Synapsin I Regulates Glutamate Release from Rat Brain Synaptosomes

Introduction of the dephosphorylated from of synapsin I into rat brain synaptosomes using freeze-thaw (transient) permeabilization significantly decreased the K(+)-induced release of glutamate. In contrast, introduction of synapsin I that had been phosphorylated by Ca2+/calmodulin-dependent protein kinase II was without effect on glutamate release. Addition of dephosphosynapsin I after freeze-thaw treatment also had no effect. Thus, the action of synapsin I was dependent on the phosphorylation state of synapsin I and on its entry into the synaptosomes. Our results implicate synapsin I as an important component in the regulation of neurotransmitter release in the mammalian nervous system.     

Saposins (Sphingolipid Activator Proteins) in the Twitcher Mutant Mouse

The twitcher mutant mouse, the animal model of Krabbe disease (human globoid cell leukodystrophy), is characterized by apparent deficiency of galactosylceramide beta-galactosidase activity. Saposin A and C, the heat-stable small sphingolipid activator glycoproteins, stimulate the activity of galactosylceramide beta-galactosidase as well as glucosylceramide beta-glucoside. The role of these saposins in the twitcher mutation was investigated. Boiled supernatant fractions, which contained saposins, were prepared from homogenates of twitcher brain, liver, kidney, and spleen. These preparations showed an almost identical effect on the activity of purified glucosylceramide beta-glucosidase (measured by hydrolysis of 4-methylumbelliferyl-beta-glucoside) with similar preparations from control tissues. The effect on the activity of galactosylceramide beta-galactosidase as well as 4-methylumbelliferyl-beta-glucoside beta-glucosidase in the twitcher brain and liver homogenates by authentic saposin A and C was similar to that in control tissues. These results suggest that the twitcher mutation does not affect the concentrations of saposin A or C or their interaction with galactosylceramide beta-galactosidase.     

Internal AU-rich elements modulate activity of two competing 3' splice sites in plant nuclei

In vivo analyses using an autonomously replicating Agrobacterium/geminivirus vector have enabled identification of AU-rich intronic elements critical for 5′ and 3′ splice site selection in dicot plant nuclei and development of a model for pre-mRNA intron recognition in plant nuclei. To determine the minimal length, spacing and nucleotide compositions constraining recognition of the 3′ boundary of an intron, two or four nucleotide substitutions have been introduced into the two AU-rich elements located between 50 and 66 nucleotides upstream from the 3′ splice site of maize Adh 1 intron 3. In each case tested, substitutions in the distal left element (?62 to ?66) inactivate the downstream 3′ splice site at ?1 more effectively than substitutions in the proximal right element (?50 to ?55). Guanosine or cytosine substitutions in either element reduce recognition of the ?1 site significantly; adenosine substitutions have a less severe effect. Mutations in both of these AU elements additively block recognition of the downstream 3′ splice site. The strong additive effect of these mutations supports a model in which short sets of AU islands bind interactive factors and cooperatively modulate usage of the downstream splice site. In contrast to the uridine requirements documented for the 3′ terminus of plant introns, adenosines are partially interchangeable with uridines within this internal region of the intron.     

An Assessment of Oxidative Damage to Proteins, Lipids, and DNA in Brain from Patients with Alzheimer's Disease

Abstract: Oxidative stress may contribute to neuronal loss in Alzheimer's disease (AD). The present study compares the levels of oxidative damage to proteins, lipids, and DNA bases from seven different brain areas of AD and matched control tissues by using a range of techniques. No differences in levels of lipid peroxidation were found in any of the brain regions by using two different assay systems. Overall, there was a trend for protein carbonyl levels to be increased in AD in frontal, occipital, parietal, and temporal lobe, middle temporal gyrus, and hippocampus, but a significant difference was found only in the parietal lobe. Gas chromatography-mass spectrometry was used to measure products of damage to all four DNA bases. Increased levels of some (8-hydroxyadenine, 8-hydroxyguanine, thymine glycol, Fapy-guanine, 5-hydroxyuracil, and Fapy-adenine), but not all, oxidized DNA bases were observed in parietal, temporal, occipital, and frontal lobe, superior temporal gyrus, and hippocampus. The baseline level of oxidative DNA damage in the temporal lobe was higher than in other brain regions in both control and AD brain. The finding of increased oxidative damage to protein and DNA strengthens the possibility that oxidative damage may play a role in the pathogenesis of AD in at least some key brain regions.     

