Nanni Din

UCP2 and UCP3 rise in starved rat skeletal muscle but mitochondrial proton conductance is unchanged.

The relationship between UCP2 and UCP3 expression and mitochondrial proton conductance of rat skeletal muscle was examined. Rats were starved for 24 h and the levels of UCP2 and UCP3 mRNA and UCP3 protein were determined by Northern and Western blots. Proton conductance was measured by titrating mitochondrial respiration rate and membrane potential with malonate. Starvation increased UCP2 and UCP3 mRNA levels more than 5‐fold and 4‐fold, respectively, and UCP3 protein levels by 2‐fold. However, proton conductance remained unchanged. These results suggest either that Northern and Western blots do not reflect the levels of active protein or that these UCPs do not catalyse the basal proton conductance in skeletal muscle mitochondria.

Prolactin and oestrogen synergistically regulate gene expression and proliferation of breast cancer cells

The pituitary hormone prolactin (PRL) plays an important role in mammary gland development. It was also suggested to contribute to breast cancer progression. In vivo data strongly supported a crucial role of PRL in promoting tumour growth; however, PRL demonstrated only a weak, if any, pro-proliferative effect on cancer cells in vitro. Several recent studies indicated that PRL action in vivo may be influenced by the hormonal milieu, e.g. other growth factors such as 17beta-oestradiol (E(2)). Here, we explored the potential interplay between PRL and E(2) in regulation of gene expression and cell growth. PRL alone induced either a weak or no proliferative response of T47D and BT-483 cells respectively, while it drastically enhanced cell proliferation in E(2)-stimulated cultures. Affymetrix microarray analysis revealed 12 genes to be regulated by E(2), while 57 genes were regulated by PRL in T47D cells. Most of the PRL-regulated genes (42/57) were not previously described as PRL target genes, e.g. WT1 and IER3. One hundred and five genes were found to be regulated upon PRL/E(2) co-treatment: highest up-regulation was found for EGR3, RUNX2, EGR1, MAFF, GLIPR1, IER3, SOCS3, WT1 and AREG. PRL and E(2) synergised to regulate EGR3, while multiple genes were regulated additively. These data show a novel interplay between PRL and E(2) to modulate gene regulation in breast cancer cells.

Design and synthesis of novel PPARα/γ/δ triple activators using a known PPARα/γ dual activator as structural template

Abstract Using a known dual PPARα/γ activator (5) as a structural template, SAR evaluations led to the identification of triple PPARα/γ/δ activators (18–20) with equal potency and efficacy on all three receptors. These compounds could become useful tools for studying the combined biological effects of PPARα/γ/δ activation.

A new Hind III restriction fragment length polymorphism in the hemophilia A locus

SummaryUsing a fragment of the cDNA for human coagulation factor VIII as a hybridization probe, we have detected a new polymorphic Hind III site in intron 19 of the factor VIII gene. The frequency of the minor allele is 0.30. This polymorphism shows strong linkage disequilibrium with a previously described BclI polymorphism in intron 18.

Vacuolar and extracellular maturation of Saccharomyces cerevisiae proteinase A.

The vacuolar aspartyl protease proteinase A (PrA) of Saccharomyces cerevisiae is encoded as a preproenzyme by the PEP4 gene and transported to the vacuole via the secretory route. Upon arrival of the proenzyme proPrA to the vacuole, active mature 42 kDa PrA is generated by specific proteolysis involving the vacuolar endoprotease proteinase B (PrB). Vacuolar activation of proPrA can also take place in mutants lacking PrB activity (prb1). Here an active 43 kDa species termed pseudoPrA is formed, probably by an autocatalytic process. When the PEP4 gene is overexpressed in wild‐type cells, mature PrA can be found in the growth medium. We have found that prb1 strains overexpressing PEP4 can form pseudoPrA extracellularly. N‐terminal amino acid sequence determination of extracellular, as well as vacuolar pseudoPrA showed that it contains nine amino acids of the propeptide, indicating a cleavage between Phe67 and Ser68 of the preproenzyme. This cleavage site is in accordance with the known substrate preference for PrA, supporting the notion that pseudoPrA is formed by autoactivation. When a multicopy PEP4 transformant of a prb1 mutant was grown in the presence of the aspartyl protease inhibitor pepstatin A, a significant level of proPrA was found in the growth medium. Our analyses show that overexpression of PEP4 leads to the secretion of proPrA to the growth medium where the zymogen is converted to pseudoPrA or mature PrA in a manner similar to the vacuolar processing reactions. Amino acid sequencing of secreted proPrA confirmed the predicted cleavage by signal peptidase between Ala22 and Lys23 of the preproenzyme.

