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The intestine is characterized by an environment in which host requirements for nutrient and water absorption are consequently paired with the requirements to establish tolerance to the outside environment. To better understand how the intestine functions in health and disease, large efforts have been made to characterize the identity and composition of cells from different intestinal regions 1-8 . However, the robustness, nature of adaptability and extent of resilience of the transcriptional landscape and cellular underpinning of the intestine in space are still poorly understood. Here we generated an integrated resource of the spatial and cellular landscape of the murine intestine in the steady and perturbed states. Leveraging these data, we demonstrated that the spatial landscape of the intestine was robust to the influence of the microbiota and was adaptable in a spatially restricted manner. Deploying a model of spatiotemporal acute inflammation, we demonstrated that both robust and adaptable features of the landscape were resilient. Moreover, highlighting the physiological relevance and value of our dataset, we identified a region of the middle colon characterized by an immune-driven multicellular spatial adaptation of structural cells to the microbiota. Our results demonstrate that intestinal regionalization is characterized by robust and resilient structural cell states and that the intestine can adapt to environmental stress in a spatially controlled manner through the crosstalk between immunity and structural cell homeostasis., Competing Interests: Competing interests: R.J.X. is a co-founder of Jnana Therapeutics, board director at MoonLake Immunotherapeutics and a consultant to Nestlé, and serves on the advisory board of Magnet Biomedicine; these organizations had no role in the study., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Odours used by insects for foraging and mating are carried by the air. Insects induce airflows around them by flapping their wings, and the distribution of these airflows may strongly influence odour source localisation. The flightless silkworm moth, Bombyx mori, has been a prominent insect model for olfactory research. However, although there have been numerous studies on antenna morphology and its fluid dynamics, neurophysiology, and localisation algorithms, the airflow manipulation of the B. mori by fanning has not been thoroughly investigated. In this study, we performed computational fluid dynamics (CFD) analyses of flapping B. mori to analyse this mechanism in depth. A three-dimensional simulation using reconstructed wing kinematics was used to investigate the effects of B. mori fanning on locomotion and pheromone capture. The fanning of the B. mori was found to generate an aerodynamic force on the scale of its weight through an aerodynamic mechanism similar to that of flying insects. Our simulations further indicate that the B. mori guides particles from its anterior direction within the ~ 60° horizontally by wing fanning. Hence, if it detects pheromones during fanning, the pheromone can be concluded to originate from the direction the head is pointing. The anisotropy in the sampling volume enables the B. mori to orient to the pheromone plume direction. These results provide new insights into insect behaviour and offer design guidelines for robots for odour source localisation., (© 2024. The Author(s).)

We conducted a large-scale online survey in February 2023 to investigate the public's perceptions of COVID-19 infection and fatality risks in Japan. We identified two key findings. First, univariate analysis comparing perceived and actual risk suggested overestimation and nonnegligible underestimation of COVID-19 risk. Second, multivariate logistic regression analyses revealed that age, income, education levels, health status, information sources, and experiences related to COVID-19 were associated with risk perceptions. Given that risk perceptions are closely correlated with daily socioeconomic activities and well-being, it is important for policy-makers and public health experts to understand how to communicate COVID-19 risk to the public effectively., (© 2024. The Author(s).)

During the 3 years of the COVID-19 pandemic, Japanese children had to live with strict mitigation measures at school, such as eating school lunches silently and wearing masks during physical exercise classes, even after those mitigation measures have been relaxed worldwide. Excursions and other school events were frequently cancelled, especially in 2020 and 2021. This study conducts a retrospective survey on school experiences to understand how the strict mitigation measures were related to children's mental health and well-being. Results revealed school excursion cancellation to be associated with a higher risk of developing depressive symptoms {odds ratio [OR] 1.543 [95% confidence interval (CI) 1.109-2.148]}, and high cancellation rate of other school events to be associated with dissatisfaction in school experience [OR 1.650 (95% CI 1.222-2.228)]. In the subsample analysis, we found that girls and children with no extracurricular activities tended to exhibit depressive symptoms due to the cancellation of school excursions. Overall, the study demonstrated that persistent strict mitigation measures at schools might be a key factor in understanding children's mental health and psychological well-being during a long-lasting pandemic., (© 2024. The Author(s).)

Optogenetics enables precise regulation of intracellular signaling in target cells. However, the application of optogenetics to induce the differentiation of precursor cells and generate mature cells with specific functions has not yet been fully explored. Here, we focused on osteoclasts, which play an important role in bone remodeling, to develop a novel optogenetics tool, Opto-RANK, which can manipulate intracellular signals involved in osteoclast differentiation and maturation using blue light. We engineered Opto-RANK variants, Opto-RANKc and Opto-RANKm, and generated stable cell lines through retroviral transduction. Differentiation was induced by blue light, and various assays were conducted for functional analysis. Osteoclast precursor cells expressing Opto-RANK differentiated into multinucleated giant cells on light exposure and displayed upregulation of genes normally induced in differentiated osteoclasts. Furthermore, the differentiated cells exhibited bone-resorbing activities, with the possibility of spatial control of the resorption by targeted light illumination. These results suggested that Opto-RANK cells differentiated by light possess the features of osteoclasts, both morphological and functional. Thus, Opto-RANK should be useful for detailed spatiotemporal analysis of intracellular signaling during osteoclast differentiation and the development of new therapies for various bone diseases., (© 2024. The Author(s).)

In Japan an original pathological classification of IgA nephropathy was used, while Oxford classification of IgA nephropathy was used globally. The Oxford classification requires ≥ 8 glomeruli while the Japanese classification requires ≥ 10. Ninety-nine patients diagnosed with IgA nephropathy were included. To determine the accuracy of histological staging, we calculated the posterior probability using Bayes' theorem and adopted three model of prior distribution. First, the actual staging distribution was reclassified using the beta distribution (reclassified distribution). Second a model with the same distribution (actual distribution) as the actual staging was used. Third, a model assuming that all cases are equally distributed (equal distribution) was used. The median number of collected glomeruli was 12 (8-19). There were 33 cases (33%) wherein the glomerular count was ≤ 9. When only cases with ≥ 10 glomeruli were included, the median posterior probability was 91% (74-99) (actual distribution, 90% [74-98]; equal distribution, 85% [73-96]). Even among the 33 cases with ≤ 9 glomeruli, there were approximately 7 cases in which the posterior probability was ≥ 90% for each model. Using Bayesian probabilistic analysis, it was possible to evaluate the histologic classification of IgA nephropathy, even when the number of obtained glomeruli was ≤ 9., (© 2023. The Author(s).)

