Illuminating G-Protein-Coupling Selectivity of GPCRs.

Heterotrimetic G proteins consist of four subfamilies (Gs, Gi/o, Gq/11, and G12/13) that mediate signaling via G-protein-coupled receptors (GPCRs), principally by receptors binding Gα C termini. G-protein-coupling profiles govern GPCR-induced cellular responses, yet receptor sequence selectivity determinants remain elusive. Here, we systematically quantified ligand-induced interactions between 148 GPCRs and all 11 unique Gα subunit C termini. For each receptor, we probed chimeric Gα subunit activation via a transforming growth factor-α (TGF-α) shedding response in HEK293 cells lacking endogenous Gq/11 and G12/13 proteins, and complemented G-protein-coupling profiles through a NanoBiT-G-protein dissociation assay. Interrogation of the dataset identified sequence-based coupling specificity features, inside and outside the transmembrane domain, which we used to develop a coupling predictor that outperforms previous methods. We used the predictor to engineer designer GPCRs selectively coupled to G12. This dataset of fine-tuned signaling mechanisms for diverse GPCRs is a valuable resource for research in GPCR signaling.

Heterotrimeric G Protein Subunit Gαq Is a Master Switch for Gßγ-Mediated Calcium Mobilization by Gi-Coupled GPCRs.

Mechanisms that control mobilization of cytosolic calcium [Ca2+]i are key for regulation of numerous eukaryotic cell functions. One such paradigmatic mechanism involves activation of phospholipase Cß (PLCß) enzymes by G protein ßγ subunits from activated Gαi-Gßγ heterotrimers. Here, we report identification of a master switch to enable this control for PLCß enzymes in living cells. We find that the Gαi-Gßγ-PLCß-Ca2+ signaling module is entirely dependent on the presence of active Gαq. If Gαq is pharmacologically inhibited or genetically ablated, Gßγ can bind to PLCß but does not elicit Ca2+ signals. Removal of an auto-inhibitory linker that occludes the active site of the enzyme is required and sufficient to empower "stand-alone control" of PLCß by Gßγ. This dependence of Gi-Gßγ-Ca2+ on Gαq places an entire signaling branch of G-protein-coupled receptors (GPCRs) under hierarchical control of Gq and changes our understanding of how Gi-GPCRs trigger [Ca2+]i via PLCß enzymes.

Discovery of an anti-malarial compound, burnettiene A, with a multidrug-sensitive Saccharomyces cerevisiae screening system based on mitochondrial function inhibitory activity.

In this paper, we describe our discovery of burnettiene A (1) as an anti-malarial compound from the culture broth of Lecanicillium primulinum (Current name: Flavocillium primulinum) FKI-6715 strain utilizing our original multidrug-sensitive yeast system. This polyene-decalin polyketide natural product was originally isolated as an anti-fungal active compound from Aspergillus burnettii. However, the anti-fungal activity of 1 has been revealed in only one fungal species for and the mechanism of action of 1 remains unknown. After the validation of mitochondrial function inhibitory of 1, we envisioned a new anti-malarial drug discovery platform based on mitochondrial function inhibitory activity. We evaluated anti-malarial activity and 1 showed anti-malarial activity against Plasmodium falciparum FCR3 (chloroquine sensitive) and K1 strain (chloroquine resistant). Our study revealed the utility of our original screening system based on a multidrug-sensitive yeast and mitochondrial function inhibitory activity for the discovery of new anti-malarial drug candidates.

Cost-Effectiveness Analysis of FOLFIRI-Based First-Line Regimens for Metastatic Colorectal Cancer Using Clinical Decision Analysis.

