Accumulation of cadmium ions in the methylotrophic yeast Hansenula polymorpha

Intracellular cadmium (Cd²⁺) ion accumulation and the ability to produce specific Cd²⁺ ion chelators was studied in the methylotrophic yeast Hansenula polymorpha. Only one type of Cd²⁺ intracellular chelators, glutathione (GSH), was identified, which suggests that sequestration of this heavy metal in H. polymorpha occurs similarly to that found in Saccharomyces cerevisiae, but different to Schizosaccharomys pombe and Candida glabrata which both synthesize phytochelatins. Cd²⁺ ion uptake in the H. polymorpha wild-type strains appeared to be an energy dependent process. It was found that Δgsh2 mutants, impaired in the first step of GSH biosynthesis, are characterized by increase in net Cd²⁺ ion uptake by the cells, whereas Δgsh1/Δmet1 and Δggt1 mutants impaired in sulfate assimilation and GSH catabolism, respectively, lost the ability to accumulate Cd²⁺ intracellularly. Apparently H. polymorpha, similarly to S. cerevisiae, forms a Cd-GSH complex in the cytoplasm, which in turn regulates Cd²⁺ uptake. Genes GSH1/MET1 and GGT1 are involved in maturation and metabolism of cellular Cd-GSH complex, respectively. Transport of [³H]N-ethylmaleimide-S-glutathione ([³H]NEM-SG) conjugate into crude membrane vesicules, purified from the wild-type cells of H. polymorpha appeared to be MgATP dependent, uncoupler insensitive and vanadate sensitive. We suggest that MgATP dependent transporter involved in Cd-GSH uptake in H. polymorpha, is similar to S. cerevisiae Ycf1-mediated vacuolar transporter responsible for accumulation of organic GS-conjugates and Cd-GSH complex.     

Mutation breeding of chitosanase-producing strain Bacillus sp. S65 by low-energy ion implantation

In order to obtain an industrial strain with higher chitosanase yield, the wild strain Bacillus sp. S65 cells were mutated by a novel mutagen, nitrogen ion beam, with energy of 15 keV and dose ranging from 2.6 × 10¹⁴ to 5.2 × 10¹⁵ ions/cm². One mutant, s65F5 with high yield of chitosanase was isolated. Results showed that the production of chitosanase of s65F5 was dramatically increased from 4.1 U/ml in s65 to 25 U/ml by ion beam implantation, while the fermentation time was shortened from 72 to 56 h, both of which greatly increased efficiency and reduced the cost of industrial production. Besides, the mutagenic effects of low-energy ion beam on survival rate showed characteristic down–up–down pattern, which was different from the traditional mutagens such as UV and γ-ray and the possible mutation mechanism was discussed.     

Changes in DNA base sequences in the mutant of Arabidopsis thaliana induced by low-energy N+ implantation

To reveal the mutation effect of low-energy ion implantation on Arabidopsis thaliana in vivo, T80II, a stable dwarf mutant, derived from the seeds irradiated by 30 keV N⁺ with the dose of 80×10¹⁵ ions/cm² was used for Random Amplified Polymorphic DNA (RAPD) and base sequence analysis. The results indicated that among total 397 RAPD bands observed, 52 bands in T80II were different from those of wild type showing a variation frequency 13.1%. In comparison with the sequences of A. thalianain GenBank, the RAPD fragments in T80II were changed greatly in base sequences with an average rate of one base change per 16.8 bases. The types of base changes included base transition, transversion, deletion and insertion. Among the 275 base changes detected, single base substitutions (97.09%) occurred more frequently than base deletions and insertions (2.91%). And the frequency of base transitions (66.55%) was higher than that of base transversions (30.55%). Adenine, thymine, guanine or cytosine could be replaced by any of other three bases in cloned DNA fragments in T80II. It seems that thymine was more sensitive to the irradiation than other bases. The flanking sequences of the base changes in RAPD fragments in T80II were analyzed and the mutational “hotspot” induced by low-energy ion implantation was discussed.     

