High hydrostatic pressure induces slow contraction in mouse cardiomyocytes.

Cardiomyocytes are contractile cells that regulate heart contraction. Ca2+ flux via Ca2+ channels activates actomyosin interactions, leading to cardiomyocyte contraction, which is modulated by physical factors (e.g., stretch, shear stress, and hydrostatic pressure). We evaluated the mechanism triggering slow contractions using a high-pressure microscope to characterize changes in cell morphology and intracellular Ca2+ concentration ([Ca2+]i) in mouse cardiomyocytes exposed to high hydrostatic pressures. We found that cardiomyocytes contracted slowly without an acute transient increase in [Ca2+]i, while a myosin ATPase inhibitor interrupted pressure-induced slow contractions. Furthermore, transmission electron microscopy showed that, although the sarcomere length was shortened upon the application of 20 MPa, this pressure did not collapse cellular structures such as the sarcolemma and sarcomeres. Our results suggest that pressure-induced slow contractions in cardiomyocytes are driven by the activation of actomyosin interactions without an acute transient increase in [Ca2+]i.

Ectopic expression of a rice plasma membrane intrinsic protein (OsPIP1;3) promotes plant growth and water uptake.

Plasma membrane intrinsic proteins (PIPs) are known to be major facilitators of the movement of a number of substrates across cell membranes. From a drought-resistant cultivar of Oryza sativa (rice), we isolated an OsPIP1;3 gene single-nucleotide polymorphism (SNP) that is mostly expressed in rice roots and is strongly responsive to drought stress. Immunocytochemistry showed that OsPIP1;3 majorly accumulated on the proximal end of the endodermis and the cell surface around the xylem. Expression of GFP-OsPIP1;3 alone in Xenopus oocytes or rice protoplasts showed OsPIP1;3 mislocalization in the endoplasmic reticulum (ER)-like neighborhood, whereas co-expression of OsPIP2;2 recruited OsPIP1;3 to the plasma membrane and led to a significant enhancement of water permeability in oocytes. Moreover, reconstitution of 10×His-OsPIP1;3 in liposomes demonstrated water channel activity, as revealed by stopped-flow light scattering. Intriguingly, by patch-clamp technique, we detected significant NO3- conductance of OsPIP1;3 in mammalian cells. To investigate the physiological functions of OsPIP1;3, we ectopically expressed the OsPIP1;3 gene in Nicotiana benthamiana (tobacco). The transgenic tobacco plants exhibited higher photosynthesis rates, root hydraulic conductivity (Lpr ) and water-use efficiency, resulting in a greater biomass and a higher resistance to water deficit than the wild-type did. Further experiments suggested that heterologous expression of OsPIP1;3 in cyanobacterium altered bacterial growth under different conditions of CO2 gas supply. Overall, besides shedding light on the multiple functions played by OsPIP1;3, this work provides insights into the translational value of plant AQPs.

Effects of a single dose of tablets containing lactononadecapeptide on cognitive function in healthy adults: a randomized, double-blind, cross-over, placebo-controlled trial.

Lactononadecapeptide (LNDP; NIPPLTQTPVVVPPFLQPE) is a memory-improving peptide. The current study aimed to determine the effects of a single dose of tablets containing LNDP on cognitive function in healthy Japanese men aged 30-59 years. A randomized, double-blind, cross-over, placebo-controlled trial was conducted in participants randomly assigned to receive LNDP or placebo tablets. The Uchida-Kraepelin test was used to induce cognitive load in participants as a model of work load. Cognitive function was evaluated using the Japanese version of the CNS Vital Signs. Composite memory and verbal memory were significantly higher following consumption of LNDP than placebo tablets. Carryover effects were observed in attention and concentration domains so that period 1 data was analyzed. LNDP consumption led to higher processing speed, executive function, and cognitive flexibility than placebo. Thus, supplementation with a single dose of LNDP tablets may improve cognitive functions including memory, attention, concentration, and information processing in daily life.

Effect of Daily Intake of Green Tea Catechins on Cognitive Function in Middle-Aged and Older Subjects: A Randomized, Placebo-Controlled Study.

