The synthesis of 17-alkoxycarbonyl- and 17-carboxamido-13alpha-estra-1,3,5(10),16-tetraene derivatives via palladium-catalyzed carbonylation reactions

17-Alkoxycarbonyl- and 17-carboxamido-13alpha-estra-1,3,5(10),16-tetraenes were synthesized from the 17-iodo-13alpha-estra-1,3,5(10),16-tetraene derivative in palladium-catalyzed alkoxycarbonylation and aminocarbonylation reactions, respectively. The synthesis of the 17-iodo-16-ene derivative, used as substrate, is based on the transformation of the 17-keto derivative (epiestrone methyl ether) to hydrazone, which was treated with iodine in the presence of a base (1,1,3,3-tetramethyl guanidine). 17-Carboxamides were obtained in good yields (up to 88%) not only with simple alkyl/aryl amines but also with amino acid methyl esters as N-nucleophiles. The use of alcohols as O-nucleophiles in alkoxycarbonylation resulted in the corresponding 17-esters; however, yields of synthetic interest were obtained only with methanol.     

2,3-benzodiazepine-type AMPA receptor antagonists and their neuroprotective effects

AMPA receptors are fast ligand-gated members of glutamate receptors in neuronal and many types of non-neuronal cells. The heterotetramer complexes are assembled from four subunits (GluR1-4) in region-, development- and function-selective patterns. Each subunit contains three extracellular domains (a large amino terminal domain, an agonist-binding domain and a transducer domain), and three transmembrane segments with a loop (pore forming domain), as well as the intracellular carboxy terminal tail (traffic and conductance regulatory domain). The binding of the agonist (excitatory amino acids and their derivatives) initiates conformational realignments, which transmit to the transducer domain and membrane spanning segments to gate the channel permeable to Na+, K+ and more or less to Ca2+. Several 2,3-benzodiazepines act as non-competitive antagonists of the AMPA receptor (termed also negative allosteric modulators), which are thought to bind to the transducer domains and inhibit channel gating. Analysing their effects in vitro, it has been possible to recognize a structure-activity relationship, and to describe the critical parts of the molecules involved in their action at AMPA receptors. Blockade of AMPA receptors can protect the brain from apoptotic and necrotic cell death by preventing neuronal excitotoxicity during pathophysiological activation of glutamatergic neurons. Animal experiments provided evidence for the potential usefulness of non-competitive AMPA antagonists in the treatment of human ischemic and neurodegenerative disorders including stroke, multiple sclerosis, Parkinson's disease, periventricular leukomalacia and motoneuron disease. 2,3-benzodiazepine AMPA antagonists can protect against seizures, decrease levodopa-induced dyskinesia in animal models of Parkinson's disease demonstrating their utility for the treatment of a variety of CNS disorders.     

Biogenesis of cytosolic ribosomes requires the essential iron-sulphur protein Rli1p and mitochondria

Mitochondria perform a central function in the biogenesis of cellular iron-sulphur (Fe/S) proteins. It is unknown to date why this biosynthetic pathway is indispensable for life, the more so as no essential mitochondrial Fe/S proteins are known. Here, we show that the soluble ATP-binding cassette (ABC) protein Rli1p carries N-terminal Fe/S clusters that require the mitochondrial and cytosolic Fe/S protein biogenesis machineries for assembly. Mutations in critical cysteine residues of Rli1p abolish association with Fe/S clusters and lead to loss of cell viability. Hence, the essential character of Fe/S clusters in Rli1p explains the indispensable character of mitochondria in eukaryotes. We further report that Rli1p is associated with ribosomes and with Hcr1p, a protein involved in rRNA processing and translation initiation. Depletion of Rli1p causes a nuclear export defect of the small and large ribosomal subunits and subsequently a translational arrest. Thus, ribosome biogenesis and function are intimately linked to the crucial role of mitochondria in the maturation of the essential Fe/S protein Rli1p.     

Mapping modifiers affecting muscularity of the myostatin mutant (Mstn(Cmpt-dl1Abc)) compact mouse

The hypermuscular Compact phenotype was first noted in a line of mice selected for high body weight and protein content. A new line, based on mice showing the Compact phenotype, was formed and selected for maximum expression of the Compact phenotype. Previously we mapped and identified a 12-bp deletion in the myostatin gene, denoted Mstn(Cmpt-dl1Abc), which can be considered as a major gene responsible for the hypermuscular phenotype. Genetic analysis revealed that full expression of the hypermuscular phenotype requires the action of modifier loci in addition to Mstn(Cmpt-dl1Abc). To map these modifier loci, an interspecific F(2) population was generated between Comp9, an inbred line homozygous for Mstn(Cmpt-dl1Abc), and CAST/Ei, an inbred line generated from Mus musculus castaneus. Selective DNA pooling and genotyping, separately by gender, was carried out within a subpopulation of the F(2) consisting of individuals homozygous for Mstn(Cmpt-dl1Abc). Significant association with hypermuscularity at a false discovery rate (FDR) of 0.05 was found for markers on chromosomes 3, 5, 7, 11, 16, and X. In all cases, the marker allele derived from the Comp9 parent showed a higher frequency in the hypermuscular group and the CAST/Ei allele in the normal group. The modifier loci apparently exerted their effects on muscularity only in the presence of Mstn(Cmpt-dl1Abc).     

