Search

Photosynthetic light reactions establish electron flow in the chloroplast's thylakoid membranes, leading to the production of the ATP and NADPH that participate in carbon fixation. Two modes of electron flow exist-linear electron flow (LEF) from water to NADP(+) via photosystem (PS) II and PSI in series and cyclic electron flow (CEF) around PSI (ref. 2). Although CEF is essential for satisfying the varying demand for ATP, the exact molecule(s) and operational site are as yet unclear. In the green alga Chlamydomonas reinhardtii, the electron flow shifts from LEF to CEF on preferential excitation of PSII (ref. 3), which is brought about by an energy balancing mechanism between PSII and PSI (state transitions). Here, we isolated a protein supercomplex composed of PSI with its own light-harvesting complex (LHCI), the PSII light-harvesting complex (LHCII), the cytochrome b(6)f complex (Cyt bf), ferredoxin (Fd)-NADPH oxidoreductase (FNR), and the integral membrane protein PGRL1 (ref. 5) from C. reinhardtii cells under PSII-favouring conditions. Spectroscopic analyses indicated that on illumination, reducing equivalents from downstream of PSI were transferred to Cyt bf, whereas oxidised PSI was re-reduced by reducing equivalents from Cyt bf, indicating that this supercomplex is engaged in CEF (Supplementary Fig. 1). Thus, formation and dissociation of the PSI-LHCI-LHCII-FNR-Cyt bf-PGRL1 supercomplex not only controlled the energy balance of the two photosystems, but also switched the mode of photosynthetic electron flow.

Aims: Twin–twin transfusion syndrome (TTTS) complicated with absent or reversed end-diastolic flow in the umbilical artery (UA-AREDF) of the donor has a high perinatal mortality rate. To improve the prenatal outcome, we introduced and modified the technique of sequential selective laser photocoagulation of communicating vessels (SQLPCV), and assessed the clinical efficacy. Methods: The modified SQLPCV was designed with the following order of coagulation: (i) artery-to-artery (AA) anastomoses; (ii) venous-to-venous anastomoses; (iii) artery-to-venous anastomoses from donor to recipient; and (iv) artery-to-venous anastomoses from recipient to donor. TTTS patients with UA-AREDF of donors were recruited, and the perinatal outcome and its association with the types of anastomoses were compared in patients who underwent the standard selective laser method (SLPCV). Results: Twenty-three patients underwent modified SQLPCV and 29 underwent SLPCV. Total intrauterine fetal death (IUFD) was significantly lower in modified SQLPCV than in SLPCV (9% vs 38%; P

Objective To validate the Quintero stage III subclassification for twin–twin transfusion syndrome (TTTS) based on visibility of the bladder of the donor twin. Methods Between July 2002 and August 2006, there were 131 pregnant Japanese women affected by severe TTTS before 26 weeks' gestation, treated with fetoscopic laser surgery at five centers in Japan, whose pregnancies continued beyond 22 weeks. Outcome data were available in all cases and surviving infants were followed up for at least 6 years. This study focused on the Stage III TTTS patients. These were subclassified into Stage III atypical (abnormal Doppler flow with visible donor bladder) and Stage III classical (abnormal Doppler flow with non-visible donor bladder) groups. Perioperative data and postnatal outcomes were compared between the groups. Results Seven Stage I, 22 Stage II, 82 Stage III and 20 Stage IV pregnancies continued beyond 22 weeks. There was a significantly higher incidence of absent or reversed end-diastolic velocity in the umbilical artery (UA-AREDV) of the donor in Stage III atypical than in Stage III classical patients (83.8% vs. 53.3%, P = 0.004). Stage III atypical cases also had a significantly higher incidence of arterioarterial (AA) anastomoses (72.9% vs. 17.8%, P < 0.001) and intrauterine fetal demise (IUFD) of the donor (43.2% vs. 13.3%, P = 0.002). However, there were no differences in overall survival or in abnormal brain scans of surviving infants. Donors with both UA-AREDV and AA anastomoses had a significantly higher incidence of IUFD compared with the others (53.3%, P < 0.001). Conclusions Quintero stage III atypical was characterized by a high incidence of AA anastomoses and UA-AREDV of the donor, resulting in IUFD. Subclassification of Stage III based on visibility of the bladder of the donor twin was adequate for and compatible with differentiating prognosis and pathophysiology. Copyright © 2008 ISUOG. Published by John Wiley & Sons, Ltd.

