YIPF3 and YIPF4 regulate autophagic turnover of the Golgi apparatus.

The degradation of organelles by autophagy is essential for cellular homeostasis. The Golgi apparatus has recently been demonstrated to be degraded by autophagy, but little is known about how the Golgi is recognized by the forming autophagosome. Using quantitative proteomic analysis and two novel Golgiphagy reporter systems, we found that the five-pass transmembrane Golgi-resident proteins YIPF3 and YIPF4 constitute a Golgiphagy receptor. The interaction of this complex with LC3B, GABARAP, and GABARAPL1 is dependent on a LIR motif within YIPF3 and putative phosphorylation sites immediately upstream; the stability of the complex is governed by YIPF4. Expression of a YIPF3 protein containing a mutated LIR motif caused an elongated Golgi morphology, indicating the importance of Golgi turnover via selective autophagy. The reporter assays reported here may be readily adapted to different experimental contexts to help deepen our understanding of Golgiphagy.

Glomerular Endothelial Cell Receptor ADGRF5 and the Integrity of the Glomerular Filtration Barrier.

BACKGROUND: Glomerular endothelial cells are recognized to be important for maintaining the glomerular filtration barrier. ADGRF5, an adhesion G protein-coupled receptor, has been suggested to be involved in endothelial cell function. However, the role of ADGRF5 in the glomerular filtration barrier integrity remains elusive. METHODS: Cellular expression of ADGRF5 in mouse glomerulus was determined by histological analyses. The impact of ADGRF5 deletion on the glomerular morphology, kidney function, and glomerular endothelial gene/protein expression was then analyzed using ADGRF5 knockout (Adgrf5-/-) mice and human primary glomerular endothelial cells. RESULTS: ADGRF5 was specifically expressed in the capillary endothelial cells within the glomerulus. Adgrf5-/- mice developed albuminuria and impaired kidney function with morphological defects in the glomeruli, namely glomerular hypertrophy, glomerular basement membrane splitting and thickening, diaphragmed fenestration and detachment of the glomerular endothelial cells, and mesangial interposition. These defects were accompanied by the altered expression of genes responsible for glomerular basement membrane organization (type IV collagens and laminins) and Krüppel-like factor 2 (Klf2) in glomerular endothelial cells. Moreover, ADGRF5 knockdown decreased COL4A3 and COL4A4 expression and increased KLF2 expression in human primary glomerular endothelial cells. CONCLUSIONS: The loss of ADGRF5 resulted in altered gene expression in glomerular endothelial cells, and perturbed the structure and permselectivity of the glomerular filtration barrier.

Muscle stiffening is associated with muscle mechanoreflex-mediated cardioacceleration.

PURPOSE: Although the muscle mechanoreflex is an important mediator to cardiovascular regulation during exercise, its modulation factors remain relatively unknown. Therefore, the purpose of this study was to investigate the effect of muscle stiffness on the muscle mechanoreflex. METHODS: Participants were divided based on their median muscle stiffness (2.00 Nm/mm) into a low group (n = 15) and a high group (n = 15), and the muscle mechanoreflex was compared between the groups. After a 15-min rest in the supine position, heart rate (HR), blood pressure (BP), stroke volume (SV), and cardiac output (CO) were measured at rest for 3 min and during static passive dorsiflexion (SPD) at 20° for 1 min. Following a 15-min re-rest, muscle stiffness and passive resistive torque were evaluated in the distal end of the muscle belly of the medial gastrocnemius. RESULTS: Peak relative changes in HR (low group: 6 ± 4% and high group: 12 ± 4%) and CO (low group: 8 ± 10% and high group: 13 ± 9%) were greater in the high group than in the low group (both, P < 0.05). A significant positive correlation was found between resistive torque during SPD and muscle stiffness and peak relative changes in HR (r = 0.51 and 0.61, both P < 0.05). However, there was no correlation between muscle elongation during SPD and peak relative changes in HR (r = - 0.23, P = 0.20). CONCLUSION: These findings suggest that muscle stiffness may be modulatory factor of muscle mechanoreflex.

