Iron Deficiency Promotes the Lack of Photosynthetic Cytochrome c550 and Affects the Binding of the Luminal Extrinsic Subunits to Photosystem II in the Diatom Phaeodactylum tricornutum.

In the diatom Phaeodactylum tricornutum, iron limitation promotes a decrease in the content of photosystem II, as determined by measurements of oxygen-evolving activity, thermoluminescence, chlorophyll fluorescence analyses and protein quantification methods. Thermoluminescence experiments also indicate that iron limitation induces subtle changes in the energetics of the recombination reaction between reduced QB and the S2/S3 states of the water-splitting machinery. However, electron transfer from QA to QB, involving non-heme iron, seems not to be significantly inhibited. Moreover, iron deficiency promotes a severe decrease in the content of the extrinsic PsbV/cytochrome c550 subunit of photosystem II, which appears in eukaryotic algae from the red photosynthetic lineage (including diatoms) but is absent in green algae and plants. The decline in the content of cytochrome c550 under iron-limiting conditions is accompanied by a decrease in the binding of this protein to photosystem II, and also of the extrinsic PsbO subunit. We propose that the lack of cytochrome c550, induced by iron deficiency, specifically affects the binding of other extrinsic subunits of photosystem II, as previously described in cyanobacterial PsbV mutants.

The afterglow photosynthetic luminescence.

The afterglow (AG) photosynthetic luminescence is a long-lived chlorophyll fluorescence emitted from PSII after the illumination of photosynthetic materials by FR or white light and placed in darkness. The AG emission corresponds to the fraction of PSII centers in the S2/3 QB non-radiative state immediately after pre-illumination, in which the arrival of an electron transferred from stroma along cyclic/chlororespiratory pathway(s) produces the S2/3 QB - radiative state that emits luminescence. This emission can be optimally recorded by a linear temperature gradient as sharp thermoluminescence (TL) band peaking at about 45°C. The AG emission recorded by TL technique has been proposed as a simple non-invasive tool to investigate the chloroplast energetic state and some of its metabolism processes as cyclic transport of electrons around PSI, chlororespiration or photorespiration. On the other hand, this emission has demonstrated to be a useful probe to study the effect of various stress conditions in photosynthetic materials.

The heterologous expression of a plastocyanin in the diatom Phaeodactylum tricornutum improves cell growth under iron-deficient conditions.

We have investigated if the heterologous expression of a functional green alga plastocyanin in the diatom Phaeodactylum tricornutum can improve photosynthetic activity and cell growth. Previous in vitro assays showed that a single-mutant of the plastocyanin from the green algae Chlamydomonas reinhardtii is effective in reducing P. tricornutum photosystem I. In this study, in vivo assays with P. tricornutum strains expressing this plastocyanin indicate that even the relatively low intracellular concentrations of holo-plastocyanin detected (≈4 µM) are enough to promote an increased growth (up to 60%) under iron-deficient conditions as compared with the WT strain, measured as higher cell densities, content in pigments and active photosystem I, global photosynthetic rates per cell, and even cell volume. In addition, the presence of plastocyanin as an additional photosynthetic electron carrier seems to decrease the over-reduction of the plastoquinone pool. Consequently, it promotes an improvement in the maximum quantum yield of both photosystem II and I, together with a decrease in the acceptor side photoinhibition of photosystem II-also associated to a reduced oxidative stress-a decrease in the peroxidation of membrane lipids in the choroplast, and a lower degree of limitation on the donor side of photosystem I. Thus the heterologous plastocyanin appears to act as a functional electron carrier, alternative to the native cytochrome c6 , under iron-limiting conditions.

The afterglow thermoluminescence band as an indicator of changes in the photorespiratory metabolism of the model legume Lotus japonicus.

