Individual and group housing in N-type Ca2+ channel alpha1B subunit-deficient mice differently affect motor activity.

N-type Ca2+ channel alpha1B-deficient mice have increased activity (ambulation, repetitive behavior, and rearing combined), suggesting contribution by the N-type Ca2+ channel, localized in the plasma membrane and essential for neurotransmitter release, on motor activity. We evaluated the effect of a 6-wk postweaning period of either individual or group housing on the activity displayed in a novel environment with or without previous habituation. Without habituation, male homozygous alpha1B-deficient mice showed significantly higher activity than wild-type controls, with no influence of the housing condition. When habituated, hyperactivity was seen in individually housed but not group-housed homozygous alpha1B-deficient mice. The results indicate that controlling for housing condition can be important when phenotypically analyzing mutant mice.

Chronic intracerebroventricular administration of relaxin-3 increases body weight in rats.

Bolus-administered intracerebroventricular (ICV) relaxin-3 has been reported to increase feeding. In this study, to examine the role of relaxin-3 signaling in energy homeostasis, we studied the effects of chronically administered ICV relaxin-3 on body weight gain and locomotor activity in rats. Two groups of animals received vehicle or relaxin-3 at 600 pmol/head/day, delivered with Alzet osmotic minipumps. In animals receiving relaxin-3, food consumption and weight gain were statistically significantly higher than those in the vehicle group during the 14-day infusion. During the light phase on days 2 and 7 and the dark phase on days 3 and 8, there was no difference in locomotor activity between the two groups. Plasma concentrations of leptin and insulin in rats chronically injected with relaxin-3 were significantly higher than in the vehicle-injected controls. These results indicate that relaxin-3 up-regulates food intake, leading to an increase of body weight and that relaxin-3 antagonists might be candidate antiobesity agents.

Enhanced expression of Ca2+ channel alpha1A and beta4 subunits and phosphorylated tyrosine hydroxylase in the adrenal gland of N-type Ca2+ channel alpha1B subunit-deficient mice with a CBA/JN genetic background.

Electrophysiologic studies have demonstrated that adrenal medulla chromaffin cells express voltage-dependent P/Q-, N-, L-, and R-type Ca2+ channels and that these channels regulate release of norepinephrine and epinephrine. However, N-type Ca2+ channel alpha1B-deficient mice with a CBA/JN background show normal plasma norepinephrine and epinephrine levels, presumably owing to compensation by other gene(s). To examine the expression patterns of the P/Q-type alpha1A, L-type alpha1C/alpha1D, and R-type alpha1E, beta1, beta2, beta3, and beta4 subunits, as well as of tyrosine hydroxylase (Th), dopamine beta hydroxylase (Dbh), and phenylethanolamine-N-methyltransferase (Pnmt) in the adrenal gland of alpha1B-deficient mice, we used real-time quantitative reverse transcription-polymerase chain reaction and Western blot analyses. The expression levels of alpha1A, beta4, Th, and Th phosphorylated at serine 40 were higher in homozygous mice than in wild-type and heterozygous mice, but the expression levels of alpha1C, alpha1D, alpha1E, beta1, beta2, beta3, Dbh, and Pnmt did not differ among wild-type, heterozygous, and homozygous mice. These results suggest that the compensatory mechanisms to maintain normal levels of epinephrine and norepinephrine in the adrenal gland of N-type Ca2+ channel alpha1B-deficient mice include increased expression of alpha1A and beta4 subunits and increased catecholamine biosynthetic activity.

Altered nociceptive response in ADAM11-deficient mice.

Mice that lack A Disintegrin And Metalloprotease 11 (ADAM11) protein showed normal responses to stimuli in the von Frey test and the hot plate test, but showed reduced responses in the formalin paw test and acetic acid writhing test. Our results indicate that the cell adhesion-related molecule ADAM11 may play a role in pain transmission and in inflammatory regulation mechanisms underlying changes in the threshold for pain perception.

Deficits in spatial learning and motor coordination in ADAM11-deficient mice.