Loss of the adhesion G-protein coupled receptor ADGRF5 in mice induces airway inflammation and the expression of CCL2 in lung endothelial cells.

BACKGROUND: Adhesion G-protein coupled receptor F5 (ADGRF5) was recently identified as an essential regulator of pulmonary surfactant homeostasis in alveolar type II cells. We previously showed that in addition to abnormal surfactant accumulation, Adgrf5-deficient (Adgrf5-/-) mice exhibit emphysema-like signs, suggesting a possible role for ADGRF5 in immune regulation. Here, we extended the phenotypic analysis of Adgrf5-/- mice to help understand its biological role in the lung, and especially in immune regulation. METHODS: Histological features of lungs were evaluated by Alcian blue and Masson's trichrome staining. Quantitative real-time PCR (qPCR) and western blot analyses were performed to analyze the differential expression of genes/proteins related to airway inflammation in lungs between wildtype and Adgrf5-/- mice. Acid-base status was assessed by performing blood gas tests and urine pH measurements. Inflammatory cell counting was performed using Giemsa-stained bronchoalveolar lavage cells. Serum IgE concentrations were determined by enzyme-linked immunosorbent assay. The expression of Ccl2, S100a8, S100a9, and Saa3 in primary lung endothelial cells (ECs) was determined by qPCR and/or western blotting. Finally, the effect of administrating RS504393 to 2-week-old Adgrf5-/- mice on gene expression in the lungs was analyzed by qPCR. RESULTS: Adgrf5-/- mice exhibited several features of chronic airway inflammation (mucous cell metaplasia, mucus hyperproduction, subepithelial fibrosis, respiratory acidosis, high serum IgE, mast cell accumulation, and neutrophilia) in parallel with elevated expression of genes involved in mucous cell metaplasia (Muc5ac, Muc5b, Slc26a4, and Clca1), fibrosis (Tgfb1, Col1a1, Fn1, and Tnc), and type 2 immune response (Il4, Il5, Il13, IL-25, and IL-33) at 12 and/or 30 weeks of age. In contrast, mRNA expression of Ccl2, S100a8, and S100a9 was upregulated in embryonic or neonatal Adgrf5-/- lungs as well as in lung ECs of Adgrf5-/- mice at 1 week of age. RS504393 treatment suppressed the upregulation of S100a8, S100a9, Slc26a4, and Il5 in Adgrf5-/- lungs. CONCLUSIONS: Targeted disruption of ADGRF5 results in the development of airway inflammation, which is likely mediated by the type 2 immune response and possibly CCL2-mediated inflammation. ADGRF5 also has a potential role in the regulation of genes encoding CCL2 in lung ECs, thereby maintaining immune homeostasis.

YIPF1, YIPF2, and YIPF6 are medial-/trans-Golgi and trans-Golgi network-localized Yip domain family proteins, which play a role in the Golgi reassembly and glycan synthesis.

In this study, we attempted to explore the function of three uncharacterized mammalian homologs of yeast Yip domain family proteins-YIPF6, a homolog of Yip1p, and YIPF1 and YIPF2, which are homologs of Yif1p. Immunofluorescence staining revealed that YIPF1, YIPF2, and YIPF6 mainly localize in the medial-/trans-Golgi and also partially in the trans-Golgi network (TGN). On treatment with brefeldin A (BFA), the homologs co-migrated partly with medial-/trans-Golgi markers and also with a TGN marker in earlier time point, but finally redistributed within cytoplasmic punctate structures that were distinct from medial-/trans-Golgi and the TGN markers. YIPF6 formed a stable complex separately with YIPF1 and YIPF2, and knockdown of YIPF6 reduced YIPF1 and YIPF2 levels. These results suggest that YIPF6 forms complexes with YIPF1 and YIPF2 for their stable expression and localization within the Golgi apparatus. Knockdown experiments showed that YIPF1 and YIPF2, by contrast, are not necessary for the expression and localization of YIPF6. The structure of the Golgi apparatus and its disassembly after BFA treatment were not significantly affected by the knockdown of YIPF1, YIPF2, or YIPF6. However, reassembly of the Golgi apparatus after the removal of BFA was markedly delayed by the knockdown of YIPF1 and YIPF2, but not by that of YIPF6. These results strongly suggest that free YIPF6 after disassociating with YIPF1 and YIPF2 interferes with the reassembly of the Golgi apparatus. Knockdown of YIPF1 and YIPF2, but not that of YIPF6, also reduced intracellular glycans in HT-29 cells. Thus, we confirmed that YIPF1, YIPF2, and YIPF6 play a significant role in supporting normal glycan synthesis.

