides digested from each protein was analyzed by high performance liquid chromatography interfaced with electrospray ionization tandem quadrupole mass spectrometry or by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Some of the peptides were isolated and their sequences were determined by gas phase Edman degradation. RNA transcripts extracted from the photoreceptor layer or the whole retina were subjected to Northern blot analysis as well as to reverse transcriptase-polymerase chain reaction amplification of probes for the successful selection of cDNA clones. These data permit both the identification of virtually any protein detectable on a two-dimensional gel, and also enable the corresponding cDNA clone to be selected. We have validated this approach by identifying aspartate aminotransferase and creatine kinase from the populations of abundant photoreceptor layer proteins. Both aspartate aminotransferase and creatine kinase are of mitochondrial origin and are thought to play crucial roles in photoreceptor functions by producing glutamate and ATP, respectively. We also identified two photoreceptor layer specific proteins: an acidic and high molecular weight protein, interphotoreceptor retinoid-binding protein, and an acidic and small molecular weight protein, recoverin.The technique presented here will allow vision researchers to discover and identify the proteins that are expressed specifically or abundantly in the photoreceptor cell as well as the proteins that undergo post-translational modification or modulation in expression under a defined biological condition. With the use of this technology, we anticipate that a researcher who knows only the 2-D gel position of a protein of interest can identify the protein, isolate a cDNA clone, and move into molecular genetic studies. Moreover, this streamlined technology will enable one to assemble a catalog of photoreceptor proteins using a minute amount of materials in a short period of time. We believe that such a catalog will serve as a valuable resource for vision investigators and will accelerate the rate of research progress.

Effect of chronic exposure to ammonia on alterations of proteins and immunoglobulins in sea bass ( Dicentrarchus labrax) serum

The relationship between serum protein, immunoglobulin concentrations and protein molecular weight profile (PMWP) alterations of sea bass (131.3 ± 4.3 g) reared in sea water with sublethal concentrations of ammonia was studied over two periods totalling 116 days. During the exposure period (62 days) the first group (group 1) lived in sea water with 0.204 mg.L -1unionized ammonia nitrogen equivalent to 12 % of the lethal concentration for 50 % of a population exposed for 96 h to ammonia (96-h LC 50), whilst the second group (group 2) lived in sea water with 0.340 mg.L -1UIA-N equivalent to 20 % of the 96-h LC 50 of ammonia. Then, the two groups were left for a recovery period (54 days) in the same water as the control group (group 0). The determination of the total immunoglobulin (Ig) concentration was carried out by enzyme-linked immunosorbant assay (ELISA). Gel filtration columns were used for the serum PMWP. Serum Ig concentration of the exposed fish (group 2: 1.76 ± 0.43 ; group 1: 1.19 ± 0.33 mg.mL -1) was lower than the control group (3.39 ± 1.01 mg.mL -1) after 21 days of exposure period but this difference was reduced at the end of this period (day 62) and treated group Ig concentrations switched to higher than the control group after the recovery period (day 116, group 2: 9.75 ± 1.84 mg.mL -1; group 1: 7.50 ± 1.22 mg.mL -1group 0: 6.38 ± 1.13 mg.mL -1). In fact, at the end of the experiment, the cumulative Ig production difference between fish exposed to ammonia and the control was less than 10 %. Although a similar evolution of the Ig serum occurred with protein concentration, the serum protein concentration deficit of group 1 was restored at the end of the exposure period (group 2: 46.49 ± 2.34 mg.mL -1; group 0: 46.74 ± 1.97 mg.mL -1) and the cumulative production during the experiment was not significantly different between group 1 and group 0. However, this remained lower for group 2. During the exposure period, the PMWP of treated fish moved towards smaller molecular weight proteins. This alteration of the PMWP showed that the 0.2-kDa fraction increased, and another fraction appeared at the end of the exposure period. However, at the end of the recovery period, no difference was found between the PMWPs of control and treated fish. The alterations of the serum protein and Ig of fish reared, for a limited period (2 months), in water with sublethal concentrations of ammonia (20 % 96-h LC 50 or less) should disappear completely after a few months in normal rearing conditions.

Recent advances in statins and the kidney

Experimental and clinical studies have suggested a correlation between the progression of renal disease and dyslipidemia. Indeed, apolipoprotein B-containing lipoproteins have been demonstrated to be an independent risk factor for the progression of renal disease in humans. Interventional strategies in experimental models of renal disease have clearly demonstrated a beneficial effect on renal structure and function in a variety of models of renal disease. Investigations into the mechanisms whereby reduction of lipids by lipid-lowering agents benefits renal disease have suggested that the 3-hydroxy-3-methylglutaryl coenzyme reductase inhibitors, the so-called statin class of lipid-lowering agents, may have additional effects on the biology of inflammation that are germane to the progression of renal disease. Both in vivo and in vitro studies that investigated secondary mechanisms of statin effects are reviewed. In addition, new studies that investigated the effects on novel cellular mechanisms are presented. Lipid-lowering agents appear to have biologically important effects in modulating a variety of intracellular signaling systems involved in cell proliferation, inflammatory responses that involve macrophage adhesion, recruitment, and maturation. In addition, the effects on fibrogenesis have been recently defined. These latter effects may influence not only the development of glomerulosclerosis, but also interstitial fibrosis. These potentially major effects of lipid-lowering agents appear to be related to the effects on intracellular synthesis of nonsterol isoprenoids, which are involved in prenylation of critical small molecular weight proteins involved in cell signal transduction. In addition to the beneficial effects of the reduction in serum lipids, statins and other lipid-lowering agents may influence important intracellular pathways that are involved in the inflammatory and fibrogenic responses, which are common components of many forms of progressive renal injury.