Carbonic anhydrases in normal gastrointestinal tract and gastrointestinal tumours

Carbonic anhydrases (CAs) catalyse the hydration of C02 to bicarbonate at physiological pH. This chemical interconversion is crucial since HCO3- is the substrate for several biosynthetic reactions. This review is focused on the distribution and role of CA isoenzymes in both normal and pathological gastrointestinal (GI) tract tissues. It has been known for many years that CAs are widely present in the GI tract and play important roles in several physiological functions such as production of saliva, gastric acid, bile, and pancreatic juice as well as in absorption of salt and water in intestine. New information suggests that these enzymes participate in several processes that were not envisioned earlier. Especially, the recent reports on plasma membrane-bound isoenzymes IX and XII have raised considerable interest since they were reported to participate in cancer invasion and spread. They are induced by tumour hypoxia and may also play a role in von Hippel-Lindau (VHL)-mediated carcinogenesis.

Expression of von Hippel-Lindau tumor suppressor and tumor-associated carbonic anhydrases IX and XII in normal and neoplastic colorectal mucosa

AIM:To analyze possible relationships between CA IX/ CA XII and pVHL expression in normal and neoplastic colorectal mucosa. METHODS: Immunohistochemical staining of 42 tissue specimens obtained from 17 cancer patients was performed to evaluate the distribution and semi-quantitatively assess the levels of CA IX, CA XII and pVHL. VHL mRNAs from 14 fresh-frozen tumors was amplified by RT-PCR and subjected to sequencing. CA9 and G412mRNA levels were analyzed by semi-quantitative RT-PCR in comparison with VEGFas an indicator of hypoxia that uncouples the pVHL control. RESULTS: Tumor tissues were associated with a borderline increase of CA IX staining signal and slight but significant decrease of CA XII immunoreactivity, whereas no association was found for pVHL. Sequence analysis of RT-PCR-amplified VHL mRNAs revealed no deletions/ mutations, suggesting that they were VHL-competent. We did not observe any correlation between pVHL and CA IX/CA XII proteins as well as between MEGFand CA9 mRNAs, but the tumor-associated changes in mRNA levels of VEGFand CA12 showed a significant inverse relationship. CONCLUSION: Our results indicate that CA9and CA12 are regulated by different intratumoral factors and that lack of apparent relationship between the levels of CA IX/CA XII and pVHL cannot be fully assigned to uncoupling of negative regulatory function of pVHL by tumor hypoxia signified by induced VEGF transcription. The interplay between the functional pVHL and CA IX/CA XII in colorectal tumors seems rather complex and is not evident merely at the expression levels.

Asymmetric expression of CA2 and CA13 linked to calcification in the bilateral mandibular condyles cause crossed beaks in chickens

Crossed beak is a complex mode of inheritance with prevalence ranging from 0.2 to 7.4% in at least 12 chicken strains worldwide.To reveal the intrinsic factors causing crossed beaks,genes expression patterns in bilateral mandibular condyle between affected and normal birds were characterized by RNA sequencing analysis in the present studies.Crossed beak was induced by short length of unilateral mandibular ramus,and a total of 110differentially expressed genes were up-or down-regulated in the affected(short)mandibular condyle side as compared to the normal side.Carbonic anhydrase 2(CA2)and Carbonic anhydrase 13(CA13)were enriched in the carbonate dehydratase activity,and high-expressed in mandibular condyle and osteoblasts(P<0.05).However,both were low-expressed in short mandibular condyle side of affected birds(P<0.05).The carbonate dehydratase inhibitor experiments confirmed that there is positive association between the calcification and carbonic anhydrase isoenzymes.Quantitative analysis with cetylpyridinium chloride showed a decrease in calcification when the cells were transfected with an anti-CA13 shRNA.Our research suggested that CA2 and CA13 are down-calcified in shortside mandibular condyle,and caused mandibular ramus to grow slowly.CA2 and CA13 have the critical role in crossed beaks by regulating calcification of mandibular condyle.

