Brain-derived neurotrophic factor treatment increases the skeletal muscle glucose transporter 4 protein expression in mice.

The purpose of the present study was to investigate whether peripheral brain-derived neurotrophic factor (BDNF) treatment induced metabolic adaptations in mouse skeletal muscle. BDNF (20 mg/kg/day) was injected subcutaneously for successive 14 days. BDNF treatment significantly reduced the total food intake and inhibited the weight gain in comparison to the control group. The glucose transporter 4 (GLUT4) protein expression in the gastrocnemius muscle was significantly increased by BDNF treatment in comparison to the control and pair-fed groups. Neither the oxidative nor the glycolytic enzyme activities in the gastrocnemius muscle changed after the BDNF treatment. These results suggest that the peripheral BDNF treatment promotes the skeletal muscle GLUT4 protein expression as well as hypophagia.

Cellular adaptations in soleus muscle during recovery after hindlimb unloading.

AIM: We used a model of chronic unloading followed by reloading to examine the apoptotic responses associated with soleus muscle atrophy and subsequent recovery. METHODS: Male Wistar rats were subjected to hindlimb unloading (HU) for 2 weeks and subsequent reloading for 0, 3, 7 and 14 days. One-half of the HU-reloaded rats were administered cyclosporine A (CsA), a calcineurin (CaN) inhibitor. RESULTS: There was fibre atrophy (73%) and a decrease in slow type I fibre/myosin heavy chain (MyHC) composition in the soleus muscle after 2 weeks of HU. Fibre size and type I MyHC composition recovered to near the age-matched control levels by recovery day 14 in non-treated, but not in CsA-treated, rats. Myonuclear number was lower and the number of apoptotic nuclei higher in 2-week HU than control rats. These values returned to control levels after 7 and 14 days of recovery, respectively, in both HU-recovery groups. After 2 weeks of HU, the levels of heat shock proteins (Hsp) 60 and 72, mitochondrial cytochrome c oxidase subunit IV (Cox IV), and peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1) proteins were lower than control. The levels of all of these proteins gradually increased to or above the control levels during cage recovery in both groups. CONCLUSION: Our results indicate that apoptotic mechanisms are involved in the modulation of myonuclear number during chronic unloading and subsequent reloading. Furthermore, it appears that CaN is related to fibre size and phenotype adaptations, but not to apoptotic responses.

Assessment of the otolith-autonomic response using spectral analysis of the R-R interval and blood pressure.

The effects of otolith stimulation on systolic blood pressure (SBP) and R-R interval fluctuations were investigated in 12 healthy subjects and 5 patients with loss of labyrinthine function. The vestibular otolith organs were stimulated by centrifugal forces, the constant rotation of a chair fixed outside of the rotation axis on the horizontal plane. The chair was fixed along the direction of centrifugal force (eccentric rotation [ECR]) or along the tangent line (eccentric lateral rotation [ECLR]). The ECR stimulates both the utricular and saccular maculae, whereas the ECLR stimulates only the utricular maculae. Spectral analysis showed that in the healthy subjects, low-frequency SBP increased significantly during ECR but not during ECLR. There was no significant increase in low-frequency SBP in patients with labyrinthine loss. In the R-R interval analysis, however, the low- and high-frequency components did not change significantly in the healthy subjects during ECR. Our findings indicate that stimulation of the otolith maculae, especially the saccular organs, predominantly produces augmentation of the alpha-sympathetic activities rather than cardiac sympathovagal outflow to the heart.

A scaffold protein in the c-Jun NH2-terminal kinase signaling pathways suppresses the extracellular signal-regulated kinase signaling pathways.