The afterglow (AG) luminescence is a delayed chlorophyll fluorescence emitted by the photosystem II that seems to reflect the level of assimilatory potential (NADPH+ATP) in chloroplast. In this work, the thermoluminescence (TL) emissions corresponding to the AG band were investigated in plants of the WT and the Ljgln2-2 photorespiratory mutant from Lotus japonicus grown under either photorespiratory (air) or non-photorespiratory (high concentration of CO2 ) conditions. TL glow curves obtained after two flashes induced the strongest overall TL emissions, which could be decomposed in two components: B band (tmax = 27-29°C) and AG band (tmax = 44-45°C). Under photorespiratory conditions, WT plants showed a ratio of 1.17 between the intensity of the AG and B bands (IAG /IB ). This ratio increased considerably under non-photorespiratory conditions (2.12). In contrast, mutant Ljgln2-2 plants grown under both conditions showed a high IAG /IB ratio, similar to that of WT plants grown under non-photorespiratory conditions. In addition, high temperature thermoluminescence (HTL) emissions associated to lipid peroxidation were also recorded. WT and Ljgln2-2 mutant plants grown under photorespiratory conditions showed both a significant HTL band, which increased significantly under non-photorespiratory conditions. The results of this work indicate that changes in the amplitude of IAG /IB ratio could be used as an in vivo indicator of alteration in the level of photorespiratory metabolism in L. japonicus chloroplasts. Moreover, the HTL results suggest that photorespiration plays some role in the protection of the chloroplast against lipid peroxidation.

Valuation of Water and Emissions in Energy Systems.

Pricing, incentives, and economic penalties (monetization) are common approaches to control and improve water usage and total direct greenhouse gas emissions (externalities) of energy and other industrial systems. We discuss that homogenous pricing for externalities provides limited flexibility for controlling and improving environmental impacts as different systems are affected differently by externalities. We use trade-off analysis and scalarization techniques to determine marginal prices for water and carbon by taking into account the actual physical and technical limits, stakeholders, and real-time conditions of the system at hand. We demonstrate that high water and emission prices might be needed to control and improve the current system of fixed price for externalities. In addition, a combined heat and power (CHP) system providing hot water and electricity to a real residential building complex is undertaken as case study to demonstrate and describe these concepts. For this CHP system, we found that carbon prices should be increased by a factor of 14 and water prices by a factor of 217 to achieve an optimal compromise between cost, water use, and emissions. Our results point towards the need to consider alternative pricing schemes such as resource bidding (as is done with electricity) that better capture technology trade-offs and push systems towards their efficiency limits.

The photosynthetic cytochrome c 550 from the diatom Phaeodactylum tricornutum.

The photosynthetic cytochrome c 550 from the marine diatom Phaeodactylum tricornutum has been purified and characterized. Cytochrome c 550 is mostly obtained from the soluble cell extract in relatively large amounts. In addition, the protein appeared to be truncated in the last hydrophobic residues of the C-terminus, both in the soluble cytochrome c 550 and in the protein extracted from the membrane fraction, as deduced by mass spectrometry analysis and the comparison with the gene sequence. Interestingly, it has been described that the C-terminus of cytochrome c 550 forms a hydrophobic finger involved in the interaction with photosystem II in cyanobacteria. Cytochrome c 550 was almost absent in solubilized photosystem II complex samples, in contrast with the PsbO and Psb31 extrinsic subunits, thus suggesting a lower affinity of cytochrome c 550 for the photosystem II complex. Under iron-limiting conditions the amount of cytochrome c 550 decreases up to about 45% as compared to iron-replete cells, pointing to an iron-regulated synthesis. Oxidized cytochrome c 550 has been characterized using continuous wave EPR and pulse techniques, including HYSCORE, and the obtained results have been interpreted in terms of the electrostatic charge distribution in the surroundings of the heme centre.

Psychosis management in patients with hiv: case report.