OBJECTIVE: Metastatic colorectal cancer with KRAS wild type is treated using a range of drug regimens, including fluorouracil, irinotecan, and Leucovorin(FOLFIRI)plus bevacizumab(Bmab), cetuximab(Cmab), or panitumumab(Pmab). The present study aimed to identify the optimal regimen using a decision analysis method, in combination with clinical and economic evidence. METHOD: A simple Markov model with a monthly cycle time was constructed. Probabilistic variables for input into the model were derived from randomized controlled trials. Direct costs for the drugs, laboratory analyses, and medical staff were calculated and used in the model. RESULTS: The expected survival times and costs of FOLFIRI alone and combination therapies were 20.9 months and 2,299,198 yen for FOLFIRI, 29.9 months and 8,929,888 yen for Bmab, 27.8 months and 11,811,849 yen for Cmab, and 22.6 months and 8,795,622 yen for Pmab. The incremental cost-effectiveness ratios to FOLFIRI were 736,743 yen/month for Bmab, 1,378,645 yen/month for Cmab, and 3,821,426 yen/month for Pmab. CONCLUSIONS: These findings suggested that these regimens were not sufficiently cost-effective, although they have excellent therapeutic efficacy. From the economic point of view, these combination regimens were inferior to FOLFIRI alone.

Preventive effect of tertiary lymphoid structures on lymph node metastasis of lung adenocarcinoma.

BACKGROUND: Ectopic lymphoid formations are called tertiary lymphoid structures (TLSs). TLSs in cancer have been reported to be associated with good prognosis and immunotherapy response. However, the relationship between TLSs and lymph node (LN) metastasis is unclear. METHODS: We analyzed 218 patients with radically resected lung adenocarcinoma. TLSs were defined as the overlap of T cell zone and B cell zone. Granzyme B + cells were defined as cytotoxic lymphocytes. We evaluated phenotypes of lymphocytes in TLSs, tumor-infiltrating lymphocytes (TILs) and LNs by immunohistochemistry. We divided the patients into mature TLS (DC-Lamp high) and immature TLS (DC-Lamp low) groups. The relationship between TLS maturation and clinicopathological factors was analyzed. RESULTS: The mature TLS group was associated with significantly lower frequency of LN metastasis (P < 0.0001) and early cancer stage (P = 0.0049). The mature TLS group had significantly more CD8 + (P = 0.0203) and Foxp3 + (P = 0.0141) cells in TILs than the immature TLS group had. Mature TLSs were independently associated with a favorable overall survival (hazard ratio [HR] = 0.17, P = 0.0220) and disease-free survival (HR = 0.54, P = 0.0436). Multivariate analysis showed that mature TLS was an independent low-risk factor for LN metastasis (odds ratio = 0.06, P = 0.0003). The number of cytotoxic lymphocytes in LNs was higher in the mature TLS group than in the immature group (20.0 vs. 15.1, P = 0.017). CONCLUSION: Mature TLSs were associated with an increased number of cytotoxic lymphocytes in draining LNs, a lower frequency of LN metastasis, and favorable outcomes. Mature TLSs may support antitumor immunity by lymphocyte activation.

Smoking enhances the expression of angiotensin-converting enzyme 2 involved in the efficiency of severe acute respiratory syndrome coronavirus 2 infection.

Smoking is one of the risk factors most closely related to the severity of coronavirus disease 2019 (COVID-19). However, the relationship between smoking history and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity is unknown. In this study, we evaluated the ACE2 expression level in the lungs of current smokers, ex-smokers, and nonsmokers. The ACE2 expression level of ex-smokers who smoked cigarettes until recently (cessation period shorter than 6 months) was higher than that of nonsmokers and ex-smokers with a long history of nonsmoking (cessation period longer than 6 months). We also showed that the efficiency of SARS-CoV-2 infection was enhanced in a manner dependent on the angiotensin-converting enzyme 2 (ACE2) expression level. Using RNA-seq analysis on the lungs of smokers, we identified that the expression of inflammatory signaling genes was correlated with ACE2 expression. Notably, with increasing duration of smoking cessation among ex-smokers, not only ACE2 expression level but also the expression levels of inflammatory signaling genes decreased. These results indicated that smoking enhances the expression levels of ACE2 and inflammatory signaling genes. Our data suggest that the efficiency of SARS-CoV-2 infection is enhanced by smoking-mediated upregulation of ACE2 expression level.

