Differentiation-associated changes in membrane proteins of mouse myeloid leukemia cells

The mouse myeloid leukemia cell line (M1) is known to differentiate in vitro into macrophages and granulocytes upon treatment with various inducer including mouse ascitic fluid. Changes of cell surface proteins during differentiation of M1 cells were analyzed by the lactoperoxidase-catalyzed radioiodination method and SDS-polycrylamide slab gel electrphoresis. Treatment of the cells with ascitic fluid changed the electrophoretic pattern of the iodinated proteins, the prominent change being the appearance of a new protein with a molecular weight of 180 000 (P180). Iodinated P180 was also detected in normal macrophages in granulocytes, which are similar to differentiated M1 cells. This protein was metabolically labeled with l-[¹⁴C]fucose, increasing with the period of the treatment. P180 was not expressed on ascitic fluid-treatment of a resistant clone of M1 cells that could not be induced to differentiate. These results indicate that P180 is a glycoprotein that is exposed on the outer surface of differentiated M1 cells, and that its expression is associated with differentiation of the cells.P180 was solubilized from ¹²⁵I-labeled macrophages with detergents bound to concanavalin A-Sepharose. This suggests that P180 is one of the receptors for concanavalin A. Therefore, P180 may contribute partly to the increases in agglutinability by concanavalin A and in the number of concanavalin A binding sites on the surface of M1 cells, which are known to be associated with differentiation of M1 cells.     

Effects of grouping and fighting on the reproductive tracts of male white-footed mice (Peromyscus leucopus)

Summary Adult, male white-footed mice (Peromyscus leucopus) were subjected to a variety of social situations ranging from isolation during the 20 day experimental period to constant contact with both females and other adult males. Contacts included grouping (three or four males per cage) and exposure to fighters (once daily for 20 minutes). The following measurements were recorded: weights of the body, testes, epididymides, vesicular glands, vesicular gland tissue (wet and dry), seminal fluid of the vesicular gland, adrenal glands, and baculum; spermatozoan reserves of the testes and epididymides. Grouping significantly affected both the weight and spermatozoan reserves of the testes and epididymides, as well as both the tissue and seminal fluid weight of the vesicular glands. The results suggested a graded effect of all treatments on the reproductive tract. In order of magnitude of the associated response, from none to greatest, the treatments may be ranked as follows: pairing with females, isolating, handling, fighting, and grouping. All reproductive parameters measured showed this general ranking, suggesting that the response to the various treatments was similar and differed only quantitatively. The results further suggested decreased secretion of LH and testosterone, although measurements of testosterone did not substantiate this conclusion. The lack of significant effects of grouping on adrenal gland weights strengthened the argument that adrenal involvement is not a necessary adjunct to the suppression of the reproductive tract in groupedPeromyscus, but the adrenal may be involved if contacts between males result in overt fighting.     

Schistosoma mansoni: Astiban-induced damage to tegument and the male reproductive system

Astiban produced structural damage in male Schistosoma mansoni (Puerto Rican strain) in mice. The degree of disorganization was directly related to the dosage administered, although initial changes in structure for the first three doses (3 × 30–40 mg/kg) varied between individual worms of the same infection. More consistent damage to the tegument, parenchyma, and reproductive organs occurred after 6 × 40 mg/kg of Astiban injections. Exposure of the subtegumentary musculature preceded appearance of an increased number of noncytoplasmic spaces in various tissues, probably a result of osmotic stress. Testicular disorganization was prominent initially in spermatozoa and spermatids, but became more generalized with drug accumulation. The sustentacular cells showed increased phagocytic activity with testicular damage. Continuous administration of drug resulted in a general distortion of the worm's morphology. However, partial recovery occurred within 22 days following cessation of drug administration.     

