Human TRPV5 and TRPV6: key players in cadmium and zinc toxicity

TRPV5 and TRPV6 are two major calcium transport pathways in the human body maintaining calcium homeostasis. TRPV5 is mainly expressed in the distal convoluted and connecting tubule where it is the major, regulated pathway for calcium reabsorption. TRPV6 serves as an important calcium entry pathway in the duodenum and the placenta. Previously, we showed that human TRPV6 (hTRPV6) transports several heavy metals. In this study we tested whether human TRPV5 (hTRPV5) also transports cadmium and zinc, and whether hTRPV5 together with hTRPV6 are involved in cadmium and zinc toxicity. The hTRPV5 mRNA and protein were expressed in HEK293 cells transiently transfected with pTagRFP-C1-hTRPV5. The overexpression of the hTRPV5 protein at the plasma membrane was revealed by cell surface biotinylation and immunofluorescence techniques. We observed that both cadmium and zinc permeate hTRPV5 in ion imaging experiments using Fura-2 or Newport Green DCF. Our results were further confirmed using whole-cell patch clamp technique. Transient overexpression of hTRPV5 or hTRPV6 sensitized cells to cadmium and zinc. Toxicity curves of cadmium and zinc were also shifted in hTRPV6 expressing HEK293 cells clones. Our results suggest that TRPV5 and TRPV6 are crucial gates controlling cadmium and zinc levels in the human body especially under low calcium dietary conditions, when these channels are maximally upregulated.

Structure and function of CinD (YtjD) of Lactococcus lactis, a copper-induced nitroreductase involved in defense against oxidative stress

In Lactococcus lactis IL1403, 14 genes are under the control of the copper-inducible CopR repressor. This so-called CopR regulon encompasses the CopR regulator, two putative CPx-type copper ATPases, a copper chaperone, and 10 additional genes of unknown function. We addressed here the function of one of these genes, ytjD, which we renamed cinD (copper-induced nitroreductase). Copper, cadmium, and silver induced cinD in vivo, as shown by real-time quantitative PCR. A knockout mutant of cinD was more sensitive to oxidative stress exerted by 4-nitroquinoline-N-oxide and copper. Purified CinD is a flavoprotein and reduced 2,6-dichlorophenolindophenol and 4-nitroquinoline-N-oxide with k(cat) values of 27 and 11 s(-1), respectively, using NADH as a reductant. CinD also exhibited significant catalase activity in vitro. The X-ray structure of CinD was resolved at 1.35 A and resembles those of other nitroreductases. CinD is thus a nitroreductase which can protect L. lactis against oxidative stress that could be exerted by nitroaromatic compounds and copper.

Copper induction of lactate oxidase of Lactococcus lactis: a novel metal stress response

Lactococcus lactis IL1403, a lactic acid bacterium widely used for food fermentation, is often exposed to stress conditions. One such condition is exposure to copper, such as in cheese making in copper vats. Copper is an essential micronutrient in prokaryotes and eukaryotes but can be toxic if in excess. Thus, copper homeostatic mechanisms, consisting chiefly of copper transporters and their regulators, have evolved in all organisms to control cytoplasmic copper levels. Using proteomics to identify novel proteins involved in the response of L. lactis IL1403 to copper, cells were exposed to 200 muM copper sulfate for 45 min, followed by resolution of the cytoplasmic fraction by two-dimensional gel electrophoresis. One protein strongly induced by copper was LctO, which was shown to be a NAD-independent lactate oxidase. It catalyzed the conversion of lactate to pyruvate in vivo and in vitro. Copper, cadmium, and silver induced LctO, as shown by real-time quantitative PCR. A copper-regulatory element was identified in the 5' region of the lctO gene and shown to interact with the CopR regulator, encoded by the unlinked copRZA operon. Induction of LctO by copper represents a novel copper stress response, and we suggest that it serves in the scavenging of molecular oxygen.

