Arabidopsis KEA2, a homolog of bacterial KefC, encodes a K(+)/H(+) antiporter with a chloroplast transit peptide

KEA genes encode putative K(+) efflux antiporters that are predominantly found in algae and plants but are rare in metazoa; however, nothing is known about their functions in eukaryotic cells. Plant KEA proteins show homology to bacterial K(+) efflux (Kef) transporters, though two members in the Arabidopsis thaliana family, AtKEA1 and AtKEA2, have acquired an extra hydrophilic domain of over 500 residues at the amino terminus. We show that AtKEA2 is highly expressed in leaves, stems and flowers, but not in roots, and that an N-terminal peptide of the protein is targeted to chloroplasts in Arabidopsis cotyledons. The full-length AtKEA2 protein was inactive when expressed in yeast; however, a truncated AtKEA2 protein (AtsKEA2) lacking the N-terminal domain complemented disruption of the Na(+)(K(+))/H(+) antiporter Nhx1p to confer hygromycin resistance and tolerance to Na(+) or K(+) stress. To test transport activity, purified truncated AtKEA2 was reconstituted in proteoliposomes containing the fluorescent probe pyranine. Monovalent cations reduced an imposed pH gradient (acid inside) indicating AtsKEA2 mediated cation/H(+) exchange with preference for K(+)=Cs(+)>Li(+)>Na(+). When a conserved Asp(721) in transmembrane helix 6 that aligns to the cation binding Asp(164) of Escherichia coli NhaA was replaced with Ala, AtsKEA2 was completely inactivated. Mutation of a Glu(835) between transmembrane helix 8 and 9 in AtsKEA2 also resulted in loss of activity suggesting this region has a regulatory role. Thus, AtKEA2 represents the founding member of a novel group of eukaryote K(+)/H(+) antiporters that modulate monovalent cation and pH homeostasis in plant chloroplasts or plastids.     

Breast cancer is associated with an increase in the activity and expression of cholinephosphotransferase in rats

AIM: The present study aims to establish that cholinephosphotransferase (CPT), the terminal enzyme for the de novo biosynthesis of phosphatidylcholine (PC), can be used as a biomarker for breast cancer in an animal model. MAIN METHODS: Breast cancer was induced by intragastric administration of dimethylbenz(a)anthracene (DMBA) in rats. The activity and expression of CPT were compared between normal breast tissues and breast tumors. To establish possible mechanistic model, we looked into other enzymes of PC biosynthesis as well as c-fos protein expression and DNA binding. KEY FINDINGS: CPT enzyme activity and its expression were significantly higher in breast cancer tissues relative to normal breast tissues. Corresponding to the increase in the CPT activity and its expression, c-fos activity and its expression were also increased in breast tumors. SIGNIFICANCE: The present study suggests that increased CPT activity and expression is associated with DMBA-induced breast cancer development.     

香蕉组织培养过程中内生菌污染的控制

研究了体外培养一种孟加拉传统香蕉(Musa spp.Cv. Kanthali)的茎尖组织。茎尖的原始细胞表面经无菌处理(0.1%HgCl2处理12min) ,接种6~15d后外植体地下茎部分仍有微生物污染(大部分是细菌) ,杀死了85%的外植体。为确定无污染培养基,将等量外植体分别浸泡在含400mg/L氨苄青霉素和200mg/L庆大霉素(两种光谱抗生素)的培养基中1h。结果表明,经抗生素处理的外植体完全没有污染,但培养3周后不能再生。进行二次继代培养后,其中一部分外植体吸收了培养基并胀大,颜色由苍白转变成浅绿或深绿。三次继代培养后数天,不再观察到外植体的生长,所有经抗生素处理过的外植体都开始死亡。在未经抗生素处理的活外植体中,单个茎发育的最佳培养基是:MS 4.0mg/L BA 0.5mg/L KT 15% CW,平均生长时间为18~21d,但再生率很低,只有30%。茎细胞增殖的最佳培养基是:MS 4.0mg/L BA 2.0mg/LIAA 15% CW,每个茎平均只萌发3~4个芽。最后,在添加0.5mg/LIBA的一半浓度的MS培养基中,体外培养茎最大生根率达到90%。     

