Multi-scale mechanical characterization of prostate cancer cell lines: Relevant biological markers to evaluate the cell metastatic potential.

BACKGROUND: Considering the importance of cellular mechanics in the birth and evolution of cancer towards increasingly aggressive stages, we compared nano-mechanical properties of non-tumoral (WPMY-1) and highly aggressive metastatic (PC-3) prostate cell lines both on cell aggregates, single cells, and membrane lipids. METHODS: Cell aggregate rheological properties were analyzed during dynamic compression stress performed on a homemade rheometer. Single cell visco-elasticity measurements were performed by Atomic Force Microscopy using a cantilever with round tip on surface-attached cells. At a molecular level, the lateral diffusion coefficient of total extracted lipids deposited as a Langmuir monolayer on an air-water interface was measured by the FRAP technique. RESULTS: At cellular pellet scale, and at single cell scale, PC-3 cells were less stiff, less viscous, and thus more prone to deformation than the WPMY-1 control. Interestingly, stress-relaxation curves indicated a two-step response, which we attributed to a differential response coming from two cell elements, successively stressed. Both responses are faster for PC-3 cells. At a molecular scale, the dynamics of the PC-3 lipid extracts are also faster than that of WPMY-1 lipid extracts. CONCLUSIONS: As the evolution of cancer towards increasingly aggressive stages is accompanied by alterations both in membrane composition and in cytoskeleton dynamical properties, we attribute differences in viscoelasticity between PC-3 and WPMY-1 cells to modifications of both elements. GENERAL SIGNIFICANCE: A decrease in stiffness and a less viscous behavior may be one of the diverse mechanisms that cancer cells adopt to cope with the various physiological conditions that they encounter.

Isolation of Mycoplasma meleagridis from chickens.

Mycoplasma meleagridis (MM) is a major cause of disease and economic loss in turkeys. Formerly it was thought that this species was very host specific and only restricted to turkey. In this study, we report on the recovery of MM from breeding flocks of chickens located near a turkey breeding unit. Ten MM field strains were isolated (by culture on Frey broth medium) from tracheal swabs of chickens displaying clinical signs of mycoplasmosis-essentially respiratory symptoms and poor performance. Assignment of the isolated field strains to MM was confirmed by a growth inhibition assay using MM-specific polyclonal antiserum and by PCR amplification targeting the 16S rRNA sequence as well as the Mm14 sequence, a MM-species-specific DNA fragment previously identified and characterized in our laboratory. The nucleotide sequence of Mm14 proved to be highly conserved among the 10 MM field strains, indicating a common source of infection. However, on the basis of slight differences in sodium dodecyl sulfate-polyacrylamide gel electrophoresis whole-cell proteins and western blot profiles, two groups of the isolated MM field strains could be distinguished. Evidence of MM infection of chickens was further provided by serology, since 13.77% (35/254) of sera proved positive to MM by either rapid serum agglutination or recombinant antigen-based enzyme-linked immunosorbent assay. In addition, sera of all chickens from which MM was isolated were positive for antibodies to MM. Collectively, the data unambiguously show that MM could infect chickens; thus, MM warrants further exploration to determine its pathogenicity in this unusual host.

The effect of methylation on some biological parameters in Salmonella enterica serovar Typhimurium.

Cell growth is tightly coupled to DNA replication and its methylation [Proc Natl Acad Sci U S A 93 (1996) 12206-12211]. In a culture medium, growing of Salmonella Typhimurium (S. Typhimurium) mutant cells (dam⁻) decreased (2.5 fold) relative to the wild type strain (dam⁺). In this study, we show that the reason for this growth deficiency is due to the DNA methylation. The absence of a Dam methyltransferase protein results in poor growth efficiency and disturbs the synchrony of replication initiation in vivo, as evaluated by flow cytometry. On the other hand, we show that lack of methylation could increase the DNA response to thermal stress (decreasing the DNA melting temperature, T(m)), and the reason for this effect is due to the methylation status and not to the number of guanine and cytosine bases (G+C) in the duplex DNA. Our results show that methylation is an epigenetic factor that may play a key role in the cell growth, the synchrony of DNA replication [C R Biologies 330 (2007) 576-580] and the stress protection.

