Differential expression of CPD1 during postnatal development in the mouse cerebellum.

Several regulated mRNAs were detected by applying differential display to the mouse cerebellum during postnatal development. One cDNA fragment, referred to as CPD1 (GenBank U89345), was characterized and cloned. Northern blots showed maximum mRNA expression at postnatal day seven (P7). The mRNA encodes a protein of 260 amino acids. In situ RT-PCR showed that CPD1 is expressed mainly in granule cells and faintly in Purkinje cells. Polyclonal rabbit antibodies and oligobodies (oligonucleotide-based synthetic antibodies) revealed a protein of 34 kDa in Western blots. Immunohistochemistry showed not only marked nuclear staining but also mild cytoplasmic localization. Granule cells undergoing active division (P4) showed very little expression of CPD1 protein, which increases from P7 to P17. CPD1, affinity-purified using a chemically synthesized oligobody inhibits the activity of protein phosphatase PP2A but not protein phosphatase PP1. Differentiated PC12 cells also showed nuclear and cytoplasmic localization. Interestingly, maximal cytoplasmic CPD1/PP2A colocalization was observed near cell membrane regions that are far from growing neurites, and on growing cones. These results suggest that CPD1 might have an important role in cerebellar development.

NF-kappaB activation is involved in regulation of cystic fibrosis transmembrane conductance regulator (CFTR) by interleukin-1beta.

Interleukin-1 beta (IL-1beta) regulates the levels of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and protein in the T84 human carcinoma cell line. Here, we studied the role of the transcription factor NF-kappaB in this regulation. Initially, T84 cells were pretreated with the NF-kappaB inhibitor pyrrolidine dithiocarbamate. Cells were then stimulated with IL-1beta, and CFTR mRNA levels were determined after 4 h by Northern blot analysis. As a result of PDTC treatment, IL-1beta stimulation of CFTR mRNA was blocked. On the other hand, daunorubicin, an NF-kappaB activator, increased the steady-state levels of CFTR mRNA. Furthermore, after treatment with IL-1beta for 1 h, cytoplasmic IkappaBalpha degradation occurred simultaneously with translocation of p65 into the nucleus. The T84 cells were also transduced with an adenoviral vector expressing a dominant negative form of IkappaBalpha, which prevents IkappaBalpha phosphorylation and the subsequent nuclear translocation of NF-kappaB. After viral transduction, the cells were stimulated with IL-1beta for 4 h, and CFTR mRNA levels were measured by Northern blot analysis. The stimulation of CFTR, induced by IL-1beta, was also blocked in the presence of the dominant negative mutant. These results indicate that NF-kappaB is involved in the pathway by which IL-1beta regulates CFTR.

Interleukin-1beta regulates CFTR expression in human intestinal T84 cells.

Cystic fibrosis is an autosomal recessive genetic disease, produced by a mutation in the CFTR gene that impairs its function as a chloride channel. In this work, we have examined the effects of interleukin-1beta (IL-1beta) on the expression of CFTR in human colonic T84 cells. Treatment of T84 cells with IL-1beta (0.25 ng/ml) for 4 h resulted in an increased CFTR expression (mRNA and protein). However, higher doses of IL-1beta (1 ng/ml and over) produced inhibition of CFTR mRNA and protein expression. The protein kinase C (PKC) inhibitors H7 (50 microM) and GF109203X (1 microM) inhibited the stimulatory effect of IL-1beta. Similar effects were seen in the presence of the protein tyrosine kinase (PTK) inhibitors genistein (60 microM) and herbymicin A (2 microM). These results suggest that some PKC isoform(s) and at least a PTK might be involved in the CFTR up-regulation induced by IL-1beta. The repression of CFTR up-regulation by cycloheximide (35.5 microM) suggests the participation of a de novo synthesized protein. Results obtained by using the RNA polymerase II inhibitor DRB (78 microM), suggest that the increased mRNA levels seen after IL-1beta treatment are not due to an increased stability of the message. We conclude that the CFTR mRNA and protein levels are modulated by IL-1beta, this cytokine being the first extracellular protein known to up-regulate CFTR gene expression.

