All3048, a DnaJ III homolog of Anabaena sp. PCC7120 mediates heat shock response in E. coli and its N-terminus J-domain stimulates DnaK ATPase activity.

Cyanobacterial DnaJ offers thermo-tolerance and effectively prevents aggregation of denatured protein in coordination with DnaK. The hypothetical protein All3048 of Anabaena sp. PCC7120 was found to be a 24 kDa DnaJ III protein with a putative J-domain at the extreme N-terminus. This paper decodes the role of All3048 in thermo-tolerance and as a co-chaperon of DnaK. Semi-quantitative and RT-PCR results showed up-accumulation of all3048 in heat, UV-B, cadmium, arsenic and salt. BL21/pET-28a-all3048, all3048(1-95) and all3048(31-128) reduced the heat stress-induced ROS generation by 40 %, 21 % and 24 % as compared to BL21/pET-28-a. Conformational properties of All3048 and its truncated variants were assessed using bis ANS, guanidine hydrochloride and acrylamide quenching. All3048(1-95), All3048 and All3048(31-128) increased DnaK ATPase activity by 8.6, 8.2, and 2.5 fold, respectively. The thermostability investigated using DSC and DSF methods affirmed the relative stability of All3048 and All3048 (31-128), whereas All3048 (1-95) was the least stable. All3048 is a novel cyanobacterial DnaJ III that imparts heat stress tolerance in E. coli; however, only the J-domain present at N-terminus was sufficient for stimulating DnaK's ATPase activity.

Functional Characterization of Alr0765, A Hypothetical Protein from Anabaena PCC 7120 Involved in Cellular Energy Status Sensing, Iron Acquisition and Abiotic Stress Management in E. coli Using Molecular, Biochemical and Computational Approaches.

BACKGROUND: Cyanobacteria are excellent model to understand the basic metabolic processes taking place in response to abiotic stress. The present study involves the characterization of a hypothetical protein Alr0765 of Anabaena PCC7120 comprising the CBS-CP12 domain and deciphering its role in abiotic stress tolerance. METHODS: Molecular cloning, heterologous expression and protein purification using affinity chromatography were performed to obtain native purified protein Alr0765. The energy sensing property of Alr0765 was inferred from its binding affinity with different ligand molecules as analyzed by FTIR and TNP-ATP binding assay. AAS and real time-PCR were applied to evaluate the iron acquisition property and cyclic voltammetry was employed to check the redox sensitivity of the target protein. Transcript levels under different abiotic stresses, as well as spot assay, CFU count, ROS level and cellular H2O2 level, were used to show the potential role of Alr0765 in abiotic stress tolerance. In-silico analysis of Alr0765 included molecular function probability analysis, multiple sequence analysis, protein domain and motif finding, secondary structure analysis, protein-ligand interaction, homologous modeling, model refinement and verification and molecular docking was performed with COFACTOR, PROMALS-3D, InterProScan, MEME, TheaDomEx, COACH, Swiss modeller, Modrefiner, PROCHECK, ERRAT, MolProbity, ProSA, TM-align, and Discovery studio, respectively. RESULTS: Transcript levels of alr0765 significantly increased by 20, 13, 15, 14.8, 12, 7, 6 and 2.5 fold when Anabaena PCC7120 treated with LC50 dose of heat, arsenic, cadmium, butachlor, salt, mannitol (drought), UV-B, and methyl viologen respectively, with respect to control (untreated). Heterologous expression resulted in 23KDa protein observed on the SDS-PAGE. Immunoblotting and MALDI-TOF-MS/MS, followed by MASCOT search analysis, confirmed the identity of the protein and ESI/MS revealed that the purified protein was a dimer. Binding possibility of Alr0765 with ATP was observed with an almost 6-fold increment in relative fluorescence during TNP-ATP binding assay with a λ max of 538 nm. FTIR spectra revealed modification in protein confirmation upon binding of Alr0765 with ATP, ADP, AMP and NADH. A 10-fold higher accumulation of iron was observed in digests of E. coli with recombinant vector after induction as compared to control, which affirms the iron acquisition property of the protein. Moreover, the generation of the redox potential of 146 mV by Alr0765 suggested its probable role in maintaining the redox status of the cell under environmental constraints. As per CFU count recombinant, E. coli BL21 cells showed about 14.7, 7.3, 6.9, 1.9, 3 and 4.9 fold higher number of colonies under heat, cadmium (CdCl2), arsenic (Na3AsO4), salt (NaCl), UV-B and drought (mannitol) respectively compared to pET21a harboring E. coli BL21 cells. Deterioration in the cellular ROS level and total cellular H2O2 concentration validated the stress tolerance ability of Alr0765. In-silico analysis unraveled novel findings and attested experimental findings in determining the role of Alr0765. CONCLUSION: Alr0765 is a novel CBS-CP12 domain protein that maintains cellular energy level and iron homeostasis which provides tolerance against multiple abiotic stresses.

