Gelatin Embedding of Central Nervous System Tissue Improves the Quality of Vibratome Sections

We have developed a procedure for Vibratome (Oxford Laboratories) sections that is particularly valuable for providing uniformly thick, well preserved CNS tissue sections for morphometric applications.     

Immunohistochemical detection of strain-specific major histocompatibility complex class I antigens in paraffin-embedded rat osteochondral tissue

Summary An immunohistochemical method using formalin-fixed, paraffin wax-embedded sections is described for detecting strain-specific major histocompatibility complex class I antigens in knee-joint tissue from DA and Lewis strains of rat. The fixed osteochondral tissues were additionally decalcified in formic acid before processing for paraffin wax embedding. For immunohistochemistry, two monoclonal antibodies, one specific for DA class I allele RT1Aa and the other for Lewis class I allele RT1A1, were used together with the avidin-biotin immunoperoxidase procedure. It was necessary to use strain-specific normal rat serum as a diluent for the antibodies to suppress cross-strain recognition. DA-specific antibody stained positively only on DA rat sections, not on Lewis rat sections, and Lewis-specific antibody stained positively only on Lewis rat sections, and not on DA. Positive staining was localized in the bone marrow, osteochondral cells and endothelium. We propose that the use of a decalcification medium may have enhanced the immunoreactivity of the tissue. The method described can be used on sections of allografts from the two strains of rat to assess morphologically the extent of cellular replacement of the graft by the host's cells.     

Micromethods in single muscle fibers. 1. Determination of catalase and superoxide dismutase

Methods have been developed for the measurements of catalase and superoxide dismutase (SOD) in single, isolated muscle fibers. These fibers are also classified according to fiber type. Catalase is determined using a fluorescent method for the measurement of hydrogen peroxide consumed. SOD measurements are carried out using a modification of established techniques whereby the inhibition of oxidation of epinephrine by SOD is assayed fluorometrically. Both enzymes may be determined in submicrogram samples of dried muscle. This approach avoids the complication of the inclusion of nonmuscle tissue with varying enzymatic activities which is frequently experienced when using homogenates of muscle, particularly diseased muscle. In addition, these techniques can be used to determine the inherent variation in SOD and catalase activities within individual fibers of the same fiber type. The Km and Vmax for catalase, determined using homogenates of human muscle, were found to be 12 mM and 1.45 mumol/min/mg dry wt, respectively. Catalase of muscle was inhibited 50% by 2 microM sodium azide. Mn-SOD contributes less than one-fifth of the total SOD activity. Therefore the activity is largely due to the Cu-Zn form of SOD. These methods are applicable to a wide variety of tissues.     

Metabolomic approaches for the identification of flavonoids associated with weed suppression in selected Hardseeded annual pasture legumes

Plant and Soil - Weed suppressive potential of annual pasture legumes has been previously described, the mechanism of interference with weeds has not been clearly elucidated. We, therefore, aimed...     

Sex-dependent structural asymmetry of the medial habenular nucleus of the chicken brain

Summary An investigation of structural asymmetry in the avian brain was conducted on the epithalamic medial habenular nucleus of the chicken. Twelve male and ten female two-day-old chickens were used for a morphometric evaluation of asymmetry. The medial habenular nucleus was measured from paraffin-wax-embedded, 8 m-thick sections by use of a semiautomatic image analyser. The volumes of the right and left medial habenula of each animal were statistically analysed (within animal experimental design). The right medial habenula in males showed significant group asymmetry. In contrast, females failed to demonstrate group bias in favour of either hemisphere. However, individual females were lateralised, with either a larger right or left medial habenula. Although individuals of both sexes were lateralised, there was no significant sex difference in volume in either the right or left medial habenula.We propose that sex-linked structural asymmetry may be influenced by steroid hormonal effects in the central nervous system, and that such asymmetry could be more prevalent in the non-mammalian vertebrate brain than previously considered.     

Duchenne muscular dystrophy and dystrophin: Sequence homology observations

Duchenne muscular dystrophy (DMD) is a genetically transmitted disease characterized by progressive muscle weakness and usually leads to death. DMD results from the absence, deficiency or dysfunction of the protein dystrophin. Analysis of protein data bases, including homology alignments and domain recognition patterns, have located highly significant correlations between dystrophin and other calcium regulating proteins. In particular, a major portion of the dystrophin sequence has been found to contain repeating units of approximately 100 amino acid residues. These repeating units were found to exhibit significant homology to troponin I. Troponin I has been found to bind to the calcium binding proteins calmodulin and troponin C. The regions of highest homology were characterized by patterns of high localization of charged amino acids and thus could represent a possible calmodulin or troponin C surface accessible binding site. Since subcellular localization studies have indicated that dystrophin is associated with the triadic junction, these findings imply that dystrophin could be involved in controlling intracellular calcium homeostasis.Special issue dedicated to Dr. Lawrence Austin.     

