Hitomi Watanabe

Glomerulonephritis induced by methicillin-sensitive Staphylococcus aureus infection.

A 57-year-old woman developed exacerbation of atopic dermatitis, fever, and nephrotic syndrome with microscopic hematuria. By bacteriological study, methicillin-sensitive Staphylococcus aureus (MSSA) was detected from each culture of pharyngeal mucus, stool, and blood samples. Renal biopsy specimens showed endocapillary proliferative glomerulonephritis with massive IgA deposition in the mesangium and along the capillary loops. After antistaphylococcal therapy with antibiotics, MSSA was negative for each culture and urinary protein decreased. Nine months after the first renal biopsy, a re-biopsy was performed, which revealed apparent disappearance of both endocapillary cell proliferation and IgA deposition. It is known that methicillin-resistant S. aureus (MRSA) infection causes glomerulonephritis through T-cell stimulation by superantigen presented by MRSA. The present results suggest that not only MRSA but also MSSA can cause this type of glomerulonephritis.

Mouse sperm undergo GPI-anchored protein release associated with lipid raft reorganization and acrosome reaction to acquire fertility

Mammalian sperm undergo several maturation steps after leaving the testis to become competent for fertilization. Important changes occur in sperm within the female reproductive tract, although the molecular mechanisms underlying these processes remain unclear. To investigate sperm membrane remodeling upon sperm maturation, we developed transgenic mouse lines carrying glycosylphosphatidylinositol (GPI)-anchored enhanced green fluorescent protein (EGFP–GPI) and traced the fate of this fluorescent protein during the fertility-acquiring process in sperm in vitro and in vivo. When the GFP-labeled sperm were treated with compounds for promoting the acrosome reaction, EGFP–GPI was released from the sperm surface crosslinked with characteristic relocation of a lipid raft marker ganglioside GM1. Sperm ejaculated into the uterus strongly expressed EGFP–GPI in the head region, whereas a part of the oviductal sperm lost fluorescence in a manner that was dependent on the presence of angiotensin-converting enzyme (ACE). Moreover, sperm on the zona pellucida of eggs in the oviduct were all found to have low levels of GFP. These results suggest that sperm undergoing GPI-anchored protein release associated with reorganization of lipid rafts and the acrosome reaction acquire fertilization potential.

Dipeptidase-Inactivated tACE Action In Vivo: Selective Inhibition of Sperm-Zona Pellucida Binding in the Mouse

Abstract The angiotensin-converting enzyme (ACE) plays a crucial role in male fertilization and is a key regulator of blood pressure. Testicular ACE (tACE), the germinal specific isozyme expressed on different promoters, exclusively carries out the role of ACE in fertility, although the site and mode of action are not well known. To investigate the contribution of tACE in fertilization, we produced transgenic mouse lines carrying a dipeptidase-inactivated mutant. Although the transgenic mice showed normal blood pressure, kidney morphology, and fertility, reduced fertilization was observed after in vitro fertilization (IVF). The sperm-zona pellucida (ZP) binding was exclusively impaired in these lines in a manner similar to that observed in an Ace knockout mouse. The dipeptidase activity was reduced in epididymal ingredients but not in the testis. Furthermore, direct application of mutant protein did not suppress sperm-ZP binding of intact sperm during IVF, implying that the dipeptidase-inactivated mutant affects sperm modification in the epididymis for ZP binding. Our results indicate that the dipeptidase-inactivated tACE acts in vivo, suggesting that tACE contributes to fertilization as a dipeptidase at least in the epididymis.

Disruption of MeCP2 attenuates circadian rhythm in CRISPR/Cas9‐based Rett syndrome model mouse

Methyl‐CpG‐binding protein 2 (Mecp2) is an X‐linked gene encoding a methylated DNA‐binding nuclear protein which regulates transcriptional activity. The mutation of MECP2 in humans is associated with Rett syndrome (RTT), a neurodevelopmental disorder. Patients with RTT frequently show abnormal sleep patterns and sleep‐associated problems, in addition to autistic symptoms, raising the possibility of circadian clock dysfunction in RTT. In this study, we investigated circadian clock function in Mecp2‐deficient mice. We successfully generated both male and female Mecp2‐deficient mice on the wild‐type C57BL/6 background and PER2Luciferase (PER2Luc) knock‐in background using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system. Generated Mecp2‐deficient mice recapitulated reduced activity in mouse models of RTT, and their activity rhythms were diminished in constant dark conditions. Furthermore, real‐time bioluminescence imaging showed that the amplitude of PER2Luc‐driven circadian oscillation was significantly attenuated in Mecp2‐deficient SCN neurons. On the other hand, in vitro circadian rhythm development assay using Mecp2‐deficient mouse embryonic stem cells (ESCs) did not show amplitude changes of PER2Luc bioluminescence rhythms. Together, these results show that Mecp2 deficiency abrogates the circadian pacemaking ability of the SCN, which may be a therapeutic target to treat the sleep problems of patients with RTT.