De-etiolation of dark-grown seedlings is a commonly used experimental system to study the mechanisms of chloroplast biogenesis, including the stacking of thylakoid membranes into grana, the response of the nuclear-chloroplast transcriptome to light, and the ordered synthesis and assembly of photosystem II (PSII). Here, we present the xeroplast to chloroplast transition during rehydration of the resurrection plant Xerophyta humilis as a novel system for studying chloroplast biogenesis, and investigate the role of light in this process. Xeroplasts are characterized by the presence of numerous large and small membrane-bound vesicles and the complete absence of thylakoid membranes. While the initial assembly of stromal thylakoid membranes occurs independently of light, the formation of grana is light dependent. Recovery of photosynthetic activity is rapid in plants rehydrated in the light and correlates with the light-dependent synthesis of the D1 protein, but does not require de novo chlorophyll biosynthesis. Light-dependent synthesis of the chlorophyll-binding protein Lhcb2 and digalactosyldiacylglycerol synthase 1 correlated with the formation of grana and with the increased PSII activity. Our results suggest that the molecular mechanisms underlying photomorphogenic development may also function in desiccation tolerance in poikilochlorophyllous resurrection plants.

Chylothorax is a relatively uncommon condition defined as an abnormal collection of lymphatic fluid within the pleural space. We are reporting the use of OK-432 for treatment of prolonged idiopathic congenital chylothorax in 2 newborn infants who failed to respond to conservative medical therapy, including octreotide injection.

State transitions, or the redistribution of light-harvesting complex II (LHCII) proteins between photosystem I (PSI) and photosystem II (PSII), balance the light-harvesting capacity of the two photosystems to optimize the efficiency of photosynthesis. Studies on the migration of LHCII proteins have focused primarily on their reassociation with PSI, but the molecular details on their dissociation from PSII have not been clear. Here, we compare the polypeptide composition, supramolecular organization, and phosphorylation of PSII complexes under PSI- and PSII-favoring conditions (State 1 and State 2, respectively). Three PSII fractions, a PSII core complex, a PSII supercomplex, and a multimer of PSII supercomplex or PSII megacomplex, were obtained from a transformant of the green alga Chlamydomonas reinhardtii carrying a His-tagged CP47. Gel filtration and single particles on electron micrographs showed that the megacomplex was predominant in State 1, whereas the core complex was predominant in State 2, indicating that LHCIIs are dissociated from PSII upon state transition. Moreover, in State 2, strongly phosphorylated LHCII type I was found in the supercomplex but not in the megacomplex. Phosphorylated minor LHCIIs (CP26 and CP29) were found only in the unbound form. The PSII subunits were most phosphorylated in the core complex. Based on these observations, we propose a model for PSII remodeling during state transitions, which involves division of the megacomplex into supercomplexes, triggered by phosphorylation of LHCII type I, followed by LHCII undocking from the supercomplex, triggered by phosphorylation of minor LHCIIs and PSII core subunits.

We report a simple and rapid protocol for large-scale purification of supercomplex consisting of photosystem I (PSI) core and light-harvesting chlorophyll a/b complexes I (LHCI) from the green alga Chlamydomonas reinhardtii. The thylakoid extracts were separated by sucrose density gradient ultracentrifugation and were subsequently purified with anion exchange column chromatography. The purified PSI-LHCI supercomplex contained eleven PSI (PsaA, PsaB, PsaC, PsaD, PsaE, PsaF, PsaG, PsaH, PsaI, PsaJ, PsaK, PsaL) and nine LHCI polypeptides, but lacked three PSI polypeptides, PsaN, PsaO and PsaP. We also tried to crystallize PSI-LHCI and were able to obtain small crystals, which showed X-ray diffraction of 10.3 A thus far. It still needs to improve the purity of the PSI-LHCI preparation and search for optimal crystallization conditions before we will be able to start analysis of crystal structures.

PsbT is a small chloroplast-encoded hydrophobic polypeptide associated with the D1/D2 heterodimer of the photosystem II (PSII) reaction center and is required for the efficient post-translational repair of photodamaged PSII. Here we addressed that role in detail in Chlamydomonas reinhardtii wild type and DeltapsbT cells by analyzing the activities of PSII, the assembly of PSII proteins, and the redox components of PSII during photoinhibition and repair. Strong illumination of cells for 15 min decreased the activities of electron transfer through PSII and Q(A) photoreduction by 50%, and it reduced the amount of atomic manganese by 20%, but it did not affect the steady-state level of PSII proteins, photoreduction of pheophytin (pheo(D1)), and the amount of bound plastoquinone (Q(A)), indicating that the decrease in PSII activity resulted mainly from inhibition of the electron transfer from pheo(D1) to Q(A). In wild type cells, we observed parallel recovery of electron transfer activity through PSII and Q(A) photoreduction, suggesting that the recovery of Q(A) activity is one of the rate-limiting steps of PSII repair. In DeltapsbT cells, the repairs of electron transfer activity through PSII and of Q(A) photoreduction activity were both impaired, but PSII protein turnover was unaffected. Moreover, about half the Q(A) was lost from the PSII core complex during purification. Since PsbT is intimately associated with the Q(A)-binding region on D2, we propose that this polypeptide enhances the efficient recovery of Q(A) photoreduction by stabilizing the structure of the Q(A)-binding region.

