Specific binding of HIV-1 nucleocapsid protein to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues.

The nucleocapsid (NC) protein of human immunodeficiency virus HIV-1 (NCp7) is responsible for packaging the viral RNA by recognizing a packaging site (PSI) on the viral RNA genome. NCp7 is a molecule of 55 amino acids containing two zinc fingers, with only the first one being highly conserved among retroviruses. The first zinc finger is flanked by two basic amino acid clusters. Here we demonstrate that chemically synthesized NCp7 specifically binds to viral RNA containing the PSI using competitive filter binding assays. Deletion of the PSI from the RNA abrogates this effect. The 35 N-terminal amino acids of NCp7, comprising the first zinc finger, are sufficient for specific RNA binding. Chemically synthesized mutants of the first zinc finger demonstrate that the amino acid residues C-C-C/H-C/H are required for specific RNA binding and zinc coordination. Amino acid residues F16 and T24, but not K20, E21 and G22, located within this zinc finger, are essential for specific RNA binding as well. The second zinc finger cannot replace the first one. Furthermore, mutations in the basic amino acid residues flanking the first zinc finger demonstrate that R3, 7, 10, 29 and 32 but not K11, 14, 33 and 34 are also essential for specific binding. Specific binding to viral RNA is also observed with recombinant NCp15 and Pr55Gag. The results demonstrate for the first time specific interaction of a retroviral NC protein with its PSI RNA in vitro.     

Characterization and sequence of a Thermomonospora fusca xylanase.

TfxA is a thermostable xylanase produced by the thermophilic soil bacterium Thermomonospora fusca. The enzyme was purified to homogeneity from the culture supernatant of Streptomyces lividans transformed by plasmid pGG92, which carries the gene for TfxA, xynA. The molecular mass of TfxA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 32 kDa. TfxA is extremely stable, retaining 96% of its activity after 18 h at 75 degrees C. It has a broad pH optimum around pH 7 and retains 80% of its maximum activity between pH 5 and 9. The native enzyme binds strongly to both cellulose and insoluble xylan even though it has no activity on cellulose. Treatment of TfxA with a T. fusca protease produced a 24-kDa catalytically active fragment that had the same N-terminal sequence as TfxA. The fragment does not bind to cellulose and binds weakly to xylan. The Vmax values for TfxA and the fragment are 600 and 540 mumol/min/mg, respectively, while the Kms are 1.1 and 2.3 mg of xylan per ml, respectively. The DNA sequence of the xynA gene was determined, and it contains an open reading frame that codes for a 42-amino-acid (42-aa) actinomycete signal peptide followed by the 32-kDa mature protein. There is a 21-aa Gly-Pro-rich region that separates the catalytic domain from an 86-aa C-terminal binding domain. The amino acid sequence of the catalytic domain of TfxA has from 40 to 72% identity with the sequence of 12 other xylanases from seven different organisms and belongs to family G.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acceptability of carrier screening for cystic fibrosis during pregnancy in a German population

A pilot project offering voluntary heterozygote screening for the F508 mutation causing cystic fibrosis (CF) to 638 pregnant women attending two antenatal clinics in the eastern part of Berlin was carried out from 1990–1993. Participation was invited using an information leaflet and inclusion in the study was conditional on written informed consent. Of those invited to participate, only one refused to be tested, on the grounds of non-acceptance of prenatal diagnosis. Eighteen pregnant women were identified as carriers of the F508 mutation. All of them and their male partners accepted counselling in which the genetics of CF, its prognosis and treatment were explained, with emphasis on the meaning of heterozygosity, the fact that carriers are healthy, and the risk of an affected fetus when only one parent is identified as a heterozygote. All partners agreed to be tested for the F508 R553X and G551D mutations and a second counselling session was carried out after this test result was available. No problems were observed during initial testing but, as in other studies, we found considerable anxiety on being given the result in all couples where the woman tested positive; this was reduced substantially by counselling and when the partner tested negative. All probands found to be carriers stated that they found screening acceptable. In contrast to the cautious statement by the German Berufsverband Medizinische Genetik and the hostile reaction from a representative of the CF self-support organisation towards community-based heterozygote screening for CF, this study shows that CF screening is generally acceptable in this German population and that it is actively taken up by most pregnant women when offered.     

