Cryo-EM structure of monomeric photosystem II at 2.78 Å resolution reveals factors important for the formation of dimer

Photosystem II (PSII) functions mainly as a dimer to catalyze the light energy conversion and water oxidation reactions. However, monomeric PSII also exists and functions in vivo in some cases. The crystal structure of monomeric PSII has been solved at 3.6 Å resolution, but it is still not clear which factors contribute to the formation of the dimer. Here, we solved the structure of PSII monomer at a resolution of 2.78 Å using cryo-electron microscopy (cryo-EM). From our cryo-EM density map, we observed apparent differences in pigments and lipids in the monomer-monomer interface between the PSII monomer and dimer. One β-carotene and two sulfoquinovosyl diacylglycerol (SQDG) molecules are found in the monomer-monomer interface of the dimer structure but not in the present monomer structure, although some SQDG and other lipid molecules are found in the analogous region of the low-resolution crystal structure of the monomer, or cryo-EM structure of an apo-PSII monomer lacking the extrinsic proteins from Synechocystis sp. PCC 6803. In the current monomer structure, a large part of the PsbO subunit was also found to be disordered. These results indicate the importance of the β-carotene, SQDG and PsbO in formation of the PSII dimer.     

Spatial variation of microtubule depolymerization in large asters

Microtubule plus-end depolymerization rate is a potentially important target of physiological regulation, but it has been challenging to measure, so its role in spatial organization is poorly understood. Here we apply a method for tracking plus ends based on time difference imaging to measure depolymerization rates in large interphase asters growing in Xenopus egg extract. We observed strong spatial regulation of depolymerization rates, which were higher in the aster interior compared with the periphery, and much less regulation of polymerization or catastrophe rates. We interpret these data in terms of a limiting component model, where aster growth results in lower levels of soluble tubulin and microtubule-associated proteins (MAPs) in the interior cytosol compared with that at the periphery. The steady-state polymer fraction of tubulin was ∼30%, so tubulin is not strongly depleted in the aster interior. We propose that the limiting component for microtubule assembly is a MAP that inhibits depolymerization, and that egg asters are tuned to low microtubule density.     

Unweighted state as a sidestep preparation improve the initiation and reaching performance for basketball players

The preparatory motion of a defensive motion in contact sport such as basketball should be small and involve landing on both feet for strict time and motion constraints. We thus proposed the movement creating a unweighted state. Ten basketball players performed a choice reaction sidestepping task with and without the voluntary, continuous vertical fluctuation movement. The results indicated that the preparatory movement shortened the time of their sidestep initiation (301 vs. 314 ms, p = 0.011) and reaching performance (883 vs. 910 ms, p = 0.018) but did not increase their peak ground reaction force or movement velocity. The mechanism of the improvement was estimated to be the following: in the preparation phase, the vertical body fluctuation created the force fluctuation; after the direction signal, the unweighted state can shorten the time required to initiate the sidestepping (unweighted: 279 ms; weighted: 322 ms, p = 0.002); around the initiation phase, the dropping down of the body and weighted state can contribute to the reaching performance. We conducted additional experiment investigating muscle–tendon-complex dynamics and muscle activity using ultrasound device and electromyography. The result suggests that the building up of active state of muscle might explain the improvement of sidestepping performance.     

Synthesis of 2-Alkynylcordycepins and Evaluation of Their Vasodilating Activity

Abstract

Based on the recently developed lithiation-mediated stannyl migration of 6-chloropurine derivatives, 2-iodocordycepin was prepared from cordycepin. The reaction of this compound with terminal alkynes was carried out to synthesize a series of 2-alkynyl derivatives. The vasodilating effect of these compounds was evaluated.     

Increase of salt dependence of halophilic nucleoside diphosphate kinase caused by a single amino acid substitution

Nucleoside diphosphate kinase (HsNDK) from an extremely halophilic archaea, Halobacterium salinarum, is composed of a homo hexamer, assembled as a trimer of basic dimeric units. It requires >2 M NaCl for refolding, although it does not require NaCl for stability or enzymatic activity below 30 °C. A HisN111L mutant with an N-terminal extension sequence containing hexa-His tag, in which Asn111 was replaced with Leu, was designed to be less stable between basic dimeric units. This mutant can lose between 6 and 12 hydrogen bonds between basic dimeric units in the hexamer structure. The HisN111L mutant had enhanced salt requirements for enzymatic activity and refolding even though the secondary structure of the HisN111L mutant was confirmed to be similar to the control, HisNDK, in low and high salt solutions using circular dichroism. We reported previously that G114R and D148C mutants, which had enhanced interactions between basic dimeric units, showed facilitated refolding and stabilization in low salt solution. The results of this study help to elucidate the process for engineering industrial enzymes by controlling subunit–subunit interactions through mutations.     

Apoptosis inhibitor of macrophage (AIM) expression in alveolar macrophages in COPD

Background

Marked accumulation of alveolar macrophages (AM) conferred by apoptosis resistance has been implicated in pathogenesis of chronic obstructive pulmonary disease (COPD). Apoptosis inhibitor of macrophage (AIM), has been shown to be produced by mature tissue macrophages and AIM demonstrates anti-apoptotic property against multiple apoptosis-inducing stimuli. Accordingly, we attempt to determine if AIM is expressed in AM and whether AIM is involved in the regulation of apoptosis in the setting of cigarette smoke extract (CSE) exposure.

Methods

Immunohistochemical evaluations of AIM were performed. Immunostaining was assessed by counting total and positively staining AM numbers in each case (n = 5 in control, n = 5 in non-COPD smoker, n = 5 in COPD). AM were isolated from bronchoalveolar lavage fluid (BALF). The changes of AIM expression levels in response to CSE exposure in AM were evaluated. Knock-down of anti-apoptotic Bcl-xL was mediated by siRNA transfection. U937 monocyte-macrophage cell line was used to explore the anti-apoptotic properties of AIM.