Streptozotocin (STZ), an anti-cancer drug that is primarily used to treat neuroendocrine tumors (NETs) in clinical settings, is incorporated into pancreatic β-cells or proximal tubular epithelial cells through the glucose transporter, GLUT2. However, its cytotoxic effects on kidney cells have been underestimated and the underlying mechanisms remain unclear. We herein demonstrated that DNA damage and subsequent p53 signaling were responsible for the development of STZ-induced tubular epithelial injury. We detected tubular epithelial DNA damage in NET patients treated with STZ. Unbiased transcriptomics of STZ-treated tubular epithelial cells in vitro showed the activation of the p53 signaling pathway. STZ induced DNA damage and activated p53 signaling in vivo in a dose-dependent manner, resulting in reduced membrane transporters. The pharmacological inhibition of p53 and sodium-glucose transporter 2 (SGLT2) mitigated STZ-induced epithelial injury. However, the cytotoxic effects of STZ on pancreatic β-cells were preserved in SGLT2 inhibitor-treated mice. The present results demonstrate the proximal tubular-specific cytotoxicity of STZ and the underlying mechanisms in vivo. Since the cytotoxic effects of STZ against β-cells were not impaired by dapagliflozin, pretreatment with an SGLT2 inhibitor has potential as a preventative remedy for kidney injury in NET patients treated with STZ., (© 2023. The Author(s).)

Benign paroxysmal positional vertigo (BPPV) is associated with menopause and/or osteopenia. Morphological changes in the otoconial layer have been reported after ovariectomy (OVX). Moreover, hormone replacement therapy decreases BPPV risk. However, knowledge concerning the effect of hormonal therapy on the otoconial changes caused by estrogen deficiency is limited. We aimed to examine the effect of hormonal therapy on otoconial changes caused by estrogen deficiency. We hypothesized that hormonal therapy could reduce otoconial changes caused by OVX. Eight-week-old C57BL/6 mice were divided into four groups: sham operation with implantation of vehicle (sham + v), OVX with implantation of vehicle (OVX + v), OVX with implantation of estradiol (E2) (OVX + E2), and OVX with implantation of raloxifene (RAL) (OVX + RAL) groups. Otoconial layer volume was measured by micro-CT at 4 weeks after OVX or the sham operation. The otic bullae were removed; immunohistochemistry was performed for estrogen receptor alpha and 4-hydroxynonenal. Otoconial layer volume was significantly higher in the OVX + v than in the sham + v group. E2 and RAL significantly reduced these changes in the endometrial layer. The staining of estrogen receptor alpha and 4-hydroxynonenal were stronger in the OVX + v than in the sham + v group but equal in the sham + v, OVX + E2, and OVX + RAL groups. These results indicate that E2 and RAL are effective against morphological changes of the otoconial layer caused by estrogen deficiency via oxidative stress reduction., (© 2022. The Author(s).)

Recently, whole-exome sequencing (WES) has been used for genetic diagnoses of patients who remain otherwise undiagnosed. WES was performed in 177 Japanese patients with undiagnosed conditions who were referred to the Tokai regional branch of the Initiative on Rare and Undiagnosed Diseases (IRUD) (TOKAI-IRUD). This study included only patients who had not previously received genome-wide testing. Review meetings with specialists in various medical fields were held to evaluate the genetic diagnosis in each case, which was based on the guidelines of the American College of Medical Genetics and Genomics. WES identified diagnostic single-nucleotide variants in 66 patients and copy number variants (CNVs) in 11 patients. Additionally, a patient was diagnosed with Angelman syndrome with a complex clinical phenotype upon detection of a paternally derived uniparental disomy (UPD) [upd(15)pat] wherein the patient carried a homozygous DUOX2 p.E520D variant in the UPD region. Functional analysis confirmed that this DUOX2 variant was a loss-of-function missense substitution and the primary cause of congenital hypothyroidism. A significantly higher proportion of genetic diagnoses was achieved compared to previous reports (44%, 78/177 vs. 24-35%, respectively), probably due to detailed discussions and the higher rate of CNV detection., (© 2022. The Author(s).)

Kidney hypertrophy is a common clinical feature in patients with diabetes and is associated with poor renal outcomes. Initial cell proliferation followed by cellular hypertrophy are considered the responsible mechanisms for diabetic kidney hypertrophy. However, whether similar responses against hyperglycemia continue in the chronic phase in diabetes is unclear. We performed lineage tracing analysis of proximal tubular epithelia using novel type 2 diabetic mice with a tamoxifen-inducible proximal tubule-specific fluorescent reporter. Clonal analysis of proximal tubular epithelia demonstrated that the labeled epithelia proliferated in type 2 diabetic mice. Based on the histological analysis and protein/DNA ratio of sorted labeled tubular epithelia, there was no evidence of cellular hypertrophy in type 2 diabetic mice. Lineage tracing and histological analyses of streptozocin-induced type 1 diabetes also revealed that cellular proliferation occurs in the chronic phase of type 1 diabetes induction. According to our study, epithelial proliferation accompanied by SGLT2 upregulation, rather than cellular hypertrophy, predominantly occurs in the hypertrophic kidney in both type 1 and type 2 diabetes. An increased number of SGLT2+ tubular epithelia may be an adaptive response against hyperglycemia, and linked to the hyper-reabsorption of sodium and glucose observed in type 2 diabetes patients., (© 2022. The Author(s).)

