Recurrent Serous Borderline Tumor After Fertility-Sparing Surgery Following Twin Pregnancy and Resumption of Menstruation.

Gynecologic malignancies sometimes affect women before menopause. Aggressive treatments, such as surgery, chemotherapy, and/or radiotherapy, often lead to premature menopause. Hormone replacement therapy (HRT), typically used for managing menopause-associated health issues, may be limited by tumor sensitivity to estrogen. Here, we present a case of a 37-year-old woman seeking fertility, who was diagnosed with a serous borderline ovarian tumor (BOT). Fertility-preserving surgery and in-vitro fertilization resulted in a twin pregnancy. During a postpartum amenorrheic period, there was no recurrence. However, she experienced a rapid recurrence of the disease following the resumption of menstruation and underwent radical surgery. This rapid recurrence after menstruation resumed suggests potential estrogen sensitivity. Close postoperative monitoring has been ongoing without HRT.

Impact of Menopause and the Menstrual Cycle on Oxidative Stress in Japanese Women.

Although estrogen possesses both pro- and anti-oxidant properties, its overall role in oxidative stress among women remains unclear, particularly since the influence of exogenously administered estrogen during previous studies differed by dose, administration route, and estrogen type. The aim of this study was to elucidate the effects of endogenous estrogen on oxidative stress in women. Thus, we performed a non-interventional observational study of healthy postmenopausal (n = 71) and premenopausal (n = 72) female volunteers. Serum levels of derivatives of reactive oxygen metabolites (d-ROMs, which are collectively a marker of oxidative stress), as well as the biological antioxidant potential (BAP, an indicator of antioxidant capacity), were compared between (1) pre- versus post-menopausal women, and (2) premenopausal women in early follicular versus mid-luteal phases of their menstrual cycles. We found that serum d-ROMs and BAP values in postmenopausal women were significantly higher than those in premenopausal women. Moreover, the d-ROM levels were significantly correlated with serum copper concentrations. However, neither d-ROMs nor BAP values were significantly affected by the menstrual cycle phase, although changes in d-ROMs between the follicular and luteal phases were significantly correlated with copper concentration shifts. These data indicate that postmenopausal hypoestrogenism is associated with elevated oxidative stress, although regular fluctuations of estrogen levels during the menstrual cycle do not influence oxidative stress.

Successful laparoscopic management of acute abdominal pain due to spontaneous rupture of subserosal vessels overlying a uterine fibroid: a case report and surgical video.

BACKGROUND: Acute abdomen comprises several emergencies. Hemoperitoneum associated with uterine fibroids, which can present as acute abdominal pain, is rare and difficult to diagnose. Especially, spontaneous hemorrhage from the rupture of the superficial vessels overlying a uterine fibroid is extremely rare, and its diagnosis and management have not been established. CASE PRESENTATION: We report a case of a 55-year-old woman who presented at our hospital with acute abdomen. After performing a computed tomography scan, we conducted a laparoscopic examination and diagnosed hemoperitoneum of ambiguous origin. We treated the patient surgically, performing a laparoscopic myomectomy to remove the origin of the hemorrhage. The patient recovered well. CONCLUSIONS: We report a case of hemoperitoneum of ambiguous origin that was diagnosed laparoscopically and treated by laparoscopic myomectomy to remove the origin of the hemorrhage. Surgeons should rapidly diagnose and manage acute abdominal pain in women with a history of uterine fibroids to prevent severe morbidity or even mortality. Therefore, laparoscopic surgery is recommended in patients with stable hemodynamics.

A High Risk of Missing Congenital Cytomegalovirus-Associated Hearing Loss through Newborn Hearing Screening in Japan.