An approach for live imaging of first cleavage in mouse embryos using fluorescent chemical probes for DNA, microtubules, and microfilaments.

Purpose: Dynamic morphological changes in the chromosome and cytoskeleton occur in mammals and humans during early embryonic development, and abnormalities such as embryonic chromosomal aneuploidy occur when development does not proceed normally. Visualization of the intracellular organelles and cytoskeleton allows elucidation of the development of early mammalian embryos. The behavior of the DNA and cytoskeleton in early mammalian embryos has conventionally been observed by injecting target molecule mRNAs, incorporating a fluorescent substance-expressing gene, into embryos. In this study, we visualized the chronological behavior of male and female chromosome condensation in mouse embryos, beginning in the two-pronuclear zygote, through the first division to the two-cell stage, using fluorescent chemical probes to visualize the behavior of DNA, microtubules, and microfilaments. Method: Mouse two-pronuclear stage embryo were immersed in medium containing fluorescent chemical probes to visualize DNA, microtubules, and microfilaments. Observation was performed with a confocal microscope. Results: This method allowed us to observe how chromosome segregation errors in first somatic cell divisions in mouse embryos and enabled dynamic analysis of a phenomenon called lagging chromosomes. Conclusions: By applying this method, we can observe any stage of embryonic development, which may provide new insights into embryonic development in other mammals.

[A Patient with Small Intestinal Leiomyosarcoma and Intussusception Who Underwent Single Port Surgery].

The patient was a 33-year-old female. She was referred to our hospital from a previous clinic with abdominal pain and vomiting. Contrast-enhanced abdominal CT revealed intussusception, leading to a diagnosis of intussusception with a small intestinal tumor as an advanced part. Intestinal obstruction symptoms were observed, and emergency surgery was performed on the same day. As a technique, single port surgery was selected, and laparoscopic reduction was attempted. However, it was ineffective. The site of intussusception was induced outside the body through a small laparotomy wound, and reduction was performed using Hutchinson's procedure. As an adjacent lymph node was markedly swollen, the mesentery involving this lymph node was dissected in a fan shape, and the tumor was extirpated. The tumor measured 40 mm in long diameter, being a hemicircular, protruding lesion. Histologically, disarray of short spindle tumor cells was observed. Immunostaining showed Kit- and DOG1-negtive reactions and partially α-SMA and desmin-positive reactions, suggesting leiomyosarcoma. With the establishment of an immunostaining-test-based classification, leiomyosarcoma is currently rare. In this study, we report a patient in whom single port surgery for intussusception related to small intestinal leiomyosarcoma was successful.

Interleukin-38 promotes tumor growth through regulation of CD8+ tumor-infiltrating lymphocytes in lung cancer tumor microenvironment.

BACKGROUND: Interleukin (IL)-38 was discovered in 2001 and is a member of the IL-1 family of cytokines. IL-38 shows anti-inflammatory activity in several inflammatory diseases. In lung adenocarcinoma, we previously demonstrated that high IL-38 expression in tumor cells was associated with poor prognosis. However, the role of IL-38 in the tumor microenvironment has not been clarified. METHODS: IL-38-plasmid-transfected Lewis lung carcinoma cells (LLC-IL38) and empty vector-transfected LLC cells (LLC-vector) were established. Cell proliferation in vitro and tumor growth in vivo were examined, and immunohistochemical staining was used to assess tumor-infiltrating lymphocytes (TILs). A CD8+ lymphocyte depletion model was established to show the association between IL-38 and CD8+ lymphocytes. Moreover, we examined the association between IL-38 expression and CD8+ TILs in human samples, analyzing immunohistochemical staining in 226 patients with radically resected lung adenocarcinoma. RESULTS: Tumor growth of LLC-IL38 in vivo was significantly increased compared with that of LLC-vector, although cell proliferation of LLC-IL38 in vitro was lower than that of LLC-vector. CD8+ TILs were significantly decreased in LLC-IL38 tumor compared with LLC-vector tumor. The difference in tumor growth between LLC-IL38 and LLC-vector became insignificant after depletion of CD8+ lymphocytes. In immunohistochemical staining in tissues from patients with lung adenocarcinoma, multivariate analysis showed high IL-38 expression was an independent negative predicter of high density of CD8+ TILs. CONCLUSION: We demonstrated that high IL-38 expression in tumor cells was significantly associated with reduction of CD8+ TILs and tumor progression. These results suggest that IL-38 could be a therapeutic target for lung cancer.