State transition in photosynthesis is a short-term balancing mechanism of energy distribution between photosystem I (PSI) and photosystem II (PSII). When PSII is preferentially excited (state 2), a pool of mobile light-harvesting complex II (LHCII) antenna proteins is thought to migrate from PSII to PSI, but biochemical evidence for a physical association between LHCII proteins and PSI in state 2 is weak. Here, using the green alga Chlamydomonas reinhardtii , which has a high capacity for state transitions, we report the isolation of PSI-light-harvesting complex I (LHCI) super-complexes from cells locked into state 1 and state 2. We solubilized the thylakoid membranes with a mild detergent, separated the proteins by sucrose density gradient centrifugation, and subjected gradient fractions to gel-filtration chromatography. Three LHCII polypeptides were associated with a PSI-LHCI supercomplex only in state 2; we identified them as two minor monomeric LHCII proteins (CP26 and CP29) and one previously unreported major LHCII protein type II, or LhcbM5. These three LHCII proteins, in addition to the major trimeric LHCII proteins, were phosphorylated upon transition to state 2. The corresponding phylogenetic tree indicates that among the LHCII proteins associated with PSII, these three LHCII proteins are the most similar to the LHC proteins for PSI (LHCI). Our results are important because CP26, CP29, and LhcbM5, which have been viewed as belonging solely to the PSII complex, are now postulated to shuttle between PSI and PSII during state transitions, thereby acting as docking sites for the trimeric LHCII proteins in both PSI and PSII.

OBJECTIVES: to evaluate the impact of laser therapy on inter-twin discordance in twin-to-twin transfusion syndrome (TTTS). METHODS: biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), femur length (FL) and estimated fetal weight were prospectively collected during a five-year period (1999 to 2004). The inter-twin discordance was expressed as a percentage of the largest twin's measurements. The measurements were made the day before laser, twice following laser and after delivery. The mean values of discordance in measurements and in fetal weight were calculated. ANOVA was used to compare mean values. RESULTS: the mean (SD) discordance for BPD, HC, AC, FL and estimated fetal weight the day before laser were 8.53% (5.28), 8.75% (2.76), 16.19% (4.85), 12.92% (5.13) and 28.50% (6.46) respectively. At the at 2nd ultrasound assessment after surgery were 4.37% (3.55), 3.73% (2.71), 8.90% (4.42), 6.61% (4.99) and 19.11% (8.01) respectively; and at birth the weight discordance was 18.55% (8.74). There was a significant decrease in discordance for HC and AC for each ultrasound assessment. CONCLUSIONS: there was a decrease in fetal growth discordance following laser therapy in TTTS. These changes might be related to re-adaptation of blood flow following laser therapy. OBJETIVOS: avaliar impacto da terapia a laser no crescimento fetal na Sindrome de transfusão feto-fetal (STFF). MÉTODOS: diâmetro biparietal (DBP), circunferência cefálica (CC) e abdominal (CA), comprimento do fêmur (CF) e peso fetal foram colhidos prospectivamente no período de cinco anos (1999 a 2004). A discordância entre os gêmeos foi expressa como porcentagem da medida do maior. As medidas foram feitas um dia antes do laser, duas vezes após e depois do nascimento. A cada exame e pós-parto foram calculadas médias das discordâncias entre medidas e peso fetal. ANOVA foi usada para comparar as médias. RESULTADOS: a discordância média (SD) para DBP, CC, CA, CF e peso fetal um dia antes do laser foi 8,53% (5,28), 8,75% (2,76), 16,19% (4,85), 12,92% (5,13) e 28,50% (6,46). No segundo exame pós-laser os resultados foram 4,37% (3,55), 3,73% (2,71), 8,90% (4,42), 6,61% (4,99) e 19,11% (8,01); e a discordância do peso fetal ao nascimento foi 18,55% (8,74). Houve diminuição significativa na discordância para CC e CA em cada exame. CONCLUSÕES: houve redução significativa na discordância do crescimento fetal após o laser na STFF. Estas alterações podem estar relacionadas à readaptação dos fluxos sangüíneos após tratamento.

Objective. The purpose of this study was to evaluate fetal growth patterns after laser therapy in twin-to-twin transfusion syndrome (TTTS). Methods. All cases with delivery after 24 weeks from pregnancies complicated by TITS treated by laser therapy between January 1999 and September 2004 were included. Fetal biometric parameters and estimated fetal weight were measured by sonography at least twice before laser therapy and twice after laser therapy. The interval between sonographic measurements was at least 2 weeks. The study population was divided into group I, which consisted of pregnancies with 2 liveborn neonates, and group II, which consisted of pregnancies resulting in only 1 liveborn neonate. The z scores for biometric measurements were calculated in all cases accordingly and compared for each twin across gestational age by analysis of variance and between groups by a t test. In group I, the intertwin discordance was also assessed, and analysis of variance was used to compare mean values across gestational age. Results. Ninety-eight cases with 51 single and 47 double liveborn neonates were included. Across time, z scores of the recipients significantly decreased, whereas there was no change in z scores of the donors. The z scores were not statistically different between groups. There was a significant decrease in fetal discordance after laser therapy in group I. Conclusions. The fetal growth pattern in TITS can change after laser treatment. There is a slowdown in the recipient growth and maintained growth in the donor twin. Both mechanisms lead to a decrease in intertwin discordance.

