Evaluation of QSAR models for predicting mutagenicity: outcome of the Second Ames/QSAR international challenge project.

Quantitative structure-activity relationship (QSAR) models are powerful in silico tools for predicting the mutagenicity of unstable compounds, impurities and metabolites that are difficult to examine using the Ames test. Ideally, Ames/QSAR models for regulatory use should demonstrate high sensitivity, low false-negative rate and wide coverage of chemical space. To promote superior model development, the Division of Genetics and Mutagenesis, National Institute of Health Sciences, Japan (DGM/NIHS), conducted the Second Ames/QSAR International Challenge Project (2020-2022) as a successor to the First Project (2014-2017), with 21 teams from 11 countries participating. The DGM/NIHS provided a curated training dataset of approximately 12,000 chemicals and a trial dataset of approximately 1,600 chemicals, and each participating team predicted the Ames mutagenicity of each trial chemical using various Ames/QSAR models. The DGM/NIHS then provided the Ames test results for trial chemicals to assist in model improvement. Although overall model performance on the Second Project was not superior to that on the First, models from the eight teams participating in both projects achieved higher sensitivity than models from teams participating in only the Second Project. Thus, these evaluations have facilitated the development of QSAR models.

NFKBIL1 confers resistance to experimental autoimmune arthritis through the regulation of dendritic cell functions.

We and others have reported that human NF-κB inhibitor-like-1 (NFKBIL1) was a putative susceptible gene for autoimmune diseases such as rheumatoid arthritis (RA). However, its precise role in the pathogenesis of RA is still largely unknown. In this study, we generated transgenic mice expressing human NFKBIL1 (NFKBIL1-Tg) and examined whether NFKBIL1 plays some role(s) in the development of autoimmune arthritis. In both a collagen-induced arthritis model and a collagen antibody-induced arthritis model, NFKBIL1-Tg mice showed resistance to arthritis compared to control mice, indicating that the gene product of NFKBIL1 was involved in the control of thusly induced arthritis. Total spleen cells of NFKBIL1-Tg mouse showed decreased proliferation to mitogenic stimuli, consistent with its resistance to arthritis. Unexpectedly, purified T cells of NFKBIL1-Tg mouse showed increased proliferation and cytokine production. This apparent discrepancy was accounted for by the impaired functions of antigen-presenting cells of NFKBIL1-Tg mouse; both T/B cell-depleted spleen cells and bone marrow-derived dendritic cells of the Tg mouse induced less prominent proliferation and IL-2 production of T cells. Furthermore, dendritic cells (DCs) derived from NFKBIL1-Tg mouse showed lower expression of co-stimulatory molecules and decreased production of inflammatory cytokines when they were activated by lipopolysaccharide. Taken together, these results indicated that NFKBIL1 affected the pathogenesis of RA at least in part through the regulation of DC functions.

Mapping of susceptibility locus for endometriosis within the HLA region using microsatellite markers in Japanese women.

Endometriosis is a female disorder characterized by the presence of uterine endometrial tissue in ectopic loci. Previous studies reported a higher prevalence of particular human leukocyte antigen (HLA) in endometriosis. In order to confirm the association between endometriosis and the HLA region, 15 polymorphic microsatellite markers distributed in the HLA class II to class III region were subjected to association analysis by polymerase chain reaction (PCR)-based DNA typing of 89 patients and 136 healthy controls. Statistical analysis of the allelic frequency at each microsatellite locus showed that there were no statistically significant differences in the allele frequency distributions between the cases and controls. This finding suggests that the etiology of endometriosis does not involve the HLA class II genomic region and a portion of class III genomic region in the Japanese population.

Role of capsaicin-sensitive afferent neurons in receptive relaxation induced by gastric distension in rats.

We investigated the role of capsaicin-sensitive afferent neurons in receptive relaxation of the rat stomach in response to distension. Under urethane anesthesia, a balloon with barostat was inserted through the pylorus and placed in the forestomach. Isobaric distension was performed in a stepwise increment of 2 mmHg, each lasting for 2 min, while the corresponding intragastric volume was recorded. Gastric distension produced the intraballoon volume in a progressive manner with saturation, suggesting the occurrence of receptive relaxation of the stomach during distension. Intragastric application of capsaicin significantly enhanced the degree of receptive relaxation. The capsaicin-induced enhancement of receptive relaxation was totally abolished by bilateral vagotomy as well as chemical ablation of capsaicin-sensitive afferent neurons. Likewise, the enhanced receptive relaxation in response to stomach distension was also significantly attenuated by pretreatment of the animals with NG-nitro-L-arginine methyl ester (L-NAME, an inhibitor of nitric oxide (NO) synthase), calcitonin gene related peptide (CGRP)8-37 (CGRP antagonist), indomethacin and ONO-8711 (EP1 receptor antagonist). These results suggest that capsaicin significantly enhanced the receptive relaxation induced by gastric distention through both vagal nerves and capsaicin-sensitive afferent neurons. This process is intervened by endogenous NO and CGRP in addition to prostaglandins (PGs), and the effect of PGs may be mediated by EP1 receptors.

