Factors affecting the survival of E. coli in a river

The relationships between physical, chemical and microbial characteristics of an aquatic ecosystem and the survival of E. coli have been studied. Two conditions of the ecosystem (warm and cold) are considered. T₉₀ (time necessary for 90% of a bacterial population to die) in the warm situation shows an inverse exponential relationship with water temperature. Besides the direct relationship temperature-T₉₀, there is an indirect effect of temperature upon T₉₀ through the natural microflora of the water. The relationships between temperature and the heterotrophic population, and between the heterotrophic population and the bacterial consumers (P.F.U.), are exponential and linear, respectively.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Improved high-affinity binding of 3H-apomorphine with a subcellular membrane fraction of mammalian brain tissue

The binding of low concentrations of [³H](?)apomorphine to preparations of calf and rat forebrain tissue was evaluated. Fractionation of crude homogenates to prepare a membrane fraction (P₄) of striatal or caudate homogenates increased the proportion of saturable to total binding from 33% to over 80%, and increased the apparent density of binding sites from 94 to 681 fmol/mg protein. Binding in calf caudate P₄ tissue was protein-dependent and optimal at pH = 7.0 to 7.5, and T = 20 to 25°C; at higher temperatures tissue binding sites appeared to degrade. The half-time of association and dissociation at 22°C were, respectively, 14.0 and 18.5 min; equilibration was complete in 60 min. Kinetic characteristics of high-affinity binding obtained from association and dissociation constants and from saturation isotherms were similar (K_d = 2.1 to 3.4 nM). The pharmacology of competition for ³H-APO suggests selectivity for dopamine-agonist interactions. These results indicate that the P₄ membrane preparation may be useful for the evaluation of dopamine-agonist binding sites or “receptors.”     

Movement patterns of Antillean manatees in Chetumal Bay (Mexico) and coastal Belize: A challenge for regional conservation

Information from 15 satellite‐tracked Antillean manatees (Trichechus manatus manatus) was analyzed in order to assess individual movements, home ranges, and high‐use areas for conservation decisions. Manatees were captured in Chetumal Bay, Mexico, and tagged with Argos‐monitored satellite transmitters. Location of the manatees and physical characteristics were assessed to describe habitat properties. Most manatees traveled to freshwater sources. The Maximum Area Size (MAS) for each manatee was determined using the observation‐area method. Additional kernel densities of 95% home range and 50% Center of Activity (COA) were also calculated, with manatees having 1–3 COAs. Manatees exhibited two different movement patterns: remaining in Chetumal Bay, and long‐distance (up to 240 km in 89 d). The residence time in Chetumal Bay was higher for females (89.6% of time) than for males (72.0%), but the daily travel rate (0.4–0.5 km/d) was similar for both sexes. Most of the COAs fell within Natural Protected Areas (NPA). However, manatees also travel for long distances into unprotected areas, where they face uncontrolled boat traffic, fishing activities, and habitat loss. Conservation of movement corridors may promote long‐distance movements and facilitate genetic exchange.     

In Vitro Study of Taenia solium Postoncospheral Form

Background

The transitional period between the oncosphere and the cysticercus of Taenia solium is the postoncospheral (PO) form, which has not yet been completely characterized. The aim of this work was to standardize a method to obtain T. solium PO forms by in vitro cultivation. We studied the morphology of the PO form and compared the expression of antigenic proteins among the PO form, oncosphere, and cysticerci stages.

Methodology/Principal Findings

T. solium activated oncospheres were co-cultured with ten cell lines to obtain PO forms, which we studied at three stages of development–days 15, 30, and 60. A high percentage (32%) of PO forms was obtained using HCT-8 cells in comparison to the other cell lines. The morphology was observed by bright field, scanning, and transmission electron microscopy. Morphology of the PO form changed over time, with the six hooks commonly seen in the oncosphere stage disappearing in the PO forms, and vesicles and microtriches observed in the tegument. The PO forms grew as they aged, reaching a diameter of 2.5 mm at 60 days of culture. 15–30 day PO forms developed into mature cysticerci when inoculated into rats. Antigenic proteins expressed in the PO forms are also expressed by the oncosphere and cysticerci stages, with more cysticerci antigenic proteins expressed as the PO forms ages.

Conclusions/Significance

This is the first report of an in vitro production method of T. solium PO forms. The changes observed in protein expression may be useful in identifying new targets for vaccine development. In vitro culture of PO form will aid in understanding the host-parasite relationship, since the structural changes of the developing PO forms may reflect the parasite’s immunoprotective mechanisms. A wider application of this method could significantly reduce the use of animals, and thus the costs and time required for further experimental investigations.     