It remains unclear to what extent newborn hearing screening (NHS) detects congenital cytomegalovirus (cCMV)-associated sensorineural hearing loss (SNHL) in Japan. This study aimed to clarify the NHS results and audiological characteristics of patients with cCMV-associated SNHL. A total of 541 individuals with unilateral or bilateral hearing loss of unknown etiology were examined for cCMV infection. cCMV infection was defined by the presence of CMV DNA in the dried umbilical cord detected using real-time quantitative PCR. NHS results and audiological data were retrospectively obtained from medical records. Forty-four cases (8.1%) were positive for cCMV infection. Of them, 33 cases underwent NHS and 13 cases (39.4%) passed NHS bilaterally. The pure-tone audiograms of 21 patients were obtained. There were seven cases of unilateral SNHL, five cases of asymmetric bilateral SNHL, and nine cases of symmetric bilateral SNHL. cCMV-related hearing loss is highly heterogeneous, and there is a high risk of missing this condition through NHS.

Investigation of the hearing levels of siblings affected by a single GJB2 variant: Possibility of genetic modifiers.

OBJECTIVE: Variants in GJB2 can cause autosomal recessive deafness (DFNB1). There is evidence for genotype-phenotype correlations of GJB2 variants; however, several genotypes can cause varying levels of hearing loss likely attributable to differences in genetic or environmental background. As siblings share approximately 50% of their genetic background and usually have a common environmental background, analysis of phenotypes of siblings with a specific GJB2 variant may reveal factors relevant to phenotypic variation. There have been no previous analyses of differences in hearing among siblings carrying a single GJB2 genotype. Here, we investigated hearing differences between siblings with a single GJB2 variant, which can cause various levels of hearing loss. METHODS: We examined hearing levels in 16 pairs of siblings homozygous for the c.235delC variant of GJB2. Differences in hearing acuity between sibling pairs were detected by auditory evaluation. RESULTS: Average differences in acoustic threshold >30 dB were observed between five pairs of siblings, whereas the remaining 11 pairs had average threshold values within approximately 10 dB of one another. Hearing loss varied from moderate to profound. CONCLUSION: Our results indicate that auditory acuity associated with homozygosity for GJB2 c.235delC can vary in degree; however, in approximately 70% of younger siblings, it was approximately the same as that in the first child, despite a diverse spectrum of hearing loss among different families. These results suggest that differences in genetic background may modify the phenotype associated with homozygous GJB2 c.235delC.

Neuroendocrine Carcinoma of Uterine Cervix: Stereotactic Radiotherapy for Brain Metastasis and Stereotactic Body Radiotherapy for Renal and Pancreatic Metastases.

A case of cervical neuroendocrine carcinoma (NEC) of the uterine cervix (NECUC) was presented. After total hysterectomy with bilateral salpingo-oophorectomy and adjuvant chemotherapy, a left renal tumor and a pancreatic lesion developed and were both diagnosed on pathological examination as metastases from NEC. In addition, a brainstem metastasis causing neurologic signs developed. The brain lesion was treated by stereotactic radiotherapy (SRT) and the renal and pancreatic lesions by stereotactic body radiotherapy (SBRT). Despite control of the renal and pancreatic lesions, multiple small lung metastases developed later. Recurrence and newly developed brain metastases were treated by repeat stereotactic radiosurgery (SRS)/SRT successfully. Chemotherapy was continued and controlled the lung metastases until three and a half years after the initial operation of the uterus.

Differences in hearing levels between siblings with hearing loss caused by GJB2 mutations.

OBJECTIVE: Hearing loss caused by GJB2 mutations is inherited in an autosomal recessive manner (DFNB1); thus siblings of an affected child have a 25% chance of also being affected. Hearing loss among subsequent siblings carrying the same GJB2 mutation is a concern for parents and a frequent topic of enquiry during genetic counseling. Evidence exists for genotype-phenotype correlations of GJB2 mutations; however, no analysis of differences in hearing among siblings, in whom the common genetic background may decrease variation, has been reported. The purpose of the present study was to investigate hearing differences between siblings with identical GJB2 mutations. METHODS: We examined the hearing levels of 12 pairs of siblings; each pair had the same pathogenic GJB2 mutations. Differences in hearing acuity between sibling pairs detected by auditory evaluation. RESULTS: No significant correlation was detected between the average hearing levels of first and second affected siblings. Average differences in acoustic threshold >30 dB were observed between four pairs of siblings, whereas the remaining eight pairs had average threshold values within 20 dB of one another. CONCLUSION: Our results indicate that auditory acuity would be expected to approximate that found in the first child in approximately 70% of subsequent children with GJB2-mediated hearing loss, whereas 30% of subsequent siblings would have average differences of >30 dB.

