Absence of the myelin-associated glycoprotein (MAG) and the neural cell adhesion molecule (N-CAM) interferes with the maintenance,but not with the formation of peripheral myelin

We have previously shown that mice deficient in the gene for the myelin-associated glycoprotein (MAG) develop normal myelin in the peripheral nerves, but show axon and myelin degeneration at eight months of age, suggesting that MAG is involved in the maintenance of axon-Schwann cell integrity. The search for molecules that might replace MAG during myelination revealed an overexpression of the neural cell adhesion molecule (N-CAM) at those aspects where MAG is detectable in wild type mice. To test whether N-CAM might compensate for MAG during myelination in MAG-deficient mice, double mutants deficient in both MAG and N-CAM (MAG⁻/N-CAM⁻mice) were generated by cross-breeding the single mutants. Whereas alterations of myelin development were not detectable in either of the single or double mutants, degeneration of myelin and axons occurred approximately 4 weeks earlier in MAG⁻/N-CAM⁻than in MAG⁻mutants. Furthermore, at 8 weeks of age, single fiber preparation and electron microscopy revealed that the number of profiles indicative of degeneration was substantially increased in MAG⁻/N-CAM⁻mutants when compared to MAG⁻mice. These data suggest that in MAG-deficient mice N-CAM does not compensate for MAG in myelin formation but partially substitutes for it in the maintenance of axon-myelin integrity. Received: 20 May 1996 / Accepted: 19 July 1996     

Vitrification of one-cell mouse embryos in cryotubes

Preventing intracellular ice formation is essential to cryopreserve cells. Prevention can be achieved by converting cell water into a non-crystalline glass, that is, to vitrify. The prevailing belief is that to achieve vitrification, cells must be suspended in a solution containing a high concentration of glass-inducing solutes and cooled rapidly. In this study, we vitrified 1-cell mouse embryos and examined the effect of the cooling rate, the warming rate, and the concentration of cryoprotectant on cell survival. Embryos were vitrified in cryotubes. The vitrification solutions used were EFS20, EFS30, and EFS40, which contained ethylene glycol (20, 30 and 40% v/v, respectively), Ficoll (24%, 21%, and 18% w/v, respectively) and sucrose (0.4 0.35, and 0.3 M, respectively). A 5-μl EFS solution suspended with 1-cell embryos was placed in a cryotube. After 2 min in an EFS solution at 23 °C, embryos were vitrified by direct immersion into liquid nitrogen. The sample was warmed at 34 °C/min, 4,600 °C/min and 6,600 °C/min. With EFS40, the survival was low regardless of the warming rate. With EFS30 and EFS20, survival was also low when the warming rate was low, but increased with higher warming rates, likely due to prevention of intracellular ice formation. When 1-cell embryos were vitrified with EFS20 and warmed rapidly, almost all of the embryos developed to blastocysts in vitro. Moreover, when vitrified 1-cell embryos were transferred to recipients at the 2-cell stage, 43% of them developed to term. In conclusion, we developed a vitrification method for 1-cell mouse embryos by rapid warming using cryotubes.     

Mitochondrial toxicity and caspase activation in HIV pregnant women

To assess the impact of HIV‐infection and highly active anti‐retroviral treatment in mitochondria and apoptotic activation of caspases during pregnancy and their association with adverse perinatal outcome. Changes of mitochondrial parameters and apoptotic caspase activation in maternal peripheral blood mononuclear cells were compared at first trimester of pregnancy and delivery in 27 HIV‐infected and ‐treated pregnant women versus 24 uninfected pregnant controls. We correlated immunovirological, therapeutic and perinatal outcome with experimental findings: mitochondrial DNA (mtDNA) content, mitochondrial protein synthesis, mitochondrial function and apoptotic caspase activation. The HIV pregnancies showed increased adverse perinatal outcome (OR: 4.81 [1.14–20.16]; P < 0.05) and decreased mtDNA content (42.66 ± 5.94%, P < 0.01) compared to controls, even higher in naïve participants. This depletion caused a correlated decrease in mitochondrial protein synthesis (12.82 ± 5.73%, P < 0.01) and function (20.50 ± 10.14%, P < 0.001), not observed in controls. Along pregnancy, apoptotic caspase‐3 activation increased 63.64 ± 45.45% in controls (P < 0.001) and 100.00 ± 47.37% in HIV‐pregnancies (P < 0.001), in correlation with longer exposure to nucleoside analogues. HIV‐infected women showed increased obstetric problems and declined genetic and functional mitochondrial parameters during pregnancy, especially those firstly exposed to anti‐retrovirals. The apoptotic activation of caspases along pregnancy is emphasized in HIV pregnancies promoted by nucleoside analogues. However, we could not demonstrate direct mitochondrial or apoptotic implication in adverse obstetric outcome probably because of the reduced sample size.     