Fluid intake restores retinal blood flow early after exhaustive exercise in healthy subjects.

PURPOSE: It remains unclear whether rehydration restores retinal blood flow reduced by exhaustive exercise. We investigated the effect of fluid intake on retinal blood flow after exhaustive exercise. METHODS: Blood flow in the inferior (ITRA) and superior temporal retinal arterioles (STRA) was measured before and after incremental cycling exercise until exhaustion in 13 healthy males. After the exercise, the subjects rested without drinking (control condition: CON) or with drinking an electrolyte containing water (rehydrate condition: REH) and were followed up for a period of 120 min. To assess the hydration state, the body mass was measured, and venous blood samples were collected and plasma volume (PV) was calculated. RESULTS: Body mass decreased in CON after the trial [- 1.1 ± 0.1% (mean ± SE), p < 0.05]. PV was lower in CON than in REH during recovery. The ITRA and STRA blood flows decreased immediately after exercise from the resting baseline (ITRA; - 23 ± 4% in REH and - 30 ± 4% in CON, p < 0.05). The ITRA blood flow recovered baseline level at 15 min of recovery in REH (- 9 ± 3%, p = 0.5), but it remained reduced in CON (-14 ± 3%, p < 0.05). The STRA blood flow was higher in REH than in CON at 15 min (2 ± 3 vs. - 5 ± 3%, p < 0.05). CONCLUSIONS: The results of this study suggest that the reduction in retinal blood flow induced by exhaustive exercise can be recovered early by rehydration.

Ubiquitin System.

Ever since the discovery of ubiquitin in 1975[...].

Influence of Different Loads on Force-Time Characteristics during Back Squats.

The deceleration sub-phase during the back squat (BSQ) makes it difficult to stimulate the muscles throughout the full range of motion, and it has only been reported for one load during BSQ. The purpose of this study was to investigate whether a deceleration sub-phase occurs during BSQ with different loads and to assess the influence of load on the deceleration sub-phase duration and negative impulse during the deceleration sub-phase. Sixteen healthy men (mean ± standard deviation: age: 25 ± 3 years; height: 1.73 ± 0.07 m; mass: 83.2 ± 16.1 kg; BSQ one repetition maximum (1RM): 163.8 ± 36.6 kg; BSQ 1RM/body weight: 2.0 ± 0.4) who had performed resistance training for at least 1 year were recruited for this study. The subjects performed parallel BSQ with 0%, 12%, 27%, 42%, 56%, 71%, and 85% of each 1RM on a force plate in a random order. The deceleration sub-phase duration and negative impulse during the deceleration sub-phase were calculated from force-time data. The absolute durations of the deceleration sub-phase were not significantly different according to load except for 27% 1RM and 85% 1RM (p = 0.01). However, as the load increased from 12 to 85% 1RM, the relative duration of the deceleration sub-phase decreased (p < 0.05). The negative impulse during the deceleration sub-phase also increased from 0 to 42% 1RM (p < 0.05). A deceleration sub-phase occurs regardless of the load (0%-85% 1RM), and a large portion of the deceleration sub-phase occupied the concentric phase, with low-moderate loads, and a large amount of negative impulse occurred during the short deceleration sub-phase with a high load.

JNK2 controls fragmentation of the Golgi complex and the G2/M transition through phosphorylation of GRASP65.

