Inflammation in neurodegenerative disease--a double-edged sword

Inflammation is a defense reaction against diverse insults, designed to remove noxious agents and to inhibit their detrimental effects. It consists of a dazzling array of molecular and cellular mechanisms and an intricate network of controls to keep them in check. In neurodegenerative diseases, inflammation may be triggered by the accumulation of proteins with abnormal conformations or by signals emanating from injured neurons. Given the multiple functions of many inflammatory factors, it has been difficult to pinpoint their roles in specific (patho)physiological situations. Studies of genetically modified mice and of molecular pathways in activated glia are beginning to shed light on this issue. Altered expression of different inflammatory factors can either promote or counteract neurodegenerative processes. Since many inflammatory responses are beneficial, directing and instructing the inflammatory machinery may be a better therapeutic objective than suppressing it.     

Acyl CoA profiles of transgenic plants that accumulate medium-chain fatty acids indicate inefficient storage lipid synthesis in developing oilseeds

Several Brassica napus lines transformed with genes responsible for the synthesis of medium- or long-chain fatty acids were examined to determine limiting factor(s) for the subsequent accumulation of these fatty acids in seed lipids. Examination of a decanoic acid (10:0) accumulating line revealed a disproportionately high concentration of 10:0 CoA during seed development compared to long-chain acyl CoAs isolated from the same tissues, suggesting that poor incorporation of 10:0 CoA into seed lipids limits 10:0 fatty acid accumulation. This relationship was also seen for dodecanoyl (12:0) CoA and fatty acid in a high 12:0 line, but not for octadecanoic (18:0) CoA and fatty acid in a high 18:0 line. Comparison of 10:0 CoA and fatty acid proportions from seeds at different developmental stages for transgenic B. napus and Cuphea hookeriana, the source plant for the medium-chain thioesterase and 3-ketoacyl-ACP synthase transgenes, revealed that C. hookeriana incorporates 10:0 CoA into seed lipids more efficiently than transgenic B. napus. Furthermore, beta-oxidation and glyoxylate cycle activities were not increased above wild type levels during seed development in the 8:0/10:0 line, suggesting that lipid catabolism was not being induced in response to the elevated 10:0 CoA concentrations. Taken together, these data suggest that transgenic plants that are engineered to synthesize medium-chain fatty acids may lack the necessary mechanisms, such as specific acyltransferases, to incorporate these fatty acids efficiently into seed lipids.     

Distribution of basement membrane pores in bronchus revealed by microscopy following epithelial removal

The basement membrane of the bronchial epithelium separates the epithelial and mesenchymal compartments. Basement membrane pores allow cells to cross this boundary. We present a method for preparation of samples of human basement membrane allowing us easy visualisation and characterisation of the distribution and persistence of these pores. Columnar epithelial cells were removed from airway samples with gentle scraping with a circular glass coverslip. In contrast, the underlying basal cells required incubation once in dithiothreitol and twice in ethylenediaminetetraacetic acid. Scanning electron microscopy (SEM) at each stage of the epithelial stripping process showed the selective removal of epithelial cells with eventual visualisation of the pores. Using confocal microscopy on blocks of viable tissue, pores were shown to persist in culture for at least 5 days, despite the presence of viable cells in the submucosa. The distribution of pores in tissues determined by SEM was compared to simulations of three distribution patterns (random, clumped, and distributed). The pattern of pores in the samples was consistent with a random distribution. We suggest that basement membrane pores can be generated by the passage of infiltrating cells into the epithelium providing a network suitable for intraepithelial surveillance.     

Biaxial failure behavior of bovine tibial trabecular bone

Multiaxial failure properties of trabecular bone are important for modeling of whole bone fracture and can provide insight into structure-function relationships. There is currently no consensus on the most appropriate form of multiaxial yield criterion for trabecular bone. Using experimentally validated, high-resolution, non-linear finite element models, biaxial plain strain boundary conditions were applied to seven bovine tibial specimens. The dependence of multiaxial yield properties on volume fraction was investigated to quantify the interspecimen heterogeneity in yield stresses and strains. Two specimens were further analyzed to determine the yield properties for a wide range of biaxial strain loading conditions. The locations and quantities of tissue level yielding were compared for on-axis, transverse, and biaxial apparent level yielding to elucidate the micromechanical failure mechanisms. As reported for uniaxial loading of trabecular bone, the yield strains in multiaxial loading did not depend on volume fraction, whereas the yield stresses did. Micromechanical analysis indicated that the failure mechanisms in the on-axis and transverse loading directions were mostly independent. Consistent with this, the biaxial yield properties were best described by independent curves for on-axis and transverse loading. These findings establish that the multiaxial failure of trabecular bone is predominantly governed by the strain along the loading direction, requiring separate analytical expressions for each orthotropic axis to capture the apparent level yield behavior.     

