Transponder-induced sarcoma in the heterozygous p53+/- mouse.

Heterozygous p53+/- transgenic mice are being studied for utility as a short-term alternative model to the 2-yr rodent carcinogenicity bioassay. During a 26-wk study to assess the potential carcinogenicity of oxymetholone using p-cresidine as a positive control, glass/polypropylene microchips (radio transponder identification devices) were subcutaneously implanted into male and female p53+/- mice. During week 15, the first palpable mass was clinically observed at an implant site. This rapidly growing mass virtually quadrupled in size by week 25. Microscopic examination of all implant sites revealed that 18 of 177 animals had a subcutaneous histologically malignant sarcoma. The neoplasms were characterized as undifferentiated sarcomas unrelated to drug treatment, as indicated by the relatively even distribution among dose groups, including controls. An unusual preneoplastic mesenchymal change characterized by the term "mesenchymal dysplasia" was present in most groups and was considered to be a prodromal change to sarcoma development. The tumors were observed to arise from dysplastic mesenchymal tissue that developed within the tissue capsule surrounding the transponder. The preneoplastic changes, including mesenchymal dysplasia, appeared to arise at the transponder's plastic anchoring barb and then progressed as a neoplasm to eventually surround the entire microchip. Capsule membrane endothelialization, inflammation, mesenchymal basophilia and dysplasia, and sarcoma were considered unequivocal preneoplastic/neoplastic responses to the transponder and were not related to treatment with either oxymetholone or p-cresidine.     

Loss of P53 heterozygosity is not responsible for the small colony thymidine kinase mutant phenotype in L5178Y mouse lymphoma cells

The mouse lymphoma L5178Y Tk+/- 3.7.2C assay is a well-characterized in vitro system used for the study of somatic cell mutation. It was determined that this cell line has a heterozygous mutation in exon 5 of Trp53. Based on this assumption that the cell line is heterozygous for the Trp53 gene, it was postulated that the small colony thymidine kinase (Tk) mutant phenotype may be due to a newly induced mutation/deletion in both the Trp53 and Tk1 alleles. The resultant Tk-/- mutants would also be Trp53+/0 or Trp53+/+ and would lose their ability to grow at normal rates. Subsequently, we published our evaluation of the Trp53 status in L5178Y cells. This analysis included sequencing of Trp53 exon 4 and determined that the mouse lymphoma cell line has a mutation in both of the Trp53 alleles and, therefore, no wild-type Trp53 allele in either Tk+/- cells or Tk-/- mutants. Because the cells have no wild-type Trp53, it is not possible that the small colony phenotype results from a newly induced loss of both functional Trp53 and Tk. To determine whether small colonies might, however, include the deletion of both Trp53 and Tk we evaluated, using microsatellite marker analysis, a series of small colony mutants. We also utilized in situ hybridization to determine that the Trp53 alleles are, in fact, in their normal chromosome 11 location in Tk+/- 3.7.2C mouse lymphoma cells. From all of these analyses we can conclude that the small colony mutant phenotype is not caused by deletion of both Trp53 and Tk1.     

Na+ channel expression and neuronal function in the Na+/H+ exchanger 1 null mutant mouse

Mice lacking Na(+)/H(+) exchanger 1 (NHE1) suffer from recurrent seizures and die early postnatally. Although the mechanisms for seizures are not well established, our previous electrophysiological work has shown that neuronal excitability and Na(+) current density are increased in hippocampal CA1 neurons of these mutant mice. However, it is unknown whether this increased density is related to altered expression or functional regulation of Na(+) channels. In this work, we asked three questions: is the increased excitability limited to CA1 neurons, is the increased Na(+) current density related to an increased Na(+) channel expression, and, if so, which Na(+) channel subtype(s) is upregulated? Using neurophysiological, autoradiographic, and immunoblotting techniques, we showed that both CA1 and cortical neurons have an increase in membrane excitability and Na(+) current density; Na(+) channel density is selectively upregulated in the hippocampus and cortex (P < 0.05); and Na(+) channel subtype I is significantly increased in the hippocampus and Na(+) channel subtype II is increased in the cortex. Our results demonstrate that mice lacking NHE1 upregulate their Na(+) channel expression in the hippocampal and cortical regions selectively; this leads to an increase in Na(+) current density and membrane excitability. We speculate that neuronal overexcitability due to Na(+) channel upregulation in the hippocampus and cortex forms the basis of epileptic seizures in NHE1 mutant mice.     