Correction: Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph+ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Standardisation and consensus guidelines for minimal residual disease assessment in Philadelphia-positive acute lymphoblastic leukemia (Ph + ALL) by real-time quantitative reverse transcriptase PCR of e1a2 BCR-ABL1.

Minimal residual disease (MRD) is a powerful prognostic factor in acute lymphoblastic leukemia (ALL) and is used for patient stratification and treatment decisions, but its precise role in Philadelphia chromosome positive ALL is less clear. This uncertainty results largely from methodological differences relating to the use of real-time quantitative PCR (qRT-PCR) to measure BCR-ABL1 transcript levels for MRD analysis. We here describe the first results by the EURO-MRD consortium on standardization of qRT-PCR for the e1a2 BCR-ABL1 transcript in Ph + ALL, designed to overcome the lack of standardisation of laboratory procedures and data interpretation. Standardised use of EAC primer/probe sets and of centrally prepared plasmid standards had the greatest impact on reducing interlaboratory variability. In QC1 the proportion of analyses with BCR-ABL1/ABL1 ratios within half a log difference were 40/67 (60%) and 52/67 (78%) at 10-3 and 36/67 (53%) and 53/67 (79%) at 10-4BCR-ABL1/ABL1. Standardized RNA extraction, cDNA synthesis and cycler platforms did not improve results further, whereas stringent application of technical criteria for assay quality and uniform criteria for data interpretation and reporting were essential. We provide detailed laboratory recommendations for the standardized MRD analysis in routine diagnostic settings and in multicenter clinical trials for Ph + ALL.

Alr2954 of Anabaena sp. PCC 7120 with ADP-ribose pyrophosphatase activity bestows abiotic stress tolerance in Escherichia coli.

In silico derived properties on experimental validation revealed that hypothetical protein Alr2954 of Anabaena sp. PCC7120 is ADP-ribose pyrophosphatase, which belongs to nudix hydrolase superfamily. Presence of ADP-ribose binding site was attested by ADP-ribose pyrophosphatase activity (K m 44.71 ± 8.043 mM, V max 7.128 ± 0.417 µmol min-1 mg protein-1, and K cat/K m 9.438 × 104 µM-1 min-1). Besides ADP-ribose, the enzyme efficiently hydrolyzed various nucleoside phosphatases such as 8-oxo-dGDP, 8-oxo-dADP, 8-oxo-dGTP, 8-oxo-dATP, GDP-mannose, ADP-glucose, and NADH. qRT-PCR analysis of alr2954 showed significant expression under different abiotic stresses reconfirming its role in stress tolerance. Thus, Alr2954 qualifies to be a member of nudix hydrolase superfamily, which serves as ADP-ribose pyrophosphatase and assists in multiple abiotic stress tolerance.

Exploring the membrane proteome of the diazotropic cyanobacterium Anabaena PCC7120 through gel-based proteomics and in silico approaches.

