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Fukutin, a product of the causative gene of Fukuyama congenital muscular dystrophy (FCMD), is known to be responsible for basement membrane formation. Patients with FCMD exhibit not only muscular dystrophy but also central nervous system abnormalities, including polymicrogyria and neurofibrillary tangles (NFTs) in the cerebral cortex. The formation of NFTs cannot be explained by basement membrane disorganization. To determine the involvement of fukutin in the NFT formation, we performed molecular pathological investigations using autopsied human brains and cultured neurons of a cell line (SH-SY5Y). In human brains, NFTs, identified with an antibody against phosphorylated tau (p-tau), were observed in FCMD patients but not age-matched control subjects and were localized in cortical neurons lacking somatic immunoreactivity for glutamic acid decarboxylase (GAD), a marker of inhibitory neurons. In FCMD brains, NFTs were mainly distributed in lesions of polymicrogyria. Immunofluorescence staining revealed the colocalization of immunoreactivities for p-tau and phosphorylated glycogen synthase kinase-3β (GSK-3β), a potential tau kinase, in the somatic cytoplasm of SH-SY5Y cells; both the immunoreactivities were increased by fukutin knockdown and reduced by fukutin overexpression. Western blot analysis using SH-SY5Y cells revealed consistent results. Enzyme-linked immunosorbent assay (ELISA) confirmed the binding affinity of fukutin to tau and GSK-3β in SH-SY5Y cells. In the human brains, the density of GAD-immunoreactive neurons in the frontal cortex was significantly higher in the FCMD group than in the control group. GAD immunoreactivity on Western blots of SH-SY5Y cells was significantly increased by fukutin knockdown. On immunofluorescence staining, immunoreactivities for fukutin and GAD were colocalized in the somatic cytoplasm of the human brains and SH-SY5Y cells, whereas those for fukutin and synaptophysin were colocalized in the neuropil of the human brains and the cytoplasm of SH-SY5Y cells. ELISA confirmed the binding affinity of fukutin to GAD and synaptophysin in SH-SY5Y cells. The present results provide in vivo and in vitro evidence for novel properties of fukutin as follows: (i) there is an inverse relationship between fukutin expression and GSK-3β/tau phosphorylation in neurons; (ii) fukutin binds to GSK-3β and tau; (iii) tau phosphorylation occurs in non-GAD-immunoreactive neurons in FCMD brains; (iv) neuronal GAD expression is upregulated in the absence of fukutin; and (v) fukutin binds to GAD and synaptophysin in presynaptic vesicles of neurons.

Pituicytoma is a rare neoplasm, arising in the posterior pituitary or in the hypophyseal stalk, and its cytological findings have not yet been well-described. We have experienced a case of pituicytoma, which was difficult to diagnose intraoperatively, because of its cellular pleomorphism. A tumor measuring 18 mm in maximum diameter was found at the sella turcica in a Japanese woman in her forties. Both intraoperative crush cytology and histology of the resected tumor showed pleomorphic spindle or round cells, including multinucleated cells. Tumor cells were positive for TTF-1, S-100 protein, and vimentin, partially positive for glial fibrillary acidic protein and epithelial membrane antigen, and negative for synaptophysin, hormones of the anterior pituitary gland, CD34, Olig2, PAX8, and napsin A. Ki-67 labeling index was 2.0%. Tumors included in the differential diagnosis in general are pituitary adenoma, craniopharyngioma, germinoma, and metastatic tumor on the radiological standpoint, and pilocytic astrocytoma and meningioma on the cytological standpoint. However, our case was difficult to differentiate especially from high-grade glioma only by morphology, and immunohistochemistry including TTF-1 was helpful.

A vast body of evidence implicates increased oxidative stress and extracellular glutamate accumulation in the pathomechanism of sporadic amyotrophic lateral sclerosis (ALS). Cystine/glutamate antiporter (xCT) carries extracellular cystine uptake and intracellular glutamate release (cystine/glutamate exchange) in the presence of oxidative stress. The aim of the present study was to determine the involvement of xCT in ALS. Immunohistochemical observations in the spinal cord sections demonstrated that xCT was mainly expressed in astrocytes, with staining more intense in 12 sporadic ALS patients as compared to 12 age-matched control individuals. Western blot and densitometric analyses of the spinal cord samples revealed that the relative value of xCT/s-actin optical density ratio was significantly higher in the ALS group as compared to the control group. Next, we conducted cell culture experiments using a human astrocytoma-derived cell line (1321N1) and a mouse motor neuron/neuroblastoma hybrid cell line (NSC34). In 1321N1 cells, the normalized xCT expression levels in cell lysates were significantly increased by H2 O2 treatment. Glutamate concentrations in 1321 N1 cell culture-conditioned media were significantly elevated by H2 O2 treatment, and the H2 O2 -driven elevations were completely canceled by the xCT inhibitor erastin pretreatment. In motor neuron-differentiated NSC34 cells (NSC34d cells), both the normalized xCT expression levels in the cell lysates and glutamate concentrations in the cell-conditioned media were constant with or without H2 O2 treatment. The present results provide in vivo and in vitro evidence that astrocytes upregulate xCT expression to release glutamate in response to increased oxidative stress associated with ALS, contributing to extracellular glutamate accumulation.

