Increase in nuclear phosphatidylinositol 3-kinase activity and phosphatidylinositol (3,4,5) trisphosphate synthesis precede PKC-zeta translocation to the nucleus of NGF-treated PC12 cells.

We and others have previously demonstrated the existence of an autonomous nuclear polyphosphoinositide cycle that generates second messengers such as diacylglycerol (DAG), capable of attracting to the nucleus specific protein kinase C (PKC) isoforms (Neri et al. (1998) J. Biol. Chem. 273, 29738-29744). Recently, however, nuclei have also been shown to contain the enzymes responsible for the synthesis of the non-canonical 3-phosphorylated inositides. To clarify a possible role of this peculiar class of inositol lipids we have examined the question of whether nerve growth factor (NGF) induces PKC-zeta nuclear translocation in PC12 cells and whether this translocation is dependent on nuclear phosphatidylinositol 3-kinase (PI 3-K) activity and its product, phosphatidylinositol 3,4, 5-trisphosphate [PtdIns(3,4,5)P(3)]. NGF increased both the amount and the enzyme activity of immunoprecipitable PI 3-K in PC12 cell nuclei. Activation of the enzyme, but not its translocation, was blocked by PI 3-K inhibitors wortmannin and LY294002. Treatment of PC12 cells for 9 min with NGF led to an increase in the nuclear levels of PtdIns(3,4,5)P(3). Maximal translocation of PKC-zeta from the cytoplasm to the nucleus (as evaluated by immunoblotting, enzyme activity, and confocal microscopy) occurred after 12 min of exposure to NGF and was completely abrogated by either wortmannin or LY294002. In contrast, these two inhibitors did not block nuclear translocation of the conventional, DAG-sensitive, PKC-alpha. On the other hand, the specific phosphatidylinositol phospholipase C inhibitor, 1-O-octadeyl-2-O-methyl-sn-glycero-3-phosphocholine, was unable to abrogate nuclear translocation of the DAG-insensitive PKC-zeta. These data suggest that a nuclear increase in PI 3-K activity and PtdIns(3,4,5)P(3) production are necessary for the subsequent nuclear translocation of PKC-zeta. Furthermore, they point to the likelihood that PKC-zeta is a putative nuclear downstream target of PI 3-K during NGF-promoted neural differentiation.-Neri, L. M., Martelli, A. M., Borgatti, P., Colamussi, M. L., Marchisio, M., Capitani, S. Increase in nuclear phosphatidylinositol 3-kinase activity and phosphatidylinositol (3,4, 5) trisphosphate synthesis precede PKC-zeta translocation to the nucleus of NGF-treated PC12 cells.     

Cloning and characterisation of a procorticotrophin-releasing hormone in the IZD-MB-0503 immunocyte line from the insect Mamestra brassicae

The cloning and characterisation of a procorticotrophin-releasing hormone (proCRH) and the related CRH fragment in the IZD-MB-0503 cell line from the leptidopteran Mamestra brassicae were performed. PCR amplification of the genomic DNA reveals a fragment of 276 bp, while inverse PCR shows the presence of a gene consisting of 1153 bp. The comparison of the insect genomic proCRH gene with proCRH cDNA obtained by RACE shows the presence of three introns. There was a 61% identity with the corresponding coding sequence in Tilapia mossambica, and a 65.2% identity with the human proCRH coding sequence.     

Subtype-specific translocation of diacylglycerol kinase alpha and gamma and its correlation with protein kinase C