The mutant genotype is the main determinant of the metabolic phenotype in phenylalanine hydroxylase deficiency.

Phenylketonuria and mild hyperphenylalaninemias are allelic disorders caused by mutations in the phenylalanine hydroxylase (PAH) gene. Following identification of the disease-causing mutation in 11 PAH-deficient patients, we tested the activity of the mutant gene products in an eukaryotic expression system. Two mutations markedly reduced PAH activity (A259V and L333F), one mutation mildly altered the enzyme activity (E390G), while the majority of mutant genotypes reduced the in vitro expression of PAH activity to 15-30% of controls. Comparing the predicted residual activity derived from expression studies to the clinical phenotypes of our PAH-deficient patients, we found that homozygosity for the L333F and E390G mutations resulted in severe and mild PAH deficiencies, respectively, both in vivo and in vitro, while compound heterozygosity (L333F/E390G) resulted in an intermediate dietary tolerance. Similarly, in vitro expression studies largely predicted dietary tolerance in compound heterozygotes for the A259V/IVS12nt1 (typical PKU), A259V/A403V, G218V/I65T, and G218V/R158Q mutations (mild variants). Taken together, these results support the view that expression studies are useful in predicting residual enzyme activity and that the mutant genotype at the PAH locus is the major determinant of metabolic phenotype in hyperphenylalaninemias.     

Unresponsiveness of CD4-8+/- thymocytes to lectin stimulation in LEC mutant rats.

A mutant strain of rat, LEC, shows a novel arrest of T-cell maturation from CD4+8+ to CD4+8-, but not to CD4-8+ cells in the thymus. The responsible mutant locus is designated the thid, which was acted upon in a recessive manner of inheritance. We found that LEC rat thymocytes failed to respond to interleukin (IL)-1, IL-6 and IL-7 in the presence of the mitogenic lectins, Allo A or concanavalin A (Con A). The unresponsiveness appeared to be due to unresponsiveness to the lectin stimulation rather than because of cytokine stimulation. Normal rat CD4-8+/- (consisting of CD4-8+ and CD4-8- thymocytes), CD4+/-8- (consisting of CD4+8- and CD4-8- thymocytes), and CD4-8- thymocyte subsets normally responded to mitogenic stimulation, while CD4+8+ thymocytes did not. In contrast, all LEC rat CD4-8+/-, CD4+/-8-, CD4-8- and CD4-8+ thymocytes did not respond to the mitogenic stimulation, suggesting that the unresponsiveness of the CD4-8+/- thymocytes seems to be responsible for the unresponsiveness of whole thymocytes in LEC rats. LEC rat CD4-8+/- thymocytes normally expressed Con A receptor (R), lymphocyte function-associated antigen-1 (LFA-1), and CD45, which are thought to be important molecules for lectin stimulation. When backcross rats from (F344xLEC)F1xLEC were examined, the phenotype for the thid mutation correlated with the [3H]thymidine deoxyribose (TdR) incorporation level in response to Con A stimulation; thymocytes from backcross rats showing +/thid phenotype responded to Con A stimulation normally, whereas thymocytes from backcross rats showing thid/thid phenotype showed significantly lower responsiveness compared with +/thid rats. However, in WKAH.C-thid congenic rat thymocytes that carry the thid mutation, the responsiveness to mitogenic stimulation was comparable to that of normal rat thymocytes. These results suggest that a defect in responsiveness to mitogenic stimulation in LEC rat thymocytes is controlled by multiple genetic loci and the thid locus is one of the important loci for the development of this abnormal phenotype.     

A functionally active complement system is present in uterine secretion of the mouse prior to implantation.