Paraganglioma is a neuroendocrine tumor arising from extra-adrenal sites in the peripheral nervous system. Although malignant paraganglioma is known to metastasize to bones, including vertebral bodies, there is little literature on the compressive myelopathy accompanied by sphincter dysfunction; to our knowledge, only 12 cases have been reported. Moreover, neuropathological investigations of the spinal cord in this state have not been well-documented. This autopsy report describes a 55-year-old man with malignant paraganglioma and compression myelopathy caused by vertebral metastasis. The present case showed a gradual numbness and a sudden onset of irreversible paraplegia with sphincter dysfunction, which were not palliated these neurologic dysfunctions despite radiotherapy. Computed tomography (CT) revealed multiple metastases to the bones, lymph nodes, and lungs when he was diagnosed with malignant paraganglioma. At the same time, he had numbness, and magnetic resonance imaging (MRI) showed multiple diffuse metastatic lesions in the vertebral bodies. Following abrupt onset of paralysis, MRI showed fractured third and sixth thoracic vertebral bodies. An autopsy revealed residual vertebral metastases with fractures of the third and sixth thoracic vertebral bodies, resulting in compressive myelopathy at the fourth thoracic segment, which was characterized by complete spinal cord destruction. Destructive spinal cord lesion-induced secondary degeneration was observed in the gracile fasciculus at the rostral side and in the pyramidal tract at the caudal side, which showed Wallerian degeneration. Such pathology was consistent with the presenting neurological symptoms, including paraplegia and somatic sensory loss below the fourth thoracic spinal cord segment. Although it is difficult to identify the pathognomonic morphological changes responsible for the sphincter dysfunction, the present case suggests a supranuclear dysregulation of the somatosensory and central autonomic nervous systems involved in urination and defecation. Based on a review of the literature and the features of the present case, paraganglioma can metastasize aggressively even with a low pathological grading. This case of vertebral metastasis as a result of malignant paraganglioma may not be extraordinary but the autopsy report is rare. This autopsy revealed transverse myelopathy as a result of malignant vertebral metastasis of malignant paraganglioma.

We report the neuropathology of a patient with a family history of amyotrophic lateral sclerosis (ALS) and a p.N345K mutation in the transactivation response DNA-binding protein 43 kDa (TDP-43) gene (TARDBP). A 62-year-old man had bulbar palsy with progressive weakness in the extremities. Neurological examination revealed evident upper motor neuron signs and lower motor neuron involvement corroborated by needle electromyography. The patient was diagnosed as having probable ALS according to the revised El Escorial diagnostic criteria and was eventually diagnosed with familial ALS. At 65 years of age, respiratory failure became critical, and artificial ventilation was initiated. At 70 years of age, the patient died from a urinary tract infection. Histopathological investigation showed Bunina bodies in the remaining motor neurons and anterolateral funicular myelin pallor in the spinal cord. TDP-43-positive cytoplasmic inclusions were quite rare in the spinal cord motor neurons, being predominantly present in the glial cells (especially astrocytes) of the spinal cord anterior horn. Although the reason for the preferential vulnerability of spinal glial cells to TARDBP mutations remains unclear, our findings indicate that TARDBP p.N345K mutation could have an influence on the topography of TDP-43 aggregation.

Aim The optimal antibiotic regimen for preterm premature rupture of membrane (pPROM) is still unclear. This study aimed to determine the effects of ampicillin-sulbactam (SBT/ABPC) and azithromycin (AZM) on the incidence of bronchopulmonary dysplasia (BPD). Methods This retrospective study included women with singleton gestations and a diagnosis of pPROM between 22 and 27 weeks of gestation. In patients presenting with a high risk of intra-amniotic infection between January 2011 and May 2013, piperacillin or cefmetazole + clindamycin (regimen 1 group; n = 11) was administered, whereas SBT/ABPC and AZM (regimen 2 group; n = 11) were administered in patients presenting a similar risk between June 2013 and May 2016. Results The incidence of moderate or severe infant BPD in the regimen 2 group was significantly lower than that in the regimen 1 group, even when adjusted for gestational age at the time of rupture of membrane, with an odds ratio (95% confidence interval) of 0.02 (1.8 × 10-5 -0.33). The incidence of BPD and total days on mechanical ventilation were significantly lower in the regimen 2 group than in the regimen 1 group. No significant differences were seen in other morbidities. Conclusion In patients with pPROM between 22 and 27 weeks of gestation, the administration of SBT/ABPC and AZM may improve the perinatal outcomes.

We present a case of hepatolenticular degeneration, so-called Wilson's disease (WD), in a 31-year-old Japanese man with broader deposition of copper in the liver, kidney and brain. The liver showed severe cirrhotic changes with macronodular pseudolobule formation, but there was little difference in immunohistochemical expression patterns of the copper transporter ATP7B between the control and present case. In the brain, there were both WD-related lesions such as the scattering of Opalski cells and changes caused by hepatic encephalopathy including the appearance of Alzheimer type II glia. Of note, we identified copper deposits in the systemic organs, including hepatocytes, renal tubules, and in broad areas of the brain. Surprisingly, as a result of further pursuit, copper accumulation in the brain was rarely identified in neuronal cells, but in Olig2-positive glial cells with double immunohistochemical staining. Together, this rare autopsied case suggests a novel cellular candidate affected by abnormal copper metabolism and the necessity to perform the systemic examination of copper deposition in WD.

The activating mutation of MYD88 has been identified in diffuse large B-cell lymphoma (DLBCL). We investigated the mutational status and both the gene amplification and protein expression of MYD88 in 23 cases of testicular DLBCL. To detect the MYD88 mutations, we employed the allele-specific PCR and Sanger sequencing. MYD88 gene amplification and protein expression were analyzed by quantitative PCR and by immunohistochemistry, respectively. There were 17 cases of primary testicular DLBCL: 94% (16/17) exhibited a non-Germinal center B-cell (non-GCB) subtype, 82% (14/17) showed the MYD88 L265P, and 65% (11/17) had intense expression of MYD88. When compared with normal lymph nodes, the MYD88 is significantly amplified in primary testicular DLBCL. However, the amplification status showed no correlation with its mutational status or protein expression. Moreover, neither the MYD88 mutational status nor the expression pattern affected overall survival. Six cases were secondary testicular DLBCL with an 83% (5/6) and an 80% (4/5) incidence of the non-GCB subtype and of the MYD88 L265P, respectively. In conclusion, we demonstrated a high prevalence of the non-GCB subtype and the common MYD88 L265P in both primary and secondary testicular DLBCL. Our data suggest that the MYD88 mutation is a fairly consistent genetic feature in testicular DLBCL.