In mammalian cells, the Golgi complex is composed of stacks that are connected by membranous tubules. During G2, the Golgi complex is disassembled into isolated stacks. This process is required for entry into mitosis, indicating that the correct inheritance of the organelle is monitored by a 'Golgi mitotic checkpoint'. However, the regulation and the molecular mechanisms underlying this Golgi disassembly are still poorly understood. Here, we show that JNK2 has a crucial role in the G2-specific separation of the Golgi stacks through phosphorylation of Ser277 of the Golgi-stacking protein GRASP65 (also known as GORASP1). Inhibition of JNK2 by RNA interference or by treatment with three unrelated JNK inhibitors causes a potent and persistent cell cycle block in G2. JNK activity becomes dispensable for mitotic entry if the Golgi complex is disassembled by brefeldin A treatment or by GRASP65 depletion. Finally, measurement of the Golgi fluorescence recovery after photobleaching demonstrates that JNK is required for the cleavage of the tubules connecting Golgi stacks. Our findings reveal that a JNK2-GRASP65 signalling axis has a crucial role in coupling Golgi inheritance and G2/M transition.

Functional characterisation of the YIPF protein family in mammalian cells.

In this study, we carry out a systematic characterisation of the YIPF family of proteins with respect to their subcellular localisation profile, membrane topology and functional effects on the endomembrane system. YIPF proteins primarily localise to the Golgi complex and can be grouped into trans-Golgi-localising YIPFs (YIPF1 and YIPF2) and cis-Golgi-localising YIPFs (YIPF3, YIPF4 and YIPF5), with YIPF6 and YIPF7 showing a broader profile being distributed throughout the Golgi stack. YIPF proteins have a long soluble N-terminal region, which is orientated towards the cytosol, followed by 5 closely stacked transmembrane domains, and a C terminus, orientated towards the lumen of the Golgi. The significance of YIPF proteins for the maintenance of the morphology of the Golgi was tested by RNA interference, revealing a number of specific morphological changes to this organelle on their depletion. We propose a role for this family of proteins in regulating membrane dynamics in the endomembrane system.

Targeted Disruption of Ig-Hepta/Gpr116 Causes Emphysema-like Symptoms That Are Associated with Alveolar Macrophage Activation.

Ig-Hepta/GPR116 is a member of the G protein-coupled receptor family predominantly expressed in the alveolar type II epithelial cells of the lung. Previous studies have shown that Ig-Hepta is essential for lung surfactant homeostasis, and loss of its function results in high accumulation of surfactant lipids and proteins in the alveolar space. Ig-Hepta knock-out (Ig-Hepta(-/-)) mice also exhibit emphysema-like symptoms, including accumulation of foamy alveolar macrophages (AMs), but its pathogenic mechanism is unknown. Here, we show that the bronchoalveolar lavage fluid obtained from Ig-Hepta(-/-) mice contains high levels of inflammatory mediators, lipid hydroperoxides, and matrix metalloproteinases (MMPs), which are produced by AMs. Accumulation of reactive oxygen species was observed in the AMs of Ig-Hepta(-/-) mice in an age-dependent manner. In addition, nuclear factor-κB (NF-κB) is activated and translocated into the nuclei of the AMs of Ig-Hepta(-/-) mice. Release of MMP-2 and MMP-9 from the AMs was strongly inhibited by treatment with inhibitors of oxidants and NF-κB. We also found that the level of monocyte chemotactic protein-1 is increased in the embryonic lungs of Ig-Hepta(-/-) mice at 18.5 days postcoitum, when AMs are not accumulated and activated. These results suggest that Ig-Hepta plays an important role in regulating macrophage immune responses, and its deficiency leads to local inflammation in the lung, where AMs produce excessive amounts of reactive oxygen species and up-regulate MMPs through the NF-κB signaling pathway.

USP19-Mediated Deubiquitination Facilitates the Stabilization of HRD1 Ubiquitin Ligase.