Deterioration in Distortion Product Otoacoustic Emissions in Auditory Neuropathy Patients With Distinct Clinical and Genetic Backgrounds.

OBJECTIVES: Auditory neuropathy (AN) is a clinical disorder characterized by the absence of auditory brainstem response and presence of otoacoustic emissions. A gradual loss of otoacoustic emissions has been reported for some cases of AN. Such cases could be diagnosed as cochlear hearing loss and lead to misunderstanding of the pathology when patients first visit clinics after the loss of otoacoustic emissions. The purpose of this study was to investigate the time course of changes in distortion product otoacoustic emissions (DPOAEs) in association with patients' genetic and clinical backgrounds, including the use of hearing aids. DESIGN: DPOAE measurements from 31 patients with AN were assessed. Genetic analyses for GJB2, OTOF, and mitochondrial m.1555A> G and m.3243A> G mutations were conducted for all cases, and the analyses for CDH23 and OPA1 were conducted for the selected cases. Patients who were younger than 10 years of age at the time of AN diagnosis were designated as the pediatric AN group (22 cases), and those who were 18 years of age or older were designated as the adult AN group (9 cases). DPOAE was measured at least twice in all patients. The response rate for DPOAEs was defined and analyzed. RESULTS: The pediatric AN group comprised 10 patients with OTOF mutations, 1 with GJB2 mutations, 1 with OPA1 mutation, and 10 with indefinite causes. Twelve ears (27%) showed no change in DPOAE, 20 ears (46%) showed a decrease in DPOAE, and 12 ears (27%) lost DPOAE. Loss of DPOAE occurred in one ear (2%) at 0 years of age and four ears (9%) at 1 year of age. The time courses of DPOAEs in patients with OTOF mutations were divided into those with early loss and those with no change, indicating that the mechanism for deterioration of DPOAEs includes not only the OTOF mutations but also other common modifier factors. Most, but not all, AN patients who used hearing aids showed deterioration of DPOAEs after the start of using hearing aids. A few AN patients also showed deterioration of DPOAEs before using hearing aids. The adult AN group comprised 2 patients with OPA1 mutations, 2 with OTOF mutations, and 5 with indefinite causes. Four ears (22%) showed no change in DPOAE, 13 ears (72%) showed a decrease, and one ear (6%) showed a loss of DPOAE. Although the ratio of DPOAE decrease was higher in the adult AN group than in the pediatric AN group, the ratio of DPOAE loss was lower in the adult AN group. DPOAE was not lost in all four ears with OPA1 mutations and in all four ears with OTOF mutations in the adult group. CONCLUSIONS: DPOAE was decreased or lost in approximately 70% of pediatric and about 80% of adult AN patients. Eleven percent of pediatric AN patients lost DPOAEs by 1 year of age. Genetic factors were thought to have influenced the time course of DPOAEs in the pediatric AN group. In most adult AN patients, DPOAE was rarely lost regardless of the genetic cause.

Effect of anthocyanin-rich bilberry extract on bone metabolism in ovariectomized rats.

Menopause is associated with increased oxidative stress, which serves a role, in part, in the pathogenesis of postmenopausal bone loss. Fruits and vegetables are rich in antioxidative nutrients and phytochemicals. Berries are a natural source of anthocyanins, and their intake may improve bone health. The aim of the present study was to determine the effect of an anthocyanin-rich bilberry extract (VME) on bone metabolism in an ovariectomized (Ovx) rat. Female Sprague-Dawley rats (12 weeks old) were randomly divided into the following four groups: Baseline, Sham, Ovx and Ovx+VME (n=8-12 rats per group). Rats in the Baseline group were sacrificed immediately, while those in the other groups were subjected to either sham operation (Sham) or bilateral Ovx (Ovx and Ovx+VME). Rats in the Ovx+VME group were administered VME daily at a dose of 500 mg/kg body weight. At 8 weeks after surgery, bone mass and bone histomorphometry were evaluated. The femur bone mineral density (BMD) in the Ovx group was significantly lower than that in the Sham group (P<0.01). Supplementation of VME in the Ovx rats did not result in an increase in BMD. Histomorphometric analyses revealed that Ovx resulted in decreased measures of bone volume and trabecular number and increased measures of osteoid volume, mineralizing surface and bone formation rates (all P<0.01), whereas VME had no significant effects on these parameters. The present findings indicate that VME did not alter bone metabolism in Ovx rats, suggesting that consumption of VME may not be helpful in preventing postmenopausal bone loss.