Identification by differential display of a mRNA specifically induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in T84 human colon carcinoma cells.

12-o-Tetradecanoylphorbol-13-Acetate (TPA) down-regulates the expression of the gene responsible for cystic fibrosis (CFTR). To understand the mechanism by which TPA down-regulates CFTR, we decided to study genes specifically induced by this phorbol ester in T84 human colon carcinoma cells, which highly express CFTR, using differential display. Several strategies that allowed us to overcome false-positive reactions in differential displays are described. We have detected different cDNAs obtained from mRNAs specifically induced by TPA. A cDNA fragment corresponding to a mRNA of approximately 2.2 kb was sequenced. Part of this sequence has been reported by others in GenBank and corresponds to a cDNA from a human lung library. The function is unknown and does not have any significant homology with other sequences. It is expressed after two hrs in T84 cells treated with TPA, reaching a maximum response by four hrs. The dose-response curve shows increased mRNA levels starting at 10 ng/ml of TPA and reaching a maximum by 50 ng/ml TPA (10-fold stimulation over control). This mRNA shows a rapid and large response to TPA and it might be involved in the differentiation of T84 cells induced by TPA.

Microcephaly, characteristic facies, joint abnormalities, and deficient leucocyte chemotaxis: a further case of the syndrome of Say et al.

We report on a 13 year old boy with microcephaly, sloping forehead, prominent nose, scoliosis, and flexion contractures involving the elbows and knees. The patient showed severe mental and growth retardation. Since birth and up to the present he has suffered from multiple and varied infections. Immunological studies showed a marked decrease in leucocyte chemotaxis. Clinical and laboratory findings confirm the similarity of this case to the two brothers described by Say et al. We have not found any descriptions of similar patients. The purpose of this paper is to contribute to the phenotypic delineation of this syndrome and to highlight the need for immunological investigation in patients with multiple congenital malformations.

Developmental regulation of Thy 1.2 rate of synthesis in the mouse cerebellum.

Thy 1.2 is a well-known major cell surface glycoprotein of the central nervous system (CNS). However, the regulation of the expression of this molecule as well as its function are yet to be determined. To approach these problems we studied the synthesis of the molecule in the developing cerebellum of wild-type and staggerer mutant mice. We found the appearance of a [35S]-methionine-labeled band detected with specific Sepharose 4B-bound monoclonal antibodies (Mabs). The Thy 1.2 activity increases progressively from postnatal day 9 (P9), reaching the highest rate at P12, subsequently decreasing sharply at P13, and remaining relatively low up to P16 in the wild type. Comparison of these data to the rates of total protein synthesis reveals a selective developmental regulation of Thy 1.2 expression, at least at the translational level. This correlates quite well with the timing of synaptic stabilization between parallel fibers and Purkinje cell dendritic spines. Furthermore, at P12 Thy 1.2 protein is preferentially located in the synaptosomal fraction. The parallel fiber:Purkinje cell synapsis is not stabilized in the staggerer mutant mouse. At P12 Thy 1.2 synthesis is 30% of the wild type, indicating that the translational regulation of Thy 1.2 is altered in the staggerer mutation.

The kallikrein-kinin system in the colon and lung fluid from a cribriform degeneration (cri) mutant mouse.

The cribriform degeneration (cri) mutant mouse was widely studied in regard to the electrolyte and kallikrein metabolism because of its potentiality as a cystic fibrosis (CF) genetic animal model. In this paper the activity of the kallikrein-kinin system, and the kininase activity and glycoproteins concentration in colon and pulmonary lavage fluid (PLF) in homozygous mutant (cri/cri) and control sibling mice are described. The mutant mice showed a diminished kininogenase and kininase activity and glycoproteins concentrations in both studied organs. It is concluded that a kallikrein-kinin system alteration could be responsible of the cri/cri electrolyte defect.

Glucagon secretion and alpha-cell sensitivity to somatostatin in genetically diabetic mice C57BL/KsJ-mdb.