We previously reported that c-Jun NH(2)-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1) functions as a putative scaffold factor in the JNK mitogen-activated protein kinase (MAPK) cascades. In that study we also found MEK1 and Raf-1, which are involved in the extracellular signal-regulated kinase (ERK) MAPK cascades, bind to JSAP1. Here we have defined the regions of JSAP1 responsible for the interactions with MEK1 and Raf-1. Both of the binding regions were mapped to the COOH-terminal region (residues 1054-1305) of JSAP1. We next examined the effect of overexpressing JSAP1 on the activation of ERK by phorbol 12-myristate 13-acetate in transfected COS-7 cells and found that JSAP1 inhibits ERK's activation and that the COOH-terminal region of JSAP1 was required for the inhibition. Finally, we investigated the molecular mechanism of JSAP1's inhibitory function and showed that JSAP1 prevents MEK1 phosphorylation and activation by Raf-1, resulting in the suppression of the activation of ERK. Taken together, these results suggest that JSAP1 is involved both in the JNK cascades, as a scaffolding factor, and the ERK cascades, as a suppressor.

c-Abl tyrosine kinase is not essential for ataxia telangiectasia mutated functions in chromosomal maintenance.

c-Abl is activated by DNA damage in an ataxia telangiectasia mutated (ATM)-dependent manner and plays important roles in growth arrest and apoptosis induced by DNA damage. c-Abl also interacts physically and functionally with Rad51, a key molecule in homologous recombinational (HR) DNA repair. To study further the roles of c-Abl in HR DNA repair, we generated c-Abl(-/-) and ATM(-/-)/c-Abl(-/-) mutant cell lines from a chicken B lymphocyte DT40 cell line, comparing the phenotypes of these mutants to those of ATM(-/-) DT40 cells that we had created previously. We found that the time course of radiation-induced Rad51 focus formation is abnormal in ATM(-/-) DT40 cells, consistent with the observation that ATM(-/-) DT40 cells display hypersensitivity to ionizing radiation and highly elevated frequencies of both spontaneous and radiation-induced chromosomal aberrations. In contrast, c-Abl(-/-) cells did not show these ATM-related defects in their cellular response to radiation, nor did the disruption of c-Abl in ATM(-/-) DT40 cells exacerbate these ATM-related defects. However, c-Abl(-/-) DT40 cells, but not ATM(-/-) DT40 cells, were resistant to radiation-induced apoptosis, indicating an important role for c-Abl in this cellular response to ionizing radiation. These results therefore indicate that, although ATM plays an important role in genome maintenance, c-Abl is not essential for this ATM function. These findings suggest that c-Abl and ATM play important roles in the maintenance of the cell homeostasis in response to DNA damage that are, at least in part, independent.

JSAP1, a novel jun N-terminal protein kinase (JNK)-binding protein that functions as a Scaffold factor in the JNK signaling pathway.

The major components of the mitogen-activated protein kinase (MAPK) cascades are MAPK, MAPK kinase (MAPKK), and MAPKK kinase (MAPKKK). Recent rapid progress in identifying members of MAPK cascades suggests that a number of such signaling pathways exist in cells. To date, however, how the specificity and efficiency of the MAPK cascades is maintained is poorly understood. Here, we have identified a novel mouse protein, termed Jun N-terminal protein kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1), by a yeast two-hybrid screen, using JNK3 MAPK as the bait. Of the mammalian MAPKs tested (JNK1, JNK2, JNK3, ERK2, and p38alpha), JSAP1 preferentially coprecipitated with the JNKs in cotransfected COS-7 cells. JNK3 showed a higher binding affinity for JSAP1, compared with JNK1 and JNK2. In similar cotransfection studies, JSAP1 also interacted with SEK1 MAPKK and MEKK1 MAPKKK, which are involved in the JNK cascades. The regions of JSAP1 that bound JNK, SEK1, and MEKK1 were distinct from one another. JNK and MEKK1 also bound JSAP1 in vitro, suggesting that these interactions are direct. In contrast, only the activated form of SEK1 associated with JSAP1 in cotransfected COS-7 cells. The unstimulated SEK1 bound to MEKK1; thus, SEK1 might indirectly associate with JSAP1 through MEKK1. Although JSAP1 coprecipitated with MEK1 MAPKK and Raf-1 MAPKKK, and not MKK6 or MKK7 MAPKK, in cotransfected COS-7 cells, MEK1 and Raf-1 do not interfere with the binding of SEK1 and MEKK1 to JSAP1, respectively. Overexpression of full-length JSAP1 in COS-7 cells led to a considerable enhancement of JNK3 activation, and modest enhancement of JNK1 and JNK2 activation, by the MEKK1-SEK1 pathway. Deletion of the JNK- or MEKK1-binding regions resulted in a significant reduction in the enhancement of the JNK3 activation in COS-7 cells. These results suggest that JSAP1 functions as a scaffold protein in the JNK3 cascade. We also discuss a scaffolding role for JSAP1 in the JNK1 and JNK2 cascades.

