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The endothelial glycocalyx is a key component of the glomerular filtration barrier. We have shown that matrix metalloproteinase (MMP)-mediated syndecan 4 shedding is a mechanism of glomerular endothelial glycocalyx damage in vitro, resulting in increased albumin permeability. Here we sought to determine whether this mechanism is important in early diabetic kidney disease, by studying streptozotocin-induced type 1 diabetes in DBA2/J mice. Diabetic mice were albuminuric, had increased glomerular albumin permeability and endothelial glycocalyx damage. Syndecan 4 mRNA expression was found to be upregulated in isolated glomeruli and in flow cytometry-sorted glomerular endothelial cells. In contrast, glomerular endothelial luminal surface syndecan 4 and Marasmium oreades agglutinin lectin labelling measurements were reduced in the diabetic mice. Similarly, syndecan 4 protein expression was significantly decreased in isolated glomeruli but increased in plasma and urine, suggesting syndecan 4 shedding. Mmp-2, 9 and 14 mRNA expression were upregulated in isolated glomeruli, suggesting a possible mechanism of glycocalyx damage and albuminuria. We therefore characterised in detail the activity of MMP-2 and 9 and found significant increases in kidney cortex, plasma and urine. Treatment with MMP-2/9 inhibitor I for 21 days, started six weeks after diabetes induction, restored endothelial glycocalyx depth and coverage and attenuated diabetes-induced albuminuria and reduced glomerular albumin permeability. MMP inhibitor treatment significantly attenuated glomerular endothelial and plasma syndecan 4 shedding and inhibited plasma MMP activity. Thus, our studies confirm the importance of MMPs in endothelial glycocalyx damage and albuminuria in early diabetes and demonstrate that this pathway is amenable to therapeutic intervention. Hence, treatments targeted at glycocalyx protection by MMP inhibition may be of benefit in diabetic kidney disease., Graphical abstract

Lipid Phosphate phosphatase 3 (LPP3), encoded by the Plpp3 gene, is an enzyme that dephosphorylates the bioactive lipid mediator lysophosphatidic acid (LPA). To study the role of LPP3 in the myocardium, we generated a cardiac specific Plpp3 deficient mouse strain. Although these mice were viable at birth in contrast to global Plpp3 knockout mice, they showed increased mortality ~ 8 months. LPP3 deficient mice had enlarged hearts with reduced left ventricular performance as seen by echocardiography. Cardiac specific Plpp3 deficient mice had longer ventricular effective refractory periods compared to their Plpp3 littermates. We observed that lack of Lpp3 enhanced cardiomyocyte hypertrophy based on analysis of cell surface area. We found that lack of Lpp3 signaling was mediated through the activation of Rho and phospho-ERK pathways. There are increased levels of fetal genes Natriuretic Peptide A and B (Nppa and Nppb) expression indicating myocardial dysfunction. These mice also demonstrate mitochondrial dysfunction as evidenced by a significant decrease (P < 0.001) in the basal oxygen consumption rate, mitochondrial ATP production, and spare respiratory capacity as measured through mitochondrial bioenergetics. Histology and transmission electron microscopy of these hearts showed disrupted sarcomere organization and intercalated disc, with a prominent disruption of the cristae and vacuole formation in the mitochondria. Our findings suggest that LPA/LPP3-signaling nexus plays an important role in normal function of cardiomyocytes., Graphical abstract fx1, Highlights • PLPP3 plays a prominent role in the heart compared to other isoforms of PLPP. • Lack of PLPP3 results in deteriorating cardiac function. • PLPP3 regulates LPA signaling in cardiomyocytes. • Presence of PLPP3 is required for optimal mitochondrial function. • Increased free radical production is mitigated with activated PLPP3.

The bioactive lysophosphatidic acid (LPA) plays a well-known role in atherosclerotic disease, whereas, its role in myocardial function remains virtually unexplored. Following acute myocardial infarction, serum LPA concentration rises by six-fold over control human subjects, suggesting LPA may contribute to the pathogenesis of myocardial infarction. LPA production involves hydrolysis of lysophosphatidylcholine by the secreted enzyme autotaxin, whereas lipid phosphate phosphatase-3 (LPP3) catalyzes LPA dephosphorylation to generate lipid products that are not receptor active. We present the first evidence that cardiac ischemia/reperfusion (I/R) injury enhances myocardial autotaxin levels and decreases myocardial LPP3 expression, and this is associated with increased serum LPA levels. Upon reperfusion, reactive oxygen species production arises as a burst of superoxide from mitochondria following I/R injury. The redox-sensitive transcription factor NFAT has been shown to bind to the autotaxin promoter and induce its expression. Therefore, we looked at the autotaxin and LPP3 regulation in mice following I/R injury in the myocardium. After 1h ligation followed by 3h reperfusion in the myocardium, we observed a 3-fold increase in the autotaxin protein levels, whereas LPP3 protein levels were significantly downregulated as observed through Western blot analysis in these myocardial ischemic tissues. Autotaxin and miR-92a mRNA expression levels were significantly upregulated, whereas KLF2 and LPP3 mRNA expressions were significantly downregulated following I/R injury at 24 hours. Western blot analysis showed a 3-fold increase autotaxin protein levels and immunohistochemistry of human infarct tissues at 24 hours showed disruption of the sarcomere with decreased LPP3 staining. We found that I/R injury transactivates miR-92a, and inhibit KLF2, an upstream activator of LPP3. Taken together, us in vivo data, from the myocardial I/R injury and human infarct tissues, suggest that regulation of autotaxin and LPP3 activity might cause the rise in serum LPA levels as reported with acute myocardial infarct patients

