Adenylyl Cyclases: mRNA and Characteristics of Enzyme Activity in Three Areas of Brain

Abstract: Arachidonic acid and oleoylacetylglycerol enhance depolarization-evoked glutamate release from hippocampal mossy fiber nerve endings. It was proposed this is a Ca²⁺-dependent effect and that protein kinase C is involved. Here we report that arachidonic acid and oleoylacetylglycerol synergistically potentiate the glutamate release induced by the Ca²⁺ ionophore ionomycin. The Ca²⁺ dependence of this effect was established, as removal of Ca²⁺ eliminated evoked release and the lipid-dependent potentiation. Also, Ca²⁺ channel blockers attenuated ionomycin- and KCI-evoked exocytosis, as well as the facilitating effects of the lipid mediators. Although facilitation required Ca²⁺, it may not involve an enhancement of evoked Ca²⁺ accumulation, because ionomycin-dependent glutamate release was potentiated under conditions that did not increase ionomycin-induced Ca²⁺ accumulation. Also, the facilitation may not depend on inhibition of K⁺ efflux, because enhanced release was observed in the presence of increasing concentrations of 4-aminopyridine and diazoxide did not reduce the lipid-dependent potentiation of exocytosis. In contrast, disruption of cytoskeleton organization with cytochalasin D occluded the lipid-dependent facilitations of both KCI- and ionomycin-evoked glutamate release. In addition, arachidonic acid plus glutamatergic or cholinergic agonists enhanced glutamate release, whereas a role for protein kinase C in the potentiation of exocytosis was substantiated using kinase inhibitors. It appears that the lipid-dependent facilitation of glutamate release from mossy fiber nerve endings requires Ca²⁺ and involves multiple presynaptic effects, some of which depend on protein kinase C.     

Assessment of Amyloid β Protein in Cerebrospinal Fluid as an Aid in the Diagnosis of Alzheimer's Disease

Abstract: The principal constituent of amyloid plaques found in the brains of individuals with Alzheimer's disease (AD) is a 39–42-amino-acid protein, amyloid β protein (Aβ). This study examined whether the measurement of Aβ levels in CSF has diagnostic value. There were 108 subjects enrolled in this prospective study: AD (n = 39), non-AD controls (dementing diseases/syndromes; n = 20), and other (n = 49). CSF was obtained by lumbar puncture, and Aβ concentrations were determined using a dual monoclonal antibody immunoradiometric sandwich assay. The mean Aβ value for the AD group (15.9 ± 6.8 ng/ml) was not significantly different from that for the non-AD control group (13.0 ± 7.1 ng/ml; p = 0.07), and substantial overlap in results were observed. Aβ values did not correlate with age ( r = −0.05, p = 0.59), severity of cognitive impairment ( r = 0.22, p = 0.21), or duration of AD symptoms ( r = 0.14, p = 0.45). These findings are in conflict with other reports in the literature; discrepant results could be due to the instability of Aβ in CSF. Aβ immunoreactivity decays rapidly under certain conditions, particularly multiple freeze/thaw cycles. Use of a stabilizing sample treatment buffer at the time of lumbar puncture allows storage of CSF without loss of Aβ reactivity. In conclusion, the total CSF Aβ level is not a useful marker for current diagnosis of AD.     

Complete replication in vitro of tobacco mosaic virus RNA by a template-dependent, membrane-bound RNA polymerase.

A crude membrane-bound RNA polymerase, obtained by differential centrifugation of extracts of tomato leaves infected with tobacco mosaic tobamovirus (tomato strain L) TMV-L), was purified by sucrose density gradient centrifugation. Removal of the endogenous RNA template with micrococcal nuclease rendered the polymerase template dependent and template specific. The polymerase was primer independent and able to initiate RNA synthesis on templates containing the 3'-terminal sequences of the TMV-L positive or negative strands. TMV-vulgare RNA was a less efficient template, while RNAs of cucumber mosaic cucumovirus and red clover necrotic mosaic dianthovirus, or 5'-terminal sequences of TMV-L positive or negative strands, did not act as templates for the polymerase. A main product of the reaction with TMV-L genomic RNA as a template, carried out in the presence of [alpha-32P]UTP, was genomic-length single-stranded RNA. This was shown to be the positive strand and uniformly labelled along its length, demonstrating complete replication of TMV-L RNA. Genomic-length double-stranded RNA, labelled in both strands, and small amounts of RNAs corresponding to the single- and double-stranded forms of the coat protein subgenomic mRNA were also formed. Antibodies to N-terminal and C-terminal portions of the 126-kDa protein detected the 126-kDa protein and the 183-kDa readthrough protein in purified RNA polymerase preparations, whereas antibodies to the readthrough portion of the 183-kDa protein detected only the 183-kDa protein. All three antibodies inhibited the template-dependent RNA polymerase, but none of them had any effect on the template-bound enzyme.     

K252a Modulates the Expression of Nerve Growth Factor-Dependent Capsaicin Sensitivity and Substance P Levels in Cultured Adult Rat Dorsal Root Ganglion Neurones

Abstract: K252a, an inhibitor of trk phosphorylation and nerve growth factor signal transduction in PC12 cells, blocked nerve growth factor-induced responses in cultured adult rat dorsal root ganglion sensory neurones. The nerve growth factor-dependent appearance of capsaicin sensitivity and accumulation of the neuropeptide substance P were inhibited when dorsal root ganglion neurones were grown in the presence of low concentrations (100 n M ) of K252a. At higher concentrations (3 µ M ), however, K252a stimulated the development of capsaicin sensitivity and the accumulation of substance P even in the absence of nerve growth factor. By using a wide dose range, therefore, we showed that K252a could either inhibit or mimic nerve growth factor's actions on sensory neurones. These results may explain the apparent paradox in the literature that some groups show a blocking effect of K252a on nerve growth factor-dependent survival of dorsal root ganglion sensory neurones, whereas others report that K252a can substitute for nerve growth factor or other trophic factors and promote neuronal survival.     

