Divalent cations modify adsorption of 5′-AMP onto precipitated calcium phosphate: A model for cation modulation of adsorptive processes in primitive aqueous environments

The adsorption of 5′-AMP onto precipitated calcium phosphate (CaPi) requires the presence of soluble calcium and this dependence exhibits a Michaelian-like behavior. This result suggests that the formation of a complex between 5′-AMP and free Ca²⁺ (CaAMP) is a prelude to the adsorption of the nucleotide in the solid matrix. At concentrations one order of magnitude higher, Mn²⁺ and Mg²⁺ can substitute for soluble Ca²⁺ in the adsorption of 5′-AMP onto solid CaPi. However, when added simultaneously with 5′-AMP to a heterogeneous mixture that contains CaPi and soluble Ca²⁺, Mn²⁺ and Mg²⁺ inhibit the adsorption of 5′-AMP in a concentration-dependent manner. This suggests the formation of complexes that are much less effective for 5′-AMP adsorption than the CaAMP complex. On the other hand, Mn²⁺ and Mg²⁺ cannot promote desorption of the nucleotide attached to the precipitate in the presence of soluble Ca²⁺ if they are added after adsorption has attained equilibrium. Although desorption of 5′-AMP can be obtained by a sequential dilution of the soluble phase with buffer and no nucleotide in a process that obeys a Langmuir equation, the lack of effect of Mn²⁺ or Mg²⁺ when adsorption has attained its maximal value suggests strong interactions between the CaAMP complex and the solid matrix when adsorption equilibrium is reached. The divalent cations present in the matrix also participate with different selectivity in the attachment of the CaAMP complex, indicating that a cation-exchange mechanism could have acted in the modulation of adsorptive/desorptive processes involving biomonomers and phosphate surfaces in primitive aqueous environments. Received: 11 December 1995 / Accepted: 5 April 1996     

Involvement of Intracellular or Extracellular Calcium in Activation of Tyrosine Hydroxylase Gene Expression in PC12 Cells

Abstract: The relationship between elevations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) by different mechanisms and tyrosine hydroxylase (TH) gene expression was examined. Depolarization by an elevated K⁺ concentration triggered rapid and sustained increases in [Ca²⁺]_i from a basal level of ～50 to 110–150 nM and three- to fourfold elevations in TH mRNA levels, requiring extracellular calcium but not inositol 1,4,5-trisphosphate (IP₃). On the other hand, bradykinin or thapsigargin, both of which induce release of intracellular calcium stores via IP₃ or inhibition of Ca²⁺-ATPase, rapidly elevated [Ca²⁺]_i to >200 nM and increased TH gene expression (three-to fivefold). Confocal imaging showed that the elevations in [Ca²⁺]_i in each case occurred throughout the cyto- and nucleoplasm. The initial rise in [Ca²⁺]_i due to either bradykinin or thapsigargin, which did not require extracellular calcium, was sufficient to initiate the events leading to increased TH expression. Consistent with this, the effects of bradykinin on TH expression were inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid or 3,4,5-trimethoxybenzoic acid 8-(diethylamino)-octyl ester which chelates or inhibits the release of intracellular calcium, respectively. Bradykinin required a rise in [Ca²⁺]_i for <10 min, as opposed to 10–30 min for depolarization to increase TH mRNA levels. These results demonstrate that although each of these treatments increased TH gene expression by raising [Ca²⁺]_i, there are important differences among them in terms of the magnitude of elevated [Ca²⁺]_i, requirements for extracellular calcium or release of intracellular calcium stores, and duration of elevated [Ca²⁺]_i, indicating the involvement of different calcium signaling pathways leading to regulation of TH gene expression.     

