Linkage disequilibrium analysis in Machado-Joseph disease patients of different ethnic origins

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration originally described in families of Portuguese-Azorean ancestry. The hypothesis that its present world distribution could result from the spread of an original founder mutation has been raised. To test this possibility we have conducted a linkage disequilibrium study of markers segregating with the MJD1 locus in a total of 64 unrelated families of different geographical origins. Significant association was detected between the MJD1 locus and marker alleles at loci D14S280, D14S1050 and D14S81. All affected individuals, except one Chinese family, had allele 3 (237 bp) at D14S280. This finding is consistent with a founder effect in our MJD population. However, distinct haplotypes were observed in patients originating from the two Azorean islands showing the highest disease prevalence; therefore, the possible existence of more than one founder mutation can not be excluded with the markers currently available. Received: 27 February 1996 / Revised: 4 June 1996     

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.     

Complete development of Cryptosporidium parvum in MDBK cells

Abstract Sporozoites of Cryptosporidium parvum excysted in vitro from bovine oocysts were incubated with monolayers of Madin-Darby bovine kidney cells. The extent of parasite colonisation was monitored by light microscopy and immunofluorescence. Electron microscopy confirmed the complete development and replication of C. parvum within Madin-Darby bovine kidney cells.     

Neural control of the expression of a Ca2+-activated K+ channel involved in the induction of myotonic-like characteristics

Summary 1. Expression of the apamin-sensitive K⁺ channel (SK⁺) in rat skeletal muscle is neurally regulated. The regulatory effect of the nerve over the expression of some muscle ion channels has been attributed to the electrical activity triggered by the nerve and/or to a trophic effect of some molecules transported from the soma to the axonal endings. 2. SK⁺ channels apparently are involved in myotonic dystrophy (MD), therefore understanding the factors that regulate their expression may ultimately have important clinical relevance. 3. To establish if axoplasmic transport is involved in this process, we used two experimental approaches in adult rats: (a) Both sciatic nerves were severed, leaving a short or a long nerve stump attached to the anterior tibialis (AT). (b) Colchicine or vinblastine (VBL), two axonal transport blockers of different potencies, was applied on one leg to the sciatic nerve. To determine whether electrical activity affects the expression of SK⁺ channels, denervated AT were directly stimulated. The corresponding contralateral muscles were used as controls. 4. With these experimental conditions we measured (a) apamin binding to muscle membranes, (b) muscle contractile characteristics, and (c) electromyographic activity. 5. In the short- and long-nerve stump experiments, 5 days after denervation¹²⁵I-apamin binding to AT membranes was 2.0 times higher in the short-stump side. This difference disappeared at longer times. The delayed expression of SK⁺ channels in the muscle left with a longer nerve stump can be attributed to the extra axoplasm contained in the longer stump, which maintains a normally repressive signal for a longer period of time. Ten to 15 days after application of axonal transport blockers we found that the muscle half-relaxation time increased in the drug-treated side and apamin partially reverted the prolonged relaxation. Myotonic-like discharges specifically blockable by apamin were always present in the drug-treated leg.¹²⁵I-Apamin binding, which is undetectable in a microsomal preparation from hind leg control muscles, was increased in the drug-treated preparations. Apamin binding to denervated and stimulated AT muscles was lower than in the contralateral unstimulated muscles [3.3±1.0 vs 6.8±0.8 (n=4) fmol/mg protein]. 6. Our results demonstrate that electrical activity and axoplasmic transport are involved in the control of expression of SK⁺ in rat skeletal muscle. However, the increased expression of this channel induces myotonic-like characteristics that are reversed by apamin. This myotonic activity could be a model for MD.     

Modulation of maize roots H+-ATPase by sulfated polysaccharides

Vesicles derived from maize roots retain a membrane bound H⁺-ATPase that is able to pump H⁺ at the expense of ATP hydrolysis. In this work it is shown that heparin, fucose-branched chondroitin sulfate and dextran sulfate 8000 promote a shift of the H⁺-ATPase optimum pH from 6.0 to 7.0. This shift is a result of a dual effect of the sulfated polysaccharides, inhibition at pH 6.0 and activation at pH 7.O. At pH 6.0 dextran 8000 promotes an increase of the apparent K_m for ATP from 0.28 to 0.95 mM and a decrease of the V_max from 14.5 to 7.1 mol P_i/mg · 30 min^–1. At pH 7.0 dextran 8000 promotes an increase in V_max from 6.7 to 11.7 mol Pi/mg · 30 min^–1. In the presence of lysophosphatidylcholine the inhibitory effect of the sulfated polysaccharides observed at pH 6.0 was not altered but the activation of pH 7.0 decreased. It was found that in the presence of sulfated polysaccharides the ATPase became highly sensitive to K⁺ and Na⁺. Both the inhibition at pH 6.0 and the activation promoted by the polysaccharide were antagonized by monovalent cations (K⁺>Na⁺Li⁺).Abbreviations Mops 4-morpholinopropanesulfonic acid - EDTA ethylenediaminetetraacetic acid - ACMA 9-amino-6-chloro-2-methoxyacridine - FCCP carbonyl cyanide p(trifluoromethoxy)-phenylhyrazone     