Human immunodeficiency virus (HIV) infection can cause neuropsychiatric disorders such as cognitive impairment, behavioural difficulties or psychiatric symptoms –for instance, mania and psychosis. HIV patients with psychiatric comorbidities need an appropriate treatment which tackles the HIV infection as much as the particular mental symptoms. Here we present the case of a patient suffering from delusions, which turned out to be caused by encephalitis secondary to a previously unknown HIV infection. A review of psychosis in HIV-infected patients is also presented. This review is focused on the epidemiology, etiopathogenesis and clinical presentation of HIV-induced psychosis, as well as the recommended pharmacological treatment (antiretroviral therapy and antipsychotic medication) and the expected treatment response. We also present wide information concerning pharmacological interactions between antiretroviral and antipsychotic medications that we hope will help the clinician to better manage this complex condition.

Mutations of Cytochrome b559 and PsbJ on and near the QC Site in Photosystem II Influence the Regulation of Short-Term Light Response and Photosynthetic Growth of the Cyanobacterium Synechocystis sp. PCC 6803.

The characteristic features of two types of short-term light adaptations of the photosynthetic apparatus of the cyanobacterium Synechocystis sp. PCC 6803, state transition and blue-green light-induced fluorescence quenching, were compared in wild-type and cytochrome b559 and PsbJ mutant cells with mutations on and near the QC site in photosystem II (PSII). All mutant cells grew photoautotrophically and assembled stable PSII. Thermoluminescence emission experiments showed a decrease in the stability of the S3QB(-)/S2QB(-) charge pairs in the A16FJ, S28Aß, and V32Fß mutant cells. When dark-adapted wild-type and mutant cells were illuminated by medium-intensity blue light, the increase in the PSII fluorescence yield (indicating a transition to state 1) was more prominent in mutant than wild-type cells. Strong blue-light conditions induced a quenching of fluorescence corresponding to nonphotochemical fluorescence quenching (NPQ). The extension of NPQ decreased significantly in the mutants, and the kinetics appeared to be affected. When similar measures were repeated on an orange carotenoid protein (OCP)-deficient background, little or no quenching was observed, which confirms that the decrease in fluorescence under strong blue light corresponded to the OCP-dependent NPQ. Immunoblot results showed that the attenuated effect of blue light-induced NPQ in mutant cells was not due to a lack of OCP. Photosynthetic growth and biomass production were greater for A16FJ, S28Aß, and V32Fß mutant cells than for wild-type cells under normal growth conditions. Our results suggest that mutations of cytochrome b559 and PsbJ on and near the QC site of PSII may modulate the short-term light response in cyanobacteria.

Risk factor assessment and counselling for 12 months reduces metabolic and cardiovascular risk in overweight or obese patients with schizophrenia spectrum disorders: The CRESSOB study.

BACKGROUND: Metabolic syndrome (MS) and cardiovascular risk factors (CRF) have been associated with patients with schizophrenia. The main objective is to assess the evolution of CRF and prevalence of MS for 12 months in a cohort of overweight patients diagnosed with schizophrenia schizophreniform disorder or schizoaffective disorder in which the recommendations for the assessment and control of metabolic and cardiovascular risk were applied. METHODS: The Control of Metabolic and Cardiovascular Risk in Patients with Schizophrenia and Overweight (CRESSOB) study is a 12-month, observational, prospective, open-label, multicentre, naturalistic study including 109 community mental health clinics of Spain. The study included a total of 403 patients, of whom we could collect all variables related to CRF and MS in 366 patients. Of these 366 patients, 286 completed the follow-up, (baseline, months 3, 6 and 12) where they underwent a complete physical examination and a blood test (glucose, cholesterol and triglycerides), they were asked about their health-related habits (smoking, diet and exercise) and they were given a series of recommendations to prevent cardiovascular risk and MS. RESULTS: A total of 403 patients were included, 63% men, mean age (mean; (SD)) 40.5 (10.5) years. After 12 months, the study showed statistically significant decrease in weight (p<0.0001), waist circumference (p<0.0001), BMI (p<0.0001), blood glucose (p=0.0034), total cholesterol (p<0.0001), HDL cholesterol (p=0.02), LDL cholesterol (p=0.0023) and triglycerides (p=0.0005). There was a significant reduction in the percentage of smokers (p=0.0057) and in the risk of heart disease at 10 years (p=0.0353). CONCLUSION: Overweight patients with schizophrenia who receive appropriate medical care, including CRF monitoring and control of health-related habits experience improvements with regard to most CRFs.

