E-cadherin germline missense mutations and cell phenotype: evidence for the independence of cell invasion on the motile capabilities of the cells

In Hereditary Diffuse Gastric Cancer syndrome, E-cadherin germline mutations of the missense type harbour significant functional consequences. In this study, we have characterised the effect of T340A, A617T, A634V and V832M E-cadherin germline missense mutations on cell morphology, motility and proliferation. Wild-type E-cadherin and A617T expressing cells have an epithelial-like morphology, with polarised cells migrating unidirectionally. T340A and A634V expressing cells, fibroblast-like, have a high motile phenotype. We show that this phenotype is dependent on an increased level of active RhoA. V832M expressing cells grow in piled-up structure of round cells, as an effect of the disturbance of the binding between alpha-catenin and beta-catenin. The destabilisation of the adhesion complex is shown to hamper the motile capabilities of these cells. We did not observe any effect of the E-cadherin mutations on cell proliferation. We show the existence of a genotype-phenotype correlation between different E-cadherin mutations and cell behaviour. However, we demonstrate that the ability of cells expressing the different E-cadherin mutations to invade is independent on their motile capabilities, providing evidence that motility is neither necessary nor sufficient for cells to invade. Our data give new insights into the understanding of the mechanisms linking invasion and E-cadherin mutations in diffuse gastric cancer.     

Pregnancy with frozen-thawed and fresh testicular biopsy after motile and immotile sperm microinjection, using the mechanical touch technique to assess viability

BACKGROUND: There are few reports of pregnancy using immotile sperm, and none using a purely mechanical assessment of viability. METHODS: In this pilot study, we retrospectively analysed 66 cycles in 61 patients with determinant male factor, recording rates of fertilization, implantation, normal pregnancy and take-home babies achieved with ICSI. Sperm selection was based on morphologically normal appearance under the inverted microscope. Viability of immotile spermatozoa was assessed by the mechanical touch technique to observe tail flexibility and tail shape recovery. RESULTS: Of 17 ICSI cycles using frozen-thawed testicular sperm, six microinjected with immotile and 11 with motile sperm, we achieved fertilization rates of 65.7 and 74.3%, respectively, and five pregnancies (two and three, respectively). Of 49 ICSI cycles using fresh testicular sperm, 10 microinjected with immotile and 39 with motile sperm, we achieved fertilization rates of 73.4 and 64.4%, respectively, and 12 pregnancies (three and nine, respectively). CONCLUSIONS: Immotile (fresh and frozen-thawed) testicular sperm of normal morphological appearance can be used to achieve clinical pregnancy with ICSI. Our results strongly suggest that immotile sperm viability can be assessed by the mechanical touch technique.     

Modulation of mechanosensory responses by motoneurons that regulate skin surface topology in the leech

Central regulation of somatosensory signals has been extensively studied, but little is known about their regulation in the periphery. Given the widespread exposure of the skin sensory terminals to the environment, it is of interest to explore how somatosensory sensitivity is affected by changes in properties of the skin. In the leech, the annuli that subdivide the skin can be erected under the control of the annulus erector (AE) motoneurons. To analyze whether this surface change influences mechanosensory sensitivity, we studied the responses of low threshold mechanosensory T cells to mechanical stimulation of the skin as AE motoneurons were activated. In segments of the body wall connected to the corresponding ganglion and submerged in an aqueous environment, T cells responded to localized bubbling on the skin and to water flow parallel to its surface. Excitation of AE motoneurons diminished these responses in a way that depended on the motoneuron firing frequency. Video recordings established that the range of AE firing frequencies that produced effective annulus erection coincided with that influencing T cell responses. In isolated ganglia, AE firing had no effect on T cell excitability, suggesting that annulus erection diminished T cell responsiveness to mechanical input. Counteracting this effect, mechanosensory inputs inhibited AE motoneurons. However, because depolarization of AE cells caused a decrease in their input resistance, the more active the motoneuron, the less sensitive it became to inhibitory signals. Thus when brought to fire, AE motoneurons would stay "committed" to a high activity level, and this would limit sensory responsiveness to incoming mechanical signals.     

Metabolic polymorphisms and the micronucleus frequency in buccal epithelium of adolescents living in an urban environment

Micronuclei and other biomarkers were evaluated in oral cells from 11- to 16-year-old girls living in a foster home in the central area of México City. Variables analyzed for possible association with these biomarkers include smoking habits, body mass index, metabolic polymorphisms for NAT1 and GSTM1 and whether the cells were obtained from the cheek or pharynx. The results indicated that individuals having the NAT1*10 homozygous genotype showed a significant increase in chromatin buds and binucleated cells. When the damage in the cheek was compared with damage in the pharynx, a significant increase in micronuclei and binucleated cells was found for the latter tissue in all the individuals analyzed.     

Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion

Alphaviruses have the ability to induce cell-cell fusion after exposure to acid pH. This observation has served as an article of proof that these membrane-containing viruses infect cells by fusion of the virus membrane with a host cell membrane upon exposure to acid pH after incorporation into a cell endosome. We have investigated the requirements for the induction of virus-mediated, low pH-induced cell-cell fusion and cell-virus fusion. We have correlated the pH requirements for this process to structural changes they produce in the virus by electron cryo-microscopy. We found that exposure to acid pH was required to establish conditions for membrane fusion but that membrane fusion did not occur until return to neutral pH. Electron cryo-microscopy revealed dramatic changes in the structure of the virion as it was moved to acid pH and then returned to neutral pH. None of these treatments resulted in the disassembly of the virus protein icosahedral shell that is a requisite for the process of virus membrane-cell membrane fusion. The appearance of a prominent protruding structure upon exposure to acid pH and its disappearance upon return to neutral pH suggested that the production of a "pore"-like structure at the fivefold axis may facilitate cell penetration as has been proposed for polio (J. Virol. 74 (2000) 1342) and human rhino virus (Mol. Cell 10 (2002) 317). This transient structural change also provided an explanation for how membrane fusion occurs after return to neutral pH. Examination of virus-cell complexes at neutral pH supported the contention that infection occurs at the cell surface at neutral pH by the production of a virus structure that breaches the plasma membrane bilayer. These data suggest an alternative route of infection for Sindbis virus that occurs by a process that does not involve membrane fusion and does not require disassembly of the virus protein shell.     

Looking for ferns: optimization of digestion pretreatment in fluorescence in situ hybridization (FISH) technique on paraffin-embedded tissues.

Fluorescence in situ hybridization (FISH) is a useful cytogenetic technique for the detection of chromosome aberrations. However, applying this technique routinely on paraffin-embedded tissue is hampered by technical problems. The efficiency of hybridization is influenced by formalin fixation time, and this may vary considerably between specimens. We present a simple method for improving hybridization by microscopically monitoring the time of enzymatic digestion. To establish optimal digestion time, enzymatic digestion was stopped at 3-minute intervals for biopsies and 10-minute intervals for autopsies in 24 paraffin-embedded samples. At every stop, tissue morphology was examined under light microscopy to determine if observed changes could be correlated with subsequent FISH results. The appearance of fernlike formations was found to mark the optimal digestion time that produced the strongest hybridization signals. Using this method of digestion time control, an additional 41 cases were evaluated for FISH with various types of probe. Monitoring under the microscope could be more spaced if the morphology did not change after the first visual control and could be adapted to the type of sample (in general, endoscopic samples, total digestion time of about 10 min; routine biopsies, 15 to 30 min; autopsy samples, 20 to 40 min). In every case, the appearance of the fernlike pattern correlated with proper hybridization signal. Monitoring digestion time for the appearance of fernlike structures is a useful method for improving reproducibility of FISH technique on paraffin-embedded samples. It is particularly useful when dealing with samples under heterogeneous fixation conditions (consultations, autopsies, etc.), because it eliminates the need for repetition.     

The oxidizing agent tertiary butyl hydroperoxide induces disturbances in spindle organization, c-meiosis, and aneuploidy in mouse oocytes

It has been recently proposed that a concomitant generationof oxidative stress of oocytes with increasing maternal agemay be a major factor responsible for the age-related increasein aneuploid conceptions. As a preliminary step in the testingof this hypothesis, we need to confirm that oxidative stressin itself can induce errors in chromosome segregation. In orderto achieve this goal, germinal vesicle (GV)-stage mouse oocytesfrom unstimulated ICR and (C57BLxCBA) F₁ hybrid female micewere matured in vitro for 9 h for metaphase I (MI) oocytes or16 h for metaphase II (MII) oocytes in the presence of varyingconcentrations of the oxidizing agent tertiary-butyl hydroperoxide(tBH). MII oocytes from (C57BLxCBA) F₁ hybrid mice were fixedand C-banded for karyotyping analysis. MI and MII oocytes fromICR mice were fixed and stained with the DNAfluorescent probe4',6-diamidino-2-phenylindole (DAPI) to detect abnormalitiesin chromosomal distribution. Meiosis I and meiosis II spindlesfrom ICR mice were visualized by confocal immunofluorescencemicroscopy. Data from these experiments demonstrate that in-vitroexposure of mouse oocytes to tBH during meiosis I reduces thelength (pole-to-pole distance) and width (diameter at the equatorof the spindle) of meiosis I and meiosis II spindles. This reductionis associated with an increase in the percentage of oocytesshowing chromosome scattering and clumping on the MII plate,and of aneuploidy (hyperhaploidy) in MII oocytes. However, tBHat the concentrations used in the present study has only a minimalnegative effect on the frequency of meiotic maturation. Theseresults suggest that oxidative stress during meiotic maturationin vitro may induce chromosomal errors that are undetectablein the living oocyte and whose developmental consequences maybecome manifest after fertilization. aneuploidy/meiosis/mouse oxidative stress/spindle/tertiary butyl hydroperoxide     