BACKGROUND: ADAM11 is a member of the ADAM gene family and is mainly expressed in the nervous system. It is thought to be an adhesion molecule, since it has a disintegrin-like domain related to cell-cell or cell-matrix interactions. To elucidate the physiological functions of ADAM11, we generated ADAM11-deficient mice by means of gene targeting. RESULTS: ADAM11-deficient mice were apparently normal, and survived more than one year with no major histological abnormalities in the brain or spinal cord. Because ADAM11 is highly expressed in the hippocampus and cerebellum, we have examined ADAM11 mutant mice for learning using visual and hidden water maze tasks, and their motor coordination using a rotating rod task. Our results showed that their visual water maze task results are normal, but the hidden water maze and rotating rod task skills are impaired in ADAM11-deficient mice. CONCLUSION: Our results indicate that ADAM11 mutation does not affect cell migration and differentiation during development, but affects learning and motor coordination. Thus, ADAM11 might play an important signalling or structural role as a cell adhesion molecule at the synapse, and may thus participate in synaptic regulation underlying behavioural changes.

Genetic background influences P/Q-type Ca2+ channel alpha1A subunit mRNA expression in olfactory bulb and reproductive ability of N-type Ca2+ channel alpha1B subunit-deficient mice.

The Ca2+ channel alpha1B subunit is a pore-forming component capable of generating N-type Ca2+ channel activity. Although the N-type Ca2+ channel plays a role in a variety of neuronal functions, alpha1B-deficient mice did not show apparent behavioral abnormality. In a previous study, we observed a compensatory increase of mRNA expression of the P/Q-type Ca2+ channel alpha1A subunit gene in olfactory bulb of alpha1B-deficient mice with a CBA x C57BL/6 background; these mice showed a normal reproductive ability. In this study, we found that the mRNA expression level of the alpha1A subunit was the same in olfactory bulb of wild, heterozygous, and homozygous alpha1B-deficient mice with a CBA/JN background, and the homozygous male mice produced no offspring. These results suggest that the genetic background influences alpha1A subunit mRNA expression and reproductive ability in alpha1B-deficient mice.

Specificity of immobilized metal affinity-based IMAC/C18 tip enrichment of phosphopeptides for protein phosphorylation analysis.

We have developed a simple, highly specific enrichment procedure for phosphopeptides, by increasing the specificity of an immobilized metal affinity column (IMAC) without using any chemical reaction. The method employs a biphasic IMAC-C18 tip, in which IMAC beads are packed on an Empore C18 disk in a 200-microL pipet tip. Phosphopeptides are separated from non-phosphopeptides on the IMAC in an optimized solvent without any chemical reaction, then desorbed from the IMAC using a phosphate buffer, reconcentrated, and desalted on the C18 disk. The increase in selectivity was achieved by (a) using a strong acid to discriminate phosphates from carboxyl groups of peptides and (b) using a high concentration of acetonitrile to remove hydrophobic non-phosphopeptides. The entire procedure was optimized by using known phosphoproteins such as Akt1 kinase and protein kinase A. Although it was difficult to detect phosphopeptides in MALDI-MS spectra of tryptic peptide mixtures before enrichment, after the IMAC procedure, we could successfully detect phosphopeptides with almost no non-phosphopeptides. Next, we constructed an array of IMAC-IMAC/C18 tips, such that number of arrayed tips on a 96-well plate could easily be changed depending on the loading amount of sample. Applying this approach to mouse forebrain resulted in the identification of 162 phosphopeptides (166 phosphorylation sites) from 135 proteins using nano-LC/MS.

Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein.

To estimate absolute protein contents in complex mixtures, we previously defined a protein abundance index (PAI) as the number of observed peptides divided by the number of observable peptides per protein (Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Large-scale proteomic analysis of the human spliceosome. Genome. Res. 12, 1231-1245). Here we report that PAI values obtained at different concentrations of serum albumin show a linear relationship with the logarithm of protein concentration in LC-MS/MS experiments. This was also the case for 46 proteins in a mouse whole cell lysate. For absolute quantitation, PAI was converted to exponentially modified PAI (emPAI), equal to 10PAI minus one, which is proportional to protein content in a protein mixture. For the 46 proteins in the whole lysate, the deviation percentages of the emPAI-based abundances from the actual values were within 63% on average, similar or better than determination of abundance by protein staining. emPAI was applied to comprehensive protein expression analysis and to a comparison study between gene and protein expression in a human cancer cell line, HCT116. The values of emPAI are easily calculated and add important quantitation information to proteomic experiments; therefore we suggest that they should be reported in large scale proteomic identification projects.