Electrophilic aldehyde (4-hydroxynonenal; 4-HNE), formed after lipid peroxidation, is a mediator of mitochondrial dysfunction and implicated in both the pathogenesis and the progression of cardiovascular disease. Manganese superoxide dismutase (MnSOD), a nuclear-encoded antioxidant enzyme, catalyzes the dismutation of superoxide radicals (O2•-) in mitochondria. To study the role of MnSOD in the myocardium, we generated a cardiomyocyte-specific SOD2 (SOD2Δ) deficient mouse strain. Unlike global SOD2 knockout mice, SOD2Δ mice reached adolescence; however, they die at ~4 months of age due to heart failure. Ultrastructural analysis of SOD2Δ hearts revealed altered mitochondrial architecture, with prominent disruption of the cristae and vacuole formation. Noninvasive echocardiographic measurements in SOD2Δ mice showed dilated cardiomyopathic features such as decreased ejection fraction and fractional shortening along with increased left ventricular internal diameter. An increased incidence of ventricular tachycardia was observed during electrophysiological studies of the heart in SOD2Δ mice. Oxidative phosphorylation (OXPHOS) measurement using a Seahorse XF analyzer in SOD2Δ neonatal cardiomyocytes and adult cardiac mitochondria displayed reduced O2 consumption, particularly during basal conditions and after the addition of FCCP (H+ ionophore/uncoupler), compared to that in SOD2fl hearts. Measurement of extracellular acidification (ECAR) to examine glycolysis in these cells showed a pattern precisely opposite that of the oxygen consumption rate (OCR) among SOD2Δ mice compared to their SOD2fl littermates. Analysis of the activity of the electron transport chain complex identified a reduction in Complex I and Complex V activity in SOD2Δ compared to SOD2fl mice. We demonstrated that a deficiency of SOD2 increases reactive oxygen species (ROS), leading to subsequent overproduction of 4-HNE inside mitochondria. Mechanistically, proteins in the mitochondrial respiratory chain complex and TCA cycle (NDUFS2, SDHA, ATP5B, and DLD) were the target of 4-HNE adduction in SOD2Δ hearts. Our findings suggest that the SOD2 mediated 4-HNE signaling nexus may play an important role in cardiomyopathy., Graphical abstract Image 1, Highlights • SOD2 is essential for cardiomyocyte function; its deficiency causes lethal dilated cardiomyopathy. • SOD2 deficiency disturbs mitochondrial architecture in the heart. • SOD2 is required for optimum mitochondrial respiration. • Deficiency of SOD2 leads to the production of 4-HNE within mitochondria. • 4-HNE targets proteins of the mitochondrial respiratory complex and TCA cycle.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in soils and sediments, particularly in urbanized environments in which the concentrations of 16 (or so) PAHs are regulated. Distinguishing among the numerous PAH sources is of practical and legal concern and thereby is often an objective of environmental forensic chemistry studies. Studies of prospective sources and impacted soils and sediments that rely upon the 16 U.S. EPA Priority Pollutant PAHs are disadvantaged, as these few compounds generally lack the specificity to distinguish among different PAH sources in the environment. Advances in analytical and interpretive methods over several decades have shown that different PAH sources can be more defensibly distinguished using modified EPA Method 8270 that, among other improvements, measure many other polycyclic aromatic compounds (PACs) that co-occur with the Priority Pollutant PAHs in different sources and in the environment. The PACs include variously-alkylated PAHs and polycyclic aromatic sulfu...

One of the proteins most frequently found in neuropathological lesions is the ubiquitin binding protein p62 (sequestosome 1). Post-mortem analysis of p62 is a defining diagnostic marker in several neurodegenerative diseases including amyotrophic lateral sclerosis and inclusion body myositis. Since p62 functions in protein degradation pathways including autophagy, the build-up of p62-positive inclusions suggests defects in protein clearance. p62 was expressed unilaterally in the rat substantia nigra with an adeno-associated virus vector (AAV9) in order to study p62 neuropathology. Inclusions formed within neurons from several days to several weeks after gene transfer. By electron microscopy, the inclusions were found to contain packed 10 nm thick filaments, and mitochondria cristae structure was disrupted, resulting in the formation of empty spaces. In corollary cell culture transfections, p62 clearly impaired mitochondrial function. To probe for potential effects on macroautophagy, we co-expressed p62 with a double fluorescent tagged reporter for the autophagosome protein LC3 in the rat. p62 induced a dramatic and specific dissociation of the two tags. By 12 weeks, a rotational behavior phenotype manifested, consistent with a significant loss of dopaminergic neurons analyzed post-mortem. p62 overexpression resulted in a progressive and robust pathology model with neuronal inclusions and neurodegeneration. p62 gene transfer could be a novel methodological probe to disrupt mitochondrial function or autophagy in the brain and other tissues in vivo.

Detailed chemical analysis of petroleum – often referred to as “chemical fingerprinting” – has played a vital role in the identification of oil from accidental spills in the marine and riverine environments (e.g., sediments). The analytical tools used to identify and quantify the spilled oil must provide sufficient chemical resolution to separate the oil from any pre-existing (historical or naturally occurring) background hydrocarbons ubiquitous in many environments. Qualitative chemical fingerprinting analysis is a visual comparison between various spectroscopic or chromatographic fingerprints and is most successfully applied when the oil signature is unweathered and/or not mixed with other hydrocarbon sources (e.g., background). Quantitative chemical fingerprinting is the use of target compound calibration standards during the chemical analysis (e.g., GC/MS) of the sample to quantify the concentrations of source diagnostic hydrocarbons such as polycyclic aromatic hydrocarbons and biomarkers (e.g., triterpanes and steranes). These quantitative results can then be used to develop interpretive tools, such as diagnostic ratios, that allow the forensic scientist to compare spill and source oils and also to develop mixing models capable of allocating hydrocarbons derived from the spilled oil versus any background hydrocarbon signatures. Although the utility of diagnostic ratios has been discussed extensively in regard to oil spill identification in the scientific literature, the same is not true for the development and application hydrocarbon mixing models at oil spill sites. This chapter examines the applications and limitations of qualitative and quantitative oil fingerprinting methods and provides four oil spill case studies where mixing models were used to identify and or allocate liability in complex mixtures of spilled oils and various forms of background hydrocarbons.