Susceptibility locus for non-obstructive azoospermia is localized within the HLA-DR/DQ subregion: primary role of DQB1*0604.

Non-obstructive azoospermia is a male infertility characterized by no or little sperm in semen as a result of a congenital dysfunction in spermatogenesis. Previous studies have reported a higher prevalence of particular human leukocyte antigen (HLA) antigens in non-obstructive azoospermia. As the expression of the RING3 gene located in the HLA class II region was predominant in the testis, mainly around spermatids and pachytene spermatocytes, it is tempting to speculate that RING3 is one of the strong candidate genes responsible for the pathogenesis of the disease. In this study, the genetic polymorphism in the RING3 gene was investigated by the direct sequencing technique. As a result, a total of 14 single nucleotide polymorphisms were identified. Among them, six were localized in the coding region but none of them was accompanied by an amino-acid substitution. No significant difference in the allelic distribution at these 14 polymorphic sites was observed between the patients and healthy controls, suggesting that the susceptible gene for non-obstructive azoospermia is not the RING3 gene. Then, in order to map the susceptibility locus for non-obstructive azoospermia precisely within the HLA region, 11 polymorphic microsatellite markers distributed from the SACM2L gene just outside the HLA class II region (187 kb telomeric of the DPB1 gene) to the OTF3 gene in the HLA class I region were subjected to association analysis in the patients. Statistical analysis of distribution in the allelic frequency at each microsatellite locus demonstrated that the pathogenic gene for non-obstructive azoospermia is located within the HLA-DR/DQ subregion. In fact, DRB1*1302 and DQB1*0604 were found to be strongly associated with non-obstructive azoospermia by polymerase chain reaction-based DNA typing. Further, haplotype analysis suggested that the DQB1*0604 allele may play a decisive role in the pathogenesis of non-obstructive azoospermia.

New polymorphic microsatellite markers in the human MHC class III region.

The human major histocompatibility complex (MHC) class III region spanning approximately 760 kb is characterized by a remarkably high gene density with 59 expressed genes (one gene every 12.9 kb). Recently, susceptibility loci to numerous diseases, such as Graves disease, Crohn disease, and SLE have been suggested to be localized to this region, as assessed by associations mainly with genetic polymorphisms of TNF and TNF-linked microsatellite loci. However, it has been difficult to precisely localize these susceptibility loci to a single gene due to a paucity to date of polymorphic markers in the HLA class III region. To facilitate disease mapping within this region, we have analyzed 2 approximately 5 bases short tandem repeats (microsatellites) in this region. A total of 297 microsatellites were identified from the genomic sequence, consisting of 69 di-, 62 tri-, 107 tetra-, and 59 penta-nucleotide repeats. It was noted that among them as many as 17 microsatellites were located within the coding sequence of expressed genes (NOTCH4, PBX2, RAGE, G16, LPAAT, PPT2, TNXB, P450-CYP21B, G9a, HSP70-2, HSP70-1, HSP-hom, MuTSH5 and BAT2). Eight microsatellite repeats were collected as polymorphic markers due to their high number of alleles (11.9 on average) as well as their high polymorphic content value (PIC) (0.63). By combining the 38 and the 22 polymorphic microsatellites we have previously collected in the HLA class I and class II regions, respectively, we have now established a total of 68 novel genetic markers which are uniformly interspersed with a high density of one every 63.3 kb throughout the HLA region. This collection of polymorphic microsatellites will enable us to search for the location of any disease susceptible loci within the HLA region by association analysis.

New polymorphic microsatellite markers in the human MHC class II region.

The human major histocompatibility complex (MHC) class II region spans approximately 1.1 Mb and presently contains over 30 functional genes Susceptibility loci to numerous diseases, mainly of autoimmune nature are known to map to the this region, as assessed by associations with particular HLA class II alleles. However, it has been difficult to precisely localize these susceptibility loci to a single gene, for example DQB1 or DRB1, due to the tight linkage disequilibrium observed in the HLA class II region. To facilitate disease mapping within this region, we have analyzed 2 to approximately 5 bases short tandem repeats (microsatellites) in this same region. A total of 494 microsatellites were identified from the genomic sequence of the HLA class II region. These consist of 158 di-, 65 tri-, 163 tetra-, and 108 pent-nucleotide repeats, out of which four were located within the coding sequence of expressed genes (Daxx, BING1, RXRB and COL11A2). Twenty-two repeats were selected as polymorphic markers due to their high (average) number of alleles (8.9) as well as their high polymorphic content value (PIC) (0.58). These novel polymorphic microsatellites will provide useful genetic markers in HLA-related research, such as genetic mapping of HLA class II-associated diseases, transplantation matching, population genetics, identification of recombination hot spots as well as linkage disequilibrium studies.

