MORPHOLOGICAL ASSESSMENT OF HORSESHOE CRABS (TACHYPLEUS TRIDENTATUS) BY USING MAGNETIC RESONANCE IMAGING.

The population of Tachypleus tridentatus, a horseshoe crab, has decreased sharply in Japan. With the exception of a few reports regarding the morphological features of T. tridentatus, a three-dimensional analysis of its inner anatomical structures has never been documented. Here, the anatomical structures in two T. tridentatus specimens were examined using magnetic resonance imaging (MRI). In the larger of the two specimens, the three-dimensional T2-weighted image clearly revealed muscles, digestive and urinary systems, ovary, and brain; however, the cardiovascular system was poorly visualized. In the smaller specimen, cardiovascular, digestive, and urinary systems were identifiable. In the three-dimensional T1-weighted images, tissue contrast was low, making it challenging to distinguish the individual organs. These results suggest that MRI is a powerful tool for visualizing the inner anatomical structures of horseshoe crabs.

A cross-sectional study of the psychological impact of the COVID-19 pandemic on undergraduate and graduate students in Japan.

BACKGROUND: Due to the COVID-19 pandemic, a state of emergency was declared in Japan and university classes were suspended, causing concern about the deterioration of the mental health of isolated students. This study aimed to understand students' mental health status during the COVID-19 pandemic and suggest measures to prevent depressive anxiety among them. METHOD: Undergraduate and graduate students at one national and two private universities in the Kansai region were surveyed. The Kessler Psychological Distress Scale-6 was used to assess the students' mental health. Questions were included to assess students' awareness of COVID-19 and changes in lifestyle habits, including drinking, smoking, gaming, and other addictive habits. The University of Tokyo Health Sociology's version of the Sense of Coherence Scale was used to assess the ability to cope with stressors. RESULTS: More than 50% of undergraduate and graduate students felt more than mild depressive anxiety and approximately 11% felt severe depressive anxiety, indicating that anxiety about the future worsened the levels of depressive anxiety. Life with reversed day and night schedules was associated with the worsening of depressive anxiety levels, but a high sense of coherence was associated with decreased levels of depressive anxiety. CONCLUSION: COVID-19 pandemic triggered isolation which led to worsening the mental health of undergraduate and graduate students. Psychological support for lifestyle and a sense of coherence is necessary to prevent mental health deterioration among isolated students. LIMITATIONS: As we were unable to contact all students, the sample bias may affect interpretation of the data.

Analysis of the alphaB-crystallin domain responsible for inhibiting tubulin aggregation.

The cytoskeleton has a unique property such that changes of conformation result in polymerization into a filamentous form. alphaB-Crystallin, a small heat shock protein (sHsp), has chaperone activities for various substrates, including proteins constituting the cytoskeleton, such as actin; intermediate filament; and tubulin. However, it is not clear whether the "alpha-crystallin domain" common to sHsps also has chaperone activity for the protein cytoskeleton. To investigate the possibility that the C-terminal alpha-crystallin domain of alpha-crystallin has the aggregation-preventing ability for tubulin, we constructed an N-terminal domain deletion mutant of alphaB-crystallin. We characterized its structural properties and chaperone activities. Far-ultraviolet (UV) circular dichroism measurements showed that secondary structure in the alpha-crystallin domain of the deletion mutant is maintained. Ultracentrifuge analysis of molecular masses indicated that the deletion mutant formed smaller oligomers than did the full-length protein. Chaperone activity assays demonstrated that the N-terminal domain deletion mutant suppressed heat-induced aggregation of tubulin well. Comparison of chaperone activities for 2 other substrates (citrate synthase and alcohol dehydrogenase) showed that it was less effective in the suppression of their aggregation. These results show that alphaB-crystallin recognizes a variety of substrates and especially that alpha-crystallin domain binds free cytoskeletal proteins. We suggest that this feature would be advantageous in its functional role of holding or folding multiple proteins denatured simultaneously under stress conditions.

The decrease of the cytoskeleton tubulin follows the decrease of the associating molecular chaperone alphaB-crystallin in unloaded soleus muscle atrophy without stretch.

