Structure of porphobilinogen synthase from Pseudomonas aeruginosa in complex with 5-fluorolevulinic acid suggests a double Schiff base mechanism

The seeds of jack fruit (Artocarpus integrifolia) contain two tetrameric lectins, jacalin and artocarpin. Jacalin was the first lectin found to exhibit the beta-prism I fold, which is characteristic of the Moraceae plant lectin family. Jacalin contains two polypeptide chains produced by a post-translational proteolysis which has been shown to be crucial for generating its specificity for galactose. Artocarpin is a single chain protein with considerable sequence similarity with jacalin. It, however, exhibits many properties different from those of jacalin. In particular, it is specific to mannose. The structures of two crystal forms, form I and form II, of the native lectin have been determined at 2.4 and 2.5 A resolution, respectively. The structure of the lectin complexed with methyl-alpha-mannose, has also been determined at 2.9 A resolution. The structure is similar to jacalin, although differences exist in details. The crystal structures and detailed modelling studies indicate that the following differences between the carbohydrate binding sites of artocarpin and jacalin are responsible for the difference in the specificities of the two lectins. Firstly, artocarpin does not contain, unlike jacalin, an N terminus generated by post-translational proteolysis. Secondly, there is no aromatic residue in the binding site of artocarpin whereas there are four in that of jacalin. A comparison with similar lectins of known structures or sequences, suggests that, in general, stacking interactions with aromatic residues are important for the binding of galactose while such interactions are usually absent in the carbohydrate binding sites of mannose-specific lectins with the beta-prism I fold.     

Biosynthetic studies on the alpha-glucosidase inhibitor acarbose: the chemical synthesis of dTDP-4-amino-4,6-dideoxy-alpha-D-glucose

To study the biosynthesis of the pseudotetrasaccharide acarbose, dTDP-4-amino-4,6-dideoxy-alpha-D-glucose (3) was prepared from galactose in 16 steps. After initial protecting-group manipulations, the 6-position of galactose was deoxygenated by hydride displacement of a tosylate. Similarly a tosyl group at the 4-position was displaced upon reaction with sodium azide. Conversion of the resulting glycoside to a glycosyl phosphate was accomplished by reaction of a glycosyl trichloroacetimidate with dibenzyl phosphate. Subsequent removal of the benzyl protecting groups and reduction of the azide by hydrogenation and coupling with an activated nucleoside phosphate gave dTDP-4-amino-4,6-dideoxy-alpha-D-glucose.     

Effects of L-carnitine on mechanical recovery of isolated rat hearts in relation to the perfusion with glucose and palmitate

The purpose of this study was to investigate the effects of L-carnitine on the hemodynamic parameters of Langendorff hearts. Isolated rat hearts were perfused with various solutions containing high or low concentrations of fatty acids, additional glucose or no glucose, and L-carnitine or no L-carnitine. The most interesting part of the experiments was the behaviour of the hearts in the reperfusion period after no-flow ischemia of 20 min. The results were: (1) With glucose and high fatty acid concentrations the hearts showed an improved recovery of the left ventricular functions in the reperfusion period compared with low fatty acid concentrations. Without glucose the left ventricular pressure is much lower in the reperfusion period. (2) Addition of L-carnitine improved the recovery of the ischemically damaged hearts. This improvement is especially impressive at low fatty acid concentrations. L-carnitine addition at high fatty acid concentrations but without glucose strongly improved reperfusion behaviour. (3) The coronary flow is increased by 2 experimental conditions: (i) perfusion at low levels of fatty acids, carnitine and with glucose and (ii) high levels of fatty acids and carnitine but without glucose. These findings suggest that supplementation of L-carnitine has a beneficial effect on the isolated heart under various conditions, and possibly on specific human heart diseases.     

Influence of different culture conditions on sarcoplasmic reticular calcium transport in isolated neonatal rat cardiomyocytes

This study investigates sarcoplasmic reticulum (SR) calcium-(Ca²⁺) transport ATPase (SERCA2a) and phospholamban (PLB) in cultured spontaneously contracting neonatal rat cardiomyocytes (CM) to ascertain the function of both SR proteins under various culture conditions. The two major SR proteins were readily detectable in cultured CM by immunofluorescent microscopy using specific anti-SERCA2 and anti-PLB antibodies. Double labeling technique revealed that PLB-positive CM also labeled with anti-SERCA2. Coexpression of SERCA2 and PLB in CM was supported by measurement of cell homogenate oxalate-supported Ca²⁺ uptake which was completely inhibited by thapsigargin and stimulated by protein kinase A-catalyzed phosphorylation. Under serum-free conditions, incubation of CM with the SERCA2a expression modulator 3,3,5-triiodo-L-thyronine (100 nM, 72 h) resulted in elevated Ca²⁺ uptake of +33%. Specific Ca²⁺ uptake activity was not altered if insulin was omitted from the serum-free culture medium but total SR Ca²⁺ transport activity was reduced under this culture condition. The results indicate that primary culture of spontaneously contracting neonatal rat CM can be employed as a useful model system for investigating both short- and long-term mechanisms determining the Ca²⁺ re-uptake function of the SR under defined culture conditions.     

