Genetic analysis of the vaccinia virus I6 telomere-binding protein uncovers a key role in genome encapsidation

The linear, double-stranded DNA genome of vaccinia virus contains covalently closed hairpin termini. These hairpin termini comprise a terminal loop and an A+T-rich duplex stem that has 12 extrahelical bases. DeMasi et al. have shown previously that proteins present in infected cells and in virions form distinct complexes with the telomeric hairpins and that these interactions require the extrahelical bases. The vaccinia virus I6 protein was identified as the protein showing the greatest specificity and affinity for interaction with the viral hairpins (J. DeMasi, S. Du, D. Lennon, and P. Traktman, J. Virol. 75:10090-10105, 2001). To gain insight into the role of I6 in vivo, we generated eight recombinant viruses bearing altered alleles of I6 in which clusters of charged amino acids were changed to alanine residues. One allele (temperature-sensitive I6-12 [tsI6-12]) conferred a tight ts phenotype and was used to examine the stage(s) of the viral life cycle that was affected at the nonpermissive temperature. Gene expression, DNA replication, and genome resolution proceeded normally in this mutant. However, proteolytic processing of structural proteins, which accompanies virus maturation, was incomplete. Electron microscopic studies confirmed a severe block in morphogenesis in which immature, but no mature, virions were observed. Instead, aberrant spherical virions and large crystalloids were seen. When purified, these aberrant virions were found to have normal protein content but to be devoid of viral DNA. We propose that the binding of I6 to viral telomeres directs genome encapsidation into the virus particle.     

A Ciprofloxacin Extended Release Tablet Based on Swellable Drug Polyelectrolyte Matrices

The aim of this work was the development of extended release tablets of 500 mg of ciprofloxacin based on swellable drug polyelectrolyte matrices (SDPM). A set of complexes of carbomer, ciprofloxacin and sodium, (CB–Cip)₅₀Na _x , having a molar ratio Cip/CB acid groups of 0.5 and variable proportions of Na⁺ was used to prepare SDPM. Characterization of complexes by FT-IR, powder X-ray diffraction and thermal analysis revealed that Cip, in its protonated form, is ionically bonded to the functional groups of CB. Rates of fluid uptake of (CB–Cip)₅₀Na _x matrices as well as Cip release in simulated gastric fluid were modulated by changes in the proportion of Na⁺ incorporated in the complexes. A direct correlation between fluid uptake and delivery rate was observed along the series of matrices. Release rates were modulated from 1.4 mg/min to 25 mg/min in going from (CB–Cip)₅₀Na₁₀ to (CB–Cip)₅₀Na₁₄. The analysis of kinetic data suggest that rates of swelling, ionic pair dissociation and drug diffusion play a role in the kinetic control of delivery. Complexes were satisfactorily prepared and processed together with small amounts of antiadherent and lubricant excipients to obtain a series of extended release SDPM tablets through the current tableting technology processes. Cip release from matrices was widely modulated by the composition of the complexes yielding a flexible system that allows selecting a composition that releases in 120 min 90% of the dose in simulated gastric fluid.     

Solution structure of contryphan-R, a naturally occurring disulfide-bridged octapeptide containing D-tryptophan: comparison with protein loops.

Contryphan-R is a disulfide-constrained octapeptide containing a D-tryptophan that was isolated recently from venom of the cone shell Conus radiatus. The polypeptide is present in two forms in solution due to cis-trans isomerization at hydroxyproline 3. The solution structure of the major form of this unusual polypeptide, determined from NMR data, consists of a well-defined fold containing a non-hydrogen-bonded chain reversal from Gly1 to Glu5, which includes a cis-hydroxyproline and a D-Trp, and a type I beta-turn from Glu5 to Cys8. The presence of a putative salt bridge between the Glu5 carboxyl group and the N-terminal ammonium group is investigated by using various solvation models during energy minimization and is compared with the results of a pH titration. A comparison of the structure of contryphan-R with other cyclic peptide structures highlights some of the key structural determinants of these peptides and suggests that the contryphan-R fold could be exploited as a scaffold onto which unrelated protein binding surfaces could be grafted. Comparison with small disulfide-bridged loops in larger proteins shows that contryphan-R is similar to a commonly occurring loop structure found in proteins.     

