Attenuated airway hyperresponsiveness and mucus secretion in HDM-exposed Igf1r-deficient mice.

BACKGROUND: Asthma is a common chronic lung disease characterized by airflow obstruction, airway hyperresponsiveness (AHR), and airway inflammation. IGFs have been reported to play a role in asthma, but little is known about how the insulin-like growth factor 1 receptor (IGF1R) affects asthma pathobiology. METHODS: Female Igf1r-deficient and control mice were intranasally challenged with house dust mite (HDM) extract or PBS five days per week for four weeks. Lung function measurements, and bronchoalveolar lavage fluid (BALF), serum, and lungs were collected on day 28 for further cellular, histological, and molecular analysis. RESULTS: Following HDM exposure, the control mice responded with a marked AHR and airway inflammation. The Igf1r-deficient mice exhibited an increased expression of the IGF system and surfactant genes, which were decreased in a similar manner for control and Igf1r-deficient mice after HDM exposure. On the other hand, the Igf1r-deficient mice exhibited no AHR, and a selective decrease in blood and BALF eosinophils, lung Il13 levels, collagen, and smooth muscle, as well as a significant depletion of goblet cell metaplasia and mucus secretion markers after HDM exposure. The Igf1r-deficient mice displayed a distinctly thinner epithelial layer than control mice, but this was not altered by HDM. CONCLUSIONS: Herein, we demonstrate by the first time that the Igf1r plays an important role in murine asthma, mediating both AHR and mucus secretion after HDM exposure. Thus, our study identifies IGF1R as a potential therapeutic target, not only for asthma but also for hypersecretory airway diseases.

Luminescent pentafluorophenyl-cycloplatinated complexes: synthesis, characterization, photophysics, cytotoxicity and cellular imaging.

Luminescent mono(pentafluorophenyl) cycloplatinated complexes [Pt(C^N-κC,N)(HC^N-κN)(C6F5)] [HC^N = Hthpy (2-(2-thienyl)pyridine) 2a, Hbt (2-phenylbenzothiazole) 2b, Hpq (2-phenylquinoline) 2c] have been prepared by C­H activation of a HC^N ligand in the corresponding [Pt(HC^N-κN)2(C6F5)2] (1a, 1b, 1c) complexes. Complexes 2 evolve in DMSO solution into solvate complexes and we present here successful routes for the synthesis of [Pt(C^N)(C6F5)(DMSO)] (C^N = thpy 3a, bt 3b). They have been fully characterized (X-ray for 1a, 1c, 2b, 3a and 3b), their electronic absorption and emission properties have been investigated and DFT and TD-DFT calculations for 1a, 1c, 2b and 3a have been carried out. Complexes 3a, 3b and [Pt(ppy)(C6F5)(DMSO)] 4 (Hppy = 2-phenylpyridine) show remarkable stability in a mixed DMSO-cellular medium and their cytotoxicity towards the human lung tumor (A549) and bronchial epithelial non-tumorigenic (NL20) cell lines has been evaluated by MTS assays. Their cellular localization in A549 and NL20 human cells and in mouse embryonic fibroblasts obtained from lungs (LMEFs) has also been investigated by fluorescence microscopy.

Delayed inner ear maturation and neuronal loss in postnatal Igf-1-deficient mice.

Insulin-like growth factor-1 (IGF-1) has been shown to play a key role during embryonic and postnatal development of the CNS, but its effect on a sensory organ has not been studied in vivo. Therefore, we examined cochlear growth, differentiation, and maturation in Igf-1 gene knock-out mice at postnatal days 5 (P5), P8, and P20 by using stereological methods and immunohistochemistry. Mutant mice showed reduction in size of the cochlea and cochlear ganglion. An immature tectorial membrane and a significant decrease in the number and size of auditory neurons were also evident at P20. IGF-1-deficient cochlear neurons showed increased caspase-3-mediated apoptosis, along with aberrant expression of the early neural markers nestin and Islet 1/2. Cochlear ganglion and fibers innervating the sensory cells of the organ of Corti presented decreased levels of neurofilament and myelin P(0) in P20 mouse mutants. In addition, an abnormal synaptophysin expression in the somata of cochlear ganglion neurons and sensory hair cells suggested the persistence of an immature pattern of synapses distribution in the organ of Corti of these animals. These results demonstrate that lack of IGF-1 in mice severely affects postnatal survival, differentiation, and maturation of the cochlear ganglion cells and causes abnormal innervation of the sensory cells in the organ of Corti.

