Different oligosaccharides accumulate in the brain and urine of a cat with α-mannosidosis: structure determination of five brain-derived and seventeen urinary oligosaccharides

Five brain-derived and 17 urinary oligomannose-type oligosaccharides were isolated by ion-exchange chromatography on Mono Q or Dowex, followed by HPLC on Lichrosorb-NH₂ from a Persian cat suffering from -mannosidosis. The structures ofthe carbohydrate chains were determined by 500- or 600-MHz¹H-NMR spectroscopy. Different oligosaccharide patterns were found in brain and urine. 99% of the urinary oligosaccharides possess an (1-6)-linked mannose residue attached to -mannose, whereas only 5% of the brain-derived oligosaccharides contain such a residue. Furthermore, of the urinary carbohydrate chains 71% end with Man1-4GlcNAc1-4GlcNAc and 29% end with Man1-4GlcNAc, whereas the corresponding amounts are 23% and 77%, respectively, for the brain-derived oligosaccharides.Abbreviations MLEV-17 composite pulse devised by M. Levitt - HOHAHA homonuclear Hartman-Hahn spectroscopy - TPPI time-proportional phase incrementation - 2D two dimensional - GlcNAc N-acetylglucosamine - Man mannose - Fuc fucose     

Expression of two cytochromes P450 involved in carcinogen activation in a human colon cell line

Cytochrome P450 is known to cause carcinogen activation and correspondingly increased cancer risk in animal models. In order to determine whether P450 in the colon may be involved in cancer development in the human, the human colon cell line LS174T was examined for the presence of various cytochromes P450. Two isozymes of P450 were identified in the human cell line. Expression of P450IAl or IA2 was increased by treatment of the cell line with benzanthracene; the induction was demonstrated by an increase in RNA hybridizing to a probe for P4501Al and by ethoxyresorufin deethylation activity. Western analysis of microsomes isolated from human colon tissue also demonstrated the presence of P4501A1, as well as a form which cross-reacted to an antibody to human P450IIC9. Another isozyme, P450IIE1, was identified by polymerase chain reaction amplification of RNA from LS174T cells. These results underscore the presence of cytochromes P450 in colonic tissue and provide a basis for the involvement of isozyme-specific P450 mediated reactions in carcinogenesis of the colon.Some of the data presented here were taken from a thesis submitted by D.K.H. in partial fulfillment of the requirements for the Ph.D. degree in the University of Texas Graduate School of Biomedical Sciences.     

Ca2+ transport activities of inside-out vesicles prepared from density-separated erythrocytes from rat and human

The plasma membrane Ca²⁺ ATPase in erythrocytes is vital for the maintenance of intracellular Ca²⁺ levels. Since the cytoplasmic Ca²⁺ concentration is elevated in older erythrocytes, the properties of the Ca²⁺ transport ATPase were examined during cell aging using inside-out vesicles (IOVs) prepared from density-separated, young (less dense, Ey) and old (more dense, Eo) rat and human erythrocytes. The transport of Ca²⁺ and the coupled hydrolysis of ATP were measured using radiolabeled substrates. The calmodulin-independent Ca²⁺ transport activity (Ey, 38.8 vs. Eo, 23.3 nmols/min/mg IOV protein) and the Ca²⁺ dependent ATP phosphohydrolase activity (Ey, 53.5 vs. Eo, 48.8 nmols/min/mg protein) were greater in IOVs prepared from younger (less dense) rat erythrocytes. The calmodulin-independent Ca²⁺ transport activity in IOVs from human erythrocytes was 12.9 nmols/min/mg IOV protein for Ey and 10.7 nmols/min/mg IOV protein for Eo. Inside-out vesicles from older (more dense) cells exhibited a lower pumping efficiency as determined by the calculated stoichiometry, molecule of Ca²⁺ transported per molecule of ATP hydrolyzed (rat: Ey, 0.74 vs. Eo, 0.49; human: Ey, 1.22 vs. Eo, 0.77). The enzymatic activity of rat and human Ey IOVs was labile. The Ca²⁺ transport activity in Ey but not Eo IOVs rapidly declined during cold storage (4°C). The decrease in Ca²⁺ transport activity during aging may accentuate the age-related decline in several erythrocytic properties.Abbreviations IOV Inside-Out Vesicles - Ey Erythrocytes enriched with young (less dense) cells - Eo Erythrocytes enriched with old (more dense) cells - ACEase Acetylcholinesterase     

Evidence for pyroglutamyl peptidase I and prolyl endopeptidase activities in the rat insulinoma cell line RINm 5F: lack of relationship with TRH metabolism