Summary YdiV is an EAL-like protein that acts as a post-transcriptional, negative regulator of the flagellar master transcriptional activator complex, FlhD4C2, in Salmonella enterica to couple flagellar gene expression to nutrient availability. Mutants defective in ClpXP protease no longer exhibit YdiV-dependent inhibition of FlhD4C2-dependent transcription under moderate YdiV expression conditions. ClpXP protease degrades FlhD4C2, and this degradation is accelerated in the presence of YdiV. YdiV complexed with both free and DNA-bound FlhD4C2; and stripped FlhD4C2 from DNA. A L22H substitution in FlhD was isolated as insensitive to YdiV inhibition. The FlhD L22H substitution prevented the interaction of YdiV with free FlhD4C2 and the ability of YdiV to release FlhD4C2 bound to DNA. These results demonstrate that YdiV prevents FlhD4C2-dependent flagellar gene transcription and acts as a putative adaptor to target FlhD4C2 for ClpXP-dependent proteolysis. Our results suggest that YdiV is an EAL-like protein that has evolved from a dicyclic-GMP phosphodiesterase into a dual-function regulatory protein that connects flagellar gene expression to nutrient starvation.

Fukutin is the gene responsible for Fukuyama-type congenital muscular dystrophy (FCMD), an autosomal recessive disease associated with central nervous system (CNS) and eye anomalies. Fukutin is involved in basement membrane formation via the glycosylation of α-dystroglycan (α-DG), and hypoglycosylation of α-DG provokes the muscular, CNS and eye lesions of FCMD. Astrocytes play an important role in the pathogenesis of the CNS lesions, but the post-transcriptional regulation of fukutin mRNA has not been elucidated. In this study, we investigated the characteristics of fukutin mRNA using an astrocytoma cell line that expresses fukutin and glycosylated α-DG. The glycosylation of α-DG was considered to be increased by over-expression of fukutin and decreased by knockdown of fukutin. Knockdown of Musashi-1, one of the RNA-binding proteins involved in the regulation of neuronal differentiation, induced a decrease in fukutin mRNA. Immunoprecipitation and ELISA-based RNA-binding assay demonstrated possible binding between fukutin mRNA and Musashi-1 protein. A relationship between fukutin mRNA and vimentin protein was also proposed. In situ hybridization for fukutin mRNA showed a positive cytoplasmic reaction including cytoplasmic processes. From these results, fukutin mRNA is suggested to be a localized mRNA up-regulated by Musashi-1 and to be a component of a mRNA-protein complex which includes Musashi-1 and (presumably) vimentin proteins.

Several studies have suggested the involvement of neuroinflammation in the pathomechanism of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). We recently demonstrated increased levels of protein-bound 4-hydroxy-2-nonenal (HNE) as a highly reactive lipid peroxidation product and cytosolic phospholipase A(2) (cPLA(2)) as a proinflammatory enzyme in glial cells as well as motor neurons in the spinal cord of sporadic ALS patients. However, a link between HNE and cPLA(2) in ALS remains to be determined. To address this issue, we investigated effects of HNE stimulation on the state of cPLA(2) expression in cultured microglial cell line (Ra2). Exposure of Ra2 cells to HNE significantly increased expression levels of cPLA(2) and its activated form phosphorylated at amino acid residue S(505) (p-cPLA(2)) on immunoblots. Pretreatment of Ra2 cells with the antioxidant N-acetylcysteine, the extracellular signal-regulated kinase (ERK) inhibitor PD98059 or the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 prevented the HNE-induced increased expression of cPLA(2) and p-cPLA(2). Immunocytochemical analysis revealed that staining for p-cPLA(2) in Ra2 cells was localized in the cytoplasm and more intense in the HNE-stimulated group than in the vehicle group. The present results provide in vitro evidence that HNE upregulates and phosphorylates cPLA(2) in microglia via the ERK and p38 MAPK pathways.

Signal transducer and activator of transcription-3 (STAT3) is a member of the proinflammatory transcription factor STAT family. Several studies have documented implications for neuroinflammation in amyotrophic lateral sclerosis (ALS). We recently demonstrated activation of STAT3 in spinal cords obtained at autopsy from sporadic ALS patients. To determine the involvement of STAT3 and effects of pioglitazone on STAT3 activity in familial ALS with superoxide dismutase-1 (SOD1) mutation, we performed immunoblot and immunohistochemical analyses of the active form of STAT3 (p-STAT3) in spinal cords from mice overexpressing mutant SOD1 (ALS mice) and nontransgenic littermates (control mice). Immunoblot analysis delineated significant increases in nuclear p-STAT3 levels in non-treated ALS mice as compared with pioglitazone-treated ALS mice and non-treated and pioglitazone-treated control mice. Immunohistochemical analysis revealed prominent p-STAT3 accumulations in the nucleus of motor neurons, reactive astrocytes and activated microglia in non-treated ALS mice but not pioglitazone-treated ALS mice and non-treated and pioglitazone-treated control mice. The present results provide in vivo evidence for increased phosphorylative activation and nuclear translocation of STAT3 in motor neurons and glia in mouse motor neuron disease, suggesting a common pathological process between sporadic and SOD1-mutated familial forms of ALS. Moreover, it is likely that pioglitazone may exert inhibitory effects on STAT3-mediated proinflammtory mechanisms in this disease.