等离子体浸没N+注入豌豆种子的生长发育效应初报

就等离子体浸没离子注入豌豆休眠种子后 ,对其生长发育的遗传学效应进行了研究。结果表明 ,经处理后的种子的萌发率明显下降 ,出苗、生长发育均呈滞后现象 ,且降低了株高。 2 0min为半致死剂量。提出等离子体浸设离子注入可以作为诱变育种的一种经济、有效的诱变方法。     

A Novel Approach to Isolation and Functional Characterization of Genomic DNA Sequences from the Methylotrophic Yeast Hansenula polymorpha

A novel approach to isolation and functional characterization of the Hansenula polymorpha genes basing on the use of two strains of different origin is described. One of these strains is better suited for the isolation of genomic DNA fragments, while the other is preferable for their functional analysis. Thirty three genomic sequences governing expression of a reporter protein have been isolated. Analysis of the sequence encoding a homolog of the Saccharomyces cerevisiae cofilin revealed two introns. Another isolated DNA fragment encoded a homolog of the S. cerevisiae Vps10p. Disruption of the corresponding gene resulted in secretion of a vacuolar protein, carboxypeptidase Y, into the culture medium.     

A highly efficient integrated chromatographic procedure for the purification of recombinant hepatitis B surface antigen from Hansenula polymorpha

The high expression level of recombinant hepatitis B surface antigen obtained from Hansenula polymorpha yeast cell (Hans-HBsAg) made it possible to produce HBsAg vaccine in a large scale and by cost-effective process. However, the present available purification process was somewhat tedious, time-consuming and difficult to scale up. To improve the purification efficiency and simplify the purification process, an integrated chromatographic process was developed and optimized. The downstream process included ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and gel filtration chromatography (GFC). A series of chromatographic adsorbents were evaluated for their performances on the purification of Hans-HBsAg, and then the suitable adsorbents for IEC and HIC were screened out, respectively. After clarification by centrifugation, the supernatant of cell disruption (SCD) was purified by standard chromatographic steps, IEC on DEAE Sepharose FF, HIC on Butyl-S-QZT and GFC on Sepharose 4FF. Furthermore, HBsAg recovery, purification factor (PF) and purity during the downstream process were evaluated with enzyme-linked immunosorption assay (ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance size-exclusion chromatography (HPSEC). The results demonstrated that in the scale of 550ml SCD, the total HBsAg recovery and PF of the whole procedure were about 21.0+/-0.9% and 80.7+/-8.4 (n=3) respectively, with the purity of above 99%. This new downstream process was efficient, reproducible and relatively easy to be scaled up.     

Enhanced utilization-rate of methanol during growth on a mixed substrate: A continuous culture study with Hansenula polymorpha

The yeast Hansenula polymorpha was grown in a chemostat using either methanol or sorbitol as substrate or a mixture of both. Methanol alone could be utilized up to a dilution rate (D) of 0.18 h^-1, and sorbitol allowed growth at D's higher than 0.52 h^-1. In combination with sorbitol, methanol was completely utilized in the mixture even up to a D of 0.3 h^-1, and partially utilized at higher D's, To elucidate the basis of methanol utilization at high D's, enzyme activities on the single substrates and on the substrate mixture were compared. At D's above 0.3 h^-1 an increase of formate dehydrogenase activity was evident, an enzyme involved in the oxidation of methanol to carbon dioxide. It was concluded that at high D's large amounts of methanol were oxidized to generate energy. This was proved with ¹⁴C-methanol, and it was found that in the range of partial methanol utilization approximately 75% of methanol was converted to carbon dioxide and 25% incorporated into cell material.Abbreviation D dilution rate     