A large upstream region is not necessary for gene expression or hypersensitive site formation at the mouse beta -globin locus.

Developmental expression at the beta-globin locus is regulated in part by the locus control region, a region upstream of the genes containing at least five major DNase I hypersensitive sites (HSs) in mammalian erythrocytes. Sequences farther 5' of these HSs are conserved in mouse and human, and both loci are embedded within a cluster of functional odorant receptor genes. In humans, distant upstream sequences have been implicated in regulation of the beta-globin genes. In this study, the role of the 5'-most HSs and their adjacent sequence was investigated by deletion of an 11-kb region from the mouse locus, including 5'HS 4.2, 5'HS 5, 5'HS 6, and the 5'beta1 odorant receptor gene. Mice that were homozygous for this deletion were fully viable, and no significant effect on adult beta-globin gene expression was seen. 5'HSs 1-4, which are located downstream of the deletion, were still present in the mutant mice. In addition, two new upstream HSs, HS -60.7 and HS -62.5, were found in erythroid tissue of both wild-type and mutant mice. Therefore, although the possibility of a minor role still exists, neither the HSs nor the other regions deleted in this study are essential for beta-globin gene expression, and it is unlikely that chromatin structure is affected either upstream or downstream of the deletion. This is the largest deletion at the mouse locus control region to show no apparent phenotype, and focuses attention on the possible contribution of sequences even farther upstream.

Regulation of cell fate decision of undifferentiated spermatogonia by GDNF.

The molecular control of self-renewal and differentiation of stem cells has remained enigmatic. Transgenic loss-of-function and overexpression models now show that the dosage of glial cell line-derived neurotrophic factor (GDNF), produced by Sertoli cells, regulates cell fate decisions of undifferentiated spermatogonial cells that include the stem cells for spermatogenesis. Gene-targeted mice with one GDNF-null allele show depletion of stem cell reserves, whereas mice overexpressing GDNF show accumulation of undifferentiated spermatogonia. They are unable to respond properly to differentiation signals and undergo apoptosis upon retinoic acid treatment. Nonmetastatic testicular tumors are regularly formed in older GDNF-overexpressing mice. Thus, GDNF contributes to paracrine regulation of spermatogonial self-renewal and differentiation.

Defects in inositol 1,4,5-trisphosphate receptor expression, Ca(2+) signaling, and insulin secretion in the anx7(+/-) knockout mouse.

The mammalian anx7 gene codes for a Ca(2+)-activated GTPase, which supports Ca(2+)/GTP-dependent secretion events and Ca(2+) channel activities in vitro and in vivo. To test whether anx7 might be involved in Ca(2+) signaling in secreting pancreatic beta cells, we knocked out the anx7 gene in the mouse and tested the insulin-secretory properties of the beta cells. The nullizygous anx7 (-/-) phenotype is lethal at embryonic day 10 because of cerebral hemorrhage. However, the heterozygous anx7 (+/-) mouse, although expressing only low levels of ANX7 protein, is viable and fertile. The anx7 (+/-) phenotype is associated with a substantial defect in insulin secretion, although the insulin content of the islets, is 8- to 10-fold higher in the mutants than in the normal littermate control. We infer from electrophysiological studies that both glucose-stimulated secretion and voltage-dependent Ca(2+) channel functions are normal. However, electrooptical recordings indicate that the (+/-) mutation has caused a change in the ability of inositol 1,4,5-trisphosphate (IP(3))-generating agonists to release intracellular calcium. The principle molecular consequence of lower anx7 expression is a profound reduction in IP(3) receptor expression and function in pancreatic islets. The profound increase in islets, beta cell number, and size may be a means of compensating for less efficient insulin secretion by individual defective pancreatic beta cells. This is a direct demonstration of a connection between glucose-activated insulin secretion and Ca(2+) signaling through IP(3)-sensitive Ca(2+) stores.

Morphological alterations in the peripheral and central nervous systems of mice lacking glial cell line-derived neurotrophic factor (GDNF): immunohistochemical studies.