Novel pseudo-staphylococcal cassette chromosome mec element (ψSCCmec57395) in methicillin-resistant Staphylococcus pseudintermedius CC45

Genetic characterization of methicillin-resistant Staphylococcus pseudintermedius (MRSP) from Thailand and Israel revealed the presence of a predominant atypical clonal lineage which was not typeable by SmaI-PFGE and SCCmec typing. All the atypical isolates (n = 34) belonged to CC45 (30 ST45 and 2 ST179 isolates, 1 ST57 isolate, and 1 ST85 isolate). The isolates originated from healthy and diseased dogs and cats, as well as from the environment of one clinic. Cfr9I-pulsed-field gel electrophoresis (Cfr9I-PFGE) and dru typing permitted the further distinction of CC45 isolates from the two different countries. Microarray analysis identified genes that confer resistance to β-lactams (mecA; blaZ), aminoglycosides [aac(6')-Ie-aph(2')-Ia; aph(3')-III; ant(6)-Ia], macrolides and lincosamides [erm(B)], tetracyclines [tet(M)], trimethoprim [dfr(G)], streptothricin (sat4), and chloramphenicol (catpC221). Fluoroquinolone resistance was attributed to specific amino acid substitutions, i.e., Ser84Leu in GyrA and Ser80Ile and Asp84Asn in GrlA. A novel pseudo-staphylococcal cassette chromosome (ΨSCCmec57395) element was identified in MRSP strain 57395 (sequence type ST45) by whole-genome sequencing. The 12,282-bp ΨSCCmec57395 element contained a class C1 mec gene complex but no ccr genes. In addition to the methicillin resistance gene mecA, ΨSCCmec57395 also carried determinants of resistance to heavy metals, such as arsenic, cadmium, and copper. Bsu36I restriction analysis of the ΨSCCmec57395 element amplified by long-range PCR revealed the presence of ΨSCCmec57395 in the 33 additional isolates of MRSP CC45. The ΨSCCmec57395 element represents a new class of SCCmec and has been identified in MRSP of CC45, which is a predominant clonal lineage in Israel and Thailand.

Chondrocytes expressing intracellular collagen type II enter the cell cycle and co-express collagen type I in monolayer culture.

For autologous chondrocyte transplantation, articular chondrocytes are harvested from cartilage tissue and expanded in vitro in monolayer culture. We aimed to characterize with a cellular resolution the synthesis of collagen type II (COL2) and collagen type I (COL1) during expansion in order to further understand why these cells lose the potential to form cartilage tissue when re-introduced into a microenvironment that supports chondrogenesis. During expansion for six passages, levels of transcripts encoding COL2 decreased to <0.1%, whereas transcript levels encoding COL1 increased 370-fold as compared to primary chondrocytes. Flow cytometry for intracellular proteins revealed that chondrocytes acquired a COL2/COL1-double positive phenotype during expansion, and the COL2 positive cells were able to enter the cell cycle. While the fraction of COL2 positive cells decreased from 70% to <2% in primary chondrocytes to passage six cells, the fraction of COL1 positive cells increased from <1% to >95%. In parallel to the decrease of the fraction of COL2 positive cells, the cells' potential to form cartilage-like tissue in pellet cultures steadily decreased. Intracellular staining for COL2 enables for characterization of chondrocyte lineage cells in more detail with a cellular resolution, and it may allow predicting the effectiveness of expanded chondrocytes to form cartilage-like tissue.