Thyrotropin-R eleasing hormone (TRH)-degrading pyroglutamyl peptidase I(PGP I) and prolyl endopeptidase (PE) activities have been demonstrated in rat insulinoma RINm 5F cell line. These two enzymes catalyze the conversion of TRH to Histydyl-Proline-Diketopiperazine and to acid TRH respectively.After cell fractionation, we found all the PGP I and PE activities in the cytosolic fraction. The membranebound PGP II activity is not detectable in the RINm 5F cells. Further investigations on these two cytosolic enzymes show that pyroglutamyl- and proline-containing peptides are inhibitors of each TRH-degrading enzyme.Gelfiltration chromatography on Sephadex G100 shows that PGP I and PE activity have an apparent molecular mass of about 18 kDa and 57 kDa, respectively. Kinetic analysis with TRH as substrate, gives a Km of 44 µM and 235 µM, and a Vmax of 1.49 and 8.80 pmoUmin/µg protein for PGP I and PE, respectively. Immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH levels in the cell line extracts are 2.2 ± 0.9, 22.5 ± 11.1 and 28.7 ± 14.6pg/10⁶ cells, respectively. When cells have been incubated for 2 to 72 hours with a P. E. inhibitor (Z-Gly-Pro-CHN₂) at 5 × 10^–7M, both cell PGP I and PE activities are inhibited. No change in the cellular content of immunoreactive TRH, His-Pro-Diketopiperazine and acid TRH have been observed in treated cells.These data suggest that TRH is not degraded by cytosolic, unspecific PGP I and PE enzymes in RINm 5F. The finding that these cells contain 10 and 13 times more His-Pro-Diketopiperazine and acid TRH than TRH may be an indirect evidence for the existence of another precursor than TRH for these two peptides or of the possibility that TRH can be degraded by other peptidases.Abbreviations TRH Thyrotropin-Releasing Hormone or Thyroliberin - His-Pro-DKP Histidyl-ProlineDiketopiperazine - TRH-OH acid TRH or deamidated TRH - LH-RH Luteinizing Hormone-Releasing Hormone - Z-Gly-Pro-CHN₂ N-benzyloxycarboxyl-Gly-Pro-diazomethylketone - PGP Pyroglutamyl Peptidase, PGP I (EC 3.4.19.3) and PGP II (EC 3.4.19.-) - PE Prolyl Endopeptidase or post-proline cleaving enzyme (EC 3.4.21.26)     

Human and mouse S-protein mRNA detected in Northern blot experiments and evidence for the gene encoding S-protein in mammals by Southern blot analysis

Summary Human S-protein is a serum glycoprotein that binds and inhibits the activated complement complex, mediates coagulation through interaction with antithrombin III and plasminogen activator inhibitor I, and also functions as a cell adhesion protein through interactions with extracellular matrix and cell plasma membranes. A full length cDNA clone for human S-protein was isolated from a lambda gt11 cDNA library of mRNA from the HepG2 hepatocellular carcinoma cell line using mixed oligonucleotide sequences predicted from the amino-terminal amino acid sequence of human S-protein. The cDNA clone in lambda was subcloned into pUC18 for Southern and Northern blot experiments. Hybridization with radiolabeled human S-protein cDNA revealed a single copy gene encoding S-protein in human and mouse genomic DNA. In addition, the S-protein gene was detected in monkey, rat, dog, cow and rabbit genomic DNA. A 1.7 Kb mRNA for S-protein was detected in RNA from human liver and from the PLC/PRF5 human hepatoma cell line. No S-protein mRNA was detected in mRNA from human lung, placenta, or leukocytes or in total RNA from cultured human embryonal rhabdomyosarcoma (RD cell line) or cultured human fibroblasts from embryonic lung (IMR90 cell line) and neonatal foreskin. A 1.6 Kb mRNA for S-protein was detected in mRNA from mouse liver and brain. No S-protein mRNA was detected in mRNA from mouse skeletal muscle, kidney, heart or testis.     

Differential cytotoxicity and mycotoxin content among isolates of Fusarium moniliforme

Curvularia lunata was found causing (disseminated phaeohyphomycosis among a group of Nezara viridula (Insecta:Heteroptera) parasitizing vegetable crop Vigna unguiculata. Dark lesions were seen on pronotum and abdominal sterna. Experimental lesions were produced by applying 0.1 ml of 6.2 × 10⁸cfu/ml^–1 on abdominal sterna. Histopathology revealed that almost all internal organs and tissues showed extensive damage. It is interesting to note that C. lunata exhibited predeliction for chitinous tissues and elicited cellular immune response by granulocytes (phagocytosis). This is the first report of phaeohyphomycosis of an insect, extending the disease to invertebrates.A promising research career of one of the authors (Mrs. Vinita Dubey) was cut short by untimely death. This paper is dedicated to her memory.     