Expression pattern of voltage-dependent calcium channel alpha1 and beta subunits in adrenal gland of N-type Ca2+ channel alpha1B subunit gene-deficient mice.

The Ca2+ channel alpha1B subunit is a pore-forming component capable of generating N-type Ca2+ channel activity. Although N-type Ca2+ channel plays a role in a variety of neuronal functions, alpha1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca2+ channel alpha1 or beta subunit. In this study, we studied the levels of alpha1A, alpha1C, alpha1D, C1E, beta1, beta2, beta3 and beta4 mRNAs in adrenal gland of alpha1B-deficient mice. The alpha1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of alpha1c, alpha1D, alpha1E, beta1, beta2, beta3 and beta4 was found among wild, heterozygous and homozygous mice. The protein level of alpha1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5'-upstream region of alpha1A gene, we examined lacZ expression in alpha1B-deficient x alpha1A6.3-lacZ mice (carrying a 6.3-kb 5'-upstream fragment of alpha1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous alpha1B-deficient x alpha1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of alpha1B-deficient mice is that compensation by alpha1A subunit occurs and that 6.3-kb 5'-upstream region of alpha1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells.

Increased glucose tolerance in N-type Ca2+ channel alpha(1B)-subunit gene-deficient mice.

The voltage-dependent N-type Ca2+ channel is localized in the plasma membrane of insulin-releasing beta-cells and glucagon-releasing alpha-cells in the islets of Langerhans in the pancreas. To examine the contribution of N-type Ca2+ channel to glucose homeostasis, we performed glucose tolerance and insulin tolerance tests with N-type Ca2+ channel alpha(1B)-subunit-deficient mice on a normal or high-fat diet. The fasting glucose level in homozygous mice on the normal diet was significantly lower than those in wild and heterozygous mice. In glucose tolerance tests, the homozygous mice showed a higher glucose clearance rate and a similar pattern of insulin levels to those of wild and heterozygous mice. In insulin tolerance tests, glucose clearance rates showed no significant difference among wild, heterozygous and homozygous mice. In animals on the high-fat diet, food consumption was the same among wild, heterozygous and homozygous mice, but body weight gain was reduced in homozygous mice. After 8 weeks of the high-fat diet, homozygous mice showed lower fasting glucose levels and exhibited higher glucose clearance and lower insulin levels than wild or heterozygous mice in glucose tolerance tests. Glucose clearance rates showed no significant difference among wild, heterozygous and homozygous mice in insulin tolerance tests. After 10 weeks of the high-fat diet, the alpha(1B)-deficient homozygous mice showed lower lipid deposition in liver and lower plasma glucagon, leptin and triglyceride levels than wild or heterozygous mice. These results suggest that N-type Ca2+ channels play a role in insulin and glucagon release, and that N-type Ca2+ channel alpha(1B)-subunit deficient mice show improved glucose tolerance without any change in insulin sensitivity. Thus, N-type Ca2+ channel blockers might be candidate anti-diabetic/anti-obesity agents.

Ataxia and peripheral nerve hypomyelination in ADAM22-deficient mice.