Lipocalin 2 binds to membrane phosphatidylethanolamine to induce lipid raft movement in a PKA-dependent manner and modulates sperm maturation

Mammalian sperm undergo multiple maturation steps after leaving the testis in order to become competent for fertilization, but the molecular mechanisms underlying this process remain unclear. In terms of identifying factors crucial for these processes in vivo, we found that lipocalin 2 (Lcn2), which is known as an innate immune factor inhibiting bacterial and malarial growth, can modulate sperm maturation. Most sperm that migrated to the oviduct of wild-type females underwent lipid raft reorganization and glycosylphosphatidylinositol-anchored protein shedding, which are signatures of sperm maturation, but few did so in Lcn2 null mice. Furthermore, we found that LCN2 binds to membrane phosphatidylethanolamine to reinforce lipid raft reorganization via a PKA-dependent mechanism and promotes sperm to acquire fertility by facilitating cholesterol efflux. These observations imply that mammals possess a mode for sperm maturation in addition to the albumin-mediated pathway.

Relationship between Filtration Coefficients of Microvasculature and Levels of Atrial Natriuretic Peptide or Echocardiographic Measurements

Background/Aims: Assessing the volume status of hemodialysis (HD) patients and determining their adequate dry weight (DW) present great challenges for physicians involved in HD. In this study the relationship between standardized filtration coefficients of microvasculature (Lpst) and the plasma atrial natriuretic peptide (ANP) levels or echocardiographic measurements (UCGm) were clarified. The aim of this study was to evaluate the possibility of utilizing Lpst as one of the tools for assessing volume status of patients undergoing HD. Methods: 52 patients on maintenance HD were examined. Lpst was calculated by utilizing continuous measurements of blood volume during HD by means of monitoring changes of hematocrit with CRIT-LINETM. Plasma ANP levels were measured shortly after HD. Plasma ANP levels were elevated beyond the normal limit in 32 patients (Hi group) and were within the normal range in the remaining 20 patients (Lo group). UCGm were performed within 1 month prior to the study. Inferior vena cava diameters in quiet expiration (IVCe) were dilated in 21 patients (Hivc group) and were within the normal range in the remaining 31 patients (Livc group). Lpst was compared with plasma ANP level and UCGm. Results: Lpst in Lo group were significantly lower than those in the Hi group (0.83 ± 0.19 vs. 2.64 ± 2.73 ml/mm Hg/min; p < 0.001). Lpst correlated significantly with plasma ANP levels (r = 0.613; p < 0.001). Lpst in the Livc group were significantly lower than those in the Hivc group (1.33± 1.61 vs. 2.85 ± 2.88 ml/mm Hg/min; p < 0.001). Lpst also correlated with IVCe (r = 0.630; p < 0.001). The receiver operating characteristic (ROC) curves for high plasma ANP level and for dilated IVCe were significant for Lpst. Area under the ROC curve for elevated ANP was 0.909 (95% confidence interval (CI) 0.834–0.985) and for dilated IVCe was 0.833 (95% CI 0.724–0.941). Conclusion: We conclude that there exists a significant association between Lpst and plasma ANP levels at the end of a dialysis session. There is a possibility that high plasma ANP levels cause elevation of Lpst. Besides ANP, Lpst significantly correlated with IVCe. These results suggested that Lpst can be utilized as one of the tools for assessing volume status of patients undergoing HD.

Simulation of post-dialysis urea rebound using regional flow model

BackgroundA regional flow model (RFM) can establish the missing link between hemodynamics and solute removal. We tried to simulate post-dialysis urea rebound using a RFM for the purpose of evaluating the validity of this model.MethodsEight patients on maintenance hemodialysis with negligible renal function were investigated. The parameters of the RFM were estimated so as to fit the calculated values of urea nitrogen to the measured values during a dialysis session. The estimated parameters were total urea distribution volume (TUV), systemic blood flow (Qsys), flow fraction (fQH) and volume fraction (fVH) of the high-flow system. Thirteen types of parameter sets were used for the estimation. The urea rebound at 60 min after a dialysis session (Creb) and the rebound ratio (RR) were calculated using these estimated parameters. The accuracy of the calculated Creb and RR was assessed.ResultsThe accuracy of Creb and RR determined using estimated TUV, by taking Qsys as systemic blood flow calculated from ultrasonic echo cardiogram (Qucg), fQH as 0.8, and fVH as 0.2, was insufficient (method 1a). The accuracy of these values was significantly increased by taking fQH as 0.85 (method 1b). The estimation of Qsys with TUV did not improve the accuracy of Creb and RR (methods 2a and 2b). The estimation of fQH, fVH, and TUV (method 8) increased the accuracy of Creb and RR significantly compared with method 1a, but not compared with method 1b. Even with method 1b or method 8, the percentage RR was less than 90% in two patients.ConclusionsBy taking fQH as 0.85, an acceptably accurate simulation of urea rebound can be accomplished with the necessity to estimate only TUV. The simulation was not significantly improved by the estimation of Qsys, fQH, and fVH. The RFM is useful in practice, although it has some limitations.