Epidemiological studies in Japan, including the Nakajima study and the Tsurugaya study, have indicated that green tea consumption may improve cognitive impairment. Catechins, which are typical polyphenols contained in green tea, have been reported to have antioxidative, anti-inflammatory, and neuroprotective effects. However, their impact on human cognitive function remains unclear. Therefore, we performed a double-blind, randomized, controlled study to investigate the effect of 336.4 mg of decaffeinated green tea catechins (GTC) on cognitive function after a single dose and after 12 weeks of daily intake. This study included Japanese adults between the ages of 50 and 69 years with a Mini-Mental State Examination Japanese version score of >24 and self-assessed cognitive decline. The Cognitrax testing battery was used to evaluate cognitive function. The incorrect response rate on the Continuous Performance Test significantly decreased after a single dose of GTC. After 12 weeks of daily GTC intake, the response time for Part 4 of the 4-part Continuous Performance Test, which is a two-back test, was shortened. These results suggest that daily intake of GTC might have beneficial effects on working memory.

Addition of urea and thiourea to electrophoresis sample buffer improves efficiency of protein extraction from TCA/acetone-treated smooth muscle tissues for phos-tag SDS-PAGE.

Phosphorylation analysis by using phos-tag technique has been reported to be suitable for highly sensitive quantification of smooth muscle myosin regulatory light chain (LC20 ) phosphorylation. However, there is another factor that will affect the sensitivity of phosphorylation analysis, that is, protein extraction. Here, we optimized the conditions for total protein extraction out of trichloroacetic acid (TCA)-fixed tissues. Standard SDS sample buffer extracted less LC20 , actin and myosin phosphatase targeting subunit 1 (MYPT1) from TCA/acetone treated ciliary muscle strips. On the other hand, sample buffer containing urea and thiourea in addition to lithium dodecyl sulfate (LDS) or SDS extracted those proteins more efficiently, and thus increased the detection sensitivity up to 4-5 fold. Phos-tag SDS-PAGE separated dephosphorylated and phosphorylated LC20 s extracted in LDS/urea/thiourea sample buffer to the same extent as those in standard SDS buffer. We have concluded that LDS (or SDS) /urea/thiourea sample buffer is suitable for highly sensitive phosphorylation analysis in smooth muscle, especially when it is treated with TCA/acetone.

Disease flare patterns and predictors of systemic lupus erythematosus in a monocentric cohort of 423 Japanese patients during a long-term follow-up: The JUDE study.

OBJECTIVE: To clarify the clinical features of systemic lupus erythematosus (SLE) patients, factors associated with flares, and changes over time. METHODS: Patients having SLE with a visiting history were entered into the Juntendo University Database of Erythematosus. We included 423 cases in the long-term follow-up analysis, and 383 cases were followed for 10 years after the initiation of any therapeutic intervention (comparative analysis: 1973-1982, 82 cases; 1983-1992, 141, and 1993-2002, 160). We assessed changes in the patients' background characteristics, disease symptoms, flare rates, etc. RESULTS: Among the 423 cases, the mean follow-up period was 25.9 years, and mean number of flares was 0.51. Of those, 31.9% had ≥1 flares. Thrombocytopenia at onset contributed to the flares. For disease symptoms at onset, a recent trend in increasing thrombocytopenia was observed. The combination rate of immunosuppressive agents for diseases other than lupus nephritis was slightly increased, and there was no improvement until the first flare or in the flare rate. CONCLUSIONS: Thrombocytopenia at onset is predictive factor for flares. Since SLE is a diverse disease with varying symptoms at recurrence, the treatment guidelines should be improved for thrombocytopenia from a long-term perspective.

Dynamic regulation of the root hydraulic conductivity of barley plants in response to salinity/osmotic stress.