The use of ultrasonography (USG) during pregnancy provides a tremendous amount of valuable information about fetal and maternal well-being. However, in some cases the image quality may be poor due to maternal fat tissue, fetal position, or some other reason. Magnetic resonance imaging (MRI) can provide clear images from multiple angles without X-ray exposure. We believe that it is important to evaluate the usefulness of MRI for diagnostic purposes during pregnancy. While MRI can provide different information to what can be obtained from USG, it is not always superior to USG. The clinical indications for MRI during pregnancy are limited but well defined. Fetal MRI is a valuable complement to USG especially in the further evaluation of problems first detected by USG. When MRI is performed, we must consider its purpose and accordingly determine the appropriate procedure to provide the most precise and useful diagnostic information.

Arabidopsis yellow variegated1 (VAR1) and VAR2 are separate loci that encode similar chloroplast FtsH proteases. To date, FtsH is the best-characterized protease in thylakoid membranes involved in the turnover of photosynthetic protein complexes. It comprises a protein family that is encoded by 12 different nuclear genes in Arabidopsis. We show here that nine FtsH proteins are located in the chloroplasts. Mutations in either VAR1 or VAR2 cause typical leaf variegation and sensitivity to photoinhibition. By contrast, none of these phenotypes was observed in T-DNA insertion mutants in other ftsH genes (ftsh1, ftsh6, and ftsh8) closely related to VAR1 and VAR2. This finding suggests that VAR1 and VAR2 play a predominant role in the photosystem II repair cycle in thylakoid membranes. By generating VAR1- and VAR2-specific antibodies, we found that loss of either VAR1 or VAR2 results in the decreased accumulation of the other. Thus, the genetic nonredundancy between VAR1 and VAR2 could be attributed to their coordinated regulation at the protein level. These observations led us to examine whether VAR1 and VAR2 form a complex. Sucrose density gradient and gel filtration analyses revealed a complex of approximately 400 to 450 kD, probably representing a hexamer. Furthermore, VAR1 and VAR2 were shown to coprecipitate by immunoprecipitation using VAR1- and VAR2-specific antibodies. The majority of VAR1 appears to exist as heterocomplexes with VAR2, whereas VAR2 may be present as homocomplexes as well. Based on these results, we conclude that VAR1 and VAR2 are the major components of an FtsH complex involved in the repair of photodamaged proteins in thylakoid membranes.

To investigate the relationship between fetoplacental leptin secretion and blood gases.We measured the levels of umbilical arterial and venous leptin, umbilical cord gas, umbilical venous blood glucose, and estradiol-17beta (E2) in 89 pregnant women. Correlation between the leptin levels and other variables (gestational age, birth weight, maternal body weight, height, body mass index, maternal body weight gain, placental weight, umbilical cord gas data, and levels of umbilical venous blood glucose and E2) were examined statistically.Umbilical arterial and venous leptin levels were 7.64 +/- 12.76 and 7.76 +/- 13.17 (ng/mL), respectively, correlating positively with carbon dioxide pressure levels (r = 0.446, P.001; r = 0.406, P.001, respectively) and correlating inversely with pH (r = -0.337, P =.001; r = -0.247, P =.019, respectively). Umbilical venous glucose, E2, and other factors did not correlate with leptin levels.Leptin secretion into the fetoplacental circulation may be associated with fetal hypercapnia, suggesting two important roles for leptin: one for basal control of fetal fat tissue and one as an acute stress-related hormone.

Objective. The dual function of β-catenin (e.g., as an intermediate protein between adherence junctions and the microfilaments, and as a mediator of the Wnt signaling pathway) is currently known. Stabilization of β-catenin and subsequent activation of the Wnt signaling pathway are involved in the development of some malignancies. We analyzed the immunohistochemical localization of β-catenin and the somatic mutation of exon 3 of the β-catenin gene in the malignant phenotype of the uterine cervix. Methods. Immunohistochemical localization of β-catenin and mutation of exon 3 of the β-catenin gene were analyzed in 38 precancerous lesions and 43 cancerous lesions. Results. In normal cervix, β-catenin was observed around the plasma membrane of the cells in the basal and parabasal layers of the epithelium. The frequency of cytoplasmic/nuclear β-catenin expression correlated with a high histological grade of cervical intraepithelial neoplasia. Among invasive carcinomas, 11 (73%) of 15 samples showed cytoplasmic/nuclear localization to variable extents. A mutational analysis showed that mutation occurred in 7 of 68 specimens. Six cases with mutations revealed cytoplasmic/nuclear β-catenin expression, though 32 (84%) of the 38 samples showing cytoplasmic/nuclear β-catenin expression were not associated with the mutation. Conclusion. These results indicate that cytoplasmic/nuclear expression of β-catenin is associated with the malignant phenotype of the cervix, but the contribution of mutation of the β-catenin gene is limited.