It is well known that the similarity in species composition between two communities decays with the geographic distance that separates them. It is thus likely that the similarity in the dynamics of two communities also decays with distance, because the distance-decay relationship is fundamental in nature. However, the distance-decay relationships of community dynamics have not yet been revealed. We used transition matrix models to evaluate distance-decay relationships of seasonal community dynamics (from spring to summer) in rocky intertidal sessile assemblages along the Pacific coast of Japan between 31°N and 43°N. We evaluated the distance-decay relationships of whole-community dynamics and of three dynamics-related components—recruitment, disturbance, and species interaction (competition and facilitation)—for communities separated by distances ranging from several meters to thousands of kilometers. The similarity of the recruitment dynamics among communities declined rapidly with distance within the fine spatial scale, but only moderately within larger scales. The similarity of the disturbance dynamics was independent of distance, and the similarity of species interaction declined slightly with increasing distance. The similarity of whole-community dynamics declined rapidly with distance at a fine spatial scale and moderately at larger scales. The fact that the distance-decay relationship of whole-community dynamics was similar to that of recruitment may suggest that recruitment processes are the most important determinant of spatial variability of community dynamics at our study sites during the study period.

1. Although latitudinal gradients in species richness within a region are observed in a range of taxa and habitats, little is known about variability in its scale dependence or causal processes. The scale-dependent variability of latitudinal gradients in species richness can be affected by latitudinal differences in (i) the regional relative abundance distribution, and (ii) the degree of aggregated distribution (i.e., intraspecific aggregation and interspecific segregation; henceforth, the degree of aggregation) reflecting differences in ecological processes among regions, which are not mutually exclusive. 2. In rocky intertidal sessile animal assemblages along Japan's Pacific coast (between 31 degrees N and 43 degrees N), scale-dependent variability of the latitudinal gradient in species richness and its causal mechanisms were examined by explicitly incorporating three hierarchical spatial scales into the monitoring design: plots (50 x 100 cm), shores (78 to 235 m), and regions (16.7 to 42.5 km). 3. To evaluate latitudinal differences in the degree of aggregation, the degree of intraspecific aggregation at each spatial scale in each region was examined using the standardized Morishita index. Furthermore, the observed species richness was compared with the species richness expected by random sampling from the regional species pool using randomization tests. 4. Latitudinal gradients in species richness were observed at all spatial scales, but the gradients became steadily more moderate with decreasing spatial scale. The slope of the relative abundance distribution decreased with decreasing latitude. 5. Tests of an index of intraspecific aggregation and randomization tests indicated that although species richness at smaller scales differed significantly from species richness expected based on a random distribution, the degree of aggregation did not vary with latitude. Although some ecological processes (possibly species sorting) may have played a role in determining species richness at small spatial scales, the importance of these processes did not vary with latitude. 6. Thus, scale-dependent variability in the latitudinal gradient of species richness appears to be explained mainly by latitudinal differences in the regional relative abundance distribution by imposing statistical constraint caused by decreasing grain size.

Fukutin, a gene responsible for Fukuyama type congenital muscular dystrophy (FCMD), is presumably related to the glycosylation of α-dystroglycan (α-DG), involved in basement membrane formation. Hypoglycosylation of α-DG plays a key role for the pathogenesis of FCMD. On the other hand, fukutin and α-DG are also expressed in various non-neuromuscular tissues. Recently, a role of α-DG as a cancer suppressor has been proposed, because of a decrease of glycosylated α-DG in cancers. In this study, function of fukutin was investigated in two cancer cell lines, focusing on whether fukutin is involved in the glycosylation of α-DG in cancer cells and has any possible roles related to a cancer suppressor. Localization of fukutin and a result of laminin-binding assay after RNA interference suggest that fukutin may be involved in the glycosylation of α-DG in a small portion in these cancer cell lines. In Western blotting and immuno-electron microscopy, localization of fukutin in the nucleus was suggested in addition to the Golgi apparatus and/or endoplasmic reticulum. Immunohistochemically, there were more Ki-67-positive cells and more nuclear staining of phosphorylated c-jun after knockdown of fukutin in two cell lines. Fukutin appears to suppress cell proliferation through a system involving c-jun, although it is unclear this process is related to α-DG or not at present. The result may propose a possibility of another function of fukutin in addition to the glycosylation of α-DG in cancer cells.

Aplastic anaemia (AA) is defined as a pancytopenia caused by bone marrow failure, and its pathogenesis is thought to involve autoimmune processes. Several predictive markers of the response to immunosuppressive therapy (IST) have been proposed, which appear to reflect the immune pathophysiology. We prospectively investigated the presence of human leucocyte antigen (HLA)-DR15, a minor population of paroxysmal nocturnal haemoglobinuria (PNH)-type cells, and antibodies to the recently identified autoantigen postmeiotic segregation increased 1 (PMS1) in 103 children with AA enrolled in a multicentre study. In contrast to adults, children with AA did not show an increased frequency of HLA-DR15. In addition, a sensitive flow cytometric assay revealed that children with AA have a much lower prevalence of PNH-type cells (21.4%) than reported for adults with this disease. An immunoblotting assay detected anti-PMS1 antibody in 15 of 103 (14.6%) of the children. Finally, the response rate to IST was not significantly different between patients with and without DR15 (45.5% vs. 54.0%), PNH-type cells (68.2% vs. 53.1%) or anti-PMS1 antibody (40.0% vs. 59.1%). The current study did not confirm a correlation between these markers and the response to IST, suggesting that there is a difference in the pathophysiologies of adult and paediatric AA.

We report the first study of sperm whale-fall ecosystems, based on mass sinking of whale carcasses at shelf depths in the northwest Pacific. We conducted three observations over a 2-year period on replicate sperm-whale carcasses implanted at depths of 219‐254 m off the southern part of Japan from July 2003 to August 2005. The study was made possible by a mass stranding of sperm whales in January 2002, and the subsequent sinking of 12 carcasses in the waters off Cape Nomamisaki. Dense aggregations of unique chemosynthesis-based fauna had formed around the whale carcasses after 18 months (July 2003). The mytilid mussel Adipicola pacifica was the most abundant macrofaunal species and covered most of the exposed bone surfaces. The general composition of the fauna was similar to that of deep-water reducing habitats, but none of the species appearing in this study has been found at hydrothermal vents, cold seeps or deep-water whale falls. A new species of lancelet, which was the first record of the subphylum Cephalochordata from reducing environments, a new species of Osedax; a rarely encountered benthic ctenophore, and a rare gastropod species were discovered at this sperm whale-fall site. Benthic communities were similar across all the carcasses studied, although the body sizes of the whales were very different. The succession of epifaunal communities was relatively rapid and the sulphophilic stage was considerably shorter than that of other known whale falls.

