The expression of β-glucuronidase during preimplantation development of mouse embryos

β-Glucuronidase activity was measured in mouse embryos during the preimplantation period of development by using a microfluorometric assay. A 100-fold increase in activity was observed between 57 (8-cell stage) and 84 hr (morulae) of development. Activity changes between 30 and 60 hr were also significant. Genetic variants of β-glucuronidase occur between the strains of mice $\text{C57BL}\text{6J}$ and $\text{C3H}\text{HeJ}$ which differ in levels of activity and heat denaturation kinetics. Activity changes and heat denaturation kinetics of β-glucuronidase in $\text{C57BL}\text{6}\text{,}\text{C3H}\text{HeJ}$ and F₁ hybrid embryos were compared, and it was demonstrated that paternal genes were expressed during the 100-fold increase in activity and that embryonic genes may be functioning between 30 and 60 hr of development.     

Hypotonic stress upregulates β- and γ-ENaC expression through suppression of ERK by inducing MKP-1

We investigated a physiological role for ERK, a member of the MAPK family, in the hypotonic stimulation of epithelial Na(+) channel (ENaC)-mediated Na(+) reabsorption in renal epithelial A6 cells. We show that hypotonic stress causes a major dephosphorylation of ERK following a rapid transient phosphorylation. PD98059 (a MEK inhibitor) increases dephosphorylated ERK and enhances the hypotonic-stress-stimulated Na(+) reabsorption. ERK dephosphorylation is mediated by MAPK phosphatase (MKP). Hypotonic stress activates p38, which in turn induces MKP-1 and to a lesser extent MKP-3 mRNA expression. Inhibition of p38 suppresses MKP-1 induction, preventing hypotonic stress from dephosphorylating ERK. Inhibition of MKP-1 and -3 by the inhibitor NSC95397 also suppresses the hypotonicity-induced dephosphorylation of ERK. NSC95397 reduces both β- and γ-ENaC mRNA expression and ENaC-mediated Na(+) reabsorption stimulated by hypotonic stress. In contrast, pretreatment with PD98059 significantly enhances mRNA and protein expression of β- and γ-ENaC even under isotonic conditions. However, PD98059 only stimulates Na(+) reabsorption in response to hypotonic stress, suggesting that ERK inactivation by itself (i.e., under isotonic conditions) is not sufficient to stimulate Na(+) reabsorption, even though ERK inactivation enhances β- and γ-ENaC expression. Based on these results, we conclude that hypotonic stress stimulates Na(+) reabsorption through at least two signaling pathways: 1) induction of MKP-1 that suppresses ERK activity and induces β- and γ-ENaC expression, and 2) promotion of translocation of the newly synthesized ENaC to the apical membrane.     

PDGF-Rα gene expression predicts proliferation,but PDGF-A suppresses transdifferentiation of neonatal mouse lung myofibroblasts

Background

Platelet-derived growth factor A (PDGF-A) signals solely through PDGF-Rα, and is required for fibroblast proliferation and transdifferentiation (fibroblast to myofibroblast conversion) during alveolar development, because pdgfa-null mice lack both myofibroblasts and alveoli. However, these PDGF-A-mediated mechanisms remain incompletely defined. At postnatal days 4 and 12 (P4 and P12), using mouse lung fibroblasts, we examined (a) how PDGF-Rα correlates with ki67 (proliferation marker) or alpha-smooth muscle actin (αSMA, myofibroblast marker) expression, and (b) whether PDGF-A directly affects αSMA or modifies stimulation by transforming growth factor beta (TGFβ).

Methods

Using flow cytometry we examined PDGF-Rα, αSMA and Ki67 in mice which express green fluorescent protein (GFP) as a marker for PDGF-Rα expression. Using real-time RT-PCR we quantified αSMA mRNA in cultured Mlg neonatal mouse lung fibroblasts after treatment with PDGF-A, and/or TGFβ.