The expression level of the human telomerase reverse transcriptase (hTERT) is a rate-limiting determinant of telomerase activity. Several alternatively spliced variants of hTERT transcript are currently known. We have studied the expression of the splicing variants arising in the transcript encoding the reverse transcriptase domain, and have compared this to the telomerase activity in 27 endometria, 14 myometria and 18 endometrial carcinomas. Telomerase activity and the full-length hTERT transcript were observed in endometrial samples from the proliferative and early secretory phases, but not in those from the late secretory phase. Steady-state expression of the hTERT splicing variant entirely lacking exon 7 and exon 8 was observed in the endometria throughout the menstrual cycle. In the analysed myometria, this type of splicing variant was the most commonly detected, and telomerase activity occurred in only three samples. In both endometria and myometria, the expression of the full-length transcript correlated well with the telomerase activity. In each of the endometrial carcinomas, telomerase activity was detected and the full-length transcript was found together with varying combinations of deletion splicing variants. These results suggest that regulation of splicing in the transcript encoding the hTERT reverse transcriptase domain is associated with telomerase activation in uterine tissues.

PsbT is a small chloroplast-encoded hydrophobic polypeptide associated with the photosystem II (PSII) core complex. A psbT-deficient mutant (Delta psbT) of the green alga Chlamydomonas reinhardtii grows photoautotrophically, whereas its growth is significantly impaired in strong light. To understand the photosensitivity of Delta psbT, we have studied the effect of strong illumination on PSII activity and proteins. It is shown that the level of PSII activity and proteins is reduced in the Delta psbT more significantly than in wild type under strong light. When recovery of the photodamaged PSII is inhibited by a chloroplast protein synthesis inhibitor, the light-induced inactivation and degradation of PSII occur similarly in wild-type and mutant cells. On the contrary, the recovery of PSII activity after partial photoinactivation is remarkably delayed in the Delta psbT cells, suggesting that PsbT is required for efficient recovery of the photodamaged PSII complex. These results therefore present the first evidence for involvement of this small PSII polypeptide in the recovery process. Partial disintegration of the purified PSII core complex and localization of PSII proteins in the resulting PSII subcore complexes have revealed that PsbT is associated with D1/D2 heterodimer. A possible role of PsbT in the recovery process is discussed.

To study the function of the carboxyl-terminal domain of a photosystem II (PSII) reaction center polypeptide, D1, chloroplast mutants of the green alga Chlamydomonas reinhardtii have been generated in which Leu-343 and Ala-344 have been simultaneously or individually replaced by Phe and Ser, respectively. The mutants carrying these replacements individually, L343F and A344S, showed a wild-type phenotype. In contrast, the double mutant, L343FA344S, evolved O 2 at only 20–30% of the wild-type rate and was unable to grow photosynthetically. In this mutant, PSII accumulated to 60% of the wild-type level, indicating that the O 2 -evolving activity per PSII was reduced to approximately half that of the wild-type. However, the amount of Mn atom detected in the thylakoids suggested that a normal amount of Mn cluster was assembled. An investigation of the kinetics of flash-induced fluorescence yield decay revealed that the electron transfer from Q − A to Q B was not affected. When a back electron transfer from Q − A to a donor component was measured in the presence of 3-(3,4-dichlorophenol)-1,1-dimethylurea, a significantly slower component of the Q − A oxidation was detected in addition to the normal component that corresponds to the back electron transfer from the Q − A to the S 2 -state of the Mn cluster. Thermoluminescence measurements revealed that L343FA344S cells contained two functionally distinct Mn clusters. One was equivalent to that of the wild-type, while the other was incapable of water oxidation and was able to advance the transition from the S 1 -state to the S 2 -state. These results suggested that a fraction of the Mn cluster had been impaired by the L343FA344S mutation, leading to decreased O 2 evolution. We concluded that the structure of the C-terminus of D1 is critical for the formation of the Mn cluster that is capable of water oxidation, in particular, transition to higher S-states.

Because the expression level of hTERT, a catalytic subunit of human telomerase, is a rate-limiting determinant of telomerase activity, hTERT mRNA would be an excellent target of hammerhead ribozymes for the regulation of telomerase activity. We studied the efficiency of several hammerhead ribozymes targeting hTERT mRNA by transient and stable transfection procedures. To screen the potency of the ribozymes, transient ribozyme transfection and telomerase determination were performed. The ribozyme targeting 13 nucleotides downstream from the 5′-end of hTERT mRNA (13-ribozyme) exhibited the strongest telomerase-inhibitory activity, and the ribozyme to target 59 nucleotides upstream from the poly(A) tail showed clear activity. A stable transfection study confirmed that the 13-ribozyme suppressed telomerase. These observations suggest that the 13-ribozyme can regulate telomerase activity and may possess potential for cancer therapy.

