The effect of social preference on academic diligence in adolescence.

In the current study, we were interested in whether adolescents show a preference for social stimuli compared with non-social stimuli in the context of academic diligence, that is, the ability to expend effort on tedious tasks that have long-term benefits. Forty-five female adolescents (aged 11-17) and 46 female adults (aged 23-33) carried out an adapted version of the Academic Diligence Task (ADT). We created two variations of the ADT: a social ADT and non-social ADT. Individuals were required to freely split their time between an easy, boring arithmetic task and looking at a show-reel of photographs of people (in the social ADT) or landscapes (in the non-social ADT). Individuals also provided enjoyment ratings for both the arithmetic task and the set of photographs they viewed. Adolescents reported enjoying the social photographs significantly more than the non-social photographs, with the converse being true for adults. There was no significant difference in the time spent looking at the social photographs between the adolescents and adults. However, adults spent significantly more time than adolescents looking at the non-social photographs, suggesting that adolescents were less motivated to look at the non-social stimuli. Further, the correlation between self-reported enjoyment of the pictures and choice behaviour in the ADT was stronger for adults than for adolescents in the non-social condition, revealing a greater discrepancy between self-reported enjoyment and ADT choice behaviour for adolescents. Our results are discussed within the context of the development of social cognition and introspective awareness between adolescence and adulthood.

Novel implications of Lingo-1 and its signaling partners in schizophrenia.

Myelination and neurite outgrowth both occur during brain development, and their disturbance has been previously been implicated in the pathophysiology of schizophrenia. Leucine-rich repeat and immunoglobulin domain-containing protein (Lingo-1) is a potent negative regulator of axonal myelination and neurite extension. As co-factors of Lingo-1 signaling (Nogo receptor (NgR), With No Lysine (K) (WNK1) and Myelin transcription factor 1 (Myt1)) have been implicated in the genetics of schizophrenia, we explored for the first time the role of Lingo-1 signaling pathways in this disorder. Lingo-1 protein, together with its co-receptor and co-factor proteins NgR, tumor necrosis factor (TNF) receptor orphan Y (TROY), p75, WNK1 and Myt1, have never been explored in the pathogenesis of schizophrenia. We examined protein levels of Lingo-1, NgR, TROY, p75, WNK1, Myt1 and myelin basic protein (MBP) (as a marker of myelination) within the post-mortem dorsolateral prefrontal cortex (DLPFC) (37 schizophrenia patients versus 37 matched controls) and hippocampus (Cornu Ammonis, CA1 and CA3) (20 schizophrenia patients versus 20 matched controls from the same cohort). Both of these brain regions are highly disrupted in the schizophrenia pathophysiology. There were significant increases in Lingo-1 (P<0.001) and Myt1 (P=0.023) and a reduction in NgR (P<0.001) in the DLPFC in schizophrenia subjects compared with controls. There were also increases in both TROY (P=0.001) and WNK1 (P=0.011) in the CA1 of schizophrenia subjects and, in contrast to the DLPFC, there was an increase in NgR (P=0.006) in the CA3 of schizophrenia subjects compared with controls. No significant difference was reported for MBP levels (P>0.05) between the schizophrenia and control groups in the three tested regions. This is the first time that a study has shown altered Lingo-1 signaling in the schizophrenia brain. Our novel findings may present a direct application for the use of a Lingo-1 antagonist to complement current and future schizophrenia therapies.

Aging does not alter the mechanosensitivity of the p38, p70S6k, and JNK2 signaling pathways in skeletal muscle.

The capacity for skeletal muscle to recover its mass following periods of unloading (regrowth) has been reported to decline with age. Although the mechanisms responsible for the impaired regrowth are not known, it has been suggested that aged muscles have a diminished capacity to sense and subsequently respond to a given amount of mechanical stimuli (mechanosensitivity). To test this hypothesis, extensor digitorum longus muscles from young (2-3 mo) and old (26-27 mo) mice were subjected to intermittent 15% passive stretch (ex vivo) as a source of mechanical stimulation and analyzed for alterations in the phosphorylation of stress-activated protein kinase (p38), ribosomal S6 kinase (p70S6k), and the p54 jun N-terminal kinase (JNK2). The results indicated that the average magnitude of specific tension (mechanical stimuli) induced by 15% stretch was similar in muscles from young and old mice. Young and old muscles also revealed similar increases in the magnitude of mechanically induced p38, p70S6k (threonine/serine 421/424 and threonine 389), and JNK2 phosphorylation. In addition, coincubation experiments demonstrated that the release of locally acting growth factors was not sufficient for the induction of JNK2 phosphorylation, suggesting that JNK2 was activated by a mechanical rather than a mechanical/growth factor-dependent mechanism. Taken together, the results of this study demonstrate that aging does not alter the mechanosensitivity of the p38, p70S6k, and JNK2 signaling pathways in skeletal muscle.