In previous studies in C57BL/KsJ mdb/mdb mice, we observed alterations in glucose-induced insulin secretion in vitro, and a defective inhibitory effect of somatostatin on insulin secretion. In this work we studied glucagon secretion patterns under arginine-glucose stimulation, in perifused pancreatic slices from genetically diabetic mice aged 20 to 90 days. We also explored whether alpha-cells present a diminished sensitivity to somatostatin. Results showed that: a) in mdb/mdb mice aged 20 to 90 days, glucagon secretion patterns exhibited basal hypersecretion and a diminished first peak; b) somatostatin inhibited stimulated-glucagon secretion below baseline values in mdb/mdb mice aged 20 to 30 days. In later stages (40 to 90 days), somatostatin exerted a lower inhibitory effect since glucagon levels remain above basal values. This could indicate a progressive impairment in alpha-cell sensitivity to somatostatin, as it was previously observed in beta-cells.

Glucagon secretion and alpha-cell sensitivity to somatostatin in genetically diabetic mice C57BL/KsJ-mdb.

In previous studies in C57BL/KsJ mdb/mdb mice, we observed alterations in glucose-induced insulin secretion in vitro, and a defective inhibitory effect of somatostatin on insulin secretion. In this work we studied glucagon secretion patterns under arginine-glucose stimulation, in perifused pancreatic slices from genetically diabetic mice aged 20 to 90 days. We also explored whether alpha-cells present a diminished sensitivity to somatostatin. Results showed that: a) in mdb/mdb mice aged 20 to 90 days, glucagon secretion patterns exhibited basal hypersecretion and a diminished first peak; b) somatostatin inhibited stimulated-glucagon secretion below baseline values in mdb/mdb mice aged 20 to 30 days. In later stages (40 to 90 days), somatostatin exerted a lower inhibitory effect since glucagon levels remain above basal values. This could indicate a progressive impairment in alpha-cell sensitivity to somatostatin, as it was previously observed in beta-cells.

The kallikrein kinin system in pulmonary lavage fluid of reserpinized rats.

Kallikrein content in lavage of the respiratory tract was determined by a specific RIA. The results show an immunological identity of the lung kallikrein with the standard rat urinary kallikrein. The levels of immunoreactive lung fluid kallikrein were significantly lower in rats injected with reserpine.

First phase of insulin secretion stimulated by glucose plus theophylline and inhibitory effect of somatostatin in genetically diabetic mice (C57BL/KsJ-mdb).

In a previous study in C57BL/KsJ mdb/mdb mice aged 4 to 12 days we observed a diminished first phase of glucose-induced insulin secretion in vitro, and alterations in the inhibitory effect of somatostatin on insulin secretion. This study explores, using perifused pancreatic slices, whether the reduced B-cell responsiveness to somatostatin in mdb/mdb mice can be overcome upon induction of a biphasic insulin release by using theophylline. Under these conditions our results show: (1) in mdb/mdb mice aged 4 to 6 days, the restoration of the first peak of insulin secretion overcomes the reduced B-cell sensitivity to somatostatin; and (2) in mdb/mdb mice aged 7 to 12 days, the addition of theophylline only causes a partial restoration of B-cell responsiveness to somatostatin, suggesting that other mechanisms could be involved in the progressive impairement of B-cell sensitivity to somatostatin inhibitory effect.

Kallikrein-like esteroprotease activity and kininase activity in submandibular gland from a mutant mouse with human cystic fibrosis like alterations.

The submandibular gland of cri/cri and control mice were compared for their activity of glandular kallikrein like esteroprotease and kininase. Esteroprotease activity is significantly reduced in cri/cri mice with respect to control, with an increased kininase activity in cri/cri mice. Since previous work showed an electrolyte abnormality in the salivary glands of this mutant mouse (1) a possible relationship between this alteration with the low activity of cellular esteroprotease and the high kininase activity is suggested.

