Novel sterically hindered cannabinoid CB1 receptor ligands

In the present study, 11 novel N-(3,3-diphenyl)propyl-2,2-diphenylacetamide derivatives (4a-d and 9a-g) and six triphenylacetamides (10a-c and 11a-c) were synthesized and tested as ligands of cannabinoid CB(1) and CB(2) receptors. All compounds exhibited affinity for CB(1) and CB(2) receptors. Four compounds (4b, 9a, 9b, and 11a) showed selectivity for CB(1) versus CB(2) receptors, although only the N-(3,3-diphenyl)propyl-2,2-diphenylacetamide (4b) can be considered a potent CB(1) ligand (K(i)=58 nM). It was 140-fold selective over CB(2) receptors (K(i)=7800 nM) and behaved as an inverse agonist by stimulating forskolin-induced cAMP formation in mouse N18TG2 neuroblastoma cells. This compound is the first of a novel class of tetraphenyl CB(1) ligands that, in view of its easy synthesis and high affinity for CB(1) receptors and despite its sterical hindrance, will be useful for the design of new blockers of this therapeutically exploitable receptor type.     

βA and βthal DNA haplotypes in Sicily

Summary A close association between specific restriction fragment polymorphism patterns and specific mutations in Mediterranean people with thalassemia has been demonstrated by Kazazian et al. (1984). This finding is useful to characterize the number and types of mutations in each ethnic group for setting up prenatal diagnosis in the first trimester of pregnancy by the oligonucleotide technique. For this reason we studied 99 ^thal and 46 ^A chromosomes in the Sicilian population. We found seven different cleavage patterns, not considering two new haplotypes so far uncharacterized. Many of the patients (68.3%) were genetic compounds for different haplotypes while only 31.7% were haplotype homozygotes. They may still be thalassemia compound heterozygotes. These findings confirm the molecular basis of the heterogeneity of thalassemia in Sicily.     

NONO enhances mRNA processing of super‐enhancer‐associated GATA2 and HAND2 genes in neuroblastoma

High‐risk neuroblastoma patients have poor survival rates and require better therapeutic options. High expression of a multifunctional DNA and RNA‐binding protein, NONO, in neuroblastoma is associated with poor patient outcome; however, there is little understanding of the mechanism of NONO‐dependent oncogenic gene regulatory activity in neuroblastoma. Here, we used cell imaging, biochemical and genome‐wide molecular analysis to reveal complex NONO‐dependent regulation of gene expression. NONO forms RNA‐ and DNA‐tethered condensates throughout the nucleus and undergoes phase separation in vitro, modulated by nucleic acid binding. CLIP analyses show that NONO mainly binds to the 5′ end of pre‐mRNAs and modulates pre‐mRNA processing, dependent on its RNA‐binding activity. NONO regulates super‐enhancer‐associated genes, including HAND2 and GATA2. Abrogating NONO RNA binding, or phase separation activity, results in decreased expression of HAND2 and GATA2. Thus, future development of agents that target RNA‐binding activity of NONO may have therapeutic potential in this cancer context.     

Production of functionally active <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> isopenicillin N synthase in the yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis>

Background  

β-Lactams like penicillin and cephalosporin are among the oldest known antibiotics used against bacterial infections. Industrially, penicillin is produced by the filamentous fungus Penicillium chrysogenum. Our goal is to introduce the entire penicillin biosynthesis pathway into the methylotrophic yeast Hansenula polymorpha. Yeast species have the advantage of being versatile, easy to handle and cultivate, and possess superior fermentation properties relative to filamentous fungi. One of the fundamental challenges is to produce functionally active enzyme in H. polymorpha.     

A novel amino acid substitution in the reactive site of a congenital variant antithrombin. Antithrombin pescara, ARG393 to pro, caused by a CGT to CCT mutation

Antithrombin is a plasma protein inhibitor that can be grouped within a serine proteinase inhibitor superfamily. Antithrombin Pescara is a functional variant of antithrombin found in a family with a high incidence of thrombosis. Preliminary functional analysis has suggested that the abnormality resides in the reactive site rather than in the heparin binding domain of the molecule. Accordingly, we have isolated the variant from plasma using heparin-Sepharose chromatography, followed by chromatography upon thrombin-Sepharose to remove the normal antithrombin that is present (the propositus is heterozygous for the variant). The variant protein was reduced, S-carboxy-methylated, and fragmented with CNBr. A pool ("CNBr pool 4") containing the reactive site region was isolated by reverse-phase high performance liquid chromatography and sequentially treated with trypsin and V8 protease. Fast atom bombardment-mass spectrometric analysis of this subdigest identified a novel peptide of mass 1708. Four steps of Edman degradation together with further analysis by fast atom bombardment-mass spectroscopy identified the NH2-terminal sequence of this peptide as Ala-Ala-Ala-Ser. The mass of the novel peptide and its changing mass in response to Edman degradation are only compatible with its identity as Ala382-Arg399, with the reactive site Arg393 replaced by Pro. Using specific oligonucleotide hybridization, we demonstrated that the molecular defect of antithrombin Pescara is caused by a CGT to CCT mutation in codon 393. These findings may be of broad interest, as other members of the serine protease inhibitor superfamily contain arginine at their reactive sites and may be expected to undergo a similar mutation.