Tyramine-modified pectins via periodate oxidation for soybean hull peroxidase induced hydrogel formation and immobilization

Pectin was modified by oxidation with sodium periodate at molar ratios of 2.5, 5, 10, 15 and 20 mol% and reductive amination with tyramine and sodium cyanoborohydride afterwards. Concentration of tyramine groups within modified pectin ranged from 54.5 to 538 μmol/g of dry pectin while concentration of ionizable groups ranged from 3.0 to 4.0 mmol/g of dry polymer compared to 1.5 mmol/g before modification due to the introduction of amino group. All tyramine-pectins showed exceptional gelling properties and could form hydrogel both by cross-linking of carboxyl groups with calcium or by cross-linking phenol groups with peroxidase in the presence of hydrogen peroxide. These hydrogels were tested as carriers for soybean hull peroxidase (SHP) immobilization within microbeads formed in an emulsion based enzymatic polymerization reaction. SHP immobilized within tyramine-pectin microbeads had an increased thermal and organic solvent stability compared to the soluble enzyme. Immobilized SHP was more active in acidic pH region and had slightly decreased K _m value of 2.61 mM compared to the soluble enzyme. After 7 cycles of repeated use in batch reactor for pyrogallol oxidation microbeads, immobilized SHP retained half of the initial activity.

     

Histamine catabolism in Pseudomonas putida U: identification of the genes,catabolic enzymes and regulators

In this study, the catabolic pathway required for the degradation of the biogenic amine histamine (Hin) was genetically and biochemically characterized in Pseudomonas putida U. The 11 proteins (HinABCDGHFLIJK) that participate in this pathway are encoded by genes belonging to three loci hin1, hin2 and hin3 and by the gene hinK. The enzymes HinABCD catalyze the transport and oxidative deamination of histamine to 4‐imidazoleacetic acid (ImAA). This reaction is coupled to those of other well‐known enzymatic systems (DadXAR and CoxBA‐C) that ensure both the recovery of the pyruvate required for Hin deamination and the genesis of the energy needed for Hin uptake. The proteins HinGHFLKIJ catalyze the sequential transformation of ImAA to fumaric acid via N₂‐formylisoasparagine, formylaspartic acid and aspartic acid. The identified Hin pathway encompasses all the genes and proteins (transporters, energizing systems, catabolic enzymes and regulators) needed for the biological degradation of Hin. Our work was facilitated by the design and isolation of genetically engineered strains that degrade Hin or ImAA and of mutants that accumulate Ala, Asp and Hin catabolites. The implications of this research with respect to potential biotechnological applications are discussed.     

Two new 4-Cys conotoxins (framework 14) of the vermivorous snail Conus austini from the Gulf of Mexico with activity in the central nervous system of mice

As part of continuing studies of the venom components present in Conus austini (syn.: Conus cancellatus), a vermivorous cone snail collected in the western Gulf of Mexico, Mexico, two major peptides, as14a and as14b, were purified and characterized. Their amino acid sequences were determined by automatic Edman sequencing after reduction and alkylation. Their molecular masses, established by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, confirmed the chemical analyses and indicated that as14a and as14b have free C-termini. Each peptide contains 4-Cys residues arranged in a pattern (C-C-C-C, framework 14). The primary structure of as14a is GGVGRCIYNCMNSGGGLNFIQCKTMCY (experimental monoisotopic mass 2883.92Da; calculated monoisotopic mass 2884.20Da), whereas that of as14b is RWDVDQCIYYCLNGVVGYSYTECQTMCT (experimental monoisotopic mass 3308.63Da; calculated monoisotopic mass 3308.34Da). Both purified peptides elicited scratching and grooming activity in mice, and as14b also caused body and rear limb extension and tail curling immediately upon injection. The high sequence similarity of peptide as14a with peptide vil14a from the vermivorous C. villepinii suggests that the former might block K+ channels.     

