A rat gene encoding heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

There are at least 3 isozymes of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, a bifunctional enzyme which catalyzes the synthesis and degradation of fructose 2,6-bisphosphate. A 22-kb rat gene that encodes the heart isozyme has been identified and compared with the 55-kb rat gene encoding the liver and muscle isozymes which had been described earlier. Although these 2 genes include 12 successive similar exons, they contain dissimilar exons at both ends, consistent with the occurrence of different regulatory domains at the N- and C-termini in the 3 isozymes.     

Structural and Functional Impact of SRP54 Mutations Causing Severe Congenital Neutropenia

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A locus involved in kanamycin, chloramphenicol and L-serine resistance is located in the bglY-galU region of the Escherichia coli K12 chromosome

Summary Spontaneous mutants of Escherichia coli K12 displaying an increased level of the kanamycin resistance conferred by plasmid pGR71 were selected. Several mutants obtained in this way apparently carry large chromosomal deletions extending into galU and/or bglY (27 min). This positive selection of deletions allowed detection of a new locus located between galU and bglY. Deletions of this locus are responsible for increased resistance to kanamycin (Irk), decreased resistance to l-serine in minimal medium (Drs) and decreased resistance to chloramphenicol (Drc) when a cat gene is present in the bacteria.     

Cloning and expression in Escherichia coli of a rat hepatoma cell cDNA coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

In liver, the 470-residue bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) catalyses the synthesis and degradation of fructose 2,6-bisphosphate, a potent stimulator of glycolysis. In rat hepatoma (HTC) cells, this enzyme has kinetic, antigenic, and regulatory properties, such as insensitivity to cyclic AMP-dependent protein kinase and lack of associated FBPase-2 activity, that differ from those in liver. To compare the sequence of the HTC enzyme with that of the liver enzyme, we have cloned the corresponding fully-coding cDNA from HTC cells. This cDNA predicts a protein of 448 residues in which the first 32 residues of liver PFK-2/FBPase-2 including the cyclic AMP target sequence have been replaced by a unique N-terminal decapeptide. The rest of the protein is identical with the liver enzyme. An N-terminally truncated recombinant peptide of 380 residues containing the PFK-2 and FBPase-2 domains was expressed in Escherichia coli as a beta-galactosidase fusion protein. It was recognized by anti-PFK-2 antibodies but its enzymic activities were barely detectable. In contrast, a cDNA fully-coding for the HTC enzyme could be expressed in E. coli as a beta-galactosidase-free peptide that exhibited both PFK-2 and FBPase-2 activities. This peptide had those PFK-2 kinetic properties of the HTC enzyme that differ from the liver enzyme. These data, together with immunoblot experiments, suggest that the lack of associated FBPase-2 activity in HTC cells results from a post-translational modification of the enzyme rather than from the difference in amino acid sequence. As well as this peculiar type of PFK-2/FBPase-2 mRNA, HTC cells also contained low concentrations of the liver-type mRNA. Unlike in liver, neither mRNA was induced by dexamethasone in these cells.     

New informative polymorphism at the DXS304 locus,a close distal marker for the fragile X locus

Summary The polymorphic DNA marker DXS304 detected by probe U6.2 has recently been shown to be closer to the fragile X locus than previously available markers. Its usefulness has however been limited by its relatively low heterozygosity. We have isolated, by cosmid cloning, a 67 kilobase region around probe U6.2 and have characterized a new probe (U6.2-20E) that detects BanI and BstEII restriction fragment length polymorphisms (RFLPs). The BanI RFLP has a heterozygosity of 0.49 and is in partial linkage disequilibrium with the previously described polymorphism, with a combined heterozygosity of 0.63. Furthermore, we have found that the U6.2 original probe, which probably detects an insertion-deletion polymorphism, is also informative in BanI digests. Thus, the two informative RFLPs at the DXS304 locus can be conveniently tested in a single hybridization with a single digest. An updated linkage analysis confirms that DXS304 is distal to the fragile X locus. This informative locus can now be used effectively for genetic mapping of the Xq27–q28 region, and for diagnostic applications in fragile X or Hunter syndrome families.     

A mechanical analysis of the closed Hancock heart valve prosthesis

In order to obtain mechanical specifications for the design of an artificial leaflet valve prosthesis, a geometrically non-linear numerical model is developed of a closed Hancock leaflet valve prosthesis. In this model, the fibre reinforcement of the leaflet and the viscoelastic properties of frame and leaflets are incorporated. The calculations are primarily restricted to 1/6 part of the valve and a time varying pressure load is applied. The calculations are verified experimentally by measuring the commissure displacements and leaflet centre displacement of a Hancock valve. The numerically obtained commissure displacements are found to be linearly dependent on the pressure load, and the slope of the curves is hardly dependent on loading type and loading velocity. Experimentally a difference is found between the three commissure displacements, which is also predicted numerically using a simplified asymmetric total valve model. Besides, experimentally a clear dependency of commissure displacements on frame size is found. For the leaflet centre displacement, a qualitative agreement exists between numerical prediction and experimental result, although the numerical predicted values are systematically higher. The numerically obtained stress distributions revealed that the maximum von Mises intensity in the membranes occurs in the vicinity of the commissure in the free leaflet area (0.2 N mm-2). Wrinkling of the membranes may occur in the coaptation area near the leaflet suspension. The maximum fibre stress is found near the aortic ring in the fibres which form the boundaries of the coaptation area (0.64 N mm-2). These locations seem to correlate with some common regions of tissue valve failure.