The role of the high potential form of the cytochrome b559: Study of Thermosynechococcus elongatus mutants.

Cytochrome b559 is an essential component of the photosystem II reaction center in photosynthetic oxygen-evolving organisms, but its function still remains unclear. The use of photosystem II preparations from Thermosynechococcus elongatus of high integrity and activity allowed us to measure for the first time the influence of cytochrome b559 mutations on its midpoint redox potential and on the reduction of the cytochrome b559 by the plastoquinone pool (or QB). In this work, five mutants having a mutation in the α-subunit (I14A, I14S, R18S, I27A and I27T) and one in the ß-subunit (F32Y) of cytochrome b559 have been investigated. All the mutations led to a destabilization of the high potential form of the cytochrome b559. The midpoint redox potential of the high potential form was significantly altered in the αR18S and αI27T mutant strains. The αR18S strain also showed a high sensitivity to photoinhibitory illumination and an altered oxidase activity. This was suggested by measurements of light induced oxidation and dark re-reduction of the cytochrome b559 showing that under conditions of a non-functional water oxidation system, once the cytochrome is oxidized by P680(+), the yield of its reduction by QB or the PQ pool was smaller and the kinetic slower in the αR18S mutant than in the wild-type strain. Thus, the extremely positive redox potential of the high potential form of cytochrome b559 could be necessary to ensure efficient oxidation of the PQ pool and to function as an electron reservoir replacing the water oxidation system when it is not operating.

Spectroscopic and functional characterization of cyanobacterium Synechocystis PCC 6803 mutants on the cytoplasmic-side of cytochrome b559 in photosystem II.

We performed spectroscopic and functional characterization on cyanobacterium Synechocystis PCC6803 with mutations of charged residues of the cytoplasmic side of cytochrome (Cyt) b559 in photosystem II (PSII). All of the mutant cells grew photoautotrophically and assembled stable PSII. However, R7Eα, R17Eα and R17Lß mutant cells grew significantly slower and were more susceptible to photoinhibition than wild-type cells. The adverse effects of the arginine mutations on the activity and the stability of PSII were in the following order (R17Lß>R7Eα>R17Eα and R17Aα). All these arginine mutants exhibited normal period-four oscillation in oxygen yield. Thermoluminescence characteristics indicated a slight decrease in the stability of the S3QB(-)/S2QB(-) charge pairs in the R7Eα and R17Lß mutant cells. R7Eα and R17Lß PSII core complexes contained predominantly the low potential form of Cyt b559. EPR results indicated the displacement of one of the two axial ligands to the heme of Cyt b559 in R7Eα and R17Lß mutant reaction centers. Our results demonstrate that the electrostatic interactions between these arginine residues and the heme propionates of Cyt b559 are important to the structure and redox properties of Cyt b559. In addition, the blue light-induced nonphotochemical quenching was significantly attenuated and its recovery was accelerated in the R7Lα and R17Lß mutant cells. Furthermore, ultra performance liquid chromatography-mass spectrometry results showed that the PQ pool was more reduced in the R7Eα and R17Lß mutant cells than wild-type cells in the dark. Our data support a functional role of Cyt b559 in protection of PSII under photoinhibition conditions in vivo.

[Prevalence of metabolic syndrome in Spanish patients with schizophrenia and overweight. The CRESSOB Study]. / Prevalencia del síndrome metabólico en pacientes españoles con esquizofrenia y sobrepeso. El estudio CRESSOB.

