A covalent linkage between the gene 5 DNA polymerase of bacteriophage T7 and Escherichia coli thioredoxin,the processivity factor: fate of thioredoxin during DNA synthesis

Gene 5 protein (gp5) of bacteriophage T7 is a non-processive DNA polymerase, which acquires high processivity by binding to Escherichia coli thioredoxin. The gene 5 protein-thioredoxin complex (gp5/trx) polymerizes thousands of nucleotides before dissociating from a primer-template. We have engineered a disulfide linkage between the gene 5 protein and thioredoxin within the binding surface of the two proteins. The polymerase activity of the covalently linked complex (gp5-S-S-trx) is similar to that of gp5/trx on poly(dA)/oligo(dT). However, gp5-S-S-trx has only one third the polymerase activity of gp5/trx on single-stranded M13 DNA. gp5-S-S-trx has difficulty polymerizing nucleotides through sites of secondary structure on M13 DNA and stalls at these sites, resulting in lower processivity. However, gp5-S-S-trx has an identical processivity and rate of elongation when E. coli single-stranded DNA-binding protein (SSB protein) is used to remove secondary structure from M13 DNA. Upon completing synthesis on a DNA template lacking secondary structure, both complexes recycle intact, without dissociation of the processivity factor, to initiate synthesis on a new DNA template. However, a complex stalled at secondary structure becomes unstable, and both subunits dissociate from each other as the polymerase prematurely releases from M13 DNA.     

The cell adhesion molecule CEACAM1-L is a substrate of caspase-3-mediated cleavage in apoptotic mouse intestinal cells

The CEACAM1 cell adhesion molecule is a member of the carcinoembryonic antigen family. In the mouse, four distinct isoforms are generated by alternative splicing. These encode either two or four immunoglobulin domains linked through a transmembrane domain to a cytoplasmic domain that encompasses either a short 10-amino acid tail or a longer one of 73 amino acids. Inclusion of exon 7, well conserved in evolution, generates the long cytoplasmic domain. A potential caspase recognition site in mouse, rat, and human CEACAM1-L also becomes available within the peptide encoded by exon 7. We used CEACAM1-L-transfected mouse colon carcinoma CT51 cells treated with three different apoptotic agents to study its fate during cell death. We found that CEACAM1-L is cleaved resulting in rapid degradation of most of its 8-kDa cytoplasmic domain. Caspase-mediated cleavage was demonstrated using purified recombinant caspases. The long cytoplasmic domain was cleaved specifically by caspase-3 in vitro but not by caspase-7 or -8. Moreover cleavage of CEACAM1-L in apoptotic cells was blocked by addition of a selective caspase-3 inhibitor to the cultures. Using point and deletion mutants, the conserved DQRD motif in the membrane-proximal cytoplasmic domain was identified as a caspase cleavage site. We also show that once CEACAM1-L is caspase-cleaved it becomes a stronger adhesion molecule than both the shorter and the longer expressing isoforms.     

Identification of a novel genetically controlled step in mycorrhizal colonization: plant resistance to infection by fungal spores but not extra-radical hyphae

Vesicular arbuscular mycorrhizal fungi infect plants by means of both spores and vegetative hyphae at early stages of symbiosis. Using 2500 M2 fast-neutron-mutagenized seeds of the miniature tomato (Lycopersicon esculentum) cultivar, Micro-Tom, we isolated a mutant, M161, that is able to resist colonization in the presence of Glomus intraradices spores. The myc(-) phenotype of the mutant was stable for nine generations, and found to segregate as a single Mendelian recessive locus. The mutant exhibited morphological and growth-pattern characteristics similar to those of wild-type plants. Alterations of light intensity and day/night temperatures did not eliminate the myc(-) characteristic. Resistance to mycorrhizal fungal infection and colonization was also evident following inoculation with the fungi Glomus mosseae and Gigaspora margarita. Normal colonization of M161 was evident when mutant plants were grown together with arbuscular mycorrhizal-inoculated wild-type plants in the same growth medium. During evaluation of the pre-infection stages in the mutant rhizosphere, spore germination and appressoria formation of G. intraradices were lower by 45 and 70%, respectively, than the rates obtained with wild-type plants. These results reveal a novel, genetically controlled step in the arbuscular mycorrhizal colonization process, governed by at least one gene, which significantly reduces key steps in pre-mycorrhizal infection stages.     

RNA world: catalysis abets binding, but not vice versa

The recent selection of a complex ribozyme capable of general polymerization on a template in trans has revealed how catalysts may have arisen from one another in the RNA world.     