UNLABELLED: This paper focuses on the gel-based membrane proteomics from diazotrophic cyanobacterium Anabaena PCC7120 by modifying the protocol of Hall et al. [1]. The bioinformatic analysis revealed that 59 (29 integral, 30 peripheral) of the 67 proteins identified were membrane proteins. Of the 29 integral proteins, except Alr0834, the remaining 28 contained 1-12 transmembrane helices. Sixteen integral proteins harboring signal peptides (Sec/TAT/LipoP) suggest that protein targeting in Anabaena involves both sec-dependent and sec-independent pathways. While majority of photosynthesis and respiration proteins (21 of 24) were confined to broad pH gradient the hypothetical and unknown (12 of 13), and cell envelope proteins (3 of 3) preferred the narrow pH range. Of the 5 transporters and binding proteins, Na(+)/H(+)-exchanging protein and Alr2372 were present in broad, pstS1 and cmpD in narrow and cmpA was common to both pH ranges. The distribution of proteins across pH gradient, thus clearly indicates the functional and structural diversity in membrane proteome of Anabaena. It requires mention that protochlorophyllide oxido-reductase, Na(+)/H(+)-exchanging protein, All1355, Alr2055, Alr3514, Alr2903 and Alr2751 were new entries to the 2DE membrane protein profile of Anabaena. This study demonstrates suitability of the modified protocol for the study of membrane protein from filamentous cyanobacteria. SIGNIFICANCE: Anabaena sp. PCC7120 is used as a model organism due to its agriculture significance as biofertilizer, close resemblance with higher plant chloroplast and availability of full genome sequence. Although cytosolic proteome has been explored a lot membrane proteins are still understudied as they are notoriously difficult to display using 2-D technology. Identification and characterization of these proteins is therefore required to elucidate and understand cellular mechanisms. The purpose of this study was to develop a protocol suitable for membrane protein extraction from Anabaena. Additionally, by homology comparison or domain assignment a possible function could be ascribed to novel uncharacterized proteins which will serve as a useful reference for further detailed studies of membrane system in filamentous cyanobacteria. Resolution of membrane proteins ranging from least (single transmembrane helix) to highly hydrophobic (several transmembrane helices) one on 2D gels recommends the gel based approach for identification of membrane proteomics from filamentous cyanobacteria. This article is part of a Special Issue entitled: Proteomics in India.

Cadmium toxicity in diazotrophic Anabaena spp. adjudged by hasty up-accumulation of transporter and signaling and severe down-accumulation of nitrogen metabolism proteins.

Present study demonstrates interspecies variation in proteome and survival strategy of three Anabaena species i.e., Anabaena L31, Anabaena sp. PCC 7120 and Anabaena doliolum subjected to respective LC50 doses of Cd at 0, 1, 3, 5 and 7day intervals. The proteome coverage with 452 differentially accumulated proteins unveiled species and time specific expression and interaction network of proteins involved in important cellular functions. Statistical analysis of protein abundance across Cd-treated proteomes clustered their co-expression pattern into four groups viz., (i) early (days 1 and 3) accumulated proteins, (ii) proteins up-accumulated for longer duration, (iii) late (days 5 and 7) accumulated proteins, and (iv) mostly down-accumulated proteins. Appreciable growth of Cd treated A L31 over other two species may be ascribed to proteins contained in the first and second groups (belonging to energy and carbohydrate metabolism (TK, G6-PI, PGD, FBA, PPA, ATP synthase)), sulfur metabolism (GR, GST, PGDH, PAPS reductase, GDC-P, and SAM synthetase), fatty acid metabolism (AspD, PspA, SQD-1), phosphorous metabolism (PhoD, PstB and SQD1), molecular chaperones (Gro-EL, FKBP-type peptidylprolyl isomerase), and antioxidative defense enzymes (SOD-A, catalase). Anabaena sp. PCC 7120 harboring proteins largely from the third group qualified as a late accumulator and A. doliolum housing majority of proteins from the fourth group emerged as the most sensitive species. Thus early up-accumulation of transporter and signaling category proteins and drastic reduction of nitrogen assimilation proteins could be taken as a vital indicator of cadmium toxicity in Anabaena spp. This article is part of a Special Issue entitled: Proteomics in India.