Growing evidence documents oxidative stress involvement in ALS. We previously demonstrated accumulation of a protein-bound form of the highly toxic lipid peroxidation product crotonaldehyde (CRA) in the spinal cord of sporadic ALS patients. In the present study, to the determine the role for CRA in the disease processes of superoxide dismutase-1 (SOD1) mutation-associated familial ALS (FALS), we performed immunohistochemical and semi-quantitative cell count analyses of protein-bound CRA (P-CRA) in the spinal cord of SOD1-mutated FALS and its transgenic mouse model. Immunohistochemical analysis revealed increased P-CRA immunoreactivity in the spinal cord of the FALS patients and the transgenic mice compared to their respective controls. In the FALS patients, P-CRA immunoreactivity was localized in almost all of the chromatolytic motor neurons, neurofilamentous conglomerates, spheroids, cordlike swollen axons, reactive astrocytes and microglia, and the surrounding neuropil in the affected areas represented by the anterior horns. In the transgenic mice, P-CRA immunoreactivity was localized in only a few ventral horn glia in the presymptomatic stage, in almost all of the vacuolated motor neurons and cordlike swollen axons and some of the ventral horn reactive astrocytes and microglia in the onset stage, and in many of the ventral horn reactive astrocytes and microglia in the advanced stage. Cell count analysis on mouse spinal cord sections disclosed a statistically significant increase in the density of P-CRA-immunoreactive glia in the ventral horns of the young to old G93A mice compared to the age-matched control mice. The present results indicate that enhanced CRA formation occurs in motor neurons and reactive glia in the spinal cord of SOD1-mutated FALS and its transgenic mouse model as well as sporadic ALS, sug- gesting implications for CRA in the pathomechanism common to these forms of ALS.

The DnaK chaperone binds non-specifically to many unfolded polypeptides and also binds selectively to specific substrates. Although its involvement in targeting the unfolded polypeptides to assist proper folding is well documented, less is known about its role in targeting the folded polypeptides. We demonstrate that DnaK regulates the expression of the Salmonella flagellar regulon by modulating the FlhD and FlhC proteins, which function as master regulators at the apex of a transcription hierarchy comprising three classes of genes. FlhD and FlhC form an FlhD2C2 complex that activates sigma70 promoter of class 2 genes. In DeltadnaK cells, FlhD and FlhC proteins seemed to be assembled into hetero-tetrameric FlhD2C2 but the complex was not fully active in class 2 gene transcription, suggesting that the DnaK chaperone is involved in activating native FlhD2C2 complex into a regulator of flagellar regulon expression. This is the first time that involvement of the DnaK chaperone machinery in activating folded oligomerized proteins has been demonstrated.

We evaluated the efficacy of CS2022 (the Lon protease-deficient mutant strain of Salmonella enterica serovar Typhimurium) as a candidate live oral vaccine strain against subsequent oral challenge with a virulent strain administered to BALB/c and C57BL/6 mice. CS2022 persistently resided in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum of both strains of mice after a single oral inoculation with 1 x 10(8) colony-forming units. Finally, CS2022 almost disappeared from each tissue sample by week 12 in BALB/c mice, whereas CS2022 still resided in each tissue type at week 12 after inoculation of C57BL/6 mice. A significant increase in the serovar Typhimurium lipopolysaccharide-specific secretory immunoglobulin A (s-IgA), as measured for one of the mucosal immune responses, was detected in bile and intestinal samples of both strains of immunized mice at week 4 after immunization. In addition, the expression of gamma interferon mRNA in the spleens of both strains of immunized mice, especially those of C57BL/6 mice, was significantly increased at week 4 after immunization and was boosted during the following 5 days after the challenge was administered to the mice. Furthermore, peritoneal macrophages isolated from immunized mice at week 4 after immunization exhibited an increase in intracellular killing activity against both virulent and avirulent Salmonella. The present results suggested that salmonellae-specific s-IgA on the mucosal surfaces induced by immunization with CS2022 generally prevented mice from succumbing to an oral challenge with a virulent strain. Simultaneously, CS2022 promoted the protective immunity associated with macrophages in both strains of mice.

Several lines of evidence indicate the clonal nature of juvenile myelomonocytic leukaemia (JMML), involving myeloid, erythroid, megakaryocyte and B-lymphoid lineages. However, it is unclear whether the T-lymphocyte lineage is involved. We demonstrated that cells from six patients with JMML repopulated in non-obese diabetic/severe combined immunodeficient/gammac(null) mice and differentiated into granulocytes, monocytes, erythrocytes, B lymphocytes, T lymphocytes and natural killer cells. The percentage of human CD45 antigen-positive cells ranged from 41% to 73% in the murine bone marrow 12 weeks after transplantation. To examine the involvement of lymphocyte subpopulations, we purified human CD3(+), CD19(+) and CD56(+) cells from murine bone marrow cells transplanted from a patient with monosomy 7. Fluorescence in situ hybridization (FISH) showed the clonal marker in 96-100% of purified CD3(+), CD19(+) and CD56(+) subpopulations. These findings support the concept that JMML originates in transplantable multilineage haematopoietic stem cells. This novel murine xenotransplant model should be useful for investigating the nature of stem cells and testing new therapies for patients with JMML.