Application of Nitrate, Ammonium, or Urea Changes the Concentrations of Ureides, Urea, Amino Acids and Other Metabolites in Xylem Sap and in the Organs of Soybean Plants (Glycine max (L.) Merr.).

Soybean (Glycine max (L.) Merr.) plants form root nodules and fix atmospheric dinitrogen, while also utilizing the combined nitrogen absorbed from roots. In this study, nodulated soybean plants were supplied with 5 mM N nitrate, ammonium, or urea for 3 days, and the changes in metabolite concentrations in the xylem sap and each organ were analyzed. The ureide concentration in the xylem sap was the highest in the control plants that were supplied with an N-free nutrient solution, but nitrate and asparagine were the principal compounds in the xylem sap with nitrate treatment. The metabolite concentrations in both the xylem sap and each organ were similar between the ammonium and urea treatments. Considerable amounts of urea were present in the xylem sap and all the organs among all the treatments. Positive correlations were observed between the ureides and urea concentrations in the xylem sap as well as in the roots and leaves, although no correlations were observed between the urea and arginine concentrations, suggesting that urea may have originated from ureide degradation in soybean plants, possibly in the roots. This is the first finding of the possibility of ureide degradation to urea in the underground organs of soybean plants.

Combined Evaluation of Tumor-Infiltrating CD8 + and FoxP3 + Lymphocytes Provides Accurate Prognosis in Stage IA Lung Adenocarcinoma.

BACKGROUND: Immunotherapy has become a standard treatment option for non-small cell lung cancer (NSCLC), with the tumor microenvironment attracting significant attention. CD8 + and forkhead box protein P3 + (FoxP3 +) tumor-infiltrating lymphocytes (TILs) influence the tumor microenvironment, but the clinical significance of CD8 + and FoxP3 + TILs in stage IA lung adenocarcinoma (LAD) is poorly understood. METHODS: We analyzed 203 patients with stage IA primary LAD who had undergone surgery at Kyushu University from January 2003 to December 2012. We evaluated CD8 + and FoxP3 + TILs by immunohistochemistry. We set the cutoff values at 50 cells/0.04 mm2 for CD8 + TILs and 20 cells/0.04 mm2 for FoxP3 + TILs, respectively. We divided the patients into four groups: CD8-Low/FoxP3-Low; CD8-High/FoxP3-Low; CD8-Low/FoxP3-High; and CD8-High/FoxP3-High. We compared clinical outcomes among them. Programmed cell death ligand-1 (PD-L1) expression by tumor cells was also evaluated as previously reported. RESULTS: Respectively, 104 (51.2%), 46 (22.7%), 22 (10.8%), and 31 (15.3%) patients were classified as CD8-Low/FoxP3-Low, CD8-High/FoxP3-Low, CD8-Low/FoxP3-High, and CD8-High/FoxP3-High. Both disease-free survival (DFS) and overall survival (OS) were significantly worse in the CD8-Low/FoxP3-High group than the other groups (5-year DFS: 66.3% vs. 90.5%; P = 0.0007, 5-year OS: 90.9% vs. 97.0%; P = 0.0077). In the multivariate analysis, CD8-Low/FoxP3-High and PD-L1 expression were independent prognostic factors of DFS, and lymphatic invasion, surgical procedure, and PD-L1 expression were independent prognostic factors of OS. CONCLUSIONS: CD8-Low/FoxP3-High was an independent prognostic factor of DFS (hazard ratio: 3.22; 95% confidence interval: 1.321-7.179; P = 0.0121) in stage IA LAD. Immunosuppressive conditions were associated with poor prognosis in stage IA LAD.

