Recombinant RNA polymerase: inducible overexpression, purification and assembly of Escherichia coli rpo gene products

The genes, rpoA, rpoB and rpoC of Escherichia coli, which encode the RNA polymerase alpha-, beta- and beta'-subunits, respectively, have been individually placed on expression plasmids under the control of the bacteriophage T7 promoter. Induction of the T7 RNA polymerase gene in host cells harboring each of the three plasmids resulted in the extensive overproduction of the three polypeptides. The overproduced subunits were purified and assembled into a functional enzyme, whose specific activity and dependence on the sigma-factor were indistinguishable from native RNA polymerase purified by conventional methods.     

Protein import through the nuclear pore complex is a multistep process

The transport of macromolecules across the nuclear envelope is mediated by the nuclear pore complex (NPC). Using cryo-electron microscopy and image processing we have mapped the interaction of three specific gold probes with the NPC and obtained projection maps of two possible intermediates in nuclear import. The probes used in these experiments were (a) mAb-414, which cross-reacts with Xenopus nucleoporins containing O-linked N-acetyl glucosamines; (b) wheat germ agglutinin, a transport inhibitor; and (c) nucleoplasmin, a transport substrate. Strong binding sites of the three probes are circularly arrayed on NPCs between radii of 100 and 125 A and may be coextensive. These results suggest that nucleoplasmin-gold (NP-gold) can form at least three distinct complexes with a central transport assembly of the NPC, which may represent intermediates of a multistep protein import pathway. Initially, NP-gold appears to bind at multiple sites located around the periphery of the closed NPC transporter and also directly over the center where it can dock. In a subsequent step NP-gold is translocated through the nuclear pore.     

Seasonal nitrogen and carbon concentrations in white,brown and woody fine roots of sugar maple (Acer saccharum Marsh)

Small diameter (<1.0-mm) Acer saccharum Marsh roots were separated into white, brown and woody development state classes and analyzed for total N and C concentrations in April, July and October of 1988. White roots had greater concentrations of N and C than either brown or woody roots at each sampling date, and the N concentration of brown roots was consistently greater than that of woody roots. There were no temporal changes in N concentrations in any of the roots. C was slightly elevated in mid-summer in all three classes of roots. The data suggest the possible existence of an N translocation mechanism in ageing and developing fine roots. More research should be undertaken to establish the mechanisms of N loss in developing fine roots.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Molecular genetic studies of Creutzfeldt-Jakob disease

Genetic study of over 200 cases of Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), fatal familial insomnia (FFI), and kuru have brought a reliable body of evidence that the familial forms of CJD and all known cases of GSS and FFI are linked to germline mutations in the coding region of the PRNP gene on chromosome 20, either point substitutions or expansion of the number of repeat units. No pathogenic mutations have so far been found in sporadic or infectious forms of CJD, although there are features of genetic predisposition in iatrogenic CJD and kuru. In FFI and familial CJD, clinically and pathologically distinct syndromes that are both linked to the 178^Asp→Asn substitution, phenotypic expression is dependent on a polymorphism at codon 129. Synthetic peptides homologous to several regions of PrP spontaneously form insoluble amyloid fibrils with unique morphological characteristics and polymerization tendencies. Peptides homologous to mutated regions of PrP exhibit enhanced fibrilogenic properties and, if mixed with the wild-type peptide, produce even more abundant and larger fibrous aggregates. A similar process in vivo may lead to amyloid accumulation and disease, and transmission of “baby fibrils” may induce disease in other hosts.     

Murine FGF-4 gene expression is spatially restricted within embryonic skeletal muscle and other tissues

Fibroblast growth factors are believed to play many distinct roles in vertebrate development, owing to their ability to stimulate cell growth, prevent cell death, determine cell fate, and inhibit terminal differentiation in a variety of in vitro culture systems. We have used in situ hybridization to localize fibroblast growth factor-4 (FGF-4, also termed HST and K-FGF) gene expression in 7.5 to 16.5 day gestation mouse embryos. Seven discrete sites of gene expression were detected: (1) primitive streak (E7.5–8.5); (2) paraxial presomitic mesoderm in the trunk (E7.5–11.5); (3) primitive neuroectoderm (E8.0–8.5); (4) pharyngeal pouch endoderm (E8.5–9.5); (5) branchial arch ectoderm (E8.5–9.5); (6) limb apical ectoderm (E10.5–12.5), and (7) skeletal myoblast groups (E9.5–13.5). FGF-4 gene expression is spatially restricted within many of these sites. The profile of FGF-4 gene expression among skeletal muscle groups is overlapping, but distinct, from that of FGF-5, thereby revealing myoblast heterogeneity at the molecular level and suggesting distinct roles for multiple FGFs in muscle development.     

Monocarbonyl Analogs of Curcumin with Potential to Treat Colorectal Cancer

Curcumin has a plethora of biological properties, making this compound potentially effective in the treatment of several diseases, including cancer. However, curcumin clinical use is compromised by its poor pharmacokinetics, being crucial to find novel analogs with better pharmacokinetic and pharmacological properties. Here, we aimed to evaluate the stability, bioavailability and pharmacokinetic profiles of monocarbonyl analogs of curcumin. A small library of monocarbonyl analogs of curcumin 1a–q was synthesized. Lipophilicity and stability in physiological conditions were both assessed by HPLC-UV, while two different methods assessed the electrophilic character of each compound monitored by NMR and by UV-spectroscopy. The potential therapeutic effect of the analogs 1a–q was evaluated in human colon carcinoma cells and toxicity in immortalized hepatocytes. Our results showed that the curcumin analog 1e is a promising agent against colorectal cancer, with improved stability and efficacy/safety profile.