Prevalence of TECTA mutation in patients with mid-frequency sensorineural hearing loss.

BACKGROUND: To date, 102 genes have been reported as responsible for non-syndromic hearing loss, some of which are associated with specific audiogram features. Four genes have been reported as causative for mid-frequency sensorineural hearing loss (MFSNHL), among which TECTA is the most frequently reported; however, the prevalence of TECTA mutations is unknown. To elucidate the prevalence of TECTA mutation in MFSNHL and clarify genotype-phenotype correlations, we analyzed the genetic and clinical features of patients with MFSNHL. METHODS: Subjects with bilateral non-syndromic hearing loss were prescreened for GJB2 and m.1555A > G and m.3243A > G mitochondrial DNA mutations, and patients with inner ear malformations were excluded. We selected MFSNHL patients whose audiograms met the U-shaped criterion proposed by the GENDEAF study group, along with those with shallow U-shaped audiograms, for TECTA analysis. All TECTA exons were analyzed by Sanger sequencing. Novel missense variants were classified as possibly pathogenic, non-pathogenic, and variants of uncertain significance, based on genetic data. To evaluate novel possibly pathogenic variants, we predicted changes in protein structure by molecular modeling. RESULTS: Pathogenic and possibly pathogenic variants of TECTA were found in 4 (6.0%) of 67 patients with MFSNHL. In patients with U-shaped audiograms, none (0%) of 21 had pathogenic or possibly pathogenic variants. In patients with shallow U-shaped audiograms, four (8.7%) of 46 had pathogenic or possibly pathogenic variants. Two novel possibly pathogenic variants were identified and two previously reported mutations were considered as variant of unknown significance. The clinical features of patients with pathogenic and possibly pathogenic variants were consistent with those in previous studies. Pathogenic or possibly pathogenic variants were identified in 3 of 23 families (13.0%) which have the family histories compatible with autosomal dominant and 1 of 44 families (2.3%) which have the family histories compatible with sporadic or autosomal recessive. CONCLUSIONS: TECTA mutations were identified in 6.0% of MFSNHL. These mutations were more frequent in patients with shallow U-shaped audiograms than those with U-shaped audiograms, and in families which have the family histories compatible with autosomal dominant than those with the family histories compatible with sporadic or autosomal recessive.

Frequency and specific characteristics of the incomplete partition type III anomaly in children.

OBJECTIVES/HYPOTHESIS: To determine the frequency of the incomplete partition type III anomaly and the genetic and clinical features associated with POU3F4 mutations in children with hearing loss. STUDY DESIGN: Retrospective case series from 2000 to 2014 at the National Hospital Organization Tokyo Medical Center and collaborating hospitals. METHODS: A total of 1,004 patients (from 938 families) who had hearing loss by 10 years of age and had undergone computed tomography scanning of their temporal bones were enrolled in this genetic, clinical, and radiological study. RESULTS: The incomplete partition type III anomaly was identified in six patients (0.6%), each of whom had an enlargement of the vestibular aqueduct at the end close to the vestibule. The six patients also had POU3F4 variants, and a genetic analysis revealed frameshift deletions in three patients, a missense variant in two patients of the same family, and a large deletion in one patient. Three of the six patients with POU3F4 variants were sporadic cases, and in one patient the genetic mutation occurred de novo. CONCLUSIONS: It was indicated that POU3F4 mutations can be predicted by incomplete partition type III anomaly by radiological examination of the inner ear. All six of the patients showed mixed hearing loss, but none showed fluctuations in hearing, which may be related to the lack of vestibular aqueduct enlargement at the operculum. LEVEL OF EVIDENCE: 4 Laryngoscope, 127:1663-1669, 2017.