Nucleotide sequence of the ring3 gene in the class II region of the mouse MHC and its abundant expression in testicular germ cells.

The RING3 (NAT) gene is the first and only locus with no obvious function associated with the immune system in the class II region of the human major histocompatibility complex. This gene is a homologue of the Drosophila homeotic gene female sterile homeotic (fsh) and encodes a nuclear serine-threonine kinase. To study more about the physiological function of the RING3 gene, we isolated a mouse homologue from a genomic library, determined its gene structure, and investigated its expression profile. The mouse Ring3 gene spans approximately 8 kb and consists of 12 exons encoding a 798-amino-acid protein, sharing as high as 96% amino acid identity with the human RING3 protein. Northern hybridization revealed that the Ring3 gene abundantly produced 3.8- and 3.0-kb transcripts in the testis but was weakly expressed with 4.6- and 3.8-kb transcripts in somatic tissues. It appears that testis-specific 3.0-kb transcript gives rise to a smaller size Ring3 protein resulting from the usage of the second ATG codon for translational initiation compared to the almost ubiquitous 4.6-kb transcript. In RNAs isolated from fractionated testicular germ cells, the two testicular mRNAs were detected exclusively in the fractions containing a large population of round spermatids and pachytene spermatocytes. Furthermore, in situ hybridization on cross sections of seminiferous tubules in the testis showed that the expression of the Ring3 gene was initiated at the pachytene spermatocyte stage during meiosis and persisted throughout the round spermatid stage during spermiogenesis. These results suggest that the Ring3 gene plays an important role in spermatogenesis.

Changing directions of forthcoming arm movements: neuronal activity in the presupplementary and supplementary motor area of monkey cerebral cortex.

1. To understand roles played by two cortical motor areas, the presupplementary motor area (pre-SMA) and supplementary motor area (SMA), in changing planned movements voluntarily, cellular activity was examined in two monkeys (Macaca fuscata) trained to perform an arm-reaching task in which they were asked to press one of two target buttons (right or left) in three different task modes. 2. In the first mode (visual), monkeys were visually instructed to result and press either a right or left key in response to a forth coming trigger signal. In the second mode (stay), monkeys were required to wait for the trigger signal and press the same target key as pressed in preceding trials. In the third mode (shift), a 50 Hz auditory cue instructed the monkey to shift the target of the future reach from the previous target to the previous nontarget. 3. While the monkeys were performing this task, we recorded 399 task-related cellular activities from the SMA and the pre-SMA. Among them, we found a group of neurons that exhibited activity changes related specifically to shift trials (shift-related cells). The following properties characterized these 112 neurons. First, they exhibited activity changes after the onset of the 50-Hz auditory cue and before the movement execution when the monkeys were required to change the direction of forthcoming movement. Second, they were not active when the monkeys pressed the same key without changing the direction of the movements. Third, they were not active when the monkeys received the 50-Hz auditory cue but failed to change the direction of the movements by mistake. These observations indicate that the activity of shift-related cells is related to the redirection of the forthcoming movements, but not to the auditory instruction itself or to the location of the target key or the direction of the forthcoming movements. 4. Although infrequently, monkeys made errors in the stay trials and changed directions of the reach voluntarily. In that case, a considerably high proportion of shift-related neurons (12 of 19) exhibited significant activity changes long before initiation of the reach movement. These long-lasting activities were not observed during the preparatory period in correct stay trials, but resembled the shift-related activity observed when the target shift was made toward the same direction. Thus these activity changes were considered to be also related to the process of changing the intended movements voluntarily. 5. We found another population of neurons that showed activity modulation when the target shift was induced by the visual instruction in visual trials (visually guided shift-related neurons). These neurons were active when the light-emitting diode (LED) guided the forthcoming reach to the previous nontarget but not to the previous target. Therefore their activity was not a simple visual response to the LED per se. A majority of them also showed shift-related activity in shift trials (19 of 22 in monkey 2). 6. Neurons exhibiting the shift-related activity were distributed differentially among the two areas. In the pre-SMA, 31% of the neurons recorded showed the shift-related activity, whereas in the SMA, only 7% showed such an activity. These results suggest that pre-SMA and SMA play differential roles in updating the motor plans in accordance with current requirements.

Neuronal activity in the primate supplementary, pre-supplementary and premotor cortex during externally and internally instructed sequential movements.

