Permeability to phi chi 174 bacteriophages in polyolephin membrane condoms

Membranes used for the manufacture of condoms eventually can develop tiny pores, thereby decreasing dramatically their effectiveness as a physical barrier against the transmission of infectious agents. A technique was designed that was based on the ability of bacteriophage viruses to trespass membranes and to infect certain bacteria species, and then developing lysis plaques in the colonies of the host bacteria. The effectiveness of 60 polyolefin condoms in preventing the diffusion of the bacteriophage phi chi 174(ATCC13706-B1), 27 nm diameter, was compared to 20 latex condoms. Physiological conditions such as pressure, pH, superficial tension, length, time of exposure and viral titre were simulated. A pressurization system was designed, in which compressed air was injected simultaneously to ten condoms. Four of the 60 polyolefin condoms and one of the 20 latex condoms were permeable to the virus. Therefore, at least 93% of the condoms evaluated were able to contain the virus. The difference in permeability between the two types of membranes was not statistically significant (P = 0.79).     

Is root growth under phosphorus deficiency affected by source or sink limitations?

Reduced net photosynthesis (Pn) and decreasing shoot and root biomass are typical effects of phosphorus deficiency in plants. Lower biomass accumulation could be the result of reduced Pn (source limitation), but may also be due to direct negative effects of low P availability on growth (sink limitation). Because of the principal importance of root growth for P uptake, this study specifically examined the question whether source or sink limitations were responsible for reduced root growth rates under P deficiency. Rice plants were grown in nutrient solutions with four levels of P supply and at two light treatments and the effect of Pxlight treatments on growth and carbohydrate distribution was observed. Plants had up to 70% higher Pn when grown with natural (high) light compared with low light. Higher Pn, however, did not lead to additional growth under P deficiency, suggesting that assimilate supply from source leaves to roots was not a limiting factor under P deficiency. This was supported by observations that root starch concentrations increased in P-deficient roots. The comparison of two genotypes with different tolerance to P deficiency showed that the more tolerant one preferentially distributed P to roots where the additional P stimulated root growth and, ultimately, P uptake. The results therefore suggest that source limitation is of little importance under P deficiency. Even at highly sub-optimal tissue P concentrations of below 0.7 mg P g(-1) dry weight, plants were able to produce enough assimilates to sustain growth rates that were directly limited by low P availability.     

Endoreduplication induced in cultured Chinese hamster cells by different anti-topoisomerase II chemicals. Evidence for the essential contribution of the enzyme to chromosome segregation

With the ultimate purpose of testing the hypothesis that, as shown in yeast mutants, any malfunction of DNA topoisomerase II might result in aberrant mitosis due to defective chromosome segregation, we have chosen three chemicals of different nature, recently reported to catalytically inhibit the enzyme. The endpoint selected to assess any negative effect on the ability of topoisomerase II to properly carry out decatenation of fully replicated chromosomes in the G2/M phase of the cell cycle was the presence of metaphases showing diplochromosomes as a result of endoreduplication, i.e. two successive rounds of DNA replication without intervening mitosis. The anti-topoisomerase drugs selected were the anthracycline antibiotic and antineoplastic agent aclarubicin, the respiratory venom sodium azide, and 9-aminoacridine, a chemical compound with planar topology capable of intercalation between DNA bases. Our results show that the three chemicals tested are able to induce endoreduplication to different degrees. These observations seem to lend support to the proposal that topoisomerase II plays a central role in chromosome segregation in mammalian cells.     

Tau protein binds single-stranded DNA sequence specifically--the proof obtained in vitro with non-equilibrium capillary electrophoresis of equilibrium mixtures

Tau is a microtubule-associated protein, which plays an important role in physiology and pathology of neurons. Tau has been recently reported to bind double-stranded DNA (dsDNA) but not to bind single-stranded DNA (ssDNA) [Cell. Mol. Life Sci. 2003, 60, 413-421]. Here, we prove that tau binds not only dsDNA but also ssDNA. This finding was facilitated by using two kinetic capillary electrophoresis methods: (i) non-equilibrium capillary electrophoresis of equilibrium mixtures (NECEEM); (ii) affinity-mediated NECEEM. Using the new approach, we observed, for the first time, that tau could induce dissociation of strands in dsDNA by binding one of them in a sequence-specific fashion. Moreover, we determined the equilibrium dissociation constants for all tau-DNA complexes studied.     

Role of water in aging of human butyrylcholinesterase inhibited by echothiophate: the crystal structure suggests two alternative mechanisms of aging

Organophosphorus poisons (OP) bind covalently to the active-site serine of cholinesterases. The inhibited enzyme can usually be reactivated with powerful nucleophiles such as oximes. However, the covalently bound OP can undergo a suicide reaction (termed aging) yielding nonreactivatable enzyme. In human butyrylcholinesterase (hBChE), aging involves the residues His438 and Glu197 that are proximal to the active-site serine (Ser198). The mechanism of aging is known in detail for the nerve gases soman, sarin, and tabun as well as the pesticide metabolite isomalathion. Aging of soman- and sarin-inhibited acetylcholinesterase occurs by C-O bond cleavage, whereas that of tabun- and isomalathion-inhibited acetylcholinesterase occurs by P-N and P-S bond cleavage, respectively. In this work, the crystal structures of hBChE inhibited by the ophthalmic reagents echothiophate (nonaged and aged) and diisopropylfluorophosphate (aged) were solved and refined to 2.1, 2.25, and 2.2 A resolution, respectively. No appreciable shift in the position of the catalytic triad histidine was observed between the aged and nonaged conjugates of hBChE. This absence of shift contrasts with the aged and nonaged crystal structures of Torpedo californica acetylcholinesterase inhibited by the nerve agent VX. The nonaged hBChE structure shows one water molecule interacting with Glu197 and the catalytic triad histidine (His438). Interestingly, this water molecule is ideally positioned to promote aging by two mechanisms: breaking either a C-O bond or a P-O bond. Pesticides and certain stereoisomers of nerve agents are expected to undergo aging by breaking the P-O bond.     