In vitro capacitation of spermatozoa in different strains of mice: the acrosome reaction

La reacción acrosómica normal se midió como parámetro indirecto para determinar la capacitación in vitro de espermatozoides en diferentes cepas de ratones. Se utilizaron tres cepas: FI (BALB/c x C57BL/6J-bg). C57BL/6J-bg y C57BL/6J-small. Se hallaron diferencias significativas entre las tres cepas (p<0,05). Se realizó la fertilización in vitro utilizando una concentración de espermatozoides relativamente baja (10 espermatozoide/ml), a fin de averiguar si las diferencias en el porcentaje de espermatozoides que han sufrido reacción acrosómica a los 120min de incubación se correlaciona con las diferencias en las tasas de fertilidad. Se encontraron diferencias en la fertilización (p<0,05), pero sólo en algunas cepas ésto podría ser debido a diferencias en el porcentaje de espermatozoides sin acrosomas (capacitación). Cuando la concentración de espermatozoides fue aumentada a 10 espermatozoide/ml, las diferencias desaparecieron (p>0,05). No se detectaron diferencias entre las cepas utilizadas con respecto a la motilidad o hiperactividad después de la capacitación. La misma cinética de reacción acrosómica se halló en las cepas C57BL/6J-bg y C57BL/6J-small, pero fue diferente en el híbrido. También se observó un alto número (4%) (p<0,05) de espermatozoides morfológicamente anormales en las cepas endocriadas, con respecto a la híbrida (1%)

In vitro capacitation of spermatozoa in different strains of mice: the acrosome reaction

La reacción acrosómica normal se midió como parámetro indirecto para determinar la capacitación in vitro de espermatozoides en diferentes cepas de ratones. Se utilizaron tres cepas: FI (BALB/c x C57BL/6J-bg). C57BL/6J-bg y C57BL/6J-small. Se hallaron diferencias significativas entre las tres cepas (p<0,05). Se realizó la fertilización in vitro utilizando una concentración de espermatozoides relativamente baja (10 espermatozoide/ml), a fin de averiguar si las diferencias en el porcentaje de espermatozoides que han sufrido reacción acrosómica a los 120min de incubación se correlaciona con las diferencias en las tasas de fertilidad. Se encontraron diferencias en la fertilización (p<0,05), pero sólo en algunas cepas ésto podría ser debido a diferencias en el porcentaje de espermatozoides sin acrosomas (capacitación). Cuando la concentración de espermatozoides fue aumentada a 10 espermatozoide/ml, las diferencias desaparecieron (p>0,05). No se detectaron diferencias entre las cepas utilizadas con respecto a la motilidad o hiperactividad después de la capacitación. La misma cinética de reacción acrosómica se halló en las cepas C57BL/6J-bg y C57BL/6J-small, pero fue diferente en el híbrido. También se observó un alto número (4%) (p<0,05) de espermatozoides morfológicamente anormales en las cepas endocriadas, con respecto a la híbrida (1%) (AU)

Increase in insulin secretion induced by plasma from mice injected with allogeneic lymphocytes.

Plasma from BALB/c mice bled 90 minutes after allogeneic lymphocyte injection significantly rises glucose induced insulin secretion. This rise is observed in pancreas either from non-treated or from allogeneized mice. This rise is time and dose-dependent. An 1/40 dilution is enough to bring about a significant increase on insulin secretion. This effect is seen when mice are bled between 60 and 180 minutes after injection with a maximum effect at 90-120 minutes. Plasma from BALB/c mice injected with C57BL/6 J lymphocytes rises insulin secretion from BALB/c, C57BL/6 J, C3h and C57BL/KsJ mice pancreas. Plasma from streptozotocin diabetic BALB/c mice and from genetically diabetic C57BL/KsJ mdb-mdb mice injected with allogeneic lymphocytes stimulates glucose induced insulin secretion but to a lesser extent than plasma from normal non-diabetic mice does.

Bethanechol-induced restricted stimulation of pancreatic juice secretion in mice.

Flow and enzymatic output from mouse pancreas were studied "in vivo" under bethanechol stimulation. A biphasic dose response curve was found in both, enzymatic output and juice flow, implicating that exocrine pancreatic juice secretion was also involved in restricted stimulation phenomenon.