Synthesis and receptor-binding examinations of the normal and 13-epi-D-homoestrones and their 3-methyl ethers

An effective epimerization of the normal estrone 3-methyl and 3-benzyl ethers by using o-phenylenediamine and AcOH made the possibility for facile entry into the 13alpha-estrone series. Combination of this synthetic methodology with an isolation step carried out by means of the Girard-P reagent, the corresponding ethers of 13-epi-estrone were obtained in excellent yields. The 3-hydroxy and 3-methoxy D-homoestrone derivatives in both the normal and the 13alpha-estrone series were then synthesized and tested in vitro in a radioligand-binding assay. The estrogen receptor recognizes these compounds, but their relative binding affinities (RBAs) are lower than that of the reference compound 3,17beta-estradiol. The progesterone receptor-binding affinities of the four D-homo derivatives were also tested showing low values for 13alpha-D-homoestrone and its 3-methyl ether. Pharmacologically, these 13alpha-D-homoestrone derivatives are estrogen receptor-selective molecules.     

Calretinin and calbindin D28k have different domain organizations

The domain organization of calretinin (CR) was predicted to involve all six EF-hand motifs (labeled I to VI) condensed into a single domain, as characterized for calbindin D28k (Calb), the closest homolog of calretinin. Unperturbed (1)H,(15)N HSQC NMR spectra of a (15)N-labeled calretinin fragment (CR III-VI, residues 100-271) in the presence of the unlabeled complimentary fragment (CR I-II, residues 1-100) show that these fragments do not interact. Size exclusion chromatography and affinity chromatography data support this conclusion. The HSQC spectrum of (15)N-labeled CR is similar to the overlaid spectra of individual (15)N-labeled CR fragments (CR I-II and CR III-VI), also suggesting that these regions do not interact within intact CR. In contrast to these observations, but in accordance with the Calb studies, we observed interactions between other CR fragments: CR I (1-60) with CR II-VI (61-271), and CR I-III (1-142) with CR IV-VI (145-271). We conclude that CR is formed from at least two independent domains consisting of CR I-II and CR III-VI. The differences in domain organization of Calb and CR may explain the specific target interaction of Calb with caspase-3. Most importantly, the comparison of CR and Calb domain organizations questions the value of homologous modeling of EF-hand proteins, and perhaps of other protein families.     

PNA-DNA chimeras containing 5-alkynyl-pyrimidine PNA units. Synthesis, binding properties, and enzymatic stability

Three chimeric dimer synthons (oeg_t(NH)T, oeg_up(NH)T and oeg_uh(NH)T) containing thymine (t), 5-(1-propynyl)-uracil (up) and 5-(1-hexyn-1-yl)-uracil (uh) PNA units with N-(2-hydroxyethyl)glycine (oeg) backbone were synthesized in solution and incorporated into T20 oligonucleotide analogues, using standard P-amidite chemistry. Insertion of dimer blocks led to destabilization of duplexes with dA20 target. The smallest Tm drops were found for chimeras containing oeg_up(NH)T dimers. Incorporation of the chimeric synthons into the 3'-end of T20 brought about growing resistance to 3'-exonucleolytic (SV PDE) cleavage in the order of oeg_t(NH)T < oeg_up(NH)T < oeg_uh(NH)T. Due to different endonuclease activities of 3'- and 5'-exonucleases applied, placing of five consecutive dimers at the 5'-terminus resulted in a relatively smaller, but also side-chain dependent, stabilization towards the hydrolysis by 5'-exonuclease (BS PDE). Neither exonucleases (SV and BS PDE) nor an endonuclease (Nuclease P1) could hydrolyse the unnatural phosphodiester bond linking the 3'-OH of thymidine to the terminal OH of N-(2-hydroxyethyl)glycine PNA backbone.     

Properties of the basal calcium influx in human red blood cells

The basal (45)Ca(2+) influx in human red blood cells (RBC) into intact RBC was measured. (45)Ca(2+) was equilibrated with cells with t(1/2)=15-20 s and the influx reached the steady state value in about 90-100 s and the steady state level was 1.5+/-0.2 micromol/l(packed cells) (n=6) at 37 degrees C. The average value of the Ca(2+) influx rate was 43.2+/-8.9 micromol/l(packed cells) hour. The rate of the basal influx was pH-dependent with a pH optimum at pH 7.0 and on the temperature with the temperature optimum at 25 degrees C. The basal Ca(2+) influx was saturable with Ca(2+) up to 5 mmol/l but at higher extracellular Ca(2+) concentrations caused further increase of basal Ca(2+) influx. The (45)Ca(2+) influx was stimulated by addition of submicromolar concentrations of phorbol esters (phorbol 12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and forskolin. Uncoupler (3,3',4',5-tetrachloro-salicylanilide (TCS) 10(-6)-10(-5) mol/l) inhibited in part the Ca(2+) influx. The results show that the basal Ca(2+) influx is mediated by a carrier and is under control of intracellular regulatory circuits. The effect of uncoupler shows that the Ca(2+) influx is in part driven by the proton-motive force and indicates that the influx and efflux of Ca(2+) are coupled via the RBC H(+) homeostasis.