离子束介导甘蓝全DNA转化拟南芥菜的分子分析

30 keV的Ar+离子束在1.5×1017 ions/cm2的注入剂量下介导外源甘蓝全DNA导入模式植物拟南芥菜,在94株转化当代植株中,有6株表型产生变异。以其中的一株(T-5)作为研究对象,用80条10碱基随机引物对该株和其子代变异株基因组作随机扩增的多态性DNA分析,引物S176 在T-5和其变异子代T-5-2中扩增出了相同分子量的变异新条带T-5S176-620。T-5S176-620的碱基序列和拟南芥菜基因组序列进行同源比对,结果表明该片段不属于拟南芥菜基因组,Southern杂交实验证明该片段来自供体甘蓝基因组。但是,根据T-5S176-620序列设计的引物不能从甘蓝基因组中扩增出预期长度的DNA片段,结合离子束介导外源全DNA转化的特点和过程,探讨了其中可能的机制。     

Silver ion (Ag+)-Induced increases in cell membrane K+ and Na+ permeability in the renal proximal tubule: Reversal by thiol reagents

Summary The initial mechanisms of injury to the proximal tubule following exposure to nephrotoxic heavy metals are not well established. We studied the immediate effects of silver (Ag⁺) on K⁺ transport and respiration with extracellular K⁺ and O₂ electrodes in suspensions of renal cortical tubules. Addition of silver nitrate (AgNO₃) to tubules suspended in bicarbonate Ringer's solution caused a rapid, dose-dependent net K⁺ efflux (K _m =10^–4 m,V _max=379 nmol K⁺/min/mg protein) which was not inhibited by furosemide, barium chloride, quinine, tetraethylammonium, or tolbutamide. An increase in the ouabain-sensitive oxygen consumption rate (QO₂) (13.9±1.1 to 25.7±4.4 nmol O₂/min/mg,P<0.001), was observed 19 sec after the K⁺ efflux induced by AgNO₃ (10^–4 m), suggesting a delayed increase in Na⁺ entry into the cell. Ouabain-insensitive QO₂, nystatin-stimulated QO₂, and CCCP-uncoupled QO₂ were not significantly affected, indicating preserved function of the Na⁺, K⁺-ATPase and mitochondria. External addition of the thiol reagents dithiothreitol (1mm) and reduced glutathione (1mm) prevented and/or immediately reversed the effects on K⁺ transport and QO₂. We conclude that Ag⁺ causes early changes in the permeability of the cell membrane to K⁺ and then to Na⁺ at concentrations that do not limit Na⁺, K⁺-ATPase activity or mitochondrial function. These alterations are likely the result of a reversible interaction of Ag⁺ with sulfhydryl groups of cell membrane proteins and may represent initial cytotoxic effects common to other sulfhydryl-reactive heavy metals on the proximal tubule.     

Crystal Structures of Sodium-Bound Annexin A4

Annexin A4 (Anx4) possesses four repeat domains with one Ca²⁺-binding site (CBS) in each domain. In this study, we resolved two crystal structures of the Na⁺-bound form at high resolution (1.58 and 1.35 Å). This is the first report that Anx4 binds the Na⁺ ion in CBSs. Electron density maps, valence screening, and atomic absorbance spectrometry confirmed that Anx4 bound the Na⁺ ion. One structure (1.58 Å) bound the Na⁺ ion in CBS I, whereas another structure (1.35 Å) bound the Na⁺ ion in CBS II and CBS III. We compared the two Na⁺-bound forms by superimposing their C^α traces. The C^α atoms of CBS III largely moved by coordination of the Na⁺ ion. In the C^α atoms of CBS I, however, little change resulted from Na⁺-coordination. Only the side chain of Glu71 was moved by Na⁺-coordination in CBS I. These results indicate that Anx4 also binds not only Ca²⁺ but also Na⁺ ion in the CBS.     