Factors influencing endometrial thickness in postmenopausal women.

BACKGROUND: Cut-off values for endometrial thickness (ET) in asymptomatic postmenopausal woman have been standardized. However, there are no comprehensive studies to document how various factors can influence the ET after the age of menopause. AIM: To study the various factors influencing the ET in postmenopausal women. SUBJECTS AND METHODS: This was a prospective observational study. A total of 110 postmenopausal women underwent detailed history taking, clinical examination, and transvaginal scan for uterine volume and ovarian volume. The volumes were calculated by using ellipsoid formula: Width × thickness × height × 0.523. The variation in ET with respect to the influencing factors such as age, duration of menopause, parity, body mass index (BMI), medical illness like diabetes/hypertension, drugs like tamoxifen, presence of myoma, uterine volume, ovarian volume, and serum estradiol (in selected patients) were measured. Descriptive analysis was performed using SPSS software (version 16, Chicago II, USA) to obtain mean, standard deviation (SD), 95% confidence intervals (CIs) and inter quartile ranges. Comparison of means was carried out using analysis of variance. RESULTS: The mean (SD) age of the patients was 55.4 (6.91) years (95% CI, 54.1, 56.7). The mean (SD) age at menopause was 47.95 (3.90) years (95% CI, 47.2, 48.7) and the mean (SD) duration of menopause was 7.27 (6.65) years (95% CI, 6.01, 8.53). The mean (SD) ET was 3.8 (2.3) mm (95% CI, 3.36, 4.23). Medical illness like diabetes and hypertension did not alter the ET. ET increased as BMI increased and it was statistically significant. The presence of myoma increased uterine volume significantly and was associated with thick endometrial stripe. Similarly, whenever the ovaries were visualized and as the ovarian volume increased, there was an increase in ET. When ET was > 4 mm (n = 37), they were offered endocel, of which 16 agreed to undergo the procedure. None were found to have endometrial cancer. CONCLUSION: This study suggests that parity, BMI, presence of myoma, tamoxifen usage, uterine volume, ovarian volume and serum estradiol influence the ET in postmenopausal women.

Unusual facial cleft in Fryns syndrome: defect of stomodeum?

Unusual facial cleft in Fryns syndrome: defect of stomodeum?: We report on a fetus with Fryns syndrome. The facial cleft was unusual. There was bilateral cleft lip with cleft palate. The intermaxillary segment was connected through the base of a mound in the midline to the lower lip. We believe this is an atypical facial cleft in Fryns syndrome and likely represents a defective stomodeum.

Impact of aluminium, fluoride and fluoroaluminate complex on ATPase activity of Nostoc linckia and Chlorella vulgaris.

This study demonstrates a pH-dependent inhibition of Mg(2+)- and Ca(2+)-ATPase activities of Nostoc linckia and Chlorella vulgaris exposed to AlCl3, AlF3, NaF and AlCl3+NaF together. AlF3 and the combination of AlCl3+NaF were more inhibitory to both the enzymes as compared with AlCl3 and NaF. Toxicity of the test compounds increased with increasing acidity. Interaction of AlCl3+NaF was additive on N. linckia and C. vulgaris, respectively, at pH 7.5 and 6.8, and synergistic at pH 6.0 and 4.5. In the presence of 60 and 100 microM PO4(3-) an increased NaF concentration (in the AlCl3+NaF combination) was required to produce the same degree of inhibition in ATP synthesis and ATPase activity. Toxicity of fluoroaluminate was reduced in the presence of EDTA and citrate. Except for beryllium to some extent, combinations of cadmium, cobalt, iron, manganese, tin and zinc with fluoride were not as effective as aluminium in inhibiting the ATPase activity. The presence of a 100 kDa protein band in SDS-PAGE of both control as well as AlCl3+NaF-treated samples suggested that AlF4- inhibits the ATPase activity by acting as a functional barrier without affecting the structure of the enzyme.

