Nightlife clusters of coronavirus disease in Tokyo between March and April 2020.

We analysed associations between exposure to nightlife businesses and severe acute respiratory syndrome coronavirus 2 PCR test results at a tertiary hospital in Tokyo between March and April 2020. A nightlife group was defined as those who had worked at or visited the businesses. We included 1517 individuals; 196 (12.9%) were categorised as the nightlife group. After propensity score matching, the proportion of positive PCR tests in the nightlife group was significantly higher than that in the non-nightlife group (nightlife, 63.8%; non-nightlife, 23.0%; P < 0.001). An inclusive approach to mitigate risks related to the businesses needs to be identified.

Quantification of changes in metamorphopsia and retinal contraction in eyes with spontaneous separation of idiopathic epiretinal membrane.

BACKGROUND: To quantify changes in metamorphopsia and retinal contraction in eyes with idiopathic epiretinal membrane (ERM) before and after a spontaneous separation of ERM. METHODS: Among 92 eyes of 92 patients with idiopathic ERM who were followed up at our hospital, 5 eyes of 5 patients had experienced a spontaneous separation of ERM during the follow-up period. Patient's metamorphopsia was assessed horizontally and vertically by a metamorphopsia chart developed by our group, M-CHARTS, to obtain the horizontal (MH) and vertical (MV) metamorphopsia scores. Difference in the scores before and after the membrane separation represents change in patient's metamorphopsia. Changes in retinal contraction were also evaluated horizontally and vertically with our original software for fundus image analysis. The difference between M-CHARTS scores and distances of retinal vessel movements with before and after membrane separation were measured. RESULTS: All five subjects showed a decrease in the retinal contraction. Improved visual acuity was observed in three subjects, and no change was seen in the other two. Four subjects obtained better metamorphopsia scores after the membrane separation, while the other one was not detected with metamorphopsia by M-CHARTS either before or after the separation. In subjects with an improved MV, horizontal retinal movement was seen larger than the vertical movement. Similarly, the subjects with an improved MH indicated a larger vertical retinal movement than the horizontal movement. CONCLUSIONS: The direction of patient's metamorphopsia closely associated with the direction of retinal contraction before and after a spontaneous separation of ERM.

Hemophagocytic histiocytic sarcoma in a Japanese black cow.

At necropsy, an 11-year-old Japanese Black cow with anemia, leukocytopenia, and progressive hind limb ataxia had marked diffuse splenomegaly and multiple masses in the thoracic vertebrae. Histologically, neoplastic erythrophagocytic histiocytes were in the splenic red pulp, vertebral masses, and blood vessels of the liver and lungs. The spinal cord was compressed by the vertebral masses. Clinicopathological, macroscopic, and histologic findings were consistent with hemophagocytic histiocytic sarcoma. Vertebral involvement with spinal cord compression and resultant hind limb ataxia is an unusual presentation for this tumor, which has been described mainly in dogs and cats.

Diabetic neuropathies in brain are induced by deficiency of BDNF.

Diabetes is known to be one of the risk factors for dementia; however, neuropathic changes in the brain of patients with the disease have not been completely revealed. So in the present study, we investigated the brain function of rats with diabetes induced by streptozotocin (STZ), one of the most commonly used animal models for diabetes. In the diabetic rats, immediately working memory performance was impaired in the Y-maze task and neuronal cytoskeleton proteins such as calbindin, synaptophysin, and syntaxin were reduced. Furthermore, morphological observation by Golgi staining showed a decrease in the number of basal dendrites and abnormality of spine structure. Next, we measured the content of brain-derived neurotrophic factor (BDNF) in the diabetic brain, because BDNF is one of the essential proteins for the maintenance of neuronal functions including synapse function and neuronal transmissions. In the diabetic brains, both protein and mRNA levels of BDNF were severely reduced. These results suggest that, in diabetes, synapse dysfunction is, at least in part, caused by a failure of BDNF synthesis in the brain.

Transforming growth factor-beta1 enhances expression of brain-derived neurotrophic factor and its receptor, TrkB, in neurons cultured from rat cerebral cortex.