We examined the translocation of diacylglycerol kinase (DGK) alpha and gamma fused with green fluorescent protein in living Chinese hamster ovary K1 cells (CHO-K1) and investigated temporal and spatial correlations between DGK and protein kinase C (PKC) when both kinases are overexpressed. DGKalpha and gamma were present throughout the cytoplasm of CHO-K1 cells. Tetradecanoylphorbol 13-acetate (TPA) induced irreversible translocation of DGKgamma, but not DGKalpha, from the cytoplasm to the plasma membrane. The (TPA)-induced translocation of DGKgamma was inhibited by the mutation of C1A but not C1B domain of DGKgamma and was not inhibited by staurosporine. Arachidonic acid induced reversible translocation of DGKgamma from the cytoplasm to the plasma membrane, whereas DGKalpha showed irreversible translocation to the plasma membrane and the Golgi network. Purinergic stimulation induced reversible translocation of both DGKgamma and alpha to the plasma membrane. The timing of the ATP-induced translocation of DGKgamma roughly coincided with that of PKCgamma re-translocation from the membrane to the cytoplasm. Furthermore, re-translocation of PKCgamma was obviously hastened by co-expression with DGKgamma and was blocked by an inhibitor of DGK (R59022). These results indicate that DGK shows subtype-specific translocation depending on extracellular signals and suggest that PKC and DGK are orchestrated temporally and spatially in the signal transduction.     

Intracellular trafficking and release of intact edible mushroom lectin from HT29 human colon cancer cells.

Our previous studies have shown that the Galbeta1-3GalNAcalpha- (Thomsen-Friedenreich antigen)-binding lectin from the common edible mushroom Agaricus bisporus (ABL) reversibly inhibits cell proliferation, and this effect is a consequence of inhibition of nuclear localization sequence-dependent nuclear protein import after ABL internalization [Yu, L.G., Fernig, D.G., White, M.R.H., Spiller, D.G., Appleton, P., Evans, R.C., Grierson, I., Smith, J.A., Davies, H., Gerasimenko, O.V., Petersen, O.H., Milton, J.D. & Rhodes, J.M. (1999) J. Biol. Chem. 274, 4890-4899]. Here, we have investigated further the intracellular trafficking and fate of ABL after internalization in HT29 human colon cancer cells. Internalization of 125I-ABL occurred within 30 min of the lectin being bound to the cell surface. Subcellular fractionation after pulse labelling of the cells with 125I-ABL for 2 h at 4 degrees C followed by culture of the cells at 37 degrees C demonstrated a steady increase in radioactivity in a crude nuclear extract. The radioactivity in this extract reached a maximum after 10 h and declined after 20 h. Release of ABL from the cell, after pulse labelling, was assessed using both fluorescein isothiocyanate-labelled ABL and 125I-ABL and was slow, with a t1/2 of 48 h. Most of the 125I-ABL both inside cells and in the medium remained intact, as determined by trichloroacetic acid precipitation and SDS/PAGE, and after 48 h only 22 +/- 2% of ABL in the medium and 14 +/- 2% inside the cells was degraded. This study suggests that the reversibility of the antiproliferative effect of ABL is associated with its release from cells after internalization. The internalization and subsequent slow release, with little degradation of ABL, reflects the tendency of lectins to resist biodegradation and implies that other endogenous or exogenous lectins may be processed in this way by intestinal epithelial cells.     

Role of the sonchus yellow net virus N protein in formation of nuclear viroplasms

Sonchus yellow net virus is a plant nucleorhabdovirus whose nucleocapsid (N), phosphoprotein (P), and polymerase (L) proteins form large viroplasms in the nuclei of infected plants (C. R. F. Martins, J. A. Johnson, D. M. Lawrence, T. J. Choi, A. Pisi, S. L. Tobin, D. Lapidus, J. D. O. Wagner, S. Ruzin, K. McDonald, and A. O. Jackson, J. Virol. 72:5669-5679, 1998). When expressed alone, the N protein localizes to the nuclei of plant and yeast (Saccharomyces cerevisiae) cells and the P protein is distributed throughout the cells, but coexpression of N and P results in formation of subnuclear viroplasm-like foci (M. M. Goodin, J. Austin, R. Tobias, M. Fujita, C. Morales, and A. O. Jackson, J. Virol. 75:9393-9406, 2001; M. M. Goodin, R. G. Dietzgen, D. Schichnes, S. Ruzin, and A. O. Jackson, Plant J. 31:375-383, 2002). We now show that the N protein and various fluorescent derivatives form similar subnuclear foci in plant cells and that homologous interactions mediated by a helix-loop-helix region near the amino terminus are required for formation of the foci. Mutations within the helix-loop-helix region also interfere with N- and P-protein interactions that are required for N and P colocalization in the subnuclear foci. Affinity purification of N proteins harboring single mutations within the motif revealed that Tyr40 is critical for N-N and N-P interactions. Additional in vitro binding assays also indicated that the N protein binds to yeast and plant importin alpha homologues, whereas mutations in the carboxy-terminal nuclear localization signal abrogate importin alpha binding. The P protein did not bind to the importin alpha homologues, suggesting that the N and P proteins use different pathways for nuclear entry. Our results in toto support a model suggesting that during infection, the N and P proteins enter the nucleus independently, that viroplasm formation requires homologous N-protein interactions, and that P protein targeting to the viroplasm requires N-P protein interactions that occur after N and P protein import into the nucleus.     