Quality control of photosystem II: direct imaging of the changes in the thylakoid structure and distribution of FtsH proteases in spinach chloroplasts under light stress.

Under light stress, the reaction center-binding protein D1 of PSII is photo-oxidatively damaged and removed from PSII complexes by proteases located in the chloroplast. A protease considered to be responsible for degradation of the damaged D1 protein is the metalloprotease FtsH. We showed previously that the active hexameric FtsH protease is abundant at the grana margin and the grana end membranes, and this homo-complex removes the photodamaged D1 protein in the grana. Here, we showed a change in the distribution of FtsH in spinach thylakoids during excessive illumination by transmission electron microscopy (TEM) and immunogold labeling of FtsH. The change in distribution of the protease was accompanied by structural changes to the thylakoids, which we detected using spinach leaves by TEM after chemical fixation of the samples. Quantitative analyses showed several characteristic changes in the structure of the thylakoids, including shrinkage of the grana, outward bending of the marginal portions of the thylakoids and an increase in the height of the grana stacks under excessive illumination. The increase in the height of the grana stacks may include swelling of the thylakoids and an increase in the partition gaps between the thylakoids. These data strongly suggest that excessive illumination induces partial unstacking of the thylakoids, which enables FtsH to access easily the photodamaged D1 protein. Finally three-dimensional tomography of the grana was recorded to observe the effect of light stress on the overall structure of the thylakoids.

Quality control of PSII: behavior of PSII in the highly crowded grana thylakoids under excessive light.

The grana thylakoids of higher plant chloroplasts are crowded with PSII and the associated light-harvesting complexes (LHCIIs). They constitute supercomplexes, and often form semi-crystalline arrays in the grana. The crowded condition of the grana may be necessary for efficient trapping of excitation energy by LHCII under weak light, but it might hinder proper movement of LHCII necessary for reversible aggregation of LHCII in the energy-dependent quenching of Chl fluorescence under moderate high light. When the thylakoids are illuminated with extreme high light, the reaction center-binding D1 protein of PSII is photodamaged, and the damaged protein migrates to the grana margins for degradation and subsequent repair. In both moderate and extreme high-light conditions, fluidity of the thylakoid membrane is crucial. In this review, we first provide an overview of photoprotective processes, then discuss changes in membrane fluidity and mobility of the protein complexes in the grana under excessive light, which are closely associated with photoprotection of PSII. We hypothesize that reversible aggregation of LHCII, which is necessary to avoid light stress under moderate high light, and swift turnover of the photodamaged D1 protein under extreme high light are threatened by irreversible protein aggregation induced by reactive oxygen species in photochemical reactions.

Quality control of Photosystem II: reversible and irreversible protein aggregation decides the fate of Photosystem II under excessive illumination.

In response to excessive light, the thylakoid membranes of higher plant chloroplasts show dynamic changes including the degradation and reassembly of proteins, a change in the distribution of proteins, and large-scale structural changes such as unstacking of the grana. Here, we examined the aggregation of light-harvesting chlorophyll-protein complexes and Photosystem II core subunits of spinach thylakoid membranes under light stress with 77K chlorophyll fluorescence; aggregation of these proteins was found to proceed with increasing light intensity. Measurement of changes in the fluidity of thylakoid membranes with fluorescence polarization of diphenylhexatriene showed that membrane fluidity increased at a light intensity of 500-1,000 µmol photons m(-) (2) s(-) (1), and decreased at very high light intensity (1,500 µmol photons m(-) (2) s(-) (1)). The aggregation of light-harvesting complexes at moderately high light intensity is known to be reversible, while that of Photosystem II core subunits at extremely high light intensity is irreversible. It is likely that the reversibility of protein aggregation is closely related to membrane fluidity: increases in fluidity should stimulate reversible protein aggregation, whereas irreversible protein aggregation might decrease membrane fluidity. When spinach leaves were pre-illuminated with moderately high light intensity, the qE component of non-photochemical quenching and the optimum quantum yield of Photosystem II increased, indicating that Photosystem II/light-harvesting complexes rearranged in the thylakoid membranes to optimize Photosystem II activity. Transmission electron microscopy revealed that the thylakoids underwent partial unstacking under these light stress conditions. Thus, protein aggregation is involved in thylakoid dynamics and regulates photochemical reactions, thereby deciding the fate of Photosystem II.