Crystal structure of the complex of plasminogen activator inhibitor 2 with a peptide mimicking the reactive center loop.

The structure of the serpin, plasminogen activator inhibitor type-2 (PAI-2), in a complex with a peptide mimicking its reactive center loop (RCL) has been determined at 1.6-A resolution. The structure shows the relaxed state serpin structure with a prominent six-stranded beta-sheet. Clear electron density is seen for all residues in the peptide. The P1 residue of the peptide binds to a well defined pocket at the base of PAI-2 that may be important in determining the specificity of protease inhibition. The stressed-to-relaxed state (S --> R) transition in PAI-2 can be modeled as the relative motion between a quasirigid core domain and a smaller segment comprising helix hF and beta-strands s1A, s2A, and s3A. A comparison of the Ramachandran plots of the stressed and relaxed state PAI-2 structures reveals the location of several hinge regions connecting these two domains. The hinge regions cluster in three locations on the structure, ensuring a cooperative S --> R transition. We hypothesize that the hinge formed by the conserved Gly(206) on beta-strand s3A in the breach region of PAI-2 effects the S --> R transition by altering its backbone torsion angles. This torsional change is due to the binding of the P14 threonine of the RCL to the open breach region of PAI-2.

Generation and characterization of E1/E2a/E3/E4-deficient adenoviral vectors encoding human factor VIII.

The use of adenoviral vectors for gene therapy has been limited due to host immune responses directed toward the vector and/or transgene and vector toxicity. To decrease adenoviral vector immunogenicity and toxicity, we attenuated viral gene expression by eliminating E1, E2a, E3, and E4 early genes from the adenoviral backbone. Two highly attenuated, fourth-generation (Av4) E1/E2a/E3/E4-deficient adenoviral vectors encoding human factor VIII (FVIII) under the control of a liver-specific albumin promoter were generated. One Av4 vector (Av4DeltaE4FVIII) was deficient in the entire E4 coding region and the second vector contained a deletion of the E4 region, except for open reading frame 3 (orf 3; Av4orf3FVIII). The Av4 vectors were compared to an E1/E2a/E3-deficient third-generation vector (Av3H8101) containing an analogous transgene expression cassette in vitro and in vivo following intravenous administration in hemophiliac mice. In vitro transduction of Hep3B cells revealed at all three vectors expressed functional FVIII. However, the Av4DeltaE4FVIII vector could not be scaled-up for in vivo studies. Both Av3H8101 and Av4orf3FVIII initially expressed similar levels of FVIII in hemophiliac mice. However, at 3 months, animals treated with the Av4orf3FVIII vector no longer expressed FVIII while Av3H8101-treated mice displayed persistent FVIII expression. Liver enzyme analyses of plasma samples revealed that the Av4orf3FVIII vector was significantly less hepatotoxic than the Av3H8101 vector. These data demonstrate that further attenuation of the adenoviral vector backbone by removal of the majority of the E4 coding region significantly diminished vector toxicity; however, the duration of transgene expression was reduced.

Systemic delivery of an adenoviral vector encoding canine factor VIII results in short-term phenotypic correction, inhibitor development, and biphasic liver toxicity in hemophilia A dogs.