Results

The intensity of GFP-fluorescence enabled us to distinguish three groups of fibroblasts which exhibited absent, lower, or higher levels of PDGF-Rα. At P4, more of the higher than lower PDGF-Rα + fibroblasts contained Ki67 (Ki67+), and Ki67+ fibroblasts predominated in the αSMA + but not the αSMA- population. By P12, Ki67+ fibroblasts comprised a minority in both the PDGF-Rα + and αSMA+ populations. At P4, most Ki67+ fibroblasts were PDGF-Rα + and αSMA- whereas at P12, most Ki67+ fibroblasts were PDGF-Rα- and αSMA-. More of the PDGF-Rα + than - fibroblasts contained αSMA at both P4 and P12. In the lung, proximate αSMA was more abundant around nuclei in cells expressing high than low levels of PDGF-Rα at both P4 and P12. Nuclear SMAD 2/3 declined from P4 to P12 in PDGF-Rα-, but not in PDGF-Rα + cells. In Mlg fibroblasts, αSMA mRNA increased after exposure to TGFβ, but declined after treatment with PDGF-A.

Conclusion

During both septal eruption (P4) and elongation (P12), alveolar PDGF-Rα may enhance the propensity of fibroblasts to transdifferentiate rather than directly stimulate αSMA, which preferentially localizes to non-proliferating fibroblasts. In accordance, PDGF-Rα more dominantly influences fibroblast proliferation at P4 than at P12. In the lung, TGFβ may overshadow the antagonistic effects of PDGF-A/PDGF-Rα signaling, enhancing αSMA-abundance in PDGF-Rα-expressing fibroblasts.     

Microenvironment-regulated gene expression,morphology, and in vivo performance of mouse pancreatic β-cells

Cell behavior is determined by intrinsic characteristics and complex interactions with microenvironments. This study demonstrated the performance of a murine pancreatic β-cell line, MIN-6, cultured on tissue-culture polystyrene (TCPS), gelatin, type I collagen, and type IV collagen dishes. MIN-6 cells aggregated as clusters on gelatin, type I collagen, and type IV collagen, which was different from the epithelial morphology of cells grown on TCPS. The diameter and survival rate of aggregated cells did not differ significantly regardless of whether the cells were grown on gelatin or type I collagen, while smaller clusters were observed on type IV collagen. Compared with the monolayers on TCPS, the clusters had a higher insulin stimulation index. The mRNA expression levels of Ins1, Pdx-1, NeuroD1 and connexin 36 were upregulated in clusters relative to monolayers. Conversely, E-cadherin and MafA were downregulated when cells were grown on type IV collagen. Monolayers or cell aggregates grown on type IV collagen were subsequently transplanted into diabetic C57BL/6 mice. Animals that received both monolayers and clusters had decreased blood glucose levels and regained body weight. However, the area under curve for the intraperitoneal glucose tolerance test showed that clusters exhibited superior in vivo performance. This study reveals that a type IV collagen substrate promotes β-cell clustering, regulates gene expression and enhances in vivo performance.     

Moderate exercise leads to decreased expression of β1 and β2 integrins on leucocytes

 Intravascular adhesion of leucocytes plays a role in the pathogenesis of acute and chronic vascular disease. Regular aerobic exercise seems to protect against vascular disease. Since leucocyte adhesion is mediated by integrins, we tested the hypothesis that surface expression of the integrin adhesive receptors LFA-1 (cd11a/cd18), MAC-1 (cd11b/cd18), gp 150/95 (cd11c/cd18), and VLA-4 (cd29/cd49) is decreased by moderate endurance exercise. Surface expression of integrins was measured by FACS analysis in 19 healthy subjects (16 males, 3 females, 36.6 ± 8.7 years, 177.1 ± 7.5 cm, 70.3 ± 8.1 kg) before and after submaximal exercise (3 h run) using monoclonal antibodies against cd11a, cd11b, cd11c, cd18, cd29 and cd49. In addition, we compared resting integrin expression in this group with a group of sedentary subjects (19 males, 6 females, 29.3 ± 5.3 years). White blood cell count increased from 5300 ml^–1 to 9740 ml^–1 during exercise (P<0.001). Nevertheless, the expression (indicated by the mean log fluorescence) of cd11a (94 ± 24 vs. 78 ± 14) and cd18 (128 ± 31 vs. 102 ± 21) on lymphocytes and of cd11a (104 ± 25 vs. 85 ± 16), cd11c (497 ± 171 vs. 408 ± 126) cd29 (109 ± 16 vs. 89 ± 16), cd49 (69± 8 vs. 54 ± 11) on monocytes was decreased after exercise (all P<0.05). In contrast, integrin expression on granulocytes was not altered by exercise. Comparison of exercising and sedentary subjects showed a significantly decreased expression of integrins in exercising subjects. Our results demonstrate that moderate exercise leads to decreased expression of integrin receptors on leucocytes. This decreased expression of adhesion molecules may result in decreased adhesion and infiltration of leucocytes into the vessel wall. This phenomenon may play a role in the beneficial effect of moderate exercise in prevention of acute and chronic vascular disease. Accepted: 18 March 1997     