Myxoma virus M11L blocks apoptosis through inhibition of conformational activation of Bax at the mitochondria

Many viruses inhibit or retard apoptosis, a strategy that subverts one of the most ancient antiviral mechanisms. M11L, a myxoma virus-encoded antiapoptotic protein, has been previously shown to localize to mitochondria and block apoptosis of virus-infected cells (H. Everett, M. Barry, S. F. Lee, X. J. Sun, K. Graham, J. Stone, R. C. Bleackley, and G. McFadden, J. Exp. Med. 191:1487-1498, 2000; H. Everett, M. Barry, X. Sun, S. F. Lee, C. Frantz, L. G. Berthiaume, G. McFadden, and R. C. Bleackley, J. Exp. Med. 196:1127-1139, 2002; and G. Wang, J. W. Barrett, S. H. Nazarian, H. Everett, X. Gao, C. Bleackley, K. Colwill, M. F. Moran, and G. McFadden, J. Virol. 78:7097-7111, 2004). This protection from apoptosis involves constitutive-forming inhibitory complexes with the peripheral benzodiazepine receptor and Bak on the outer mitochondrial membrane. Here, we extend the study to investigate the interference of M11L with Bax activation during the process of apoptosis. Myxoma virus infection triggers an early apoptotic signal that induces rapid Bax translocation from cytoplasm to mitochondria, despite the existence of various viral antiapoptotic proteins. However, in the presence of M11L, the structural activation of Bax at the mitochondrial membrane, which is characterized by the occurrence of a Bax conformational change, is blocked in both M11L-expressing myxoma-infected cells and M11L-transfected cells under apoptotic stimulation. In addition, inducible binding of M11L to the mitochondrially localized Bax is detected in myxoma virus-infected cells and in M11L/Bax-cotransfected cells as measured by immunoprecipitation and tandem affinity purification analysis, respectively. Importantly, this inducible Bax/M11L interaction is independent of Bak, demonstrated by the complete block of Bax-mediated apoptosis in myxoma-infected cells that lack Bak expression. Our findings reveal that myxoma M11L modulates apoptosis by multiple independent strategies which all contribute to the blockade of apoptosis at the mitochondrial checkpoint.     

Single nucleotide polymorphisms in the corticotrophin-releasing hormone and pro-opiomelancortin genes are associated with growth and carcass yield in beef cattle

A single nucleotide polymorphism (SNP) in the corticotrophin-releasing hormone gene (CRH C22G) alters the fourth amino acid in the signal sequence from proline to arginine. Two other SNPs (CRH A145G and C240G) occur in the propeptide region at residue positions 45 and 77, respectively, that result in serine/asparagine and histidine/aspartic acid substitutions respectively. These SNPs, as well as SNPs in pro-opiomelancortin (POMC), leptin (LEP) and melanocortin-4 receptor (MC4R), were evaluated for associations with average daily gain, end-of-test rib-eye area, shipping weight and hot carcass weight in a group of 256 steers using a general linear model. The CRH C22G SNP was associated with end-of-test rib-eye area (P < 0.034) and hot carcass weight (P < 0.0015). The SNP in POMC was associated with shipping weight (P < 0.0078) and hot carcass weight (P = 0.006) while it approached significance for average daily gain (P < 0.07). The SNP in MC4R approached significance for hot carcass weight (P < 0.085) while no significance was observed between the leptin SNP and the above listed traits. Because both CRH and POMC regulate appetite, potential interaction effects between these two genes were assessed. The absence of an interaction effect between CRH and POMC with hot carcass weight suggests that these genes act independently to increase carcass yield. These gene effects used singularly or together could result in an economic benefit to the beef industry.     