Morphological and biochemical changes of salmonella hadar exposed to aqueous garlic extract / Cambios morfológicos y bioquímicos de salmonella hadar expuestas a extracto acuoso de ajo

The effect on Salmonella hadar growth was investigated using fresh sterile liquid medium (Pronadisa, Hispanlab) containing aqueous garlic extract (AGE) at different concentration (0, 11, 12, and 13 mg/ml). The garlic extract added at these final concentrations had a bacteriostatic effect on Salmonella hadar. The effect of these bacteriostatic concentration of AGE on the growth of the tested serovar, revealed a pattern of inhibition characterized by: (i) a transitory inhibition phase whose duration was proportional to AGE concentration (ii) a resumed growth phase which showed a lower rate of growth than in uninhibited controls, and (iii) an entry into stationary phase at a lower culture density. The minimal inhibitory concentration and minimum bactericidal concentrations were very close, garlic MIC was 12 mg/ml and the MBC was 14 mg/ml. Among enzymatic activities followed with the API-ZYM system, significant changes during the inhibition phase were detected. These biochemical changes represent an adaptative response towards the garlic stress. Some cellular enzymatic activities disappeared, whereas others were induced or maintained after AGE addition. TEM images of the samples treated with the bacteriostatic concentration of AGE (12 mg/ml) revealed the rupture of cell walls and nonhomogeneous disposition of cytoplasmic materials within treated bacteria.

El efecto sobre el crecimiento de Salmonella hadar fue investigado utilizando un medio líquido estéril fresco (Pronadisa, Hispanlab) conteniendo el extracto acuoso de ajo (EAA) en diferentes concentraciones (0, 11, 12 y 13 mg/ml). El extracto de ajo añadido con estas concentraciones tuvo un efecto bacteriostático sobre Salmonella hadar. La prueba serovar reveló un patrón de inhibición caracterizado por: (i) una fase de inhibición transitoria cuya duración fue proporcional a la concentración de EAA, (ii) una reanudación de la fase de crecimiento, la cual mostró una tasa más baja de crecimiento que controles sin inhibición, y (iii) una ingreso en fase estacionaria con una menor densidad de cultivo. La concentración mínima inhibitoria (CMI) y la concentración mínima bactericida (CMB) fueron muy cercanas, la CMI de ajo fue de 12 mg/ml y la CMB fue de 14 mg/ml. Las actividades enzimáticas seguidas con el sistema API-ZYM, mostraron cambios significativos durante la fase de inhibición. Estos cambios bioquímicos representan una respuesta adaptativa al estrés del ajo. Algunas actividades enzimáticas celulares desaparecieron, mientras que otras fueron inducidas o mantenidas después de la adición de EAA. Las imágenes de MET de las muestras tratadas con la concentración del bacteriostático EAA (12 mg/ml) revelaron la ruptura de las paredes celulares y la disposición no homogénea de materiales citoplasmáticos dentro de las bacterias tratadas.

Modeling of the full-length Escherichia coli SeqA protein, in complex with DNA.

The Escherichia coli SeqA protein, a negative regulator of chromosome DNA replication, prevents the overinitiation of replication within one cell cycle by binding to hemimethylated GATC sequences in the replication origin, oriC. In addition to the hemimethylated DNA-binding activity, the SeqA protein has a self-association activity, which is also considered to be essential for its regulatory function in replication initiation. To study the SeqA protein biological activity, we performed a SeqA protein model to examine its architecture. SeqA has a bipartite structure composed of a large and small lobe. The SeqA spatial conformation contributes to its ability to bind to a pair of hemimethylated GATC sequences and to its cooperative behavior.

Effect of repeated in vivo passage (in mice) on Salmonella typhimurium dam mutant virulence and fitness.

Numerous studies have shown that Salmonella typhimurium dam mutants are highly attenuated for virulence in mice. In vivo studies have also shown that, on oral and intraperitoneal administration, low number of these mutants is able to colonize and persist in target organs. So, they must sense and overcome a myriad of host killing mechanisms. Our goal was to evaluate the effect of in vivo passage, in mice, on S. typhimurium dam mutant virulence and fitness. Swiss albino mice were used for the determination of LD50 and enumeration of bacteria recovered from liver eight days postinfection. Our results indicate that LD50 values of re-isolated mutants were at least two to three-fold lower than those of control strains. Strains re-isolated from liver showed decreased in vitro sensitivity toward sodium deoxycholate and H(2)O(2) than control strains. In addition, the number of re-isolated mutants colonizing spleen and liver was relatively higher than control strains. According to these results, we suggest that persistence of S. typhimurium dam mutants within target organs can increase their in vivo virulence in mice.