Plasma membrane lipids in the resurrection plant Ramonda serbica following dehydration and rehydration

Plants of Ramonda serbica were dehydrated to 3.6% relative water content (RWC) by withholding water for 3 weeks, afterwards the plants were rehydrated for 1 week to 93.8% RWC. Plasma membranes were isolated from leaves using a two-phase aqueous polymer partition system. Compared with well-hydrated (control) leaves, dehydrated leaves suffered a reduction of about 75% in their plasma membrane lipid content, which returned to the control level following rewatering. Also the lipid to protein ratio decreased after dehydration, almost regaining the initial value after rehydration. Lipids extracted from the plasma membrane of fully-hydrated leaves were characterized by a high level of free sterols and a much lower level of phospholipids. Smaller amounts of cerebrosides, acylated steryl glycosides and steryl glycosides were also detected. The main phospholipids of control leaves were phosphatidylcholine and phosphatidylethanolamine, whereas sitosterol was the free sterol present in the highest amount. Following dehydration, leaf plasma membrane lipids showed a constant level of free sterols and a reduction in phospholipids compared with the well-hydrated leaves. Both phosphatidylcholine and phosphatidylethanolamine decreased following dehydration, their molar ratio remaining unchanged. Among free sterols, the remarkably high cholesterol level present in the control leaves (about 14 mol%) increased 2-fold as a result of dehydration. Dehydration caused a general decrease in the unsaturation level of individual phospholipids and total lipids as well. Upon rehydration the lipid composition of leaf plasma membranes restored very quickly approaching the levels of well-hydrated leaves.     

Involvement of focal adhesion kinase in inhibition of motility of human breast cancer cells by sphingosine 1-phosphate

Sphingosine 1-phosphate (SPP), a bioactive sphingolipid metabolite, inhibits chemoinvasiveness of the aggressive, estrogen-independent MDA-MB-231 human breast cancer cell line. As in many other cell types, SPP stimulated proliferation of MDA-MB-231 cells, albeit to a lesser extent. Treatment of MDA-MB-231 cells with SPP had no significant effect on their adhesiveness to Matrigel, and only high concentrations of SPP partially inhibited matrix metalloproteinase-2 activation induced by Con A. However, SPP at a concentration that strongly inhibited invasiveness also markedly reduced chemotactic motility. To investigate the molecular mechanisms by which SPP interferes with cell motility, we examined tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important for organization of focal adhesions and cell motility. SPP rapidly increased tyrosine phosphorylation of FAK and paxillin and of the paxillin-associated protein Crk. Overexpression of FAK and kinase-defective FAK in MDA-MB-231 cells resulted in a slight increase in motility without affecting the inhibitory effect of SPP, whereas expression of FAK with a mutation of the major autophosphorylation site (F397) abolished the inhibitory effect of SPP on cell motility. In contrast, the phosphoinositide 3'-kinase inhibitor, wortmannin, inhibited chemotactic motility in both vector and FAK-F397-transfected cells. Our results suggest that autophosphorylation of FAK on Y397 may play an important role in SPP signaling leading to decreased cell motility.     

A novel conus peptide ligand for K+ channels

Voltage-gated ion channels determine the membrane excitability of cells. Although many Conus peptides that interact with voltage-gated Na(+) and Ca(2+) channels have been characterized, relatively few have been identified that interact with K(+) channels. We describe a novel Conus peptide that interacts with the Shaker K(+) channel, kappaM-conotoxin RIIIK from Conus radiatus. The peptide was chemically synthesized. Although kappaM-conotoxin RIIIK is structurally similar to the mu-conotoxins that are sodium channel blockers, it does not affect any of the sodium channels tested, but blocks Shaker K(+) channels. Studies using Shaker K(+) channel mutants with single residue substitutions reveal that the peptide interacts with the pore region of the channel. Introduction of a negative charge at residue 427 (K427D) greatly increases the affinity of the toxin, whereas the substitutions at two other residues, Phe(425) and Thr(449), drastically reduced toxin affinity. Based on the Shaker results, a teleost homolog of the Shaker K(+) channel, TSha1 was identified as a kappaM-conotoxin RIIIK target. Binding of kappaM-conotoxin RIIIK is state-dependent, with an IC(50) of 20 nm for the closed state and 60 nm at 0 mV for the open state of TSha1 channels.     

Cancer vaccines: between the idea and the reality

Whether vaccines are designed to prepare the immune system for the encounter with a pathogen or with cancer, certain common challenges need to be faced, such as what antigen and what adjuvant to use, what type of immune response to generate and how to make it long lasting. Cancer, additionally, presents several unique hurdles. Cancer vaccines must overcome immune suppression exerted by the tumour, by previous therapy or by the effects of advanced age of the patient. If used for cancer prevention, vaccines must elicit effective long-term memory without the potential of causing autoimmunity. This article addresses the common and the unique challenges to cancer vaccines and the progress that has been made in meeting them. Considering how refractory cancer has been to standard therapy, efforts to achieve immune control of this disease are well justified.