Testicular Angiotensin-Converting Enzyme with Different Glycan Modification: Characterization on Glycosylphosphatidylinositol-Anchored Protein Releasing and Dipeptidase Activities

We have previously found that the angiotensin-converting enzyme (ACE) carries GPI-anchored protein releasing activity (GPIase) as well as dipeptidase activity. Testicular ACE (tACE), the male germinal specific isozyme, plays a crucial role in male fertilization. The amino-terminal region of this isozyme is different from that of somatic isozyme (sACE) and contains potential O-linked glycosylation sites. By multiple mutagenesis after an in silico prediction, amino acid residues acquiring O-glycans were assigned. Both GPIase and dipeptidase activities were compared between O-glycan null mutant and wild-type molecules, but no differences were found. Furthermore, the wild-type tACE was produced in two different cells (COS7 and CHO) and its activities compared. The GPIase activity, but not dipeptidase, was apparently higher for CHO-derived molecule than COS7. Sensitivity to neuraminidase and O-glycosidase digestions and the profile of glycosylation were quite different between these two molecules. Moreover, serial digestions with neuraminidase and O-glycosidase have no influence on GPIase activity of both molecules, suggesting that the sialylation and the presence of O-glycan has no influence on tACE enzyme activities, while the set of glycans modulate GPIase activity.

An In Vitro ES Cell-Based Clock Recapitulation Assay Model Identifies CK2α as an Endogenous Clock Regulator

We previously reported emergence and disappearance of circadian molecular oscillations during differentiation of mouse embryonic stem (ES) cells and reprogramming of differentiated cells, respectively. Here we present a robust and stringent in vitro circadian clock formation assay that recapitulates in vivo circadian phenotypes. This assay system first confirmed that a mutant ES cell line lacking Casein Kinase I delta (CKIδ) induced ∼3 hours longer period-length of circadian rhythm than the wild type, which was compatible with recently reported results using CKIδ null mice. In addition, this assay system also revealed that a Casein Kinase 2 alpha subunit (CK2α) homozygous mutant ES cell line developed significantly longer (about 2.5 hours) periods of circadian clock oscillations after in vitro or in vivo differentiation. Moreover, revertant ES cell lines in which mutagenic vector sequences were deleted showed nearly wild type periods after differentiation, indicating that the abnormal circadian period of the mutant ES cell line originated from the mutation in the CK2α gene. Since CK2α deficient mice are embryonic lethal, this in vitro assay system represents the genetic evidence showing an essential role of CK2α in the mammalian circadian clock. This assay was successfully applied for the phenotype analysis of homozygous mutant ES cells, demonstrating that an ES cell-based in vitro assay is available for circadian genetic screening.

Scleraxis is required for maturation of tissue domains for proper integration of the musculoskeletal system

Scleraxis (Scx) is a basic helix-loop-helix transcription factor that is expressed persistently in tendons/ligaments, but transiently in entheseal cartilage. In this study, we generated a novel ScxCre knock-in (KI) allele, by in-frame replacement of most of Scx exon 1 with Cre recombinase (Cre), to drive Cre expression using Scx promoter and to inactivate the endogenous Scx. Reflecting the intensity and duration of endogenous expression, Cre-mediated excision occurs in tendinous and ligamentous tissues persistently expressing Scx. Expression of tenomodulin, a marker of mature tenocytes and ligamentocytes, was almost absent in tendons and ligaments of ScxCre/Cre KI mice lacking Scx to indicate defective maturation. In homozygotes, the transiently Scx-expressing entheseal regions such as the rib cage, patella cartilage, and calcaneus were small and defective and cartilaginous tuberosity was missing. Decreased Sox9 expression and phosphorylation of Smad1/5 and Smad3 were also observed in the developing entheseal cartilage, patella, and deltoid tuberosity of ScxCre/Cre KI mice. These results highlighted the functional importance of both transient and persistent expression domains of Scx for proper integration of the musculoskeletal components.