Baculovirus-Induced Fast-Acting Innate Immunity Kills Liver-Stage Plasmodium.

Baculovirus (BV), an enveloped insect virus with a circular dsDNA genome, possesses unique characteristics that induce strong innate immune responses in mammalian cells. In this study, we show that BV administration in BALB/c mice not only provides complete protection against a subsequent Plasmodium berghei sporozoite infection for up to 7 d after the injection but also eliminates existing liver-stage parasites completely. The elimination of sporozoites by BV was superior to that by primaquine, and this effect occurred in a TLR9-independent manner. At 6 h after BV administration, IFN-α and IFN-γ were robustly produced in the serum, and RNA transcripts of IFN-stimulated genes were markedly upregulated in the liver compared with control mice. The in vivo passive transfer of serum after BV administration effectively eliminated liver-stage parasites, and IFN-α neutralization abolished this effect, indicating that the BV liver-stage parasite-killing mechanism is downstream of the type I IFN signaling pathway. These findings provide evidence that BV-induced, fast-acting innate immunity completely kills liver-stage parasites and, thus, may lead to new malaria drug and vaccine strategies.

Protein 19F-labeling using transglutaminase for the NMR study of intermolecular interactions.

The preparation of stable isotope-labeled proteins is important for NMR studies, however, it is often hampered in the case of eukaryotic proteins which are not readily expressed in Escherichia coli. Such proteins are often conveniently investigated following post-expression chemical isotope tagging. Enzymatic 15N-labeling of glutamine side chains using transglutaminase (TGase) has been applied to several proteins for NMR studies. 19F-labeling is useful for interaction studies due to its high NMR sensitivity and susceptibility. Here, 19F-labeling of glutamine side chains using TGase and 2,2,2-trifluoroethylamine hydrochloride was established for use in an NMR study. This enzymatic 19F-labeling readily provided NMR detection of protein-drug and protein-protein interactions with complexes of about 100 kDa since the surface residues provided a good substrate for TGase. The 19F-labeling method was 3.5-fold more sensitive than 15N-labeling, and could be combined with other chemical modification techniques such as lysine 13C-methylation. 13C-dimethylated-19F-labeled FKBP12 provided more accurate information concerning the FK506 binding site.

[Examination of Measures for Preventing Exposure in Nurses Who Handle Cyclophosphamide].

Health hazards due to long-term exposure to anticancer drugs have been reported among health care professionals. In Yamagata Prefectural Central Hospital, constant use of personal protective equipment(gloves and mask with face shield)is mandatory, but there is no clear description of the protective gown. To verify the exposure status of nurses while handling cyclophosphamide and the usefulness of a protective gown as a protective measure, urinary concentration of cyclophosphamide was measured for nurses who handled cyclophosphamide. No cyclophosphamide was detected in the urine samples collected from nurses who handled cyclophosphamide while wearing protective gowns or in the samples collected from nurses who handled cyclophosphamide without protective gowns. This finding suggests that gloves and a mask with a face shield are sufficient for preventing exposure to cyclophosphamide. However, considering that only experienced nurses were included as subjects in this study, we cannot conclude that a protective gown is unnecessary, because inexperienced nurses may be exposed to cyclophosphamide. Our study's findings may be one reference to examine measures for preventing exposure in nurses.

Characterization of co-fired sodium-ion conductive Na2Ni2TeO6 and Na2Zn2TeO6 with honeycomb layer structure.