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     

Plant regeneration from somatic embryos ofTaxus brevifolia

Summary Taxusbrevifolia is the source of paclitaxel (Taxol®), an anticancer drug. A method for regeneration ofTaxus brevifolia from immature zygotic embryos via somatic embryogenesis is described. Embryogenic callus tissues were obtained by culturing immature zygotic embryos on Lloyd and McCown medium (MCM) supplemented with 160 M 2,4-dichlorophenoxyacetic acid (2,4-D) + 5 M benzylaminopurine (BA) + 5 M naphthaleneacetic acid (NAA) for 4 weeks. Putative embryoids were obtained following transfer of cultures to MCM medium supplemented with 4 M BA + 5 M kinetin + 1 M NAA for 6 to 8 weeks. Conversion of embryos was obtained on MCM medium supplemented with 40 M abscisic acid (ABA) + 1% activated charcoal. Development of bipolar structures with recognizable shoot and root apices was observed in somatic embryos. Five percent of somatic embryos were regenerated into plantlets on half-strength growth regulator-free MCM medium.     

Cytokinins and fruit development in the kiwifruit (Actinidia deliciosa). I. Changes during fruit development

The cytokinin content in fruit tissue of the kiwifruit ( Actinidia deliciosa [A. Chev.] C. F. Liang et A. R. Ferguson var. deliciosa cv. Hayward) was monitored during fruit development to identify which cytokinins were present and if they were linked with specific stages of fruit growth. Cytokinins were isolated and purified by column chromatography and high-performance liquid chromatography and quantified by radioimmunoassay. A novel HPLC step utilising an amine column was successfully introduced as a preparative step in the separation of the O - and 9-glucosides from the free bases and ribosides. The radioimmunoassay results were validated, and the different cytokinins identified, by gas chromatography-mass spectrometry. Cytokinins detected in fruit included the cytokinin free bases, zeatin and isopentenyladenine, their ribosides, nucleotides and both O - and 9-glucosides. Both qualitative and quantitative changes of the cytokinins occurred during fruit development. A decrease in cytokinin concentration occurred after anthesis (from 342 pmol g⁻¹ fresh weight at anthesis to 41 pmol g⁻¹ fresh weight 27 days after anthesis). A large increase in cytokinin concentration and content per fruit occurred as the fruit reached commercial maturity (to 1900 pmol g⁻¹ fresh weight). Individual cytokinins showed quite different patterns. Zeatin, in particular, showed a peak in concentration (13 pmol g⁻¹ fresh weight) 11 days after anthesis that correlated with the beginning of the cell division phase of fruit growth. The accumulation of cytokinin (mostly zeatin riboside or zeatin nucleotide) in mature fruit may be of significance for the postharvest storage of kiwifruit fruit.     

Complete sequence of a new HLA-B35 allele found in a tribe of Mapuche Indians in the south of Argentina

The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U17107. The nameB*3509 was officially assigned by the WHO Nomenclature Committee in December 1994     

A Familial Factor Independent of CAG Repeat Length Influences Age at Onset of Machado-Joseph Disease

Machado-Joseph disease (MJD) is a late-onset, progressive, neurodegenerative disorder caused by the expansion of an unstable trinucleotide (CAG) repeat sequence in a novel gene (MJD1) on chromosome 14. Previous studies showed that age at onset is negatively correlated with the number of CAG repeat units, but only part of the variation in onset age is explained by CAG repeat length. Ages at onset and CAG repeat lengths of 136 MJD patients from 23 kindreds of Portuguese descent were analyzed, to determine whether familial factors independent of CAG repeat length modulate age at onset of MJD. Correlation among sibs for onset age adjusted for CAG repeat length was .43, which indicates that an environmental or genetic factor common to sibs influences onset age. Positive correlations were also observed for avuncular (r = .22) and first-cousin pairs (r = .28), which supports the hypothesis that a genetic factor is influencing age at onset. Commingling analysis of onset ages adjusted for CAG repeat length identified three distributions in this population of affected individuals. Further studies of a much larger sample are needed to determine whether these distributions represent the influence of a genetic or environmental factor.     

Effect of medium composition on 1-octen-3-ol formation in submerged cultures of Pleurotus pulmonarius

The effect of nitrogen and fatty-acid-rich substrates on the production of 1-octen-3-ol by the edible fungus Pleurotus pulmonarius, during growth in both shaken flask and fermentor cultures, and in-vitro, in post-harvested mycelium, was studied. Addition of soybean flour and soybean oil to the growth medium enhanced 1-octen-3-ol production about sevenfold and doubled the fungal biomass, as compared to that obtained from P. pulmonarius cultured on a defined synthetic medium. A clear relationship between the production of 1-octen-3-ol and lipoxygenase activity was found during the growth of mushroom pellets. The highest in-vitro generation of 1-octen-3-ol was obtained upon addition of exogenous linoleic acid and pure O₂ to pellets grown with soybean fluor and soybean oil. This generation was even higher than that of fruiting bodies exposed to the same conditions. These results suggest that lipoxygenase activity and, subsequently, 1-octen-3-ol biosynthesis in P. pulmonarius are enhanced by the presence of substrates containing fatty acids in the growth medium. Correspondence to: D. Levanon