Preimplantation genetic analysis of translocations: case-specific probes for interphase cell analysis

Carriers of balanced translocations show an increased risk of infertility and spontaneous abortions, because of errors in gametogenesis, and constitute a significant fraction of patients seeking assisted reproduction. The objective of this study was to design approaches for preimplantation diagnosis of chromosome translocations and to apply such techniques to the selection of chromosomally normal or balanced embryos prior to their transfer to the mother’s womb. Three slightly different approaches were assessed by means of chromosome-specific, non-isotopically labeled DNA probes and an assay based on fluorescence in situ hybridization- to score and characterize chromosomes in single blastomeres biopsied from embryos on their third day of development. The three approaches were used for preimplantation genetic diagnosis involving four couples who had enrolled in our IVF program and in which one of the partners was a carrier of one of the following translocations: 46,XX,t(12;20)(p13.1;q13.3), 46,XY,t(3;4) (p24;p15), 45,XY,der(14;15)(10q;10q), and 46,XY,t(6;11) (p22.1;p15.3). A total of 33 embryos were analyzed, of which 25 (75.8%) were found to be either unbalanced or otherwise chromosomally abnormal. Only a single embryo could be transferred to patients A and D, whereas three embryos were transferred to patient B in a total of two IVF cycles. Transfer of two embryos to patient C resulted in an ongoing pregnancy. Re-analysis of non-transferred embryos with additional probes confirmed the initial results in 95% (20/21) of the cases. In conclusion, case-specific translocation tests can be applied to any translocation carrier for the selection of normal or chromosomally balanced embryos prior to embryo transfer. This is expected significantly to increase the success rates in IVF cycles of translocation carriers, while preventing the spontaneous abortion or birth of abnormal offspring. Received: 13 January 1998 / Accepted: 24 March 1998     

Carboxypeptidase D (gp180), a Golgi-Resident Protein, Functions in the Attachment and Entry of Avian Hepatitis B Viruses

Carboxypeptidase D (gp180), one of many candidate receptors proposed for hepatitis B viruses (HBVs), was examined and found to be the actual cellular receptor for avian HBVs. This conclusion was based on the following observations: (i) gp180 was the only host protein that bound with high affinity to the pre-S ectodomain of the large duck hepatitis B virus (DHBV) envelope protein, which is known to be essential for virus infection; (ii) a pre-S subdomain which determines physical binding to gp180 was found to coincide with a domain functionally defined in infection competition experiments as a receptor binding domain; (iii) soluble gp180, lacking the membrane anchor, efficiently inhibited DHBV infection; (iv) efficient interspecies gp180–pre-S interaction was limited to the natural hosts of avian hepadnaviruses; and (v) expression of gp180 in a heterologous hepatoma cell line mediated cellular attachment and subsequent internalization of fluorescently labeled viral particles into vesicular structures. However, gp180 expression did not render transfected heterologous cells permissive for productive infection, suggesting that a species-specific coreceptor is required for fusion to complete viral entry. In contrast to the case for known virus receptors, gp180 was not detected on the hepatocyte cell surface but was found to be concentrated in the Golgi apparatus, from where it functions by cycling to and from the plasma membrane.     

The behaviour of the autonomous maize transposable element En/Spm in Arabidopsis thaliana allows efficient mutagenesis

The behavior of the autonomous maize transposable element En/Spm of maize was studied in Arabidopsis. Transgenic Arabidopsis plants carrying En-1 elements were propagated for 12 generations using a single seed descent procedure. The distribution and activity of the En-1 element was monitored using Southern DNA hybridisations in generations 1, 6 and 12. In the first generation the highest number of En-1 insertions per line was 7, which increased to 20 in generation 12. The average number of En-1 insertions increased only slightly in the population, due to a gradual accumulation of segregants that lost the transposable element. During the development of the En-1 mutagenised population the element remained active even in the high-copy lines. In situ hybridisation demonstrated that multiple En-1 insertions were distributed over all Arabidopsis chromosomes. From the initial En-1 mutagenised populations many unstable gene mutations were recovered, indicating that En-1 can be used as a efficient tool for gene tagging in Arabidopsis.