Controlled assembly and single electron charging of monolayer protected Au 144 clusters: an electrochemistry and scanning tunneling spectroscopy study

Single gold particles may serve as room temperature single electron memory units because of their size dependent electronic level spacing. Here, we present a proof-of-concept study by electrochemically controlled scanning probe experiments performed on tailor-made Au particles of narrow dispersity. In particular, the charge transport characteristics through chemically synthesized hexane-1-thiol and 4-pyridylbenzene-1-thiol mixed monolayer protected Au144 clusters (MPCs) by differential pulse voltammetry (DPV) and electrochemical scanning tunneling spectroscopy (EC-STS) are reported. The pyridyl groups exposed by the Au-MPCs enable their immobilization on Pt(111) substrates. By varying the humidity during their deposition, samples coated by stacks of compact monolayers of Au-MPCs or decorated with individual, laterally separated Au-MPCs are obtained. DPV experiments with stacked monolayers of Au144-MPCs and EC-STS experiments with laterally separated individual Au144-MPCs are performed both in aqueous and ionic liquid electrolytes. Lower capacitance values were observed for individual clusters compared to ensemble clusters. This trend remains the same irrespective of the composition of the electrolyte surrounding the Au144-MPC. However, the resolution of the energy level spacing of the single clusters is strongly affected by the proximity of neighboring particles.

Selective Transport of Zinc, Manganese, Nickel, Cobalt and Cadmium in the Root System and Transfer to the Leaves in Young Wheat Plants

• Background and Aims The uptake, translocation and redistribution of the heavy metals zinc, manganese, nickel, cobalt and cadmium are relevant for plant nutrition as well as for the quality of harvested plant products. The long-distance transport of these heavy metals within the root system and the release to the shoot in young wheat (Triticum aestivum ‘Arina’) plants were investigated. • Methods After the application of 65Zn, 54Mn, 63Ni, 57Co and 109Cd for 24 h to one seminal root (the other seminal roots being excised) of 54-h-old wheat seedlings, the labelled plants were incubated for several days in hydroponic culture on a medium without radionuclides. • Key Results The content of 65Zn decreased quickly in the labelled part of the root. After the transfer of 65Zn from the roots to the shoot, a further redistribution in the phloem from older to younger leaves was observed. In contrast to 65Zn, 109Cd was released more slowly from the roots to the leaves and was subsequently redistributed in the phloem to the youngest leaves only at trace levels. The content of 63Ni decreased quickly in the labelled part of the root, moving to the newly formed parts of the root system and also accumulating transiently in the expanding leaves. The 54Mn content decreased quickly in the labelled part of the root and increased simultaneously in leaf 1. A strong retention in the labelled part of the root was observed after supplying 57Co. • Conclusions The dynamics of redistribution of 65Zn, 54Mn, 63Ni, 57Co and 109Cd differed considerably. The rapid redistribution of 63Ni from older to younger leaves throughout the experiment indicated a high mobility in the phloem, while 54Mn was mobile only in the xylem and 57Co was retained in the labelled root without being loaded into the xylem.

Redistribution of Nickel, Cobalt, Manganese, Zinc, and Cadmium via the Phloem in Young and Maturing Wheat

The phloem mobility of heavy metals is relevant to the redistribution of micronutrients and pollutants and, ultimately, to the quality of harvested plant parts. The relative mobility in wheat may vary considerably between different cations. In the study reported here, radio-labeled nickel (Ni), cobalt (Co), manganese (Mn), zinc (Zn) and cadmium (Cd) were introduced into either intact young winter wheat (Triticum aestivum L. cv. Arina) via a leaf flap, or detached maturing shoots via the cut stem. Elements fed into the lamina of the second leaf of 21-day-old plants were translocated to the younger (expanding) leaves and to the roots but not or only in trace amounts to the first (already fully expanded) leaf. The 63Ni and 65Zn were exported more rapidly compared with the other heavy metals. Most of 54Mn was retained in the originally labeled leaf. The peduncle of some maturing shoots was steam-girdled below the ear to distinguish between xylem and phloem transport. This phloem interruption reduced the content of 63Ni in the ear to about 25%. Intermediate effects were observed for 65Zn, 57Co, and 109Cd. Total 54Mn accumulation in the ear was hardly affected by steam-girdling, indicating a transport of this element within the xylem to the ear. These results suggest that the relative phloem mobility of Ni and Zn in young wheat plants and in maturing wheat shoots is higher than the mobility of Co and Cd, whereas the mobility of Mn is very low.

