Immunolocalization of tenascin and cellular fibronectins in diverse glomerulopathies

Frozen samples of minimal change glomerulopathy (MCG), and of membranous, segmental and diffuse lupus glomerulonephritis (MGN, SGN, DLGN) were studied to assess the distribution of tenascin (Ten), and the extradomains A and B (EDA-and EDB-) and oncofetal (Onc-) isoforms of cellular fibronectin (cFn). Cryosections were immunostained by the ABC method with specific monoclonal antibodies. In MCG, mesangial Ten and EDA-cFn reactions were increased. In MGN, mesangial Ten and EDA-cFn staining was enhanced except in segmental scars; convincing reactions were seen in cases with membranous transformation; spikes stained strongly. In SGN, variably intense staining for Ten and all cFn isoforms was seen in glomerular necrosis, proliferation and crescents; parietal epithelium EDA-cFn staining was noted. In DLGN, strong and extensive mesangial Ten and EDA-cFn staining was seen as were focal EDB-and Onc-cFn reactions. Parietal cells with and without crescents stained variably with all Mabs. Obsolete glomeruli were unreactive save for rare periglomerular Ten rims. Interstitial inflammation and fibrosis in MGN, SGN and DLGN had moderate to strong Ten and EDA-cFn staining with rare traces of EDB-and Onc-cFn. We conclude that enhanced Ten and EDA-cFn is a potentially reversible response to glomerular injury whereas the expression of EDB-and Onc-cFn apparently result from necrosis and/or cellular proliferation which lead to scarring. And, while mesangial cells are the major source of these molecules, epithelial cells might also partake in their synthesis.     

Low concentrations of primaquine inhibit degradation but not receptor-mediated endocytosis of asialoorosomucoid by HepG2 cells

Asialoorosomucoid (ASOR) is internalized and degraded by HepG2 cells after binding to the asialoglycoprotein (ASGP) receptor, internalization through the coated pit/coated vesicle pathway, and trafficking to lysosomes. Primaquine, an 8-aminoquinoline antimalarial compound, inhibits ASOR degradation at concentrations greater than 0.2 mM by neutralizing intracellular acid compartments. This leads to alterations in surface receptor number, receptor-ligand dissociation, and receptor recycling. We have investigated the effects of primaquine on 125I-ASOR uptake and degradation as a function of primaquine concentration and duration of exposure. Concentrations below those required for neutralization of acidic compartments block 125I-ASOR degradation in HepG2 cells and lead to intracellular ligand accumulation. This effect is maximal at 80 microM primaquine. The intracellular 125I-ASOR is undegraded, dissociated from the ASGP receptor, and contained within vesicular compartments distinct from lysosomes, plasma membrane, or endosomes. In addition, the effect of 80 microM primaquine on 125I-ASOR degradation is very slowly reversible (greater than 6 h), in contrast to primaquine's rapidly reversible effect on receptor recycling and ligand uptake (10 min). Furthermore, the effect is ligand-specific. 125I-asialofetuin, another ASGP receptor ligand, is internalized and degraded in lysosomes at normal rates in HepG2 cells exposed to 80 microM primaquine. These findings indicate that primaquine has multiple effects on the uptake and degradation of ligand occurring in the endosome-lysosome pathway. These effects of primaquine differ in their concentration-dependence, site of action, reversibility, and ligand selectivity.     

Phenotypic alteration in retroviral gene expression by leukemia-resistant thymocytes differentiating in leukemia-susceptible recipients

(AKR x NZB)F1 mice possess the dominant genes, Akv-1, Akv-2, Nzv-1a and Nzv-2a, which determine the expression of ecotropic and xenotropic viruses. Nevertheless, their thymic lymphocytes fail to produce these agents, and these mice are resistant to leukemia. We investigated the mechanism of this cell-specific restriction in radiation chimeras. (AKR x NZB)F1 thymocytes that had differentiated in lethally irradiated AKR recipients produced high levels of ecotropic and xenotropic viruses and showed marked amplification of MuLV antigen expression. Polytropic viruses could also be isolated from such thymocytes. These virological changes in chimeric thymocytes were donor- and host-specific and occurred only when (AKR x NZB)F1 bone marrow cells were inoculated into AKR recipients. This inductive capacity of the host environment could be detected in irradiated AKR recipients as early as age 2 months. The phenotypic changes brought about in leukemia-resistant (AKR x NZB)F1 thymocytes by the leukemia-susceptible AKR thymic microenvironment may be the result of a three-component inductive system.     

Genetic monitoring as a promising tool for conservation and management

In response to ever-increasing anthropogenic changes to natural ecosystems, regional, national and international organizations have established guidelines for monitoring biological diversity. Most monitoring programs, however, do not take full advantage of the potential afforded by molecular genetic markers, which can provide information relevant to both ecological and evolutionary time frames, while costing less and being more sensitive and reliable than traditional monitoring approaches. As several molecular and computational approaches are relatively new, many technical and theoretical issues remain to be resolved. Here, we illustrate how DNA and population genetic data can provide valuable information, often unattainable via other approaches, for monitoring species of management, conservation and ecological interest.     