Response to antihypertensive drugs in patients with chronic kidney disease (CKD) has great interindividual variability. Adrenomedullin (ADM) is produced abundantly in hypertension, but clearance is very rapid. Mid-regional proADM (MR-proADM) produced from an ADM precursor is considered a surrogate biomarker for quantification of ADM. We investigated the association of MR-proADM with antihypertensive resistance in CKD patients with poor blood pressure (BP) control. This cross-sectional study analyzed 33 CKD patients with poor BP control defined as failure to achieve target BP despite at least two classes of antihypertensive drugs. Treatment intensity score was calculated to facilitate comparability of antihypertensive regimens across subjects taking different drugs. Plasma MR-proADM concentration was measured using ultra-performance liquid chromatography coupled with tandem mass spectrometry. Plasma MR-proADM concentration correlated with estimated glomerular filtration rate (eGFR) (r =  - 0.777, p < 0.001). Treatment intensity score correlated positively with plasma MR-proADM concentration (r = 0.355, p = 0.043), and the correlation was further enhanced after correction by weight (r = 0.538, p = 0.001). Single and multiple regression analysis identified MR-proADM concentration (p = 0.005) as independently associated with weight-corrected treatment intensity score. MR-proADM may be useful as a biomarker to determine the therapeutic intensity of antihypertensive drugs in CKD patients with poor BP control., (© 2021. The Author(s).)

Cisplatin is a commonly used anticancer drug, but nephrotoxicity is a dose-limiting adverse effect. Recent experimental and clinical observations have demonstrated that multiple injections of cisplatin induce the transition to chronic kidney disease; however, the underlying mechanisms remain unclear. We found that multiple injections of higher doses of cisplatin in a shorter interval affected the severity of kidney injury, causing kidney fibrosis to develop at a later time point. An additional injection of cisplatin during the recovery period after a prior injury, when proximal tubule epithelia are actively proliferating, induced substantial tubular injury by inducing more severe DNA damage than that induced by a single injection. Lineage tracing analysis of proximal tubular epithelia demonstrated that the tubular epithelia that underwent multiple rounds of cell division after multiple injections of cisplatin existed at the chronic phase, and these populations often expressed vcam1 + , suggesting the induction of proinflammatory failed-repair tubular epithelia. Our study revealed that as cisplatin exerts cytotoxic effects on actively proliferating cells, additional cisplatin injections before the completion of tubular repair exacerbates kidney injury through cumulative DNA damage. Appropriate both the setting of dosage and dosing intervals, with careful monitoring, are essential to prevent nephrotoxicity of repeated cisplatin treatment in cancer patients., (© 2021. The Author(s).)

Since the treatment window of thrombolytic therapy for stroke is limited, new therapy remains to be developed. We have recently developed low-intensity pulsed ultrasound (LIPUS) therapy to improve cognitive dysfunction in mouse models of vascular dementia and Alzheimer's disease. Here, we further aimed to examine whether our LIPUS therapy improves neurological recovery from ischemic stroke, and if so, to elucidate the mechanisms involved. In a mouse model of middle cerebral artery occlusion (MCAO), we applied LIPUS (32 cycles, 193 mW/cm 2 ) to the whole brain 3 times in the first week (days 1, 3, and 5) after MCAO. We evaluated neurological functions using behavioral tests and performed histological analyses. Furthermore, to elucidate how LIPUS works within the injured brain, we also tested the effects of LIPUS in endothelial nitric oxide synthase (eNOS)-deficient (eNOS -/- ) mice. In wild-type mice, the LIPUS therapy markedly improved neurological functions in the tightrope and rotarod tests at 28 days after MCAO. Histological analyses showed that the LIPUS therapy significantly increased the numbers of CD31-positive blood vessels in the perifocal lesion and doublecortin (DCX)-positive neurons in the ischemic striatum, indicating the angio-neurogenesis effects of the therapy. Importantly, these beneficial effects of the LIPUS therapy were totally absent in eNOS -/- mice. No adverse effects of the LIPUS therapy were noted. These results indicate that the LIPUS therapy improves neurological functions after stroke through enhanced neuro-angiogenesis in mice in vivo in an eNOS-dependent manner, suggesting that it could a novel and non-invasive therapeutic option for stroke.

The DNA damage response after kidney injury induces cell cycle arrest in renal tubular epithelial cells, resulting in the secretion of pro-fibrotic cytokines, thereby promoting interstitial fibrosis in a paracrine manner. Phosphorylation of ataxia-telangiectasia mutated (ATM) is the initial step in the DNA damage response and subsequent cell cycle arrest; however, the effects of ATM inhibition on the injured kidney have not been explored. Pharmacological ATM inhibition by KU55933 in cisplatin-treated mice did not ameliorate, but instead exacerbated cisplatin-induced DNA damage and tubular injury, thereby increasing mortality. Analysis of isolated tubular epithelia by FACS from bigenic SLC34a1-CreERt2; R26tdTomato proximal tubular-specific reporter mice revealed that KU55933 upregulated p53 and subsequent pro-apoptotic signaling in tubular epithelia of cisplatin-treated mice, leading to marked mitochondrial injury and apoptosis. In addition, KU55933 attenuated several DNA repair processes after cisplatin treatment, including single-strand DNA repair and Fanconi anemia pathways, suggesting that DNA repair after dual treatment of cisplatin and KU55933 was not sufficient to prevent the cisplatin-induced tubular injury. Our study suggested that ATM inhibition does not increase DNA repair after cisplatin-induced DNA damage and exacerbates tubular injury through the upregulation of p53-dependent pro-apoptotic signaling. Acute kidney injury must be carefully monitored when ATM inhibitors become available in clinical practice in the future.

Objective: The objective of this study was to describe a novel amplitude-integrated electroencephalography (aEEG) pattern in infants with hypoxic-ischemic encephalopathy (HIE) and to assess the clinical significance., Methods: The aEEG traces of infants with HIE who were treated with therapeutic hypothermia (TH) from 2012 to 2017 were analyzed. A pseudo-sawtooth (PST) pattern was defined as a periodic increase of the upper and/or lower margin of the trace on aEEG without showing seizure activities on conventional EEG (CEEG)., Results: Of the 46 infants, 6 (13%) had the PST pattern. The PST pattern appeared following a flat trace or a continuous low-voltage pattern and was followed by a burst-suppression pattern. On CEEG, the PST pattern consists of alternating cycles of low-voltage irregular activities and almost flat tracing. The PST pattern was associated with neuroimaging abnormalities and with various degrees of neurodevelopmental outcomes. Positive predictive values of the PST or worse pattern for adverse outcomes were high at 12 h after birth., Conclusion: A novel aEEG background pattern in infants with HIE was reported. The PST pattern likely indicates a suppressed background pattern and may be linked to unfavorable outcomes. Further multicenter validation study is needed to clarify its clinical significance.