Complex mixtures of volatile organic chemical (VOC) vapors can exist over subsurface accumulations of non-aqueous phase liquids (NAPLs) and contaminated soils. The ability to predict the relative soil gas chemical composition arising from such NAPLs is relevant to studies of the sources and fate of soil gas, and in assessing the possible intrusion of soil gas chemical constituents to indoor air. However, it is difficult to directly compare the chemical ‘fingerprints’ of NAPL and vapor because the differences are significant in liquid-gas partitioning coefficients among the many volatile hydrocarbons that compose the liquid NAPL. In this article, detailed chemical characterization data from analysis of liquid NAPLs are used to calculate equilibrium vapor phase distributions of 66 diagnostic paraffin, isoparaffin, aromatic, naphthene, and olefin (PIANO) compounds. Measured NAPL and predicted vapor phase chemical composition are presented for a diverse group of 10 hydrocarbon products that include crude oil,...

Virtually all environmental forensics investigations focus on addressing questions pertaining to the nature, source, age, and ownership of site-related contamination. Contamination, particularly at complex historic sites, is usually a multifarious mixture of both organic and inorganic chemicals. Thus, the forensic investigator is typically faced with “unravelling” a complicated mixture of chemicals into component parts in order to better link the chemicals to their historic origins and differentiate them from often similar types and sources of contaminants. This chapter describes advanced methods of chemical analyses that have evolved, and continue to be refined by environmental chemists to address the specific needs of the forensic investigator and focuses on arguably some of the most important organic contaminants commonly encountered in terrestrial and sediment investigations: petroleum hydrocarbons and polycyclic aromatic hydrocarbons (PAH). The details of advanced methods for the measurement of these chemicals in multiple media (water, soils, sediment, air, and biological tissues) are presented. Laboratory techniques, including sample preparation, instrumental analysis, and quality control and quality assurance procedures are presented so that the reader can readily adapt forensic measurement techniques to suit his or her specific site investigation activities. Case studies are presented throughout the text that demonstrate the application of advanced methods of chemical analysis to varying kinds of complicated, real world forensic instigations.

The source(s) of the non-aqueous phase liquid (NAPL) in the downtown area of Mandan, ND, has been investigated through a two-part study in which the detailed chemical compositions of: 1) the NAPLs from the downtown area and remote areas on a nearby rail yard (phase I), and 2) residual petroleum in vadose zone and capillary fringe soils from borings near suspected sources (phase II) have been determined. The NAPL and soils are shown to contain variably weathered middle distillate fuels. The distributions of n-alkylcyclohexanes (CHs) and relative abundance of sulfur-containing aromatics (alkyl-dibenzothiophenes), both of which are stable over the various degrees of weathering exhibited by the NAPLs in the study area, were of particular utility in characterizing these fuels. The data indicate that multiple types of middle distillate fuels exist(ed) in the downtown area, establishing multiple sources and releases to the subsurface.

Petroleum biomarkers are ubiquitous hydrocarbons that occur in crude oils and are derived from formerly living organisms whose organic materials gave rise to the crude oil over geologic time. Biomarkers' specificity and resistance to weathering renders them useful in chemical fingerprinting of petroleum contamination in the environment. The biomarkers most commonly used in forensic issues, viz., tri- and penta-cyclic triterpanes and regular or rearranged steranes, are too high boiling for use in fingerprinting middle distillate petroleum products. The present article describes a suite of “low boiling” biomarkers (i.e., drimane-based bicyclic sesquiterpanes) that boil within the distillate fuel range (n-C13 to n-C16; ∼ 240°C to 285°C). GC/MS analysis of 24 dispensed (fresh) distillate fuels (Jet A, JP-5, and diesel fuel/fuel oil #2) demonstrate the specificity of sesquiterpane patterns in fuels of different origins. Analysis of fresh diesel fuel #2 and its associated, variably weathered NAPLs demonstrate t...

Automotive gasoline is a common petroleum product found at contaminated terrestrial sites. The need to recognize and distinguish different types of gasoline(s) that may be present at a contaminated site and the need to determine the relative proportion of inputs from different sources, are often critical components of environmental 'forensic' investigations. Historically, identification and differentiation of automotive gasolines (particularly when weathered) has been hampered by analytical limitations of existing methods, notably US EPA Method 8260. In this article, we describe a modified EPA Method 8260 that is suitable for environmental investigations involving gasoline (and other light petroleum products). In the modified EPA 8260 method, 109 analytes that can occur in automotive gasoline are quantified in nonaqueous liquid samples (NAPL), water, and soil matrices. The accuracy and precision of the method is demonstrated through comparative analysis using several NIST SRM gasoline standards and replic...

Troglitazone is a peroxisome proliferator-activated receptor-γ agonist that has been shown to halt mesangium expansion in experimental models of type 2 diabetes mellitus and to act directly on rat mesangial cells. Because glutamine serves as the precursor for cellular biosynthetic processes, we asked whether troglitazone would inhibit mesangial cell glutamine metabolism under these conditions. Confluent monolayers of rat mesangial cells were incubated in RPMI medium in the presence of troglitazone or vehicle (DMSO). Troglitazone effected a dose-dependent reduction in glutamine utilization and in alanine formation, associated with a decrease in monolayer collagen-glycosaminoglycan content. Despite the reduced glutamine uptake, ammonium formation did not decrease, consistent with increased glutamate flux through the deamination pathway. Assayable activity of the alanine aminotransferase decreased by 63%, whereas assayable glutamate dehydrogenase remained unchanged. In control monolayers, the sum of ammonium plus alanine plus glutamate nitrogen released accounted for

Item does not contain fulltext Heparan sulfate proteoglycans have been shown to modulate podocyte adhesion to--and pedicel organization on--the glomerular basement membrane. Recent studies showed that foot process effacement developed in a mutant mouse model whose podocytes were unable to assemble heparan sulfate glycosaminoglycan chains. This study, a further refinement, explored the role of heparan N-sulfation on podocyte behavior. A novel mutant mouse (Ndst1(-/-)) was developed, having podocyte-specific deletion of Ndst1, the enzyme responsible for N-sulfation of heparan sulfate chains. Podocytes having this mutation had foot process effacement and abnormal adhesion to Bowman's capsule. Although glomerular hypertrophy did develop in the kidneys of mutant animals, mesangial expansion was not seen. The lack of heparan N-sulfation did not affect the expression of agrin or perlecan proteoglycan core proteins. Loss of N-sulfation did not result in significant proteinuria, but the increase in the albumin/creatinine ratio was coincident with the development of the enlarged lysosomes in the proximal tubules. Thus, although the renal phenotype of the Ndst1(-/-) mouse is mild, the data show that heparan chain N-sulfation plays a key role in podocyte organization.