PLIP, a Novel Splice Variant of Tip60, Interacts with Group IV Cytosolic Phospholipase A2, Induces Apoptosis, and Potentiates Prostaglandin Production

The group IV cytosolic phospholipase A(2) (cPLA(2)) has been localized to the nucleus (M. R. Sierra-Honigmann, J. R. Bradley, and J. S. Pober, Lab. Investig. 74:684-695, 1996) and is known to translocate from the cytosolic compartment to the nuclear membrane (S. Glover, M. S. de Carvalho, T. Bayburt, M. Jonas, E. Chi, C. C. Leslie, and M. H. Gelb, J. Biol. Chem. 270:15359-15367, 1995; A. R. Schievella, M. K. Regier, W. L. Smith, and L. L. Lin, J. Biol. Chem. 270:30749-30754, 1995). We hypothesized that nuclear proteins interact with cPLA(2) and participate in the functional effects of this translocation. We have identified a nuclear protein, cPLA(2)-interacting protein (PLIP), a splice variant of human Tip60, which interacts with the amino terminal region of cPLA(2). Like Tip60, PLIP cDNA includes the MYST domain containing a C2HC zinc finger and well-conserved similarities to acetyltransferases. Both PLIP and Tip60 coimmunoprecipitate and colocalize with cPLA(2) within the nuclei of transfected COS cells. A polyclonal antibody raised to PLIP recognizes both PLIP and Tip60. Endogenous Tip60 and/or PLIP in rat mesangial cells is localized to the nucleus in response to serum deprivation. Nuclear localization coincides temporally with apoptosis. PLIP expression, mediated by adenoviral gene transfer, potentiates serum deprivation-induced prostaglandin E(2) (PGE(2)) production and apoptosis in mouse mesangial cells from cPLA(2)(+/+) mice but not in mesangial cells derived from cPLA(2)(-/-) mice. Thus PLIP, a splice variant of Tip60, interacts with cPLA(2) and potentiates cPLA(2)-mediated PGE(2) production and apoptosis.     

Reviews

The Flavonoids. Advances in Research since 1980. Ed. by J. B. H arborne .
Hungry crops : a guide to nutrient deficiencies in field crops By N. J. G rundon .
Microbial ecology: organisms, habitats, activities. By H einz S tolp .
Evolutionary Biology of the Fungi. Ed. by A. D. M. R ayner , C. M. B rasier and D. M oore .
Micro-algal Biotechnology . Edited by M.A. B orowitzka and L. J. B orowitzka and L. J. B orowitzka .
Experimental Phycology: A laboratory manual Edited by C hristopher S. L obban , D avid J. C hapman , and B runo P. K remer .
100 Families of Flowering Plants. By M. H ickey and C. K ing .
Differentiation Patterns in Higher Plants. Edited by K. M. U rbanska .
The Living Tundra. By Yu. I. C hernov , Translated by D. L öve .
North American Terrestrial Vegetation. Edited by M. G. B arbour & W. D. B illings .
Biogeography and Quaternary History in Tropical Latin America. By T. C. W hitmore and G. T. P rance (Editors).
Biogeographical Evolution of the Malay Archipelago . By T. C. W hitmore (Editor).
Postglacial Vegetation of Canada. By J. C. R ichie .     