Evaluation of the mouse lymphoma tk assay (microwell method) as an alternative to the in vitro chromosomal aberration test.

In order to evaluate the utility of the mouse lymphoma assay (MLA) for detecting in vitro clastogens and spindle poisons and to compare it with the in vitro chromosomal aberration test (CA), we conducted an international collaborative study of the MLA that included 45 Japanese laboratories and seven overseas laboratories under the cooperation of the Ministry of Health and Welfare of Japan and the Japanese Pharmaceutical Manufacturer's Association. We examined 40 chemicals; 33 were reportedly positive in the CA but negative in the bacterial reverse mutation assay, six were negative in both assays and one was positive in both. We assayed mutations of the thymidine kinase (TK) locus (tk) of L5178Y tk +/- mouse lymphoma cells using the microwell method. According to our standard protocol, cells were exposed to the chemical for 3 h, cultured for 2 days and TK-deficient mutants were expressed in 96-well plates under trifluorothymidine. Each chemical was coded and tested by two or three laboratories. Among the 34 CA-positive chemicals, positive MLA results were obtained for 20 and negative results were obtained for nine. The remaining five chemicals were inconclusive or equivocal because of discrepant inter-laboratory results or reproduced discrepant results, respectively. Among the six CA-negative chemicals, one was negative in the MLA, two were positive and three were inconclusive. Thus, the MLA could detect only 59% (20/34) of CA-positive chemicals. We concluded that the MLA was not as sensitive as the CA. Some MLA-negative chemicals evoked positive responses in the CA only after long continuous treatment. These might also be genotoxic in the MLA with long continuous treatment. Improvement of the MLA protocol, including alteration of the duration of the treatment, might render the MLA as sensitive as the CA.

Detection of in vitro clastogens and spindle poisons by the mouse lymphoma assay using the microwell method: interim report of an international collaborative study.

Under the auspices of the Ministry of Health and Welfare of Japan and the Japanese Pharmaceutical Manufacturer Association, a collaborative study of the mouse lymphoma assay (MLA) was conducted by 42 Japanese laboratories and seven overseas laboratories to clarify the performance of the MLA for the detection of in vitro clastogens and spindle poisons. Twenty-one chemicals that were positive in in vitro chromosomal aberration assays (CA) but negative in bacterial reverse mutation assays (BRM) were examined by the MLA using the microwell method. All chemicals were coded, and each chemical was tested by two or three laboratories. Positive responses were obtained with 14 chemicals: mitomycin C (an internal positive control), arsenic trioxide, cadmium sulphate, chlorendic acid, cytosine arabinoside, diethylstilbestrol, eugenol, 5-fluorouracil, griseofulvin, hexamethyl phosphoramide, hydroxyurea, methotrexate, monocrotaline and pentachloroethane. Two chemicals (benzene and chlorodibromomethane) showed positive responses in one of two laboratories and were judged probably positive chemicals. Three chemicals (bromodichloromethane, isophorone and tetrachloroethane) were inconclusive because of a marginal response in one laboratory and a negative response in the other. Urethane was judged probably negative because two laboratories out of three showed clear negative responses. Dideoxycytidine (DDC) was a clear negative chemical in this study. The present results showed that 75.0% of the test chemicals (15/20, excluding mitomycin C) were positive, 15.0% (3/20) were inconclusive, and 10.0% (2/20) were negative. This suggests that the MLA may detect a majority of CA-positive chemicals. The inconclusive chemicals, however, are critical for the judgement of the MLA potential to detect clastogens. The findings that DDC was clearly negative suggests that the MLA may not be able to detect some clastogens. To clarify these issues, we began the second phase of the collaborative study with other BRM-negative and CA-positive chemicals.