Glial cell line-derived neurotrophic factor (GDNF) is a member of the TGF-beta superfamily of growth factors with neurotrophic activity on midbrain dopaminergic neurons and on developing and mature motoneurons of the brainstem and spinal cord. To investigate the extent of GDNF dependency of central and peripheral nervous structures during development, we have performed an immunohistochemical analysis of sections from the whole head including brain, peripheral ganglia, developing teeth and tongue, as well as intestines, in mutant mice lacking a part of the third exon that encodes the GDNF protein. As described previously, these null-mutated mice lack most of the enteric nerve plexus and are subject to agenesis or severe dysgenesis of the kidneys. In the present communication, we examined the development of vibrissae and incisor and molar teeth, as well as the innervation of these structures, and found no differences between null-mutated and control mice. A decrease in the immunohistochemical labeling intensity with tyrosine hydroxylase was observed in the superior cervical ganglion (SCG), as well as in the pontine nucleus locus coeruleus, and the sympathetic innervation of blood vessels and glands in the head was significantly decreased. None of the brain nuclei studied exhibited any significant decreases in the total number of neurons, but the packing density of neurons in the nucleus locus coeruleus was decreased. These data indicate that GDNF might be one neurotrophic factor that contributes to the development of central and peripheral noradrenergic neurons.

Defects in enteric innervation and kidney development in mice lacking GDNF.

Glial-lial-cell-line-derived neurotrophic factor (GDNF) has been isolated as neurotrophic factor for midbrain dopaminergic neurons. Because of its neurotrophic activity on a wide range of neuronal populations in vitro and in vivo, GDNF is being considered as a potential therapeutic agent for neuronal disorders. During mammalian development, it is expressed not only in the nervous system, but also very prominently in the metanephric kidney and the gastrointestinal tract, suggesting possible functions during organogenesis. We have investigated the role of GDNF during development by generating a null mutation in the murine GDNF locus, and found that mutant mice show kidney agenesis or dysgenesis and defective enteric innervation. We demonstrate that GDNF induces ureter bud formation and branching during metanephros development, and is essential for proper innervation of the gastrointestinal tract.

Efficient in vivo manipulation of mouse genomic sequences at the zygote stage.

We describe a transgenic mouse line carrying the cre transgene under the control of the adenovirus EIIa promoter that targets expression of the Cre recombinase to the early mouse embryo. To assess the ability of this recombinase to excise loxP-flanked DNA sequences at early stages of development, we bred EIIa-cre transgenic mice to two different mouse lines carrying loxP-flanked target sequences: (i) a strain with a single gene-targeted neomycin resistance gene flanked by 1oxP sites and (ii) a transgenic line carrying multiple transgene copies with internal loxP sites. Mating either of these loxP-carrying mouse lines to EIIa-cre mice resulted in first generation progeny in which the loxP-flanked sequences had been efficiently deleted from all tissues tested, including the germ cells. Interbreeding of these first generation progeny resulted in efficient germ-line transmission of the deletion to subsequent generations. These results demonstrate a method by which loxP-flanked DNA sequences can be efficiently deleted in the early mouse embryo. Potential applications of this approach are discussed, including reduction of multicopy transgene loci to produce single-copy transgenic lines and introduction of a variety of subtle mutations into the line.

An apoptotic defect in lens differentiation caused by human p53 is rescued by a mutant allele.

If deprived of wild-type p53 function, the body loses a guardian that protects against cancer. Restoration of p53 function has, therefore, been proposed as a means of counteracting oncogenesis. This concept of therapy requires prior knowledge with regard to proper balance of p53 function in a given target tissue. We have addressed this problem by targeting expression of the wild-type human p53 gene to the lens, a tissue entirely composed of epithelial cells that differentiate into elongated fiber cells. Transgenic mice expressing wild-type human p53 develop microphthalmia as a result of a defect in fiber formation that sets in shortly after birth. We see apoptotic cells that fail to undergo proper differentiation. In an effort to directly link the observed lens phenotype to the activity of the wild-type human p53 transgene, we also generated mice expressing a mutant human p53 allele that lacks wild-type function. A normal lens phenotype is restored in double transgenic animals that carry both wild-type and mutant human p53 alleles. Our study highlights the difficulties that can arise if p53 levels are improperly balanced in a differentiating tissue.