Bioleaching of vanadium from stone coal vanadium ore by Bacillus mucilaginosus:Influencing factors and mechanism

Vanadium and its derivatives are used in various industries,including steel,metallurgy,pharmaceuticals,and aerospace engineering.Although China has massive reserves of stone coal resources,these resources have low grades.Therefore,the effective extraction and recovery of metallic vanadium from stone coal is an important way to realize the efficient resource utilization of stone coal vanadium ore.Herein,Bacillus mucilaginosus was selected as the leaching strain.The vanadium leaching rate reached 35.5%after 20 d of bioleaching under optimal operating conditions.The cumulative vanadium leaching rate in the contact group reached 35.5%,which was higher than that in the noncontact group(9.3%).The metabolites of B.mucilaginosus,such as oxalic,tartaric,citric,and malic acids,dominated in bioleaching,accounting for 73.8%of the vanadium leaching rate.Interestingly,during leaching,the presence of stone coal stimulated the expression of carbonic anhydrase in bacterial cells,and enzyme activity increased by 1.335-1.905 U.Enzyme activity positively promoted the production of metabolite organic acids,and total organic acid content increased by 39.31 mg·L^(-1),resulting in a reduction of 2.51 in the pH of the leaching system with stone coal.This effect favored the leaching of vanadium from stone coal.Atomic force microscopy illustrated that bacterial leaching exacerbated corrosion on the surface of stone coal beyond 10 nm.Our study provides a clear and promising strategy for exploring the bioleaching mechanism from the perspective of microbial enzyme activity and metabolites.

Carbon Capture and Storage in the Biofouling Tubeworm Ficopomatus Enigmaticus

The study investigates the potential of the serpulid tubeworm Ficopomatus sp.,sourced from Jesolo Lido(Venice,Italy),for CCS(Carbon Capture and Storage).Specimens were processed to extract bioactive compounds using a 50%polypropylene glycol-water solvent system.Extracts were analysed and purified using column gel filtration chromatography,with fractions identified by TLC(Thin Layer Chromatography)and further characterized for antioxidant and antibacterial activities.Antioxidant activity was detected via DPPH(2,2-Diphenyl-1-Picrylhydrazyl)spraying on TLC plates,while antibacterial activity was evaluated using the antibiogram paper disk diffusion method.Enzyme activity in the fractions was previously confirmed through a bromothymol blue test followed by spectrophotometric analysis.The primary goal was to explore the CCS potential,using an experimental module involving Arduino Uno embedded microprocessor for the CO_(2) measurement and to confirm the conversion into insoluble carbonates(storage).The most active fraction,identified as S1,showed significant CCS action,confirmed by microscopic observation of calcareous deposits on treated sponges.These findings suggest that Ficopomatus sp.can be used in CCS research highlighting its potential for biotechnological applications in mitigating climate change.The paper underscores the importance of marine organisms in CCS and offers insights into innovative strategies for environmental conservation and carbon management.

Plant Carbonic Anhydrases： Structures, Locations, Evolution, and Physiological Roles

Carbonic anhydrases （CAs） are zinc metalloenzymes that catalyze the interconversion of CO2 and HCO3- and are ubiquitous in nature. Higher plants contain three evolutionarily distinct CA families, αCAs, 13CAs, and γCAs, where each family is represented by multiple isoforms in all species. Alternative splicing of CA transcripts appears common; consequently, the number of functional CA isoforms in a species may exceed the number of genes. CAs are expressed in numerous plant tissues and in different cellular locations. The most prevalent CAs are those in the chloroplast, cytosol, and mitochondria. This diversity in location is paralleled in the many physiological and biochemical roles that CAs play in plants. In this review, the number and types of CAs in C3, C4, and crassulacean acid metabolism （CAM） plants are considered, and the roles of the α and yCAs are briefly discussed. The remainder of the review focuses on plant βCAs and includes the identification of homologs between species using phylogenetic approaches, a consideration of the inter- and intracellular localization of the proteins, along with the evidence for alternative splice forms. Current understanding of βCA tissue-specific expression patterns and what controls them are reviewed, and the physiological roles for which βCAs have been implicated are presented.

