The solution structure of the Ga(III)-bleomycin A2 complex resolved by NMR and molecular modeling; interaction with d(CCAGGCCTGG)

The solution structure of the Ga(III)-bleomycin A2 complex (GaBLM) has been determined using 2D NMR methods in combination with molecular dynamics calculations. Complete assignment of the amide and amine protons, observation of 80 NOEs and measurement of 15 (3)JH(-H) coupling constants provided us with a well-defined structure using a restrained simulated annealing protocol. On the basis of distance and dihedral angle constraints agreement, along with potential energy considerations, the favored model is a five-coordinate complex with the primary amine of beta-aminoalanine holding the axial position of a distorted tetragonal pyramid. The disaccharide moiety of GaBLM is not a ligand, sharing the same side of the equatorial plane with the axial amine ligand. Titration of the self-complementary oligonucleotide d(CCAGGCCTGG) with GaBLM results in the formation of only one 1:1 complex in slow exchange on the NMR time scale. Our data indicate that the bithiazole moiety intercalates between the C6*G15 and C7*G14 base pairs, in a similar mode to that reported by earlier studies. Structural implications and comparisons to other metallo-bleomycins are discussed.     

A nonparametric Bayesian modeling approach for cytogenetic dosimetry

In cytogenetic dosimetry, samples of cell cultures are exposed to a range of doses of a given agent. In each sample at each dose level, some measure of cell disability is recorded. The objective is to develop models that explain cell response to dose. Such models can be used to predict response at unobserved doses. More important, such models can provide inference for unknown exposure doses given the observed responses. Typically, cell disability is viewed as a Poisson count, but in the present work, a more appropriate response is a categorical classification. In the literature, modeling in this case is very limited. What exists is purely parametric. We propose a fully Bayesian nonparametric approach to this problem. We offer comparison with a parametric model through a simulation study and the analysis of a real dataset modeling blood cultures exposed to radiation where classification is with regard to number of micronuclei per cell.     

Genome sequence and analysis of the oral bacterium Fusobacterium nucleatum strain ATCC 25586

We present a complete DNA sequence and metabolic analysis of the dominant oral bacterium Fusobacterium nucleatum. Although not considered a major dental pathogen on its own, this anaerobe facilitates the aggregation and establishment of several other species including the dental pathogens Porphyromonas gingivalis and Bacteroides forsythus. The F. nucleatum strain ATCC 25586 genome was assembled from shotgun sequences and analyzed using the ERGO bioinformatics suite (http://www.integratedgenomics.com). The genome contains 2.17 Mb encoding 2,067 open reading frames, organized on a single circular chromosome with 27% GC content. Despite its taxonomic position among the gram-negative bacteria, several features of its core metabolism are similar to that of gram-positive Clostridium spp., Enterococcus spp., and Lactococcus spp. The genome analysis has revealed several key aspects of the pathways of organic acid, amino acid, carbohydrate, and lipid metabolism. Nine very-high-molecular-weight outer membrane proteins are predicted from the sequence, none of which has been reported in the literature. More than 137 transporters for the uptake of a variety of substrates such as peptides, sugars, metal ions, and cofactors have been identified. Biosynthetic pathways exist for only three amino acids: glutamate, aspartate, and asparagine. The remaining amino acids are imported as such or as di- or oligopeptides that are subsequently degraded in the cytoplasm. A principal source of energy appears to be the fermentation of glutamate to butyrate. Additionally, desulfuration of cysteine and methionine yields ammonia, H(2)S, methyl mercaptan, and butyrate, which are capable of arresting fibroblast growth, thus preventing wound healing and aiding penetration of the gingival epithelium. The metabolic capabilities of F. nucleatum revealed by its genome are therefore consistent with its specialized niche in the mouth.     

Synthesis of isomaltooligosaccharides and oligodextrans in a recycle membrane bioreactor by the combined use of dextransucrase and dextranase

A recycle ultrafiltration membrane reactor was used to develop a continuous synthesis process for the production of isomaltooligosaccharides (IMO) from sucrose, using the enzymes dextransucrase and dextranase. A variety of membranes were tested and the parameters affecting reactor stability, productivity, and product molecular weight distribution were investigated. Enzyme inactivation in the reactor was reduced with the use of a non-ionic surfactant but its use had severe adverse effects on the membrane pore size and porosity. During continuous isomaltooligosaccharide synthesis, dextransucrase inactivation was shown to occur as a result of the dextranase activity and it was dependent mainly on the substrate availability in the reactor and the hydrolytic activity of dextranase. Substrate and dextranase concentrations (50-200 mg/mL(-1) and 10-30 U/mL(-1), respectively) affected permeate fluxes, reactor productivity, and product average molecular weight. The oligodextrans and isomaltooligosaccharides formed had molecular weights lower than in batch synthesis reactions but they largely consisted of oligosaccharides with a degree of polymerization (DP) greater than 5, depending on the synthesis conditions. No significant rejection of the sugars formed was shown by the membranes and permeate flux was dependent on tangential flow velocity.     