BACKGROUND: ADAM22 is a member of the ADAM gene family, but the fact that it is expressed only in the nervous systems makes it unique. ADAM22's sequence similarity to other ADAMs suggests it to be an integrin binder and thus to have a role in cell-cell or cell-matrix interactions. To elucidate the physiological functions of ADAM22, we employed gene targeting to generate ADAM22 knockout mice. RESULTS: ADAM22-deficient mice were produced in a good accordance with the Mendelian ratio and appeared normal at birth. After one week, severe ataxia was observed, and all homozygotes died before weaning, probably due to convulsions. No major histological abnormalities were detected in the cerebral cortex or cerebellum of the homozygous mutants; however, marked hypomyelination of the peripheral nerves was observed. CONCLUSION: The results of our study demonstrate that ADAM22 is closely involved in the correct functioning of the nervous system. Further analysis of ADAM22 will provide clues to understanding the mechanisms of human diseases such as epileptic seizures and peripheral neuropathy.

Quantitative mouse brain proteomics using culture-derived isotope tags as internal standards.

An important challenge for proteomics is to be able to compare absolute protein levels across biological samples. Here we introduce an approach based on the use of culture-derived isotope tags (CDITs) for quantitative tissue proteome analysis. We cultured Neuro2A cells in a stable isotope-enriched medium and mixed them with mouse brain samples to serve as internal standards. Using CDITs, we identified and quantified a total of 1,000 proteins, 97-98% of which were expressed in both mouse whole brain and Neuro2A cells. CDITs also allow comprehensive and absolute protein quantification. Synthetic unlabeled peptides were used to quantify the corresponding proteins labeled with stable isotopes in Neuro2A cells, and the results were used to obtain the absolute amounts of 103 proteins in mouse whole brain. The expression levels correlated well with those in Neuro2A cells. Thus, the use of CDITs allows both relative and absolute quantitative proteome studies.

Pattern of compensatory expression of voltage-dependent Ca2+ channel alpha1 and beta subunits in brain of N-type Ca2+ channel alpha1B subunit gene-deficient mice with a CBA/JN genetic background.

The Ca(2+) channel alpha(1B) subunit is a pore-forming component capable of generating N-type Ca(2+) channel activity. Although the N-type Ca(2+) channel plays a role in a variety of neuronal functions, alpha(1B)-deficient mice with a CBA/JN genetic background show no apparent behavioral or anatomical-histological abnormality, presumably owing to compensation by other Ca(2+) channels. In this study, we examined the mRNA expression of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits in the olfactory bulb, cerebral cortex, hippocampus and cerebellum of alpha(1B)-deficient mice. We found that the mRNA expression levels of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits were the same in the olfactory bulbs of wild, heterozygous and homozygous alpha(1B)-deficient mice. In the cerebral cortex, alpha(1A) mRNA in homozygous alpha(1B)-deficient mice was expressed at a higher level than in wild or heterozygous mice, but no difference in the expression levels of the alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits was found among wild, heterozygous and homozygous mice. In hippocampus and cerebellum, beta(4) mRNA in homozygous alpha(1B)-deficient mice was expressed at a higher level than in wild or heterozygous mice, but no difference in the expression levels of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2) and beta(3) subunits was found among wild, heterozygous and homozygous mice. These results suggest that the compensatory mechanisms differ in different brain regions of alpha(1B)-deficient mice with a CBA/JN genetic background.

Efficient in-gel digestion procedure using 5-cyclohexyl-1-pentyl-beta-D-maltoside as an additive for gel-based membrane proteomics.

A cycloalkyl aliphatic saccharide, 5-cyclohexyl-1-pentyl-beta-D-maltoside (CYMAL-5), was evaluated as a novel additive in a high-throughput in-gel protein digestion system using 96-well plates. Addition of 0.1% CYMAL-5 (final concentration) during trypsin treatment was compatible with both matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis, and gave a better digestion efficiency than n-octylglucoside, which we previously reported. In-gel reduction and alkylation of Cys residues under denaturing conditions also improved the sequence coverage of peptides. In-gel tryptic digestion with the optimum combination of 0.5 mm thick gels, negative staining, alkylation under denaturing conditions (6 M guanidine hydrochloride), and digestion in the presence of CYMAL-5, gave excellent performance especially for membrane protein analysis, where recovery of hydrophobic peptides was markedly enhanced. The new protocol is simple and convenient, and should be widely applicable to gel-based proteomics.

Synthesis and evaluation of novel pyrimido-acridone, -phenoxadine, and -carbazole as topoisomerase II inhibitors.