Synthesis and Characterization of Hyperbranched Polyethylenes Made with Nickel‐α‐Diimine Catalysts

The polymerization of ethylene in the presence of 1,4‐bis(2,6‐diisopropylphenyl)acenaphthenediiminenickel(II) dichloride ( 1 ) and methylaluminoxane (MAO) gives hyperbranched polyethylene (HBPE) in appropriate reaction conditions. The system 1 /MAO is active in solvents like toluene or hexane at temperatures as high as 80 °C and ethylene pressures ranging from 1 to 15 atm. The polyethylenes obtained show high molecular weights (up to 467 kg · mol^?1) and more than 218 branches per 1 000 backbone carbon atoms, qualifying these materials as hyperbranched. Dynamic‐mechanical thermal analysis (DMTA) of these materials shows high β‐transitions, directly related to the branch content of these polyethylenes.

DMTA analysis of polyethylenes obtained with 1 /MAO at 0, 30, and 50 °C (corresponding to entries 1, 2 and 3).     

Dual role of interleukin-4 (IL-4) in pulmonary paracoccidioidomycosis: endogenous IL-4 can induce protection or exacerbation of disease depending on the host genetic pattern

Resistance to paracoccidioidomycosis, the most important endemic mycosis in Latin America, is thought to be primarily mediated by cellular immunity and the production of gamma interferon. To assess the role of interleukin-4 (IL-4), a Th2 cytokine, pulmonary paracoccidioidomycosis in IL-4-depleted susceptible (B10.A) and intermediate (C57BL/6) mice was studied. Two different protocols were used to neutralize endogenous IL-4 in B10.A mice: 1 mg of anti-IL-4 monoclonal antibody (MAb)/week and 8 mg 1 day before intratracheal infection with 10(6) Paracoccidioides brasiliensis yeast cells. Unexpectedly, both protocols enhanced pulmonary infection but did not alter the levels of pulmonary cytokines and specific antibodies. Since in a previous work it was verified that C57BL/6 mice genetically deficient in IL-4 were more resistant to P. brasiliensis infection, we also investigated the effect of IL-4 depletion in this mouse strain. Treatment with the MAb at 1 mg/week led to less severe pulmonary disease associated with impaired synthesis of Th2 cytokines in the lungs and liver of control C57BL/6 mice. Conversely, in IL-4-depleted C57BL/6 mice, increased levels of tumor necrosis factor alpha and IL-12 were found in the lungs and liver, respectively. In addition, higher levels of immunoglobulin G2a (IgG2a) and lower levels of IgG1 antibodies were produced by IL-4-depleted mice than by control mice. Lung pathologic findings were equivalent in IL-4-depleted and untreated B10.A mice. In IL-4-depleted C57BL/6 mice, however, smaller and well-organized granulomas replaced the more extensive lesions that developed in untreated mice. These results clearly showed that IL-4 can have a protective or a disease-promoting effect in pulmonary paracoccidioidomycosis depending on the genetic background of the host.     

Are Anti-Beta2-Glycoprotein-I Antibodies Markers for Recurrent Pregnancy Loss in Lupus Anticoagulant/Anticardiolipin Seronegative Women?

Problem  Anti-beta₂-Glicoprotein-1 antibodies (anti-β₂GPI-ab) have been related to recurrent miscarriage (RM) with conflicting results. The aim was to evaluate the role of anti-β₂-GPI-ab as unique biological marker in RM related to antiphospholipid (aPL).
Method of study  A cohort study that included 59 cases, divided in two groups, was designed: group 1 comprised 43 pregnant women with 'obstetric' antiphospholipid syndrome (APS) and group 2 included 16 cases with similar complaints but only having repeatedly anti-β₂-GPI-ab. Previous thrombosis and/or inherited thrombophilia were excluded. Lupus anticoagulant, anticardiolipin antibodies (aCA), anti-β₂-GPI-ab, and other autoantibodies were analyzed. Miscarriages, premature births, pre-eclampsia, live births, placental and systemic thromboses were studied.
Results  No differences in previous obstetric complications were detected ( P  =   1.00–0.164). After the treatment, differences in number of obstetric complications were not seen ( P  =   1.00). Live births were similar in two groups (88.4% and 93.7%; P  =   1.00). Placental thrombosis was equal in both groups, 93.3% versus 80% ( P  =   1.00).
Conclusion  These results suggest that anti-β₂-GPI-ab may be considered a biological marker for obstetric APS.