Population doublings and percentage of S100-positive cells as predictors of in vitro chondrogenicity of expanded human articular chondrocytes

The aim of this study was to investigate the interconnection between the processes of proliferation, dedifferentiation, and intrinsic redifferentiation (chondrogenic) capacities of human articular chondrocyte (HAC), and to identify markers linking HAC dedifferentiation status with their chondrogenic potential. Cumulative population doublings (PD) of HAC expanded in monolayer culture were determined, and a threshold range of 3.57-4.19 PD was identified as indicative of HAC loss of intrinsic chondrogenic capacity in pellets incubated without added chondrogenic factors. While several specific gene and surface markers defined early HAC dedifferentiation process, no clear correlation with the loss of intrinsic chondrogenic potential could be established. CD90 expression during HAC monolayer culture revealed two subpopulations, with sorted CD90-negative cells showing lower proliferative capacity and higher chondrogenic potential compared to CD90-positive cells. Although these data further validated PD as critical for in vitro chondrogenesis, due to the early shift in expression, CD90 could not be considered for predicting chondrogenic potential of HAC expanded for several weeks. In contrast, an excellent mathematically modeled correlation was established between PD and the decline of HAC expressing the intracellular marker S100, providing a direct link between the number of cell divisions and dedifferentiation/loss of intrinsic chondrogenic capacity. Based on the dynamics of S100-positive HAC during expansion, we propose asymmetric cell division as a potential mechanism of HAC dedifferentiation, and S100 as a marker to assess chondrogenicity of HAC during expansion, of potential value for cell-based cartilage repair treatments.

Regulation and structure of YahD, a copper-inducible / serine hydrolase of Lactococcus lactis IL1403

Lactococcus lactis IL1403 is a lactic acid bacterium that is used widely for food fermentation. Copper homeostasis in this organism chiefly involves copper secretion by the CopA copper ATPase. This enzyme is under the control of the CopR transcriptional regulator. CopR not only controls its own expression and that of CopA, but also that of an additional three operons and two monocistronic genes. One of the genes under the control of CopR, yahD, encodes an α/β-hydrolase. YahD expression was induced by copper and cadmium, but not by other metals or oxidative or nitrosative stress. The three-dimensional structure of YahD was determined by X-ray crystallography to a resolution of 1.88 Å. The protein was found to adopt an α/β-hydrolase fold with the characteristic Ser-His-Asp catalytic triad. Functional testing of YahD for a wide range of substrates for esterases, lipases, epoxide hydrolases, phospholipases, amidases and proteases was, however, unsuccessful. A copper-inducible serine hydrolase has not been described previously and YahD appears to be a new functional member of this enzyme family.

Structure-activity relationship and mechanism of action studies of manzamine analogues for the control of neuroinflammation and cerebral infections

Structure-activity relationship studies were carried out by chemical modification of manzamine A (1), 8-hydroxymanzamine A (2), manzamine F (14), and ircinal isolated from the sponge Acanthostrongylophora. The derived analogues were evaluated for antimalarial, antimicrobial, and antineuroinflammatory activities. Several modified products exhibited potent and improved in vitro antineuroinflammatory, antimicrobial, and antimalarial activity. 1 showed improved activity against malaria compared to chloroquine in both multi- and single-dose in vivo experiments. The significant antimalarial potential was revealed by a 100% cure rate of malaria in mice with one administration of 100 mg/kg of 1. The potent antineuroinflammatory activity of the manzamines will provide great benefit for the prevention and treatment of cerebral infections (e.g., Cryptococcus and Plasmodium). In addition, 1 was shown to permeate across the blood-brain barrier (BBB) in an in vitro model using a MDR-MDCK monolayer. Docking studies support that 2 binds to the ATP-noncompetitive pocket of glycogen synthesis kinase-3beta (GSK-3beta), which is a putative target of manzamines. On the basis of the results presented here, it will be possible to initiate rational drug design efforts around this natural product scaffold for the treatment of several different diseases.