The effects of transforming growth factor (TGF)-beta1 on expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, TrkB, in neurons cultured from the cerebral cortex of 18-day-old embryonic rats were examined. BDNF mRNA was significantly increased from 24-48 hr after the TGF-beta1 treatment over 20 ng/ml. Accumulation of BDNF protein in the culture medium was also potentiated by TGF-beta1, although the intracellular content of BDNF was nearly unchanged. The enhancement of BDNF mRNA expression was suppressed by the co-presence of decorin, a small TGF-beta-binding proteoglycan that inhibits the biological activities of TGF-betas. mRNA expression of full-length TrkB, the bioactive high-affinity receptor for BDNF, was also upregulated after treatment with TGF-beta1. These observations suggest that: 1) TGF-beta1 potentiates BDNF/TrkB autocrine or local paracrine system; and 2) the neurotrophic activity of TGF-beta1 is partly responsible for the BDNF induced by TGF-beta1 itself. To test this latter possibility, we examined the neuronal survival activity of TGF-beta1 with or without K252a, a selective inhibitor of Trk family tyrosine kinases. TGF-beta1 significantly enhanced neuronal survival, but the co-presence of K252a completely suppressed the activity, demonstrating the involvement of Trk receptor signaling in TGF-beta1-mediated neuronal survival in cultured rat cortical neurons. These results seem to be in line with recent findings by other investigators that some neurotrophic factors including BDNF require TGF-betas as a cofactor to exert their neurotrophic activities.

Administration of FGF-2 to embryonic mouse brain induces hydrocephalic brain morphology and aberrant differentiation of neurons in the postnatal cerebral cortex.

Fibroblast growth factor-2 (FGF-2) was injected into mouse cerebral ventricles at embryonic day (E) 14 in utero and its effects on developing brain morphology and expression of various cell- or differentiation-associated protein markers in the cerebral cortex were examined. High doses of FGF-2 (200 or 300 ng) caused encephalic alternations such as deformation of the calvarium, enlargement of the ventricular spaces, and thinning of the cerebral cortex. There was no gross abnormality in the alignment of the cerebral neuronal layers, however, both cell number and cell density of the upper layers (II/III) and the lower layers (IV-VI) of the cerebral cortex were increased. Brain-derived neurotrophic factor (BDNF), tyrosine hydroxylase, nestin, and microtubule-associated protein 2 were aberrantly or ectopically expressed in the deep areas of the cerebral cortex. A substantial number of these cells coexpressed these antigens. These observations demonstrate that a subpopulation of neurons in the cortical deep layer abnormally differentiated or partly sustained their immature state following a single administration of FGF-2 at E14. Developmental analysis of localization of BDNF-positive cells suggested that the abnormality started around P5. Furthermore, cell migration was not affected by FGF-2 administration. FGF-2 seems to play predominant roles in the proliferation of neuronal precursors and in neuronal differentiation in the developing mouse cerebral cortex even at relatively late stages of brain neurogenesis.

An association study between polymorphism of L1CAM gene and schizophrenia in a Japanese sample.

L1CAM, a neural cell adhesion molecule, plays an important role in the development of the central nervous system. The human L1CAM gene is located in Xq28. Mutations in the gene are responsible for a wide spectrum of neurological abnormalities and mental retardation. Schizophrenia may result from early neurodevelopmental abnormalities. We screened 30 male and 30 female schizophrenic patients for their genomic sequence of the L1CAM gene in order to determine the DNA sequence variations. Three novel variations located in exon 18 (10564 G > A, GG/AA at codon 758), intron 11 (8575 A > C), and intron 25 (13504 C > T) were detected. An association study of the identified polymorphisms was then performed in a Japanese sample of 152 male and 115 female patients with schizophrenia and 121 male and 114 female control subjects. A statistically significant increase in the count of the 13504 T-allele was observed in the male patients, compared to the male controls, with no differences in the variations of exon 18 or intron 11. There was no statistically significant change in the distribution of allele or genotype of any variations in the female schizophrenics, in comparison with the female controls. These results suggest that the polymorphism in intron 25 plays a role in the genetic predisposition of male schizophrenia in the Japanese sample.

No association of two missense variations of the benzodiazepine receptor (peripheral) gene and mood disorders in a Japanese sample.

The benzodiazepine receptor (peripheral) (BZRP) plays an important role in the steroid syntheses of the adrenal glands and brain, which is possibly involved in the pathophysiology of mood disorders. We evaluated an association study between two missense variations of the BZRP gene and mood disorders in a Japanese sample. However, no statistically significant associations with either bipolar disorders or depressive disorders were observed in the allele frequencies, genotype counts, or haplotype distributions for the two variations, although the present sample size had a moderate power (0.46-0.86). These results do not suggest that the BZRP gene plays a role in the genetic predisposition of affective disorders.

Balance of two secretion pathways of nerve growth factor in PC12 cells changes during the progression of their differentiation, with a decrease in constitutive secretion in more differentiated cells.