Combined effect of bicarbonate and water in photosynthetic oxygen evolution and carbon neutrality

Carbon neutrality is widely concerned and highly valued by many countries.Biosphere has always maintained the balance between oxidized organic substances and assimilated organic matter,resulting in netzero carbon dioxide(CO_(2)) emissions and maintaining its own carbon neutrality.Nature has set a good example for human beings to coordinate oxygen(O_(2)) balance and CO_(2)balance,and achieve carbon neutrality.How does photosynthetic oxygen evolution initiate carbon and water neutrality?My synthesis shows that photo system Ⅱ functions as carbonic anhydrase to catalyze the reaction of CO_(2)hydration under physiological conditions,and CO_(2)hydration coupled with chemical equilibrium,H^(+)+HCO_(3)^(-)→1/2O_(2)+2e^(-)+2H^(+)+CO_(2),occurs in a photosystem Ⅱ corecomplex.Meanwhile,I focused on the revisiting of four classical heavy oxygen(O^(18)) labeling experiments and found that bicarbonate can promote photo synthetic oxygen evolution,and that photo synthetic oxygen evolution can alternately come from bicarbonate and water,not only water.Bicarbonate photolysis and water photolysis account for half of the photo synthetic oxygen evolution respectively,which can well explain the bicarbonate effect,Dole effect and plants’ environmental adaptability.Photosynthetic oxygen evolution initiated the journey of water metabolism and carbon metabolism in nature,which led to the coupling as 1:1(mol/mol) stoichiometric relationship between the reduction of CO_(2)and oxidation of organic carbon,coordinated the evolution of the atmosphere,hydrosphere,lithosphere and biosphere,and realized "carbon neutrality" in the whole Earth system.

Inorganic Carbon Utilization in Some Marine Phytoplankton Species

In order to learn the ways and possible utilization mechanisms of dissolved inorganic carbon (DIC) in marine phytoplankton species under carbon-replete or -limited conditions, the activity of extracellular carbonic anhydrase (CA) was assayed in different pH, CO 2 and DIC concentrations. Extracellular CA in Amphidinium carterae and Prorocentrum minimum was detected under carbon-replete conditions, while in Melosira sp., Phaeodactylum tricornutum, Skeletonema costatum, Thalassiosira rotula, Emiliania huxleyi and Pleurochrysis carterae, CA activity was assayed under conditions of carbon limitation. No CA activity was found even under carbon-limited conditions in Chaetoceros compressus, Glenodinium foliaceum, Coccolithus pelagicus, Gephrocapsa oceanica and Heterosigma akashiwo. In species without extracellular CA activity, the direct HCO - 3 uptake was investigated using a pH drift technique and the anion exchange inhibitor 4′4′-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) in a closed system. The result showed that direct HCO - 3 transport might occur by an anion exchange mechanism in species Coc. pelagicus and G. oceanica. Of the 13 species investigated, only H. akashiwo did not have the potential for direct uptake or extracellular CA-catalyzed HCO - 3 utilization.

乳腺癌分子亚型中CAIX的表达及临床意义

目的:探讨乳腺癌各分子亚型中碳酸酐酶IX(carbonic anhydrase IX,CAIX)的表达及临床意义。方法:采用免疫组织化学检测CAIX在4种不同乳腺癌分子亚型中的表达水平并分析其与临床病理参数的关系。结果:CAIX在管腔A型、管腔B型、HER-2过表达型和基底样型乳腺癌中的阳性表达率分别为6.65%(28/421)、12.50%(7/56)、60.61%(40/66)和32.31%(84/260)。ER阴性的HER-2过表达型及基底样型乳腺癌组织中,CAIX的阳性表达明显高于ER阳性的管腔A型和管腔B型(P<0.001),并且HER-2过表达型乳腺癌组织CAIX的阳性表达率明显高于基底样型相比,差异有统计学意义。CAIX与乳腺癌的组织学分级、淋巴结转移、临床分期及高增殖指数相关(P<0.05),而与患者年龄、是否绝经及肿瘤大小无关。结论:CAIX在不同乳腺癌分子亚型中的表达具有明显差异,可能与ER阴性乳腺癌关系更密切,并在乳腺癌的侵袭、转移中发挥重要作用,可能成为乳腺癌浸润、进展及预后评估的指标。

Malaria parasite carbonic anhydrase:inhibition of aromatic/heterocyclic sulfonamides and its therapeutic potential