Occurrence of premature chromosome condensation in mouse spermatogonia treated with busulfan.

Busulfan induction of premature chromosome condensation (PCC) was examined in mouse spermatogonia. Adult (C3H X SWV) F1 male mice were injected intraperitoneally with busulfan at 10 or 30 mg/kg of body weight and killed 18-72 h later. Polyploid-like mitoses were frequent (ca. 1/4 of all spermatogonial mitoses examined) in both the untreated and treated groups. Most of these were considered to have been derived from normal spermatogonia. In busulfan-treated mice, polyploid-like mitoses with PCC were seen. The frequency of PCC-containing mitoses increased with increasing dose and exposure time. A possible interpretation for PCC induction in mouse spermatogonia is discussed.

Induction of micronuclei in mouse fetal liver after exposure in utero to N-methyl-N'-nitro-N-nitrosoguanidine.

The effects of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) on the induction of micronuclei were examined in mouse fetuses exposed in utero. By this study, MNNG was proved to be mutagenic in vivo. The frequency of micronucleated erythrocytes (MNEs) in fetal liver peaked at 18 h after a single intraperitoneal injection into pregnant mice on day 13 of gestation. Then, to examine the effects of administration routes on the induction of micronuclei, the chemical was given by various routes, and the percentage of MNEs (%MNEs) in fetuses were examined 18 h after treatment. The %MNEs after administration of MNNG intraperitoneally, subcutaneously, intravenously, and orally was 4.7, 1.9, 0.8 and 0.3, respectively. The control value was 0.3. In the intraperitoneally treated mice, %MNEs for fetuses in the uterine horn located nearer the injection site was higher than that in the other. In addition, in the intraperitoneally treated mice, there was a tendency for the higher %MNEs to occur in the fetuses located near the injection site. Together with the results on the distribution of MNNG in mice (Frei and Lawley, 1976), these findings suggest that MNNG might be inactivated in the maternal systemic circulation and that the agent which induces micronuclei might be distributed to the fetuses by diffusion.

Effect of quinonyl-N-acetyl muramyl dipeptide on immune responses in tumor-bearing mice.

The efficacy of 6-O-QS-10-N-acetyl muramyl-L-valyl-D-isoglutamine methyl ester (quinonyl-MDP-66) for restoring impaired immune status was examined in mice bearing Lewis lung carcinoma. Quinonyl-MDP-66 suspended in phosphate-buffered saline was shown to restore the depressed allogeneic cell-mediated cytotoxicity of spleen cells from mice with Lewis lung carcinoma when the chemical was injected twice intraperitoneally, intravenously, or intratumorally. However, primary tumor size and the numbers of lung metastases were not affected when quinonyl-MDP-66 was administered under the present experimental conditions. Intraperitoneal injection of quinonyl-MDP-66 in mice with Lewis lung carcinoma enhanced host resistance to Listeria monocytogenes infection.

Studies on micronuclei time response and on the effects of multiple treatments of mutagens on induction of micronuclei.

Micronuclei time response and the effects of multiple treatments of mutagens on induction of micronuclei were studied. In the time-response investigation, mice were treated once with each of 5 mutagens, then killed at various times. The bone marrow was examined for the presence of micronucleated erythrocytes (MNEs). The maximal frequencies for MNEs occurred around 30 h post treatment for all mutagens tested. To examine the effects of multiple treatments, the frequencies of MNEs observed after a single- or a 5-treatment schedule were compared for 9 mutagens. Both treatment schedules were equally sensitive in detecting alkylating agents and spindle poisons, whereas the 5-treatment schedule was more sensitive for anti-metabolites. The 5-treatment schedule was particularly effective for detecting the anti-metabolites 5-fluorouracil and methotrexate, which require longer than 30 h to induce micronuclei (Maier and Schmid, 1976). These results suggest that it is practicable to sample at 30 h in the single-treatment schedule, and seem to support the usefulness of the 5-treatment schedule in screening tests.