Sephadex beads were placed carefully in the uterus on days 2 and 3 and left for 6 to 8 h to absorb uterine secretion. The beads were then removed with volatile silicon oil and mounted on small pieces of nitrocellulose paper. Immuno-staining of these bead blots showed they contained the complement components C1q, C3, C4, and C5. We demonstrated that complement component C3 in the uterine secretion could be activated and deposited on model immune complexes, and also that antibody-coated erythrocytes were lysed in utero, that is, a membrane attack complex was produced. Thus, the mouse uterine secretion at the preimplantation stage contains a functionally active complement system.     

Procarbazine is a potent mutagen at the heterozygous thymidine kinase (tk+/-) locus of mouse lymphoma assay

Procarbazine (Natulan®) is a potent inducer of gene mutationsat the heterozygous tk^+/– locus in L5178Y mouse lymphomacells in the presence of Aroclor-induced rat liver s9 metabolicactivation ({small tilde} 10^–3 mutant frequency at 10µg/ml) while exerting a far weaker effect in the absenceof s9. This mutagenicity is fairly robust with respect to thequantitative composition of the s9 mix and to variations inmouse lymphoma assay protocols (soft agar cloning versus ‘microwell’assays). The high proportion of small colony tk^–/–mutants induced by procarbazine together with the far weakermuta-genic response at the hemizygous hgprt locus in these samecells is interpreted in terms of a chromosomal or mufti-genemutational mechanism. Although procarbazine is clastogenic invivo, it does not appear to be so under standard protocols usingcultured human lymphocytes (±s9). It is not yet clearwhy this should be so, especially in light of its apparent clasto-genicityin mouse lymphoma cells.     

A new type of chondrodystrophic mutant in the mouse.

Stumpy is a new chondrodystrophic mutant in the mouse. The condition is inherited as a fully penetrant Mendelian recessive, and is not allelic with brachymorphic, achondroplasia, or stubby-three similar, previously described mutants. No chromosomal position has yet been assigned to the gene. Phenotypically, stumpy mice are chondrodystrophic dwarfs with all cartilage-formed bones in the skeleton affected. The condition differs from the usual chondrodystrophy described in mice in that proximal elements in the limbs are affected more than distal ones.     

Down-regulation of delta-opioid receptors in Na+/H+ exchanger 1 null mutant mouse brain with epilepsy

Mice lacking Na+/H+ exchanger 1 (NHE1) show a unique epilepsy phenotype although the underlying mechanisms remain unclear. Since expression of delta-opioid receptor (DOR) may be involved in control of epileptic activity, we conducted immunohistochemistry and autoradiography to investigate whether DOR expression is dys-regulated in the brain of NHE1 null mouse. Immunohistochemistry showed a decline in DOR expression in hippocampus and cortex. Autoradiographic results confirmed that the density of DOR was decreased in most cortical and hippocampal regions such as striate and temporal cortex, hippocampal CA1 and CA3 regions (reduced by 27.7 +/- 6.4%, 29.4 +/- 5.1%, 40.7 +/- 4.4% and 20.6 +/- 5.7%, respectively, P < 0.05). These data demonstrate that NHE1 null mutation leads to a reduction of DOR expression in the cortical and hippocampal regions, which provides a new clue for the genesis of epilepsy.     

Behavioral phenotype of the reeler mutant mouse: effects of RELN gene dosage and social isolation

Reeler (rl/rl) and reeler/wild-type (+/rl) mice synthesize Reln at subnormal rates, as do patients with schizophrenia, bipolar disorder, and autism, thereby forming the basis for a Reln hypothesis for vulnerability to these psychopathologies and justifying attention to the behavioral phenotypes of Reln-deficient mice. Tests of gait, emotionality, social aggression, spatial working memory, novel-object detection, fear conditioning, and sensorimotor reflex modulation revealed the behavioral phenotype of rl/rl, but not +/rl, mice to be different from that of wild-type (+/+) mice. These results reveal no effect of Reln gene dosage and provide significant challenges to both the Reln and the neurodevelopmental hypotheses of the etiology of major psychopathologies.     