This study recorded the activity of neurons in the (i) supplementary motor area (SMA), (ii) pre-SMA (the motor area immediately rostral to the SMA), (iii) premotor cortex (PMC) and (iv) primary motor cortex (MI), while the monkey performed a conditional sequential motor task that ensures sequencing of multiple movements to the same manipulandum. This paradigm was chosen in order to prevent the participation of spatial cues in prompting the correct motor sequence. Three different movements (turn-push-pull) were performed under two task conditions: (i) internally determined (I): the monkey had to generate a pre-determined sequence from memory and without visual guidance; (ii) externally triggered (E): the correct sequence of movements was performed by following lights illuminated one after the other. Neuronal activity during the following periods were analyzed: instruction (300 ms following the onset of the auditory instruction signal); delay (interval between the end of the instruction period or the termination of the previous movement and the movement trigger); premovement (interval between the trigger signal and the movement onset); movement (interval between the mechanically-sensed movement onset and the completion of the movement) and reward (500 ms period centered at the time of reward delivery). Pre-SMA neurons were generally more active during the delay and premovement as compared to the movement, instruction and reward periods. Activity in the pre-SMA was more related to E during the pre-movement period, but exhibited a preferential relationship to I in the movement period. SMA neurons were more active when the sequential motor task was internally generated.(ABSTRACT TRUNCATED AT 250 WORDS)

Change of retinal correspondence in monocularly reduced intensity of stimulus.

We analyzed the retinal correspondence with a phase difference haploscope on reducing the stimulus intensity of a monocular image with a series of neutral density filters. Ninety-one exotropes were examined by this method. Five cases changed from normal to anomalous correspondence when the stimulation of the strabismic eye was reduced. Two cases showed anomalous correspondence without a filter, but normal correspondence with a reduced stimulus of the normal eye. All these seven cases showed normal correspondence with any other test. The results suggest that masked anomalous correspondence becomes manifest in some cases when the strabismic eye is stimulated more intensely than the normal eye. These cases display both normal and anomalous correspondence and this condition may be called dual correspondence.

[Study of retinal correspondence in esotropia with phase difference haploscope].

There is some concern regarding the evaluated depth of anomalous correspondence, because we often experience paradoxical results. We have already reported that, in intermittent exotropia, we sometimes found dual correspondence with a phase difference haploscope (PDH) reducing the stimulus intensity of either image with a series of neutral density filters. In this study, we analyzed the retinal correspondence in esotropes with the same procedure. 27 esotropes were examined. 11 cases had normal correspondence and 16 cases anomalous correspondence in PDH. All of the 11 normal correspondence cases retained normal correspondence when we reduced the stimulus intensity of either image of fixating eyes or squinting eyes. In the 16 anomalous correspondence cases in PDH, 2 cases showed normal correspondence when the stimulus intensity of the fixating eyes was reduced. On the other hand, 3 cases showed normal correspondence when the stimulus intensity of the squinting eyes was reduced. In fixed heterotropia, both the fovea and the part of the retina of the squinting eye which received the same images as the fovea of the fixating eye (fixating point), were suppressed by the fixating eye. We consider that, in some cases with anomalous retinal correspondence in PDH, when the stimulus intensity of the fixating eyes or the squinting eyes is reduced, the balance of the sensitivity of the foveas and the fixating points of the squinting eyes change, and show normal correspondence.

A motor area rostral to the supplementary motor area (presupplementary motor area) in the monkey: neuronal activity during a learned motor task.

1. The rostromesial agranular frontal cortex of macaque monkey (Macaca fuscata), traditionally defined as the supplementary motor area (SMA), was studied using various physiological techniques to delineate two different areas rostrocaudally. 2. Field and unitary responses to electrical stimulation of the primary motor cortex were distinct in the caudal part, but minimal or absent in the rostral part. Intracortical microstimulation readily evoked limb or orofacial movements in the caudal part, but only infrequently in the rostral part. Neuronal responses to visual stimuli prevailed in the rostral part, but somatosensory responses were rare. The opposite was true in the caudal part. 3. The rostral part, roughly corresponding to area 6a beta, was operationally defined as the presupplementary motor area (pre-SMA). The caudal part was redefined as the SMA proper. 4. Single-cell activity in the pre-SMA was quantitatively compared with that in the SMA proper in relation to a trained motor task. 5. Phasic responses to visual cue signals indicating the direction of forthcoming arm-reaching movement were more abundant in the pre-SMA. 6. Activity changes during the preparatory period, which lasted until the occurrence of the trigger signal for the reaching movement, were more frequent in the pre-SMA. 7. Phasic, movement-related activity was more frequent in the SMA, and its onset was often time locked to the movement onset. In the pre-SMA, the occurrences of response time locked to the movement-trigger signal were more frequent than in the SMA. 8. Among neurons in both areas, directional selectivity was found in all the cue, preparatory, and movement-related responses.