Urocortin, but not corticotropin-releasing hormone (CRH), activates the mitogen-activated protein kinase signal transduction pathway in human pregnant myometrium: an effect mediated via R1alpha and R2beta CRH receptor subtypes and stimulation of Gq-proteins

CRH and CRH-related peptides such as urocortin mediate their actions in the human myometrium via activation of two distinct classes of CRH receptors, R1 and R2. These heptahelical receptors are able to stimulate a number of different intracellular signals; one key mediator of G protein-activated intracellular signaling is the cascade of p42/p44, mitogen-activated protein kinase (MAPK). We therefore hypothesized that activation of MAPK might mediate CRH and or/urocortin actions in the myometrium. In cultured human pregnant myometrial cells, urocortin but not CRH was able to induce MAPK phosphorylation and activation, suggesting that in the human myometrium these two peptides have distinct actions and biological roles. To identify the particular receptor subtypes mediating this phenomenon, all known CRH receptors present in the human myometrial cells were stably expressed individually in HEK293 and CHO cells, and their ability to activate MAPK was tested. The R1alpha and R2beta, but not the R1beta, R1c, or R1d, receptor subtypes were able to mediate urocortin-induced MAPK activation. The signaling components were further investigated; activation of Gs, Go, or Gi proteins did not appear to be involved, but activation of Gq with subsequent production of inositol triphosphates (IP3) and protein kinase C (PKC) activation correlated with MAPK phosphorylation. Studies on Gq protein activation using [alpha-32P]-GTP-gamma-azidoanilide and IP3 production in cells expressing the R1alpha or R2beta CRH receptors demonstrated that urocortin was 10 times more potent than CRH. Moreover, urocortin (UCN) generated peak responses that were 50-70% greater than CRH in activating the Gq protein and stimulating IP3 production. In conclusion, UCN acting thought multiple receptor subtypes can stimulate myometrial MAPK via induction of the Gq/phospholipase C/IP3/PKC pathway, whereas CRH-induced activation of this pathway appears to be insufficient to achieve MAPK activation.     

Cell cycle regulation of glucocorticoid receptor function.

Glucocorticoid receptor (GR) nuclear translocation, transactivation and phosphorylation were examined during the cell cycle in mouse L cell fibroblasts. Glucocorticoid-dependent transactivation of the mouse mammary tumor virus promoter was observed in G0 and S phase synchronized L cells, but not in G2 synchronized cells. G2 effects were selective on the glucocorticoid hormone signal transduction pathway, since glucocorticoid but not heavy metal induction of the endogenous Metallothionein-1 gene was also impaired in G2 synchronized cells. GRs that translocate to the nucleus of G2 synchronized cells in response to dexamethasone treatment were not efficiently retained there and redistributed to the cytoplasmic compartment. In contrast, GRs bound by the glucocorticoid antagonist RU486 were efficiently retained within nuclei of G2 synchronized cells. Inefficient nuclear retention was observed for both dexamethasone- and RU486-bound GRs in L cells that actively progress through G2 following release from an S phase arrest. Finally, site-specific alterations in GR phosphorylation were observed in G2 synchronized cells suggesting that cell cycle regulation of specific protein kinases and phosphatases could influence nuclear retention, recycling and transactivation activity of the GR.     

Rhodiola rosea L. modulates inflammatory processes in a CRH-activated BV2 cell model

BackgroundRhodiola rosea L. (Crassulaceae) has been used for years in the traditional medicine of several countries as an adaptogen drug, able to preserve homeostasis in response to stress stimuli. Currently R. rosea roots and rhizome are classified as a traditional herbal medicinal product for temporary relief of symptoms of stress, such as fatigue and sensation of weakness by the European Medicines Agency.Hypothesis/PurposeIncreasing evidences suggest the involvement of neuroinflammation in response to stress. However, whether the modulation of neuroinflammatory parameters could be involved in the anti-stress effect of R. rosea has been barely studied. Thus, the aim of this work is to investigate the possible modulation of molecular inflammatory processes elicited by a R. rosea roots and rhizome ethanolic extract in an in vitro model of corticotropin releasing hormone (CRH)-stimulated BV2 microglial cells.MethodsBV2 cells were stimulated with CRH 100 nM and changes in cell viability, cytokines production and heat shock protein 70 (HSP70) levels were evaluated. Intracellular pathways related to inflammation, such as nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) nuclear translocation and mitogen-activated protein kinases (MAPK) activation were also analyzed.ResultsWe found that R. rosea extract (2.7% m/m rosavin and 1% m/m salidroside) 20 µg/ml was able to counteract the neuroinflammatory effect of CRH by inhibiting NF-κB nuclear translocation with a mechanism of action involving the modulation of mitogen-activated protein kinase-activated protein kinase 2 (MKK2), extracellular signal-regulated kinase 1/2 (ERK 1/2) and c-Jun n-terminal kinase (JNK), resulting in a reduction of HSP70 expression.ConclusionThis work expands the knowledge of the intracellular mechanisms involved in R. rosea anti-stress activity and may be useful for the study of other adaptogen drugs.     