Canine hemophilia A closely mimics the human disease and has been used previously in the development of factor VIII (FVIII) protein replacement products. FVIII-deficient dogs were studied to evaluate an in vivo gene therapy approach using an E1/E2a/E3-deficient adenoviral vector encoding canine FVIII. Results demonstrated a high level of expression of the canine protein and complete phenotypic correction of the coagulation defect in all 4 treated animals. However, FVIII expression was short-term, lasting 5 to 10 days following vector infusion. All 4 dogs displayed a biphasic liver toxicity, a transient drop in platelets, and development of anticanine FVIII antibody. Canine FVIII inhibitor development was transient in 2 of the 4 treated animals. These data demonstrate that systemic delivery of attenuated adenoviral vectors resulted in liver toxicity and hematologic changes. Therefore, the development of further attenuated adenoviral vectors encoding canine FVIII will be required to improve vector safety and reduce the risk of immunologic sequelae, and may allow achievement of sustained phenotypic correction of canine hemophilia A.

Development of a facile method for high throughput screening with reporter gene assays.

This report describes a facile methodology for high throughput screening with stable mammalian cell reporter gene assays. We have adapted a 96-well adherent cell method to an assay in which cells propagated in suspension are dispensed into 96- or 384-well plates containing test compounds in 100% DMSO. The validation of a stable CHO cell line that expresses 6xCRE-luciferase for use as a reporter gene host cell line is described. The reporter gene, when expressed in this particular CHO cell line, appears to respond specifically to modulation of cAMP levels, thus the cell line is appropriate for screening and pharmacological analysis of Galpha(s)- and Galpha(i)-coupled seven-transmembrane receptors. The development of the new suspension cell assay in both 96- and 384-well formats was performed using a derivative of the CHO host reporter cell line that was stably transfected with human melanocortin-1 receptor. The response of this cell line to NDP-alpha-melanocyte-stimulating hormone and forskolin was nearly identical between the adherent and suspension methods. The new method offers improvements in cost, throughput, cell culture effort, compound stability, accuracy of compound delivery, and hands-on time. The 384-well assay can be performed at high capacity in any laboratory without the use of expensive automation systems such that a single person can screen 100 plates per day with 3.5-4 h hands-on time. Although the system has been validated using Galpha(s)-coupled receptor-mediated activation of a cAMP response element, the method can be applied to other types of targets and/or transcriptional response elements.

In vivo evaluation of a novel epitope-tagged human factor VIII-encoding adenoviral vector.

Haemophilia A is caused by a deficiency in coagulation factor VIII (FVIII) and is an attractive target for gene therapy. Adenoviral vectors encoding a human B-domain deleted (BDD) FVIII cDNA have been shown previously to mediate expression of high levels of human FVIII and correct the bleeding defect in haemophiliac mice and dogs. While vector assessment in a non-human primate model would have a significant preclinical benefit, a haemophiliac non-human primate model is not available, and assays that distinguish human FVIII from monkey FVIII have not been developed successfully. As a first step to enable vector evaluation in non-human primates, we have constructed an epitope-tagged FVIII molecule by the addition of 16 amino-acids to the carboxy terminus of the BDD protein (BDD-E). Following vector administration to normal mice, therapeutic levels of BDD-E FVIII were expressed for at least 20 weeks. Treatment of haemophiliac mice revealed that the BDD-E protein was biologically active in vivo. To distinguish the BDD-E protein from non-human primate FVIII, a sensitive immunoprecipitation/Western assay was developed that reproducibly detected 1 ng mL-1 of the epitope-tagged human FVIII in the presence of monkey plasma. These data demonstrate that the addition of an epitope tag had no effect on FVIII function or immunogenicity, and suggest that the BDD-E vector will be an effective reagent for non-human primate studies.

Development of class-switched, affinity-matured monoclonal antibodies following a 7-day immunization schedule.

Previously we reported making high-affinity monoclonal antibodies (MAbs) 13 days after the onset of Repetitive Immunizations Multiple Sites (RIMMS) strategy. The Ig subclass variety and affinity of these antibodies suggested that maturational processes had already begun within draining lymph nodes. We now demonstrate that this diversity can in fact be captured as early as Day 7. In the work reported here, somatic fusion of immune lymphocytes isolated from peripheral lymph nodes resulted in the isolation of affinity-matured MAbs reactive with cytosine deaminase. This model further demonstrates and substantiates at a cellular level the rapid development and maturation of T-cell-dependent B-cell responses occurring within draining lymph nodes following antigen challenge.

Tissue-specific mutant frequencies and mutational spectra in cyclophosphamide-treated lacI transgenic mice.