As part of a series of studies to discover new topoisomerase II inhibitors, novel pyrimidoacridones, pyrimidophenoxadines, and pyrimidocarbazoles were synthesized, and in vitro and in vivo antitumor activities and DNA-protein and/or DNA-topoisomerase II cross-linking activity as an indicator of topoisomerase II-DNA cleavable complex formation were evaluated. The pyrimidocarbazoles possessed high in vitro and in vivo potencies. Compound 26 (ER-37326), 8-acetyl-2-[2-(dimethylamino)ethyl]-1H-pyrimido[5,6,1-jk]carbazole-1,3(2H)-dione, showed in vitro growth inhibitory activity with respective IC(50) values of 0.049 microM and 0.35 microM against mouse leukemia P388 and human oral cancer KB. In vivo, this compound inhibited the tumor growth of mouse sarcoma M5076 implanted into mice with T/C values of 42% and 13% at 3.13 and 6.25 mg/kg/d respectively without significantly affecting the body weight. In addition, compound 26 (ER-37326) increased the formation of DNA-topoisomerase II cross-linking in P388 cells.

Expression of a human SOCS protein, HSOCP-1, in peripheral blood eosinophils from patients with atopic dermatitis.

To identify new genes related to atopic dermatitis (AD), we screened for differentially expressed genes in peripheral blood eosinophils derived from AD patients and healthy volunteers. RNA was prepared from peripheral blood eosinophils obtained from both AD patients and healthy volunteers, and the expression of various genes was monitored using fluorescent differential display and real-time RT-PCR. One of the expressed sequence tags (ESTs) was expressed at a significantly higher level in AD patients than in healthy volunteers. A full-length cDNA was identified that encoded a human suppressor of cytokine signaling (SOCS) protein, HSOCP-1, also named hASB-8. The expression of HSOCP-1 was increased in cultured peripheral blood eosinophils after IL-4 stimulation, and overexpression of HSOCP-1 caused cell death in an eosinophil cell line, AML14.3D10. p34(SEI-1) was identified as a HSOCP-1-interacting protein by a yeast two-hybrid system. It is a protein that also interacts with the cyclin-dependent kinase inhibitor p16(INK4), suggesting that HSOCP-1 is involved in cell cycle control and apoptosis.

Increased expression of P/Q-type Ca2+ channel alpha1A subunit mRNA in cerebellum of N-type Ca2+ channel alpha1B subunit gene-deficient mice.

The Ca(2+) channel alpha(1B) subunit is a pore-forming component capable of generating N-type Ca(2+) channel activity. Although the N-type Ca(2+) channel plays a role in a variety of neuronal functions, alpha(1B)-deficient mice show normal behavior, presumably owing to compensation by the other Ca(2+) channels. In this study, we examined the mRNA expression of the P/Q-type Ca(2+) channel alpha(1A) subunit in cerebellum of alpha(1B)-deficient mice. The alpha(1A) subunit mRNA in homozygous alpha(1B)-deficient mice was expressed at a significantly higher level than in wild or heterozygous mice. To examine whether the increased expression is induced by a cis-regulatory element within the 5'-upstream region of the alpha(1A) subunit gene, we examined lacZ expression in alpha(1B)-deficient x alpha(1A)3.0-lacZ mice (carrying a 3.0-kb 5'-upstream fragment of the alpha(1A) subunit gene fused to Escherichia coli lacZ reporter gene), which express lacZ in granule but not in Purkinje cells, and in alpha(1B)-deficient x alpha(1A)6.3-lacZ mice (carrying a 6.3-kb 5'-upstream region fused to lacZ gene), which express lacZ in Purkinje but not in granule cells. The levels of lacZ expression in homozygous alpha(1B)-deficient x alpha(1A)3.0-lacZ mice were significantly higher than in wild or heterozygous mice, but no difference in lacZ expression level was found among wild, heterozygous and homozygous alpha(1B)-deficient x alpha(1A)6.3-lacZ mice. Therefore, a possible explanation of the normal behavior of alpha(1B)-deficient mice is that compensation by alpha(1A) subunit gene occurs and that the 3.0-kb 5'-upstream region of alpha(1A) subunit gene contains an enhancer cis-element(s) for compensation in cerebellar granule cells.