INTRODUCTION: Metabolic syndrome (MS) (visceral obesity, dyslipidemia, hyperglycemia, and hypertension), has become one of the major public-health challenges worldwide. Patients with schizophrenia are more likely to suffer from MS than the general population. OBJECTIVE: The primary aim of this study was to analyze the prevalence of MS in Spanish patients with schizophrenia and overweight and to compare the best method to calculate the MS prevalence in this population. A secondary aim of the CRESSOB study was to determine whether the presence of the metabolic syndrome (MS) is associated or not with clinical remission of schizophrenia. METHODS: The Control of Metabolic and Cardiovascular Risk in Patients with Schizophrenia and Overweight (CRESSOB) study is a 12-month, prospective, naturalistic study including 110 community mental health clinics selected at random. Each site enrolled four consecutive patients with a diagnosis of schizophrenia, according to DSM-IV TR criteria, and who were overweight (Body Mass Index (BMI) >25 kg/m2). To assess the prevalence of MS we analyzed the baseline results of the CRESSOB study. The National Cholesterol Education Program (NCEP-ATP III), the International Diabetes Federation (IDF) and the American Heart Association/National Heart, Lung, and Blood Institute (AHA/NHLBI) definitions were used to establish the presence of MS. The Positive and Negative Syndrome Scale (PANSS) was used to determine the percentage of patients in remission. Psychosocial functioning was measured by the Global Assessment of Functioning (GAF) scale. RESULTS: A total of 391 patients were enrolled in the study (mean age 40.5 years, 63.8% men). 75.9% of the patients did not meet criteria for remission, using the selected PANSS items. The mean GAF score was 52.7 (Standard Deviation (SD) 15.4). Overall, 59.0% of males and 58.3% of females fulfilled the NCEP-ATP III criteria, 71.1% of males and 65.8% of females fulfilled the IDF criteria and 70.1% of males and 65.1% of females fulfilled the AHA/ NHLBI criteria. The patients who fulfilled remission criteria were younger, had a lower BMI, and a higher GAF score. CONCLUSIONS: MS is highly prevalent in Spanish patients with schizophrenia who are overweight. Given that metabolic syndrome is an important risk factor for cardiovascular disease, these patients should receive appropriate clinical monitoring for this syndrome.

Tubulo-interstitial inflammation increases the risk of graft loss after the recurrence of IgA nephropathy.

Background: Immunoglobulin A nephropathy (IgAN) is the most frequent recurrent disease in kidney transplant recipients and its recurrence contributes to reducing graft survival. Several variables at the time of recurrence have been associated with a higher risk of graft loss. The presence of clinical or subclinical inflammation has been associated with a higher risk of kidney graft loss, but it is not precisely known how it influences the outcome of patients with recurrent IgAN. Methods: We performed a multicentre retrospective study including kidney transplant recipients with biopsy-proven recurrence of IgAN in which Banff and Oxford classification scores were available. 'Tubulo-interstitial inflammation' (TII) was defined when 't' or 'i' were ≥2. The main endpoint was progression to chronic kidney disease (CKD) stage 5 or to death censored-graft loss (CKD5/DCGL). Results: A total of 119 kidney transplant recipients with IgAN recurrence were included and 23 of them showed TII. Median follow-up was 102.9 months and 39 (32.8%) patients reached CKD5/DCGL. TII related to a higher risk of CKD5/DCGL (3 years 18.0% vs 45.3%, log-rank 7.588, P = .006). After multivariate analysis, TII remained related to the risk of CKD5/DCGL (HR 2.344, 95% CI 1.119-4.910, P = .024) independently of other histologic and clinical variables. Conclusions: In kidney transplant recipients with IgAN recurrence, TII contributes to increasing the risk of CKD5/DCGL independently of previously well-known variables. We suggest adding TII along with the Oxford classification to the clinical variables to identify recurrent IgAN patients at increased risk of graft loss who might benefit from intensified immunosuppression or specific IgAN therapies.

Photosynthetic cytochrome c550.

