Volume 381, Issue 4

Oligodeoxynucleotides designed to form intramolecular triple helices are widely used as model systems in thermodynamic and structural studies. We now report results from UV, Raman and NMR experiments demonstrating that the strand polarity, which also determines the orientation of the connecting loops, has a considerable impact on the formation and stability of pyr pur pyr triple helices. There are two types of monomolecular triplexes that can be defined by the location of their purine tract at either the 5['] or 3[']end of the sequence. We have examined four pairs of oligonucleotides with the same base composition but with reversed polarity that can fold into intramolecular triple helices with seven base triplets and two T4 loops under appropriate conditions. UV spectroscopic monitoring of thermal denaturation indicates a consistently higher thermal stability for the 5[']sequences at pH 5.0 in the absence of Mg[2+] ions. Raman spectra provide evidence for the formation of triple helices at pH 5 for oligomers with purine tracts located at either the 5['] or 3[']end of the sequence. However, NMR measurements reveal considerable differences in the secondary structures formed by the two types of oligonucleotides. Thus, at acidic pH significant structural heterogeneity is observed for the 3[']sequences. Employing selectively 15labeled oligomers, NMR experiments indicate a folding pattern for the competing structures that at least partially changes both Hoogsteen and WatsonCrick basebase interactions.

A 55 kDa nuclear localization signal binding protein (p55) is involved in the transport of the goat uterine estrogen receptor from the cytoplasm to the nuclear pore complex (NPC). p55 forms a complex with a 12 kDa protein (p12) which in turn becomes docked at the NPC. The present study reports on the purification and functional characterization of p12. Both p55 and p12 are Mg[2+]dependent ATPases. The proteinprotein interactions that take place between these two molecules at the NPC cause an enhancement in the net ATPase activity associated with the protein complex. Presumably, this enhanced ATPase function helps in the final nuclear entry of the estrogen receptor; p55 remains associated with p12 at the nuclear entry site under these conditions.

The effect of nucleotides on binding of the B2 kinin (BK) receptor agonist [[3]H]BK and the antagonist [[3]H]NPC17731 to particulate fractions of human foreskin fibroblasts was studied. At 0C, particulate fractions exhibited a single class of binding sites with a K of 2.3n for [[3]H]BK and a K of 3.8n for the antagonist [[3]H]NPC17731. Incubation with radioligands at 37C for 5 min gave a reduction of agonist, as well as antagonist, binding that was between 0 40% depending on the preparation, even in the absence of guanosine nucleotides. As shown by Scatchard analysis, this reduction in specific binding was due to a shift in the affinity of at least a fraction of the receptors. The presence at 37C of the guanine nucleotides GTP, GDP and their poorly hydrolyzable analogs left [[3]H] NPC17731 binding unaffected, but reduced the receptor affinity for [[3]H]BK to a K of about 15 n. The maximal number of receptors, however, was unchanged. This affinity change was strongly dependent on the presence of bivalent cations, in particular Mg[2+]. It was reversed by incubation at 0C. The rank order of the guanosine nucleotides for [[3]H]BK binding reduction was GTP[γS] = Gppp [greater than] GTP = GDP [greater than] GDP[βS]. GMP, ATP, ADP and AMP showed no influence on agonist binding. A model for the interaction of the B2 kinin receptor with G proteins is discussed.