Proteins are secreted from animal cells by either a constitutive or a regulated pathway. When cDNA of nerve growth factor (NGF) was introduced into PC12 cells, these cells produced and secreted active NGF, where NGF was secreted not only in constitutive but also in activity-dependent regulated way according to the results of pulse-chase and ELISA studies. The regulated secretion was caused by depolarization, cyclic AMP analogue, or beta-adrenergic agonist but not by glutamate or carbachol. Because these transfected cells differentiated into a morphology indistinguishable from that incubated with NGF protein, we next compared the secretion pathways of NGF from PC12 cells at different stages of the differentiation. NGF was secreted in both constitutive and regulated way at 2 and 7 days after the transfection of NGF-cDNA, but the constitutive secretion of NGF from the more differentiated cells of Day 7 was decreased and mature NGF tended to accumulate in the cells. These results indicate that the neurotrophin secretion mechanism is intimately regulated in the course of the differentiation of PC12 cells. Such a change in the protein secretion pathway might have an profound role in the development of neurons.

An association study between two missense variations of the benzodiazepine receptor (peripheral) gene and schizophrenia in a Japanese sample.

The benzodiazepine receptor (peripheral) (BZRP) mainly localized on glial cells plays a role in neurosteroid synthesis, and increases with glial proliferation. We have recently reported a significant decrease in the density of BZRP labeled by [3H] PK 11195 in the postmortem brain of chronic schizophrenics, suggesting that dysfunctions of the BZRP are involved in the pathophysiology of schizophrenia. We screened 11 patients with schizophrenia and 10 controls, which were used in a previous postmortem study, for their genomic sequences of the BZRP gene in order to find DNA sequence variations. One novel missense polymorphism (His162Arg) and another previously reported missense mutation (Ala147Thr) were detected. An association study of the identified variations was then performed in an extended Japanese sample of 304 schizophrenic patients and 369 controls. While there was an increased tendency in the frequency of the 162Arg allele of schizophrenics compared to that of the controls (p = 0.0603), no statistically significant association with schizophrenia was observed in the Ala147Thr allele (p = 0.1016). These results do not suggest that the two missense polymorphisms play a major role in the genetic predisposition of schizophrenia in the Japanese sample.

Induction of a physiologically active brain-derived neurotrophic factor in the infant rat brain by peripheral administration of 4-methylcatechol.

Effects of 4-methylcatechol (4MC), a known potent stimulator of nerve growth factor (NGF) synthesis, on expression of brain-derived neurotrophic factor (BDNF) mRNA and BDNF-like immunoreactivity (BDNF-LI) was investigated in infant rat brains. A single intraperitoneal administration of 4MC caused transient increases in the levels of BDNF mRNA and BDNF-LI in neurons of the cerebral cortex from 1 to 3 h and 3 to 12 h, respectively, after the injection. Repetitive injections of 4MC to newborn rats (12-h intervals for 10 days) caused a marked and dose-dependent elevation of the level of BDNF mRNA in the whole brain besides elevating the number of cells containing calbindin D-28 and enhancing its immunoreactive intensity in the pyriform cortex and hippocampus. These findings demonstrate that 4MC stimulates de novo synthesis of BDNF in the infant rat brain, resulting in acceleration of the developmental expression of calbindin D-28.

4-methylcatechol increases brain-derived neurotrophic factor content and mRNA expression in cultured brain cells and in rat brain in vivo.

Practical use of brain-derived neurotrophic factor (BDNF) as therapy is limited by two serious problems, i.e., its inability to cross the blood-brain barrier and its instability in the bloodstream. In the present study, we investigated the effects of 4-methylcatechol (4-MC), which stimulates nerve growth factor synthesis and protects against peripheral neuropathies in rats, on BDNF content and mRNA expression in cultured brain cells and in vivo in the rat brain. 4-MC elevated BDNF content in culture media of both rat astrocytes and neurons with different dose-response relations. The increase in BDNF mRNA level was correlated with the increase in BDNF content, demonstrating that 4-MC can stimulate BDNF synthesis of both neurons and astrocytes. Then we examined the in vivo effects of 4-MC. First, we found that ventricularly administered 4-MC facilitated an increase in the BDNF content in the cerebral cortex and hippocampus in association with its diffusion into the brain parenchyma. Second, i.p. administration of 4-MC enhanced BDNF mRNA expression in the infant rat brain, in which the blood-brain has not yet fully been established. These results demonstrate that 4-MC, once delivered into the brain, can stimulate BDNF synthesis.