Molecular cloning of a Mycoplasma meleagridis-specific antigenic domain endowed with a serodiagnostic potential.

A recombinant phage library harbouring Mycoplasma meleagridis (MM) genomic DNA fragments was generated in the bacteriophage lambda gt11 expression vector. The library was screened for expression of MM specific antigens with a polyclonal antiserum that had been preadsorbed with antigens of the most common unrelated avian mycoplasma species. A 49-amino acid antigenic domain unique to MM was isolated, expressed in Escherichia coli, and its serodiagnostic potential was demonstrated. An antiserum raised against this MM-specific antigenic domain recognized a cluster of seven membrane-associated MM proteins with molecular masses ranging from 34 to 75 kDa. Overall, this study resulted in the identification of a potent serodiagnostic tool and revealed the complex antigenic nature of MM.

DnaA protein dependent denaturation of negative supercoiled oriC DNA minicircles.

The DnaA protein binds specifically and tightly to oriC supercoiled 641 bp minicircle DNA. The binding of the initiator promoted a partial unwinding of the superhelical oriC minicircle (Mc-oriC). Open complexes are detected either by a change in the linking number or by the sensitivity to the attack of a single strand specific Bal 31 nuclease. The open complex is found only in the presence of the DnaA protein.

Competition between the replication initiator DnaA and the sequestration factor SeqA for binding to the hemimethylated chromosomal origin of E. coli in vitro.

BACKGROUND: Following replication initiation, the replication origin (oriC) in Escherichia coli enters a hemimethylated state at Dam methylation sites which are recognized by the SeqA protein. SeqA binds preferentially to hemimethylated GATC sequences of DNA in vitro. SeqA is essential for the synchronous initiation of chromosome replication from oriC copies in vivo. RESULTS: We show that: (i) purified SeqA binds AT-rich and 13-mers regions and two DnaA boxes, R1 and M, of hemimethylated oriC. (ii) SeqA inhibits the in vitro replication of a hemimethylated oriC plasmid more efficiently than the fully methylated, (iii) SeqA inhibits competitive binding of DnaA protein to the regions of the hemimethylated oriC plasmid, explaining the mechanism of its inhibitory effect. The inhibition of DnaA binding by SeqA also occurs efficiently on a small hemimethylated oriC fragment containing both R1 and M DnaA boxes, but not the 13-mer region. CONCLUSIONS: SeqA binds strongly the long region from the AT-rich region to the M DnaA box of the hemimethylated oriC DNA and releases DnaA molecules from the long region.

Initiation of DNA replication in delta cya mutants of Escherichia coli K12.

The purified DnaA protein has a high affinity for cyclic AMP (cAMP). Using equilibrium dialysis, we determined the K(A) value for cAMP as 0.819 muM(-1). The number of cAMP binding sites per DnaA protein molecule was calculated to be 1.04. This binding was quite specific for cAMP. ATP was also bound by DnaA protein and inhibited cAMP binding. This inhibition was non-competitive in nature with an inhibition constant (K(i)) of about 8.25 muM. However, in vivo we have found not only that the DnaA protein level is reduced in a cyclase deletion mutant strain, Delta++ cya, but also that DnaA protein is not degraded. The Delta cya mutants of E. coli are unable to continue DNA synthesis in the absence of de novo protein synthesis and the initiation of DNA replication in these mutants takes place from oriC.

Parental strand recognition of the DNA replication origin by the outer membrane in Escherichia coli.

The outer membrane of Escherichia coli binds the origin of DNA replication (oriC) only when it is hemimethylated. We report here the results of a footprinting analysis with the outer membrane which demonstrate that its interaction with oriC occurs mainly at the left moiety of the minimal oriC, where 10 out of 11 Dam methylation sites are concentrated. Two regions, flanking the Integration Host Factor (IHF) sites, are preferentially recognized at the minimum membrane concentration at which oriC plasmid replication is inhibited in vitro. We have identified the putative proteins involved in hemimethylated oriC binding and cloned one of the corresponding genes (hobH). The purified LacZ-HobH fusion protein specifically binds oriC DNA at the same preferential sites as the membrane. A mutant of the hobH gene reveals partial asynchronous initiation of DNA replication.