TNF-alpha and IL-1 beta are not essential to the inflammatory response in LPS-induced airway disease

To determine the role of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta in the lower respiratory tract inflammatory response after inhalation of lipopolysaccharide (LPS), we conducted inhalation exposure studies in mice lacking expression of TNF-alpha and/or IL-1 receptor type 1 and in mice with functional blockade of these cytokines using adenoviral vector delivery of soluble receptors to one or both cytokines. Alterations in airway physiology were assessed by pulmonary function testing before and immediately after 4 h of LPS exposure, and the cellular inflammatory response was measured by whole lung lavage and assessment of inflammatory cytokine protein and mRNA expression. Airway resistance after LPS exposure was similarly increased in all groups of mice without evidence that blockade of either or both cytokines was protective from this response. Additionally, all groups of mice demonstrated significant increases in lung lavage fluid cellularity with a complete shift in the population of cells to a predominantly neutrophilic infiltrate as well as elevation in inflammatory cytokine protein and mRNA levels. There were no significant differences between the groups in measures of lung inflammation. These results indicate that TNF-alpha and IL-1 beta do not appear to have an essential role in mediating the physiological or inflammatory response to inhaled LPS.     

Comprehensive proteome analysis of ovarian cancers using liquid phase separation, mass mapping and tandem mass spectrometry: a strategy for identification of candidate cancer biomarkers

A two-dimensional (2-D) liquid phase separation method, liquid isoelectric focusing followed by nonporous reversed-phase high performance liquid chromatography (HPLC), was used to separate proteins from human ovarian epithelial whole cell lysates. HPLC eluent was interfaced on-line to an electrospray ionization (ESI) time of flight (TOF) mass spectrometer to obtain accurate intact protein molecular weights (Mr). 2-D protein expression maps were generated displaying protein isoelectric point (pI) versus intact protein Mr. Resulting 2-D images effectively displayed quantitative differential protein expression in ovarian cancer cells versus non-neoplastic ovarian epithelial cells. Protein peak fractions were collected from the HPLC eluent, enzymatically digested, and analyzed by matrix-assisted laser desorption/ionization (MALDI) TOF-mass spectrometry (MS) peptide mass fingerprinting and by MALDI-quadrupole TOF tandem mass spectrometry peptide sequencing. Interlysate comparisons of differential protein expression between two ovarian adenocarcinoma cell lines, ES2 and MDAH-2774, and ovarian surface epithelial cells was performed. Five pI fractions from each sample were selected for comparative study and over 300 unique proteins were positively identified from the 2-D liquid expression maps using MS, which covered around 60% of proteins detected by on-line ESI-TOF-MS. This represents one of the most comprehensive proteomic analyses of ovarian cancer samples to date. Protein bands with significant up- or down-regulation in one cell line versus another as viewed in the 2-D expression maps were identified. This strategy may prove useful in identifying novel ovarian cancer marker proteins.     

Thrombin attenuation is neuroprotective in the injured rat optic nerve

The functional loss that often follows injury of the mammalian CNS has been attributed not only to the immediate neural loss, but also to secondary neuronal degeneration caused by toxic biochemical mediators in the environment of the injured nerve. We report here that a high thrombin content, produced as a result of injury-induced activation of prothrombin, appears to be an important mediator of secondary damage. Measurement of post-traumatic neuronal survival in vivo revealed that post-traumatic local application of the thrombin inhibitor N-alpha-(2-naphthylsulphonylglycyl)-4-(D,L)-amidinophenylalanine piperidide acetate in the rat optic nerve subjected to mild partial crush injury left twice as many retinal ganglion cells with functioning axons as in controls. Thus, by readjusting thrombin activity, thereby possibly obtaining a moderate post-traumatic increase and thus gaining the benefit of thrombin without its toxic effects, it may be possible to create an environment that is more favourable for post-traumatic survival.     

PPAR-γ as a therapeutic target in cardiovascular disease: evidence and uncertainty

Peroxisome proliferator-activated receptor γ (PPAR-γ) is a key regulator of fatty acid metabolism, promoting its storage in adipose tissue and reducing circulating concentrations of free fatty acids. Activation of PPAR-γ has favorable effects on measures of adipocyte function, insulin sensitivity, lipoprotein metabolism, and vascular structure and function. Despite these effects, clinical trials of thiazolidinedione PPAR-γ activators have not provided conclusive evidence that they reduce cardiovascular morbidity and mortality. The apparent disparity between effects on laboratory measurements and clinical outcomes may be related to limitations of clinical trials, adverse effects of PPAR-γ activation, or off-target effects of thiazolidinedione agents. This review addresses these issues from a clinician's perspective and highlights several ongoing clinical trials that may help to clarify the therapeutic role of PPAR-γ activators in cardiovascular disease.