The trace addition of rare earth (RE) elements in Mg alloys can modify the extrusion texture, leading to the formation of RE texture and thus improved formability. The interrupted extrusion experiment as well as electron back-scatter diffraction (EBSD) characterization was conducted in Mg-1.5Zn-0.5Gd (wt.%) alloy to unveil the dominant dynamic recrystallization (DRX) mechanism and its correlation with the formation of RE texture during extrusion. The results indicate that continuous DRX (CDRX) dominated the microstructural development. Fresh DRXed grains with 30° [0001] grain boundaries preferentially nucleated in unDRXed grains with [10[Formula: see text]0] basal fiber orientation via CDRX, showing preferred selection of [2[Formula: see text][Formula: see text]0] basal fiber orientation rather than RE texture orientation. Consequently, CDRX contributed to the weakening of [10[Formula: see text]0] basal fiber texture and had a more significant effect on the formation of [2[Formula: see text][Formula: see text]0] basal fiber component than that of RE texture component. Besides, the preferred growth of recrystallized grains with RE texture orientation was confirmed to occur during static annealing after extrusion, which is inferred as the key reason for the formation of RE texture in dilute Mg-RE alloys.

Upon invasion, Plasmodium falciparum exports hundreds of proteins across its surrounding parasitophorous vacuole membrane (PVM) to remodel the infected erythrocyte. Although this phenomenon is crucial for the parasite growth and virulence, elucidation of precise steps in the export pathway is still required. A translocon protein complex, PTEX, is the only known pathway that mediates passage of exported proteins across the PVM. P. falciparum Parasitophorous Vacuolar protein 1 (PfPV1), a previously reported parasitophorous vacuole (PV) protein, is considered essential for parasite growth. In this study, we characterized PfPV1 as a novel merozoite dense granule protein. Structured illumination microscopy (SIM) analyses demonstrated that PfPV1 partially co-localized with EXP2, suggesting the protein could be a PTEX accessory molecule. Furthermore, PfPV1 and exported protein PTP5 co-immunoprecipitated with anti-PfPV1 antibody. Surface plasmon resonance (SPR) confirmed the proteins' direct interaction. Additionally, we identified a PfPV1 High-affinity Region (PHR) at the C-terminal side of PTP5 where PfPV1 dominantly bound. SIM analysis demonstrated an export arrest of PTP5ΔPHR, a PTP5 mutant lacking PHR, suggesting PHR is essential for PTP5 export to the infected erythrocyte cytosol. The overall results suggest that PfPV1, a novel dense granule protein, plays an important role in protein export at PV.

Mosquitoes exhibit unusual wing kinematics; their long, slender wings flap at remarkably high frequencies for their size (>800 Hz)and with lower stroke amplitudes than any other insect group. This shifts weight support away from the translation-dominated, aerodynamic mechanisms used by most insects, as well as by helicopters and aeroplanes, towards poorly understood rotational mechanisms that occur when pitching at the end of each half-stroke. Here we report free-flight mosquito wing kinematics, solve the full Navier-Stokes equations using computational fluid dynamics with overset grids, and validate our results with in vivo flow measurements. We show that, although mosquitoes use familiar separated flow patterns, much of the aerodynamic force that supports their weight is generated in a manner unlike any previously described for a flying animal. There are three key features: leading-edge vortices (a well-known mechanism that appears to be almost ubiquitous in insect flight), trailing-edge vortices caused by a form of wake capture at stroke reversal, and rotational drag. The two new elements are largely independent of the wing velocity, instead relying on rapid changes in the pitch angle (wing rotation) at the end of each half-stroke, and they are therefore relatively immune to the shallow flapping amplitude. Moreover, these mechanisms are particularly well suited to high aspect ratio mosquito wings.

The effect of long period stacking ordered (LPSO) phase and γ' precipitates on the ageing behavior and mechanical properties of the extruded Mg-8.2Gd-3.8Y-1.0Zn-0.4Zr (wt.%) alloy was investigated. The results show that more β' phases precipitate during ageing treatment in the LPSO phase containing alloy so that the LPSO phase containing alloy exhibits a higher age-hardening response than the γ' precipitates containing alloy. The precipitation strengthening induced by β' precipitates is the greatest contributor to the strength of the peak-aged LPSO-containing alloys. Higher strength is achieved in γ' precipitates containing alloy due to the more effective strengthening induced by dense nanoscale γ' precipitates than LPSO phases as well as the higher volume fraction of coarse unrecrystallized grains with strong basal texture. The extruded alloy containing γ' precipitates after T5 peak-ageing treatment shows ultra-high tensile yield strength of 462 MPa, high ultimate tensile strength of 520 MPa, and superior elongation to failure of 10.6%.

High performance Mg-8.2Gd-3.8Y-1.0Zn-0.4Zr alloy with high strength and excellent ductility has been successfully developed by hot extrusion. The effect of plate-shaped long period stacking ordered (LPSO) phases and solute-segregated stacking faults (SFs) on the dynamically recrystallization (DRX) behavior was analyzed. The plate-shaped LPSO phases stimulate the DRX by particle stimulated nucleation mechanism, leading to higher DRX ratio and weaker basal texture. While for the alloy with dense fine SFs inside the original grains, discontinuous DRX initially occurs at the original grain boundaries, and the DRX is obviously restricted. Consequently, alloy containing dense SFs exhibits higher strength but lower ductility compared with alloy with plated-shaped LPSO phases., Competing Interests: The authors declare no competing financial interests.