A mutation in the catalytic domain of pp60v-src is responsible for the host- and temperature-dependent phenotype of the Rous sarcoma virus mutant tsLA33-1.

We have analyzed a host- and temperature-dependent mutant of Rous sarcoma virus in order to learn more about the nature of mutations which lead to a host range phenotype. We have cloned and sequenced the v-src genes from this mutant, tsLA33-1, and from its presumed parent, tsLA33. Both the tsLA33 and the tsLA33-1 pp60v-src proteins contain multiple mutations. The tsLA33 v-src gene product has amino acid alterations at four positions. In the tsLA33-1 v-src gene product, two of these four mutations have reverted to wild type. We have constructed chimeras between the two mutant v-src gene products and between each mutant and the Prague A v-src gene product. To assess the contribution of each amino acid change to the transformation phenotypes of tsLA33 and tsLA33-1, we expressed the hybrid proteins in both chicken embryo fibroblasts and Rat-3 fibroblasts. Additionally, we have measured the protein tyrosine kinase activity of chimeras constructed between the tsLA33 and tsLA33-1 pp60v-src proteins. Our results indicate that mutations in the catalytic domain of each protein are the principal determinants of the transforming ability and protein tyrosine kinase activity of the tsLA33 and tsLA33-1 pp60v-src proteins.     

Oxymetholone: III. Evaluation in the p53+/- transgenic mouse model.

Oxymetholone has been identified as a suspected nongenotoxic carcinogen and has recently completed testing in a conventional National Toxicology Program (NTP) 2-yr rodent bioassay program. As a synthetic androgen with a limited historical database in toxicology, oxymetholone is an ideal candidate for prospective examination of the performance of short-term transgenic mouse models in the detection of carcinogenic activity. In the present series of 3 articles, studies are described where oxymetholone was evaluated prior to disclosure of the results of the NTP 2-yr bioassay. The accompanying articles provide evidence showing that oxymetholone is devoid of mutagenic activity yet elicits a positive carcinogenic response in the Tg.AC transgenic mouse model. In the present study, oxymetholone was administered by oral gavage to p53 heterozygous male and female mice for 26 wk at doses of 125, 625, and 1,250 mg/kg/day. The vehicle was 0.5% aqueous methylcellulose. Positive controls consisted of mice treated daily by oral gavage with 200 or 400 mg/kg/day of p-cresidine in corn oil. The oxymetholone-treated females showed significantly increased body weight gain and clitoral enlargement attributable to drug treatment. In addition, significant alterations in kidney, liver, and testis weights were attributable to oxymetholone. However, there were no neoplastic lesions that were attributable to oxymetholone in either sex. p-Cresidine produced unequivocal bladder neoplasms in both sexes at the high dose and in males at the lower dose. The absence of a neoplastic response with oxymetholone is consistent with the selectivity of the p53-/- mouse model for detecting carcinogens that act by genotoxic mechanisms.     

Soluble erythropoietin receptor is present in the mouse brain and is required for the ventilatory acclimatization to hypoxia

While erythropoietin (Epo) and its receptor (EpoR) have been widely investigated in brain, the expression and function of the soluble Epo receptor (sEpoR) remain unknown. Here we demonstrate that sEpoR, a negative regulator of Epo's binding to the EpoR, is present in the mouse brain and is down-regulated by 62% after exposure to normobaric chronic hypoxia (10% O₂ for 3 days). Furthermore, while normoxic minute ventilation increased by 58% in control mice following hypoxic acclimatization, sEpoR infusion in brain during the hypoxic challenge efficiently reduced brain Epo concentration and abolished the ventilatory acclimatization to hypoxia (VAH). These observations imply that hypoxic downregulation of sEpoR is required for adequate ventilatory acclimatization to hypoxia, thereby underlying the function of Epo as a key factor regulating oxygen delivery not only by its classical activity on red blood cell production, but also by regulating ventilation.     

A novel mutant phenotype implicates dicephalic in cyst formation in the Drosophila ovary.