Salmonella pathogenicity island 1 (SPI1) enables infecting Salmonella to cross the small intestinal barrier and to escape phagocytosis by inducing apoptosis. Several environmental signals and transcriptional regulators modulate the expression of hilA, which encodes a protein playing a central role in the regulatory hierarchy of SPI1 gene expression. We have previously shown that Lon, a stress-induced ATP-dependent protease, is a negative regulator of hilA, suggesting that it targets factors required for activating hilA expression. To elucidate the mechanisms by which Lon protease negatively regulates SPI1 transcription, we looked for its substrate proteins. We found that HilC and HilD, which are positive regulators of hilA expression, accumulate in Lon-depleted cells, and that the enhancement of SPI1 expression that occurs in a lon-disrupted mutant is not observed in the lon hilC hilD triple null mutant. Furthermore, we demonstrated that the half-lives of HilC and HilD are, respectively, about 12 times and three times longer in the Lon-depleted mutant, than in the Lon+ cells, suggesting that Lon targets both of HilC and HilD. In view of these findings, we suggest that the regulation of SPI1 expression is negatively controlled through degradation of the HilC and HilD transcriptional regulators by Lon.

Natural prey composition and prey selectivity of the muricid snail Muricodrupa fusca (Kuster), which forages on a wide range of prey, was investigated. Natural prey composition was evaluated through correcting the apparent diet (the result of observations of the feeding behavior) by the handling time, which was determined by laboratory analysis. The apparent diet and the natural diet should generally differ because prey items that require a longer handling time will be observed with higher frequencies. Multiple regression equations were derived to relate the handling time to prey size, predator size and water temperature. A large discrepancy in the apparent versus natural diet of M. fusca was found in prey species composition and prey size. They foraged on at least 11 species of sessile and mobile prey including six limpet species, and mainly preyed on Siphonaria spp. and Lottia spp. in the field. By comparing the percentage of the natural diet to that of prey abundance in the field, M. fusca preferred Siphonaria spp. and Lottia spp. and did not select the other prey species. The selectivity of this predator was explained by optimal foraging theory and antipredator defenses of some limpet species. The relationships among attacking methods, handling time and prey selectivity are also examined.

A 65-year-old man was diagnosed with granulomatosis with polyangiitis (GPA) at the age of 47, when cytoplasmic anti-neutrophil cytoplasmic antibody (C-ANCA) serology was positive, and he had multiple nodular shadows in both lungs. He had been treated with prednisolone, cyclophosphamide (CPA) and plasma exchange. At the age of 64, a nodular shadow was newly detected in the right lower lung field and serum tumour marker increased. Subsequent positron emission tomography/computed tomography scan demonstrated accumulations of fluorodexyglucose (FDG) in the same area, mediastinum lymph nodes, thoracic wall, right iliac bone, and right retroperitoneum. The diagnosis of squamous cell lung cancer cT2bN2M1b Stage4 was made with endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA). There are no reports of cases that lung cancer has developed with GPA during the long-term treatment with CPA. We suggest that in such patients, the differential diagnosis should include not only the relapse of GPA, but also the rare possibility of development of carcinomas.

— Nitrogen and noble gas isotopic compositions and C abundance of ureilites were analyzed using a stepwise combustion technique. Four Antarctic ureilites, ALHA77257, Asuka 881931, Yamato 791538 and Yamato 790981 were analyzed. Multiple N isotopic components were observed in these ureilites. The δ15N values of these N components ranged from +160 to −120%. The minimum δ15N values of typically −120% were observed at combustion temperatures at 700–900 °C where large amounts of C were released. A heavy N component was observed in only two ureilites, ALHA77257 and Asuka 881931. Silicate-enriched fractions and C-concentrated fractions were prepared for these two ureilites. We conclude that both the light N and the heavy N are trapped in the carbonaceous vein minerals. The lack of correction between the N/C ratio and the 36Ar/C ratio suggests that the primary carrier phase of the light N does not correspond to that of the planetary noble gases. We consider that the isotopically heavy N, which was observed in this study, is related to the heavy N observed among polymict ureilites. Small amounts (

We conducted prenatal diagnosis by haplotype analysis, using newly developed microsatellite markers, in eight Fukuyama type congenital muscular dystrophy (FCMD) families. In addition to six new families, two previously reported families were re-examined by haplotype analysis including detection of an ancestral founder haplotype (138–183–301) for 3 microsatellite markers closest to the FCMD gene, designated D9S2105–D9S2107–D9S172, the distances of which from the FCMD gene are presumed to be ∼140, ∼20, and ∼280 kb, respectively. Five fetuses from five families were diagnosed as nonaffected, and were subsequently confirmed to be healthy. Three fetuses of the other three families were diagnosed as having a high probability of being affected by FCMD. In the prenatal diagnosis conducted for these eight families, the ancestral founder allele was observed in 13 of 16 (81%) FCMD-bearing chromosomes. Detection of the ancestral haplotype facilitated achieving accurate prenatal diagnosis of FCMD. The brains of all three fetuses prenatally diagnosed as FCMD-affected showed the initial stage of cortical dysplasia, strong evidence of FCMD. Am. J. Med. Genet. 77:310–316, 1998. © 1998 Wiley-Liss, Inc.

The H20mAb recognizing the 60-kilodalton protein, which existed in the outer membrane and was induced by heat shock at 42 C, was established. The molecule recognized with the mAb was a heat-shock protein 60 (HSP60) of Helicobacter pylori. To understand the role of HSP60 on the cell surface of H. pylori, whether or not H20mAb affects the growth of H. pylori was investigated. When bacteria were cultured with H20mAb, growth was markedly inhibited after 24 hr, although an initial 5 hr-incubation with the mAb induced no significant inhibition of H. pylori growth. The 24- and 48 hr growth of the bacteria after washing to remove the mAb at 5 hr was also inhibited though the inhibitory effect was not strong. In electron microscopical analysis, the spots with high electron density in the cytoplasm of the bacteria treated with H20mAb were increased, depending on the length of incubation time from 5 to 24 hr. After 24 hr treatment with H20mAb, bacterial destruction was also observed, indicating bactericidal activity by H20mAb. These results suggest that the HSP60 on the cell surface of H. pylori might have an essential role in the growth of the bacteria.

