The effect of vanadate on glucose transport and metabolism in rat small intestine

The effect of sodium orthovanadate on the absorption, transmural transport and metabolism of glucose was studied by perfusion of isolated loops of rat jejunum in vitro. The presence of 1 mM vanadate in the serosal medium diminished absorption from 539 +/- 19 (n = 12) to 246 +/- 19 (P less than 0.001) mumol/h per g dry weight and transmural transport from 333 +/- 17 to 14 +/- 19 (P less than 0.001) mumol/h per g dry weight, whereas glucose utilisation was unaffected. The rate of release of lactate into the serosal medium was also diminished from 168 +/- 14 to 75 +/- 5 mumol/h per g dry weight (P less than 0.001). The observed rates were linear with respect to time and vanadate was effective within 5 min. In contrast, the rate of release of lactate into the luminal perfusate was strongly enhanced. Moreover, the progress curve showed a positive transient with an apparent lag time of 18.0 +/- 0.3 min, during which the rate increased to a value 9.2-times that of the control. Under the final steady-state conditions, the ratio of mucosal to serosal lactate production was 5.2 +/- 0.2 compared with 0.25 +/- 0.06 for the control, so that the effect of vanadate was to reverse the vectorial disposition of lactate. The concentration dependence of the effect of vanadate on absorption and metabolism was similar to that observed for the inhibition by vanadate of Na+/K+-ATPase activity in mucosal homogenates. The results are discussed in terms of the dissipation of transmembrane Na+ gradients as a result of the inhibition of the Na+/K+-ATPase.     

Host-parasite associations of Eimeria spp. (Apicomplexa:Eimeriidae) in kangaroos and wallabies of the genus Macropus (Marsupialia:Macropodidae)

Faecal samples from 514 kangaroos and wallabies representing 12 species of the genus Macropus were examined for oocysts of Eimeria spp. Six species of Eimeria were redescribed from their type hosts, and on the basis of finding homologous oocysts in the faeces of other Macropus spp., host ranges for these coccidia were extended. Eimeria hestermani Mykytowycz, 1964 is redescribed from M. giganteus (eastern grey kangaroo) and is described from M. fuliginosus (western grey kangaroo), M. rufogriseus (red-necked wallaby), M. dorsalis (black-striped wallaby), and M. eugenii (tammar wallaby). E. toganmainensis Mykytowycz, 1964 is redescribed from M. rufus (red kangaroo) and the host range is extended to M. giganteus, M. fuliginosus, M. rufogriseus and M. eugenii. E. wilcanniensis Mykytowycz, 1964 is redescribed from M. rufus, and the host range is extended to M. giganteus, M. fuliginosus and M. robustus (euro or wallaroo). E. macropodis Wenyon & Scott, 1925 is redescribed from M. rufogriseus, and is described from M. giganteus, M. fuliginosus, M. rufus, M. irma (western brush wallaby), M. parryi (whip-tailed wallaby), M. dorsalis, M. eugenii, and M. parma (parma wallaby). E. fausti Yakimoff & Matschoulsky, 1936, E. cunnamullensis Mykytowycz, 1964 and E. purchasei Mykytowycz, 1964 are synonymized with E. macropodis. E. marsupialium Yakimoff & Matschoulsky, 1936 is redescribed from M. giganteus, and from M. fuliginosus. E. gungahlinensis Mykytowycz, 1964 is redescribed from M. fuliginosus, and from M. giganteus. Seven new species of Eimeria are described. E. flindersi, new species, is described from M. eugenii, M. rufogriseus, and M. antilopinus (antilopine wallaroo). E. prionotemni, new species, is described from M. eugenii, M. parryi, M. rufogriseus, M. agilis (agile wallaby) and M. dorsalis. E. mykytowyczi, new species, is described from M. agilis, M. antilopinus, and M. parryi. E. parryi, new species, is described from M. parryi. E. yathongensis, new species, is described from M. fuliginosus and M. giganteus. E. parma, new species, is described from M. parma, and E. desmaresti, new species, is described from M. rufogriseus. E. kogoni Mykytowycz, 1964, and E. rufusi Prasad, 1960 are considered species inquirendae. The host-parasite associations of these coccidia, and of similar species of Eimeria in other genera of Macropodoid marsupials, are discussed in relation to the postulated phylogeny of the hosts.     

Expression of 9E3 mRNA is associated with mitogenicity, phosphorylation, and morphological alteration in chicken embryo fibroblasts.