Intestinal epithelial cells (IECs) play an indispensable role in maintaining body fluid balance partly through their ability to regulate anion/fluid secretion. Yet in various inflammatory gastrointestinal diseases, over-secretion of anions results in symptoms such as severe diarrhoea. Endogenous mediators, such as vasoactive intestinal peptide or prostaglandin E 2 (PGE 2 ), regulate intestinal anion/fluid secretion, but their direct effect on purified human IECs has never been described in detail. Based on a previously described intestinal organoid swelling model, we established a 3D-scanner-assisted quantification method to evaluate the anion/fluid secretory response of cultured human IECs. Among various endogenous secretagogues, we found that PGE 2 had the lowest EC 50 value with regard to the induction of swelling of the jejunal and colonic organoids. This PGE 2 -mediated swelling response was dependent on environmental Cl - concentrations as well as on several channels and transporters as shown by a series of chemical inhibitor studies. The concomitant presence of various inflammatory cytokines with PGE 2 failed to modulate the PGE 2 -mediated organoid swelling response. Therefore, the present study features PGE 2 as a direct and robust mediator of anion/fluid secretion by IECs in the human intestine.

Cilnidipine, an N/L-type calcium channel blocker, has been reported to inhibit sympathetic nerve activity and has a greater renoprotective effect than L-type calcium channel blockers. To investigate the hypothesis that cilnidipine might ameliorate advanced hypertensive nephropathy and inhibit the renal renin-angiotensin-aldosterone system, cilnidipine (1 mg per kg per day) or amlodipine (1 mg per kg per day) was administered to uninephrectomized deoxycorticosterone (DOCA)-salt hypertensive rats (DOCA-salt) for 4 weeks by gavage. Although the blood pressure in the DOCA-salt group was higher than that of control, neither cilnidipine nor amlodipine had any effect on the increase in blood pressure in the DOCA-salt group. The DOCA (40 mg per kg per week, subcutaneously (s.c.)) and salt (1% NaCl in drinking water) treatment significantly aggravated the levels of urinary protein excretion and creatinine clearance and increased glomerulosclerosis and collagen deposition in the tubulointerstitial area of the kidney. These effects were attenuated by cilnidipine treatment. Reverse transcription-polymerase chain reaction analysis revealed that the renal expression of mRNA for collagen I/IV and transforming growth factor-β was enhanced in the DOCA-salt group and that the overexpression of these molecules was suppressed by cilnidipine. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived superoxide production in the kidney and urinary norepinephrine excretion, which were enhanced in the DOCA-salt group, were suppressed by cilnidipine. Cilnidipine also decreased the activity and expression of angiotensin-converting enzyme (ACE) and the aldosterone concentration in the renal homogenate. Although neither cilnidipine nor amlodipine had any effect on the increased blood pressure in the DOCA-salt group, these renal changes were not induced by treatment with amlodipine. In conclusion, cilnidipine inhibited renal dysfunction, sympathetic nerve activity and renal renin-angiotensin-aldosterone system in the DOCA-salt group.

The roles of aldosterone in the progression of heart failure have not been fully elucidated. This study examined whether aldosterone nongenomically activates reactive oxygen species (ROS) production, causing myocyte apoptosis. Addition of aldosterone to neonatal rat cardiac myocytes caused the activation of NADPH oxidase and intracellular ROS production in a dose-dependent manner (10-(9)-10(-7) mol/L). NADPH oxidase activation was evident as soon as 5 min after aldosterone treatment. Neither an inhibitor for nuclear transcription (actinomycin D) nor an inhibitor of new protein synthesis (cycloheximide) blocked this rapid activation, and specific binding of aldosterone to plasma membrane fraction was inhibited by eplerenone, suggesting a nongenomic mechanism. Aldosterone did not affect the mRNA or protein levels of NOX2, which is a major subunit of NADPH oxidase in myocytes, after 48 h. Nuclear staining with DAPI showed that aldosterone (10(-7) mol/L) increased the myocyte apoptosis (2.3 fold, p<0.001), coincident with the activation of caspase-3 (1.4 fold, p<0.05), compared with the serum-deprived control after 48 h. Aldosterone also induced phosphorylation of apoptosis signal-regulating kinase 1 (ASK1). These effects of aldosterone on myocyte ROS accumulation, ASK1 activation, and apoptosis were abolished by eplerenone, a mineralocorticoid receptor (MR) antagonist, apocynin, an inhibitor of NADPH oxidase activation, and tempol, a free radical scavenger, but by neither RU486, a glucocorticoid receptor antagonist, nor butylated hydroxyanisol (BHA), a mitochondrial ROS scavenger. In conclusion, aldosterone-mediated ROS production is blocked by eplerenone and induced by the nongenomic activation of NADPH oxidase, leading to myocyte apoptosis associated with ASK1 activation. These proapoptotic actions of aldosterone may play a role in the progression of heart failure.

In the course of our screening for inhibitors of hyaluronic acid (HA) binding to cellular receptor CD44, a novel inhibitor, F-19848 A, was isolated from the cultured broth of the fungus strain Dacrymyces sp. SANK 20204. This compound inhibited the binding of CD44 and HA with an IC50 value of 23.5 microM and CD44-dependent HA degradation was inhibited with an IC50 value of 98.6 microM in a cell-based assay. The structure was elucidated by physico-chemical properties, analysis of spectral data, and decomposition experiments.

To reappraise the cutoff level of abdominal circumference (AC) for diagnosis of visceral obesity in Japanese, we examined the association of visceral fat deposition with other constituents of metabolic syndrome and atherosclerotic cardiovascular disease (ASCD). CT was used for determination of visceral-fat area (VFA), subcutaneous-fat area (SFA) and AC on CT (AC(CT)) in 420 Japanese patients with (n=180) or without ASCD (n=240). VFA cutoff levels were calculated by receiver operating characteristic (ROC) analysis. AC(CT) correlated with VFA (r=0.828), SFA (r=0.795), and AC measured with an anthropometric tape (AC(M), r=0.96). The VFA cutoff levels yielding the maximum sensitivity and specificity to predict two or more components of metabolic syndrome were 92 cm(2) in males and 63 cm(2) in females, which correspond to AC(M) values of 83 cm and 78 cm, respectively. The male AC(M) cutoff level was similar to the AC in current Japanese criteria (85 cm), but the female AC(M) cutoff level was considerably smaller than the criteria, and this change in cutoff level increased the prevalence of metabolic syndrome in females three-fold. The cutoff levels of VFA for predicting presence of ASCD were 98 cm(2) in males and 75 cm(2) in females, corresponding to AC(M) values of 84 cm and 80 cm, respectively. The present results obtained by CT support the validity of the current Japanese criteria for visceral obesity in males but not in females. AC(M) of 78 cm appears to be a cutoff level suitable for diagnosing visceral obesity in Japanese females, though further confirmation is needed.