This report describes cerebral cortical dysplasia in a 20 week fetus, aborted as a result of the prenatal genetic analysis which provided evidence of Fukuyama congenital muscular dystrophy (FCMD). The histological appearance of the brain of this fetus was similar to that of previously described cases. However, cortical dysplasia was less severe in this 20 week fetus, and the interesting finding was the existence of a leptomeningeal lesion probably preceding dysplasia. In this case, abnormal findings were found mainly in the cerebral surface, and maturation of the brain seemed appropriate for the gestational age. Furthermore, periodic acid-methenamine-silver-positive linear structure of the cerebral surface was disrupted irregularly. These findings suggest that defects in the pial-glial barrier of the cerebral surface may play a cardinal role for the genesis of cortical dysplasia. It is suggested that the extracortical abnormal tissue would be detectable morphologically at least in the 12-13th week because cells considered as subpial granular cells were contained in the lesion. This report on the brain of a fetus with FCMD provides information on relatively early central nervous system alterations in this disease, and may be of importance in clarifying their pathogenesis.

To elucidate the pathogenesis of adenovirus type 12 (Ad12)-induced rat retinal tumor, an experimental animal model of human retinoblastoma (RB), DNA analysis, in situ hybridization and immunohistochemistry were performed. The adenovirus oncogene E1A was detected in the host genome by Southern blot hybridization. Examined retinal tissues did not show any histological changes, but the number of retinal cells immunoreactive with an antibody to proliferating cell nuclear antigen (PCNA) increased with the course of study. In in situ hybridization, E1A gene expression was recognized at the inner granular layer of the retina at an early stage after virus inoculation, and subsequently, N-myc gene expression was recognized at the same region. No alteration was found in the retinoblastoma susceptibility gene (Rb gene) expression. The product of the virus oncogene integrated into the host genome could induce an increase in N-myc expression, without any abnormality of the Rb gene itself. Results from the present study could be useful in clarifying the tumorigenesis of this experimental model.

To determine amino acid sequences of the epitopes recognized by monoclonal antibodies (mAbs) 3C8 and 5C3 directed against Yersinia enterocolitica heat-shock protein (HSP60), a dot blot analysis was performed using synthesized peptides of Y. enterocolitica HSP60 such as peptides p316-342, p327-359, p340-366, p316-326, p316-321, p319-323, and p321-326 which represent positions of amino acids in Y. enterocolitica HSP60. The dot blot analysis revealed that 5C3 mAb reacted with p316-342, p316-326 and p321-326, and 3C8 mAb p316-342 and p316-326. These results indicate that the epitopes recognized by the mAbs were associated with eleven amino acids, Asp Leu Gly Gln Ala Lys Arg Val Val Ile Asn, of p316-326. The sequence homology between p316-326 of Y. enterocolitica HSP60 and the rest of the HSP60 family suggests that the five amino acids of Lys, Arg, Val, Ile and Asn, which are highly conserved in the HSP60 family, might be related with the epitope recognized by 3C8. In contrast, it was also demonstrated that three amino acids of Leu, Gly and Val, which are not well conserved in the HSP60 family, might be related to the epitope recognized by 5C3.

Yersinia enterocolitica is a facultative intracellular pathogen which invades to epithelial cells and survives in phagocytes. Since the internal environment of phagocytes should be stressful conditions for the phagocytosed Yersinia, the bacteria should respond to protect themselves from otherwise lethal results. We analyzed the stress-induced proteins which possibly contribute to survival of Yersinia within the phagocytes. Y. enterocolitica was radiolabeled during the growth in macrophage-like J774-1 cells, and the bacterial proteins were analyzed by two-dimensional gel electrophoresis. At least 16 proteins were selectively induced in response to phagocytosis, and several out of 16 proteins were also induced by heat shock at 42 C or oxidative stresses in vitro. Of those, two major stress proteins were identified to be homologues of DnaK and CRPA by immunoblotting analysis. These results have indicated that Y. enterocolitica exhibits a global stress response to the hostile environment in the phagocytosed macrophage.

Mouse monoclonal antibodies (MAbs) showing distinct reactivity against the 60-kilodalton (kDa) antigen heat shock protein of Yersinia enterocolitica, designated cross-reacting protein antigen (CRPA), have previously been established. The reactivities of these MAbs (5C3 and 3C8) against mouse and human host cells were studied by Western blotting and flow cytometric analysis. The results indicated that epitopes on the bacterial 60-kDa heat shock protein are present on various molecules in mouse spleen cells and human B cells. An epitope recognized by MAb 5C3 was expressed on the mouse and human host cell surface, and an epitope recognized by MAb 3C8 was also expressed on the human host cell surface.

Phagocytosis with macrophages provides a specialized mechanism for regulated ingestion and intracellular destruction of bacteria. Bacteria are first engulfed by endocytosis into a phagosome. The fusion of phagosomes and lysosomes releases toxic products that kill most bacteria and degrade them into fragments. Debris from dead bacteria is then released by exocytosis. However, some bacteria that survive within host phagocytes have evolved strategies to escape the bactericidal mechanisms associated with phagocytosis: i) antiphagocytosis (Yersinia), ii) escaping from the phagosome into cytoplasm (Listeria), and iii) remodeling their phagosome by inhibiting the maturation of phagosomes (Salmonella, Mycobacterium, Legionella). In this review, I first summarize various strategies by bacteria to avoid phagocytosis by emphasizing the steps that have been subverted by bacteria. Then, I highlight the mechanisms for surviving phagocytosis by Salmonella, with a focus on the induction of macrophage-apoptosis and modulation of membrane traffic in host cells.