Quantitation of cell kinetic responses using simultaneous flow cytometric measurements of DNA and nuclear protein

A rapid procedure was developed for the simultaneous flow cytometric analysis of nuclear protein using fluorescein isothiocyanate, and DNA using propidium iodide in isolated nuclei. The staining procedure did not involve centrifugation and was easily adapted to the staining of human peripheral blood lymphocytes stimulated with phytohemagglutinin, EL4 murine lymphoid tumor cells in suspension culture, and R3327-G rat prostatic adenocarcinoma solid tumor specimens. Histograms of unstimulated and PHA-stimulated HPBL perturbed by actinomycin D, hydroxyurea, 3H-TdR, colcemid, or hydroxyurea + colcemid showed that 1) resting, noncycling G1 (G1Q) cells are distinguished from late G1 (G1AB) cells, 2) early G2 (G2A) cells are distinguished from late G2 (G2B) cells, and 3) mitotic cells are distinguished from G2 cells. Treatment with hydroxyurea resulted in a build-up of cells having high nuclear protein content and 2C DNA content (G1AB), while incubation with 3H-TdR caused an increase in the number of cells with high nuclear protein content and 4C DNA content (G2B). Colcemid-blocked mitotic cells were identified as having low nuclear protein content (lower than G2A nuclei) and 4C DNA content. The nuclear DNA/protein histograms of untreated and colcemid-treated log-phase EL4 cells provided information concerning G1A, G1B, S, G2A, G2B, and M. The method was also used to quantitate the response of androgen-sensitive rat prostatic R3327-G tumors to androgen deprivation following castration. Sample preparation and staining for correlated nuclear DNA/protein measurements takes approximately the same amount of time as for single parameter nuclear DNA measurements.     

Reviews

Advances in Botanical Research . Vol. 14. Ed. by J. A. C allow .
Inorganic Nitrogen Metabolism . Ed. by W. R. U llrich , P. J. A paricio , P. J. S yrett and F. C astillo .
A Centry od Nitrogen Fixation Research . Ed. by F. J. B ergersen and J. R. P ostgate .
Horizons in Lichenology . Ed. by D. H. D alby , D. L. H awksworth and S. L. J ury .
Introduction to Ecological Biochemistry , By J. B. H arborne .
Russell's Soil Conditions and Plant Growth, Ed. by A lan W ild .
Disease and Plant Population Biology . By J. J. B urdon.
Molecular Determinants of Plant Diseases . Ed. by S. N ishimura , C. P. V ance and N. D oke.
British Fungus Flora, Agrics and Boleti 5. Strophariaceae & Coprinaceae P.P.: Hypholoma, Melanotus, Psilocybe, Stropharia, Lacrymaria & Panaeolus, Psilocybe, Stropharia, Lacrymaria & Panaeolus, By R W atling and M. N. G regory .
Mechanisms of Woody Plant Defenses Against Insects . Ed. by W. J. M attson , J. L evieux and C. B ernard -D agan .
Air Pollution and Acid Rain. The Biological Impact . By A. W ellburn .
The Australian Paniceae (Paniceae) . By R. D. W erster .
Kew Index for 1986 . Compiled by R.A. D avies and K.M. L loyd .
Kew Index for 1987 . Compiled by R. A. D avies and K. M. L loyd .
Index Kewensis Supplement XVII . Compiled by J. L. M. P inner , T. A. B ence , R. A. D avies and K. M. L loyd .
Index Kewensis Supplement XVIII . Compiled by J. L. M. P inner , T. A. B ence , R. A. D avies and K. M. L loyd . Edited by R. A. D avies .
The Botany of Mangroves . By P. B. T omlinson .
Floweroing Plants in West Africa . By M argaret S teentoft .
Plants in Danger . By S. D. D avies     