Renin-angiotensin system (RAS) inhibitors are likely to reduce the development of atrial fibrillation by preventing atrial fibrosis. Strain rate (SR) imaging is a novel echocardiographic technique to quantify left atrial (LA) function. However, it has not been determined whether SR imaging is applicable for detection of LA dysfunction in hypertensive (HT) patients. We used SR imaging to assess alteration in LA function in HT patients and its modification by RAS inhibitors. SR imaging was performed in 80 HT patients and 50 age-matched normotensive (NT) subjects. HT patients were divided into two groups according to left ventricular hypertrophy (LVH) and LA dilatation. Peak SR was measured at each LA segment (septal, lateral, posterior, anterior, and inferior) and mean peak systolic SR (SR-LAs) was calculated by averaging data in each segment. Mean SR-LAs levels in the dilated LA group (1.97+/-0.45 s(-1), n=25) and non-dilated LA group (2.15+/-0.57 s(-1), n=55) were significantly (p<0.05) lower than that in NT subjects (2.53+/-0.71 s(-1)). Irrespective of the presence or absence of LVH, mean SR-LAs in HT patients was lower than that in NT subjects. When RAS inhibitors were used, the mean SR-LAs level in the non-dilated LA group was equivalent to that in NT subjects. In HT patients, mean SR-LAs, an index of LA reservoir function, decreases before development of LA enlargement and LVH. Treatment with RAS inhibitors appears to preserve LA reservoir function in HT patients without dilated LA. SR imaging can detect LA dysfunction in HT patients and is useful for evaluation of the therapeutic benefit on LA reservoir function.

In the course of our screening for binding inhibitors of CD44 and hyaluronic acid, five active compounds, F-16438 A, B, E, F and G were found and isolated from the cultured broth of a fungal strain, Gloeoporus dichrous SANK 30502. The structures of these compounds except for F-16438 G were elucidated by physico-chemical and spectral data to be new compounds related to caloporoside; F-16438 G was identified to be the 6'-malonylated derivative of caloporoside.

In an attempt to obtain inhibitors of hyaluronic acid (HA) binding to its receptor, CD44, we established an efficient assay method to detect and quantify binding using fluorescein-labeled HA and HEK293 cells stably expressing CD44. As a result of the screening of culture broths of microorganisms, we found fungus strain Gloeoporus dichrous SANK 30502 produced inhibitory activity in this new assay. Five compounds, F-16438 A, B, E, F and G, were isolated from the fermentation broths, and their IC50 values were determined to be 10.3, 13.5, 27.3, 12.0 and 13.0 microM, respectively. F-16438 A, B, E, F and G are the first reported inhibitors of binding HA to CD44. F-16438 A, B, E and F have novel structures.

Calcium channel blockers have been shown to limit the progression of atherosclerosis and decrease the incidence of cardiovascular events. To investigate vasoprotective effects beyond the blood pressure-lowering effects of these agents, amlodipine (10(-6) mol/) and manidipine (10(-6) mol/l) were used to pretreat angiotensin (Ang) II-stimulated rat cultured aortic endothelial cells. A 3-h period of Ang II treatment enhanced superoxide generation and the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase protein, as detected by dihydroethidium staining and Western blotting, respectively. Pretreatment with amlodipine or manidipine attenuated the increased production of superoxide and the overexpression of NADPH oxidase. The enhanced expression of heme oxygenase-1 (HO-1) mRNA induced by Ang II was further increased by amlodipine, whereas pretreatment with manidipine led to a reduction in the expression of HO-1. Furthermore, Ang II increased vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), and monocyte chemoattractant protein-1 (MCP-1) mRNA levels, as determined by reverse transcription (RT)-polymerase chain reaction (PCR). Pretreatment with either amlodipine or manidipine decreased the overexpression of VCAM-1, ICAM-1, and MCP-1. We also demonstrated that amlodipine or manidipine prevented the Ang II-induced increase in lectin-like oxidized low-density lipoprotein receptor1 (LOX-1) content, thereby restoring control levels. These observations showed that amlodipine and manidipine reduced superoxide generation by the inhibition of the overexpression of NADPH oxidase in Ang II-stimulated endothelial cells. Such antioxidant effects of these agents might in turn have led to a decrease in the expression of VCAM-1, ICAM-1 and MCP-1. The salutary effects of calcium channel blockers in atherogenesis include the inhibition of the expression of LOX-1.

Long-acting dihydropyridine calcium channel blockades have been shown to limit the progression of atherosclerosis and decrease the incidence of cardiovascular events in humans and animals. To investigate the vasoprotective effects beyond the blood pressure-lowering effects of these agents, amlodipine (20 mg/kg/ day) and manidipine (10 mg/kg/day) were administered by gavage to N(G)-nitro-L-arginine methyl ester (L-NAME)-induced hypertensive rats for 2 weeks. L-NAME treatment (0.7 mg/ml in drinking water) significantly decreased the gene and protein expression of endothelial nitric oxide synthase (eNOS) and increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, vascular cell adhesion molecule-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1) mRNA levels in the aorta, as determined by Western blotting and reverse transcription (RT)-polymerase chain reaction (PCR). Amlodipine and manidipine normalized the decreased expression of eNOS gene and protein, and attenuated the overexpression of NADPH oxidase, VCAM-1, and MCP-1 mRNA. Furthermore, amlodipine and manidipine prevented the L-NAME-induced increase in the angiotensin converting enzyme (ACE) mRNA content, thereby restoring control levels in the aorta. On the other hand, hydralazine treatment had no such effect in L-NAME treated rats. Furthermore, the increased expression of manganese superoxide dismutase (Mn-SOD) by L-NAME treatment was not affected by amlodipine, manidipine, or hydralazine. We concluded that the direct anti-inflammatory and antioxidative effects of calcium channel blockades in the aorta of rats with L-NAME-induced hypertension were not likely to have been mediated by the blood pressure-lowering action of these agents, but instead these beneficial effects appear to have been mediated by an augmentation of eNOS expression and by the inhibition of the expression of ACE.