The cytoskeletal component tubulin/microtubule commonly allows the cell to respond mechanically to the environment. The concentration of free tubulin dimer is autoregulated in the balance of free dimer and polymeric forms of microtubule (MT) protein, having an intrinsic property of "dynamic instability", and through cotranslational beta-tubulin mRNA degradation. Recently, we have demonstrated that alphaB-crystallin is a key molecule of muscle atrophy, since alphaB-crystallin has a chaperone-like-activity that suppresses tubulin aggregation and protects the MT disassembly against both Ca2+ and depolymelizing alkaloid in vitro. Most of the small heat-shock proteins (sHsps), including alphaB-crystallin, are expressed in skeletal muscle. However, no report to date has studied the changes of tubulin/MT during muscle adaptation. Here, we examined changes in tubulin content in rat soleus muscles after hindlimb suspension (HS) with/without passive stretch and the recovery. HS induced rapid decreases of soleus muscle mass, most Hsps (alphaB-crystallin, Hsp90, Hsp70, Hsp27, and p20) and tubulin contents in soleus muscle, while heat-shock cognate 70-kDa protein (Hsc70) did not decrease. Soleus muscle mass, most Hsps, and tubulin were maintained with passive stretch. After 5 days' recovery, the levels of tubulin and Hsps, but not Hsc70, were restored to control levels. The interactions of alphaB-crystallin and tubulin/MT were observed with immunoprecipitation with an anti-alpha-tubulin antibody and taxol-dependent MT assembly. Other sHsps were also associated with alphaB-crystallin and MT, whereas Hsp90 and Hsp70 did not co-precipitate with them. These data imply an interaction and close relationship between alphaB-crystallin and tubulin/MTs in muscle tissues. The amount of mRNA of alphaB-crystallin decreased with the muscle atrophy level, whereas the gene expression level of betaI-tubulin was maintained during HS. This means a significant role of post-transcriptional regulation in tubulin/MT system in muscle adaptation, whereas alphaB-crystallin and most sHsps are regulated at the transcriptional level. Additional functional contribution of alphaB-crystallin to tubulin/MTs during myotube formation was examined using C2C12 myoblast cultured cells, the alphaB-crystallin expression of which was decreased or increased. It indicated the necessity of alphaB-crystallin during microtubule reorganization. In conclusion, tubulin/MTs were revealed to be one of the substrates of alphaB-crystallin, and also serial decreases of alphaB-crystallin and tubulin/MT in early soleus muscle atrophy suggest that the chaperone effect of alphaB-crystallin on the cytoskeleton, which may be also dynamically regulated in the muscle cell, is a key mechanism for muscle adaptation and protection of the atrophy and also muscle differentiation.

A pilot study of mRNA expressions of 5-fluorouracil pathway genes in peripheral blood mononuclear cells and tumor tissues in patients with lung adenocarcinoma.

To assess whether early lung cancer prediction might be informed by an mRNA assay for 5-fluorouracil pathway genes in peripheral blood mononuclear cells (PBMNCs), we examined specimens taken from 51 adenocarcinoma patients and 38 controls (including six patients with benign tumors). PBMNCs and tumor-tissue specimens were taken for measurement of the mRNAs of various 5-fluorouracil pathway genes [thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), and orotate phosphoribosyl transferase (OPRT)]. By quantitative RT-PCR, all four mRNAs were detected in both PBMNCs and tumor tissues. In PBMNCs, TS mRNA/GAPDH mRNA levels were significantly higher in adenocarcinoma patients than in the controls, and significantly higher for pathological stages 2-4 and lymph-node involvement pN1-pN3 than for pathological stage 1 and pN0, respectively. No correlation between PBMNCs and tumor-tissue specimens was found for the level of any mRNA. Thus, the measurement of TS mRNA in PBMNCs might aid the diagnosis of lung adenocarcinoma.

The molecular chaperone HSP47 rapidly senses gravitational changes in myoblasts.

Skeletal muscle unloading induced by spaceflight or bed rest leads to muscle atrophy. It is unclear how muscle atrophy is caused and how muscles respond to microgravity. We addressed the response of collagen and its chaperone system to gravitational forces. We show here that expression of HSP47, a collagen-specific molecular chaperone, responds to gravitational changes, including microgravity and hypergravity in vitro and in vivo. By using the method hindlimb suspension of rats, which mimics microgravity conditions, we demonstrated that the expression of Hsp47 mRNA decreased within 1 day and the mRNA levels of collagen types I and IV were subsequently reduced. In contrast, hypergravity stimulated HSP47 expression. HSP47 and collagen types I and IV were localized intracellularly in the endoplasmic reticulum and/or Golgi apparatus of myoblasts, as expected. Intriguingly, Hsp47 mRNA levels in cultured myoblasts increased significantly with hypergravity treatment at 40G for 2 h, and decreased with microgravity treatment at almost 0G for 1-2 h. Collagen mRNA levels were also altered, although changes were slower and less pronounced compared with those for HSP47. The gravity-regulated HSP47 may play a role in the maintenance of the extracellular matrix by modulating collagen production at the primary stage of adaptation.

alphaB-Crystallin-coated MAP microtubule resists nocodazole and calcium-induced disassembly.

alphaB-Crystallin, one of the small heat-shock proteins, is constitutively expressed in various tissues including the lens of the eye. It has been suggested that alphaB-crystallin provides lens transparency but its function in nonlenticular tissues is unknown. It has been reported that alphaB-crystallin is involved in the stabilization and the regulation of cytoskeleton, such as intermediate filaments and actin. In this study, we investigate the possibility whether alphaB-crystallin interacts with the third cytoskeleton component, microtubules (MTs). First, we precisely observed the cellular localization of alphaB-crystallin and MT networks in L6E9 myoblast cells and found a striking coincidence between them. MTs reconstituted from cell lysate contained alphaB-crystallin. Electron micrographs clearly showed direct interactions of purified alphaB-crystallin with the surface of microtubule-associated proteins (MAPs) attached to MTs. Purified alphaB-crystallin bound to MAP-MTs in a concentration-dependent manner. However, alphaB-crystallin did not bind MTs reconstituted from purified tubulin. Finally, we observed that alphaB-crystallin increased the resistance of MTs to depolymerization in cells and in vitro. Taken together, these results suggest that one of the functions of alphaB-crystallin is to bind MTs via MAP(s) and to give the MTs resistance to disassembly.