Cytochrome c550 (cyt c550) is a membrane component of the PSII complex in cyanobacteria and some eukaryotic algae, such as red and brown algae. Cyt c550 presents a bis-histidine heme coordination which is very unusual for monoheme c-type cytochromes. In PSII, the cyt c550 with the other extrinsic proteins stabilizes the binding of Cl(-) and Ca(2+) ions to the oxygen evolving complex and protects the Mn(4)Ca cluster from attack by bulk reductants. The role (if there is one) of the heme of the cyt c550 is unknown. The low midpoint redox potential (E(m)) of the purified soluble form (from -250 to -314mV) is incompatible with a redox function in PSII. However, more positive values for the Em have been obtained for the cyt c550 bound to the PSII. A very recent work has shown an E(m) value of +200mV. These data open the possibility of a redox function for this protein in electron transfer in PSII. Despite the long distance (22Å) between cyt c550 and the nearest redox cofactor (Mn(4)Ca cluster), an electron transfer reaction between these components is possible. Some kind of protective cycle involving a soluble redox component in the lumen has also been proposed. The aim of this article is to review previous studies done on cyt c550 and to consider its function in the light of the new results obtained in recent years. The emphasis is on the physical properties of the heme and its redox properties. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

A high redox potential form of cytochrome c550 in photosystem II from Thermosynechococcus elongatus.

Cytochrome c(550) (cyt c(550)) is a component of photosystem II (PSII) from cyanobacteria, red algae, and some other eukaryotic algae. Its physiological role remains unclear. In the present work, measurements of the midpoint redox potential (E(m)) were performed using intact PSII core complexes preparations from a histidine-tagged PSII mutant strain of the thermophilic cyanobacterium Thermosynechococcus (T.) elongatus. When redox titrations were done in the absence of redox mediators, an E(m) value of +200 mV was obtained for cyt c(550). This value is ∼300 mV more positive than that previously measured in the presence of mediators (E(m) = -80 mV). The shift from the high potential form (E(m) = +200 mV) to the low potential form (E(m) = -80 mV) of cyt c(550) is attributed to conformational changes, triggered by the reduction of a component of PSII that is sequestered and out of equilibrium with the medium, most likely the Mn(4)Ca cluster. This reduction can occur when reduced low potential redox mediators are present or under highly reducing conditions even in the absence of mediators. Based on these observations, it is suggested that the E(m) of +200 mV obtained without mediators could be the physiological redox potential of the cyt c(550) in PSII. This value opens the possibility of a redox function for cyt c(550) in PSII.

Reconstitution, spectroscopy, and redox properties of the photosynthetic recombinant cytochrome b(559) from higher plants.

A study of the in vitro reconstitution of sugar beet cytochrome b(559) of the photosystem II is described. Both α and ß cytochrome subunits were first cloned and expressed in Escherichia coli. In vitro reconstitution of this cytochrome was carried out with partially purified recombinant subunits from inclusion bodies. Reconstitution with commercial heme of both (αα) and (ßß) homodimers and (αß) heterodimer was possible, the latter being more efficient. The absorption spectra of these reconstituted samples were similar to that of the native heterodimer cytochrome b(559) form. As shown by electron paramagnetic resonance and potentiometry, most of the reconstituted cytochrome corresponded to a low spin form with a midpoint redox potential +36 mV, similar to that from the native purified cytochrome b(559). Furthermore, during the expression of sugar beet and Synechocystis sp. PCC 6803 cytochrome b(559) subunits, part of the protein subunits were incorporated into the host bacterial inner membrane, but only in the case of the ß subunit from the cyanobacterium the formation of a cytochrome b(559)-like structure with the bacterial endogenous heme was observed. The reason for that surprising result is unknown. This in vivo formed (ßß) homodimer cytochrome b(559)-like structure showed similar absorption and electron paramagnetic resonance spectral properties as the native purified cytochrome b(559). A higher midpoint redox potential (+126 mV) was detected in the in vivo formed protein compared to the in vitro reconstituted form, most likely due to a more hydrophobic environment imposed by the lipid membrane surrounding the heme.

Deep brain stimulation in obsessive-compulsive disorder: Results from meta-analysis.