Although exercise is recommended for the primary prevention of hypertension, and although it is generally known to have a beneficial effect on endothelial function, working individuals often find it difficult to maintain a consistent exercise regimen. In the present study, therefore, we examined the effects of infrequently performed exercise on flow-mediated dilatation (FMD), which is an index of endothelial function, in 15 subjects with hypertension (mild hypertensives) and 10 normotensive subjects (normotensives). All subjects performed mild bicycle exercise twice a week for 12 weeks. To assess the FMD, the diameter of the brachial artery was measured using ultrasound at baseline, during reactive hyperemia, and following sublingual administration of nitroglycerin. Measurement of these parameters was performed twice, at the beginning and the end of the exercise program. At the baseline, FMD was significantly lower in the mild hypertensives than in the normotensives. Nitroglycerin-mediated dilatation (NTG-D) was similar in the two groups. The exercise decreased blood pressure in the mild hypertensives, and increased high-density lipoprotein (HDL) cholesterol in both groups. The exercise improved FMD without altering NTG-D in the mild hypertensives, but did not result in any change in the normotensives. Multiple regression analysis revealed that the elevation in FMD was positively associated with changes in HDL cholesterol, and negatively associated with changes in plasma norepinephrine and systolic blood pressure. These findings suggest that regular exercise at a low frequency improves FMD, and thereby endothelial function, and lowers blood pressure in mild hypertensives.

This study was designed to test the hypothesis that increased pressure itself could cause endothelial dysfunction and lead to decreased nitric oxide (NO) release, partly through effects on the tissue renin angiotensin system in hypertension. Cultured endothelial cells (ECs) isolated from the aortas of WKY rats were continuously exposed to a pressure of 150 mmHg in a CO2 incubator for 72 h using a pressure system, and the NOx (NO2 and NO3) and angiotensin II (Ang II) concentrations in the supernatant were measured. An Ang II type 1 receptor (AT1R) antagonist (losartan) and an Ang II type 2 receptor (AT2R) antagonist (PD123319) were added to the medium. The expression of AT1R and AT2R mRNAs was also examined. Pressure loading significantly decreased the NO release from ECs. Concomitant administration of losartan restored NO release to the level before the application of pressure (p<0.001). This effect of losartan was blocked by simultaneous administration of PD123319, bradykinin type 2 receptor antagonist, and NO synthase inhibitor (p<0.05). The Ang II concentration was increased by pressure and was further increased by losartan. The gene expression of AT1R was not changed by pressure, but AT2R mRNA was increased almost 2-fold. These results indicate that high pressure itself attenuates NO release from ECs, and that losartan improves NO release by activating the bradykinin system via AT2R stimulation. In addition, the increase of AT2R gene expression in ECs during exposure to pressure may compensate for the reduction of NO.

To investigate the effects of angiotensin converting enzyme inhibitor (ACE-I) and other antihypertensive agents on the nitric oxide (NO) release during hypertension, seven- and fourteen-week-old SHR and deoxycorticosterone acetate (DOCA)-salt rats were treated with hydralazine, manidipine (Ca antagonist) or quinapril (ACE-I) for 3 weeks to lower blood pressure. Systolic blood pressure (SBP) was measured by the tail cuff method once each week. Endothelial cells (ECs) derived from the descending aorta of the treated rats were cultured and NOx levels in culture media were measured with an NO analyzer based on the Griess reaction. In both SHR and DOCA-salt rats, antihypertensive therapy lowered SBP to levels similar to those of control rats. The only exception was quinapril treatment of DOCA-salt rats. Although NOx release by ECs derived from hypertensive rats was improved by antihypertensive therapy, the effect was most pronounced in SHR treated with quinapril. In addition, restoration of NOx release was much more remarkable in younger SHR. NOx release was significantly higher in DOCA-salt rats treated with quinapril than in control rats without reduction of SBP. These results suggest that lowering blood pressure improves release of NO by ECs during hypertension and that the time at which antihypertensive therapy is started is also important to preserve endothelial function. Furthermore, ACE-I is suggested to protect endothelial function by increasing NO production in addition to lowering blood pressure.

We examined the role of the central nervous system, and particularly the renin-angiotensin (RA) system, in the development of hypertension produced by chronic inhibition of NO synthesis. In experiment 1, Wistar rats drank either nitro-L-arginine-methyl ester (L-NAME) or tap water. Before L-NAME treatment rats were divided into 6 groups. Four of them were administered either losartan or artificial cerebroventricular fluid (a-CSF) intracerebroventricularly (i.c.v.) for 1 week using an osmotic mini pump. The other two groups were administered the same amount of losartan intravenously (i.v.). In experiment 2, cardiovascular responses to acute i.c.v. losartan and muscimol, a GABA(A) agonist, were examined in conscious L-NAME-treated rats. Finally, in experiment 3, effects of ablation of the AV3V (anteroventral third ventricle) area, known to be one of the centers of cardiovascular control, were tested in the development of L-NAME hypertension. The development of hypertension by L-NAME treatment was attenuated with chronic i.c.v. losartan in a dose-dependent manner, while i.v. losartan had no effect. One week after cessation of i.c.v. losartan, blood pressure was elevated to the same level as in a-CSF-infused, L-NAME-treated rats. Acute i.c.v. losartan produced no cardiovascular changes in either L-NAME-treated or control rats. On the other hand, although i.c.v. muscimol elicited depressor effects in both groups, these responses were significantly larger in L-NAME-treated rats. Cardiovascular responses to i.v. hexamethonium were similar in both groups. The existence of prior lesions in the AV3V area significantly attenuated the development of L-NAME-induced hypertension. These results indicate that the central RA system plays an important role in the development of hypertension produced by chronic inhibition of NO synthase. Moreover, disorder of the central GABA system, rather than that of the RA system, might be important in the maintenance of hypertension in this model.