Plasmodium falciparum(P.falciparum) is responsible for the majority of life-threatening cases of human malaria,causing 1.5-2.7 million annual deaths.The global emergence of drug-resistant malaria parasites necessitates identification and characterisation of novel drug targets and their potential inhibitors.We identified the carbonic anhydrase(CA) genes in P.falciparum.The pfGA gene encodes an α-carbonic anhydrase,a Zn^(2+)-metalloenzme,possessing catalytic properties distinct from that of the human host CA enzyme.The amino acid sequence of the pfCA enzyme is different from the analogous protozoan and human enzymes.A library of aromatic/heterocyclic sulfonamides possessing a large diversity of scaffolds were found to be very good inhibitors for the malarial enzyme at moderate-low micromolar and submicromolar inhibitions.The structure of the groups substituting the aromatic-ureido-or aromatic-azomethine fragment of the molecule and the length of the parent sulfonamide were critical parameters for the inhibitory properties of the sulfonamides.One derivative,that is,4-(3,4-dichlorophenylureido)thioureidobcnzcnesulfonamide(compound 10) was the most effective in vitro Plasmodium falciparum CA inhibitor,and was also the most effective antimalarial compound on the in vitro P.falciparum growth inhibition.The compound 10 was also effective in vivo antimalarial agent in mice infected with Plasmodium berghei,an animal model of drug testing for human malaria infection. It is therefore concluded that the sulphonamide inhibitors targeting the parasite CA may have potential for the development of novel therapies against human malaria.

Role of Carbonic Anhydrase as an Activator in Carbonate Rock Dissolution and Its Implication for Atmospheric CO_(2)Sink

The conversion of CO2 into H+ and is a relatively slow reaction. Hence, its kinetics may be rate determining in carbonate rock dissolution. Carbonic anhydrase (CA), which is widespread in nature, was used to catalyze the CO2 conversion process in dissolution experiments of limestone and dolomite. It was found that the rate of dissolution increases by a factor of about 10 after the addition of CA at a high CO2 partial pressure (Pco2) for limestone and about 3 at low Pco2 for dolomite. This shows that reappraisal is necessary for the importance of chemical weathering (including carbonate rock dissolution and silicate weathering) in the atmospheric CO2 sink and the mysterious missing sink in carbon cycling. It is doubtless that previous studies of weathering underestimated weathering rates due to the ignorance of CA as an activator in weathering, thus the contribution of weathering to the atmospheric CO2 sink is also underestimated. This finding also shows the need to examine the situ distribution and activity of CA in different waters and to investigate the role of CA in weathering.

Nitrogen in Relation to Photosynthetic Capacity and Accumulation of Osmoprotectant and Nutrients in Brassica Genotypes Grown Under Salt Stress

Different strategies of the application of nutrients are required to overcome the adverse effects of mustard （Brassica juncea L.） in response to NaCl stress. The objective of the present study was to determine if different added levels of nitrogen （N） in growth medium could alleviate the adverse effects of salt stress on photosynthetic capacity and accumulation of osmoprotectants and nutrients. 14 days mustard seedlings of salt-sensitive （cv. Chuutki） and salt-tolerant （cv. Radha） genotypes were fed with： （i） 0 mmol L^-1 NaCl ＋ 0 mg N kg^-1 sand （control）, （ii） 90 mmol L^-1 NaCl ＋ 30 mg N kg^-1 sand, （iii） 90 mmol L^-1 NaCl ＋60 mg N kg^-1 sand, （iv） 90 mmol L^-1 NaCl ＋90 mg N kg^-1 sand and （v） 90 mmol L^-1NaCl＋ 120 mg N kg^-1 sand. Under the condition of salinity stress, N application caused a significant ameliorative effect on both genotypes with respect to growth attributes [fresh weight （FW） and dry weight （DW）] and physio-biochemical parameters [percent water content （WC）, net photosynthetic rate （PN）, stomatal conductance （gs）, total chlorophyll （Ch1）, carbonic anhydrase （CA） activity and malondialdehyde （MDA）, nitrogen （N）, potassium （K） and sodium （Na） contents, and K/Na ratio] and yield attributes （number of pods/plant, seeds/pod and seed yield/plant）. The salt-tolerant genotype exhibited maximum value for growth, physio-biochemical and yield attributes at 60 mg N kg 1 sand than that of salt-sensitive genotype. These results suggest that application of N may ameliorate most of the attributes and prove to be a physiological remedy to increase the tolerance against the ill effects of salt stress in Brassicas.