The establishment of polarity in Drosophila requires the correct specification of the oocyte in early stages of oogenesis, its positioning at the posterior of the egg chamber, and signalling events between the oocyte and the adjacent posterior follicle cells. As a consequence, the anterior-posterior and the dorsal-ventral axes are fixed. The posterior localisation of the oocyte depends on cadherin-mediated adhesion between the oocyte and the follicle cells. Here we show that dicephalic mutants affect the posterior positioning of the oocyte without interfering with oocyte specification in the germarium. Unlike other mutants that also affect the posterior placement of the oocyte, dicephalic mutants affect neither gurken expression nor karyosome formation during meiosis. By analysing in detail the mutant phenotypes of dicephalic, we find that cyst formation in mutant germaria is defective and that it shares some similarities with cysts that lack DE-cadherin in the germline cells. We propose a model in which dicephalic is involved in the proper adhesion between the oocyte and the somatic follicle cells.     

A diastrophic dysplasia sulfate transporter (SLC26A2) mutant mouse: morphological and biochemical characterization of the resulting chondrodysplasia phenotype

Mutations in the diastrophic dysplasia sulfate transporter (DTDST or SLC26A2) cause a family of recessively inherited chondrodysplasias including, in order of decreasing severity, achondrogenesis 1B, atelosteogenesis 2, diastrophic dysplasia (DTD) and recessive multiple epiphyseal dysplasia. The gene encodes a widely distributed sulfate/chloride antiporter of the cell membrane whose function is crucial for the uptake of inorganic sulfate, which is needed for proteoglycan sulfation. To provide new insights in the pathogenetic mechanisms leading to skeletal and connective tissue dysplasia and to obtain an in vivo model for therapeutic approaches to DTD, we generated a Dtdst knock-in mouse with a partial loss of function of the sulfate transporter. In addition, the intronic neomycine cassette in the mutant allele contributed to the hypomorphic phenotype by inducing abnormal splicing. Homozygous mutant mice were characterized by growth retardation, skeletal dysplasia and joint contractures, thereby recapitulating essential aspects of the DTD phenotype in man. The skeletal phenotype included reduced toluidine blue staining of cartilage, chondrocytes of irregular size, delay in the formation of the secondary ossification center and osteoporosis of long bones. Impaired sulfate uptake was demonstrated in chondrocytes, osteoblasts and fibroblasts. In spite of the generalized nature of the sulfate uptake defect, significant proteoglycan undersulfation was detected only in cartilage. Chondrocyte proliferation and apoptosis studies suggested that reduced proliferation and/or lack of terminal chondrocyte differentiation might contribute to reduced bone growth. The similarity with human DTD makes this mouse strain a useful model to explore pathogenetic and therapeutic aspects of DTDST-related disorders.     

A regulatory phenotype associated with the en-am 1 mutant of Neurospora crassa

Summary We have investigated the nature of the en-am1 mutant of Neurospora crassa and have found that it affects the regulation of proline oxidase and utilisation of other nitrogen sources. This mutant is closely linked to the gln gene but not allelic with it. Data from crosses suggest that the two genes he on opposite sides of the in1 gene on linkage group VR.     

Maternal care in the African striped mouse Rhabdomys pumilio: A behaviorally flexible phenotype that is modified by experience

The development of maternal care in mammals can be influenced by the type and quality of maternal care received. Using biparental striped mice Rhabdomys pumilio, we investigated whether development of maternal care is influenced by the mother during early rearing and by an adult female's experience and that of her mate. Offspring were raised in one of three treatments, by: both parents; mothers alone; and mothers separated from the father with a barrier. Since female striped mice increase their care when raising litters alone, which influences expression of parental care of their adult sons, we expected daughters to respond like sons. However, there was no treatment effect in the development of maternal care in daughters. In subsequent experiments, experienced and inexperienced females decreased care when raising their offspring with experienced but not inexperienced males. Therefore, maternal care in striped mice is modulated in response to prevailing environmental and social conditions. © 2012 Wiley Periodicals, Inc. Dev Psychobiol 55: 265–274, 2013