Suppression of BCG cell wall induced delayed-type hypersensitivity by BCG pre-treatment. I. Induction of adherent suppressor cells by live BCG injection and their characterization.

Previous injections of live Bacillus Calmette-Guérin (BCG) in mice produced a suppression of delayed-type hypersensitivity (DTH) induced by oil-treated BCG cell walls (CW). This phenomenon was analysed by the macrophage migration inhibition (MI) test in which peritoneal exudate cells (PEC) from live BCG-injected mice were mixed with PEC from BCG CW-immunized mice, with the result that the former cells suppressed the MI activity in the latter. We considered the Mi test to be a reliable method for demonstrating the existence of suppressor cells induced by the injection of live BCG. Moreover, we found that the adherent cells of PEC possessed a suppressive effect which was retained even after treatment with either anti-mouse Ig or anti-brain associated theta (BA theta) antigen; that the PEC from mice injected with live BCG on at least the 12th day before cell harvesting showed the suppression; and that the suppression operated across the H-2 barrier.

Proteolysis of the C5b-7 complex: cleavage of the C5b and C6 subunits and its effect on the interaction of the complex with phospholipid bilayers.

The present study was conducted to gain insight into the process of assembly of the C5b-7 complex on the phospholipid bilayer. The C5b-6 complex, C7, and 2 different forms of the C5b-7 complex (the C5b-7 complex either bound to artificial phospholipid bilayers or in fluid phase) were digested with trypsin, and the resulting products were analyzed by SDS-polyacrylamide gel electrophoresis. The alpha'-chains of C5b and C6 in the C5b-6 complex and C7 were susceptible to trypsin. The formation of the C5b-7 complex from the C5b-6 complex and C7 was accompanied by alteration in susceptibility to trypsin to C7: the C7 subunit of the fluid phase complex as well as of the complex bound to phospholipid vesicles is not susceptible to trypsin. On the other hand, the alpha'-chains of C5b and C6 in the C5b-7 complex bound to phospholipid vesicles as well as in fluid phase remained accessible and susceptible to trypsin, and cleaved as in the C5b-6 complex. Upon ultracentrifugation in a sucrose density gradient, the proteolyzed C5b-7 complex in fluid phase dissociated into subunits. However, none of the subunits in the proteolyzed C5b-7 complex bound to phospholipid vesicles dissociated from vesicles upon sucrose density gradient ultracentrifugation. Thus, the molecule of the C5b-7 complex became stable and resistant to proteolytic dissociation upon binding to the phospholipid bilayer. It is proposed that each subunit of the C5b-7 complex participates in the stable interaction of the complex with the phospholipid bilayer. In addition, the possible significance of the alteration in susceptibility to trypsin of C7 associated with the formation of the C5b-7 complex is discussed.

A comparison of diameters of micronuclei induced by clastogens and by spindle poisons.

To compare the size of micronuclei induced by clastogens and by spindle poisons, bone-marrow smears were prepared 24 h after a single intraperitoneal injection of triethylenemelamine (TEM) (0.3 mg/kg) or vincristine (VCR) (0.125 mg/kg) into male mice. 100 micronucleated erythrocytes (MNEs) were randomly selected from each group and photomicrographed. Diameters of the cytoplasm (D) and the micronucleus (d) of each MNE were measured on the photographs. Relatively large micronuclei (d greater than or equal to D/4) were frequent (74%) in the VCR-treated group, and infrequent (2%) in the TEM-treated group. The frequencies of MNEs resulting in d greater than or equal to D/4 were determined for several other mutagens. All clastogens tested could be classified as TEM type, and all spindle poisons as VCR type. These findings indicate that it is possible to analyze the action site of micronucleus-inducing agents on the basis of the relative sizes of micronuclei.

A new method for the preparation of mammalian spermatogonial chromosomes.

After the seminiferous tubules were minced and washed with phosphate-buffered saline to remove sperms, spermatids and spermatocytes (Hoo and Bowles, 1971), they were repeatedly treated with 0.1% trypsin solution to liberate spermatogonia. It was concluded that the spermatogonial chromosomes can be analysed much more easily and accurately by this procedure than by previous ones.