Cloning and functional analysis of a cDNA encoding a starch synthase from potato (Solanum tuberosum L.) that is predominantly expressed in leaf tissue

Three isoforms of starch synthase (SS) were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. A cDNA encoding SSI from rice was used as a probe to clone a corresponding cDNA from potato. The deduced amino acid sequence identified the protein as an SS from potato with an M_r of 70.6 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SSI. An analysis of the expression pattern of the gene indicated that SSI is predominantly expressed in sink and source leaves, and, to a lower extent in tubers. In several independent transgenic potato lines, where the expression of SSI was repressed using the antisense approach, the activity of a specific SS isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction in the amount of this isoform of SS did not lead to any detectable changes in starch structure, probably due to the fact that this isoform only represents a minor activity in potato tubers. Received: 19 August 1998 / Accepted: 17 December 1998     

Activation of the caspase-3 apoptotic cascade in traumatic spinal cord injury.

Traumatic spinal cord injury often results in complete loss of voluntary motor and sensory function below the site of injury. The long-term neurological deficits after spinal cord trauma may be due in part to widespread apoptosis of neurons and oligodendroglia in regions distant from and relatively unaffected by the initial injury. The caspase family of cysteine proteases regulates the execution of the mammalian apoptotic cell death program. Caspase-3 cleaves several essential downstream substrates involved in the expression of the apoptotic phenotype in vitro, including gelsolin, PAK2, fodrin, nuclear lamins and the inhibitory subunit of DNA fragmentation factor. Caspase-3 activation in vitro can be triggered by upstream events, leading to the release of cytochrome c from the mitochondria and the subsequent transactivation of procaspase-9 by Apaf-1. We report here that these upstream and downstream components of the caspase-3 apoptotic pathway are activated after traumatic spinal cord injury in rats, and occur early in neurons in the injury site and hours to days later in oligodendroglia adjacent to and distant from the injury site. Given these findings, targeting the upstream events of the caspase-3 cascade has therapeutic potential in the treatment of acute traumatic injury to the spinal cord.     

In vitro synthesis of heparosan using recombinant Pasteurella multocida heparosan synthase PmHS2

In vertebrates and bacteria, heparosan the precursor of heparin is synthesized by glycosyltransferases via the stepwise addition of UDP-N-acetylglucosamine and UDP-glucuronic acid. As heparin-like molecules represent a great interest in the pharmaceutical area, the cryptic Pasteurella multocida heparosan synthase PmHS2 found to catalyze heparosan synthesis using substrate analogs has been studied. In this paper, we report an efficient way to purify PmHS2 and to maintain its activity stable during 6 months storage at −80 °C using His-tag purification and a desalting step. In the presence of 1 mM of each nucleotide sugar, purified PmHS2 synthesized polymers up to an average molecular weight of 130 kDa. With 5 mM of UDP-GlcUA and 5 mM of UDP-GlcNAc, an optimal specific activity, from 3 to 6 h of incubation, was found to be about 0.145 nmol/μg/min, and polymers up to an average of 102 kDa were synthesized in 24 h. In this study, we show that the chain length distribution of heparosan polymers can be controlled by change of the initial nucleotide sugar concentration. It was observed that low substrate concentration favors the formation of high molecular weight heparosan polymer with a low polydispersity while high substrate concentration did the opposite. Similarities in the polymerization mechanism between PmHS2, PmHS1, and PmHAS are discussed.     

Commentary: External Nasal Cartilages in Bats: Evidence for Microchiropteran Monophyly?

A recent paper by Göbbel (2000) concluded that external nasal cartilage data support microbat monophyly. However, Göbbel did not provide the results of analyses based solely on nasal cartilage data and instead mapped nasal cartilage characters onto a phylogeny from Simmons and Geisler (1998) that supported microbat monophyly. We used maximum parsimony to analyze Göbbel's (2000) data on their own and found commensurate levels of support for microbat monophyly and microbat paraphyly.     

Monoclonal antibodies block cell-cell adhesion in Dictyostelium discoideum

Of 39 monoclonal antibodies that bind the cell surface of aggregating Dictyostelium discoideum, 4 block 76-98% of cell-cell adhesion measured in an in vitro assay. The active antibodies all bind in the range of 10(6) antigenic sites/cell surface and react with more than one material on nitrocellulose blots prepared after polyacrylamide gel electrophoresis of whole aggregating cells in sodium dodecyl sulfate. Active antibodies can by grouped into two classes, each with two very similar members. Class I binds several molecules that are prominent in aggregating cells but scarce or undetectable in vegetative cells, blocks cell adhesion only in the presence of EDTA, and has no detectable effect on cell morphology. Class II binds a wide range of molecules present in both vegetative and aggregating cells, inhibits adhesion as well in the absence as in the presence of EDTA, and reversibly alters cell shape.     

Automated determination of critical oxygen concentration for routinely active fish

Synopsis We describe an automated respirometer and control system that determines critical (metabolism-limiting) oxygen concentration for routinely active fish. A microcomputer monitors fish metabolic rate as oxygen concentration in the closed respirometer declines; the critical oxygen concentration is signaled by metabolic-rate change, which is resolved via statistically based rules that consider both magnitude and consistency of rate deviations. After the critical oxygen concentration is found, data are written to disk, the respirometer is reoxygenated and another trial is initiated. This sequence can be repeated indefinitely without human intervention, allowing replicate estimates from a single fish. Ideally, metabolic rate should be calculated frequently during a trial, to minimize exposure of the fish to sub-critical oxygen concentrations. However, precision of measurement is limited by ‘noise’ related to length of time interval over which changes in oxygen concentration are determined, respirometer chamber volume, and fish respiration rate. Short time intervals lead to excessive noise, whereas long time intervals result in insufficient numbers of rate measurements. In a respirometer chamber of calculable optimum volume, measurements made by averaging oxygen readings taken very rapidly over two to three minute intervals provide a good compromise. We present data from experiments with bluegill,Lepomis macrochirus, to illustrate the method and show that critical oxygen concentrations identified by the system are consistent with estimates made by humans viewing graphs of the same experiments     

In rhizobiaceae five different species are produced by rearrangements of one genome, induced by DNA-damaging agents

Summary All bacterial strains classified into the family Rhizobiaceae can be induced to undergo a fundamental genome rearrangement. The special structure of their genome allows the formation of five distinctive phenotypes, each one adapted to a different habitat (Fig. 1).This genome rearrangement can be induced by DNA-damaging agents, UV irridiation or chemical mutagenesis. For expression, cells have to be protected against photorepair and their replication has to be reduced by stress treatment. The rearrangement process is, with special exceptions, reversible. Classes I and II comprise Agrobacteria and Rhizobia, class III nitrogen-fixing strains and classes IV and V two different carotenoid-pigmented types. One of the class V strains has been shown to be an effective legume-symbiont. DNA characteristics and inter-class hybridization results show not only that the genomes are completely reconstructed during each step of rearrangement, but also that the bacteria of all five classes are genetically correlated. In many cases the genetic label has been maintained during rearrangement into the different classes. The identity of each class is protected by a class-specific restriction and modification system, which was analyzed by phage typing experiments and by functional analysis of class-specific restriction endonucleases. We propose to designate the classes as different species of Rhizobiaceae. The unidirectional rearrangement between nodulating Rhizobia and tumorgenic Agrobacteria has been interpreted as a sequence of decreasing complexity of genomic regions coding for the plant interactions of these bacteria.