Proteomic characterization of host response to Yersinia pestis and near neighbors

Host-pathogen interactions result in protein expression changes within both the host and the pathogen. Here, results from proteomic characterization of host response following exposure to Yersinia pestis, the causative agent of plague, and to two near neighbors, Yersinia pseudotuberculosis and Yersinia enterocolitica, are reported. Human monocyte-like cells were chosen as a model for macrophage immune response to pathogen exposure. Two-dimensional electrophoresis followed by mass spectrometry was used to identify host proteins with differential expression following exposure to these three closely related Yersinia species. This comparative proteomic characterization of host response clearly shows that host protein expression patterns are distinct for the different pathogen exposures, and contributes to further understanding of Y. pestis virulence and host defense mechanisms. This work also lays the foundation for future studies aimed at defining biomarkers for presymptomatic detection of plague.     

Plasmalemmal V-H(+)-ATPases regulate intracellular pH in human lung microvascular endothelial cells

The lung endothelium layer is exposed to continuous CO(2) transit which exposes the endothelium to a substantial acid load that could be detrimental to cell function. The Na(+)/H(+) exchanger and HCO(3)(-)-dependent H(+)-transporting mechanisms regulate intracellular pH (pH(cyt)) in most cells. Cells that cope with high acid loads might require additional primary energy-dependent mechanisms. V-H(+)-ATPases localized at the plasma membranes (pmV-ATPases) have emerged as a novel pH regulatory system. We hypothesized that human lung microvascular endothelial (HLMVE) cells use pmV-ATPases, in addition to Na(+)/H(+) exchanger and HCO(3)(-)-based H(+)-transporting mechanisms, to maintain pH(cyt) homeostasis. Immunocytochemical studies revealed V-H(+)-ATPase at the plasma membrane, in addition to the predicted distribution in vacuolar compartments. Acid-loaded HLMVE cells exhibited proton fluxes in the absence of Na(+) and HCO(3)(-) that were similar to those observed in the presence of either Na(+), or Na(+) and HCO(3)(-). The Na(+)- and HCO(3)(-)-independent pH(cyt) recovery was inhibited by bafilomycin A(1), a V-H(+)-ATPase inhibitor. These studies show a Na(+)- and HCO(3)(-)-independent pH(cyt) regulatory mechanism in HLMVE cells that is mediated by pmV-ATPases.     

Structural features of the initiator of replication protein RepB encoded by the promiscuous plasmid pMV158

The promiscuous rolling circle (RC)-replicating plasmid pMV158 encodes the 210-amino-acid initiator of replication protein, RepB. The protein relaxes supercoiled cognate DNA in a topoisomeraseI-like manner. A new vector and procedure for overproduction, scaling-up, and purification of the protein has been developed. RepB purified as a hexamer in solution, as shown by analytical ultracentrifugation assays. Circular dichroism (CD) of RepB indicated that the protein has an estimated content of around 33% alpha-helices and 20% beta-strands. Characterisation of temperature-induced transitions of the protein showed an irreversible change in the spectra when the temperature was raised above 35 degrees C, indicating that the protein undergoes a conformational change that could account for the relatively high optimal temperature of the RepB-mediated cleavage.     

Role of BMP signaling and the homeoprotein Iroquois in the specification of the cranial placodal field

Different types of placodes originate at the anterior border of the neural plate but it is still an unresolved question whether individual placodes arise as distinct ectodermal specializations in situ or whether all or a subset of the placodes originate from a common preplacodal field. We have analyzed the expression and function of the homeoprotein Iro1 in Xenopus and zebrafish embryos, and we have compared its expression with several preplacodal and placodal markers. Our results indicate that the iro1 genes are expressed in the preplacodal region, being one of the earliest markers for this area. We show that an interaction between the neural plate and the epidermis is able to induce the expression of several preplacodal markers, including Xiro1, by a similar mechanism to that previously shown for neural crest induction. In addition, we analyzed the role of BMP in the specification of the preplacodal field by studying the expression of the preplacodal markers Six1, Xiro1, and several specific placodal markers. We experimentally modified the level of BMP activity by three different methods. First, we implanted beads soaked with noggin in early neurula stage Xenopus embryos; second, we injected the mRNA that encodes a dominant negative of the BMP receptor into Xenopus and zebrafish embryos; and third, we grafted cells expressing chordin into zebrafish embryos. The results obtained using all three methods show that a reduction in the level of BMP activity leads to an expansion of the preplacodal and placodal region similar to what has been described for neural crest regions. By using conditional constructs of Xiro1, we performed gain and loss of function experiments. We show that Xiro1 play an important role in the specification of both the preplacodal field as well as individual placodes. We have also used inducible dominant negative and activator constructs of Notch signaling components to analyze the role of these factors on placodal development. Our results indicate that the a precise level of BMP activity is required to induce the neural plate border, including placodes and neural crest cells, that in this border the iro1 gene is activated, and that this activation is required for the specification of the placodes.