We investigated the reactivity of P2-type honeycomb layered oxides Na2Ni2TeO6 (NNTO) and Na2Zn2TeO6 (NZTO) co-fired at the temperature from 500 °C to 800 °C. From X-ray diffraction measurements, it was found that the reaction between NNTO and NZTO is unremarkable at the temperature below 700 °C. However, when annealed at 800 °C, they formed the solid-solution phase without any secondary phases. The NNTO and NZTO composite pellets co-fired at 800 °C showed sodium-ion conductivity well above 10-4 S cm-1 at room temperature, indicating that the solid-solution phase of NNTO and NZTO has good ionic conductivity. A maximum room temperature conductivity of 7.4 × 10-4 S cm-1 was confirmed at the mixing ratio NNTO: NZTO = 0.5 : 1.5. These results can be applied to the fabrication of all-solid-state batteries using NNTO as the cathode active material and NZTO as the solid electrolyte via a simple co-sintering process.

Label-free autofluorescence and hyperspectral imaging of cerebral amyloid-ß lesions in aged squirrel monkeys.

The observation of amyloid-ß (Aß) lesions using autofluorescence in transgenic mice and human Alzheimer disease patients has been reported frequently. However, no reports verify the autofluorescence of spontaneous Aß amyloidosis in animals, to our knowledge. We validated the autofluorescence of Aß lesions in spontaneous squirrel monkey cases under label-free conditions; lesions had intense blue-white autofluorescence in fluorescence microscopy using excitation light at 400-440 nm. Thioflavin S staining and immunohistochemistry of the same specimens revealed that this blue-white autofluorescence was derived from Aß lesions. Hyperspectral analysis of these lesions revealed a characteristic spectrum with bimodal peaks at 440 and 460 nm, as reported for Aß lesions in mice. Principal component analysis using hyperspectral data specifically separated the Aß lesions from other autofluorescent substances, such as lipofuscin. A non-labeled and mechanistic detection of Aß lesions by hyperspectral imaging could provide valuable insights for developing early diagnostic techniques.

A case of difficult-to-diagnose hepatic reactive lymphoid hyperplasia finally diagnosed by using PCR analysis of IgH-gene rearrangements: a case report.

Reactive lymphoid hyperplasia (RLH) of the liver is extremely rare. Despite advancements in diagnostic imaging technology, it is still difficult to distinguish from hepatocellular carcinoma (HCC). Herein, we present a case of hepatic RLH mimicking HCC that was postoperatively diagnosed using several imaging modalities. A 78-year-old female was referred to our hospital with a positive hepatitis C virus antibody (HCV Ab) test. Ultrasonography revealed a 13 mm isoechoic lesion in segment 8 of the liver. Contrast-enhanced computed tomography (CE-CT) demonstrated arterial hyperintensity and washout during the later phase. On ethoxybenzyl magnetic resonance imaging (EOB-MRI), the lesion was hyperenhanced in the arterial phase and of low intensity in the hepatocyte phase. Although the tumor markers were all within normal limits, the pattern of contrast enhancement of the tumor on CT and MRI was consistent with that of HCC. We performed S8 segmentectomy of the liver. Histological examination of the resected specimen revealed dense lymphoid tissue of variable sizes and shapes with expanded germinal centers. Immunohistochemical examination was positive for CD3, CD10 (germinal center), and CD20, and negative for B-cell lymphoma 2 (bcl-2) (germinal center) and Epstein-Barr virus (EBV). A polymerase chain reaction (PCR) analysis of IgH-gene rearrangements revealed polyclonality. Based on these findings, hepatic RLH was diagnosed. The postoperative course was uneventful, and the patient was discharged on the 10th postoperative day. She had a good quality of life after surgery and no liver nodule recurrence was detected at the 4-month medical follow-up. Hepatic RLH is an extremely rare disease and preoperative diagnosis is difficult. This should be considered in the differential diagnosis of single small hepatic tumors. An echo-guided biopsy and careful observation of imaging may help diagnose hepatic RLH, and a PCR analysis of IgH-gene rearrangements would be necessary for the definitive diagnosis of hepatic RLH.

Skeletal Muscle Mass Index Reduction Rate as a Prognostic Indicator for Patients Undergoing Pancreatectomy for Pancreatic Cancer.