Generation of a nitric oxide signaling pathway in mesenchymal stem cells promotes endothelial lineage commitment

Enhancing differentiation of mesenchymal stem cells (MSCs) to endothelial cells may improve their ability to vascularize tissue and promote wound healing. This study describes a novel role for nitric oxide (NO) in reprogramming MSCs towards an endothelial lineage and highlights the role of Wnt signaling and epigenetic modification by NO. Rat MSCs were transduced with lentiviral vectors expressing endothelial nitric oxide synthase (pLV-eNOS) and a mutated caveolin gene (pLV-CAV-1^F92A) to enhance NO generation resulting in increased in vitro capillary tubule formation and endothelial marker gene expression. An exogenous source of NO could also stimulate CD31 expression in MSCs. NO was associated with an arterial-specific endothelial gene expression profile of Notch1, Dll4, and Hey2 and significantly reduced expression of venous markers. Wnt signaling associated with NO was evident through increased gene expression of Wnt3a and β-catenin protein, and expression of the endothelial marker Pecam-1 could be significantly reduced by treatment with the Wnt signaling inhibitor Dkk-1. The role of NO as an epigenetic modifier was evident with reduced gene expression of the methyltransferase, DNMT1, and bisulfite sequencing of the endothelial Flt1 promoter region in NO-producing MSCs showed significant demethylation compared to control cells. Finally, subcutaneous implantation of NO-producing MSCs seeded in a biomaterial scaffold (NovoSorb®) resulted in survival of transplanted cells and the formation of blood vessels. In summary, this study describes, NO as a potent endothelial programming factor which acts as an epigenetic modifier in MSCs and may provide a novel platform for vascular regenerative therapy.     

Metabolic profiling of benzoxazinoids in the roots and rhizosphere of commercial winter wheat genotypes

Plant and Soil - Integrated weed management in commercial wheat production is urgently needed due to increasing herbicide resistance and production costs. Benzoxazinoids (BXs), which include...     

Micromethods in single muscle fibers. 2. Determination of glutathione reductase and glutathione peroxidase

This paper extends the previous study for systems which control intracellular oxidative events in muscle and describes procedures suitable to assay glutathione peroxidase (GSHPx), glutathione reductase (GR), and total glutathione (GSH + GSSG) after fiber typing of individual muscle fibers. In human skeletal muscle, both GR and GSHPx activities were relatively low when compared to those of other tissue. No difference was found among fiber types (I, IIA, and IIB) with regard to GR activity, but in contrast GSHPx activity was significantly lower in type IIB fibers than in the other types. These results suggest that type IIB fibers may have a reduced ability to cope with hydroperoxides generated during oxidative stress, which, in turn, could lead to increased damage to membrane structures by lipid peroxidation or oxidation of sensitive intracellular thiol (-SH) enzymes by hydrogen peroxide. The Km of skeletal muscle GR for GSSG was 27 microM and for NADPH was 22 microM. If one assumes approximately 95% of total glutathione is present in the reduced state, then GSSG concentration would be of the order of 0.3 mmol/kg and under these conditions skeletal muscle GR would be efficient in all muscle fiber types.     

Differential response of PCNA and Cdk-A proteins and associated kinase activities to benzyladenine and abscisic acid during maize seed germination

The proliferating cell nuclear antigen (PCNA) is a protein factor required for processive DNA synthesis that is associated with G(1) cell cycle proteins. It has been demonstrated previously that, in germinating maize (Zea mays) embryonic axes, PCNA forms protein complexes with two Cdk-A proteins (32 and 36 kDa) and with a putative D-type cyclin. These complexes exhibit protein kinase activity on histone H1 and on the maize homologue of the pRB (retinoblastoma) protein. Flow cytometry has been used to study the influence of the phytohormones benzyladenine (BA) and abscisic acid (ABA) on cell cycle advancement during maize germination. It was found that, while BA accelerates the passage of cells from G(1) to G(2), ABA delays cell cycle events so that most cells seem to remain in G(1). The amounts of PCNA and Cdk-A proteins also vary according to the hormone treatment. In embryonic axes, PCNA increases rapidly during early germination in BA, compared with a gradual increase in water, while ABA treatment had only a marginal effect. However, of the two Cdk-A proteins, the 32 kDa protein is strongly reduced after 15 h of imbibition in water while this occurs later when axes are imbibed in BA or ABA. The PCNA-associated protein kinase activity in the BA and ABA treatments falls after 3 h of imbibition compared with activity in the control; however, while kinase activity in the BA treatment continues to decline during imbibition, it remains relatively constant until 24 h of imbibition in the ABA treatment. By contrast, a p13(Suc1)-associated Cdk-A kinase is activated after 15 h of imbibition under all treatments, particularly in ABA. These results suggest that, in maize, ABA delays the germination process by affecting cell cycle advancement, stopping cells mostly in a G(1) state.