Effect of nickel on certain physiological and biochemical behaviors of an acid tolerant Chlorella vulgaris.

This study concerns the inhibitory effects of acid pH and nickel on growth, nutrient (NO3- and NH4+) uptake, carbon fixation, O2 evolution, electron transport chain and enzyme (nitrate reductase and ATPase) activities of acid tolerant and wild-type strains of Chlorella vulgaris. Though a general reduction in all these variables was noticed with decreasing pH, the tolerant strain was found to be metabolically more active than the wild-type. A reduced cation (NH4+, Na+, K+ and Ca2+) uptake, coupled with a facilitated influx of anions (NH4+, PO4(3-) and HCO3-), suggested the development of a positive membrane potential in acid tolerant Chlorella. Nevertheless, a tremendous increase in ATPase activity at decreasing pH revealed the involvement of superactive ATPase in exporting H+ ions and keeping the internal pH neutral. A difference in Na+ and K+ efflux of the two strains at decreasing pH suggests there is a difference in membrane permeability. The low toxicity of Ni in the acid tolerant strain may be due to the low Ni uptake brought about by a change in membrane potential as well as in permeability. Hence, the development of superactive ATPase and a change in both membrane potential and permeability not only offers protection against acidity, but also co-tolerance to metals.

Effect of Cu and Ni on growth, mineral uptake, photosynthesis and enzyme activities of Chlorella vulgaris at different pH values.

A pH dependent reduction in growth, pigment, ATP content, O2- evolution, carbon fixation, photosynthetic electron transport system, nutrient uptake (NO3- and NH4+), nitrate reductase, and ATPase activities and increase in K+ efflux of Chlorella vulgaris was noticed following supplementation of Cu and Ni to the culture medium. PS II was found to be more sensitive to both pH and metals than PS I. Though, nitrate reductase (NR) was more sensitive to both pH and metals, the ATPase was however, more sensitive to metals but less sensitive to acidic pH. Acid pH was found to inhibit the nutrient (NO3- and NH4+) uptake and nitrate reductase in a non-competitive manner. The inhibition produced by the test metals alone was of non-competitive type for NO3- uptake, nitrate reductase and ATPase and competitive for NH4+ uptake. Acidity not only inhibited the metabolic variables directly but also through facilitated uptake of metals and increased membrane permeability. A very low sensitivity of ATPase to acidic pH seems to be responsible for the survival of algae in acid environment.

Metal induced inhibition of photosynthesis, photosynthetic electron transport chain and ATP content of Anabaena doliolum and Chlorella vulgaris: interaction with exogenous ATP.

This study demonstrates a concentration dependent inhibition of carbon fixation, O2 evolution, photosynthetic electron transport chain and ATP content of A. doliolum and C. vulgaris by Cu, Ni and Fe. Although the mode of inhibition of photosynthetic electron transport chain of both the algae was similar, PS II depicted greater sensitivity to the test metals used. The toxicity in both organisms was Cu > Ni > Fe. A. doliolum was, however, more sensitive to Cu and Ni, and C. vulgaris to Fe. Toxicity was generally dependent on metal uptake, which in turn was dependent on their concentrations in the external medium. A partial restoration of nutrient uptake, carbon fixation, and enzyme activities following supplementation of exogenous ATP suggests that ATP regulates toxicity through chelation.

Impact of spectral quality on toxicity of iron in Anabaena doliolum and Chlorella vulgaris.