Transformation of chicken embryo fibroblasts (CEF) with viruses encoding src, ros, yes, and fps as well as ras, mos, middle T, erbA and erbB, myc, and crk stimulated 9E3 mRNA expression. Treatment of CEF with agents that modulate cell shape or attachment to the substratum caused an increase in 9E3 mRNA without an increase in tyrosine phosphorylation. 9E3 mRNA was also increased in CEF in response to several agents which modulate phosphorylation, including phorbol myristic acetate, vanadate, and okadaic acid, which suggests that the rapid induction of 9E3 mRNA expression in CEF by the src protein occurs downstream of morphological or phosphorylation events.     

Functional expression of dihydropyridine-insensitive calcium channels during PC12 cell differentiation by nerve growth factor (NGF), oncogenic ras,or src tyrosine kinase

1. Recombinant retroviruses were used to introduce a temperature-sensitive v-src gene and oncogenic c-Ha-ras into PC12 cells, and stable cell lines expressing these genes were established. 2. As previously reported, expression of v-src (Alema et al., 1985) or c-Ha-ras (Noda et al., 1985) in PC12 cells results in neurite outgrowth resembling that induced by NGF. We report here that v-src but not oncogenic c-Ha-ras induces a stable morphologic neuronal differentiation similar to treatment with NGF. Oncogenic c-Ha-ras-induced neurite outgrowth is not stable with long-term culture, rather the cells revert to an undifferentiated morphology with altered cell cycle kinetics. 3. The stable neuronal phenotype induced by v-src and NGF is characterized by the functional expression of dihydropyridine-insensitive calcium currents.     

Intrinsic potential for high fetal hemoglobin production in a Druze family with β-thalassemia is due to an unlinked genetic determinant

Summary The mechanism for elevated production of fetal hemoglobin (Hb F) in a Druze patient with °-thalassemia intermedia was investigated. Heterozygous family members exhibited normal Hb F levels, suggesting that the increase in -gene expression in the propositus may be partly due to anemic stress. Erythroid progenitors of these family members cultured in vitro [burst forming units (erythroid); (BFUe)] showed elevated synthesis of Hb F, indicating the existence of a genetically determined intrinsic capacity for high Hb F production in this family. The propositus was found to be homozygous for a IVS2-position 1 mutation, on the background of Mediterranean haplotype I, which is not known to be linked to high Hb F production. Moreover, extensive molecular studies of the -globin gene cluster, including sequence analysis of the promoter regions of the -globin genes, did not reveal any cisacting mechanism that could account for the high Hb F production in the propositus. A young niece of the propositus with °-thalassemia major was recently discovered, who was homozygous for the same -globin allele and haplotype as the propositus. However, unlike her uncle, she does not have a high Hb F level and presents with a severe clinical course. Her inability to produce high Hb F suggests that the genetic determinant for increased -gene expression in the propositus is unlinked to the -globin gene cluster.     

The achondroplasia gene is not linked to the locus for neurofibromatosis 1 on chromosome 17

Summary We have investigated genetic linkage of von Recklinghausen neurofibromatosis (NF1) and achondroplasia (ACH) using chromosome-17 markers that are known to be linked to NF1. Physical proximity of the two loci was suggested by the report of a patient with mental retardation and the de novo occurrence of both NF1 and ACH. Since the chance of de novo occurrence of these two disorders in one individual is 1 in 600 million, this suggested a chromosomal deletion as a single unifying molecular event and also that the ACH and NF1 loci might be physically close. To test this, we performed linkage analysis on a three-generation family with ACH. We used seven DNA probes that are tightly linked to the NF1 locus, including DNA sequences that are known to flank the NF1 locus on the centromeric and telomeric side. We detected two recombinants between the ACH trait and markers flanking the NF1 locus. In one recombinant, the flanking markers themselves were nonrecombinant. Multi-point linkage analysis excluded the ACH locus from a region surrounding the NF1 locus that spans more than 15cM (lod score < -2). Therefore, analysis of this ACH pedigree suggests that the ACH locus is not linked to the NF1 locus on chromosome 17.     