Short-term effect on intestinal epithelial Na(+)/H(+) exchanger by Gi(alpha1,2)-coupled 5-HT(1A) and G(q/11)-coupled 5-HT(2) receptors

The present study evaluated the effect of 5-hydroxytryptamine (5-HT) on intestinal Na(+)/H(+) exchanger (NHE) activity and the cellular signaling pathways involved in T84 cells. T84 cells express endogenous NHE1 and NHE2 proteins, detected by immunoblotting, but not NHE3. The rank order for inhibition of NHE activity in acid-loaded T84 cells was 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; IC(50)=519 [465, 579] nM)>cariporide (IC(50)=630 [484, 819] nM)>amiloride (IC(50)=19 [16, 24] microM); the NHE3 inhibitor S3226 was found to be devoid of effect. This different inhibitory sensitivity indicates that both NHE1 and NHE2 isoforms may play an active role in Na(+)-dependent intracellular pH (pH(i)) recovery in T84 cells. Short-term exposure (0.5 h) of T84 cells to 5-HT increased NHE activity in a concentration-dependent manner. The stimulation induced by 5-HT (30 microM) was partially inhibited by both WAY 100135 (300 nM) and ketanserin (300 nM), antagonists of 5-HT(1A) and 5-HT(2) receptors, respectively. NHE activity was significantly increased by 8-OH-DPAT and alpha-methyl-5-HT, agonists of, respectively, 5-HT(1A) and 5-HT(2) receptors. An incubation of T84 cells with anti-G(s) and anti-G(beta) antibodies complexed with lipofectin did not prevent the 5-HT-induced stimulation of NHE activity. Overnight treatment with anti-G(ialpha1,2) and anti-G(q/11) antibodies complexed with lipofectin blocked the stimulatory effect induced by 8-OH-DPAT and alpha-methyl-5-HT, respectively. It is concluded that in T84 cells 5-HT enhances intestinal NHE activity through stimulation of G(ialpha1,2)-coupled 5-HT(1A) and G(q/11)-coupled 5-HT(2) receptors.     

Identification of Cysteine Residues in Human Cationic Amino Acid Transporter hCAT-2A That Are Targets for Inhibition by N-Ethylmaleimide

In most cells, cationic amino acids such as l-arginine, l-lysine, and l-ornithine are transported by cationic (CAT) and y⁺L (y⁺LAT) amino acid transporters. In human erythrocytes, the cysteine-modifying agent N-ethylmaleimide (NEM) has been shown to inhibit system y⁺ (most likely CAT-1), but not system y⁺L (Devés, R., Angelo, S., and Chávez, P. (1993) J. Physiol. 468, 753–766). We thus wondered if sensitivity to NEM distinguishes generally all CAT and y⁺LAT isoforms. Transport assays in Xenopus laevis oocytes established that indeed all human CATs (including the low affinity hCAT-2A), but neither y⁺LAT isoform, are inhibited by NEM. hCAT-2A inhibition was not due to reduced transporter expression in the plasma membrane, indicating that NEM reduces the intrinsic transporter activity. Individual mutation of each of the seven cysteine residues conserved in all CAT isoforms did not lead to NEM insensitivity of hCAT-2A. However, a cysteine-less mutant was no longer inhibited by NEM, suggesting that inhibition occurs through modification of more than one cysteine in hCAT-2A. Indeed, also the double mutant C33A/C273A was insensitive to NEM inhibition, whereas reintroduction of a cysteine at either position 33 or 273 in the cysteine-less mutant led to NEM sensitivity. We thus identified Cys-33 and Cys-273 in hCAT-2A as the targets of NEM inhibition. In addition, all proteins with Cys-33 mutations showed a pronounced reduction in transport activity, suggesting that, surprisingly, this residue, located in the cytoplasmic N terminus, is important for transporter function.     