The E. coli cell surface specifically prevents the initiation of DNA replication at oriC on hemimethylated DNA templates.

A particular outer membrane fraction previously defined as possessing specific affinity for the hemimethylated form of the origin of replication of the E. coli chromosome (oriC) is shown to inhibit the initiation of DNA synthesis at this site on hemimethylated DNA templates in vitro. The replication of fully methylated or unmethylated DNA templates is not affected. Also, no inhibition is observed if initiation takes place at random sites on the hemimethylated template. The key inactivation step appears to be membrane inhibition of DnaA initiator protein binding to oriC. Remethylation of the membrane-bound hemimethylated DNA results in reactivation. Our results demonstrate direct involvement of the membrane in the control of DNA replication. We propose that association/dissociation of the origin from the cell membrane is one of the control elements governing interinitiation times in E. coli.

Dam methylated and hemimethylated oriC plasmids are replicated symmetrically; a novel and general test of replication symmetry.

Deoxyadenosine methylation (dam) of the numerous GATC sequences present in the Escherichia coli origin of chromosomal replication (oriC) has been shown to be important both in vivo and in vitro for efficient initiation of DNA synthesis. Recent in vivo data suggest that initiation is only inefficient when these sequences are hemimethylated. This raises the interesting possibility that initiation may be inefficient because it only takes place on one strand of the template, i.e., replication is asymmetric on hemimethylated DNA. We tested this possibility by a novel and rapid approach which relies on the specificities of the restriction endonucleases MboI, MboII and DpnI. Although we show that replication takes place equally well on both strands of methylated and hemimethylated oriC DNA templates, the method should be applicable to the analysis of replication symmetry on most DNA templates which contain methylated deoxyadenosine GATC sequences as part of MboII restriction sites.

dam methylation and the initiation of DNA replication on oriC plasmids.

Plasmid DNA containing the replication origin of the Escherichia coli chromosome (oriC) has been shown to be inefficient as a template for DNA synthesis in vitro when isolated from dam mutants. Here, we extend this study to hemimethylated oriC plasmids and to replication in dam-3 mutant enzyme extracts. The results show that: (1) hemimethylated oriC plasmids replicate with the same low efficiency as nonmethylated DNA; (2) DNA synthesis starts at oriC regardless of the methylated state of the template; (3) replication in dam-3 enzyme extracts is inefficient because this strain is deficient in DnaA protein; and (4) consistent with this observation, the copy number of the oriC plasmid pFH271 is reduced in the dam-3 mutant. However, we have found that low DnaA protein levels in dam-3 mutants are not sufficient to explain the reduced transformation efficiency of oriC plasmids. We suggest that there must exist in vivo inhibitory factors not present or present in low quantities in vitro which specifically recognize the hemimethylated or nonmethylated forms of the oriC region.

A novel role for cAMP in the control of the activity of the E. coli chromosome replication initiator protein, DnaA.

DnaA protein interacts with cAMP with a KD of 1 microM. This interaction stimulates DnaA protein binding to the chromosome replication origin (oriC) and the mioC promoter region, protects DnaA protein from thermal inactivation, releases ADP but not ATP bound to DnaA protein, and restores normal DNA replication activity and ATPase activity in inactive ADP-DnaA protein preparations. A model is proposed in which cellular cAMP levels govern the replication activity of DnaA protein by promoting the recycling of the inactive ADP-DnaA protein form into the active ATP form.

[Recombination between the fragile site Xq27 and the gene for coagulation factor IX]. / Recombinaison entre le site fragile Xq27 et le gène du facteur IX de la coagulation.

Using the Taq I restriction polymorphism of a factor IX probe, we analysed the segregation of this gene and that of the fragile site Xq27. The ancestor of this family was a healthy carrier male. Of twelve informative meioses, at least four recombinations were detected. The hypothesis of a particular instability of the distal part the long arm of the X chromosome is reconsidered.