Evidence is presented on the effects of low and high concentrations of iron on growth, nutrient uptake (NH+4 and NO3-), photosynthesis (CO2-fixation and O2-evolution) and nitrate reductase (NR) activity of A. doliolum and C. vulgaris under monochromatic irradiation. Control cultures (not treated with FeCl3) showed maximum growth under fluorescent followed by red, yellow, blue and green lights (fluorescent greater than yellow greater than red greater than blue greater than green). The inhibition was of synergistic type under yellow and red lights at all the iron concentrations tested. However, under blue and green lights the interaction was less than additive type. All the processes studied responded in a similar manner to a particular color of light. Under fluorescent light at low Fe concentrations, stimulation of NR, 14CO2-fixation and O2-evolution was noticed in both the test organisms. However, even the lowest concentration of iron tested was inhibitory to these processes under yellow and red lights. Under blue light at 20 micrograms.ml-1 Fe, NR activity was inhibited by 98%. This study clearly demonstrates that metal toxicity to phytoplankton will be greatly affected by spectral quality, hence it will have great significance in limnological research.

Influence of chromium on some physiological variables of Anabaena doliolum: interaction with metabolic inhibitors.

The impact of 2,4-dinitrophenol and chlorophenyl dimethylurea on ATP content, carbon fixation, O2 evolution, nitrogenase activity and Cr uptake of Anabaena doliolum has been studied. 2,4-Dinitrophenol has been found to be more toxic than chlorophenyldimethylurea for all these processes. However, when Cr toxicity to above variables was assessed in their presence the interaction was less than additive. An initial (10-15 min) concentration-dependent rapid Cr uptake, followed by a slow one, indicates a biphasic uptake. A significant inhibition of Cr uptake in the presence of both these metabolic inhibitors suggests the involvement of metabolic processes in Cr uptake.

A morphometric and X-ray energy dispersive approach to monitoring pH-altered cadmium toxicity in Anabaena flos-aquae.

Cadmium toxicity and uptake as influenced by different pH values have been studied in the freshwater cyanobacterium Anabaena flos-aquae, using the techniques of morphometric analysis, x-ray energy dispersive analysis and atomic absorption spectrophotometry. A general reduction in cell dimension, thylakoid surface area, number and volume of polyhedral bodies, polyphosphate bodies, cyanophycin granules, lipid bodies, membrane limited crystalline inclusions, volume and number of wall layers and mesosomes was observed. These reductions were more pronounced in both acidic and alkaline medium than at pH 7.2. At 0.12 microM Cd, the uptake increased with alkaline pH values, and uptake was greater at pH 7.2 than at either acid or alkaline pHs. Lysis of cell wall at 1.18 microM Cd showed the following decreasing trend: pH 4.0 greater than pH 5.5 greater than pH 10.0 greater than pH 9.0 greater than pH 7.2. There was a total loss of lipid bodies at 1.18 microM Cd at all pH values listed. It is suggested that these techniques can be successfully employed for bioassay studies of metal toxicity to algae. In particular, cell wall lysis and loss of lipids by algae are good indicators of pH effects and metal toxicity in the aquatic ecosystem.

Toxicity of chromium and tin to Anabaena doliolum. Interaction with sulphur-containing amino acids and thiols.

Toxicity of chromium and tin on growth, heterocyst differentiation, nitrogenase activity and 14CO2 uptake of Anabaena doliolum and its amelioration by sulphur-containing amino acids and thiols has been studied. The final growth yield was found to be approximately 51% and 58% of control at sublethal concentration of chromium and tin respectively. Among various amino acids tested, cysteine (0.05 mM) significantly restored growth, heterocyst differentiation, nitrogenase and 14CO2 uptake of test alga. Dithiothreitol (1 mM) restored all the parameters and processes better than monothiol, mercaptoethanol. It is obvious from present investigation that sulphur-containing amino acids and thiols, viz. cysteine, methionine, cystine, mercaptoethanol and dithiothreitol, may appreciably alleviate the toxicity of heavy metals in N2-fixing cyanobacteria if present in an aquatic ecosystem.