Surface potentials near the mouth of the large-conductance K+ channel from Chara australis: A new method of testing for diffusion-limited ion flow

The kinetics of single K⁺ channels were derived for patch-clamp recordings of membrane patches excised from cytoplasmic drops from the plant, Chara australis R. Br. Specifically, the tilt effect model of MacKinnon, Latorre and Miller (1989. Biochemistry 28:8092–8099) has been used to measure the electrostatic potential (surface PD) and fixed charge at the entrances of the channel. The surface PD is derived from the difference between the trans-pore potential difference (PD) and that between the two bulk phases. The trans-pore PD is probed using three voltage-dependent properties of the channel. These are (1) the association and dissociation rates of Ca²⁺ binding to the channel, from both the cytoplasmic and vacuolar solutions. These were determined from the mean blocked and unblocked durations of the channel in the presence of either 20 mmol liter^–1 vacuolar or 1 mmol liter^–1 cytoplasmic Ca²⁺; (2) the closing rate of the channel's intrinsic gating process. This was determined from the mean channel open time in the absence of vacuolar Ca²⁺ at membrane PDs more negative than –100 mV; and (3) the effect of Mg²⁺ on channel conductance when added to solutions initially containing 3 mmol liter^–1 KCl.The voltage dependence of properties 1 and 2 shifts along the voltage axis according to the ionic strength of the bathing media, consistent with the presence of negative charge in the channel vestibules. Furthermore, the magnitude of this shift depends on the current in a manner consistent with diffusion-limited ion flow in the channel (i.e., the rate of ion diffusion in the external electrolyte limits the channel conductance). Mg²⁺ on either side of the membrane alters channel conductance in a voltage-dependent way. A novel feature of the Mg²⁺ effect is that it reverses, from a block to an enhancement, when the membrane PD is more negative than –70 mV. This reversal only appears in solutions of low ionic strength. The attenuating effect is due to voltage-dependent binding of Mg²⁺ within the pore, which presumably plugs the channel. The enhancing effect is due to screening by Mg²⁺ of surface potentials arising from diffusion-limited flow of K⁺.     

A structural basis for substrate selectivity and stereoselectivity in octopine dehydrogenase from Pecten maximus

Octopine dehydrogenase [N²-(d-1-carboxyethyl)-l-arginine:NAD⁺ oxidoreductase] (OcDH) from the adductor muscle of the great scallop Pecten maximus catalyzes the reductive condensation of l-arginine and pyruvate to octopine during escape swimming. This enzyme, which is a prototype of opine dehydrogenases (OpDHs), oxidizes glycolytically born NADH to NAD⁺, thus sustaining anaerobic ATP provision during short periods of strenuous muscular activity. In contrast to some other OpDHs, OcDH uses only l-arginine as the amino acid substrate. Here, we report the crystal structures of OcDH in complex with NADH and the binary complexes NADH/l-arginine and NADH/pyruvate, providing detailed information about the principles of substrate recognition, ligand binding and the reaction mechanism. OcDH binds its substrates through a combination of electrostatic forces and size selection, which guarantees that OcDH catalysis proceeds with substrate selectivity and stereoselectivity, giving rise to a second chiral center and exploiting a “molecular ruler” mechanism.     

A divalent-ion binding site on the 16-kDa proton channel from Nephrops norvegicus—revealed by EPR spectroscopy

As purified from the hepatopancreas of Nephrops norvegicus, the 16-kDa proton channel proteolipid is found to contain an endogenous divalent ion binding site that is occupied by Cu²⁺. The EPR spectrum has g-values and hyperfine splittings that are characteristic of type 2 Cu²⁺. The copper may be removed by extensive washing with EDTA. Titration with Ni²⁺ then induces spin-spin interactions with nitroxyl spin labels that are attached either to the unique Cys⁵⁴, or to fatty acids intercalated in the membrane. Paramagnetic relaxation enhancement by the fast-relaxing Ni²⁺ is used to characterise the binding and to estimate distances from the dipolar interactions. The Ni²⁺-binding site on the protein is situated around 14-18 Å from the spin label on Cys⁵⁴, and is at a similar distance from a lipid chain spin-labelled on the 5 C-atom, but is more remote from the C-9 and C-14 positions of the lipid chains.     