Salinity stress significantly reduces the root hydraulic conductivity (Lpr) of several plant species including barley (Hordeum vulgare). Here we characterized changes in the Lpr of barley plants in response to salinity/osmotic stress in detail using a pressure chamber. Salt-tolerant and intermediate barley cultivars, K305 and Haruna-nijyo, but not a salt-sensitive cultivar, I743, exhibited characteristic time-dependent Lpr changes induced by 100 mM NaCl. An identical response was evoked by isotonic sorbitol, indicating that this phenomenon was triggered by osmotic imbalances. Further examination of this mechanism using barley cv. Haruna-nijyo plants in combination with the use of various inhibitors suggested that various cellular processes such as protein phosphorylation/dephosphorylation and membrane internalization appear to be involved. Interestingly, the three above-mentioned barley cultivars did not exhibit a remarkable difference in root cell sap osmolality under hypertonic conditions, in contrast to the case of Lpr. The possible biological significance of the regulation of Lpr in barley plants upon salinity/osmotic stress is discussed.

CO2 transport by PIP2 aquaporins of barley.

CO2 permeability of plasma membrane intrinsic protein 2 (PIP2) aquaporins of Hordeum vulgare L. was investigated. Five PIP2 members were heterologously expressed in Xenopus laevis oocytes. CO2 permeability was determined by decrease of cytosolic pH in CO2-enriched buffer using a hydrogen ion-selective microelectrode. HvPIP2;1, HvPIP2;2, HvPIP2;3 and HvPIP2;5 facilitated CO2 transport across the oocyte cell membrane. However, HvPIP2;4 that is highly homologous to HvPIP2;3 did not. The isoleucine residue at position 254 of HvPIP2;3 was conserved in PIP2 aquaporins of barley, except HvPIP2;4, which possesses methionine instead. CO2 permeability was lost by the substitution of the Ile254 of HvPIP2;3 by methionine, while water permeability was not affected. These results suggest that PIP2 aquaporins are permeable to CO2. and the conserved isoleucine at the end of the E-loop is crucial for CO2 selectivity.

Phenotype conversion from rheumatoid arthritis to systemic lupus erythematosus by introduction of Yaa mutation into FcγRIIB-deficient C57BL/6 mice.

We previously established an IgG Fc receptor IIB (FcγRIIB)-deficient C57BL/6 (B6)-congenic mouse strain (KO1), which spontaneously develops rheumatoid arthritis (RA), but not systemic lupus erythematosus (SLE). Here, we show that when Y chromosome-linked autoimmune acceleration (Yaa) mutation was introduced in KO1 strain (KO1.Yaa), the majority of KO1.Yaa mice did not develop RA, but instead did develop SLE. This phenotype conversion did not depend on autoantibody specificity, since KO1.Yaa mice, compared with KO1, showed a marked increase in serum levels of both lupus-related and RA-related autoantibodies. The increase in frequencies of CD69(+) activated B cells and T cells, and the spontaneous splenic GC formation with T follicular helper cell generation were manifest early in life of KO1.Yaa, but not KO1 and B6.Yaa, mice. Activated CD4(+) T cells from KO1.Yaa mice showed upregulated production of IL-21 and IL-10, compared with the finding in KO1 mice, indicating the possibility that this aberrant cytokine milieu relates to the disease phenotype conversion. Thus, our model is useful to clarify the shared and the disease-specific mechanisms underlying the clinically distinct systemic autoimmune diseases RA and SLE.

Increased serum concentration of BAFF/APRIL and IgA2 subclass in patients with mixed connective tissue disease complicated by interstitial lung disease.