Abortion is incommensurable with fetal alcohol syndrome

A recent article argued for the immorality of abortion regardless of personhood status by comparing the impairment caused by fetal alcohol syndrome to the impairment caused by abortion. I argue that two of the premises in this argument fail and that, as such, one cannot reasonably attribute moral harms to abortion on the basis of the moral harms caused by fetal alcohol syndrome. The impairment argument relies on an inconsistent instantiation, which undermines the claim that personhood is irrelevant, and it does not fulfill its own ceteris paribus clause, which demands that no additional benefit be gained from abortion that would not be gained from causing fetal alcohol syndrome.     

The feeding behaviour, activity and trappability of wild female Glossina pallidipe in relation to their pregnancy cycle

Female Glossina pallidipes Austen trapped with baited NG2B traps were subjected both to detailed ovarian dissection and to nutritional analysis. Using a calibration curve derived from dissected wild-caught, laboratory-held flies, the field females were assigned by discriminant analysis to each day of the pregnancy cycle. Field females were most available to NG2B traps while carrying the first instar larva. The nutritional characteristics of trapped field females over the pregnancy cycle lead to the following main conclusions. (i) Fat levels increase most rapidly during the egg in utero stage, while CRDW increases significantly only during the larval stages, culminating in a 4 mg increase during the last day of the third larval instar. (ii) The haematin content of the flies indicates that females feed at approximately 3-day intervals and may feed on any day of the pregnancy cycle. (iii) The estimated time of feeding during the day corresponds with the observed time of peak activity, both of which are earlier in the day later in the pregnancy cycle. (iv) The rate of fat usage reveals significantly greater flight activity on day 5 of the cycle than on other days, agreeing with the high trappability on this day, and overall females appear to use fat at twice the rate of males. Reproductive data provide a much more accurate picture of the relative sampling efficiency than do nutritional data, although the latter reveal the general trends correctly.     

Stimulator of interferon genes (STING): A “new chapter” in virus-associated cancer research. Lessons from wild-derived mouse models of innate immunity

Thanks to the numerous studies that have been carried out recently in the field of cytosolic DNA sensing, STING (Stimulator of Interferon Genes) is now recognized as a key mediator of innate immune signaling. A substantial body of evidence derived from in vivo mouse models demonstrates that STING-regulated pathways underlie the pathogenesis of many diseases including infectious diseases and cancers. It has also become evident from these studies that STING is a promising therapeutic target for the treatment of cancer. However, mouse strains commonly used for modelling innate immune response against infections or tumors do not allow investigators to accurately reproduce certain specific characteristics of immune response observed in human cells. In this review, we will discuss recent data demonstrating that the use of wild-derived genetically distinct inbred mice as a model for investigation into the innate immunity signaling networks may provide valuable insight into the STING-regulated pathways specific for human cells. The maximum complexity of STING-mediated mechanisms can probably be seen in case of DNA virus-induced carcinogenesis in which STING may perform unexpected biological activities. Therefore, in another part of this review we will summarize emerging data on the role of STING in human DNA virus-related oncopathologies, with particular attention to HPV-associated cervical cancer, aiming to demonstrate that STING indeed “starts a new chapter” in research on this issue and that wild-derived mouse models of STING-mediated response to infections will probably be helpful in finding out molecular basis for clinical observations.     

Nonhistone chromatin proteins of B-lymphocytes stimulated by lipopolysaccharide Two-dimensional gel electrophoresis analysis of proteins synthesized and phoshorylated during the initiation of lymphocyte differentiation

Synthesis and phosphorylation of nonhistone chromatin and nucleoplasmic proteins during the first 24 h of activation of mouse B-lymphocytes by the B-cell mitogen lipopolysaccharide have been studied by two-dimensional gel electrophoretic analysis. Although little change occurs in the nucleoplasmic proteins, it has been shown that the incorporation of [³⁵S]methionine into nonhistone chromatin proteins is selectively stimulated. The degree of stimulation and the kinetics of synthesis are characteristic for each individual protein; some proteins exhibit increased incorporation only 4 h after addition of mitogen, while others are synthesized de novo between 8 and 24 h. After 72 h stimulation, the majority of nonhistone chromatin protein synthesis occurs in the highly differentiated lymphoblasts and plasma cells actively secreting IgM, very little synthesis taking place in the small lymphocytes. Analysis of nuclear proteins from lymphocytes stimulated for 2 h showed no selective stimulation of phosphorylation. These observations suggest that nonhistone chromatin proteins play an important role in the regulation of gene expression in B-lymphocytes.     