Brine shrimp lethality test (BST)-guided fractionation of a methanol extract of the roots of Melia azedarach resulted in the isolation of two new limonoids, 9alpha-hydroxy-12alpha-acetoxyfraxinellone (1) and 7,14-epoxy-azedarachin B (2), together with the known compounds, 12alpha-hydroxyfraxinellone (4), 9alpha-hydroxyfraxinellone (5), azedarachin B (6), and neoazedarachin B (7). The structures of 1 and 2 were elucidated by analysis of spectroscopic data and comparison of their NMR data with those of the known compounds. Compounds 1, 2 and 4-7 exhibited significant activity in the BST, in particular, azedarachin B (6) showed remarkable BST activity with an LC(50) value of 0.0098 microM.

Through our screening for novel antifungal compounds, YM-215343 was found in the culture extract of Phoma sp. QN04621. The structure of YM-215343 was determined by several spectroscopic experiments as a novel compound closely related to apiosporamide and fischerin. YM-215343 exhibited antifungal activity against the pathogenic fungi, Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus with MIC values of 2 to approximately 16 microg/ml. It also showed cytotoxicity against HeLa S3 cells with an IC50 of 3.4 microg/ml.

A pair of 5,15-bisdiazoliumylporphyrins with only two meso-substituents, 5,15-bis(1,3-dimethylimidazolium-2-yl)porphyrin 1b and 5,15-bis(1,2-dimethylpyrazolium-4-yl)porphyrin 2b, has been synthesized. The crowded porphyrin 1b intercalates between base pairs of DNA to stabilize the duplex DNA to thermal denaturation due to its high affinity for DNA. The binding of porphyrin 1b to DNA is an exothermic interaction and enthalpically driven. Porphyrin 1b interacts more strongly with poly(dA-dT)2 than with poly(dG-dC)2, and the interaction is more favorable or selective to AT-rich sites than to GC-rich sites.

Although researchers recognize that population dynamics can vary in space and time as a result of differences in biotic and abiotic conditions, spatial and temporal variability in the patterns and processes of population dynamics have not been well documented on a seasonal time frame. We quantified seasonal changes in the coverage of intertidal barnacles, Chthamalus spp., with data collected for as many as 9 years at 88 plots in five regions located along more than 1800 km of the Pacific coastline of Japan from 31° N to 43° N. To examine how seasonal changes and the spatial heterogeneity of environments can interact to influence patterns and processes of population dynamics, we analyzed the data with two models of population variability: a population dynamics model, which provides knowledge about processes that determine population growth rates; and Taylor's power law, which summarizes the relationship between the temporal mean and variance of the size of a population (temporal mean-variance relationship). We found that seasonal differences were prevalent in population growth rates, as well as in the strength and spatial scales of processes that determine population growth rates. In addition, the seasonality of these rates and processes varied between habitats at different spatial scales ranging from the scale of among-rocks within a shore to that of among-regions located in different latitudes, suggesting that the effects of seasonal environmental fluctuations on population growth can depend on the spatial heterogeneity of biotic and abiotic conditions that vary at multiple spatial scales. In contrast, the evidence for spatiotemporal differences in temporal mean-variance relationships was weak. Unlike theoretical expectations, spatiotemporal differences in the variability of population size were best explained by a unique power law, despite remarkable regional and seasonal differences in the processes that determine population growth rates. These results suggest that spatiotemporal environmental variability can affect population dynamics at multiple spatial scales but do not necessarily alter the scaling law of population size variability.

A general method for the synthesis of a series of 4,4-disubstituted thiazolidine-2,5-dithiones (1)–(6) initially consisted in reactions of α-metallated aralkyl isothiocyanates with carbon disulphide. 4-Monosubstituted thiazolidine-2,5-dithiones (7)–(9) are very susceptible to oxidation, changing rapidly into their oxidative dimers. The reason for the unexpected methylation of thiazolidine dithiones (7)–(9) became evident from the results of MNDO calculations.

This study examined the latitudinal gradient of species diversity of rocky intertidal sessile assemblages on the slopes of rocks along the Northwestern Pacific coast of Japan, located between 31°N and 43°N, by explicitly incorporating an hierarchical spatial scale into the monitoring design. The specific questions were to examine: (1) whether there is a latitudinal gradient of regional diversity, (2) how spatial components of the regional diversity (local diversity and turnover diversity) vary with latitude depending on spatial scale, and (3) whether the latitudinal gradient differs between different measures of species diversity, i.e. species richness and Simpson’s diversity index. We measured coverage and the presence or absence of all sessile organisms in a total of 150 census plots established at five shores in each of six regions. The results showed that there were clear latitudinal gradients in regional species richness and in species turnover among shores. However, these patterns were not reflected in smaller-scale local species richness. For Simpson’s diversity index, there was no evidence of latitudinal clines either in regional diversity or in spatial components. These results suggest that relative abundance of common species does not vary along latitude, while the number of rare species increases with decreasing latitude.

The Na + pump-deficient mutant, Napl, of Vibrio alginolyticus was found to lack three subunits of Na + -dependent NADH:quinone oxidoreductase complex. Although a spontaneous Na + pump positive revertant did not appear from Napl, transconjugants that recovered both the Na + pump activity and the subunits were isolated from Napl conjugated with the wild type. Moreover, the wild type was found to contain two different sizes of plasmids. These results suggest the possibility that the Na + pump is encoded by a plasmid.

The reaction of the ammonium salts of 5(2H)-isothiazolethiones with p-bromophenacyl bromide, and methyl and phenyl isocyanates afforded 5-(p-bromophenacylthio)-, and 5-(carbamoylthio)isothiazoles. From phenyl isothiocyanate and hydrazine, 2,4-pyrimidinedithione, and 3H-pyrazole-3-thione derivatives were obtained, respectively.