B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) are known to be crucial for B cell maturation and survival, and increased expression of these factors in various autoimmune diseases has been reported. Human B cells produce two IgA subclasses: IgA1 and IgA2, the latter being abundant in the distal intestine, saliva, colostrum and bronchial fluid. We investigated these parameters in patients with mixed connective tissue disease (MCTD) complicated by interstitial lung disease (ILD+), and compared them with those in MCTD patients without ILD (ILD-). Sixty-three MCTD patients were divided into two groups: 21 ILD+ patients and 42 ILD- patients. In each patient group we analyzed soluble BAFF/APRIL using ELISA, and IgA1 and IgA2 using double immunodiffusion. Furthermore, we analyzed BAFF-APRIL receptors, BCMA, BAFF-R and TACI, using flow cytometry. The ILD+ patients had significantly higher levels of BAFF/APRIL than the ILD- patients. There were significant correlations between BAFF/APRIL, BAFF/KL-6 and APRIL/KL-6. Although there was no significant inter-group difference in the serum IgA1 level, ILD+ patients had a significantly elevated IgA2 level in comparison with ILD- patients. Moreover, although there were no significant inter-group differences in the expression of BCMA, BAFF-R and TACI on B cells, the expression of BAFF-R was significantly decreased in the ILD+ patients. In recent years, relationships between BAFF/APRIL and IgA subclass have been reported. Our results suggest that an elevated level of BAFF/APRIL drives the maturation of B cells, subsequently leading to IgA2 class switching, and possibly to the development of ILD in patients with MCTD.

Fulminant myocarditis with complete atrioventricular block after mRNA COVID-19 vaccination: A case report.

A 71-year-old man was transferred urgently to our hospital after collapsing near his home post the first shot of the BNT162b2 coronavirus disease 2019 vaccine (Pfizer-BioNTech, Comirnaty®). Immediately after arrival at our hospital, cardiac arrest due to complete atrioventricular block with no ventricular escaped beats was observed on electrocardiogram. Echocardiography showed preserved left ventricular ejection fraction, however, diffuse severe hypokinesia was revealed after 3 weeks, and he died 3 months after admission because of worsening heart failure. An autopsy examination revealed eosinophilic myocarditis or hypersensitivity myocarditis with extensive fibrosis and widespread myocardial dropout throughout the heart. Learning objective: 1. Severe myocarditis occurs extremely rarely after mRNA coronavirus disease 2019 (COVID-19) vaccination. 2. Myocarditis after mRNA COVID-19 vaccination might cause complete atrioventricular block, followed by a course of decreased left ventricular ejection fraction. 3. Histologically, severe myocarditis after mRNA COVID-19 vaccination seems to present as fulminant necrotizing eosinophilic myocarditis or hypersensitivity myocarditis.

Total synthesis of aeruginosin 98B.

The first total synthesis of aeruginosin 98B was accomplished. The key step is a highly diastereoselective Pd-catalyzed intramolecular asymmetric allylic alkylation reaction of a diastereomeric mixture of allylic carbonates that is enabled by the use of racemic phosphine ligand L1.

K+ transport by the OsHKT2;4 transporter from rice with atypical Na+ transport properties and competition in permeation of K+ over Mg2+ and Ca2+ ions.

Members of class II of the HKT transporters, which have thus far only been isolated from grasses, were found to mediate Na(+)-K(+) cotransport and at high Na(+) concentrations preferred Na(+)-selective transport, depending on the ionic conditions. But the physiological functions of this K(+)-transporting class II of HKT transporters remain unknown in plants, with the exception of the unique class II Na(+) transporter OsHKT2;1. The genetically tractable rice (Oryza sativa; background Nipponbare) possesses two predicted K(+)-transporting class II HKT transporter genes, OsHKT2;3 and OsHKT2;4. In this study, we have characterized the ion selectivity of the class II rice HKT transporter OsHKT2;4 in yeast and Xenopus laevis oocytes. OsHKT2;4 rescued the growth defect of a K(+) uptake-deficient yeast mutant. Green fluorescent protein-OsHKT2;4 is targeted to the plasma membrane in transgenic plant cells. OsHKT2;4-expressing oocytes exhibited strong K(+) permeability. Interestingly, however, K(+) influx in OsHKT2;4-expressing oocytes did not require stimulation by extracellular Na(+), in contrast to other class II HKT transporters. Furthermore, OsHKT2;4-mediated currents exhibited permeabilities to both Mg(2+) and Ca(2+) in the absence of competing K(+) ions. Comparative analyses of Ca(2+) and Mg(2+) permeabilities in several HKT transporters, including Arabidopsis thaliana HKT1;1 (AtHKT1;1), Triticum aestivum HKT2;1 (TaHKT2;1), OsHKT2;1, OsHKT2;2, and OsHKT2;4, revealed that only OsHKT2;4 and to a lesser degree TaHKT2;1 mediate Mg(2+) transport. Interestingly, cation competition analyses demonstrate that the selectivity of both of these class II HKT transporters for K(+) is dominant over divalent cations, suggesting that Mg(2+) and Ca(2+) transport via OsHKT2;4 may be small and would depend on competing K(+) concentrations in plants.