Missense mutations of the interleukin-12 receptor beta 1(IL12RB1) and interferon-gamma receptor 1 (IFNGR1) genes are not associated with susceptibility to lepromatous leprosy in Korea

Interleukin-12 receptor beta 1 ( IL12RB1), interleukin-12 receptor beta 2 ( IL12RB2), and interferon gamma receptor 1 ( IFNGR1) perform important roles in the host defense against intracellular pathogens such as Mycobacteria. Several mutations within their genes have been confirmed as associated with increased susceptibility to mycobacterial infection. However, the association between mutations of the IL12RB1, IL12RB2, and IFNGR1 encoding genes and lepromatous leprosy has not been studied. This study screened for polymorphisms within IL12RB1, IL12RB2, and IFNGR1 encoding genes in the Korean populations using polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP) DNA sequencing assay, and an association study was performed using the missense mutations of 705 A/G (Q214R), 1196 G/C (G378R), 1637 G/A (A525T), and 1664 C/T (P534S) of the IL12RB1, 83 G/A (V14M), and 1443 T/C (L467P) for the IFNGR1 encoding genes. There were no differences in the genotype and allele frequencies of IL12RB1 and IFNGR1 genes between 93 lepromatous leprosy patients and 94 control subjects. In conclusion, missense mutations of 705 A/G (Q214R), 1196 G/C (G378R), 1637 G/A (A525T), 1664 C/T (P534S) of the IL12RB1, 83 G/A (V14 M), and 1443 T/C (L467P) of the IFNGR1 encoding genes have no association with the susceptibility to lepromatous leprosy in the Korean population.     

Investigation of frequency of occurrence of core Y haplogroups in residents of Moscow and St. Petersburg

The collection of DNA from 239 Moscow residents and 62 St. Petersburg residents has been investigated by means of a biochip for genotyping haplogroup markers of the Y chromosome, including M130 (C), M145 (DE), P257 (G), M69 (II), U179 (I), M304 (J), M185 (L), M231 (N), M175 (O), P224 (R), L146 (R1a), and M343 (R1b). The distribution of haplogroup frequency in populations native to Moscow and St. Petersburg has been obtained. Three subsamples that vary in the duration of residence (one, two, or three generations) were compared. The increase in J, G, and R1b frequencies may be related to immigration from the Caucasus and other regions.     

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

A pathway for tumor necrosis factor-alpha-induced Bcl10 nuclear translocation. Bcl10 is up-regulated by NF-kappaB and phosphorylated by Akt1 and then complexes with Bcl3 to enter the nucleus

Bcl10 overexpression and nuclear translocation were originally identified in mucosa-associated lymphoid tissue lymphoma with t(1;14)(p32;q32) chromosome translocation. DNA amplification of Bcl10 was also found in other solid tumors. We have recently shown that nuclear translocation of Bcl10 is a specific molecular determinant of Helicobacter pylori-independent mucosa-associated lymphoid tissue lymphoma (Kuo, S.-H., Chen, L. T., Yeh, K.-H., Wu, M. S., Hsu, H. C., Yeh, P. Y., Mao, T. L., Chen, C. L., Doong, S. L., Lin, J. T., and Cheng, A.-L. (2004) J. Clin. Oncol. 22, 3491-3497). However, the molecular mechanism of Bcl10 nuclear translocation remains unknown. In this study, we observed that tumor necrosis factor-alpha (TNFalpha) up-regulates the expression of Bcl10 and induces a fraction of Bcl10 nuclear translocation in human breast carcinoma MCF7 cells. Chromatin immunoprecipitation assays and electrophoretic mobility shift assays indicated that an NF-kappaB-binding site resides in the Bcl10 5 '-untranslated region. This study also demonstrates that Akt1, activated by TNFalpha, phosphorylates Bcl10 at Ser218 and Ser231 and that phosphorylated Bcl10 subsequently complexes with Bcl3 to enter the nucleus. Either inhibition of Akt1 or depletion of Bcl3 blocks Bcl10 nuclear translocation. In summary, these findings characterize a molecular linkage that directs Bcl10 nuclear translocation in response to TNFalpha treatment.     