The effect of mainstream and sidestream cigarette smoke exposure on oxygen defense mechanisms of guinea pig erythrocytes

We have studied the effects of short-term exposure of guinea pigs to cigarette smoke under both mainstream (MS) and sidestream (SS) conditions on the activities of major antioxidant enzymes and lipid peroxidation potential of erythrocytes. The smoke-exposed groups had an increase in the activity of superoxide dismutase (SOD), a decrease in the activities of glutathione peroxidase (GSH-Px) and NADPH generating enzymes, and no change in the activity of catalase. Furthermore, there was a significant increase in the in vitro lipid peroxidation potential of erythrocytes in both MS- and SS-exposed groups. However, the lipid peroxidation potential was higher in the MS-exposed group than that in the SS-exposed group.     

Inhibition of photosystem II activity by Cu++ ion. Choice of buffer and reagent is critical

Cupric ion (Cu⁺⁺) inhibits the rate of photosystem II electron transport and the intensity of the variable part of chl a fluorescence in isolated chloroplast thylakoids. The inhibition is markedly dependent on the nature of the buffer used in the assay medium. In MES and HEPES buffers, complete inhibition of photosystem II occurs at 30 M of Cu⁺⁺, while in Tricine no inhibition occurred even at 200 M Cu⁺⁺. In other buffers used (TES, Phosphate, Tris), the efficacy of Cu⁺⁺ inhibition is intermediate. The calculated binding constants are found to correspond to the observed I₅₀ values for the six buffers used. It is concluded that the previous reports on copper inhibition, where buffers have been used indiscriminately should be reconsidered. Certain reagents such as hydroxylamine, ascorbate and diphenyl carbazide, which react with Cu⁺⁺, should be avoided.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenol indophenol - DCMU 3-(3,4 dichlorophenyl)-1,1-dimethyl urea - DAD diaminodurene - DPC diphenyl carbazide - Fv variable chl fluorescence - HEPES N-2-hydroxyethyl piperazine sulfonic acid - I ₃₀ inhibitor concentration causing 30% inhibition of Fv - MES 2-(N-morpholino) ethane sulfonic acid - MV Methyl viologen - PS II Photosystem II - PS I Photosystem I - TES N-tris(hydroxymethyl)-methyl-2-amino sulfonic acid - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - Tricine N-tris(hydroxymethyl) ethylglycine - Tris N-tris(hydroxymethyl)amino ethane     

Plasma Proteome Database as a resource for proteomics research

Plasma is one of the best studied compartments in the human body and serves as an ideal body fluid for the diagnosis of diseases. This report provides a detailed functional annotation of all the plasma proteins identified to date. In all, gene products encoded by 3778 distinct genes were annotated based on proteins previously published in the literature as plasma proteins and the identification of multiple peptides from proteins under HUPO's Plasma Proteome Project. Our analysis revealed that 51% of these genes encoded more than one protein isoform. All single nucleotide polymorphisms involving protein-coding regions were mapped onto the protein sequences. We found a number of examples of isoform-specific subcellular localization as well as tissue expression. This database is an attempt at comprehensive annotation of a complex subproteome and is available on the web at http://www.plasmaproteomedatabase.org.     

Estrogen rescues heart failure through estrogen receptor Beta activation

Background

Recently, we showed that exogenous treatment with estrogen (E2) rescues pre-existing advanced heart failure (HF) in mice. Since most of the biological actions of E2 are mediated through the classical estrogen receptors alpha (ERα) and/or beta (ERβ), and both these receptors are present in the heart, we examined the role of ERα and ERβ in the rescue action of E2 against HF.

Methods

Severe HF was induced in male mice by transverse aortic constriction-induced pressure overload. Once the ejection fraction (EF) reached ~?35%, mice were treated with selective agonists for ERα (PPT, 850 μg/kg/day), ERβ (DPN, 850 μg/kg/day), or E2 (30 μg/kg/day) together with an ERβ-antagonist (PHTPP, 850 μg/kg/day) for 10 days.