Free fatty acid-induced hepatic insulin resistance: a potential role for protein kinase C-delta

The mechanisms of the impairment in hepatic glucose metabolism induced by free fatty acids (FFAs) and the importance of FFA oxidation in these mechanisms remain unclear. FFA-induced peripheral insulin resistance has been linked to membrane translocation of novel protein kinase C (PKC) isoforms, but the role of PKC in hepatic insulin resistance has not been assessed. To investigate the biochemical pathways that are induced by FFA in the liver and their relation to glucose metabolism in vivo, we determined endogenous glucose production (EGP), the hepatic content of citrate (product of acetyl-CoA derived from FFA oxidation and oxaloacetate), and hepatic PKC isoform translocation after 2 and 7 h Intralipid + heparin (IH) or SAL in rats. Experiments were performed in the basal state and during hyperinsulinemic clamps (insulin infusion rate, 5 mU. kg(-1). min(-1)). IH increased EGP in the basal state (P < 0.001) and during hyperinsulinemia (P < 0.001) at 2 and 7 h. Also, 7-h infusion of IH induced resistance to the suppressive effect of insulin on EGP (P < 0.05). Glycerol infusion (resulting in plasma glycerol levels similar to IH infusion) did not have any effect on EGP. IH increased hepatic citrate content by twofold, independent of the insulin levels and the duration of IH infusion. IH induced hepatic PKC-delta translocation from the cytosolic to membrane fraction in all groups. PKC-delta translocation was greater at 7 compared with 2 h (P < 0.05). In conclusion, 1) increased FFA oxidation may contribute to the FFA-induced increase in EGP in the basal state and during hyperinsulinemia but is not associated with FFA-induced hepatic insulin resistance, and 2) the progressive insulin resistance induced by FFA in the liver is associated with a progressive increase in hepatic PKC-delta translocation.     

Site-directed perturbation of protein kinase C- integrin interaction blocks carcinoma cell chemotaxis

Polarized cell movement is an essential requisite for cancer metastasis; thus, interference with the tumor cell motility machinery would significantly modify its metastatic behavior. Protein kinase C alpha (PKC alpha) has been implicated in the promotion of a migratory cell phenotype. We report that the phorbol ester-induced cell polarization and directional motility in breast carcinoma cells is determined by a 12-amino-acid motif (amino acids 313 to 325) within the PKC alpha V3 hinge domain. This motif is also required for a direct association between PKC alpha and beta 1 integrin. Efficient binding of beta 1 integrin to PKC alpha requires the presence of both NPXY motifs (Cyto-2 and Cyto-3) in the integrin distal cytoplasmic domains. A cell-permeant inhibitor based on the PKC-binding sequence of beta 1 integrin was shown to block both PKC alpha-driven and epidermal growth factor (EGF)-induced chemotaxis. When introduced as a minigene by retroviral transduction into human breast carcinoma cells, this inhibitor caused a striking reduction in chemotaxis towards an EGF gradient. Taken together, these findings identify a direct link between PKC alpha and beta 1 integrin that is critical for directed tumor cell migration. Importantly, our findings outline a new concept as to how carcinoma cell chemotaxis is enhanced and provide a conceptual basis for interfering with tumor cell dissemination.     

Identification of a mutation causing mucopolysaccharidosis type IIIA in New Zealand Huntaway dogs

Mucopolysaccharidosis type IIIA (MPS IIIA) is an autosomal recessive disease that occurs due to a deficiency of heparan sulfate sulfamidase (SGSH). The deficiency of SGSH results in the lysosomal accumulation and urinary excretion of the glycosaminoglycan heparan sulfate. The clinical severity of MPS IIIA is predominantly characterized by severe central nervous system degeneration. Naturally occurring MPS IIIA has recently been described in New Zealand Huntaway dogs, with similar disease progression and biochemical characteristics observed in severely affected MPS IIIA patients. Here, we identify the disease-causing mutation in the MPS IIIA Huntaway dog as 708-709insC. The frequency of the 708-709insC mutation in a sample group of 203 New Zealand Huntaway dogs was determined to be 3.8%. The identification of the 708-709insC mutation will permit the identification of heterozygous carriers as an initial step toward establishing a breeding colony of MPS IIIA dogs for the study of various therapeutic strategies targeted to the central nervous system.     

Exercise,free radicals,and lipid peroxidation in type 1 diabetes mellitus

Indirect biochemical techniques have solely been used to ascertain whether type 1 diabetes mellitus patients are more susceptible to resting and exercise-induced oxidative stress. To date there is no direct evidence to support the contention that type 1 diabetic patients have increased levels of free radical species. Thus, the aim of this study was to use electron spin resonance (ESR) spectroscopy in conjunction with alpha-phenyl-tert-butylnitrone (PBN) spin trapping to measure pre- and postexercise free radical concentration in the venous blood of young male patients with type 1 diabetes mellitus (HbA(1c) = 8.2 +/- 1%, n = 12) and healthy matched controls (HbA(1c) = 5.5 +/- 0.2%, n = 13). Supporting measures of lipid peroxidation (malondialdehyde and lipid hydroperoxides), ambient blood glucose and selected antioxidants were also measured. The diabetic patients presented with a comparatively greater concentration of free radicals as measured by ESR and lipid hydroperoxides (LH) compared to the healthy group (p <.05, pooled rest and exercise data), although there was no difference in malondialdehyde (MDA) concentration. alpha-Tocopherol was comparatively lower in the healthy group (p <.05, pooled rest and exercise data vs. diabetic group) due to a selective decrease during physical exercise (p <.05 vs. rest). The hyperfine coupling constants recorded from the ESR spectra (a(Nitrogen) = 1.37 mT and abeta(Hydrogen) = 0.17 mT) are suggestive of either oxygen or carbon-centered species and are consistent with literature values. We suggest that the greater concentration of oxidants seen in the diabetic group may be due to increased glucose autoxidation as a function of this pathology and/or a lower exercise-induced oxidation rate of the major lipid soluble antioxidant alpha-tocopherol. We suggest that the ESR-detected radicals are secondary species derived from decomposition of LH because these are the major initial reaction products of free radical attack on cell membranes.