Xp-duplications with and without sex reversal

Duplications in Xp including the DSS (dosage sensitive sex reversal) region cause male to female sex reversal. We investigated two patients from families with Xp duplications. The first case was one of two sisters with karyotype 46,XY, der(22), t(X;22)(p11.3;p11)mat and unambiguous female genitalia. The living sister was developmentally retarded, and showed multiple dysmorphic features and an acrocallosal syndrome. The second case was a boy with a maternally inherited direct duplication of Xp21.3-pter with the breakpoint close to the DSS locus. He had multiple abnormalities and micropenis, but otherwise unambiguous male genitalia. We performed quantitative Southern blot analysis with probes from Xp22.13 to p21.2 to define the duplicated region. Clinical, cytogenetic, and molecular data from both patients were compared with those of previously reported related cases. A comparison of the extragenital symptoms revealed no differences between patients with or without sex reversal. In both cases, the symptoms were non-specific. Among 22 patients with a duplication in Xp, nine had unambiguous female genitalia and a well-documented duplication of the DSS region. Two patients with duplication of DSS showed ambiguous external genitalia. From these data, we conclude that induction of testicular tissue may start in these patients, but that the type of genitalia depends on the degree of subsequent degeneration by a gene in DSS.     

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.     

The 21-aminosteroid U-74389F increases the number of glial fibrillary acidic protein-expressing astrocytes in the spinal cord of control and wobbler mice

Summary 1. Wobbler mice suffer an autosomal recessive mutation producing severe motoneuron degeneration and dense astrogliosis, with increased levels of glial fibrillary acidic protein (GFAP) in the spinal cord and brain stem. They have been considered animal models of amyotrophic lateral sclerosis and infantile spinal muscular atrophy. 2. Using Wobbler mice and normal littermates, we investigated the effects of the membrane-active steroid Lazaroid U-74389F on the number of GFAP-expressing astrocytes and glucocorticoid receptors (GR). Lazaroids are inhibitors of oxygen radical-induced lipid peroxidation, and proved beneficial in cases of CNS injury and ischemia. 3. Four days after pellet implantation of U-74389F into Wobbler mice, hyperplasia and hypertophy of GFAP-expressing astrocytes were apparent in the spinal cord ventral and dorsal horn, areas showing already intense astrogliosis in untreated Wobbler mice. In control mice, U-74389F also produced astrocyte hyperplasia and hypertophy in the dorsal horn and hyperplasia in the ventral-lateral funiculi of the cord. 4. Givenin vivo U-74389F did not change GR in spinal cord of Wobbler or control mice, in line with the concept that it is active in membranes but does not bind to GR. Besides, U-74390F did not compete for [³H]dexamethasone binding when addedin vitro. 5. The results suggest that stimulation of proliferation and size of GFAP-expressing astrocytes by U-74389F may be a novel mechanism of action of this compound. The Wobbler mouse may be a valuable animal model for further pharmacological testing of glucocorticoid and nonglucocorticoid steroids in neurodegenerative diseases.     

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     

α-Parvalbumin reduces depolarizationminduced elevations of cytosolic free calcium in human neuroblastoma cells

We investigated whether the expression of human α-parvalbumin affects depolarization-induced elevations of the cytosolic free calcium concentration ([Ca²⁺]_i) in human neuroblastoma SKNBE2 cells. A full length human parvalbumin cDNA was cloned by PCR from human cerebellum and transiently transfected into SKNBE2 cells. Immunofluorescence staining using an antibody raised against parvalbumin revealed a transfection efficacy of about 14%. In parvalbumin-expressing SKNBE2 cells, parvalbumin concentration determined by quantitative Western blotting amounted to 0.42 mM.Transfected SKNBE2 cells were depolarized for 2 min by 50 mM K⁺. During this period, [Ca²⁺]_i was monitored by video microfluorimetry using the Ca²⁺ indicator Fura-2. In a fraction of cells, depolarization induced a transient elevation in [Ca²⁺]_i The size of this elevation was compared with the immunofluorimetrically determined expression of parvalbumin on a cell-to-cell basis. Cells with a significant parvalbumin immunofluorescence responded to depolarization with smaller elevations in [Ca²⁺]_i than non-parvalbumin-expressing cells. Resting [Ca 2+], did not differ between parvalbumin-expressing and control cells. These observations indicate that large depolarization-induced transient elevations of [Ca²⁺]_i in neuroblastoma cells can be suppressed by parvalbumin.     

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.     

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.