Effect of vasectomy on gene expression in the epididymis of cynomolgus monkey

Vasectomy has been shown to affect the pattern of mRNA expression of P34H, a human sperm protein added to the acrosomal cap during epididymal transit. It has been reported that vasectomy alters the histology of the reproductive tract in various species as a result of the increased pressure in the epididymis. The aim of this study was to evaluate if other epididymis-specific mRNAs, which are expressed in different patterns along the duct, are altered by vasectomy as well. We analyzed the expression of P31m (a monkey homologue of human P34H) and three different HE-like (HE-l) mRNAs along the epididymis in the cynomolgus monkey (Macaca fascicularis). Sexually mature cynomolgus monkeys were vasectomized unilaterally; then the epididymides were surgically removed at different time points. The ipsilateral normal epididymis was used as a control. Histomorphometric measurements showed that the height of the epididymal epithelial cells started to be affected only at 14 wk postsurgery. However, Northern blot and in situ hybridization analysis showed that the expression pattern of P31m, HE1, and HE5-like mRNA along the epididymis was not affected by vasectomy. Only the HE2-like mRNA predominantly expressed in the normal corpus epididymidis was significantly lowered 14 wk after vasectomy. Thus, ductal obstruction differentially alters mRNA expression along the epididymis of the cynomolgus monkey.     

Adsorption of DNA to mica mediated by divalent counterions: a theoretical and experimental study

The adsorption of DNA molecules onto a flat mica surface is a necessary step to perform atomic force microscopy studies of DNA conformation and observe DNA-protein interactions in physiological environment. However, the phenomenon that pulls DNA molecules onto the surface is still not understood. This is a crucial issue because the DNA/surface interactions could affect the DNA biological functions. In this paper we develop a model that can explain the mechanism of the DNA adsorption onto mica. This model suggests that DNA attraction is due to the sharing of the DNA and mica counterions. The correlations between divalent counterions on both the negatively charged DNA and the mica surface can generate a net attraction force whereas the correlations between monovalent counterions are ineffective in the DNA attraction. DNA binding is then dependent on the fractional surface densities of the divalent and monovalent cations, which can compete for the mica surface and DNA neutralizations. In addition, the attraction can be enhanced when the mica has been pretreated by transition metal cations (Ni(2+), Zn(2+)). Mica pretreatment simultaneously enhances the DNA attraction and reduces the repulsive contribution due to the electrical double-layer force. We also perform end-to-end distance measurement of DNA chains to study the binding strength. The DNA binding strength appears to be constant for a fixed fractional surface density of the divalent cations at low ionic strength (I < 0.1 M) as predicted by the model. However, at higher ionic strength, the binding is weakened by the screening effect of the ions. Then, some equations were derived to describe the binding of a polyelectrolyte onto a charged surface. The electrostatic attraction due to the sharing of counterions is particularly effective if the polyelectrolyte and the surface have nearly the same surface charge density. This characteristic of the attraction force can explain the success of mica for performing single DNA molecule observation by AFM. In addition, we explain how a reversible binding of the DNA molecules can be obtained with a pretreated mica surface.     

Between- and within-host species selection on cytoplasmic incompatibility-inducing Wolbachia in haplodiploids

Abstract The most common effect of the endosymbiont Wolbachia is cytoplasmic incompatibility (CI), a form of postzygotic reproductive isolation that occurs in crosses where the male is infected by at least one Wolbachia strain that the female lacks. We revisited two puzzling features of Wolbachia biology: how Wolbachia can invade a new species and spread among populations, and how the association, once established in a host species, can evolve, with emphasis on the possible process of infection loss. These questions are particularly relevant in haplodiploid species, where males develop from unfertilized eggs, and females from fertilized eggs. When CI occurs in such species, fertilized eggs either die (female mortality type: FM), or develop into males (male development type: MD), raising one more question: how transition among CI types is possible. We reached the following conclusions: (1) the FM type is a better invader and should be retained preferentially after a new host is captured; (2) given the assumptions of the models, FM and MD types are selected on neither the bacterial side nor the host side; (3) selective pressures acting on both partners are more or less congruent in the FM type, but divergent in the MD type; (4) host and symbiont evolution can drive infection to extinction for all CI types, but the MD type is more susceptible to the phenomenon; and (5) under realistic conditions, transition from MD to FM type is possible. Finally, all these results suggest that the FM type should be more frequent than the MD type, which is consistent with the results obtained so far in haplodiploids.     

Characterization of the cellulolytic complex (cellulosome) of Clostridium acetobutylicum

A large cellulosomal gene cluster was identified in the recently sequenced genome of Clostridium acetobutylicum ATCC 824. Sequence analysis revealed that this cluster contains the genes for the scaffolding protein CipA, the processive endocellulase Cel48A, several endoglucanases of families 5 and 9, the mannanase Man5G, and a hydrophobic protein, OrfXp. Surprisingly, genetic organization of this large cluster is very similar to that of Clostridium cellulolyticum, the model of mesophilic clostridial cellulosomes. As C. acetobutylicum is unable to grow on cellulosic substrates, the existence of a cellulosomal gene cluster in the genome raises questions about its expression, function and evolution. Biochemical evidence for the expression of a cellulosomal protein complex was investigated. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing and Western blotting with antibodies against specific components of the C. cellulolyticum cellulosome suggest that at least four major cellulosomal proteins are present. In addition, despite the fact that no cellulolytic activities were detected, we report here the evidence for the production of a high molecular mass cellulosomal complex in C. acetobutylicum.     