Overexpression of carbonic anhydrase Ⅱ and Ki-67 proteins in prognosis of gastrointestinal stromal tumors

AIM: To investigate the expression and prognostic value of carbonic anhydrase Ⅱ (CA Ⅱ) and Ki-67 in gastrointestinal stromal tumors (GISTs). METHODS: One hundred and thirteen GIST patients admitted to Chinese People's Liberation Army General Hospital from January 2004 to December 2010 were retrospectively followed up, and immunohistochemistry was used to detect CA Ⅱ, Ki-67 and CD117 expression in tumor samples. The survival rates of the patients were analyzed using the Kaplan-Meier method. Log-rank test, χ 2 test and Cox proportional hazards model were used to determine the relationships between CA Ⅱ, Ki-67 and CD117 expression and prognostic value in GISTs. RESULTS: The survival rates at 1, 3 and 5 years were 90.0%, 82.0% and 72.0% in all patients. However, in patients with positive CA Ⅱ or Ki-67, the survival rates were 92.0%, 83.0% and 77.0% or 83.0%, 66.6% and 53.0%, respectively. Compared with the negative groups, the survival rates in the positive groups were significantly lower (CA Ⅱ log-rankP = 0.000; Ki-67 logrank P = 0.004). Multivariate Cox analysis revealed that CA Ⅱ, CD117 and Ki-67 were considerable immune factors in prognosis of GIST patients (CA Ⅱ P = 0.043; CD117 P = 0.042; Ki-67 P = 0.007). Besides, tumor diameter, mitotic rate, tumor site, depth of invasion, complete resection, intraoperative rupture, and adjuvant therapy were important prognosis predictive factors. Our study indicated that CA Ⅱ had strong expression in GISTs and the prognosis of GISTs with high CA Ⅱ expression was better than that of GISTs with low or no expression, suggesting that CA Ⅱ is both a diagnostic and prognostic biomarker for GIST. CONCLUSION: CA Ⅱ and Ki-67 are significant prognostic factors for GISTs. CA Ⅱ associated with neovascular endothelia could serve as a potential target for cancer therapy.

Carbonic anhydrase enzymes Ⅱ, Ⅶ, Ⅸ and Ⅻ in colorectal carcinomas

AIM To investigate expression of four alpha-carbonic anhydrases(CAs) in colorectal carcinomas(CRC) and compare the results with patients' survival.METHODS Colorectal carcinoma samples from 539 CRC patients and control tissues were arranged as tissue microarrays and analyzed with antibodies against CA Ⅱ, CA Ⅶ, CA Ⅸ, and CA Ⅻ. Intensity and extent of staining were both scored from 0 to 3 in each sample. These enzyme expression levels were then correlated to patients' survival and clinicopathological parameters, which were tumor differentiation grade and stage, site of tumor, patients' age, and gender. Kaplan-Meier analysis and Cox regression hazard ratio model were used to analyze survival data. RESULTS CA Ⅱ and CA Ⅻ staining intensities correlated with patients' survival in that higher expression indicated poorer prognosis. In Cox regression analysis one unit increase in the CA Ⅱ intensity increased the hazard ratio to 1.19 fold(CI: 1.04-1.37, P = 0.009). A significant correlation was also found when comparing CA Ⅻ staining intensity with survival of CRC patients(HR = 1.18, 95%CI: 1.01-1.38, P = 0.036). The extent of CA Ⅻ immunostaining did not correlate to the patients' survival(P = 0.242, Kaplan-Meier analysis). A significant interaction between age group and extent of the CA Ⅱ staining was found. Increased extent of CA Ⅱ had a significant hazard ratio among patients 65 years and older(1.42, 95%CI: 1.16-1.73, P = 0.0006). No correlations were found between CA Ⅶ(intensity P = 0.566, extent P = 0.495, Kaplan-Meier analysis), or CA Ⅸ(intensity P = 0.879, extent P = 0.315, KaplanMeier analysis) immunostaining results and survival, or the other parameters. CONCLUSION The present findings indicate that CA Ⅱ and CA Ⅻ could be useful in predicting survival in CRC.