Brain-derived neurotrophic factor prevents neuronal cell death induced by corticosterone.

Corticosterone (CORT), one of the glucocorticoids, causes neuronal damage in the hippocampus, but the mechanism(s) of action underlying its effects remains unknown. Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor that belongs to the neurotrophin family, affects the survival and/or differentiation of various types of neurons in vitro, and is able to antagonize neuronal death induced by various brain insults or neurotoxins in vivo. In this study, the effects of CORT on BDNF protein contents and mRNA expression were investigated in relation to neuronal survival/death of cultured rat hippocampal neurons, because the colocalization of BDNF with its receptor, TrkB, suggests that BDNF may exert its putative protective and trophic effects through an autocrine mechanism in the hippocampus. Administration of CORT accelerated the neuronal death that proceeds after serum deprivation, and simultaneously reduced the levels of BDNF mRNA and intracellular BDNF content. Exogenously added BDNF actually attenuated CORT-induced neuronal death, but not in the presence of K252a, an inhibitor of the tyrosine kinase activity of Trk family receptors. These observations suggest that CORT induces damage to hippocampal neurons, at least partly, via reducing their BDNF synthesis.

Endogenous neurotrophin-3 is retrogradely transported in the rat sciatic nerve.

To address the active transport of neurotrophins, nerve growth factor, brain-derived neurotrophic factor, and neurotrophin-3 in the peripheral nerves, we examined the levels of proteins and messenger RNAs in the sciatic nerve of adult rats following transection, using enzyme immunoassays and reverse transcription polymerase chain reaction method, respectively. Neurotrophin-3 protein increased one day after transection only in the distal segment next to the transection site and returned to the original level two days later. This was considered to reflect accumulation of neurotrophin-3 transported from the periphery toward the neuronal cell bodies, because the neurotrophin-3 messenger RNA level was not changed in any sciatic segments during this experimental period. An increase in brain-derived neurotrophic factor protein was observed simultaneously in both the distal and proximal stumps three days after transection. Brain-derived neurotrophic factor messenger RNA was elevated in the same stumps two days after transection, suggesting that brain-derived neurotrophic factor was produced within the transected stumps. These observations demonstrate that neurotrophin-3, like nerve growth factor, is retrogradely transported in the sciatic nerve but that brain-derived neurotrophic factor is not. This suggests that neurotrophin-3 plays a role in the conveyance of trophic signals from target organs to neurons.

Microsphere embolism-induced elevation of nerve growth factor level and appearance of nerve growth factor immunoreactivity in activated T-lymphocytes in the rat brain.

Changes in nerve growth factor (NGF) level and type of cells producing NGF were investigated in the rat brain after sustained cerebral embolism. The NGF level was determined by a two-site enzyme immunoassay specific for NGF. The cerebral cortex, striatum, and hippocampus of the embolized hemisphere maximally contained 2.4-, 2.4-, and 1.7-times higher NGF levels than the corresponding regions of the nonembolized hemisphere. A significant increase was transiently observed for 1 week in the cerebral cortex and striatum, whereas the increase was longer lasting, at least of 4 weeks' duration, in the hippocampus. To examine the localization of NGF-like immunoreactivity (NGF-LI), we used a newly developed anti-NGF peptide antiserum that specifically recognized a 30-kDa molecule(s) in the hippocampal extracts or in NGF cDNA-transfected cells, suggesting that the antibody predominantly reacted with the putative NGF precursor protein(s). NGF-LI, which was localized in neurons of the normal or non-embolized hemisphere, was reduced, and on the embolized side new signals emerged in small non-neuronal cells having a round shape. These included cells with common leukocyte antigen CD45 and T-lymphocyte antigen CD3, which did not appear in the normal or non-embolized hemisphere. NGF-LI and CD3 were colocalized in a substantial number of the cells, suggesting that some activated T-lymphocytes produce NGF for neuronal regeneration after sustained cerebral embolism.

Identification of the three non-identical subunits constituting human deoxyribonuclease II.

We purified DNase II from human liver to apparent homogeneity. The N-terminal amino acid sequences of each of three components constituting the purified mature enzyme were then separately determined by automatic Edman degradation. A combination of this chemical information and the previously reported nucleotide sequence of the cDNA encoding human DNase II [Yasuda et al. (1998) J. Biol. Chem. 273, 2610-2626] allowed detailed elucidation of the enzyme's subunit structure: human DNase II was composed of three non-identical subunits, a propeptide, proprotein and mature protein, following a signal peptide. Expression analysis of a series of deletion mutants derived from the cDNA of DNase II in COS-7 cells suggested that although a single large precursor protein may not be necessary for proteolytic maturation, the propeptide region L17-Q46 may play an essential role in generating the active form of the enzyme.