The need for identification, delineation, and differentiation of petroleum-derived contaminants in support of NRDA investigations of chronic or catastrophic release of petroleum is paramount for the equitable settlement of damages related to the incidents. Significant advances have been made over the last decade with regard to these issues for crude oil and heavy residual fuels, but less emphasis has been place on the forensic chemistry of light- and middle-distillates. Historically, identification and differentiation among similar light-distillates by standard EPA methods have been hampered by analytical limitations. In this paper the authors suggest analysis of light-distillate products begin with a chromatographic profile obtained using a modified EPA 8015 GC/FID. Subsequently, the detailed chemical composition of the distillate products is accomplished via a modified version of EPA Method 8260, examining a targeted list of diagnostic petroleum compounds in the C5 to C12 range that represent five important categories of hydrocarbons: Paraffins, Isoparaffins, Aromatics, Naphthenes, and Olefins. Detailed analysis of over 50 2–6 ring PAH and related heteroatomics from a modified EPA 8270 GC/MS analysis can be used to compliment the data set. The analysis of the full suite of hydrocarbons across a broad molecular weight range provides much of the information necessary to identify and distinguish among light- and middle-distillate products.

Rat mesangial cells in vitro synthesize a spectrum of proteoglycan species including those of the basement membrane and interstitium. Accumulation of extracellular matrix within the mesangium is an important event in the development of glomerular disease. In this report we have used indirect immunofluorescence to positively identify a number of constituents of the mesangial matrix synthesized by rat mesangial cells (RMC) in vitro including laminin, fibronectin, type IV collagen and the basement membrane heparan sulphate proteoglycan (BM-HSPG) known as perlecan. In addition, using Mab 2B5 we demonstrate that RMC synthesize a specific basement membrane chondroitin sulfate (BM-CSPG), a matrix component that in normal animals is localized in the mesangium but is not found in the pericapillary glomerular basement membrane (GBM). Further characterization of the proteoglycans synthesized by RMC in vitro revealed: (i) a second large CSPG, identified as versican; (ii) two small dermatan sulphate proteoglycans identified as biglycan and decorin, which together account for the majority Of the proteoglycans; (iii) a large HSPG-I, probably related to perlecan; and (iv) a small HSPG-II. The cell layer proteoglycans can be sub-divided into a class that are probably free in the membrane, and a class of anchored molecules of the extracellular matrix or stabilized by cytoskeletal elements.

The glomerular basement membrane and its associated cells are critical elements in the renal ultrafiltration process. Traditionally the anionic charge associated with several carbohydrate moieties in the glomerular basement membrane are thought to form a charge selective barrier that restricts the transmembrane flux of anionic proteins across the glomerular basement membrane into the urinary space. The charge selective function, along with the size selective component of the basement membrane, serves to limit the efflux of plasma proteins from the capillary lumen. Heparan sulfate glycosaminoglycans are anionically charged carbohydrate structures attached to proteoglycan core proteins and have a role in establishing the charge selective function of the glomerular basement membrane. Although there are a large number of studies in the literature that support this concept, the results of several recent studies using molecular genetic approaches to minimize the anionic charge of the glomerular basement membrane would suggest that the role of heparan sulfate glycosaminoglycans in the glomerular capillary wall are still not yet entirely resolved, suggesting that this research area still requires new and novel exploration.

We have previously reported the production of monoclonal antibodies (MAb) recognizing the core protein of a basement membrane-specific chondroitin sulfate proteoglycan (BM-CSPG). Using immunohistochemical techniques, we have shown that BM-CSPG is present in almost every basement membrane, one exception being the normal glomerular capillary basement membrane (GBM), where it is absent. In the present study of mature kidneys we examined the distribution of BM-CSPG in streptozocin-induced diabetes mellitus in rats. We found BM-CSPG atypically associated with the GBM of diabetic animals as early as 1 month after induction of diabetes mellitus. Immunoelectron microscopy (IEM) of affected capillary loops showed BM-CSPG present in the subendothelial matrix in areas of GBM thickening and absent in areas where the GBM appears to be of normal thickness. Moreover, the association of BM-CSPG with regions of the pericapillary GBM affects the morphology of the capillary endothelial cells within these areas, directly displacing the cell body from the GBM proper and causing loss of fenestrae. These new data on BM-CSPG distribution reflect abnormal glomerular extracellular matrix protein biosynthesis/turnover in diabetes and suggest that BM-CSPG in the GBM might in turn affect normal capillary structure and/or function.

We previously reported the presence of a basement membrane-specific chondroitin sulfate proteoglycan (BM-CSPG) in basement membranes of almost all adult tissues. However, an exception to this ubiquitous distribution was found in the kidney, where BM-CSPG was absent from the glomerular capillary basement membrane (GBM) but present in other basement membranes of the nephron, including collecting ducts, tubules, Bowman's capsule, and the glomerular mesangium. In light of this unique pattern of distribution and of the complex histoarchitectural reorganization occurring during nephrogenesis, the present study used light and electron microscopic immunohistochemistry to examine the distribution of BM-CSPG and basement membrane heparan sulfate proteoglycan (BM-HSPG) during prenatal and postnatal renal development in the rat. Our results show that the temporal and spatial pattern of expression of BM-CSPG during nephrogenesis is unlike that reported for other basement membrane components such as laminin, fibronectin, and BM-HSPG, all of which can be found in the earliest formed basement membranes of the vesicle-stage nephron. Although BM-CSPG is present in the basement membranes of the invading vasculature and ureteric buds, its first appearance in nephron basement membrane occurs during the late comma stage. In capillary loop-stage glomeruli of prenatal animals, BM-CSPG is present in the presumptive mesangial matrix but undetectable in the GBM. However, as postnatal glomerular maturation progresses BM-CSPG is also found in both the lamina rara interna and lamina densa of the GBM in progressively increasing amounts, being most evident in the GBM of 21-day-old animals. Micrographs of glomeruli from 42-day-old animals show that BM-CSPG gradually disappears from the GBM and, by 56 days after birth, appears to be completely absent from the GBM, its pattern of distribution resembling that of the adult animal. Our results show that BM-CSPG is not required for the initial assembly of basement membranes but may in fact serve to stabilize basement membrane structure after histoarchitectural reorganization is completed.