Background/Aim: There have been many studies on skeletal muscle depletion before surgery, and skeletal muscle depletion is a known risk factor for poor prognosis. However, reports on the association between changes in skeletal muscle mass and prognosis after surgery for pancreatic cancer are very few. Patients and Methods: The data of 137 patients who underwent pancreatectomy for pancreatic cancer between 2005 and 2022 were reviewed. Muscle areas were measured at the third lumbar vertebral level, and skeletal muscle mass index (SMI) reduction rates were calculated. Patients were divided into two groups using receiver operating characteristic (ROC) curve analysis based on the SMI reduction rate with a cutoff of 14% reduction rate. The clinicopathological factors, overall survival (OS), and recurrence-free survival (RFS) were compared between the two groups. Survival rates were analyzed both univariately and multivariately to clarify the factors associated with poor prognosis after pancreatectomy. Results: A total of 102 patients met the inclusion criteria. SMI reduction rate ≥14% significantly correlated with advanced age and higher incidence of postoperative complications. In the multivariate Cox regression analysis, preoperative prognostic nutritional index (PNI) <40 and SMI reduction rate ≥14% were significantly associated with poor OS. Tumor size ≥3.0 cm, preoperative neutrophile-lymphocyte ratio ≥3.0, and SMI reduction rate ≥14% were significantly associated with poor RFS. Conclusion: The rate of skeletal muscle mass reduction after pancreatic surgery is an independent prognostic factor for survival in patients with pancreatic cancer.

Shape of the first mitotic spindles impacts multinucleation in human embryos.

During human embryonic development, early cleavage-stage embryos are more susceptible to errors. Studies have shown that many problems occur during the first mitosis, such as direct cleavage, chromosome segregation errors, and multinucleation. However, the mechanisms whereby these errors occur during the first mitosis in human embryos remain unknown. To clarify this aspect, in the present study, we image discarded living human two-pronuclear stage zygotes using fluorescent labeling and confocal microscopy without microinjection of DNA or mRNA and investigate the association between spindle shape and nuclear abnormality during the first mitosis. We observe that the first mitotic spindles vary, and low-aspect-ratio-shaped spindles tend to lead to the formation of multiple nuclei at the 2-cell stage. Moreover, we observe defocusing poles in many of the first mitotic spindles, which are strongly associated with multinucleation. Additionally, we show that differences in the positions of the centrosomes cause spindle abnormality in the first mitosis. Furthermore, many multinuclei are modified to form mononuclei after the second mitosis because the occurrence of pole defocusing is firmly reduced. Our study will contribute markedly to research on the occurrence of mitotic errors during the early cleavage of human embryos.

Evidence for contribution of autophagy to rubisco degradation during leaf senescence in Arabidopsis thaliana.

During leaf senescence, Rubisco is gradually degraded and its components are recycled within the plant. Although Rubisco can be mobilized to the vacuole by autophagy via specific autophagic bodies, the importance of this process in Rubisco degradation has not been shown directly. Here, we monitored Rubisco autophagy during leaf senescence by fusing synthetic green fluorescent protein (sGFP) or monomeric red fluorescent protein (mRFP) with Rubisco in Arabidopsis (Arabidopsis thaliana). When attached leaves were individually exposed to darkness to promote their senescence, the fluorescence of Rubisco-sGFP was observed in the vacuolar lumen as well as chloroplasts. In addition, release of free-sGFP due to the processing of Rubisco-sGFP was observed in the vacuole of individually darkened leaves. This vacuolar transfer and processing of Rubisco-sGFP was not observed in autophagy-deficient atg5 mutants. Unlike sGFP, mRFP was resistant to proteolysis in the leaf vacuole of light-grown plants. The vacuolar transfer and processing of Rubisco-mRFP was observed at an early stage of natural leaf senescence and was also obvious in leaves naturally covered by other leaves. These results indicate that autophagy contributes substantially to Rubisco degradation during natural leaf senescence as well as dark-promoted senescence.