Modulation of the K(v)4.3 channel by syntaxin 1A

The SNARE protein syntaxin 1A (Syn1A) is known to inhibit delayed rectifier K(+) channels of the K(v)1 and K(v)2 families with heterogeneous effects on their gating properties. In this study, we explored whether Syn1A could directly modulate K(v)4.3, a rapidly inactivating K(v) channel with important roles in neuroendocrine cells and cardiac myocytes. Immunoprecipitation studies in HEK293 cells coexpressing Syn1A and K(v)4.3 revealed a direct interaction with increased trafficking to the plasma membrane without a change in channel synthesis. Paradoxically, Syn1A inhibited K(v)4.3 current density. In particular, Syn1A produced a left-shift in steady-state inactivation of K(v)4.3 without affecting either voltage dependence of activation or gating kinetics, a pattern distinct from other K(v) channels. Combined with our previous reports, our results further verify the notion that the mechanisms involved in Syn1A-K(v) interactions vary significantly between K(v) channels, thus providing a wide scope for Syn1A modulation of exocytosis and membrane excitability.     

Iron uptake and transfer from ceruloplasmin to transferrin

Background

Dietary and recycled iron are in the Fe^2 + oxidation state. However, the metal is transported in serum by transferrin as Fe^3 +. The multi-copper ferroxidase ceruloplasmin is suspected to be the missing link between acquired Fe^2 + and transported Fe^3 +.

Methods

This study uses the techniques of chemical relaxation and spectrophotometric detection.

Results

Under anaerobic conditions, ceruloplasmin captures and oxidizes two Fe^2 +. The first uptake occurs in domain 6 (< 1 ms) at the divalent iron-binding site. It is accompanied by Fe^2 + oxidation by Cu^2 +_D6. Fe^3 + is then transferred from the binding site to the holding site. Cu⁺_D6 is then re-oxidized by a Cu^2 + of the trinuclear cluster in about 200 ms. The second Fe^2 + uptake and oxidation involve domain 4 and are under the kinetic control of a 200 s change in the protein conformation. With transferrin and in the formed ceruloplasmin–transferrin adduct, two Fe^3 + are transferred from their holding sites to two C-lobes of two transferrins. The first transfer (~ 100 s) is followed by conformation changes (500 s) leading to the release of monoferric transferrin. The second transfer occurs in two steps in the 1000–10,000 second range.

Conclusion

Fe^3 + is transferred after Fe^2 + uptake and oxidation by ceruloplasmin to the C-lobe of transferrin in a protein–protein adduct. This adduct is in a permanent state of equilibrium with all the metal-free or bounded ceruloplasmin and transferrin species present in the medium.

General significance

Ceruloplasmin is a go-between dietary or recycled Fe^2 + and transferrin transported Fe^3 +.     

The nitric oxide–iron interplay in mammalian cells: Transport and storage of dinitrosyl iron complexes

Nitrogen monoxide (NO) is a vital effector and messenger molecule that plays roles in a variety of biological processes. Many of the functions of NO are mediated by its high affinity for iron (Fe) in the active centres of proteins. Indeed, NO possesses a rich coordination chemistry with this metal and the formation of dinitrosyl–dithiolato–Fe complexes (DNICs) is well known to occur intracellularly. In mammals, NO produced by activated macrophages acts as a cytotoxic effector against tumour cells by binding and releasing cancer cell Fe that is vital for proliferation. Glucose metabolism and the subsequent generation of glutathione (GSH) are critical for NO-mediated Fe efflux and this process occurs by active transport. Our previous studies showed that GSH is required for Fe mobilisation from tumour cells and we hypothesized it was effluxed with Fe as a dinitrosyl–diglutathionyl–Fe complex (DNDGIC). It is well known that Fe and GSH release from cells induces apoptosis, a crucial property for a cytotoxic effector like NO. Furthermore, NO-mediated Fe release is mediated from cells expressing the GSH transporter, multi-drug resistance protein 1 (MRP1). Interestingly, the glutathione-S-transferase (GST) enzymes act to bind DNDGICs with high affinity and some members of the GST family act as storage intermediates for these complexes. Since the GST enzymes and MRP1 form a coordinated system for removing toxic substances from cells, it is possible to hypothesize these molecules regulate NO levels by binding and transporting DNDGICs.