The induction and nature of mutations in the lacI transgene were evaluated in multiple tissues after exposure of adult male B6C3F1 lacI transgenic mice to cyclophosphamide (CP). Mice were given a single i.p. injection of 25 mg CP/kg, 100 mg CP/kg, or vehicle (PBS) and then necropsied 6 weeks after treatment to allow DNA extraction and lacI mutant recovery. Tissues evaluated included target tissues for tumorigenesis (lung, urinary bladder) and sites not susceptible to tumor formation in B6C3F1 mice (kidney, bone marrow, splenic T-lymphocytes). After exposure to the high dose of CP, a significant increase in the mutant frequency (Mf) was detected in the lungs and urinary bladders, compared to the respective tissues from vehicle-treated controls. In contrast, the Mfs in kidney, bone marrow, and splenic T cells from CP-treated mice were not significantly different from controls. The spectra of mutations in lacI from lung and urinary bladder were significantly changed after high-dose CP treatment, with a significant increase in the frequency of A. T --> T. A transversions found in both tissues and a significantly elevated frequency of deletions in the lungs. Conversely, in vehicle-treated mice, the two predominant classes of lacI mutations recovered in lung and urinary bladder were G. C --> A. T transitions at CpG sites and G. C --> T. A transversions. These CP exposures were also genotoxic as measured by the significant induction of micronuclei in peripheral blood 48 hr after exposure. These data indicate that under these study conditions, CP-induced mutations are detectable in the lacI transgene in the target tissues, but not in nontarget tissues for CP-induced cancer. With the lacI assay it is possible to study mutagenicity in a variety of critical tissues to provide mechanistic information related to genotoxicity and carcinogenicity in vivo.

Detection of cyclophosphamide-induced mutations at the Hprt but not the lacI locus in splenic lymphocytes of exposed mice.

The relative sensitivities and specificities of the endogenous Hprt gene and the lacI transgene as mutational targets were evaluated in splenic lymphocytes from male standard B6C3F1 mice (only Hprt assayed) and from lacI transgenic B6C3F1 mice treated at 6-7 weeks- of-age with the indirect-acting agent, cyclophosphamide (CP). To define the effects of the time elapsed since CP treatment on Hprt mutant frequencies (Mfs), nontransgenic mice were given single i.p. injections of 25 mg CP/kg or vehicle (PBS) alone and then necropsied 2, 4, 6, 8, or 10 weeks after treatment. Peak Mfs were found at 6 weeks postexposure, with mean Mf values ranging from 2.27 to 3.27 x 10(-5) using two different lots of CP in standard packaging (compared with mean control Mf values of 0.14 to 0.26 x 10(-5) in various experiments). To determine the dose response for Hprt Mfs, nontransgenic mice were given single doses of 0, 12.5, 25, 50, or 100 mg CP/kg and necropsied 4 weeks postexposure. These treatments produced a supralinear dose response curve for CP-induced Hprt Mfs. Based on these experiments, CP mutagenicities at Hprt and lacI were compared in transgenic mice treated with 0, 25, or 100 mg CP/kg (using another lot of CP in ISOPAC((R)) bottles; Sigma) and necropsied 6 weeks later. There was a significant increase in Hprt Mfs in treated transgenic mice (100 mg CP/kg: 0.75 +/- 0.09 x 10(-5); 25 mg CP/kg: 0.39 +/- 0.05 x 10(-5)) versus controls (0.10 +/- 0.01 x 10(-5)); however, the Mfs in lacI of lymphocytes from the same CP-treated animals were not significantly different from controls (100 mg CP/kg: 9.4 +/- 1.1 x 10(-5); 25 mg CP/kg: 6.7 +/- 0. 8 x 10(-5); control: 7.7 +/- 0.7 x 10(-5)). Hprt mutational spectra data in CP-treated transgenic and nontransgenic mice were different from those of control mice, whereas the spectra of mutations in lacI of lymphocytes from Big Blue((R)) transgenic mice were not significantly changed after CP treatment. These data indicate that, under these treatment conditions, CP-induced mutations in splenic lymphocytes were detectable in the Hprt gene but not the lacI transgene of this nontarget tissue for CP-induced cancer.

Efficient adenoviral vector transduction and expression of functional human factor VIII in cultured primary human hepatocytes.