Structural determinants of RXPA380, a potent and highly selective inhibitor of the angiotensin-converting enzyme C-domain

RXPA380 (Cbz-PhePsi[PO(2)CH]Pro-Trp-OH) was reported recently as the first highly selective inhibitor of the C-domain of somatic angiotensin-converting enzyme (ACE), able to differentiate the two active sites of somatic ACE by a selectivity factor of more than 3 orders of magnitude. The contribution of each RXPA380 residue toward this remarkable selectivity was evaluated by studying several analogues of RXPA380. This analysis revealed that both pseudo-proline and tryptophan residues in the P(1)' and P(2)' positions of RXPA380 play a critical role in the selectivity of this inhibitor for the C-domain. This selectivity is not due to a preference of the C-domain for inhibitors bearing pseudo-proline and tryptophan residues, but rather reflects the poor accommodation of these inhibitor residues by the N-domain. A model of RXPA380 in complex with the ACE C-domain, based on the crystal structure of germinal ACE, highlights residues that may contribute to RXPA380 selectivity. From this model, striking differences between the N- and C-domains of ACE are observed for residues defining the S(2)' pocket. Of the twelve residues that surround the tryptophan side chain of RXPA380 in the C-domain, five are different in the N-domain. These differences in the S(2)' composition between the N- and C-domains are suggested to contribute to RXPA380 selectivity. The structural insights provided by this study should enhance understanding of the factors controlling the selectivity of the two domains of somatic ACE and allow the design of new selective ACE inhibitors.     

Application of global sensitivity analysis to determine goals for design of experiments: an example study on antibody-producing cell cultures

Global sensitivity analysis (GSA) can be used to quantify the importance of model parameters and their interactions with respect to model output. In this study, the Sobol' method for GSA is applied to a dynamic model of monoclonal antibody-producing mammalian cell cultures in order to identify the parameters that need to be accurately determined experimentally. Our results show that most parameters have low sensitivity indices and exhibit strong interactions with one another. These parameters can be set at their nominal values and unnecessary experimentation can therefore be avoided. In contrast, certain parameters are identified as sensitive, necessitating their estimation given sufficiently rich experimental data. Moreover, parameter sensitivity varies during culture time in a biologically meaningful manner. In conclusion, GSA can serve as an excellent precursor to optimal experiment design.     

Rupture of totally implantable central venous access devices (Intraports) in patients with cancer: report of four cases

Background  

Totally implantable central venous access devices (intraports) are commonly used in cancer patients to administer chemotherapy or parenteral nutrition. Rupture of intraport is a rare complication.     

Glutathione S-transferase in the developmental stages of the insect Apis mellifera macedonica

We investigated the pattern of glutathione S-transferase (GST) activity in the course of the development of Apis mellifera macedonica. GST activity is present in all developmental stages of A. mellifera macedonica. The highest activity towards the substrate 1-chloro-2,4-dinitrobenzene (CDNB) is found in the adult stage and the lowest in the egg. The kinetic characteristics of the whole enzyme change as the insect develops. Significant changes are observed in substrate specificity, inhibitor sensitivity and V(max). The number of isoenzymes and their rate of expression vary as the insect develops. However, two main isoenzymes are present in all developmental stages, one in the alkaline area and the other in the acidic. While in the larval stage the acidic isoenzyme is expressed at a slightly higher rate (52.2% over 47.8% for the alkaline isoenzyme), in the adult stage, the rate is reversed dramatically (13.24% and 84.2%, respectively).     

Seasonal, populational and ontogenic variation in the volatile oil content and composition of individuals of Origanum vulgare subsp. Hirtum, assessed by GC headspace analysis and by SPME sampling of individual oil glands

Small-scale GC headspace analyses combined with SPME sampling of individual oil glands have been used to measure the variation in volatile content and composition in and within different oregano plants as affected by age, season and developmental state. The main monoterpenes found were p-cymene, carvacrol and their precursor gamma-terpinene. The early season preponderance of p-cymene over carvacrol was reversed as the season progressed and this pattern could also be seen at any time within the plant, from the latest leaves to be produced (low in cymene) to the earliest (high in cymene). Seedlings from the same mother plant developed this pattern at different rates. Within individual leaves the pattern was not observed, even within the youngest developing leaves. However it was found that the oil composition of individual glands within a single leaf varied considerably, most notably in respect of the production of carvacrol and its isomer thymol.