Histologic preparations of lungs from 1-, 5-, 10-, 18-, and 25-day-old postnatal and adult rats were examined immunohistochemically with antibodies specific against chondroitin sulfate (CS), basement membrane chondroitin sulfate proteoglycan (BM-CSPG), heparan sulfate proteoglycan (HSPG), entactin, and laminin. A monoclonal antibody specific for the glycosaminoglycan portion (CS) of CSPG and a monoclonal antibody against the core protein of CSPG were used in an immunoperoxidase sequence to stain extracellular matrix (ECM) components of pulmonary basement membranes (BMs). Anti-CS stained airway BM strongly and alveolar BM weakly in the adult rat lung, as well as in vascular and airway adventitia. In developing lungs, immunoreactivity was strong in all ECM sites, including BM, at day 1 postnatal, and progressively diminished thereafter except in vascular and airway adventitia. Anti-CSPG stained alveolar, airway, and vascular BMs, in addition to smooth muscle external laminae (EL), in the adult and developing rat. Immunostaining for CSPG required hyaluronidase digestion, whereas CS staining was lost with the same treatment. A polyclonal antibody to the core protein of HSPG was found to be similarly distributed to CSPG by immunoperoxidase staining in adult and developing rat lungs, with the notable exception that little immunoreactivity for HSPG was found in smooth muscle EL. Commercially obtained polyclonal antibodies to entactin and laminin gave immunostaining comparable to that seen with CSPG, except that entactin showed particular affinity for EL. These results offer a more detailed perspective on previous survey observations of CSPG, HSPG, and entactin in the rat lung, and describe the immunoreactivity of CS for the first time.(ABSTRACT TRUNCATED AT 250 WORDS)

Contains fulltext : 89710.pdf (Publisher’s version ) (Closed access) Podocytes adhere to the glomerular basement membrane by cell surface receptors. Since in other cells these adhesions are enhanced by cell surface proteoglycans, we examined the contribution of these molecules and their glycosaminoglycan side chains to podocyte adhesion by developing immortalized podocyte cell lines with (control) or without (mutant) heparan sulfate glycosaminoglycan chains. In adhesion assays control podocytes attached, spread, and migrated more efficiently compared with mutants, indicating a requirement for heparan sulfate chains in these processes. The proteoglycan syndecan-4 is known to have direct effects on cell attachment, spreading, and cytoskeletal organization. We found it localized to focal adhesions in control podocytes coincident with stress fiber formation. In mutant cells, syndecan-4 was associated with smaller focal contacts and cortical actin organization. Analysis by flow cytometry showed that mutant cells had twice the amount of surface syndecan-4 of control cells. Protein kinase Calpha, a signaling molecule bound to and activated by syndecan-4, showed a fourfold increase in membrane localization-activation than that seen in control cells. In vivo, the loss of heparan sulfate glycosaminoglycans in PEXTKO mice led to a loss of glomerular syndecan-4. Overall, our study provides further evidence for a dynamic role of cell surface heparan sulfate glycosaminoglycans in podocyte activity. 01 december 2010

Contains fulltext : 69069.pdf (Publisher’s version ) (Closed access) Podocytes synthesize the majority of the glomerular basement membrane components with some contribution from the glomerular capillary endothelial cells. The anionic charge of heparan sulfate proteoglycans is conferred by covalently attached heparan sulfate glycosaminoglycans and these are thought to provide critical charge selectivity to the glomerular basement membrane for ultrafiltration. One key component in herparan sulfate glycosaminoglycan assembly is the Ext1 gene product encoding a subunit of heparan sulfate co-polymerase. Here we knocked out Ext1 gene expression in podocytes halting polymerization of heparin sulfate glycosaminoglycans on the proteoglycan core proteins secreted by podocytes. Glomerular development occurred normally in these knockout animals but changes in podocyte morphology, such as foot process effacement, were seen as early as 1 month after birth. Immunohistochemical analysis showed a significant decrease in heparan sulfate glycosaminoglycans confirmed by ultrastructural studies using polyethyleneimine staining. Despite podocyte abnormalities and loss of heparan sulfate glycosaminoglycans, severe albuminuria did not develop in the knockout mice. We show that the presence of podocyte-secreted heparan sulfate glycosaminoglycans is not absolutely necessary to limit albuminuria suggesting the existence of other mechanisms that limit albuminuria. Heparan sulfate glycosaminoglycans appear to have functions that control podocyte behavior rather than be primarily an ultrafiltration barrier.

Basement membranes are complex extracellular matrices present at epithelial/mesenchymal interfaces of tissues. The dermal-epidermal junction has been shown to contain numerous components, some of the most well known being laminin, types IV and VII collagens, heparan sulfate proteoglycan, fibronectin, and entactin/nidogen. In this paper we show, using core protein-specific antibodies, the presence of a newly described basement membrane-specific chondroitin sulfate proteoglycan at the epithelial/ mesenchymal interface of adult rat skin. Ultrastructurally, this antigen was proven to reside primarily within the basal lamina, apparently concentrated in the lamina densa. In addition, some of the proteoglycan was also present beneath the lamina densa, associated with the reticular lamina collagen fibrils.