Hemophilia A is a severe bleeding disorder caused by a deficiency in blood coagulation factor VIII (FVIII). Adenoviral vectors containing a potent human FVIII expression cassette encoding a truncated FVIII cDNA were developed that mediated sustained FVIII expression in normal and haemophiliac mice and complete phenotypic correction of the bleeding disorder in haemophiliac mice and dogs (Connelly and Kaleko, Haemophilia, 1998; 4: 380-8). Here, we evaluated two E1/E2a/E3-deleted adenoviral vectors encoding human FVIII, one containing the full-length cDNA and the second containing a truncated cDNA lacking the B-domain. Viral vectors encoding the human full-length FVIII cDNA have not been described previously. Hepatocyte transduction was efficient and dose dependent, ranging from 50% to 100%. High levels of functional FVIII were secreted from transduced cells at amounts up to 6000 mU-1 10(6)cells-1 60 h. B-domain deleted FVIII was expressed at levels at least 8-fold higher than the full-length FVIII protein, whereas FVIII RNA levels were similar with both vectors. These data provide the first demonstration of FVIII adenoviral vector function in primary human cells and verify the potential clinical utility of adenoviral vectors for the treatment of haemophilia A.

In vivo evaluation of an adenoviral vector encoding canine factor VIII: high-level, sustained expression in hemophiliac mice.

Hemophilia A is the most common severe hereditary coagulation disorder and is caused by a deficiency in blood clotting factor VIII (FVIII). Canine hemophilia A represents an excellent large animal model that closely mimicks the human disease. In previous studies, treatment of hemophiliac dogs with an adenoviral vector encoding human FVIII resulted in complete correction of the coagulation defect and high-level FVIII expression [Connelly et al. (1996). Blood 88, 3846]. However, FVIII expression was short term, limited by a strong antibody response directed against the human protein. Human FVIII is highly immunogenic in dogs, whereas the canine protein is significantly less immunogenic. Therefore, sustained phenotypic correction of canine hemophilia A may require the expression of the canine protein. In this work, we have isolated the canine FVIII cDNA and generated an adenoviral vector encoding canine FVIII. We demonstrate expression of canine FVIII in hemophiliac mice at levels 10-fold higher than those of the human protein expressed from an analogous vector. Canine FVIII expression was sustained above human therapeutic levels (50 mU/ml) for at least 1 year in hemophiliac mice.

Evaluation of an adenoviral vector encoding full-length human factor VIII in hemophiliac mice.

Adenoviral vectors provide a promising gene therapy system for the treatment of hemophilia A. Potent vectors encoding a human factor VIII (FVIII) cDNA were developed that mediated sustained FVIII expression in normal and hemophiliac mice and complete phenotypic correction of the bleeding disorder in hemophiliac mice and dogs (Connelly and Kaleko, Haemophilia 1998; 4: 380-8). However, these studies utilized vectors encoding a truncated version of the human FVIII cDNA lacking the B-domain (BDD FVIII). In this work, an adenoviral vector encoding the human full-length (FL) FVIII cDNA was generated and characterized. While functional FL FVIII was secreted in vitro, expression of the FL protein was not detected in the plasma of vector-treated hemophiliac mice. Unexpectedly, the FL FVIII vector-treated animals demonstrated phenotypic correction of the bleeding defect as measured by a tail-clip survival study. FL FVIII protein was visualized in the mouse livers using human FVIII-specific immunohistochemical analyses. These data demonstrate that adenoviral vector-mediated in vivo expression of BDD FVIII is more efficient than that of the FL protein and that phenotypic correction can occur in the absence of detectable levels of FVIII.

Sustained phenotypic correction of murine hemophilia A by in vivo gene therapy.

Hemophilia A is caused by a deficiency of blood coagulation factor VIII (FVIII) and has been widely discussed as a candidate for gene therapy. While the natural canine model of hemophilia A has been valuable for the development of FVIII pharmaceutical products, the use of hemophiliac dogs for gene therapy studies has several limitations such as expense and the long canine generation time. The recent creation of two strains of FVIII-deficient mice provides the first small animal model of hemophilia A. Treatment of hemophiliac mice of both genotypes with potent, human FVIII-encoding adenoviral vectors resulted in expression of biologically active human FVIII at levels, which declined, but remained above the human therapeutic range for over 9 months. The duration of expression and FVIII plasma levels achieved were similar in both hemophiliac mouse strains. Treated mice readily survived tail clipping with minimal blood loss, thus showing phenotypic correction of murine hemophilia A by in vivo gene therapy.