Mouse embryos exposed to oxygen concentrations that mimic changes in the oviduct and uterus show improvement in blastocyst rate,blastocyst size,and accelerated cell division

After in vivo fertilisation, the preimplantation embryo goes through cleavage during migration along the oviduct in mammals or the fallopian tube in a woman and ends up inside the uterus. This study investigates the effect of a protocol aimed at closely reproducing that natural oxygen concentration in the oviduct (7 % O₂ from day 1 to day 3 and 2 % from day 3 to day 5), in contrast to the concentrations (5 % or 20 %) widely used in practice in ART using morphokinetic. Female mice (BI6/CBAca) were sacrificed, and zygotes were isolated 20 h after mating and randomly allocated to three parallel groups, which were grown under high atmospheric, low, or sequential oxygen concentrations. Zygotes were cultured in GTL medium (Vitrolife) and observed by the Primovision time-lapse system. Blastocyst rate at 120 h in the sequential group (91.3 %) was significantly increased over the high (76.3 %) and low (74.4 %) groups. Blastocyst size was also enlarged in the sequential group compared to the high and low groups. Moreover, cell division in the sequential group was significantly faster at almost every cleavage stage than it was in the other groups. Notably, the duration of the interims between stages also differed significantly between the groups. This study demonstrated that, in comparison to routinely used high or low oxygen conditions, oxygen concentrations mimicking changes in the oviduct and uterus significantly improve the blastocyst rate and size and accelerate cell division at several stages as well as the interims between cleavage events.     

Effect of acute sodium nitrite intoxication on some essential biometals in mouse spleen

Background and aimSodium nitrite (NaNO₂) is an inorganic salt with numerous applications in a variety of industries, as well as in medicine. Nevertheless, exposure to high levels of NaNO₂ is toxic for animals and humans. Sodium nitrite intoxication is shown to decrease the activity of major antioxidant defence enzymes which is dependent on the maintenance of specific ion equilibrium. The aim of the present study was to investigate the effect of acute NaNO₂ intoxication on the content of the essential metals iron (Fe), calcium (Ca) and zinc (Zn) in mouse spleen.MethodsMature male ICR mice were divided into four groups and subjected to acute NaNO₂ exposure by a single intraperitoneal injection of 120 mg/kg body weight. Animals in each group were sacrificed at certain time interval after treatment (1 h, 5 h, 1 day and 2 days). Spleens were excised and processed for atomic absorption spectrometry analysis of Fe, Ca and Zn content.ResultsAt the first hour after treatment, a decrease in Fe and Ca levels was observed. One day following NaNO₂ administration, Zn concentration reached its lowest value and Ca levels remained lower, compared to the untreated controls. In contrast, Fe concentration increased on the first and second day after treatment.ConclusionThe results of the present study demonstrate that acute NaNO₂ intoxication provokes changes in the endogenous levels of Fe, Ca and Zn in mouse spleen. These findings suggest disruption of the ionic balance and impact on the activity of antioxidant defence enzymes.     

Tissue distribution and subcellular localization of the family of Kidney Ankyrin Repeat Domain (KANK) proteins

Kidney Ankyrin Repeat-containing Proteins (KANKs) comprise a family of four evolutionary conserved proteins (KANK1 to 4) that localize to the belt of mature focal adhesions (FAs) where they regulate integrin-mediated adhesion, actomyosin contractility, and link FAs to the cortical microtubule stabilization complex (CMSC). The human KANK proteins were first identified in kidney and have been associated with kidney cancer and nephrotic syndrome. Here, we report the distributions and subcellular localizations of the four Kank mRNAs and proteins in mouse tissues. We found that the KANK family members display distinct and rarely overlapping expression patterns. Whereas KANK1 is expressed at the basal side of epithelial cells of all tissues tested, KANK2 expression is mainly observed at the plasma membrane and/or cytoplasm of mesenchymal cells and KANK3 exclusively in vascular and lymphatic endothelial cells. KANK4 shows the least widespread expression pattern and when present, overlaps with KANK2 in contractile cells, such as smooth muscle cells and pericytes. Our findings show that KANKs are widely expressed in a cell type-specific manner, which suggests that they have cell- and tissue-specific functions.     