Cadmium toxicity to the cornea of pregnant rats was studied using the electron microscope and x-ray microanalyzer. In in-vivo experiments, severe corneal edema occurred in pregnant dams that received intraperitoneal injections of cadmium sulphate for 4 days during gestation, but not in nonpregnant rats. Prominent swelling of mitochondria and the occurrence of intra- and inter-cellular vacuoles in the corneal endothelium were observed only in pregnant dams. In in-vitro experiments, electron-dense deposits consiting of cadmium-oxine complexes were preferentially found in swollen mitochondria of the endothelial cells. Cadmium peaks were obtained from these deposits with x-ray microanalysis. These data suggest that the corneal edema observed after administration of cadmium may imply the disturbance of pump function and barrier function of the corneal endothelium due to the primary toxic effects of this metal on mitochondria.

The distribution of two distinct populations of basement membrane proteoglycans has been monitored through hair growth development in the rat embryo and subsequent hair growth cycle. An antiserum against a small heparan sulfate proteoglycan uniformly stained the dermal-epidermal junction of embryonic rats throughout the period of hair follicle formation. On the other hand, monoclonal antibodies recognizing a basement membrane-specific chondroitin sulfate proteoglycan only weakly stained 16-d embryo dermal-epidermal junction, but strong staining was associated with hair follicle buds as they developed. Through the hair growth cycle, it was found that the heparan sulfate proteoglycan persisted around the follicles, while the chondroitin sulfate proteoglycan decreased in amount through catagen until it was undetectable at the base and dermal papilla of the telogen follicle. As anagen commenced, expression of the chondroitin sulfate proteoglycan was again demonstrated. It therefore appears that a basement membrane-specific proteoglycan shows variation in its distribution in rat skin, expression correlating with morphogenetic activity in hair follicles. It is possible that this newly described basement membrane component is involved in the complex processes of dermal-epidermal interaction that lead to skin appendage formation and growth.

The modern chemical fingerprinting analytical methods used have evolved over the past two decades, largely due to the development and increased sophistication of analytical instrumentation. The chemical fingerprinting approaches available for the identification of oil spill sources and potentially impacted samples fall into two categories: qualitative and quantitative. The qualitative approach relies upon visual comparison of various chromatographic fingerprints and is exemplified by the ASTM D3328 and D5739 methods. The cornerstone of modern petroleum fingerprinting is high-resolution capillary gas chromatography. Qualitative chemical fingerprinting analysis of spilled oil, candidate sources, and background materials can be best described as a visual comparison between various spectroscopic or chromatographic fingerprints. Such comparisons inescapably introduce a degree of subjectivity to the source identification evaluation, which is an undesirable feature of science. The quantitative approach is preferable for most oil spill investigations because the means of interpretation are more objective and robust in the sense that they facilitate numerical comparison of diagnostic details and reduce interpretation bias.

A monoclonal antibody was used in early studies to identify a novel chondroitin sulfate proteoglycan, secreted by L-2 cells, the core protein of which was approximately 100 kDa. To characterize this proteoglycan core protein at the molecular level, an L-2 cell cDNA library was probed by expression screening and solution hybridization. Northern blot analysis assigned transcript size to approximately 3.1 kilobases and, after contig assembly, the coding region of the mRNA corresponded to 2.18 kilobases. Immunoassays were performed to confirm the identity of this sequence, using a polyclonal antibody raised against an expressed fusion protein encoded by sequence representing the carboxyl half of the molecule. The antibody recognized the core protein in Western blots after prior digestion of the intact proteoglycan with chondroitinase ABC. Immunostaining tissue sections with the same antibody localized the proteoglycan to basement membranes, and expression of the entire sequence in Chinese hamster ovary K-1 cells showed that the protein encoded by the sequence secreted as a chondroitin sulfate proteoglycan. The core protein not only has motifs permitting glycosylation as a proteoglycan, but also possesses the endoplasmic reticulum retrieval signal, KDEL, which suggests that, in addition to its role as a basement membrane component, it may also participate in the secretory pathway of cells.

A coarctation hypertensive rat model was used to examine the effects of elevated blood pressure on basement membrane component synthesis by cardiac myocytes and aorta using immunohistochemistry and Northern blot analysis. Carotid arterial pressure increased immediately on coarctation, and left ventricular hypertrophy was maximal within 5 days. In immunohistochemical studies, fibronectin and laminin were increased and the basement membrane chondroitin sulfate proteoglycan decreased in both the subendothelial space and smooth muscle cell basement membranes of the aorta above the clip compared with controls, whereas only fibronectin was elevated in the aorta below the clip. No change in basement membrane staining intensity for the cardiac myocytes was observed. Alterations in steady-state mRNA levels for fibronectin and laminin in the aorta paralleled those observed by immunohistochemical analysis with regard to protein and tissue type affected as well as intensity of the changes. However, changes in mRNA levels (but not protein deposition) for perlecan and type IV collagen were also observed in aortas from hypertensive rats compared with controls. Increases in steady-state mRNA levels for all basement membrane components in the heart and vasculature peaked before maximal cardiac hypertrophy (5 days). These studies indicate that alterations in basement membrane component deposition in the hypertrophied vasculature occur at both transcriptional and translational levels and suggest that the cell attachment glycoproteins fibronectin and laminin may be important factors in the vascular response to elevated transmural pressure.

Basement membrane proteoglycans and development. Basement membranes contain distinct collagen, glycoprotein and proteoglycan species, and these exhibit considerable heterogeneity in isoform or type when different tissue types are compared. Additionally, many components are differentially expressed in organogenesis. We have considered the distributions in glomerulogenesis of two distinct basement membrane proteoglycans, a small heparan sulfate proteoglycan and a chondroitin sulfate proteoglycan (BM-CSPG). While the former was present in all kidney basement membranes through development, the latter was apparently regulated in distribution. BM-CSPG was only strongly expressed in the vasculature invading late comma stage glomeruli, and later in presumptive and mature Bowman's capsule. Over the first six to eight weeks, the capillary basement membranes contained BM-CSPG, but in gradually decreasing amounts until it became completely undetectable. The basement membrane of the adult rat glomerulus is unique in its lack of BM-CSPG. However, in diabetic rats, BM-CSPG is apparently re-expressed in the glomerular basement membrane, a potential marker for pathological changes in glomerular structure. While its function awaits elucidation, BM-CSPG may be essential for basement membrane integrity or stability and have important roles in kidney development.