Distinct patterns of expression of the β-1,4-galactosyltransferases during testicular development in the mouse

Glycosylation is one of the most important post-translational modifications and it is clear that the single step of -1,4-galactosylation is performed by a family of -1,4-galactosyltransferases (4-GalTs) and that each member of this family may play a distinct role in different tissues and cells. In this study, we characterized the gene expression of six 4-GalTs in mouse testis and analyzed the changes of galactosylation of testis glycoproteins during postnatal development. Northern blot analysis revealed that 4-GalT-I and 4-GalT-IV were expressed mainly in newborn mouse testis and that the expression of 4-GalT-II increased markedly and persisted at the highest levels in adult mouse testis. The expression of 4-GalT-III and 4-GalT-V, however, remained relatively at low levels during mouse testicular development. In contrast, the expression of 4-GalT-VI was undetectable in mouse testis. The gene expression of 4-GalT-II in mouse testis was further analyzed by in situ hybridization due to its unique expression pattern. Strong hybridization signals were detected in the seminiferous tubules and the expression varied among the different stages of spermatogenic differentiation. The distinct gene expression patterns of 4-GalTs in mouse testis could affect the differential galactosylation of testis glycoproteins, as revealed by lectin histochemistry analysis.     

Knockdown of mouse adult β-globin gene expression in MEL cells by retrovirus vector-mediated RNA interference

RNA interference (RNAi) efficiently induces sequence-specific gene silencing in mammalian cells through short interfering RNA (siRNA) of 21–23 nucleotides synthesized in vitro or expressed by DNA-based vector. However, introduction of siRNA into mammalian cells by transfection limits the application of RNAi, especially when it is necessary to generate long-term gene silencing in vivo. Virus vector-mediated RNAi provides an alternative to transfection. In the present study, we investigated such transduction system and showed that retrovirus vector-mediated RNAi can substantially down-regulate expression of mouse adult β-globin gene in MEL cells. The results suggest that retrovirus vector-delivered RNAi may find its use in functional genomics and in gene therapy.     

High-level expression of β-N-Acetylglucosaminidase BsNagZ in Pichia pastoris to obtain GlcNAc

Bioprocess and Biosystems Engineering - β-N-Acetylglucosaminidases (NAGase) can remove N-acetylglucosamine (GlcNAc) from the non-reducing end of chitin or chitosan. GlcNAc has many important...     

Developmental changes in functional expression and β-adrenergic regula-tion of If in the heart of mouse embryo

The hyperpolarization-activated current (If) plays an important role in determining the spontaneous rate of cardiac pacemaker cells. The automatic rhythmicity also exists in working cells of embryonic heart,therefore we studied developmental changes in functional expression and β-adrenergic regulation of If in embryonic mouse heart. The expression of If is high in early developmental stage (EDS) (10.5 d after coitus) ventricular myocytes, low in intermediate developmental stage (IDS) (13.5 d) atrial or ventricular myocytes and even lower in late developmental stage (LDS) (16.5 d) atrial or ventricular myocytes, indicating that these cells of the EDS embryonic heart have some properties of pacemaker cells, β-adrenergic agonist isoproterenol (ISO) stimulates If in LDS but not in EDS cardiomyocytes, indicating that the β-adrenergic regulation of If is not mature in EDS embryonic heart. But forskolin (a direct activator of adenylate cyclase) and 8-Br-cAMP (a membrane-permeable analogue of cAMP) increase the amplitude of If in EDS cells,indicating that adenylate cyclase and cAMP function fairly well at early stage of development. Furthermore,the results demonstrate that If is modulated by phosphorylation via cAMP dependent PKA both in EDS and LDS cells.     