It was previously reported that 8701-BC breast tumour cells express the gene for parathyroid hormonerelated peptide (PTHrP) and PTH/PTHrP receptor (PTHrPR) and release immunoreactive PTHrP (iPTHrP) into the extracellular medium. Since the regulation of PTHrP and PTHrPR by breast cancer cells has been poorly investigated so far, we have chosen the 8701- BC cell line as a model system to investigate whether alterations in the extracellular Ca[2+] concentration ([Ca[2+]]) and treatment with some wellknown differentiation agents for breast cells, such as dimethyl sulfoxide, hydrocortisone, progesterone, prolactin, alltrans retinoic acid and transforming growth factorβ1 might (i) modulate quantitatively the release of iPTHrP, (ii) affect the PTHrP promoter usage and mRNA splicing patterns, and (iii) modify the expression of PTHrPR. The data obtained indicate that 8701-BC cells are potentially able to utilise different start sites and mRNA splicing patterns for PTHrP transcription, and respond to variations of [Ca[2+]] and to the addition of two hormones, hydrocortisone and progesterone, with modifications in the extracellular amount of iPTHrP. Moreover, expression of PTHrPR is also modulated by changes of [Ca[2+]] or treatment with hydrocortisone. This indicates that the 8701-BC cell line is a suitable in vitro model for further studies on the complex molecular regulation of the PTHrP/PTHrPR pair in breast cancer.

Calcineurin (CN) exhibits a bimodal regulation by different concentrations of vanadyl ions (VO[2+]) in the presence of Mn[2+]. Low concentrations of VO[2+] inhibit the enzyme, with 50[] VO[2+] completely inhibiting CN activity, while high concentrations, up to 500[] VO[2+], stimulate the CN activity. A similar bimodal regulation of CN was not observed with either calcium or vanadate under the same conditions. Xband electron spin resonance spectroscopy, used to study the binding of VO[2+] to the catalytic subunit A of calcineurin, show that there are two kinds of binding sites in the A subunit.

The relationship between the structure and activity of metaand parahydroxylated monophenols was studied during their tyrosinasecatalysed hydroxylation and the ratelimiting steps of the reaction mechanism were identified. The parahydroxylated substrates permit us to study the effect of a substituent (R) in the carbon-1 position (C-1) of the benzene ring on the nucleophilic attack step, while the meta group permits a similar study of the effect on the electrophilic attack step. Substrates with a OCH[3] group on C-1, as phydroxyanisol (4HA) and mhydroxyanisol (3HA), or with a CH[2]OH group, as phydroxybenzylalcohol (4HBA) and mhydroxybenzylalcohol (3HBA), were used because the effect of the substituent (R) size was assumed to be similar. However, the electrondonating effect of the OCH[3] group means that the carbon-4 position (C-4) is favoured for nucleophilic attack (parahydroxylated substrates) or for electrophilic attack (metahydroxylated substrates). The electronattracting effect of the CH[2]OH group has the opposite effect, hindering nucleophilic (para) or electrophilic (meta) attack of C-4. The experimental data point to differences between the maximum steadystate rate (V ) of the different substrates, the value of this parameter depends on the nucleophilic and electrophilic attack. However, differences are greatest in the Michaelis constants (K ), with the metahydroxylated substrates having very large values. The catalytic efficiency k /K is much greater for the parahydroxylated substrates although it varies greatly between one substrate and the other. However, it varies much less in the metahydroxylated substrates since this parameter describes the power of the nucleophilic attack, which is weaker in the meta OH. The large increase in the K of the metahydroxylated substrates might suggest that the phenolic OH takes part in substrate binding. Since this is a weaker nucleophil than the parahydroxylated substrates, the binding constant decreases, leading to an increase in K . The catalytic efficiency of tyrosinase on a monophenol (para or meta) is directly related to the nucleophilic power of the oxygen of the phenolic OH. The oxidation step is not limiting since if this were the case, the para and meta substrates would have the same V . The small difference between the absolute values of V suggests that the rate constants of the nucleophilic and electrophilic attacks are on the same order of magnitude.