PTEN is a tumor suppressor gene and its mutation is frequently found in endometrial carcinoma. Recently, the pseudogene of PTEN has been reported to be actively transcribed in a number of cells and tissues and a potential for translation is suggested. For further understanding of the involvement of PTEN in endometrial carcinogenesis, we analysed the expressions of PTEN and the pseudogene in 36 endometrial carcinomas with special reference to the genetic status of PTEN. Mutations of PTEN were found in 42% (15/36) of the endometrial carcinomas. The transcript of the pseudogene was expressed in 6 samples (17%) of 36 endometrial carcinomas, but in none of the normal endometria. Western blot analysis showed no translated protein of the pseudogene in any of the cases. Steady level of PTEN protein expression was observed in all the cases examined. Expression level was consistent among the proliferative endometria, secretory endometria and endometrial carcinomas as long as PTEN protein was not truncated. These results indicate that PTEN is a constitutive protein in the endometrium, so that the somatic mutation of PTEN exerts a crucial effect on the endometrial carcinogenesis. In addition, the presence of the PTEN pseudogene transcript urges us caution for the mutational analysis of PTEN as well as careful choice of the probe for the detection of PTEN transcript.

Microangiopathic thrombosis, thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS), seem to occur with certain stresses, including pregnancy. This report documents the clinical outcome with or without plasma therapy and dismal outcomes of two cases with postpartum microangiopathic thrombosis. One carried a pregnancy to successful cesarean delivery and suffered from postpartum TTP/HUS followed by plasma therapy-assisted recovery. Another developed postpartum TTP/HUS and was complicated with subarachnoid hemorrhage. Submission to plasma therapy should always be considered in a woman with postpartum microangiopathic thrombosis.

To examine the effectiveness of 22-oxacalcitriol (OCT) injection on the improvement of severe osteitis fibrosa, we studied 10 hemodialyzed patients (age, 59 +/- 12 years). The initial OCT dose was 5 microg and was administered three times weekly at the end of each hemodialysis session. OCT doses (1, 3, 5, 10, 15, and 20 microg) were changed in subsequent weeks to maintain serum calcium levels at less than 11.5 mg/dL. Administration of OCT significantly suppressed serum intact parathyroid hormone (PTH) from an initial level of 1,193 +/- 584 to 775 +/- 552 pg/mL in the 24th week (n = 10). OCT increased PTH levels again to 857 +/- 635 pg/mL in the 48th week (n = 7). Among the 10 patients, 5 patients (high responders) showed more than a 50% suppression of serum intact PTH levels at the end of the study. The rest of the patients had hypercalcemia and did not receive increased OCT doses (low responders). At the start of the treatment, the only difference between high and low responders was serum calcium level. Serum calcium levels (adjusted for serum albumin level) increased from 9.7 +/- 0.7 mg/dL (n = 10) at the beginning to 10.5 +/- 0.6 mg/dL (n = 10) in the 24th week and to 11. 1 +/- 0.7 mg/dL (n = 7) in the 48th week. Six patients (1 to 6) agreed to undergo a second bone biopsy in the 24th week of OCT administration. In bone histomorphometric measurements, OCT significantly changed bone marrow fibrosis, mineralization (labeled mineralizing surface and bone formation rate), and osteoid formation (osteoid volume and thickness). In conclusion, intravenous OCT effectively suppressed PTH secretion and improved the bone histological characteristics of severe osteitis fibrosa, especially in patients with initial serum calcium levels less than 10 mg/dL. With concerns about OCT causing adynamic bone, additional bone histological data are needed to ensure the long-term safety of OCT.

Silver staining of argyrophilic proteins of nucleolar organizer regions (Ag-NOR) has been used to evaluate a hyperactive state of cells. We studied the effects of sex steroids on the Ag-NOR proteins in the rabbit uterus. Estradiol-17beta (E2) caused uterine hypertrophy and increased the number of Ag-NOR dots in epithelial and stromal cells of the endometrium and smooth muscles cells in a dose dependent manner without proliferative change in the endometrium. Progestins including progesterone, medroxyprogesterone acetate (MPA), and norethindrone following E2 caused progestational proliferation of the endometrium with an increase in the Ag-NOR number to various extents, whereas methyltestosterone, a synthetic androgen, caused only a minimal proliferative change in the endometrium without an increase in the Ag-NOR number. Western blot analysis revealed that progesterone and MPA increased the amounts of a 100 kDa protein (nucleolin) and a 37 kDa protein (B23 protein) in the endometium and a marked proliferative change, whereas E2 did not. These results suggest that although the Ag-NOR number was enhanced by both progestin and estrogen, only progestins substantially increase the Ag-NOR protein in the rabbit uterus. This implies that an increase in the amount of nucleolin and B23 protein is associated with the proliferative potential of the cells.