Myocardial TRPC6-mediated Zn2+ influx induces beneficial positive inotropy through ß-adrenoceptors.

Baroreflex control of cardiac contraction (positive inotropy) through sympathetic nerve activation is important for cardiocirculatory homeostasis. Transient receptor potential canonical subfamily (TRPC) channels are responsible for α1-adrenoceptor (α1AR)-stimulated cation entry and their upregulation is associated with pathological cardiac remodeling. Whether TRPC channels participate in physiological pump functions remains unclear. We demonstrate that TRPC6-specific Zn2+ influx potentiates ß-adrenoceptor (ßAR)-stimulated positive inotropy in rodent cardiomyocytes. Deletion of trpc6 impairs sympathetic nerve-activated positive inotropy but not chronotropy in mice. TRPC6-mediated Zn2+ influx boosts α1AR-stimulated ßAR/Gs-dependent signaling in rat cardiomyocytes by inhibiting ß-arrestin-mediated ßAR internalization. Replacing two TRPC6-specific amino acids in the pore region with TRPC3 residues diminishes the α1AR-stimulated Zn2+ influx and positive inotropic response. Pharmacological enhancement of TRPC6-mediated Zn2+ influx prevents chronic heart failure progression in mice. Our data demonstrate that TRPC6-mediated Zn2+ influx with α1AR stimulation enhances baroreflex-induced positive inotropy, which may be a new therapeutic strategy for chronic heart failure.

Mechanisms of water transport mediated by PIP aquaporins and their regulation via phosphorylation events under salinity stress in barley roots.

Water homeostasis is crucial to the growth and survival of plants under water-related stress. Plasma membrane intrinsic proteins (PIPs) have been shown to be primary channels mediating water uptake in plant cells. Here we report the water transport activity and mechanisms for the regulation of barley (Hordeum vulgare) PIP aquaporins. HvPIP2 but not HvPIP1 channels were found to show robust water transport activity when expressed alone in Xenopus laevis oocytes. However, the co-expression of HvPIP1 with HvPIP2 in oocytes resulted in significant increases in activity compared with the expression of HvPIP2 alone, suggesting the participation of HvPIP1 in water transport together with HvPIP2 presumably through heteromerization. Severe salinity stress (200 mM NaCl) significantly reduced root hydraulic conductivity (Lp(r)) and the accumulation of six of 10 HvPIP mRNAs. However, under relatively mild stress (100 mM NaCl), only a moderate reduction in Lp(r) with no significant difference in HvPIP mRNA levels was observed. Sorbitol-mediated osmotic stress equivalent to 100 and 200 mM NaCl induced nearly identical Lp(r) reductions in barley roots. Furthermore, the water transport activity in intact barley roots was suggested to require phosphorylation that is sensitive to a kinase inhibitor, staurosporine. HvPIP2s also showed water efflux activity in Xenopus oocytes, suggesting a potential ability to mediate water loss from cells under hypertonic conditions. Water transport via HvPIP aquaporins and the significance of reductions of Lp(r) in barley plants during salinity stress are discussed.

Matcha consumption maintains attentional function following a mild acute psychological stress without affecting a feeling of fatigue: A randomized placebo-controlled study in young adults.