The MLK (mixed lineage) ser/thr kinases are most closely related to the MAP kinase kinase kinase family. In addition to a kinase domain, MLK1, MLK2 and MLK3 each contain an SH3 domain, a leucine zipper domain and a potential Rac/Cdc42 GTPase-binding (CRIB) motif. The C-terminal regions of the proteins are essentially unrelated. Using yeast two-hybrid analysis and in vitro dot-blots, we show that MLK2 and MLK3 interact with the activated (GTP-bound) forms of Rac and Cdc42, with a slight preference for Rac. Transfection of MLK2 into COS cells leads to strong and constitutive activation of the JNK (c-Jun N-terminal kinase) MAP kinase cascade, but also to activation of ERK (extracellular signal-regulated kinase) and p38. When expressed in fibroblasts, MLK2 co-localizes with active, dually phosphorylated JNK1/2 to punctate structures along microtubules. In an attempt to identify proteins that affect the activity and localization of MLK2, we have screened a yeast two-hybrid cDNA library. MLK2 and MLK3 interact with members of the KIF3 family of kinesin superfamily motor proteins and with KAP3A, the putative targeting component of KIF3 motor complexes, suggesting a potential link between stress activation and motor protein function.

To determine whether angiotensin II participates in the pathogenesis of cardiac hypertrophy and impairs coronary circulation in DOCA/salt hypertension, DOCA hypertensive rats were treated with candesartan cilexetil for 8 wk. DOCA/salt hypertension was induced in Wistar rats by removing the right kidney and subcutaneously injecting deoxycorticosterone acetate once a week. Control rats were given subcutaneous injections of saline and maintained on a normal diet. After 4 wk of observation, the angiotensin II receptor antagonist candesartan cilexetil was administered by oral gavage for 8 wk to 14 rats. Systolic blood pressure was measured weekly with the tail-cuff method. After 12 wk, the rats were killed and prepared. The isolated hearts were perfused by a Langendorff apparatus at constant flow. Perfusion pressure was measured by a small-volume transducer, and perfusion flow was recorded by a drop counter. Development of hypertension was not prevented by candesartan cilexetil treatment, but development of cardiac hypertrophy was inhibited. Minimum coronary vascular resistance (MCVR) obtained upon infusing adenosine into the isolated hearts was significantly higher in DOCA/salt hypertensive rats than in sham-operated controls. The elevated MCVR in DOCA/salt hypertensive rats was decreased by the administration of candesartan cilexetil for 8 wk. Thus, candesartan cilexetil regressed cardiac hypertrophy and improved coronary vascular resistance without affecting high blood pressure. These findings suggest that angiotensin II plays an important role in the pathogenesis of cardiac hypertrophy in DOCA/salt hypertension and that cardiac hypertrophy increases coronary vascular resistance.

Insulin drives the formation of a complex between tyrosine-phosphorylated IRS-1 and SH2-containing proteins. The SH2-containing protein Grb2 also possesses adjacent SH3 domains, which bind the Ras guanine nucleotide exchange factor Sos. In this report, we examined the involvement of another SH3 binding protein, dynamin, in insulin signal transduction. SH3 domains of Grb2 as GST fusion proteins bound dynamin from lysates of CHO cells expressing wild-type insulin receptor (IR) (CHO-IR cells) in a cell-free system (in vitro). Immunoprecipitation studies using specific antibodies against Grb2 revealed that Grb2 was co-immunoprecipitated with dynamin from unstimulated CHO-IR cells. After insulin treatment of CHO-IR cells, anti-dynamin antibodies co-immunoprecipitated the IR beta-subunit and IRS-1, as tyrosine-phosphorylated proteins and PI 3-kinase activity. However, purified rat brain dynamin did not bind directly to either the IR, IRS-1 or the p85 subunit of PI 3-kinase in vitro. Together, these results suggest that in CHO-IR cells, insulin stimulates the binding of dynamin to tyrosine-phosphorylated IRS-1 via Grb2 and that IRS-1 also associates with PI 3-kinase in response to insulin. This complex formation was reconstituted in vitro using recombinant baculovirus-expressed IRS-1, GST-Grb2 fusion proteins and dynamin peptides containing proline-rich sequences. Furthermore, dynamin GTPase activity was found to be stimulated when an IRS-1-derived phosphopeptide, containing the Grb2 binding site, was added to the dynamin-Grb2 complex in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

CRE-BPa, here designated as CRE-BPa alpha, is a novel member of the CRE (cAMP response element)-binding protein CRE-BP1 family. CRE-BPa alpha has four regions highly homologous to CRE-BP1, including a putative metal finger structure and a DNA-binding domain consisting of a basic amino acid cluster and a leucine zipper. CRE-BPa specifically binds to CRE as a homodimer or heterodimer with c-Jun or CRE-BP1. Here we report three alternative splicing forms of CRE-BPa alpha: two of them, CRE-BPa beta and CRE-BPa gamma, lack the N-terminal 7 and 33 amino acids of CRE-BPa alpha, and the third one CRE-BPa delta, has 16 additional amino acids in the N-terminus and amino acids 156-508 of CRE-BPa alpha. In CAT cotransfection experiments using CV-1 cells, transient expression of each of four CRE-BPa proteins caused a 1.6- to 3.4-fold increase of CRE-dependent transcription, respectively. Interestingly, these weak trans-activating capacities of CRE-BPa proteins were enhanced 2.7- to 3.6-fold by treatment of cells with 12-O-tetradecanoyl-phorbol 13-acetate (TPA). However, CRE-BPa did not affect the TPA-induced and TRE (TPA response element)-dependent transcription. These results indicate that CRE-BPa is a CRE-dependent trans-activator, and that CRE-BPa can confer TPA inducibility on CRE. Thus, CRE-BPa has an unique characteristic of cross-talk between cAMP pathway and TPA pathway.