GJB2-associated hearing loss undetected by hearing screening of newborns.

The hearing loss caused by GJB2 mutations is usually congenital in onset, moderate to profound in degree, and non-progressive. The objective of this study was to study genotype/phenotype correlations and to document 14 children with biallelic GJB2 mutations who passed newborn hearing screening (NHS). Genetic testing for GJB2 mutations by direct sequencing was performed on 924 individuals (810 families) with hearing loss, and 204 patients (175 families) were found to carry biallelic GJB2 mutations. NHS results were obtained through medical records. A total of 18 pathological mutations were identified, which were subclassified as eight inactivating and 10 non-inactivating mutations. p.I128M and p.H73Y were identified as novel missense GJB2 mutations. Of the 14 children with biallelic GJB2 mutations who passed NHS, eight were compound heterozygotes and 3 were homozygous for the c.235delC mutation in GJB2, and the other three combinations of non-c.235delC mutations identified were p.Y136X-p.G45E/p.V37I heterozygous, c.512ins4/p.R143W heterozygous, and p.V37I/p.R143W heterozygous. These 14 cases demonstrate that the current NHS does not identify all infants with biallelic GJB2 mutations. They suggest that the frequency of non-penetrance at birth is approximately 6.9% or higher in DFNB1 patients and provide further evidence that GJB2 hearing loss may not always be congenital in onset.

Genetic analysis of PAX3 for diagnosis of Waardenburg syndrome type I.

CONCLUSION: PAX3 genetic analysis increased the diagnostic accuracy for Waardenburg syndrome type I (WS1). Analysis of the three-dimensional (3D) structure of PAX3 helped verify the pathogenicity of a missense mutation, and multiple ligation-dependent probe amplification (MLPA) analysis of PAX3 increased the sensitivity of genetic diagnosis in patients with WS1. OBJECTIVES: Clinical diagnosis of WS1 is often difficult in individual patients with isolated, mild, or non-specific symptoms. The objective of the present study was to facilitate the accurate diagnosis of WS1 through genetic analysis of PAX3 and to expand the spectrum of known PAX3 mutations. METHODS: In two Japanese families with WS1, we conducted a clinical evaluation of symptoms and genetic analysis, which involved direct sequencing, MLPA analysis, quantitative PCR of PAX3, and analysis of the predicted 3D structure of PAX3. The normal-hearing control group comprised 92 subjects who had normal hearing according to pure tone audiometry. RESULTS: In one family, direct sequencing of PAX3 identified a heterozygous mutation, p.I59F. Analysis of PAX3 3D structures indicated that this mutation distorted the DNA-binding site of PAX3. In the other family, MLPA analysis and subsequent quantitative PCR detected a large, heterozygous deletion spanning 1759-2554 kb that eliminated 12-18 genes including a whole PAX3 gene.

Quality control of photosystem II: lipid peroxidation accelerates photoinhibition under excessive illumination.