Tea is a beverage commonly consumed worldwide. Matcha is a type of green tea produced by drying and grinding tea leaves (Camellia sinensis L.) into a fine powder. Matcha contains catechin, theanine, and caffeine, which affect cognitive function. Epidemiological studies conducted in Japan have shown that green tea consumption improves cognitive impairment. Previously, we found that daily matcha intake improves attention and executive function in middle-aged and older people. However, its effect on cognitive function in younger adults remains unclear. Moreover, it is unclear which cognitive functions are impaired by stress. This study aimed to clarify whether the administration of matcha improves the attentional function of young adults after mild acute stress and which cognitive function is improved. We included 42 participants aged 25 to 34 years who consumed 2 g of matcha daily for 2 weeks. The Uchida-Kraepelin test was used to induce mild acute psychological stress. Memory, attention, facial expression recognition, working memory, visual information, and motor function were evaluated. Reaction times on the Stroop test for attentional function were significantly lower in the matcha group than in the placebo group. Correct hits in the emotion perception test increased significantly for participants in the matcha group compared to those in the placebo group. We found no significant between-group differences in the other tests. In conclusion, after 2 weeks of matcha intake, the attentional function was maintained after mild acute psychological stress. Thus, matcha might improve cognitive function during or after stress conditions in young adults.

Effects of l-Theanine on Cognitive Function in Middle-Aged and Older Subjects: A Randomized Placebo-Controlled Study.

l-theanine (γ-glutamylethylamide), an amino acid in green tea, has been shown to affect brain functions by relieving stress disorders, improving mood, and maintaining normal sleep. However, the cognitive functions for which theanine is effective are unclear. This study aimed to clarify which cognitive functions are positively affected by intake of l-theanine. A double-blind, randomized, placebo-controlled study was conducted. The subjects were Japanese men and women aged 50-69 years. Mini Mental State Examination-Japanese version score was 24 or higher. Cognitrax was used as a test battery for cognitive function. Evaluations were performed before the intervention, after a single dose of l-theanine, and after 12 weeks of regular intake. The single dose of l-theanine reduced the reaction time to attention tasks (Stroop test, Part 1), and it increased the number of correct answers and decreased the number of omission errors in working memory tasks (4-Part continuous performance test, Part 4). In conclusion, our study indicated that l-theanine may contribute to improving attention, thus enhancing working memory and executive functions. Clinical Trial No.: UMIN000033812.

Effects of Daily Matcha and Caffeine Intake on Mild Acute Psychological Stress-Related Cognitive Function in Middle-Aged and Older Adults: A Randomized Placebo-Controlled Study.

Matcha, a type of green tea, has a higher amino acid content than other types of tea. We previously examined the ability of matcha to improve cognitive function in older adults and determined that continuous matcha intake improves attention and executive function. This study aimed to compare the effects of matcha and caffeine and clarify the differences between these effects. The study was registered at the University Hospital Medical Information Network (UMIN000036578). The effect of single and continuous intake was compared, and the usefulness of continuous intake was evaluated under the stress condition. The Uchida-Kraepelin test (UKT) was used to induce mild acute stress, and the Cognitrax was used to evaluate cognitive function. A single dose of caffeine improved attentional function during or after stress loading. The reduced reaction time in the Cognitrax, observed following a single dose of matcha, was likely due to caffeine. The matcha group showed an increase in the amount of work after continuous intake, whereas the caffeine group only showed an increase in the amount of work for the UKT after a single dose. Ingesting matcha with caffeine improves both attention and work performance when suffering from psychological stress compared with caffeine alone.

Personalized Nutritional Therapy Based on Blood Data Analysis for Malaise Patients.

As medical doctors, we routinely check patient blood chemistry and CBC data to diagnose disease. However, these data and methods of analysis are very rarely used to find pre-disease conditions or treat undiagnosed malaise. Masatoshi Kaneko Ph.D. found that many pre-disease conditions and types of malaise could be detected using his unique method of blood data analysis, and could also be treated using personalized nutritional therapy as an alternative to using drugs. The authors of this article introduce personalized nutritional therapy based on blood data analysis (Kaneko's method), and present and discuss some clinical cases. In total, 253 pre-disease or undiagnosed patients were treated using this nutritional therapy approach, and most of them recovered from their chronic symptoms and pre-disease conditions. This novel nutritional therapy has the potential to help many presymptomatic and undiagnosed patients suffering from malaise.