Retinopetal neuronal system in the brain of an air-breathing teleost fish,Channa punctata

Summary Retinopetal neurons were visualised in the telencephalon and diencephalon of an air-breathing teleost fish, Channa punctata, following administration of cobaltous lysine to the optic nerve. The labelled perikarya (n=45–50) were always located on the side contralateral to the optic nerve that had received the neuronal tracer. The rostral-most back-filled cell bodies were located in the nucleus olfactoretinalis at the junction between the olfactory bulb and the telencephalon. In the area ventralis telencephali, two groups of telencephaloretinopetal neurons were identified near the ventral margin of the telencephalon. The rostral hypothalamus exhibited retrogradely labelled cells in three discrete areas of the lateral preoptic area, which was bordered medially by the nucleus praeopticus periventricularis and nucleus praeopticus, and laterally by the lateral forebrain bundle. In addition to a dorsal and a ventral group, a third population of neurons was located ventral to the lateral forebrain bundle adjacent to the optic tract. The dorsal group of neurons exhibited extensive collaterals; a few extended laterally towards the lateral forebrain bundle, whereas others ran into the dorsocentral area of the area dorsalis telencephali. A few processes extended via the anterior commissure into the telencephalon ipsilateral to the optic nerve that had been exposed to cobaltous lysine. However, the ventral cell group did not possess collaterals. In the diencephalon, retinopetal cells were visualised in the nucleus opticus dorsolateralis located in the pretectal area; these were the largest retinopetal perikarya of the brain. The caudal-most nucleus that possessed labelled somata was the retinothalamic nucleus; it contained the largest number of retinopetal cells. The limited number of widely distributed neurons in the forebrain, some with extensive collaterals, might participate in functional integration of different brain areas involved in feeding, which in this species is influenced largely by taste, not solely by vision.     

Immunohistochemical localization of porcine diazepam-binding inhibitor (DBI) to rat endocrine pancreas

Summary The occurrence of diazepam-binding inhibitor (DBI), isolated and characterized from porcine upper intestine, was examined in the pancreas of Sprague-Dawley albino rats using indirect immunofluorescence. The polypeptide was found in the endocrine Langerhans islets and, utilizing double-labelling controls, it was shown to be present within the peripherally located glucagon-containing cells. Regulation of islet hormone production may therefore be under DBI control.     

An interplexiform cell in the goldfish retina: light-evked response pattern and intracellular staining with horseradish peroxidase

Summary The light-evoked response pattern and morphology of one interplexiform cell were studied in the goldfish retina by intracellular recording and staining. The membrane potential of the cell spontaneously oscillated in the dark. In response to a brief light stimulus, the membrane potential initially gave a slow transient depolarization. During maintained light, the oscillations showed a tendency to be suppressed; the response of the cell to the offset of the stimulus was not so prominent. The perikaryon of the interplexiform cell was positioned at the proximal boundary of the inner nuclear layer. The cell had two broad layers of dendrites; one was diffuse in the inner plexiform layer, the other was more sparse in the outer plexiform layer. The morphological and electrophysiological characteristics of the cell are discussed in relation to dopaminergic interplexiform cells and the light-evoked release pattern of dopamine in the teleost retina.     

Co-distribution of annexin VI and actin in secretory ameloblasts and odontoblasts of rat incisor

Summary Annexin VI and actin were detected by immunoblot analysis in the enamel- and dentin-related portions of dental tissues. Annexin VI was found mainly in the particulate fraction whereas actin was detected in both the soluble and particulate fractions. By immunoelectron microscopy, annexin VI antibodies conjugated with colloidal gold were seen to label the mitochondria, the cytosol and the nucleus of secretory ameloblasts and odontoblasts of rat incisor. In the processes of these cell, the plasmalemmal undercoat was labeled. Antiactin antibodies labeled the desmosome-like junctions, the cytosol, and the mitochondria of the cell bodies. Extensive labeling was seen at the periphery of the Tomes' processes and odontoblast processes. These results suggest that annexin VI may play a role in Ca²⁺-regulation in the cell bodies, especially as a calcium receptor protein in the mitochondria. Moreover, annexin VI and actin seem to be co-distributed in secretory processes. Thus, these proteins might be both involved in exocytotic and endocytotic events.