Novel (E)-3-(3-Oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides as Histone Deacetylase Inhibitors: Design,Synthesis and Bioevaluation

Herein, we report the design, synthesis and evaluation of novel (E)-3-(3-oxo-4-substituted-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-N-hydroxypropenamides ( 4 a – i , 7 a – g ) targeting histone deacetylases. Three human cancer cell lines were used to test the cytotoxicity of the synthesized compounds (SW620, colon; PC-3, prostate; NCI−H23, lung cancer); inhibitory activity towards HDAC; anticancer activity; as well as their impact on the cell cycle and apoptosis. As a result, compounds 4 a – i bearing the alkyl substituents seemed to be less potent than the benzyl-containing compounds 7 a – g in all biological assays. Compounds 7 e – f were found to be the most active HDAC inhibitors with IC₅₀ of 1.498±0.020 μM and 1.794±0.159 μM, respectively. In terms of cytotoxicity and anticancer assay, 7 e and 7 f also showed good activity with IC₅₀ values in the micromolar range. In addition, the cell cycle and apoptosis of SW620 were affected by compound 7 f in almost a similar manner to that of reference compound SAHA. Docking assays were carried out for analysis the binding mode and selectivity of this compound toward 8 HDAC isoforms. Overall, our data confirmed that the inhibition of HDAC plays a pivotal role in their anticancer activity.     

The use of percoll gradients, elutriator rotor elution, and mithramycin staining for the isolation and identification of intraerythrocytic stages of plasmodium berghei

Intraerythrocytic parasites of Plasmodium vinckei and Plasmodium berghei were separated according to their developmental stages using discontinuous Percoll gradients. Contaminating nucleated blood cells such as leukocytes were removed by elutriation centrifugation. The stages were unequivocally identified in smears using a newly developed DNA-specific staining procedure with mithramycin and fluorescence microscopy. This stain can also be used to detect parasites in human blood of very low parasitemias. The combination of methods described has many possible applications in immunologic and biochemical parasite research.     

Effect of alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, on polyamine levels in rat tissues.

α-Difluoromethylornithine (RMI 71.782), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (E.C. 4.1.1.17) $\text{in vitro}$, causes a rapid, long-lasting, dose-dependent decrease of ornithine decarboxylase activity in prostate and, to a lesser extent, in thymus and testis of rats when injected intraperitoneally. Repeated doses (100 mg/kg or 1 g/kg) of α-difluoromethylornithine given to rats for two weeks markedly decreased polyamine concentrations in several rat tissues and selectively slowed down growth of ventral prostate and of thymus.     

The interaction of methemoglobin and modified poly-(hydroxyethylmethacrylates) studied by spectroscopic methods

The interaction between methemoglobin (MetHb) and macroporous matrices on the basis of polymethacrylates was investigated by means of optical and e.p.r. spectroscopy. The spectroscopic data show that the adsorption of MetHb to imidazole-containing matrices occurs by complex formation between matrix-bound imidazole and the iron of the prosthetic group, with all 4 polypeptide chains of the MetHb molecule being included in the interaction. The adsorption to hydrophobic side chains containing matrices leads, via the protein-matrix interaction, to considerable disturbances of iron protoporphyrin IX in equilibrium or formed from protein-contacts, which are of general importance with respect to the functional variablity and control, respectively, of iron porphyrins in hemoproteins. In case of matrix containing n-hexyl groups deoxyHb is oxidized by O2 to MetHb, instead of being oxygenated to HbO2. Not all prosthetic groups are able to bind N-3. With the increase in hydrophobicity of the matrix a conformational change is enforced leading in the beta-chains to the direct interaction between iron and sulfur of cysteine (beta-cys 92), as it is proved in all cytochrome P-450 and other model compounds.