Medicago truncatula,a model plant for studying the molecular genetics of theRhizobium-legume symbiosis

Medicago truncatula has all the characteristics required for a concerted analysis of nitrogen-fixing symbiosis withRhizobium using the tools of molecular biology, cellular biology and genetics.M. truncatula is a diploid and autogamous plant has a relatively small genome, and preliminary molecular analysis suggests that allelic heterozygosity is minimal compared with the cross-fertilising tetraploid alfalfa (Medicago sativa). TheM. truncatula cultivar Jemalong is nodulated by theRhizobium meliloti strain 2011, which has already served to define many of the bacterial genes involved in symbiosis with alfalfa. A genotype of Jemalong has been identified which can be regenerated after transformation byAgrobacterium, thus allowing the analysis ofin-vitro-modified genes in an homologous transgenic system. Finally, by virtue of the diploid, self-fertilising and genetically homogeneous character ofM. truncatula, it should be relatively straightforward to screen for recessive mutations in symbiotic genes, to carry out genetic analysis, and to construct an RFLP map for this plant.     

Inositol phosphates in receptor-mediated cell signaling: metabolic origins and interrelationships

We have investigated the metabolic interrelationships of the major inositol phosphates in vasopressin-stimulated WRK 1 mammary tumor cells which were labeled to equilibrium with [14C]inositol and briefly, just prior to stimulation, with [3H]inositol. A comparison of the 3H/14C ratios of these compounds with those of the cellular inositol lipids suggests that most of the known inositol mono-, bis-, tris-, and tetrakis-phosphates are derived from precursors with turnover rates similar to those of these lipids. However, Ins(3,4,5,6)P4 (which is the major inositol tetrakisphosphate to accumulate in stimulated WRK 1 cells), Ins(1,3,4,5,6)P5, and InsP6 had 3H/14C ratios of 0 in this experiment, indicating that they must have a different metabolic origin.     

Canine alpha-L-fucosidase in relation to the enzymic defect and storage products in canine fucosidosis.

Canine liver alpha-L-fucosidase was purified to apparent homogeneity by affinity chromatography on agarose-epsilon-aminohexanoyl-fucopyranosylamine. It is composed of multiple forms of a common active subunit of 45-50 kDa, which can aggregate in different combinations to form polymers, predominantly dimers. Antiserum was raised against the purified enzyme. There is negligible residual alpha-L-fucosidase in the tissues of English springer spaniels with the lysosomal storage disease fucosidosis. Although no alpha-L-fucosidase protein was detected by Western blotting or by the purification procedure in the affected tissues, some enzymically inactive cross-reacting material was detected in both normal and affected tissues. This suggests that another protein without alpha-L-fucosidase activity was co-purified with the enzyme. Dog liver alpha-L-fucosidase was precipitated by goat anti-(human liver alpha-L-fucosidase) IgG, indicating homology between the enzymes in the two species. Two purified storage products isolated from the brain of a dog with fucosidosis were used as natural substrates for various preparations of canine liver alpha-L-fucosidase. Analysis of the digestion mixtures by t.l.c. and fast-atom-bombardment mass spectrometry suggests that canine alpha-L-fucosidase acts preferentially on the alpha-(1-3)-linked fucose at the non-reducing end and that removal of alpha-(1-6)-linked asparagine-linked N-acetylglucosamine is rate-limiting in the lysosomal catabolism of fucosylated N-linked glycans.     

Synthesis of myo-inositol 1,3,4,5,6-pentakisphosphate from inositol phosphates generated by receptor activation.

myo-[3H]Inositol 1,3,4,5,6-pentakisphosphate can be made from myo-[3H]inositol 1,4,5-trisphosphate in a rat brain homogenate or soluble fraction. Although D-myo-inositol 3,4,5,6-tetrakisphosphate can be phosphorylated by a soluble rat brain enzyme to give myo-inositol 1,3,4,5,6-pentakisphosphate, it is not an intermediate in the pathway from myo-inositol 1,4,5-trisphosphate. The intermediates in the above pathway are myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4-trisphosphate and myo-inositol 1,3,4,6-tetrakisphosphate [Shears, Parry, Tang, Irvine, Michell & Kirk (1987) Biochem. J. 246, 139-147; Balla, Guillemette, Baukal & Catt (1987) J. Biol. Chem. 262, 9952-9955], and it is catalysed by soluble kinase activities of similar anion-exchange mobility and Mr value. Compounds with chromatographic and chemical properties consistent with the structures myo-inositol 1,3,4,5-tetrakisphosphate, myo-inositol 1,3,4,6-tetrakisphosphate and myo-inositol 3,4,5,6-tetrakisphosphate are present in avian erythrocytes, human 1321 N1 astrocytoma cells and primary-cultured murine bone-marrow-derived macrophages. The amounts of these inositol tetrakisphosphates rise upon muscarinic cholinergic stimulation of the astrocytoma cells or stimulation of macrophages with platelet-activating factor.