In a total of 41 endometrial tissue samples, the relationship between telomerase activity and proliferating cell nuclear antigen (PCNA) labelling index was studied. In samples of endometrium from the proliferative phase of the menstrual cycle, telomerase activity was found in 15 out of 17 cases (88%). Two samples from the early proliferative phase showed negative telomerase activity and a low PCNA labelling index. However, three out of 16 samples of early secretory phase endometrium showed telomerase activity and a PCNA labelling index. In mid- to late secretory phase endometrium, in menopausal endometrium and in decidualized endometrium induced by progesterone neither telomerase activity nor PCNA labelling was found. These results suggest that telomerase activity of the endometrium may be correlated with the proliferative potential of the epithelial cells and that its activity may be regulated by oestrogen.

The photosystem II (PSII) mediates light-induced electron transfer from water to plastoquinone and the reaction center consists of two homologous polypeptides, D1 and D2 (1). The Dus encoded by the chloroplast psbA gene, synthesized as a precursor form with carboxyl-terminal extension (pD1) and subsequently cleaved between amino acid residues at positions 344 and 345. Although the carboxyl-terminal extension is dispensable (2–4), the assembly of the oxygen-evolving complex is closely coupled with the processing of pD1 (5). The oxygen-evolving complex may be directly or indirectly: ligated by Ala-344 at carboxyl-terminus of the mature D1 since mutants of Synechocystis in which the mature form of Dl was truncated at carboxyl-terminal region are deficient in the oxygen-evolving activity (4).

The photosystem II (PSII) is a multiprotein complex consisting of more than twenty subunits and a number of cofactors (1). The reaction center is composed of two homologous subunits, D1 and D2, encoded by the chloroplast psbA and psbD genes, respectively. The apoproteins of core antenna complexes, CP47 and CP43, which are closely associated to the reaction center are the chloroplast psbB and psbC gene products, respectively. In addition, cytochrome b559, which is tightly associated to the reaction center, consists of the chloroplast psbE and psbF gene products. These polypeptides bind the cofactors of PSII. On the lumenal side of the PSII complex, there are four extrinsic proteins, the nuclear encoded psbO, psbP, psbQ and psbR gene products, which are required for optimal oxygen-evolving activity.

In carcinomas of the uterine cervix of which p53 is frequently inactivated by papilloma viruses, gene transfer of p21, effector of p53, is an alternative tool to suppress cell growth. We introduced the p21 gene into HeLa cells. The transfectant with p21 showed a significant growth retardation by blockage of G1 to S transfer of the cell cycle. This cell line showed significantly reduced anchorage-independent growth as well as attenuated telomerase activity. These data suggest that gene transfer of p21 is an effective tool to lead carcinoma cells with inactivated p53 into less malignant phenotype.

The chloroplast genes ycf3 and ycf4 from the green alga Chlamydomonas reinhardtii have been characterized. The deduced amino acid sequences of Ycf4 (197 residues) and Ycf3 (172 residues) display 41-52% and 64-78% sequence identity, respectively, with their homologues from algae, land plants and cyanobacteria. In C. reinhardtii, ycf4 and ycf3 are co-transcribed as members of the rps9-ycf4-ycf3-rps18 polycistronic transcriptional unit into RNAs of 8.0 kb and 3.0 kb corresponding to the entire unit and to rps9-ycf4-ycf3, respectively. Using biolistic transformation, ycf4 and ycf3 were disrupted with a chloroplast selectable marker cassette. Transformants lacking ycf4 or ycf3 were unable to grow photoautotrophically and were deficient in photosystem I activity. Western blot analysis showed that the photosystem I (PSI) complex does not accumulate stably in thylakoid membranes of these transformants. Ycf4 and Ycf3 were localized on thylakoid membranes but not stably associated with the PSI complex and accumulated to wild-type levels in mutants lacking PSI. RNA blot hybridizations showed that transcripts of psaA, psaB and psaC accumulate normally in these mutants and use of chimeric reporter genes revealed that Ycf3 is not required for initiation of translation of psaA and psaB mRNA. Our results indicate that Ycf3 and Ycf4 are required for stable accumulation of the PSI complex.

