Effects of brain ischemia on intermediate filaments of rat hippocampus

Neurofilaments subunits (NF-H, NF-M, NF-L) and glial fibrillary acidic protein (GFAP) were investigated in the hippocampus of rats after distinct periods of reperfusion (1 to 15 days) following 20 min of transient global forebrain ischemia in the rat. In vitro [¹⁴Ca]leucine incorporation was not altered until 48 h after the ischemic insult, however concentration of intermediate filament subunits significantly decreased in this period. Three days after the insult, leucine incorporation significantly increased while the concentration NF-H, NF-M, and NF-L were still diminished after 15 days of reperfusion. In vitro incorporation of³²P into NF-M and NF-L suffered immediately after ischemia, but returned to control values after two days of reperfusion. GFAP levels decreased immediately after ischemia but quickly recovered and significantly peaked from 7 to 10 days after the insult. These results suggest that transient ischemia followed by reperfusion causes proteolysis of intermediate filaments in the hippocampus, and that proteolysis could be facilitated by diminished phosphorylation levels of NF-M and NF-L.     

Human aging brain disorders: Role of antioxidant enzymes

In order to investigate the role of two free radical detoxificant enzymes in patients with aging brain disorders, superoxide dismutase (SOD) and catalase (CAT) activities have been measured in blood from male and female human patients of different ages with several types of aging brain disorders. When compared with activities in the normal population, we have detected: 1) SOD and CAT activities are decreased in patients with Parkinson disease. 2) SOD activity seems to be normal and CAT activity is decreased in patients with dementia. 3) In the patients with stroke, SOD activity is normal, while CAT activity is decreased. SOD activity was measured in red blood cells using the Minami and Yoshikawa method. CAT activity was measured in hemolysates by the method of Aebi. We can conclude that SOD and CAT activities in patients with Parkinson disease are decreased.     

Unusual conformational preferences of β-alanine containing cyclic peptides. VII

In the present paper we describe the synthesis, purification, and single crystal x-ray analysis of the cyclic pentapeptide cyclo-(Pro-Phe-Phe-β-Ala-β-Ala). This compound crystallizes in the orthorhombic space group P2₁2₁2₁ from methanol and adopts in the solid state an unusual conformation characterized by a cis β-Ala⁵-Pro¹ peptide bond and by an intramolecular hydrogen bond stabilizing a C₁₁- and a C₁₂-ring structure. The C₁₁, structure contains the Phe³ and the β-Ala⁴ at the corner position of the turn; it is the first observation of a type II β-turn enlargement due to the insertion of an extra methylene group of the β-alanine residue. The rest of the molecule participates in a newly characterized C₁₂-ring structure, which incorporates a β-Ala residue at position i of the turn. © 1996 John Wiley & Sons, Inc.     

Genetic Analysis of the Epidermal Differentiation Complex (EDC) on Human Chromosome 1q21: Chromosomal Orientation, New Markers, and a 6-Mb YAC Contig

The epidermal differentiation complex (EDC) unites a remarkable number of structurally, functionally, and evolutionarily related genes that play an important role in terminal differentiation of the human epidermis. It is localized within 2.05 Mb of region q21 on human chromosome 1. We have identified and characterized 24 yeast artificial chromosome (YAC) clones by mapping individual EDC genes, sequence-tagged site (STS) markers (D1S305, D1S442, D1S498, D1S1664), and 10 new region-specific probes (D1S3619–D1S3628). Here we present a contig that covers about 6 Mb of 1q21 including the entire EDC. Fluorescencein situhybridization on metaphase chromosomes with two YACs flanking the EDC determined its chromosomal orientation and established, in conjunction with physical mapping results, the following order of genes and STSs: 1cen–D1S442–D1S498–S100A10–THH–FLG–D1S1664–IVL–SPRR3–SPRR1–SPRR2–LOR–S100A9–S100A8–S100A7–S100A6–S100A5–S100A4–S100A3–S100A2–S100A1–D1S305–1qtel. These integrated physical, cytogenetic, and genetic mapping data will be useful for linkage analyses of diseases associated with region 1q21 and for the identification of novel genes and regulatory elements in the EDC.     

Genetic analysis of agronomic traits in a cross between sugarcane (Saccharum officinarum L.) and its presumed progenitor (S. robustum Brandes & Jesw. ex Grassl)