Functional importance of the carboxyl tail cysteine residues in the human D1 dopamine receptor

Abstract: To assess the importance of the cysteine residues Cys347 and Cys351 in the carboxylic tail in the human D1 dopamine receptor, seven mutant receptors were constructed by PCR. The pharmacological and functional properties of the wild‐type and mutant receptors were assessed following transient expression in COS‐7 cells. Affinities for [3H]SCH 23390 of mutant S347 (Cys347→ Gly), T348 (Tyr348→ stop), S351 (Cys351→ Gly), T351 (Cys351→ stop), T352 (Pro352→ stop), and S347/S351 (Cys347→ Gly and Cys351→ Gly) were similar to that of wild‐type receptor, whereas the expression levels were reduced up to 80%. The potency of dopaminergic antagonists for these mutant receptors was very similar to that of the wild‐type receptor. However, mutant T347 (Cys347→ stop) showed a 15–25‐fold reduced affinity for the antagonists SCH 23390, (+)‐butaclamol, and cis‐flupentixol, thus not allowing radioligand analysis. Wild‐type and mutant receptors responded dose‐dependently with similar potency to dopamine and SKF 38393 with an increased adenylyl cyclase activity. However, mutant receptors with the Cys347 residue changed or removed displayed a diminished ability to activate adenylyl cyclase. Dopamine preexposure desensitized wild‐type and mutant S351 receptors. However, mutant receptors with Cys347 replaced or the distal part of the carboxyl tail removed were unable to desensitize. Thus, Cys347 in the cytoplasmic tail of the human D1 dopamine receptor is important for the receptor in maintaining the conformation for antagonist binding, to play a crucial role in activation of adenylyl cyclase, and to be essential for agonist‐induced desensitization.

Expression of a cytomegalovirus IE-1-factor VIII cDNA hybrid gene in transgenic mice.

A construct containing the 5′ end of the human cytomegalovirus major immediate early gene fused to the human coagulation factor VIII cDNA was used to produce transgenic mice. Two out of five transgenic lines transcribed the construct. The expression was consistently seen in a limited number, of tissues and was highest in muscle tissues. This is in contrast to the almost ubiquitous activity demonstrated in earlier studies with the IE-1 enhancer/promoter. Human factor VIII protein was detected immunochemically in muscle tissues at levels several times higher than in human plasma.

The open reading frame YAL048c affects the secretion of proteinase A in S. cerevisiae.

Over‐expression of the yeast PEP4 gene encoding the vacuolar aspartic protease proteinase A (PrA) leads to saturation of the vacuolar targeting system of the cell and missorting of PrA to the growth medium. In a screen for genes affecting the secretion of over‐expressed PrA we found that multiple copies of the open reading frame (ORF) YAL048c enhanced PrA secretion. Since no function has hitherto been ascribed to YAL048c, we undertook further studies of this ORF. Deletion of YAL048c resulted in slightly reduced secretion of over‐produced PrA. Furthermore, strains deleted for YAL048c showed a growth inhibition phenotype resulting in wrinkled colony morphology when grown on rich medium containing high concentrations of calcium. YAL048c is predicted to encode a polypeptide of 662 amino acid residues containing two consensus ATP/GTP‐binding site motifs and a putative carboxy‐terminal transmembrane region. In addition, the amino acid sequence contains two putative calcium‐binding domains. The YAL048c protein may be evolutionarily conserved, as homologues exist in humans and Caenorhabditis elegans. We suggest that the YAL048c protein is involved in vesicle transport in the secretory pathway. Copyright

Clinical use of DNA markers (RFLP) in genetic counselling and prenatal diagnosis of haemophilia A and B

land with the first settlers permanently inhabiting this area in the 16th or 17th century (Forsius, Eriksson 1980). DNA was prepared from blood samples of 130 individuals from 13 families belonging to the three kindreds. Linkage studies between TCD and the probes pDP34 (locus DXYSI) and 22-33 (locus DXSZl) were performed. Very close linkage was found between DXYSl and TCD. Out of 32 carrier females with offspring, 11 (32%) were heterozygous for pDP34. (The expected value based on Western European allele frequencies is 48%). No recombinants were observed in 33 meioses. Moreover, all the affected males so far studied had the 1 Ikb allele. Only two females with offspring were heterozygous at locus DXSIZ. They belonged to different kindreds. There were no recombinants among 4 meioses. In these two kindreds all affected individuals tested had the rarer 17kb allele while in the third kindred TCD segregatedwith themore frequent lOkb allele. We suggest that the three kindreds are connected, and that no recombination between DXYSI and TCD has occurred since the 17th century in these families. This could mean that TCD and DXYSI are very close to each other. In contrast, at least one recombination between D X S l l and TCD has occurred. Prenatal diagnosis can presently be offered to 3248% of carrier women. Further studies are being performed in an effort to better characterize the linkage relationships in the vicinity of TCD and to find new probes for prenatal diagnosis.