Gene mapping via the ancestral recombination graph

We present a multilocus gene mapping method based on linkage disequilibrium, which uses the ancestral recombination graph to model the history of sequences that may harbor an influential variant. We describe the construction of a recurrence equation used to make inferences about the location of a trait-influencing mutation. We demonstrate how a Monte Carlo algorithm combined with a local importance sampling scheme can be used for mapping. We explain how to simulate the timing of events in the coalescent in the presence of recombination and mutation, which accomodates variable population size. We provide an example to illustrate the use of the method, which can be easily extended to more general situations. Although the method is computationally intensive and variation in the likelihood profiles can occur, the method offers a great deal of promise.     

Role of tryptophan residues in the recognition of mutagenic oxidized nucleotides by human antimutator MTH1 protein

The human MTH1 antimutator protein hydrolyzes mutagenic oxidized nucleotides, and thus prevents their incorporation into DNA and any subsequent mutation. We have examined its great selectivity for oxidized nucleotides by analyzing the structure of the protein and its interaction with nucleotides, as reflected in the fluorescence of its tryptophan residues. The binding of nucleotides decreased the intensity of MTH1 protein fluorescence and red-shifted the emission peak, indicating that at least one tryptophan residue is close to the binding site. Oxidized nucleotides (2-OH-dATP and 8-oxo-dGTP) produced a larger decrease in fluorescence intensity than did unoxidized nucleotides, and MTH1 protein had a much higher binding affinity for oxidized nucleotides. Deconvolution of protein fluorescence by comparison of its quenching by positively (Cs(+)) and negatively (I(-)) charged ions indicated that the MTH1 tryptophan residues are in two different environments. One class of tryptophan residues is exposed to solvent but in a negatively charged environment; the other class is partially buried. While the binding of unoxidized nucleotides quenches the fluorescence of only class 1 tryptophan residue(s), the binding of oxidized nucleotides quenched that of class 2 tryptophan residue(s) as well. This suggests that selectivity is due to additional contact between the protein and the oxidized nucleotide. Mutation analysis indicated that the tryptophan residue at position 117, which is in a negative environment, is in contact with nucleotides. The negatively charged residues in the binding site probably correlate with the finding that nucleotide binding requires metal ions and depends upon their nature. Positively charged metal ions probably act by neutralizing the negatively charged nucleotide phosphate groups. (c) 2002 Elsevier Science Ltd.     

Synergistic antitumor effect of bispecific CD19 x CD3 and CD19 x CD16 diabodies in a preclinical model of non-Hodgkin's lymphoma

To target NK cells against non-Hodgkin's lymphoma, we constructed a bispecific diabody (BsDb) with reactivity against both human CD19 and FcgammaRIII (CD16). Bacterially produced CD19 x CD16 BsDb specifically interacted with both CD19(+) and CD16(+) cells and exhibited significantly higher apparent affinity and slower dissociation from the tumor cells than from effector cells. It was able to induce specific lysis of tumor cells in the presence of isolated human NK cells or nonfractionated PBLs. The combination of the CD19 x CD16 BsDb with a previously described CD19 x CD3 BsDb and CD28 costimulation significantly increased the lytic potential of human PBLs. Treatment of SCID mice bearing an established Burkitt's lymphoma (5 mm in diameter) with human PBLs, CD19 x CD16 BsDb, CD19 x CD3 BsDb, and anti-CD28 mAb resulted in the complete elimination of tumors in 80% of animals. In contrast, mice receiving human PBLs in combination with either diabody alone showed only partial tumor regression. These data clearly demonstrate the synergistic effect of small recombinant bispecific molecules recruiting different populations of human effector cells to the same tumor target.     

Enhanced bacterial virulence through exploitation of host glycosaminoglycans

Present in the extracellular matrix and membranes of virtually all animal cells, proteoglycans (PGs) are among the first host macromolecules encountered by infectious agents. Because of their wide distribution and direct accessibility, it is not surprising that pathogenic bacteria have evolved mechanisms to exploit PGs for their own purposes, including mediating attachment to target cells. This is achieved through the expression of adhesins that recognize glycosaminoglycans (GAGs) linked to the core protein of PGs. Some pathogens, such as Bordetella pertussis and Chlamydia trachomatis, may express more than one GAG-binding adhesin. Bacterial interactions with PGs may also facilitate cell invasion or systemic dissemination, as observed for Neisseria gonorrhoeae and Mycobacterium tuberculosis respectively. More-over, pathogenic bacteria can use PGs to enhance their virulence via a shedding of PGs that leads to there lease of effectors that weaken the host defences.The exploitation of PGs by pathogenic bacteria is thus a multifaceted mechanistic process directly related to the potential virulence of a number of microorganisms.