In the endoplasmic reticulum (ER), misfolded and unfolded proteins are eliminated by a process called ER-associated protein degradation (ERAD) in order to maintain cell homeostasis. In the ERAD pathway, several ER-localized E3 ubiquitin ligases target ERAD substrate proteins for ubiquitination and subsequent proteasomal degradation. However, little is known about how the functions of the ERAD ubiquitin ligases are regulated. Recently, USP19, an ER-anchored deubiquitinating enzyme (DUB), has been suggested to be involved in the regulation of ERAD. In this study, HRD1, an ERAD ubiquitin ligase, is shown to be a novel substrate for USP19. We demonstrate that USP19 rescues HRD1 from proteasomal degradation by deubiquitination of K48-linked ubiquitin chains. In addition, the altered expression of USP19 affects the steady-state levels of HRD1. These results suggest that USP19 regulates the stability of HRD1 and provide insight into the regulatory mechanism of the ERAD ubiquitin ligases.

Ubiquitin-specific protease 19 regulates the stability of the E3 ubiquitin ligase MARCH6.

Ubiquitin-specific protease (USP)19 is a recently identified deubiquitinating enzyme (DUB) having multiple splice variants and cellular functions. One variant encodes an endoplasmic reticulum (ER)-anchored DUB that rescues misfolded transmembrane proteins from ER-associated degradation (ERAD), but the underlying mechanism remains to be elucidated. Here, we show that USP19 interacts with the ERAD-associated E3 ubiquitin ligase MARCH6. Overexpression of USP19 delayed the degradation of MARCH6, leading to an increase in its protein level. In contrast, USP19 depletion resulted in decreased expression of MARCH6. We also show that USP19 overexpression reduced ubiquitination of MARCH6, while its knockdown had the opposite effect. In particular, USP19 was found to protect MARCH6 by deubiquitination from the p97-dependent proteasomal degradation. In addition, USP19 knockdown leads to increased expression of mutant ABCB11, an ERAD substrate of MARCH6. Moreover, USP19 is itself subjected to endoproteolytic processing by DUB activity, and the processing cleaves off an N-terminal cytoplasmic region of unknown function. However, elimination of this processing had no evident effect on MARCH6 stabilization. These results suggest that USP19 is involved in the regulation of ERAD by controlling the stability of MARCH6 via deubiquitination.

Low cytoplasmic pH reduces ER-Golgi trafficking and induces disassembly of the Golgi apparatus.

The Golgi apparatus was dramatically disassembled when cells were incubated in a low pH medium. The cis-Golgi disassembled quickly, extended tubules and spread to the periphery of cells within 30 min. In contrast, medial- and trans-Golgi were fragmented in significantly larger structures of smaller numbers at a slower rate and remained largely in structures distinct from the cis-Golgi. Electron microscopy revealed the complete disassembly of the Golgi stack in low pH treated cells. The effect of low pH was reversible; the Golgi apparatus reassembled to form a normal ribbon-like structure within 1-2h after the addition of a control medium. The anterograde ER to Golgi transport and retrograde Golgi to ER transport were both reduced under low pH. Phospholipase A2 inhibitors (ONO, BEL) effectively suppressed the Golgi disassembly, suggesting that the phospholipase A2 was involved in the Golgi disassembly. Over-expression of Rab1, 2, 30, 33 and 41 also suppressed the Golgi disassembly under low pH, suggesting that they have protective role against Golgi disassembly. Low pH treatment reduced cytoplasmic pH, but not the luminal pH of the Golgi apparatus, strongly suggesting that reduction of the cytoplasmic pH triggered the Golgi disassembly. Because a lower cytoplasmic pH is induced in physiological or pathological conditions, disassembly of the Golgi apparatus and reduction of vesicular transport through the Golgi apparatus may play important roles in cell physiology and pathology. Furthermore, our findings indicated that low pH treatment can serve as an important tool to analyze the molecular mechanisms that support the structure and function of the Golgi apparatus.

Ligand-induced internalization, recycling, and resensitization of adrenomedullin receptors depend not on CLR or RAMP alone but on the receptor complex as a whole.