Expression patterns of the murine LIM class homeobox gene lim1 in the developing brain and excretory system.

We report the cloning, sequence analysis, and developmental expression pattern of lim1, a member of the LIM class homeobox gene family in the mouse. lim1 cDNA encodes a predicted 406 amino acid protein that is 93% identical with the product of the Xenopus LIM class homeobox gene Xlim1. We have characterized lim1 expression from day 8.5 post coitum onward. Northern blot analysis of RNA transcripts indicates that lim1 is expressed both during embryogenesis and in the adult brain. Analysis by whole-mount and section in situ hybridization shows lim1 expression in the central nervous system from the telencephalon through the spinal cord and in the developing excretory system including pronephric region, mesonephros, nephric duct, and metanephros. In the metanephros, lim1 is strongly expressed in renal vesicles and S-shaped bodies, and transcripts are also detected in the ureteric branches.

Timing of SV40 oncogene activation by site-specific recombination determines subsequent tumor progression during murine lens development.

We generated mice that carry copies of a dormant transgene encoding the SV40 tumor antigens. The transgenes are specifically targeted to the lens and contain features that render their expression dependent on the action of Cre, a site-specific bacteriophage DNA recombinase. Timing of oncogene activation was controlled by making Cre available either prior to, or coincident with, the onset of primary fiber differentiation in the embryonic lens vesicle. Early expression of Cre resulted in oncogene activation in undifferentiated lens epithelial cells that rapidly proliferated inside the lens capsule. By contrast, when Cre accumulation was delayed to coincide with the onset of primary lens fiber differentiation, SV40 oncogenes were activated in cells that had begun to elongate and to accumulate lens-specific crystallins. During subsequent proliferation inside the lens capsule, transformed progeny cells maintained the profile of fiber differentiation that their parent cells had acquired at the time of oncogenic conversion. Developing lens tumors were confined within the capsule of the embryonic lens. However, if the capsule was perforated in an embryonic eye in organ culture, cells rapidly grew out while still maintaining features of differentiation. Our findings show that the differentiated state of the primary target cells is an important parameter of subsequent lens oncogenesis, and that an intact lens capsule can restrict invasive neoplastic growth.

Ketomethylenebestatin: synthesis and aminopeptidase inhibition.

The synthesis of (6R,5S,2RS)-6-amino-5-hydroxy-2-isobutyl-4-oxo-7- phenylheptanoic acid (9), a carbaanalogue of the aminopeptidase (AP) inhibitor bestatin (1) is described. This synthesis was carried out by a malonic ester alkylation with the suitably protected halomethyl ketone of (2S,3R)-AHPBA*), followed by a second alkylation with isobutyl bromide of the resulting 4-ketodiester, and subsequent decarboxylation and deprotection. The inhibitory potencies of the 1:1 diastereomeric mixture 9 against AP-B, AP-M and Leu-AP were approximately 10-fold lower than those of bestatin.

Transcript levels of thymosin beta 4, an actin-sequestering peptide, in cell proliferation.

Thymosin beta 4 (beta 4) is an ubiquitous 5-kDa peptide that has been identified as an actin-sequestering peptide. In this work, Northern blot analysis was used to study the beta 4 mRNA levels during the cell cycle of rat thymocytes and hepatocytes as well as in human lymphocytes from patients with leukemia. beta 4 mRNA was found in all the stages of thymocyte and hepatocyte cell cycle, showing an increase in the S-phase which was maintained during the G2 and M phases. Incubation of splenic T-cells with concanavalin A, phorbol myristate acetate or the ionophore A23187 lead to a similar increase of beta 4 transcript during the S-phase. The increase in beta 4 mRNA observed in the G2/M boundary of the cell cycle, together with its ability to inhibit actin polymerization, suggests a possible role of beta 4 in the the morphological changes and actin redistribution occurring during the cytokinesis.

Synthesis of 5'-N-(alpha-amino-beta-mercaptoacyl)amino-5'-deoxynucleosides as potential antiviral compounds.