Hydrophobic urethanyl derivatives of 3-amidinophenylalanine methyl ester were found to be relatively potent and selective factor Xa inhibitors. These compounds consist of the argininemimetic 3-benzamidino group as P1 residue and of hydrophobic residues as potential interaction partners for the S3/S4 aryl binding site of the enzyme. Attempts to possibly identify their binding mode to factor Xa via the Xray crystal structure of a trypsin/inhibitor complex and analogy modeling on the crystal structure of factor Xa failed. However, synthesis of enantiomerically pure (R) and (S)derivatives, combined with modeling experiments, led to an hypothetical nonsubstrate like binding mode, which was fully confirmed by the remarkably inhanced inhibitory potency of derivatives in which the methyl ester was replaced by arylamides for interactions with the S3/S4 enzyme binding subsites. With adamantyloxycarbonyl(R)-3-amidinophenylalaninephenethylamide a nanomolar inhibiton was obtained, thus indicating this new class of factor Xa inhibitors as a highly promising lead structure.

PAX7, a member of the PAX transcription factor gene family, is normally expressed at high levels during development in the neural tube and in skeletal muscle precursor cells. Interestingly, PAX7 expression was also identified in tumor cells developing from these cell types. To date not much is known about the molecular mechanisms controlling the regulation of PAX7 expression. Therefore, we have cloned and sequenced part of the proximal 5[']flanking region of the human PAX7 gene. Computerbased sequence analysis identified putative binding sites for basic transcription factors. Analysis of a series of deletion constructs in different cell types suggested that a distal region containing several Eboxes might be involved in muscle specific expression of PAX7, and that a distinct proximal region can enhance basal PAX7 expression in tumor cells.

The peroxin Pex3p has been identified as an integral peroxisomal membrane protein in yeast where pex3 mutants lack peroxisomal remnant structures. Although not proven in higher organisms, a role of this gene in the early peroxisome biogenesis is suggested. We report here the cDNA cloning and the genomic structure of the mouse PEX3 gene. The 2 kb cDNA encodes a polypeptide of 372 amino acids (42 kDa). The gene spans a region of 30 kb, contains 12 exons and 11 introns and is located on band A of chromosome 10. The putative promoter region exhibits characteristic housekeeping features. PEX3 expression was identified in all tissues analyzed, with the strongest signals in liver and in testis, and could not be induced by fenofibrate. The data presented may be useful for the generation of a mouse model defective in PEX3 in order to clarify the yet unknown functional impact of disturbances in early peroxisomal membrane assembly.

A polyHistagged bradykinin (BK) B[2] receptor (pHis BKR) cDNA was constructed and expressed in COS-7 cells. The pHisBKR is suitable for both immunoprecipitation and immunoblotting with antipolyHis antibodies and can be easily purified using NiNTA columns. Immunochemical detection revealed a molecular mass of approximately 66 kDa. The pHisBKR is capable of mediating BKinduced stimulation of inositol phosphate formation as well as of mitogenactivated protein kinase (MAPK) activity. Compared with the wildtype receptor (WTBKR) the tagged receptor showed a slightly enhanced affinity towards BK but a reduced expression level. Despite these modified pharmacological properties the pHistagged BKR may be a useful tool for studying BKR modifications and signaling.

Cells of the wildtype yeast strain Zygosaccharomyces bisporus CBS 702 form αhydroxy ketones from aromatic amino acid precursors during fermentation. Pyruvate decarboxylase (PDC, E.C. 4.1.1.1), the key enzyme of this biotransformation catalysing the nonoxidative decarboxylation of pyruvate and other 2- oxoacids, was purified and characterised. The active enzyme is homotetrameric (α[4]) with a molecular mass of about 244 kDa. Activation of PDC by its substrate pyruvate results in a sigmoidal dependence of the reaction rate from substrate concentration (apparent K value 1.73 m; Hill coefficient 2.10). A cDNA library was screened using a PCRbased procedure, and a 1856 bp cDNA of PDC was identified and sequenced. The cDNA encodes a polypeptide of 563 amino acid residues (monomeric unit). Sequence alignments demonstrate high homologies ([greater than] 80%) to PDC genes from Saccharomyces cerevisiae, Kluyveromyces lactis and Kluyveromyces marxianus.