Adrenomedullins (AM) is a multifaceted distinct subfamily of peptides that belongs to the calcitonin gene-related peptide (CGRP) superfamily. These peptides exert their functional activities via associations of calcitonin receptor-like receptors (CLRs) and receptor activity-modifying proteins (RAMPs) RAMP2 and RAMP3. Recent studies established that RAMPs and CLRs can modify biochemical properties such as trafficking and glycosylation of each other. However there is very little or no understanding regarding how RAMP or CLR influence ligand-induced events of AM-receptor complex. In this study, using pufferfish homologs of CLR (mfCLR1-3) and RAMP (mfRAMP2 and mfRAMP3), we revealed that all combinations of CLR and RAMP quickly underwent ligand-induced internalization; however, their recycling rates were different as follows: mfCLR1-mfRAMP3>mfCLR2-mfRAMP3>mfCLR3-mfRAMP3. Functional receptor assay confirmed that the recycled receptors were resensitized on the plasma membrane. In contrast, a negligible amount of mfCLR1-mfRAMP2 was recycled and reconstituted. Immunocytochemistry results indicated that the lower recovery rate of mfCLR3-mfRAMP3 and mfCLR1-mfRAMP2 was correlated with higher proportion of lysosomal localization of these receptor complexes compared to the other combinations. Collectively our results indicate, for the first time, that the ligand-induced internalization, recycling, and reconstitution properties of RAMP-CLR receptor complexes depend on the receptor-complex as a whole, and not on individual CLR or RAMP alone.

Identification and properties of a novel variant of NBC4 (Na(+)/HCO(3)- co-transporter 4) that is predominantly expressed in the choroid plexus.

Secretion of HCO(3)- at the apical side of the epithelial cells of the choroid plexus is an essential step in the formation of cerebrospinal fluid. Anion conductance with a high degree of HCO(3)- permeability has been observed and suggested to be the major pathway for HCO(3)- transport across the apical membrane. Recently, it was found that NBC (Na(+)/HCO(3)- co-transporter) 4, an electrogenic member of the NBC family, was expressed in the choroid plexus. We found that a novel variant of the NBC4 [NBC4g/Slc4a5 (solute carrier family 4, sodium bicarbonate co-transporter, member 5)] is almost exclusively expressed in the apical membrane of rat choroid plexus epithelium at exceptionally high levels. RNA interference-mediated knockdown allowed the functional demonstration that NBC4g is the major player in the HCO(3)- transport across the apical membrane of the choroid plexus epithelium. When combined with a recent observation that in choroid plexus epithelial cells electrogenic NBC operates with a stoichiometry of 3:1, the results of the present study suggest that NBC4g mediates the efflux of HCO(3)- and contributes to cerebrospinal fluid production.

Polygenic Risk Score, Cardiorespiratory Fitness, and Cardiometabolic Risk Factors: WASEDA'S Health Study.

PURPOSE: This study estimated an individual's genetic liability to cardiometabolic risk factors by polygenic risk score (PRS) construction and examined whether high cardiorespiratory fitness (CRF) modifies the association between PRS and cardiometabolic risk factors. METHODS: This cross-sectional study enrolled 1,296 Japanese adults aged ≥40 years. The PRS for each cardiometabolic trait (blood lipids, glucose, hypertension, and obesity) was calculated using the LDpred2 and clumping and thresholding methods. Participants were divided into low-, intermediate-, and high-PRS groups according to PRS tertiles for each trait. CRF was quantified as peak oxygen uptake (VO 2 peak) per kg body weight. Participants were divided into low-, intermediate-, and high-CRF groups according to the tertile VO 2 peak value. RESULTS: Linear regression analysis revealed a significant interaction between PRS for triglyceride (PRS TG ) and CRF groups on serum TG levels regardless of the PRS calculation method, and attenuated the association between PRS TG and TG levels in the high-CRF group. Logistic regression analysis revealed a significant sub-additive interaction between LDpred2 PRS TG and CRF on the prevalence of high TG, indicating that high CRF attenuated the genetic predisposition to high TG. Furthermore, a significant sub-additive interaction between PRS for body mass index and CRF on obesity was detected regardless of the PRS calculation method. These significant interaction effects on high TG and obesity were diminished in the sensitivity analysis using VO 2 peak per kg fat-free mass as the CRF index. Effects of PRSs for other cardiometabolic traits were not significantly attenuated in the high-CRF group regardless of PRS calculation methods. CONCLUSIONS: The findings of the present study suggest that individuals with high CRF overcome the genetic predisposition to high TG levels and obesity.