Environmental stresses lower the efficiency of photosynthesis and sometimes cause irreversible damage to plant functions. When spinach thylakoids and Photosystem II membranes were illuminated with excessive visible light (100-1,000 µmol photons m(-1) s(-1)) for 10 min at either 20°C or 30°C, the optimum quantum yield of Photosystem II decreased as the light intensity and temperature increased. Reactive oxygen species and endogenous cationic radicals produced through a photochemical reaction at and/or near the reaction center have been implicated in the damage to the D1 protein. Here we present evidence that lipid peroxidation induced by the illumination is involved in the damage to the D1 protein and the subunits of the light-harvesting complex of Photosystem II. This is reasoned from the results that considerable lipid peroxidation occurred in the thylakoids in the light, and that lipoxygenase externally added in the dark induced inhibition of Photosystem II activity in the thylakoids, production of singlet oxygen, which was monitored by electron paramagnetic resonance spin trapping, and damage to the D1 protein, in parallel with lipid peroxidation. Modification of the subunits of the light-harvesting complex of Photosystem II by malondialdehyde as well as oxidation of the subunits was also observed. We suggest that mainly singlet oxygen formed through lipid peroxidation under light stress participates in damaging the Photosystem II subunits.

Assay of photoinhibition and heat inhibition of photosystem II in higher plants.

When thylakoids of higher plant chloroplasts are exposed to excessive light or moderate heat stress, photosystem II reaction center-binding protein D1 is damaged. The photodamage of the D1 protein is caused by reactive oxygen species, mostly singlet oxygen, and also by endogenous cationic radicals generated by the photochemical reactions of photosystem II. Moreover, it was shown recently that the damage to the D1 protein by moderate heat stress is due to reactive oxygen species produced by lipid peroxidation near photosystem II. To maintain photosystem II activity, the oxidatively damaged D1 protein must be replaced by a newly synthesized copy, and thus degradation and removal of the photo- or heat-damaged D1 protein are essential for maintaining the viability of photosystem II. In this chapter, we describe the methods for assaying photoinhibition and heat inhibition of photosystem II in higher plant materials.

Quality control of photosystem II: FtsH hexamers are localized near photosystem II at grana for the swift repair of damage.

The reaction center-binding D1 protein of Photosystem II is oxidatively damaged by excessive visible light or moderate heat stress. The metalloprotease FtsH has been suggested as responsible for the degradation of the D1 protein. We have analyzed the distribution and subunit structures of FtsH in spinach thylakoids and various membrane fractions derived from the thylakoids using clear native polyacrylamide gel electrophoresis and Western blot analysis. FtsH was found not only in the stroma thylakoids but also in the Photosystem II-enriched grana membranes. Monomeric, dimeric, and hexameric FtsH proteases were present as major subunit structures in thylakoids, whereas only hexameric FtsH proteases were detected in Triton X-100-solubilized Photosystem II membranes. Importantly, among the membrane fractions examined, hexameric FtsH proteases were most abundant in the Photosystem II membranes. In accordance with this finding, D1 degradation took place in the Photosystem II membranes under light stress. Sucrose density gradient centrifugation analysis of thylakoids and the Photosystem II membranes solubilized with n-dodecyl-ß-d-maltoside and a chemical cross-linking study of thylakoids showed localization of FtsH near the Photosystem II light-harvesting chlorophyll-protein supercomplexes in the grana. These results suggest that part of the FtsH hexamers are juxtapositioned to PSII complexes in the grana in darkness, carrying out immediate degradation of the photodamaged D1 protein under light stress.

Quality control of photosystem II: Thylakoid unstacking is necessary to avoid further damage to the D1 protein and to facilitate D1 degradation under light stress in spinach thylakoids.

Photosystem II is vulnerable to light damage. The reaction center-binding D1 protein is impaired during excessive illumination and is degraded and removed from photosystem II. Using isolated spinach thylakoids, we investigated the relationship between light-induced unstacking of thylakoids and damage to the D1 protein. Under light stress, thylakoids were expected to become unstacked so that the photodamaged photosystem II complexes in the grana and the proteases could move on the thylakoids for repair. Excessive light induced irreversible unstacking of thylakoids. By comparing the effects of light stress on stacked and unstacked thylakoids, photoinhibition of photosystem II was found to be more prominent in stacked thylakoids than in unstacked thylakoids. In accordance with this finding, EPR spin trapping measurements demonstrated higher production of hydroxyl radicals in stacked thylakoids than in unstacked thylakoids. We propose that unstacking of thylakoids has a crucial role in avoiding further damage to the D1 protein and facilitating degradation of the photodamaged D1 protein under light stress.