Inhibition of establishment of primary and micrometastatic tumors by a urokinase plasminogen activator receptor antagonist.

Tumor establishment and metastasis are dependent on extracellular matrix proteolysis, tumor cell migration, and angiogenesis. Urokinase plasminogen activator (uPA) and its receptor are essential mediators of these processes. The purpose of this study was to investigate the effect of a recombinant human uPAR antagonist on growth, establishment, and metastasis of tumors derived from human cancer cell lines. A noncatalytic recombinant protein, consisting of amino acids 1-137 of human uPA and the CH2 and CH3 regions of mouse IgG1 (uPA-IgG), was expressed, purified, and shown to bind specifically to human uPAR and to saturate the surface of human tumor cells which express uPAR. Daily i.p. administration of uPA-IgG to nude mice extended latencies of unstaged tumors derived from Lox melanoma and SW48 colon carcinoma cells by 7.7 and 5.5 days, respectively. uPA-IgG treatment did not affect the growth of Lox or KB tumors staged to 200 mg before antagonist treatment commenced. The effect of uPA-IgG on the establishment of micrometastases was assessed in SCID mice. KB head/neck tumor cells were injected in the tail vein and allowed to seed for 48 h before initiation of daily i.p. injections of uPA-IgG for 24 days. The number of lung colonies ranged between 5 and 30% of vehicle-treated mice in two separate experiments. Furthermore, a single 800 microg dose of uPA-IgG administered 1 h prior to tail vein injection of KB cells reduced lung colony formation to just 3.5% of vehicle-treated SCID mice. These data demonstrate that antagonism of uPAR arrested metastasis and inhibited the establishment of primary tumors and micrometastases. Thus, small molecule uPAR antagonists may serve as useful adjuvant agents in combination with existing cancer chemotherapy.

Psychometric evaluation of the Organizational Job Satisfaction Scale.

Findings are reported from a replicated evaluation of the psychometric properties of the Organizational Job Satisfaction Scale. Data lend strong support for the scale which assesses four dimensions of organizational job satisfaction. Principal component factor analysis of data from two independent samples of urban staff registered nurses (RNs) (n = 496; n = 532) confirmed four dimensions of pay, interaction, administration, and status. Cronbach's alpha for subscales ranged from .76 to .88. Pearson correlation coefficients for the interrelationships among subscales and an estimate of theta verified internal consistency. The Organizational Job Satisfaction Scale scores correlated significantly with a criterion measure of job enjoyment. Hypothesis testing of theoretically predictable relationships supported construct validity. Scores on the organizational job satisfaction subscales correlated significantly with automony, stress, and commitment. Item revisions and recommendations for future use of the instrument are discussed.

Evaluation of lacI mutation in germ cells and micronuclei in peripheral blood after treatment of male lacI transgenic mice with ethylnitrosourea, isopropylmethane sulfonate or methylmethane sulfonate.

Male C57B1/6 lacI transgenic mice were used to evaluate germ cell mutagenesis in vivo as part of a collaborative study. Groups of 10 mice were administered single intraperitoneal doses of ethylnitrosourea (ENU; 150 mg/kg), isopropyl methanesulfonate (IPMS; 200 mg/kg), methyl methanesulfonate (MMS; 40 mg/kg) or vehicle. Epididymal spermatozoa and testes were recovered 3 days later and DNA isolated subsequently from epididymal spermatozoa and seminiferous tubules were analyzed for lacI mutations. The mutant frequency in seminiferous tubules (average +/- SEM) increased significantly compared with untreated controls (7.2 +/- 0.7 x 10(-5) following treatment with ENU (11.7 +/- 0.8 x 10(-5), p = 0.003) or with IPMS (9.6 +/- 0.5 x 10(-5), p = 0.018) but not following treatment with MMS (8.1 +/- 0.8 x 10(-5), p = 0.213). Group mutant frequencies were not determined for epididymal spermatozoa from MMS- or IPMS-treated mice because of poor DNA recoveries. As another indicator of the genotoxicity of these alkylating agents, the frequencies of micronuclei were determined in the peripheral blood 48 h after carcinogen administration in the same transgenic mice. The micronuclei frequencies were elevated significantly (p < 0.05) by each treatment (IPMS: 1.0%; MMS: 0.94%) compared to vehicle controls (0.3%). In a separate experiment, 40 mg/kg ENU was previously found to increase the frequency of micronuclei in peripheral blood of lacI transgenic mice 48 h after treatment (3.2%; Gibson et al., 1995). These results demonstrate that the lacI transgenic mouse male germ cells are sensitive to some, but not all, mutagens under the conditions used in this experiment. Investigation of other experimental designs would offer additional perspective on the usefulness of this transgenic model for routine mutagenicity testing in germ cells.