Saccharum robustum Brandes & Jesw. ex Grassl has been suggested as the immediate progenitor species of cultivated sugarcane (S. officinarum L.) [4]. Chromosome pairing and assortment in these two species were previously studied by genetic analysis of single-dose DNA markers in parents in and 44 F₁ progeny of a cross between euploid, meiotically regular 2n=80S. officinarum LA Purple andS. robustum Mol 5829 [2]. This same population was subsequently clonally propagated and evaluated in replicated trials for quantitative traits important to sugarcane breeders. Numbers of stalks, tasseled stalks, and stalks with smut, and the average diameter of two stalks were determined one day prior to harvest. At harvest, plant material from each plot was weighed and evaluated for pol (sucrose content) and fiber percentages. Clones were significantly different (P<0.01) for all traits analyzed. Associations of 83 single-dose arbitrarily primed PCR genetic markers with quantitative trait loci (QTL) of recorded traits was determined by single-factor ANOVA, and multiple regression. QTL analysis revealed markers significantly (P<0.05) associated with the expression of each trait analyzed. Markers associated with QTL after multiple regression were tested for digenic linear × linear epistatic interactions. The various multilocus models explained between 23% and 58% of the total phenotypic variation and 32% and 76% of the genotypic variation for the various traits. Digenic interactions were uncommon. Implications for marker-assisted selection in sugarcane and sugarcane domestication are discussed.     

Platelet activating factor (PAF) stimulates release of PGI2 from inflamed rabbit gallbladder cell cultures

This study examines the hypothesis that PAF stimulates release of PGI₂ from inflamed rabbit gallbladder explant cell cultures. New Zealand white rabbits underwent bile duct ligation for 72 h (72 h BDL), or sham operation, Sham and 72 h BDL gallbladder explants were placed in culture, and the cells grown to 75% confluence. The cells were exposed to increasing concentrations of PAF for 60 min. The media analyzed for eicosanoid release by EIA and the cells analyzed for cyclooxygenase and prostacyclin synthase content by immunoblot analysis. PAF increased release of 6-keto-PGF_1α from the 72 h BDL gallbladder cell cultures in a dose-related manner which was inhibited by indomethacin preincubation by 90%. The increased 72 h BDL cell release of 6-keto-PGF_1α was not associated with changes in the content of cyclooxygenase or prostacyclin synthase. PAF did not alter eicosanoid release from sham control cell cultures. These data suggest that PAF can only up-regulate endogenous 6-keto-PGF_1α release from the 72 h BDL cells that had been previously stimulated by inflammation. PAF may thus contribute to gallbladder distention and injury by chronic stimulation of inflamed gallbladder PGI₂ release.     

Immunolocalization of alpha-transforming growth factor in the developing rat mammary gland in vivo, rat mammary cells in vitro and in human breast diseases

Summary Immunoreactive alpha-transforming growth factor (-TGF) was shown by immunocytochemistry to be present in the rat mammary gland at various stages of development, the staining being most intense in mature myoepithelial cells. -TGF was also detected in the secretions of the mammary glands of pregnant and lactating rats. -TGF in the extracts of rat mammary glands at each stage of development, and in several rat mammary cell lines and in culture medium in which they had been grown, was shown by Western blotting to consist primarily of a protein of molecular weight 50 kDa. The amount of this protein was greater in the mammary gland of the lactating rat than in resting or involuting glands. -TGF was also found in some, but not all, human breast carcinomas, and in benign hyperplastic breast diseases.     

Microtubule inhibitors differentially affect translational movement,cell surface expression,and endocytosis of transferrin receptors in K562 cells

We used quantitative fluorescence microscopy and fluorescence photobleaching recovery techniques to investigate the translational movement, cell surface expression, and endocytosis of transferrin receptors in K562 human erythroleukemia cells. Receptors were labeled with fluorescein-conjugated transferrin (FITC-Tf). Coordinated decreases in surface fluorescence counts, the photobleachig parameter K, and transferrin receptor fractional mobility were observed as FITC-Tf was cleared from the cell surface by receptor-mediated endocytosis. Based on the kinetics of decrease in these parameters, first order rate constants for FITC-Tf uptake at 37°C and 21°C were calculated to be 0.10-0.15 min^?1 and 0.02–0.03 min, respectively. K562 cells were treated with colchicine or vinblastine to investigate the role of microtubules in transferrin receptor movement and endocytosis. Treatment of cells for 1 hr with a microtubule inhibitor prevented transferrin receptor endocytosis but had no effect on the translational mobility of cell surface receptors. In contrast, drug treatment for 3 hr caused translational immobilization of cell surface receptors as well as inhibition of endocytosis. These effects were not produced by β-lumicolchicine, an inactive colchicine analog, or by cytochalasin, a microfilament inhibitor. The effect of microtuble inhibitors on transferrin receptor mobility was reversed by pretreating cells with taxol, a microtubule-stabilizing agent. Microtubule inhibitors had no effect on the translational mobility of cell surface glycophorins or phospholipids, indicating that intact microtubules were not required for translational movement of these molecules. We conclude that the translational movement of cell surface transferrin receptors is directed by a subpopulation of relatively drug-resistant microtubules. In contrast, transferrin receptor endocytosis depends on a subpopulation of microtubules that is relatively sensitive to the action of inhibitors. These results appear to demonstrate at least two functional roles for microtubules in receptor-mediated transferrin uptake in K562 cells. © 1994 Wiley-Liss, Inc.