The aim of this work is to investigate the effectiveness of Deep Brain Stimulation (DBS) in patients with severe Obsessive Compulsive Disorder (OCD) who are resistant to pharmacological treatments, focusing on obsessive compulsive, depressive and anxiety symptoms as well as global function. A systematic review and meta-analysis including 25 studies (without language restrictions) from between 2003 and 2020 was performed. A total of 303 patients were evaluated twice (before and after DBS). After DBS treatment OCD patients with resistance to pharmacological treatments showed a significant improvement of obsessive-compulsive symptoms (25 studies; SMD=2.39; 95% CI, 1.91 to 2.87; P<0.0001), depression (9 studies; SMD= 1.19; 95%CI, 0.84 to 1.54; P<0.0001), anxiety (5 studies; SMD=1.00; 95%CI, 0.32 to 1.69; P=0.004) and functionality (7 studies; SMD=-3.51; 95%CI, -5.00 to -2.02; P=0.005) measured by the standardized scales: Yale Brown Obsessive Compulsive Scale (YBOCS), Hamilton Depression Rating Scale (HAM-D), Hamilton Anxiety Rating Scale (HAM-A) and Global Assessment of Function (GAF). Publication bias were discarded by using funnel plot. The main conclusions of this meta-analysis highlight the statistically significant effectiveness of DBS in patients with severe OCD who are resistant to conventional pharmacological treatments, underlying its role in global functioning apart from obsessive-compulsive symptoms.

Coexistence of anti-topoisomerase I and anticentromere antibodies in a patient with systemic sclerosis. Efficacy of treatment combining rituximab and nintedanib. A case report.

In the diagnostic of systemic sclerosis (SSc), both anti-centromere (ACA) and anti-topoisomerase I (ATA) antibodies are considered mutually exclusive, though their coexistence has been also reported in some patients. Notably, nintedanib has been approved for the treatment of interstitial lung disease associated to SSc. Herein, we present the clinical case of a 41-year-old woman with SSc who shows an immunological seroconversion (from ACA positivity to a coexistence of ACA and ATA antibodies) together with changes in her clinical phenotype. Besides, the patient responds positively to the treatment of her lung involvement with a combination of immunomodulators and antifibrotic agents.

Nucleoplasmic calcium regulates cell proliferation through legumain.

BACKGROUND & AIMS: Nucleoplasmic Ca(2+) regulates cell growth in the liver, but the proteins through which this occurs are unknown. METHODS: We used Rapid Subtraction Hybridization (RaSH) to subtract genes in SKHep1 liver cells expressing the Ca(2+) buffer protein parvalbumin (PV) targeted to the nucleus, from genes in cells expressing a mutated form of nuclear-targeted PV which has one of two Ca(2+)-binding sites inactivated. The subtraction permitted the selection of genes whose expression was affected by a small alteration in nuclear Ca(2+) concentration. RESULTS: The asparaginyl endopeptidase legumain (LGMN) was identified in this screening. When Ca(2+) was buffered in the nucleus of SKHep1 cells, LGMN mRNA was decreased by 97%, in part by a transcriptional mechanism, and decreased expression at the protein level was observed by immunoblot and immunofluorescence. Treatment with hepatocyte growth factor increased LGMN expression. Knockdown of LGMN by siRNA decreased proliferation of SKHep1 cells by ∼50% as measured both by BrdU uptake and mitotic index, although an inhibitor of LGMN activity did not affect BrdU incorporation. A significant reduction in the fraction of cells in G2/M phase was seen as well. This was associated with increases in the expression of cyclins A and E. Furthermore, LGMN expression was increased in hepatocellular carcinoma cells relative to normal hepatocytes in the same specimens. CONCLUSIONS: These findings suggest a new role for LGMN and provide evidence that nuclear Ca(2+) signals regulate cell proliferation in part through the modulation of LGMN expression. Increased expression of LGMN may be involved in liver carcinogenesis.