Surface hydrography and phytoplankton of the Brazil-Malvinas currents confluence

Results are presented on the phytoplankton species compositionand abundance from bottle samples collected in September 1989near the confluence of the Brazil and Malvinas currents offArgentina. The phytoplankton assemblages were dominated by diatomsand dinoflagellates. A surface diatom bloom was found alongthe west side of the Brazil Current, and was dominated by Thalassiosiradelicatula Ostenfeld emend. Hasle (cell numbers up to 5.5 x10⁵ cells 1^–1) The bloom was associated with strong temperaturegradients separating Brazil and Malvinas waters, and with thepresence of a cyclonic eddy near the confluence of the currents.These features were detected in satellite imagery coincidentwith the in situ sampling dates.     

Isolation and promoter characterization of barley geneItr1 encoding trypsin inhibitor BTI-CMe: differential activity in wild-type and mutantlys3a endosperm

The geneItr1, encoding trypsin inhibitor BTI-CMe, has been obtained from a genomic library ofHordeum vulgare L. The gene has no introns and presents in its 5-upstream region 605 bp that are homologous to the long terminal repeats (LTR) of the copia-like retro-transposon Bare-1. Functional analysis of theItr1 promoter by transient expression in protoplasts derived from different barley tissues, has shown that in this system theItr1 promoter retains its endosperm specifity and thetrans-regulation mediated by theLys3a gene. The proximal promoter extending 343 bp upstream of the translation initiation ATG codon is sufficient to confer fullGUS expression and for endosperm specifity. In protoplasts derived from thelys3a mutant, Risø 1508,GUS activity was less than 5% of that obtained with the same constructs in the protoplasts of wild-type Bomi from which it derives. Gel retardation experiments, after incubation with proteins obtained from both types of endosperm nuclei, also show differential patterns. Possible reasons for these differences are discussed.Equal authours     

Bacterial expression of an active class Ib chitinase from Castanea sativa cotyledons

Ch3, an endochitinase of 32 kDa present in Castanea sativa cotyledons, showed in vitro antifungal properties when assayed against Trichoderma viride. The characterization of a cDNA clone corresponding to this protein indicated that Ch3 is a class Ib endochitinase that is synthesized as a preprotein with a signal sequence preceding the mature polypeptide. Bacterial expression of mature Ch3 fused to the leader peptide of the periplasmic protein ompT resulted in active Ch3 enzyme. A plate assay was adapted for semi-quantitative determination of chitinase activity secreted from cultured bacteria, which should facilitate the identification of mutants with altered capacity to hydrolyse chitin.     

Somatic embryogenesis,organogenesis and callus growth kinetics of flax

The effects of plant growth regulators (PGR) on calli induction, morphogenesis and somatic embryogenesis of flax were studied. The organogenic and callus formation capacity were assessed for different types of source explants. Root and shoot explants were equally good material for calli production but the former produced calli without shoot regeneration capacity. Under the experimental conditions tested, 2,4-dichlorophenoxyacetic acid (2,4-D) + zeatin was the most efficient PGR combination on calli induction and biomass production. The calli were green but with no rhizogenic capacity. In contrast, and at similar concentrations, indole-3-butyric acid (IBA) + kinetin induced white or pale green friable calli with a good root regeneration capacity (60%). A factorial experiment with different combinations of 2,4-D + zeatin + gibberellic acid (GA₃) levels revealed that the direction of explant differentiation was determined by specific PGR interactions and concentrations. The results from these experiments revealed that the morphogenetic pathway (shoot versus root differentiation) can be manipulated on flax explants by raising the 2,4-D level from 0.05 to 3.2 mg l^?1 in the induction medium. The induction and development of somatic embryos from flax explants was possible in a range of 2,4-D + zeatin concentrations surrounding 0.4 mg l^?1 2,4-D and 1.6 mg l^?1 zeatin, the most efficient growth regulator combination.