Expression and localisation of ephrin-B1 and EphB4 in steroidogenic cells in the naturally cycling mouse ovary

Ephrin receptors and ligands are membrane-bound molecules that modulate diverse cellular functions such as cell adhesion, epithelial–mesenchymal transition, motility, differentiation and proliferation. We recently reported the co-expression of ephrin-B1 and EphB4 in adult and foetal Leydig cells of the mouse testis, and thus speculated that their co-expression is a common property in gonadal steroidogenic cells. Therefore, in this study we examined the expression and localisation of ephrin-B1 and EphB4 in the naturally cycling mouse ovary, as their expression patterns in the ovary are virtually unknown. We found that ephrin-B1 and EphB4 were co-expressed in steroidogenic cells of all kinds, i.e. granulosa cells and CYP17A1-positive steroidogenic theca cells as well as in 3β-HSD-positive luteal cells and the interstitial glands; their co-expression potentially serves as a good marker to identify sex steroid-producing cells even in extra-gonadal organs/tissues. We also found that ephrin-B1 and EphB4 expression in granulosa cells was faint and strong, respectively; ephrin-B1 expression in luteal cells was weak in developing and temporally mature corpora lutea (those of the current cycle) and likely strong in regressing corpora lutea (those of the previous cycle) and EphB4 expression in luteal cells was weak in corpora lutea of the current cycle and likely faint/negative in the corpora lutea of the previous cycle. These findings suggest that a luteinising hormone surge triggers the upregulation of ephrin-B1 and downregulation of EphB4, as this expression fluctuation occurs after the surge. Overall, ephrin-B1 and EphB4 expression patterns may represent benchmarks for steroidogenic cells in the ovary.     

Selective Inhibition of Acetylcholinesterase in the Cerebellum and Hippocampus of Mice Following an Acute Treatment with Malathion

Adult male ICR mice were treated by intraperitoneal injection with 250?mg/kg of bodyweight of commercial malathion (a dose corresponding to 1/12 the LD50). After 6?h, acetylcholinesterase (AChE) activity in blood, liver, and six brain regions was determined. A statistically significant inhibition was observed in whole blood (23%), liver (21%), and, in particular, the central nervous system; the greatest degree of AChE inhibition was observed in the cerebellum (45%), followed by the hippocampus (29%). There was no significant change in AChE activity in the caudate putamen, frontal cortex, midbrain, or pons medulla. These results demonstrate that the magnitude of AChE inhibition in peripheral tissues does not accurately reflect the central-inhibitory effects of malathion on AChE activity in specific brain regions.     

CD1d-restricted NKT cells modulate placental and uterine leukocyte populations during chlamydial infection in mice

Invariant CD1d-restricted natural killer T cells play an important immunoregulatory role and can influence a broad spectrum of immunological responses including against bacterial infections. They are present at the fetal–maternal interface and although it has been reported that experimental systemic iNKT cell activation can induce mouse abortion, their role during pregnancy remain poorly understood. In the present work, using a physiological Chlamydia muridarum infection model, we have shown that, in vaginally infected pregnant mice, C. muridarum is cleared similarly in C57BL/6 wild type (WT) and CD1d^−/− mice. We have also shown that infected- as well as uninfected-CD1d^−/− mice have the same litter size as WT counterparts. Thus, CD1d-restricted cells are required neither for the resolution of chlamydial infection of the lower-genital tract, nor for the maintenance of reproductive capacity. However, unexpected differences in T cell populations were observed in uninfected pregnant females, as CD1d^−/− placentas contained significantly higher percentages of CD4⁺ and CD8⁺ T cells than WT counterparts. However, infection triggered a significant decrease in the percentages of CD4⁺ T cells in CD1d^−/− mice. In infected WT pregnant mice, the numbers of uterine CD4⁺ and CD8⁺ T cells, monocytes and granulocytes were greatly increased, changes not observed in infected CD1d^−/− mice. An increase in the percentage of CD8⁺ T cells seems independent of CD1d-restricted cells as it occurred in both WT and CD1d^−/− mice. Thus, in the steady state, the lack of CD1d-restricted NKT cells affects leukocyte populations only in the placenta. In Chlamydia-infected pregnant mice, the immune response against Chlamydia is dampened in the uterus. Our results suggest that CD1d-restricted NKT cells play a role in the recruitment or homeostasis of leukocyte populations at the maternal–fetal interface in the presence or absence of Chlamydia infection.     

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.