Phospholipase C and protein kinase C-β 2 mediate insulin-like growth factor II-dependent sphingosine kinase 1 activation

We recently reported that IGF-II binding to the IGF-II/mannose-6-phosphate (M6P) receptor activates the ERK1/2 cascade by triggering sphingosine kinase 1 (SK1)-dependent transactivation of G protein-coupled sphingosine 1-phosphate (S1P) receptors. Here, we investigated the mechanism of IGF-II/M6P receptor-dependent sphingosine kinase 1 (SK1) activation in human embryonic kidney 293 cells. Pretreating cells with protein kinase C (PKC) inhibitor, bisindolylmaleimide-I, abolished IGF-II-stimulated translocation of green fluorescent protein (GFP)-tagged SK1 to the plasma membrane and activation of endogenous SK1, implicating PKC as an upstream regulator of SK1. Using confocal microscopy to examine membrane translocation of GFP-tagged PKCα, β1, β2, δ, and ζ, we found that IGF-II induced rapid, transient, and isoform-specific translocation of GFP-PKCβ2 to the plasma membrane. Immunoblotting of endogenous PKC phosphorylation confirmed PKCβ2 activation in response to IGF-II. Similarly, IGF-II stimulation caused persistent membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, which does not dissociate from the membrane after translocation. IGF-II stimulation increased diacylglycerol (DAG) levels, the established activator of classical PKC. Interestingly, the polyunsaturated fraction of DAG was increased, indicating involvement of phosphatidyl inositol/phospholipase C (PLC). Pretreating cells with the PLC inhibitor, U73122, attenuated IGF-II-dependent DAG production and PKCβ2 phosphorylation, blocked membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, and reduced sphingosine 1-phosphate production, suggesting that PLC/PKCβ2 are upstream regulators of SK1 in the pathway. Taken together, these data provide evidence that activation of PLC and PKCβ2 by the IGF-II/M6P receptor are required for the activation of SK1.     

The structure of the gill of the trout,Salmo gairdneri (Richardson)

Summary A light and electron microscopic study was made of the structure of the gill arch, filament and secondary lamella of Salmo gairdneri R. Blood pathways through the gill were traced from serial histological sections, and from the examination of ink perfused tissue and perspex casts formed following resin injection of the circulatory system.The epithelium covering the gill consists of unspecialized, dark, chloride and mucous cells. The distribution of specialized cells appears to be related to gill function. The basement membrane underlying the epithelium consists of three layers, the inner collagen layer being continuous with the connective tissue core of the gills.Blood supply to the secondary lamellar respiratory surface is via branchial, filament and secondary lamellar arteries. Blood spaces of the secondary lamellae are delimited by pillar cells containing what appears to be contractile material. The marginal channel of each lamella is bounded distally by cells of endothelial origin. A network of lymph spaces within the filaments connects with efferent branchial arteries. Nutritionary capillaries within the filaments connect with afferent branchial arteries. No shunts between afferent and efferent filament arteries were found.Data from this study and previous physiological and histopathological studies suggest a mechanism for the control of blood flow to suit the respiratory requirements of the fish. This mechanism involves a system of recruitment of additional respiratory units and changes in overall blood flow patterns.This work formed part of a thesis submitted for the degree of Doctor of Philosophy in 1971 and for which M. M. was in receipt of a studentship from the Natural Environmental Research Council. The authors are grateful for the support given by research grants from the M.R.C (P.T.) and the N.E.R.C. (M.M.), and to Prof. G. M. Hughes in whose department the work was carried out.