During murine AA amyloidogenesis there is co-deposition of the AA peptide and the basement membrane form of heparan sulfate proteoglycan (HSPG), (1). Recent studies have also demonstrated the HSPG in 5 different types of amyloid. These include AA amyloid in inflammation (1), IAPP amyloid in diabetes (2), beta amyloid in Alzheimer’s disease (3), prion amyloid in Gerstmann-Straussler Syndrome (4), and prealbumin amyloid in familial amyloidotic polyneuropathy (5). Although HSPG appears to be a common component of several amyloid deposits, its role in the pathogenesis of amyloids is uncertain and is currently under intense investigation. For example, heparan sulfate as opposed to other glycosaminoglycans influences SAA2 to adopt a more beta pleated configuration (6), and sulfate ions influence the conformation of AA peptides in AA amyloid fibrils (7).

Heparan sulfate proteoglycans have been described as the major proteoglycan component of basement membranes. However, previous investigators have also provided evidence for the presence of chondroitin sulfate glycosaminoglycan in these structures. Recently we described the production and characterization of core protein-specific monoclonal antibodies (MAb) against a chondroitin sulfate proteoglycan (CSPG) present in Reichert's membrane, a transient extra-embryonic structure of rodents. This CSPG was also demonstrated to be present in adult rat kidney. We report here the tissue distribution of epitopes recognized by these MAb. The ubiquitous presence of these epitopes in the basement membranes of nearly all adult rat tissues demonstrates that at least one CSPG is a constituent of most basement membranes, and by virtue of its unique distribution is distinct from other chondroitin and dermatan sulfate proteoglycans previously described.

We have used two epithelial-stripping techniques in our studies of the basal lamina in the developing rat colon. The first involves prolonged osmication followed by sonication; the second uses chelation of calcium by EDTA followed by sonication. Both techniques remove the epithelium from the basal lamina; however, the EDTA/sonication technique appears to produce a cleaner adepithelial surface of the basal lamina. In addition, the fine structure of the basal lamina appears to be better preserved in specimens prepared by the EDTA/sonication technique. In contrast, the basal lamina of specimens prepared by the osmium/sonication technique had a shattered appearance that we believe is due to an increase in the fragility of the delicate fetal basal lamina.

It has been appreciated for some time that all mammalian basement membranes contain type IV collagen, laminin, entactin/nidogen and proteoglycans [ 11, but more recently it has become clear that there may be a high degree of tissue specificity in the expression of various forms of these molecules. For example, a chain of laminin known as s-laminin [2], is restricted to basement membranes which are present between two tissues, such as the kidney glomerular loop and the neuromuscular junction. In a similar way, it now appears that many forms of proteoglycan may be present in basement membranes, varying not only in core protein but also carbohydrate characteristics. Basement membrane heparan sulphate proteoglycan (HSPG) was first isolated from the murine transplantable Engelbreth-Holm-Swarm (EHS) tumour matrix [3, 41, and was found to have comparatively low buoyant density (approx. 1.3 g/ml), because of a high protein/carbohydrate ratio, the core protein being 400 kDa. As reported by us and others [ 3, 5, 61, antibodies against this HSPG showed widespread and uniform basement membrane staining of all tissues so far examined, including those of the human. Howcvcr, immunohistochemical analysis with polyclonal antibodies raised against a small HSPG with low protein/carbohydrate ratio and high buoyant density (approx. 1.5 g/ml), indicated this had a heterogeneous distribution in rat basement membranes 171. Although synthesized by most epithelia, including those of the skin and mammary gland, the small HSPG was apparently absent from visceral smooth muscle and cardiac muscle basement membranes. It is currently debated whether small basement membrane HSPGs are derived from the large HSPG by limited proteolysis. Evidence from the EHS tumour and studies on the kidney in vivo have lent support to this view [8, 91. Equally, other evidence has indicated that small HSPGs may be unique [6]. Our studies with antibodies support the case for at least some unique small HSPGs, but the problem can only clearly be resolved by protein sequence data. This is partially available for the large HSPG [lo], and we are currently cloning cDNAs from a mammary epithelial l g t l 1 library, screening with the antibodies against a small basement membrane HSPG [7]. So far, our sequence data do not coincide with those available for the large HSPG, and further important information has emerged from Northern blots. The mRNA encoding the small HSPG was found to be approximately 2.6 kb, whereas that for the large HSPG is 10I 1 kb. Therefore, we now have some evidence for the distinct nature of at least one small HSPG core protein. However, as we and others have indicated [6, 71, there may be some conservation of epitopes between members of this class of basement membrane component which can be detected immunologically. Whether these are protein or carbohydrate determinants is presently unknown. The complexity of basement membrane proteoglycans does not end with HSPGs. We have now identified a chon

Calculations of expansion coefficients from light scattering and viscometric data on heterodisperse samples of poly(acrylamide-co-sodium-acrylate) satisfactorily describe the solution properties of these polymers across the entire copolymer range, from polyacrylamide to poly(sodium acrylate). Light scattering results correlate well with the viscosity data provided that corrections are made for polymer heterodispersity and that a modified Krigbaum equation is used to estimate the unperturbed dimensions of the molecules. The expansion coefficients deduced from light scattering and Flory–Fox hydrodynamic theory were inaccurate and insensitive to fluctuations in charge density and molecular weight.

Mark Houwink coefficients and molecular size parameters are presented for 31 unfractionated samples of polyacrylamide and hydrolyzed polyacrylamide in 0.5M and 1M aqueous NaCl. Polymers investigated include polyacrylamide, plus 10, 20, 50, 70, and 100 mol % poly(sodium acrylate). Their solution characteristics were studied by gel permeation chromatography, intrinsic viscosity, and light scattering techniques. A density function and heterodispersity index was assigned to each polymer sample due to the polydispersity of the molecular weight distributions. This technique improves the intrinsic viscosity–molecular weight correlation for heterodisperse commercially synthesized polymer systems. The intrinsic viscosity of these commercial compounds will yield useful estimates of their molecular weight, in spite of their obvious heterogeneity.