Cellular response of antioxidant metalloproteins in Cu/Zn SOD transgenic mice exposed to hyperoxia

Ceruloplasmin, metallothionein, and ferritin are metal-binding proteins with potential antioxidant activity. Despite evidence that they are upregulated in pulmonary tissue after oxidative stress, little is known regarding their influence on trace metal homeostasis. In this study, we have used copper- and zinc-containing superoxide dismutase (Cu/Zn SOD) transgenic-overexpressing and gene knockout mice and hyperoxia to investigate the effects of chronic and acute oxidative stress on the expression of these metalloproteins and to identify their influence on copper, zinc, and iron homeostasis. We found that the oxidative stress-mediated induction of ceruloplasmin and metallothionein in the lung had no effect on tissue levels of copper, iron, or zinc. However, Cu/Zn SOD expression had a marked influence on hepatic copper and iron as well as circulating copper homeostasis. These results suggest that ceruloplasmin and metallothionein may function as antioxidants independent of their role in trace metal homeostasis and that Cu/Zn SOD functions in copper homeostasis via mechanisms distinct from its superoxide scavenging properties.     

A new approach to the synthesis of APTRA indicators

We have devised a general synthesis of Mg(2+) indicators which is based on the aminophenol triacetic acid (APTRA) structure. The key step is a palladium-catalyzed coupling reaction of a precursor of the APTRA ligand with a fluorescent group. This strategy resulted in new ratioable fluorescent APTRA indicators and the finding that the fluorescence response of these indicators is different for Mg(2+) and Ca(2+) in some cases. We believe that this represents a generally useful approach for combining fluorophore and chelator functionalities.     

A mathematical model for prokaryotic protein synthesis

A kinetic model for the synthesis of proteins in prokaryotes is presented and analysed. This model is based on a Markov model for the state of the DNA strand encoding the protein. The states that the DNA strand can occupy are: ready, repressed, or having a mRNA chain of length i in the process of being completed. The case i = 0 corresponds to the RNA polymerase attached, but no nucleotides attached to the chain. The Markov model consists of differential equations for the rates of change of the probabilities. The rate of production of the mRNA molecules is equal to the probability that the chain is assembled to the penultimate nucleotide, times the rate at which that nucleotide is attached. Similarly, the mRNA molecules can also be in different states, including: ready and having an amino acid chain of length j attached. The rate of protein synthesis is the rate at which the chain is completed. A Michaelis-Menten type of analysis is done, assuming that the rate of protein degradation determines the ’slow’ time, and that all the other kinetic rates are ‘fast’. In the self-regulated case, this results in a single ordinary differential equation for the protein concentration.     

In vitro and in vivo evaluations of biodegradable implants for hormone replacement therapy: Effect of system design and PK-PD relationship

This investigation evaluated the feasibility of using subdermally implantable devices fabricated by nonconventional 3-dimensional printing technology for controlled delivery of ethinyl estradiol (EE₂). In vitro release kinetics of EE₂ and in vivo pharmacokinetics pharmacodynamics in ovariectomized New Zealand White rabbits were carried out to study 3 implant prototypes: implant I (single-channel EE₂ distribution in polycaprolactone polymer core), implant II (homogeneous EE₂ distribution in polycaprolactone polymer matrix), and implant III (concentration-gradient EE₂ distribution in polycaprolactone and poly(dl-lactide-co-glycolide) (50∶50 matrix). EE₂ was found to be released from all the implants in a nonlinear pattern with an order of implant III>implant II>implant I. The noncompartmental pharmacokinetic analysis of plasma EE₂ profiles in rabbits indicated a significant difference (p>.05) in C_max, t_max, and mean residence time between implant I and implants II and III, but no difference in the area under the plasma concentration time curves calculated by trapezoidal rule (AUC) among the implants. For pharmacodynamic studies, endogenous follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were observed to be suppressed following implantation of all implants, which demonstrated that a therapeutically effective dose of EE₂ had been delivered. Furthermore, the noncompartmental analysis of plasma FSH and LH profiles in rabbits showed a significant difference (p<.05) in AUC and the mean residence time between implant III and implants I and II. A good in vivo/in vitro relationship was observed between daily amounts of EE₂ released and plasma profiles of EE₂ for all implants. This relationship suggests that plasma profiles of EE₂ could be predicted from in vitro measurement of daily amount of EE₂ released Therefore, performing in vitro drug release studies may aid in the development of an EE₂ implant with the desired in vivo release rate. Published: September 21, 2001