Expression analysis of P/Q-type Ca2+ channel alpha 1A subunit mRNA in olfactory mitral cell in N-type Ca2+ channel alpha 1B subunit gene-deficient mice.

N-type and P/Q-type Ca2+ channels play an important role in the processing of olfactory information. However, N-type Ca2+ channel alpha1B-deficient mice show normal behavior, presumably owing to compensation by other Ca2+ channels. P/Q-type Ca2+ channel alpha1A mRNA was expressed at a higher level in olfactory bulb of homozygous alpha1B-deficient mice than wild-type or heterozygous mice. LacZ expression in olfactory mitral cells of homozygous alpha1B-deficient x alpha1A1.5-lacZ mice, carrying a 1.5-kb 5'-upstream fragment of the alpha1A gene fused to the lacZ reporter gene, was increased compared to that in wild-type or heterozygous mice. Therefore, a possible explanation for the normal behavior of alpha1B-deficient mice is compensation by the alpha1A gene and that the 1.5-kb 5'-upstream region of this gene contains an enhancer cis-element for compensation in olfactory mitral cells.

Effect of genetic background on Cav2 channel alpha1 and beta subunit messenger RNA expression in cerebellum of N-type Ca2+ channel alpha1B subunit-deficient mice.

Although the N-type Ca2+ channel plays a role in a variety of neuronal functions, N-type Ca2+ channel alpha1B-deficient mice exhibit normal life span without apparent behavioral or histologic abnormalities. To examine whether the reason for their normal behavior is compensation by other Cav2 channel alpha1 or beta subunit genes and to analyze whether genetic background influences the subunit expression pattern, we studied the alpha1A, alpha1E, beta1b, beta2, beta3 and beta4 subunit mRNA levels in cerebellum of alpha1B-deficient mice with CBA x C57BL/6 or CBA/JN background. In cerebellum of the mice with a CBA x C57BL/6 background, alpha1A mRNA was expressed at a higher level than that in wild-type or heterozygous mice, but difference in the expression levels of alpha1E, beta1b, beta2, beta3 and beta4 subunits was not found among wild-type, heterozygous, and homozygous mice. In cerebellum of alpha1B-deficient mice with CBA/JN background, beta4 mRNA was expressed at a higher level than that in wild-type or heterozygous mice, but alpha1A, alpha1E, beta1b, alpha2, beta3 and transcripts were expressed at similar levels in all genotypes. Therefore, a possible explanation of the normal behavior of alpha1B-deficient mice is that Cav2 channel family members compensate for the deficiency, and that the change of functional subunit expression pattern for compensation differs in animals with different genetic backgrounds.

Analysis of gene expression in peripheral blood eosinophils from patients with atopic dermatitis by differential display.

To identify the genes related to atopic dermatitis (AD), we compared gene expression in eosinophils from AD patients and healthy volunteers. RNA was prepared from peripheral blood eosinophils. Gene expression was monitored by fluorescent differential display (DD) and real-time RT-PCR. Eighteen new sequences, including expressed sequence tags (ESTs), were expressed at higher levels in eosinophils from AD patients than in those from healthy volunteers. The functions of most of these genes are unknown. We found no correlation between the expression of a particular gene and clinical markers such as the number of eosinophils and the amount of IgE. Multivariate analysis of the gene expression data in each sample showed a very high coefficient of correlation among the copy numbers of each gene. The genes under investigation were also expressed in cultured blood eosinophils after IL-4, IL-5 and IFN-gamma stimulation. We were able to predict the function of some of the sequences by scanning for homologies within either the human or mouse genome databases. The mouse counterpart of one of these genes, intersectin 2, was expressed dramatically, as measured by ear edema, in 1-fluoro-2,4-dinitrobenzene-induced mouse contact dermatitis and in NC/Nga mouse dermatitis.