Extraction of red beet root plasma membranes with the detergent Triton X-100 at a level of 2.0% (weight/volume) resulted in the depletion of over 90% of total membrane phospholipid and the reduction of glucan synthase activity by 80 to 90%. Reconstitution of the delipidated Triton X-100, 100,000g fraction in the presence of phospholipids restored glucan synthase activity. The most effective phospholipid was phosphatidyl-ethanolamine, which restored 110 to 144% of the original activity at 0.5% (weight/volume). Glucan synthase in the phospholipid-reactivated Triton X-100-treated fraction was enriched 9-fold in specific activity relative to microsomal membranes but was unstable in digitonin. These results support the hypothesis that glucan synthase activity is regulated by its phospholipid environment.

Reichert's membrane, an extraembryonic membrane present in developing rodents, has been proposed as an in vivo model for the study of basement membranes. We have used this membrane as a source for isolation of basement membrane proteoglycans. Reichert's membranes were extracted in a guanidine/3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate buffer followed by cesium chloride density-gradient ultracentrifugation under dissociative conditions. The proteoglycans were subsequently purified from the two most dense fractions (greater than 1.3 g/ml) by ion-exchange chromatography. Mice were immunized with the proteoglycan preparation and four mAbs recognizing the core protein of a high-density, buoyant chondroitin sulfate proteoglycan were raised. Confirmation of antibody specificity was carried out by the preparation of affinity columns made from each of the mAbs. Chondroitin sulfate proteoglycans (CSPGs) were purified from both supernatant and tissue fractions of Reichert's membranes incubated in short-term organ culture in the presence of radiolabel. The resultant affinity-purified proteoglycan samples were examined by gel filtration, SDS-PAGE, and immunoblotting. This proteoglycan is of high molecular weight (Mr = 5-6 x 10(5)), with a core protein of Mr = approximately 1.5-1.6 x 10(5) and composed exclusively of chondroitin sulfate chains with an average Mr = 1.6-1.8 x 10(4). In addition, a CSPG was purified from adult rat kidney, whose core protein was also Mr = 1.6 x 10(5). The proteoglycan and its core protein were also recognized by all four mAbs. Indirect immunofluorescence of rat tissue sections stained with these antibodies reveal a widespread distribution of this proteoglycan, localized specifically to Reichert's membrane and nearly all basement membranes of rat tissues. In addition to heparan sulfate proteoglycans, it therefore appears that at least one CSPG is a widespread basement membrane component.

Mark-Houwink coefficients and radius of gyration dimensions are presented for six unfractionated cationic polyelectrolytes of the poly(dimethylamine-epichlorohydrin-ethylendiamine) type. The copolymer system was studied in .05M, .10M, and 1.0M NaCl, and .12M NaClO4 aqueous solutions. Their solution behavior, as studied by intrinsic viscosity and light-scattering technique, suggests these polymers adopt a somewhat spherical or star-like conformation in aqueous salt solutions.

The temporal and spatial deposition of extracellular matrix proteins is critical for nephrogenesis and glomerular maturation. We previously characterized leprecan as a novel chondroitin sulfate proteoglycan which has been recently shown to have prolyl hydroxylase activity. In this study, we examine the distribution of leprecan during nephrogenesis and after a hypertrophic stimulus to the adult kidney. During development, leprecan was localized to mesenchymal aggregates, early comma- and S-phase structures as determined by immunohistochemistry and in situ hybridization. Leprecan mRNA was increased in cells around the vascular cleft of the S- and comma-phase glomeruli. Expression was found in podocytes, mesangial cells, and parietal epithelial cells of loop-phase glomeruli. Leprecan mRNA was substantially decreased in the glomeruli of the adult kidney compared to the developing kidney with a uniform distribution between the glomeruli and the tubules. Within adult glomeruli, leprecan was found in the mesangium mesangial matrix, podocytes, and in Bowman's capsule. In response to glomerular hypertrophy, produced by unilateral nephrectomy, leprecan synthesis was increased in the adult kidney. We suggest that the regulated expression of leprecan during glomerular development or hypertrophy coupled with its reported prolyl hydroxylase activity plays a role during basement membrane assembly.

We studied the effect of troglitazone on cellular acid-base balance and alanine formation in isolated rat mesangial cells. Mesangial cells were grown to confluency in RPMI 1640 media on 30-mm chambers used to monitor both cellular pH using the pH-sensitive dye 2′7′-bis(2-carboxyethyl)-5,6-carboxyfluorescein and metabolic acid production as well as glutamine metabolism. Troglitazone (10 μM) induced a spontaneous cellular acidosis (6.95 ± 0.02 vs. 7.47 ± 0.04, respectively; P < 0.0001) but without an increase in lactic acid production. Alanine production was reduced 64% ( P < 0.01) consistent with inhibition of the glutamate transamination. These findings pointed to a decrease in acid extrusion rather than an increase in acid production as the underlying mechanism leading to the cellular acidosis. To test their acid extrusion capabilities, mesangial cells were acid loaded with NH[Formula: see text] and then allowed to recover in Krebs-Henseleit media or in Krebs-Henseleit media minus bicarbonate (HEPES substituted), and the recovery response (ΔpHi/min) was monitored. In the presence of 10 μM troglitazone, the recovery response to the NH[Formula: see text] acid load was virtually eliminated in the bicarbonate-buffered media (0.00 ± 0.001 vs. 0.06 ± 0.02 pHi/min, P < 0.0001 vs. control) and reduced 75% in HEPES-buffered media (0.01 ± 0.01 vs. 0.04 ± 0.02 pHi/min, P < 0.002 vs. control). These results show that troglitazone induces a spontaneous cellular acidosis resulting from a reduction in cellular acid extrusion.