Determination of fetal biparietal diameter without the use of ultrasound in squirrel monkeys

This study evaluated manual caliper measurement of fetal BPD in Saimiri through the abdomen of the dam (TBPD) for correlation with paired ultrasound measurements, prediction of delivery date, subspecies variation, prediction of pregnancy outcome, and correlation between postpartum BPD and TBPD. Regression analysis revealed a close relationship between TBPD measurements and those obtained by ultrasound (P < 0.001). TBPD for Saimiri boliviensis boliviensis increased from a mean of 14.8 ± 0.3 mm for 11 weeks prepartum to a delivery week measurement of 33.2 ± 0.2mm. Delivery-week TBPD of Saimiri boliviensis peruviensis were significantly smaller than Saimiri sciureus sciureus (P < .05). Delivery week TBPD had a correlation coefficient of 0.64 with paired post-patrum measurements. No complications were associated with the technique. Delivery date could be predicted within two weeks. TBPD is an inexpensive, safe, rapid method of approximating fetal growth during the last half of pregnancy in Saimiri.     

CRN2 binds to TIMP4 and MMP14 and promotes perivascular invasion of glioblastoma cells

CRN2 is an actin filament binding protein involved in the regulation of various cellular processes including cell migration and invasion. CRN2 has been implicated in the malignant progression of different types of human cancer. We used CRN2 knock-out mice for analyses as well as for crossbreeding with a Tp53/Pten knock-out glioblastoma mouse model. CRN2 knock-out mice were subjected to a phenotyping screen at the German Mouse Clinic. Murine glioblastoma tissue specimens as well as cultured murine brain slices and glioblastoma cell lines were investigated by immunohistochemistry, immunofluorescence, and cell biological experiments. Protein interactions were studied by immunoprecipitation, pull-down, and enzyme activity assays. CRN2 knock-out mice displayed neurological and behavioural alterations, e.g. reduced hearing sensitivity, reduced acoustic startle response, hypoactivity, and less frequent urination. While glioblastoma mice with or without the additional CRN2 knock-out allele exhibited no significant difference in their survival rates, the increased levels of CRN2 in transplanted glioblastoma cells caused a higher tumour cell encasement of murine brain slice capillaries. We identified two important factors of the tumour microenvironment, the tissue inhibitor of matrix metalloproteinase 4 (TIMP4) and the matrix metalloproteinase 14 (MMP14, synonym: MT1-MMP), as novel binding partners of CRN2. All three proteins mutually interacted and co-localised at the front of lamellipodia, and CRN2 was newly detected in exosomes. On the functional level, we demonstrate that CRN2 increased the secretion of TIMP4 as well as the catalytic activity of MMP14. Our results imply that CRN2 represents a pro-invasive effector within the tumour cell microenvironment of glioblastoma multiforme.     

Attachment of blastocysts to lens capsule: A model system for trophoblast-epithelial cell interaction on a natural basement membrane

Summary The bovine lens capsule has previously been shown to provide an optimal surface for the examination of epithelial cell interaction with a basement membrane. This native substrate has been used to investigate some initial aspects of attachment of mouse blastocysts and trophoblastic cellular outgrowth. Mouse blastocysts were presented to the cell-free humoral side of the anterior lens capsule, incubated for 72 h, and examined by scanning and transmission electron microscopy. Blastocysts hatch and attach from their zonae pellucidae by 30 h. Trophoblastic cells proliferate rapidly in a coronal direction, display extensive surface microvilli, and advance by the extension of numerous filipodia, many of which terminate with bulbous projections. These projections were shown by transmission electron microscopy to contain numerous vacuoles and polysomes. To simulate further the initial blastocyst-uterine interaction, a suspension of lens epithelial cells was introduced to the capsule and permitted to form a monolayer prior to the addition of the blastocysts. At 72 h the monolayer of lens cells remained intact. We observed that: a) lens cells appear to recede from the advancing trophoblastic cells, and b) trophoblastic cells extend beneath the monolayer of lens cells and thereby dislodge the cells from the lens capsule substrate. No infiltration of the capsule by the advancing trophoblastic cells was observed. The lens capsule appears to offer a promising system for the study of trophoblast-epithelial cell interaction on a natural basement membrane.