Frequency and spectrum of ethylnitrosourea-induced mutation at the hprt and lacI loci in splenic lymphocytes of exposed lacI transgenic mice.

The development of mouse models with the endogenous hypoxanthine-guanine phosphoribosyl transferase (hprt) gene and lacI transgene as mutational targets provides an excellent opportunity to compare the mutant frequency (Mf) and types of mutations induced in vivo in different sequence contexts. To this end, a study was conducted to determine the Mfs and spectrum of mutations induced at these loci in splenic T cells from male B6C3F1 Big Blue mice (6 weeks old) exposed to N-ethyl-N-nitrosourea (ENU). Six weeks after i.p. injection of 40 mg ENU/kg, T cells were isolated from control (n = 7) and treated (n = 8) mice for the culture of hprt mutants and for the extraction of DNA and recovery of lacI mutants. Mutations in hprt exon 3 and in lacI were quantified and analyzed using published procedures (S. W. Kohler et al., Proc. Natl. Acad. Sci. USA, 88: 7958-7962, 1991; T. R. Skopek et al., Proc. Natl. Acad. Sci. USA, 89: 7866-7870, 1992). In treated mice, the Mfs (average +/- SE) in hprt (6.0 +/- 0.2 x 10(-5)) and lacI (11.4 +/- 1.8 x 10(-5)) were approximately 16.2-fold (P = 0.006) and 3.4-fold (P = 0.009), respectively, above controls. However, the average induced Mfs (i.e., induced Mf = treatment Mf - background Mf) in hprt and lacI were similar, with the respective increases in Mf being 5.6 +/- 0.2 x 10(-5) and 8.0 +/- 2.3 x 10(-5) over background. Eleven of the 107 hprt mutants from treated Big Blue mice had mutations in exon 3, with 73% being substitutions at AxT bp. These data are similar to those observed in ENU-exposed nontransgenic B6C3F1 mice, in which 62 of 69 exon 3 mutations were substitutions at AxT bp (T. R. Skopek et al., Proc. Natl. Acad. Sci. USA, 89: 7866-7870, 1992). For comparison, the sequences of the lacI genes in two to five mutants from each mouse were determined, and a total of 75 mutations (70 different mutations) was detected. In exposed mice, 55% (24 of 44) of the mutations in lacI were substitutions at AxT bp. In controls, substitutions at AT bp comprised only 20% of the recovered mutations in either hprt exon 3 (1 of 5) or lacI (5 of 26). These data indicate that the lacI mutation assay is less sensitive than the hprt assay for detecting increases in Mf induced by ENU exposure of mice as indicated by the lower relative increase in Mf in the lacI gene, but, given a 6-week expression time, the types of mutations induced by ENU in the transgene reflect those observed in the native transcribed gene.

A rapid antibody-based test for Sec-2, a marker for the short arm of chromosome 2 of rye (2RS).

A simple monoclonal antibody-based screening test has been developed for the presence of translocations of the short arm of chromosome 2 of rye (2RS) with wheat chromosome 2B. 2RS encodes a set of about three polypeptides known as Mr 75 000 gamma-secalins. Use of the antibody test for these secalins enabled screening of several hundred seeds per day. The antibody could readily distinguish 2BL-2RS translocations and 2R substitutions from 1BL-1RS translocations or nontranslocation wheats. Use of the antibody in analysis of segregating progeny for Sec-2 in several wheat backgrounds was successful. Results with a selection of the seed population were checked using protein gel electrophoresis, with 100% correct confirmation. Key words : rye, wheat, seed proteins, translocation, diagnostic test.