Results

EF of HF mice was significantly improved to 45.3?±?2.1% with diarylpropionitrile (DPN) treatment, but not with PPT (31.1?±?2.3%). E2 failed to rescue HF in the presence of PHTPP, as there was no significant improvement in the EF at the end of the 10-day treatment (32.5?±?5.2%). The improvement of heart function in HF mice treated with ERβ agonist DPN was also associated with reduced cardiac fibrosis and increased cardiac angiogenesis, while the ERα agonist PPT had no significant effect on either cardiac fibrosis or angiogenesis. Furthermore, DPN improved hemodynamic parameters in HF mice, whereas PPT had no significant effect.

Conclusions

E2 treatment rescues pre-existing severe HF mainly through ERβ. Rescue of HF by ERβ activation is also associated with stimulation of cardiac angiogenesis, suppression of fibrosis, and restoration of hemodynamic parameters.

     

Changes in the antenna size of Photosystem I and Photosystem II in Synechococcus sp. strain PCC 7942 grown in the presence of SANDOZ 9785 — a Photosystem II inhibitor

SANDOZ 9785, also known as BASF 13.338, is a pyridazinone derivative that inhibits Photosystem II (PS II) activity leading to an imbalance in the rate of electron transport through the photosystems. Synechococcus sp. strain PCC 7942 cells grown in the presence of sublethal concentration of SANDOZ 9785 (SAN 9785) for 48 hours exhibited a 20% decrease in Chl a per cell. However, no changes were observed in the content of phycocyanin per cell, the size of the phycobilisomes or in the PS II:PS I ratio. From an estimate of PS II electron transport rate under varying light intensities and spectral qualities and analysis of room temperature Chl a fluorescence induction, it was deduced that growth of Synechococcus PCC 7942 in the presence of SAN 9785 leads to a redistribution of excitation energy in favour of PS II. Though the redistribution appears to be primarily caused by changes affecting the Chl a antenna of PS II, the extent of energetic coupling between phycobilisomes and PS II is also enhanced in SAN 9785 grown Synechococcus PCC 7942 cells. There was a reduction in the effective size of PS I antenna based on measurement of P700 photooxidation kinetics. These results indicate that when PS II is partially inhibited, the structure of photosynthetic apparatus alters to redistribute the excitation energy in favour of PS II so that the efficiency of utilization of light energy by the two photosystems is optimized. Our results suggest that under the conditions used, drastic structural changes are not essential for redistribution of excitation energy between the photosystems.Abbreviations APC Allophycocyanin - Chl a chlorophyll a - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCMU 3-(3,4-dichlorophyenyl)-1,1-dimethyl urea - DCIP 2,6-dichlorophenolindophenol - F_o fluorescence when all the reaction centres are open - f_m fluorescence yield when all the reaction centres are closed - F_v variable chlorophyll fluorescence - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulphonic Acid - I₅₀ concentration that causes 50% inhibition in activity - MV methyl viologen - pBQ para benzoquinone - PBS phycobilisome - PC phycocyanin - PS I, PS II Photosystem I, Photosystem II - P700 reaction centre Chl a of PS I - SAN 9785 SANDOZ 9785 i.e. 4-chloro-5-dimethylamino-2-phenyl-3 (2H) pyridazinone, also known as BASF 13.338     

Momordicatin purified from fruits of Momordica charantia is effective to act as a potent antileishmania agent

Aqueous extract of the green fruits of the Indian plant Momordica charantia and purified Momordicatin structurally established as 4-(o-carboethoxyphenyl) butanol were evaluated in vitro and in vivo against kala-azar caused by Leishmania donovani. 50% inhibitory concentration (IC₅₀) against Leishmania promastigotes in vitro for the crude extract and momordicatin were 0.6 mg/L and 0.02 mg/L, respectively. When administered in the hamster model of visceral leishmaniasis, 100% parasite clearance was achieved at a dose of 300 mg/kg body weight of crude extract and 10 mg/kg body weight of Momordicatin. Fe containing parasite superoxide dismutase (SOD) was totally inhibited when treated with 0.72 mg/L crude extract and 0.20 mg/L Momordicatin, respectively, whereas Cu–Zn containing SOD present in host remained unaffected. Results reveal that the mode of action of these newly found antileishmanial agents is mediated through inhibiting parasite SOD which is one of the key enzymes of the oxidative burst. It may be proposed from the present study that both crude extract of Momordica charantia and Momordicatin obtained from the fruits of the said plant may be considered as potential candidates towards developing new chemotherapeutics against leishmaniasis.