Structural and serological studies of lipopolysaccharides from proposed new serotypes (O25 and O26) of Serratia marcescens

Abstract The surface polysaccharides of the two most recently proposed O-serotype strains of Serratia marcescens , O25 and O26, were characterised in terms of their chemical structure and immunological reactions. No polymer was isolated from O25, which was shown to lack both capsular K-antigen and smooth, O-antigenic lipopolysaccharide. A neutral polysaccharide was isolated from O26 and shown to be a polymer of rhamnose and N -acetylgalactosamine of the type previously found in the O9 and O15 reference strains. Serological cross-reactions among all three strains were demonstrated by using both whole-cell enzyme-linked immunosorbent assay and immunoblotting of lipopolysaccharide resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. No acidic polysaccharide was found in O26 and this was consistent with the absence of an immunogenic capsule. Thus, neither strain qualifies for inclusion as a new serotype in either an O-typing or a K-typing scheme.     

A new improved sub-unit vaccine for plague: the basis of protection

Abstract In this study, we have determined the limit of protection achievable by immunisation with sub-units of Yersinia pestis against the development of plague in an experimental animal model. Co-immunisation with the purified culture-derived F1 and the recombinant V sub-units afforded a greater level of protection than with either sub-unit alone. The protection given by the combined sub-units was several orders of magnitude greater than that afforded by the whole cell killed (Cutter USP) vaccine and was equivalent to that achieved by vaccination with EV76, the live attenuated Y. pestis vaccine strain. However, the combined sub-unit vaccine has clear advantages over the live vaccine in terms of safety of use and absence of side-effects.     

Constitutive expression of a fruit phytoene synthase gene in transgenic tomatoes causes dwarfism by redirecting metabolites from the gibberellin pathway

Tomato plants transformed with a copy of the fruit-expressed phytoene synthase cDNA under control of the CaMV 35S promoter showed ectopic production of carotenoids. High expressers were reduced in stature. The dwarf character was inherited with an inverse relationship between expression of phytoene synthase and plant height. Severely affected plants also showed reduced chlorophyll content in young leaves. These dwarfs showed a 30-fold reduction in levels of gibberellin A₁ (GA₁) and growth was partially restored by treatment with exogenous GA₃. Qualitative and quantitative changes in carotenoids were also found. It is proposed that the dwarf phenotype results from the over-production of phytoene synthase, which converts geranylgeranyl diphosphate to phytoene and thereby diverts this intermediate away from the gibberellin (GA) and phytol biosynthetic pathways.     

Preparation of megabase-size DNA from plant nuclei

A novel technique has been developed for the preparation of high molecular weight (HMW) DNA from plant nuclei. This technique involves physical homogenization of plant tissues, nuclei isolation, embedding of the nuclei in low-melting-point agarose microbeads or plugs, and DNA purification in situ . This technique is simple, rapid, and economical, and the majority of the DNA prepared is over 5.7 Mb in size. The genomic DNA content of the HMW DNA prepared by this technique is enriched by at least threefold and the chloroplast DNA content is reduced by over twofold relative to that prepared from plant protoplasts by existing methods. The DNA is readily digestible with different restriction enzymes and partial digestions of the DNA could be reproducibly performed. This method has been successfully used for the preparation of HMW DNA from a wide range of plant taxa, including grasses, legumes, vegetables, and trees. These results demonstrate that the DNA prepared by this technique is suitable for plant genome analysis by pulsed-field gel electrophoresis and for the construction of yeast and bacterial artificial chromosomes.