The D1 protein (D1) of photosystem II (PSII) reaction center is synthesized as a precursor (pD1) and then processed at its carboxyl terminus to establish the function of water cleavage. The amino acid sequence of the carboxyl terminal extension excised by this process is poorly conserved except for a residue after the cleavage site at position of 345. We have constructed a vector for site-directed mutagenesis of the chloroplast psbA gene encoding D1 of the green alga, Chlamydomonas reinhardtii. The vector enables one to transform the chloroplasts of a psbA deletion mutant (Fud7) and directly select transformants for resistance to spectinomycin. Using this transforming vector, we have substituted Ser345 to Gly, Cys, Val and Phe in order to investigate effects of the amino acid side chain at this position on the processing rate. All of the resulting transformants exhibited the PSII activity as wild type and grew normally under photoautotrophic conditions even under strong light where rapid turnover of D1 protein is expected to occur. Western blotting analysis demonstrated that mature D1 accumulates in these transformants at wild type level. Pulse and chase labeling of chloroplast-encoded proteins using [35S] sulfate revealed that the processing of D1 precursor protein occurs in all four transformants as efficiently as in wild type, at least under the experimental conditions examined. The results suggest that either the amino acid side chain at position of 345 (+1 position) is not crucial to the enzymatic cleavage of pD1 in vivo or the apparent rate of processing in vivo is not limited by the enzymatic cleavage.

International audience; The small chloroplast open reading frame ORF43 (ycf7) of the green unicellular alga Chlamydomonas reinhardtii is cotranscribed with the psaC gene and ORF58. While ORF58 has been found only in the chloroplast genome of C.reinhardtii, ycf7 has been conserved in land plants and its sequence suggests that its product is a hydrophobic protein with a single transmembrane alpha helix. We have disrupted ORF58 and ycf7 with the aadA expression cassette by particle-gun mediated chloroplast transformation. While the ORF58::aadA transformants are indistinguishable from wild type, photoautotrophic growth of the ycf7::aadA transformants is considerably impaired. In these mutant cells, the amount of cytochrome b6f complex is reduced to 25-50% of wild-type level in mid-exponential phase, and the rate of transmembrane electron transfer per b6f complex measured in vivo under saturating light is three to four times slower than in wild type. Under subsaturating light conditions, the rate of the electron transfer reactions within the b6f complex is reduced more strongly in the mutant than in the wild type by the proton electrochemical gradient. The ycf7 product (Ycf7) is absent in mutants deficient in cytochrome b6f complex and present in highly purified b6f complex from the wild-type strain. Ycf7-less complexes appear more fragile than wild-type complexes and selectively lose the Rieske iron-sulfur protein during purification. These observations indicate that Ycf7 is an authentic subunit of the cytochrome b6f complex, which is required for its stability, accumulation and optimal efficiency. We therefore propose to rename the ycf7 gene petL.

The product of the chloroplast psbI gene is associated with the photosystem II reaction center. To gain insights into the function of this polypeptide, we have disrupted its gene in Chlamydomonas reinhardtii with an aadA expression cassette that confers resistance to spectinomycin through biolistic transformation. The transformants are still able to grow photoautotrophically in dim light, but not in high light, and they remain photosensitive when grown on acetate containing medium. The amounts of photosystem II complex and oxygen evolving activity are both reduced to 10-20% of wild-type levels in these psbI-deficient mutants. It appears that the PsbI polypeptide plays a role in the stability of photosystem II and possibly also in modulating electron transport or energy transfer in this complex.

We have engineered and analyzed a chloroplast mutant of Chlamydomonas reinhardtii that lacks ycf8, the chloroplast open reading frame 8, which is highly conserved in location and predicted amino acid sequence in land plants and C.reinhardtii. The ycf8 sequence was replaced with the aadA cassette which confers resistance to spectinomycin when expressed in the chloroplast. Although the mutant is able to grow phototrophically, photosystem II function and cell growth are impaired under stress conditions such as high light intensity and diminished chloroplast protein synthesis induced by spectinomycin. Use of an antibody generated against the ycf8 product has revealed that this hydrophobic polypeptide is associated with photosystem II, based on its severely reduced levels in various photosystem II-deficient mutants and on its copurification with photosystem II. This protein, therefore, appears to be (i) a novel photosystem II subunit and (ii) required for maintaining optimal photosystem II activity under adverse growth conditions.

Using particle gun-mediated chloroplast transformation we have disrupted the psbK gene of Chlamydomonas reinhardtii with an aadA expression cassette that confers resistance to spectinomycin. The transformants are unable to grow photoautotrophically, but they grow normally in acetate-containing medium. They are deficient in photosystem II activity as measured by fluorescence transients and O2 evolution and they accumulate less than 10% of wild-type levels of photosystem II as measured by immunochemical means. Pulse-labeling experiments indicate that the photosystem II complex is synthesized normally in the transformants. These results differ from those obtained previously with similar cyanobacterial psbK mutants that were still capable of photoautotrophic growth (Ikeuchi et al., J. Biol. Chem. 266 (1991) 1111-1115). In C. reinhardtii the psbK product is required for the stable assembly and/or stability of the photosystem II complex and essential for photoautotrophic growth. The data also suggest that the stability requirements of the photosynthetic complexes differ considerably between C. reinhardtii and cyanobacteria.