Simultaneous expression of brain-derived neurotrophic factor and neurotrophin-3 in Cajal-Retzius, subplate and ventricular progenitor cells during early development stages of the rat cerebral cortex.

To identify production sites and action targets of neurotrophins during neurogenesis, we investigated immunoreactivities of neurotrophins and their tyrosine kinase receptors in the cerebral cortex of rat embryos. Two sets of ligand-receptor systems, brain-derived neurotrophic factor/TrkB and neurotrophin-3/TrkC, were expressed simultaneously in Cajal-Retzius, subplate neurons and ventricular multipotent stem cells at embryonic days 13 and 15. Intraventricular administration of brain-derived neurotrophic factor or neurotrophin-3 at embryonic day 16 markedly modulated microtubule-associated protein II and/or Hu protein expression in different ways in the cortical plate cells by embryonic day 20. These observations indicate the involvement of autocrine and/or local paracrine action of brain-derived neurotrophic factor and/or neurotrophin-3 during formation of the cerebral cortex.

Brain-derived neurotrophic factor-like immunoreactivity in the adult rat central nervous system predominantly distributed in neurons with substantial amounts of brain-derived neurotrophic factor messenger RNA or responsiveness to brain-derived neurotrophic factor.

Distribution of brain-derived neurotrophic factor-like immunoreactivity was investigated in the adult rat brain using two types of antibodies against peptides, V2 and V4, unique to the brain-derived neurotrophic factor. Western blot analysis showed that both antibodies specifically bound brain-derived neurotrophic factor, but not other neurotrophins, and that they recognized identical molecules of 18,000 mol. wt, but not the 14,500 mol. wt mass of mature form, in extracts from the rat hippocampus. Both antibodies recognized an identical precursor form (30,000 mol. wt) in lysates of COS7 cells transfected with brain-derived neurotrophic factor gene. These indicated that both antibodies predominantly recognized identical precursor protein(s) or its derivative(s) probably because of their much higher amounts than the amount of mature protein. Immunochemical studies showed that anti-V2 predominantly stained the cytoplasm of cells; whereas the anti-V4 bound to the nucleus, suggesting that the tertiary structure of immunoreactive molecules changed depending on their location. Cell populations with the immunoreactivity were similar in most brain sections stained with either anti-V2 or anti-V4 antibodies. These results suggest that brain-derived neurotrophic factor-like immunoreactivity distributes, in most cases, in neurons responding to brain-derived neurotrophic factor and in neurons expressing abundant brain-derived neurotrophic factor messenger RNA. These, taken together with other results concerning distributions of messenger RNAs of brain-derived neurotrophic factor and TrkB, provide additional information to elucidate the function of brain-derived neurotrophic factor in the rat central nervous system.

Administration of corticosterone alters intracellular localization of brain-derived neurotrophic factor-like immunoreactivity in the rat brain.

We investigated the distribution of immunoreactivity for brain-derived neurotrophic factor (BDNF) in rat brain after peripheral administration of corticosterone or vehicle. In the immunohistochemical study, BDNF-like immunoreactivity (LI) was observed predominantly in the nucleus of the cortical and hippocampal neurons in the brain of vehicle-treated rats. In corticosterone-treated rats, BDNF-LI was markedly reduced in the nucleus and concomitantly increased in cytoplasm. Western immunoblot study also demonstrated that corticosterone significantly reduced BDNF-LI in the nuclear fraction of the cerebral cortex and hippocampus. These results indicate that corticostrone regulates the intracellular localization of BDNF and/or its derivatives in the rat brain.

BDNF and NT-3 modulate expression and threonine phosphorylation of microtubule-associated protein 2 analogues, and alter their distribution in the developing rat cerebral cortex.

Effects of brain-derived neurotrophic factor (BDNF) and neurotrophin (NT)-3 on the expression of structural or synapse-associated proteins were examined in the developing rat cerebral cortex. Following ventricular administration of BDNF or NT-3 at embryonic day (E) 16, expression of microtubule-associated protein (MAP) 2 of 280 kDa was enhanced at E18 and/or E20, and threonine phosphorylation of MAP2 analogues of 120 and 66 kDa was modulated in different ways. NT-3 basically altered the distribution of MAP2 proteins at E20. These findings suggest that NT-3 and BDNF play a role in regulating production and phosphorylation of MAP2 analogues during development of the rat cerebral cortex.