Developmental changes in functional expression and β-adrenergic regulation of If in the heart of mouse embryo

The hyperpolarization-activated current (If) plays an important role in determining the spontaneousrate of cardiac pacemaker cells. The automatic rhythmicity also exists in working cells of embryonic heart,therefore we studied developmental changes in functional expression and β-adrenergic regulation of If inembryonic mouse heart. The expression of If is high in early developmental stage (EDS) (10.5 d after coitus)ventricular myocytes, low in intermediate developmental stage (IDS) (13.5 d) atrial or ventricular myocytesand even lower in late developmental stage (LDS) (16.5 d) atrial or ventricular myocytes, indicating thatthese cells of the EDS embryonic heart have some properties of pacemaker cells. β-adrenergic agonistisoproterenol (ISO) stimulates If in LDS but not in EDS cardiomyocytes, indicating that theβ-adrenergicregulation of If is not mature in EDS embryonic heart. But forskolin (a direct activator of adenylate cyclase)and 8-Br-cAMP (a membrane-permeable analogue of cAMP) increase the amplitude of If in EDS cells,indicating that adenylate cyclase and cAMP function fairly well at early stage of development. Furthermore,the results demonstrate that If is modulated by phosphorylation via cAMP dependent PKA both in EDSand LDS cells.     

Subcellular localization of β-catenin and cadherin expression in the cap-stage enamel organ of the mouse molar

We analyzed the subcellular distribution of -catenin in the cap-stage enamel organ and compared it with the expression of E- and P-cadherin by using confocal laser microscopy. The amounts of the molecules in the cytoplasm and the nucleus showed regional variations in the enamel organ, whereas cell surface-associated -catenin was ubiquitous. In both the enamel knot and the inner dental epithelium, -catenin was detected in the cytoplasm and in the nucleus. However, the amount of nuclear -catenin was apparently higher in the enamel knot than in the inner dental epithelium. P-cadherin also gave a stronger signal in the enamel knot than in other parts of the enamel organ. In the stellate reticulum, where E-cadherin was preferentially expressed, as well as in the cervical loop and outer dental epithelium, -catenin was localized in the cytoplasm but not in the nucleus. The nuclear localization of -catenin in the enamel knot suggests a specific activation of the canonical Wnt signaling pathway. A coincident upregulation of P-cadherin was observed in this area. Altogether, these observations suggest the possibility of a linkage between cell adhesion and Wnt signaling in the enamel knot.     

Impaired myogenic constriction of the renal afferent arteriole in a mouse model of reduced βENaC expression

Previous studies demonstrate a role for β epithelial Na(+) channel (βENaC) protein as a mediator of myogenic constriction in renal interlobar arteries. However, the importance of βENaC as a mediator of myogenic constriction in renal afferent arterioles, the primary site of development of renal vascular resistance, has not been determined. We colocalized βENaC with smooth muscle α-actin in vascular smooth muscle cells in renal arterioles using immunofluorescence. To determine the importance of βENaC in myogenic constriction in renal afferent arterioles, we used a mouse model of reduced βENaC (βENaC m/m) and examined pressure-induced constrictor responses in the isolated afferent arteriole-attached glomerulus preparation. We found that, in response to a step increase in perfusion pressure from 60 to 120 mmHg, the myogenic tone increased from 4.5 ± 3.7 to 27.3 ± 5.2% in +/+ mice. In contrast, myogenic tone failed to increase with the pressure step in m/m mice (3.9 ± 0.8 to 6.9 ± 1.4%). To determine the importance of βENaC in myogenic renal blood flow (RBF) regulation, we examined the rate of change in renal vascular resistance following a step increase in perfusion pressure in volume-expanded animals. We found that, following a step increase in pressure, the rate of myogenic correction of RBF is inhibited by 75% in βENaC m/m mice. These findings demonstrate that myogenic constriction in afferent arterioles is dependent on normal expression of βENaC.