Immobilization of carbonic anhydrase for facilitated CO2 capture and separation

Carbonic anhydrase(CA)as a typical metalloenzyme in biological system can accelerate the hydration/dehydration of carbon dioxide(CO2,the major components of greenhouse gases),which performer with high selectivity,environmental friendliness and superior efficiency.However,the free form of CA is quite expensive(~RMB 3000/100 mg),unstable,and non-reusable as the free form of CA is not easy for recovery from the reaction environment,which severely limits its large-scale industrial applications.The immobilization may solve these problems at the same time.In this context,many efforts have been devoted to improving the chemical and thermal stabilities of CA through immobilization strategy.Very recently,a wide range of available inorganic,organic and hybrid compounds have been explored as carrier materials for CA immobilization,which could not only improve the tolerance of CA in hazardous environments,but also improve the efficiency and recovery to reduce the cost of large-scale application of CA.Several excellent reviews about immobilization methods and application potential of CA have been published.By contrast,in our review,we stressed on the way to better retain the biocatalytic activity of immobilized CA system based on different carrier materials and to solve the problems facing in practical operations well.The concluding remarks are presented with a perspective on constructing efficient CO2 conversion systems through rational combining CA and advanced carrier materials.

Effects of carbon anhydrase on utilization of bicarbonate in microalgae:a case study in Lake Hongfeng

A bidirectional labeling method was established to distinguish the proportions of HCO3- and CO2 utiliza- tion pathways of microalgae in Lake Hongfeng. The method was based on microalgae cultured in a medium by adding equal concentrations of NaH13CO3 with different 613C values simultaneously. The inorganic carbon sources were quantified according to the stable carbon isotope composition in the treated microalgae. The effects of extracellular carbonic anhydrase （CAex） on the HCO3 and CO2 utilization pathways were distinguished using acetazolamide, a potent membrane-impermeable carbonic anhydrase inhibitor. The results show utilization of the added HCO3- was only 8% of the total carbon sources in karst lake. The proportion of the HCO3- utilization path- way was 52% of total inorganic carbon assimilation. Therefore, in the natural water of the karst area, the microalgae used less bicarbonate that preexisted in the aqueous medium than CO2 derived from the atmosphere. CAex increased the utilization of inorganic carbon from the atmosphere. The microalgae with CAex had greater carbon sequestration capacity in this karst area.

Targeting neuronal nitric oxide synthase as a valuable strategy for the therapy of neurological disorders

The management of neurological disorders have huge and increasing human and economic costs. Despite this, there is a scarcity of effective therapeutics, and there is an extreme urgency for new and real treatments. In this short review we analyze some promising advancements in the search of new bioactive molecules targeting neuronal nitric oxide synthase （nNOS）, an enzyme deputed to the biosynthesis of nitric oxide （NO）. In different conditions of neuronal damages, this molecule is overproduced, contributing to the pathogenesis and progression of neuronal diseases. Two main approaches to modulate nNOS are discussed： a first one consisting in the direct inhibition of the enzyme by means of small organic molecules, which can be also active against other different targets involved in such diseases. A second section is dedicated to molecules able to prevent the formation of the ternary complex N-methyl-D-aspartate （NMDA）type glutamate receptors, postsynaptic density-95 （PSD95） protein-nNOS, which is necessary to activate the latter for the biosynthesis of NO.

Interactive Effect of GA_3 ,N and P Ameliorate Growth, Seed and Fibre Yield by Enhancing Photosynthetic Capacity and Carbonic Anhydrase Activity of Linseed: A Dual Purpose Crop