Associations between cardiorespiratory fitness and lifestyle-related factors with DNA methylation-based ageing clocks in older men: WASEDA'S Health Study.

DNA methylation-based age estimators (DNAm ageing clocks) are currently one of the most promising biomarkers for predicting biological age. However, the relationships between cardiorespiratory fitness (CRF), measured directly by expiratory gas analysis, and DNAm ageing clocks are largely unknown. We investigated the relationships between CRF and the age-adjusted value from the residuals of the regression of DNAm ageing clock to chronological age (DNAmAgeAcceleration: DNAmAgeAccel) and attempted to determine the relative contribution of CRF to DNAmAgeAccel in the presence of other lifestyle factors. DNA samples from 144 Japanese men aged 65-72 years were used to appraise first- (i.e., DNAmHorvath and DNAmHannum) and second- (i.e., DNAmPhenoAge, DNAmGrimAge, and DNAmFitAge) generation DNAm ageing clocks. Various surveys and measurements were conducted, including physical fitness, body composition, blood biochemical parameters, nutrient intake, smoking, alcohol consumption, disease status, sleep status, and chronotype. Both oxygen uptake at ventilatory threshold (VO2 /kg at VT) and peak oxygen uptake (VO2 /kg at Peak) showed a significant negative correlation with GrimAgeAccel, even after adjustments for chronological age and smoking and drinking status. Notably, VO2 /kg at VT and VO2 /kg at Peak above the reference value were also associated with delayed GrimAgeAccel. Multiple regression analysis showed that calf circumference, serum triglyceride, carbohydrate intake, and smoking status, rather than CRF, contributed more to GrimAgeAccel and FitAgeAccel. In conclusion, although the contribution of CRF to GrimAgeAccel and FitAgeAccel is relatively low compared to lifestyle-related factors such as smoking, the results suggest that the maintenance of CRF is associated with delayed biological ageing in older men.

MARCH7 E3 ubiquitin ligase is highly expressed in developing spermatids of rats and its possible involvement in head and tail formation.

Spermatogenesis is a highly complicated metamorphosis process of male germ cells. Recent studies have provided evidence that the ubiquitin-proteasome system plays an important role in sperm head shaping, but the underlying mechanism is less understood. In this study, we localized membrane-associated RING-CH (MARCH)7, an E3 ubiquitin ligase, in rat testis. Northern blot analysis showed that March7 mRNA is expressed ubiquitously but highly in the testis and ovary. In situ hybridization of rat testis demonstrated that March7 mRNA is expressed weakly in spermatogonia and its level is gradually increased as they develop. Immunohistochemical analysis detected MARCH7 protein expression in spermiogenic cells from late round spermatids to elongated spermatids and in epididymal spermatozoa. Moreover, MARCH7 was found to be localized to the caudal end of the developing acrosome of late round and elongating spermatids, colocalizing with ß-actin, a component of the acroplaxome. In addition, MARCH7 was also detected in the developing flagella and its expression levels were prominent in elongated spermatids. We also showed that MARCH7 catalyzes lysine 48 (K48)-linked ubiquitination. Immunolocalization studies revealed that K48-linked ubiquitin chains were detected in the heads of elongating spermatids and in the acrosome/acroplaxome, neck, midpiece and cytoplasmic lobes of elongated spermatids. These results suggest that MARCH7 is involved in spermiogenesis by regulating the structural and functional integrity of the head and tail of developing spermatids.