A unique error signature for human DNA polymerase nu

Human DNA polymerase nu (pol nu) is one of three A family polymerases conserved in vertebrates. Although its biological functions are unknown, pol nu has been implicated in DNA repair and in translesion DNA synthesis (TLS). Pol nu lacks intrinsic exonucleolytic proofreading activity and discriminates poorly against misinsertion of dNTP opposite template thymine or guanine, implying that it should copy DNA with low base substitution fidelity. To test this prediction and to comprehensively examine pol nu DNA synthesis fidelity as a clue to its function, here we describe human pol nu error rates for all 12 single base-base mismatches and for insertion and deletion errors during synthesis to copy the lacZ alpha-complementation sequence in M13mp2 DNA. Pol nu copies this DNA with average single-base insertion and deletion error rates of 7 x 10(-5) and 17 x 10(-5), respectively. This accuracy is comparable to that of replicative polymerases in the B family, lower than that of its A family homolog, human pol gamma, and much higher than that of Y family TLS polymerases. In contrast, the average single-base substitution error rate of human pol nu is 3.5 x 10(-3), which is inaccurate compared to the replicative polymerases and comparable to Y family polymerases. Interestingly, the vast majority of errors made by pol nu reflect stable misincorporation of dTMP opposite template G, at average rates that are much higher than for homologous A family members. This pol nu error is especially prevalent in sequence contexts wherein the template G is preceded by a C-G or G-C base pair, where error rates can exceed 10%. Amino acid sequence alignments based on the structures of more accurate A family polymerases suggest substantial differences in the O-helix of pol nu that could contribute to this unique error signature.     

Cysteine 111 affects coupling of single-stranded DNA binding to ATP hydrolysis in the herpes simplex virus type-1 origin-binding protein

Herpes simplex virus type-1 origin-binding protein (UL9 protein) initiates viral replication by unwinding the origins. It possesses sequence-specific DNA-binding activity, single-stranded DNA-binding activity, DNA helicase activity, and ATPase activity that is strongly stimulated by single-stranded DNA. We have examined the role of cysteines in its action as a DNA helicase. The DNA helicase and DNA-dependent ATPase activities of UL9 protein were stimulated by reducing agent and specifically inactivated by the sulfhydryl-specific reagent N-ethylmaleimide. To identify the cysteine responsible for this phenomenon, a conserved cysteine in the vicinity of the ATP-binding site (cysteine 111) was mutagenized to alanine. UL9C111A protein exhibits defects in its DNA helicase and DNA-dependent ATPase activities and was unable to support origin-specific DNA replication in vivo. A kinetic analysis indicates that these defects are due to the inability of single-stranded DNA to induce high affinity ATP binding in UL9C111A protein. The DNA-dependent ATPase activity of UL9C111A protein is resistant to N-ethylmaleimide, while its DNA helicase activity remains sensitive. Accordingly, sensitivity of UL9 protein to N-ethylmaleimide is due to at least two cysteines. Cysteine 111 is involved in coupling single-stranded DNA binding to ATP-binding and subsequent hydrolysis, while a second cysteine is involved in coupling ATP hydrolysis to DNA unwinding.     

Changes in DNA Content and Cellular Death during a Starvation-Survival Process of Escherichia coli in River Water

Abstract Four nucleoid staining procedures were compared during the starvation-survival process of Escherichia coli in river water. Only the method performed as a modification of the standard acridine orange direct procedure allowed us to visualize nucleoids during the 95 days of experimentation. Moreover, with this method the total number of cells and nucleoid-containing cells can be simultaneously enumerated. The decrease of the chromosomal DNA content of population and of the nucleoid-containing cells indicates that ghosts form and cellular death occurs throughout the starvation-survival process. A long time (<30 days) is needed for non-nucleoid-containing cells to appear in river water; plasmid DNA is also negatively affected by environmental stress. After 4 days of storage in river water, the need to increase the volume of lysed cells used for the plasmid band visualization as well as the decrease in the plasmid band intensity would indicate a decrease in the plasmid DNA content during the starvation-survival process. According to our results, both chromosomal and plasmid DNA content decrease during the starvation-survival process of E. coli in river water. Received: 13 May 1998; Accepted: 14 September 1998     

Effect of temperature and starvation upon survival strategies of Pseudomonas fluorescens CHA0: comparison with Escherichia coli

Microorganisms in aquatic systems are exposed to continuous modifications in their environmental conditions. In these systems, both autochthonous and allochthonous bacteria respond to adverse conditions by expressing viable but nonculturable phenotype. On the basis of this common response, the behaviour of a few species is extrapolated to others. We compared the survival strategies of Escherichia coli (allochthonous, mesophile bacterium) and Pseudomonas fluorescens CHA0 (ubiquitous, psychrotrophic bacteria) under nonoptimal temperature and nutrient deprivation. In the absence of nutrients, the effect of temperature on the loss of culturability did not show a common pattern. Whereas the survival of E. coli had an inverse relationship with temperature, whereas for P. fluorescens a direct relationship between temperature and T?? values was only established in the range 5-15°C, with an inverse relationship at higher temperatures. When the subproteome of the outer membrane of P. fluorescens was comparatively analysed, starvation was not the main source of change. The most relevant modifications were due to variations in temperature. OprF, the major surface protein of the genus Pseudomonas, showed a high expression in nonculturable as well as culturable populations under all the adverse situations analysed. We therefore propose OprF as a suitable marker for Pseudomonas detection in the environment.