Linseed （Linum usitatissimum L.） is an important dual-purpose, industrial crop. Its seeds are used for the extraction of oil and stem for fibres. However, the production of linseed is not going parallel with the increasing demand of its products. The present work was carried out with an aim to find out whether exogenous application of gibberellic acid （GA3） with or without graded levels of nitrogen （N） and phosphorus （P） could improve the performance of three linseed genotypes Parvati, Shekhar and Shubhra together with minimizing the costly fertilizer input and losses. Four combinations of N and P, viz., 0 mg N＋0 mg P kg-1 soil （N0P0）, N13.4 P4.46 , N26.8P8.94 and N40.2P13.4 were constituted. Half dose of each combination was applied basally at the time of sowing and remaining half dose was given at 40 d after sowing （DAS） as foliar spray along with 10-6 mol L-1 GA3 . Prior to sowing, the seeds of each linseed genotype were grouped in to two, one group of seeds was soaked in 0 mol L-1 GA3 （control） and the other group was soaked in 10-6 M GA3 solution, each for 8 hours. Treatments were comprised of （i） 0 mol L-1 GA3＋N0P0 （T0 , control）, （ii） 10-6 mol L-1 GA3 ＋ N13.4P4.46 （T1）, （iii） 10-6 mol L-1 GA 3 ＋N 26.8 P 8.94 （T2） and （iv） 10-6 mol L-1 GA3＋N40.2P13.4 （T3）. The crop performance was assessed in terms of growth, physiological and biochemical parameters at 60 and 75 DAS and yield attributes at harvest （175 DAS）. The results showed a parallel increase in most of the parameters with increasing levels of N and P. However, application of 10-6 mol L-1 GA3 in association with N26.8P8.94 proved best, it enhanced seed yield, oil yield and fibre yield by 83.3, 97.3 and 78.7%, respectively accompanied with increase in net photosynthetic rate, carbonic anhydrase activity and dry matter accumulation. Among the genotypes tested, Shubhra performed best, while Parvati the least for most of the parameters studied. Thus, combined application of 10-6 mol L-1 GA3 plus N26.8P8.94 proved best and can be recommended to exploit the linseed as a dual-purpose crop for good yield of seed and fibre.

Is bicarbonate directly used as substrate to participate in photosynthetic oxygen evolution

If the photosynthetic organisms assimilated only CO_(2) in the Archean atmosphere,hydroxide ion in the Archean seawater would not increase.If plants would not consume bicarbonate as a direct substrate during photosynthesis,it is difficult to explain the evolution of Earth's environment.To date,it is generally accepted that photosynthetic O_(2) evolution of plants come from water photolysis.However,it should be debated by evaluating the effect of bicarbonate in photosynthetic O_(2) evolution,analyzing the role of carbonic anhydrase(CA) in photosynthetic O_(2) evolution,and the relationship between thylakoid CA and photosynthetic O_(2) evolution.In the paper,I propose that bicarbonate is directly used as substrate to participate in photosynthetic O_(2) evolution.The rationality of bicarbonate photolysis of plants is discussed from the thermodynamics and evolution of Earth's environment.The isotopic evidence that bicarbonate is not the direct substrate of photosynthetic O_(2) release is reexamined,and the new explanation of bicarbonate photolysis in photosynthetic O_(2) evolution is proposed.

Hypoxia and cytokines regulate carbonic anhydrase 9 expression in hepatocellular carcinoma cells in vitro

AIM: To study the expression of carbonic anhydrase(CA) 9 in human hepatocellular carcinoma(HCC) cells.METHODS: We studied CA9 protein, CA9 m RNA and hypoxia-inducible factor-1 alpha(HIF-1α) protein levels in Hep3 B cells exposed in different parallel approaches. In one of these approaches, HCC cells were exposed to extreme in vitro hypoxia(24 h 0.1% O2) without or with interleukin(IL)-1, IL-6, tumor necrosis factoralpha(TNF-α) and transforming growth factor-beta(TGF-β) stimulation for the same hypoxic exposure time or exposed to normoxic oxygenation conditions without or with cytokine stimulation.RESULTS: The tumour cell line analysed showed a strong hypoxic CA9 m RNA expression pattern in response to prolonged severe hypoxia with cell-line specific patterns and a marked induction of CA9 protein in response to severe hypoxia. These results were paralleled by the results for HIF-1α protein under identical oxygenation conditions with a similar expression tendency to that displayed during the CA9 protein expression experimental series. Continuous stimulation with the cytokines, IL-1, IL-6, TNF-α and TGF-β, under normoxic conditions significantly increased the carbonic anhydrase 9 expression level at both the protein and m RNA level, almost doubling the CA9 m RNA and CA9 and HIF-1α protein expression levels found under hypoxia. The findings from these experiments indicated that hypoxia is a positive regulator of CA9 expression in HCC, and the four signal transduction pathways, IL-1, IL-6, TNF-α and TGF-β, positively influence CA9 expression under both normoxic and hypoxic conditions.CONCLUSION: These findings may potentially be considered in the design of anti- cancer therapeutic approaches involving hypoxia-induced or cytokine stimulatory effects on expression. In addition, they provideevidence of the stimulatory role of the examined cytokine families resulting in an increase in CA9 expression under different oxygenation conditions in human cancer, especially HCC, and on the role of the CA9 gene as a positive disease regulator in human cancer.