5'-N-(alpha-Amino-beta-mercaptoacyl)amino-5'-deoxynucleosides have been synthesized by coupling of N-formylthiazolidines derived from D- and L-penicillamine, and D- and L-cysteine to 5'-amino-5'-deoxynucleosides using the DCC/HOSu method, followed by deprotection in N HCl in MeOH under argon. Although these compounds were designed as potential anti-HIV-1 agents, none of them showed anti-HIV-1 activity in MT-4 cells or antiviral effect against some other viruses, at concentrations below the cytotoxicity threshold.

The levels of cytochrome c oxidase subunit II mRNA change during the rat T-cell development.

Steady-state levels of the mtRNA encoding the subunit II of the cytochrome c oxidase were determined at several stages of rat T-cell differentiation. Our results showed that its abundance was higher in cells representing the early steps of T-cell development, decreasing in mature T-cells. The possible implications of these findings are discussed.

Thymosin-beta 4 gene. Preliminary characterization and expression in tissues, thymic cells, and lymphocytes.

A cDNA for rat thymosin-beta 4 was used to investigate the expression of this gene in different tissues, thymic cells, and lymphocytes. Hybridization analysis of total RNA from 13 rat tissues demonstrated the presence of an 800 nucleotides-long mRNA in all the tissues surveyed, with the highest levels in spleen, thymus, and lung. Examination of thymic cells showed that the thymosin-beta 4 gene is predominantly expressed in thymocytes. The thymosin-beta 4 mRNA was also studied in Ig+ and Ig- lymphocytes, being fourfold more abundant in Ig- than Ig+ splenic lymphocytes, whereas similar levels were found in both types of blood cells. The analysis of RNA from T cells at different maturation stages evidenced slight differences in their thymosin-beta 4 mRNA content, indicating that thymosin-beta 4 gene expression is not clearly related to the differentiation process of T cells. All these results do not support the roles for thymosin-beta 4 in cellular immunity and differentiation of lymphoid cells, suggesting a more general function for this peptide. Preliminary characterization of the human beta 4 gene by restriction analysis disclosed a complicated pattern consistent with multiple genes and/or introns. The analysis of genomic DNA from different species ranging from humans to Escherichia coli showed that this gene is only highly conserved in mammals.

The expression of prothymosin alpha gene in T lymphocytes and leukemic lymphoid cells is tied to lymphocyte proliferation.

We isolated the cDNA for human prothymosin alpha (ProT alpha) from a human peripheral T-cell library using two synthetic oligonucleotides as probes. Hybridization studies with this cDNA showed that the ProT alpha mRNA is detectable in all the rat tissues studied but is most abundant in thymus and within this gland mainly synthesized by thymocytes. In the T-cell lineage, its expression is higher in proliferative immature thymocytes than in pre- and post-thymic T lymphocytes. A quite similar pattern was obtained with the proliferation-related protein proliferating cell nuclear antigen/cyclin. These data show that ProT alpha mRNA levels change with the maturation stage of T-cells. Moreover, the amount of ProT alpha transcript is increased in lymphocytes from human patients with leukemias. Our findings indicate a role for ProT alpha linked to lymphocyte proliferation.

Synthesis and antiviral activity of 5'-O-(substituted) sulfamoyl pyrimidine nucleosides.

5'-O-[N-(Aminoacyl or isobutyryl)sulfamoyl]uridines 4a-e, 5a-e and 5'-O-[N-(isopropyl)sulfamoyl]cytidines 7-9 have been synthesized and tested against herpes simplex virus type 2. Condensation of 2',3'-O-isopropylidene-5'-O-sulfamoyluridine with the N-hydroxysuccinimide esters of Boc-Gly, Boc-L-Ala, Boc-D-Ala and Boc-L-Phe, gave 4a-d which, on deprotection under acidic conditions, provided 5a-d. A similar condensation of 2',3'-di-O-acetyl-5'-O-sulfamoyluridine with the N-hydroxysuccinimide ester of isobutyric acid afforded 4e which on deacylation led to 5e. 5'-O-[N-(Isopropyl)sulfamoyl]-2',3'-O-isopropylidenecytidine (7) was prepared by reaction of 2',3'-O-isopropylidene-4-N-[(dimethylamino)methylene] cytidine with N-isopropylsulfamoyl chloride. Acidic hydrolysis of 7 provided 8 which, upon acetylation, gave the corresponding 2',3'-di-O-acetyl derivative 9. Compounds 7-9 show antiviral effect. Structure-activity relationships are discussed.