Results

The numbers of AM and AIM-positive AM were significantly increased in COPD lungs. AIM expression was demonstrated at both mRNA and protein levels in isolated AM, which was enhanced in response to CSE exposure. AIM significantly increased Bcl-xL expression levels in AM and Bcl-xL was involved in a part of anti-apoptotic mechanisms of AIM in U937 cells in the setting of CSE exposure.

Conclusions

These results suggest that AIM expression in association with cigarette smoking may be involved in accumulation of AM in COPD.     

Deficiency in Clonogenic Endometrial Mesenchymal Stem Cells in Obese Women with Reproductive Failure – a Pilot Study

Objectives

The mechanisms of obesity associated reproductive complications remain poorly understood. Endometrial mesenchymal stem-cells are critical for cyclic renewal and uterine function. Recently, W5C5⁺ cells, with high clonogenicity, capable of producing endometrial stroma in vivo, have been described. We sought to investigate the abundance and cloning efficiency of W5C5⁺ and W5C5⁻ endometrial cells in relation to Body Mass Index, age and reproductive outcome.

Design

W5C5⁺ and W5C5⁻ cells were purified from mid-luteal endometrial biopsies (n = 54) by magnetic bead separation and subjected to in vitro colony-forming assays.

Results

First trimester pregnancy losses were significantly higher in obese subjects (n = 12) compared to overweight (n = 20) and subjects with normal Body Mass Index (n = 22) (P<0.05, P<0.01, respectively). W5C5⁺ cells (%) were significantly lower in obese subjects compared to subjects with normal Body Mass Index (P<0.05). W5C5⁺ cloning efficiency was significantly lower in obese subjects compared to overweight and subjects with normal Body Mass Index (P<0.05, respectively). W5C5⁻ cloning efficiency was significantly lower in obese subjects compared to subjects with normal Body Mass Index (P<0.05). Body Mass Index was significantly negatively correlated with W5C5⁺ cloning efficiency and W5C5⁻ cloning efficiency (P<0.01, respectively), and positively correlated with first trimester loss (P<0.01). We found no significant results with age (P>0.05).

Conclusions

Our observations suggest that the regenerative capacity and plasticity of the endometrium of obese women is suboptimal, which in turn may account for the increased risk of reproductive complications associated with obesity.     

Plasma B-type Natriuretic Peptide as a Predictor of Cardiovascular Events in Subjects with Atrial Fibrillation: A Community-Based Study

Objectives

Atrial fibrillation (AF) is a significant public health issue due to its high prevalence in the general population, and is associated with an increased risk of cardiovascular (CV) events including systemic thrombo-embolism, heart failure, and coronary artery disease. The relationship between plasma B-type natriuretic peptide (BNP) and CV risk in real world AF subjects remains unknown.

Methods

The subject of the study (n = 228; mean age = 69 years) was unselected individuals with AF in a community-based population (n = 15,394; AF prevalence rate = 1.5%). The CV event free rate within each BNP tertile was estimated, and Cox regression analysis was performed to examine the relative risk of the onset of CV events among the tertiles. The prognostic ability of BNP was compared to an established risk score for embolic events (CHADS2 score). In addition, to determine the usefulness of BNP as a predictor in addition to CHADS2 score, we calculated Net Reclassification Improvement (NRI) and Integrated Discrimination Improvement (IDI) indices.

Results

During the follow-up period 58 subjects experienced CV events (52 per 1,000 person-years). The event-free ratio was significantly lower in the highest tertile (p < 0.02). After adjustment for established CV risk factors, the hazard ratio (HR) of the highest tertile was significantly higher than that of the lowest tertile (HR = 2.38; p < 0.02). The predictive abilities of plasma BNP in terms of sensitivity and specificity for general CV events were comparable to those of CHADS2 score. Adding BNP to the CHADS2 score only model improved the NRI (0.319; p < 0.05) and the IDI (0.046; p < 0.05).

Conclusion

Plasma BNP is a valuable biomarker both singly or in combination with an established scoring system for assessing general CV risk including stroke, heart failure and acute coronary syndrome in real-world AF subjects.     

Establishment of Alternative Culture Method for Spermatogonial Stem Cells Using Knockout Serum Replacement

Since spermatogonial stem cells (SSCs) are capable of both self-renewal and differentiation to daughter cells for subsequent spermatogenesis, the development of an efficient in vitro culture system is essential for studies related to spermatogenesis. Although the currently available system is serum-free and contains only chemically-defined components, it highly relies upon bovine serum albumin (BSA), a component with batch-to-batch quality variations similar to those of fetal bovine serum. Thus, we searched for an alternative BSA-free culture system that preserved the properties of SSCs. In this study, we utilized Knockout Serum Replacement (KSR) in the SSC culture medium, as a substitute for BSA. The results demonstrated that KSR supported the continuous growth of SSCs in vitro and the SSC activity in vivo without BSA, in a feeder-cell combination with mouse embryonic fibroblasts. The addition of BSA to KSR further facilitated cell cycle progression, whereas a transplantation assay revealed that the addition of BSA did not affect the number of SSCs in vivo. The combination of KSR with BSA also allowed the elimination of GFRA1 and FGF2, and the reduction of the GDNF concentration from 20 ng/ml to 5 ng/ml, while maintaining the growth rate and the expression of SSC markers. Furthermore, KSR was also useful with SSCs from non-DBA/2 strains, such as C57BL/6 and ICR. These results suggested that KSR is an effective substitute for BSA for long-term in vitro cultures of SSCs. Therefore, this method is practical for various studies related to SSCs, including spermatogenesis and germ stem cell biology.