Copper economy in Chlamydomonas: prioritized allocation and reallocation of copper to respiration vs. photosynthesis.

Inorganic elements, although required only in trace amounts, permit life and primary productivity because of their functions in catalysis. Every organism has a minimal requirement of each metal based on the intracellular abundance of proteins that use inorganic cofactors, but elemental sparing mechanisms can reduce this quota. A well-studied copper-sparing mechanism that operates in microalgae faced with copper deficiency is the replacement of the abundant copper protein plastocyanin with a heme-containing substitute, cytochrome (Cyt) c6. This switch, which is dependent on a copper-sensing transcription factor, copper response regulator 1 (CRR1), dramatically reduces the copper quota. We show here that in a situation of marginal copper availability, copper is preferentially allocated from plastocyanin, whose function is dispensable, to other more critical copper-dependent enzymes like Cyt oxidase and a ferroxidase. In the absence of an extracellular source, copper allocation to Cyt oxidase includes CRR1-dependent proteolysis of plastocyanin and quantitative recycling of the copper cofactor from plastocyanin to Cyt oxidase. Transcriptome profiling identifies a gene encoding a Zn-metalloprotease, as a candidate effecting copper recycling. One reason for the retention of genes encoding both plastocyanin and Cyt c6 in algal and cyanobacterial genomes might be because plastocyanin provides a competitive advantage in copper-depleted environments as a ready source of copper.

The Role of Altruistic Values in Motivating Underrepresented Minority Students for Biomedicine.

Understanding how cultural values influence undergraduate students' science research experiences and career interest is important in efforts to broaden participation and to diversify the biomedical research workforce. The results from our prospective longitudinal study demonstrated that underrepresented minority student (URM) research assistants who see the altruistic value of conducting biomedical research feel more psychologically involved with their research over time, which, in turn, enhances their interest in pursuing a scientific research career. These altruistic motives are uniquely influential to URM students and appear to play an important role in influencing their interest in scientific research careers. Furthermore, seeing how research can potentially affect society and help one's community does not replace typical motives for scientific discovery (e.g., passion, curiosity, achievement), which are important for all students. These findings point to simple strategies for educators, training directors, and faculty mentors to improve retention among undergraduate URM students in biomedicine and the related sciences.

Trace element status and zinc homeostasis differ in breast and formula-fed piglets.

Differences in trace element composition and bioavailability between breast milk and infant formulas may affect metal homeostasis in neonates. However, there is a paucity of controlled studies in this area. Here, piglets were fed soy infant formula (soy), cow's milk formula (milk), or were allowed to suckle from the sow from PND2 to PND21. Serum iron concentrations were higher in formula-fed compared to breastfed piglets (P < 0.05). Serum zinc values were higher in milk compared to breastfed or soy groups (P < 0.05). Zinc transporter Zip4 mRNA was elevated in small intestine of the soy compared to breastfed group (P < 0.05). Transporter Znt1 mRNA was greater in small intestine of both formula-fed groups and in liver of the milk compared to the breastfed group (P < 0.05). Metallothionein Mt1 mRNA expression was higher in small intestine and liver of milk compared to breastfed and soy groups (P < 0.05). In liver, metallothionein protein levels and protein bound zinc were also highly elevated in the milk compared to other groups (P < 0.05). mRNA encoding the hepatic zinc-regulated gene Gclc was higher in the milk than soy group (P < 0.05). ChIP assay revealed increased binding of the zinc-regulated transcription factor MTF1 to the promoters of hepatic Mt3 and Gclc genes in the milk compared to the soy group. These data provide evidence that trace element status differs in breastfed, milk-fed, and soy-fed piglets and that despite similar levels of dietary supplementation, allows strong causal inference that significant differences in serum zinc after cow's milk formula compared to soy formula consumption result in compensatory changes in expression of zinc transporters, binding proteins, and zinc-regulated genes.

Vitamin D supplementation protects against bone loss associated with chronic alcohol administration in female mice.

Chronic alcohol abuse results in decreased bone mineral density (BMD), which can lead to increased fracture risk. In contrast, low levels of alcohol have been associated with increased BMD in epidemiological studies. Alcohol's toxic skeletal effects have been suggested to involve impaired vitamin D/calcium homeostasis. Therefore, dietary vitamin D supplementation may be beneficial in reducing bone loss associated with chronic alcohol consumption. Six-week-old female C57BL/6J mice were pair-fed ethanol (EtOH)-containing liquid diets (10 or 36% total calories) for 78 days. EtOH exposure at 10% calories had no effects on any measured bone or serum parameter. EtOH consumption at 36% of calories reduced BMD and bone strength (P<0.05), decreased osteoblastogenesis, increased osteoclastogenesis, suppressed 1,25-hydroxyvitamin D3 [1,25(OH)2D3] serum concentrations (P<0.05), and increased apoptosis in bone cells compared with pair-fed controls. In a second study, female mice were pair-fed 30% EtOH diets with or without dietary supplementation with vitamin D3 (cholecalciferol; VitD) for 40 days. VitD supplementation in the EtOH diet protected against cortical bone loss, normalized alcohol-induced hypocalcaemia, and suppressed EtOH-induced expression of receptor of nuclear factor-κB ligand mRNA in bone. In vitro, pretreatment of 1,25(OH)2D3 in osteoblastic cells inhibited EtOH-induced apoptosis. In EtOH/VitD mice circulating 1,25(OH)2D3 was lower compared with mice receiving EtOH alone (P<0.05), suggesting increased sensitivity to feedback control of VitD metabolism in the kidney. These findings suggest dietary VitD supplementation may prevent skeletal toxicity in chronic drinkers by normalizing calcium homeostasis, preventing apoptosis, and suppressing EtOH-induced increases in bone resorption.

Heavy metal in children's tooth enamel: related to autism and disruptive behaviors?

To examine possible links between neurotoxicant exposure and neuropsychological disorders and child behavior, relative concentrations of lead, mercury, and manganese were examined in prenatal and postnatal enamel regions of deciduous teeth from children with Autism Spectrum Disorders (ASDs), high levels of disruptive behavior (HDB), and typically developing (TD) children. Using laser ablation inductively coupled plasma mass spectrometry, we found no significant differences in levels of these neurotoxicants for children with ASDs compared with TD children, but there was marginal significance indicating that children with ASDs have lower manganese levels. No significant differences emerged between children with HDB and TD children. The current findings challenge the notion that perinatal heavy metal exposure is a major contributor to the development of ASDs and HDB.

Loss of metal ions, disulfide reduction and mutations related to familial ALS promote formation of amyloid-like aggregates from superoxide dismutase.

Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) are one of the causes of familial amyotrophic lateral sclerosis (FALS). Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1) formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1.

Copper proteins and ferroxidases in human plasma and that of wild-type and ceruloplasmin knockout mice.

In the blood plasma of humans and rats, ceruloplasmin is the major copper-binding protein and ferroxidase, accounting for 70% of the copper present in the plasma, with the rest binding primarily to albumin and a macroglobulin. Systematic studies with fresh plasma were carried out to compare what occurs in the mouse. C57BL6 mice had half as much copper and pPD (p-phenylene diamine) oxidase activity as humans and rats, 20-40% as much ferroxidase activity as humans (determined using three different assays) and less inhibition by azide. Plasma from ceruloplasmin knockout mice had no pPD oxidase activity, but retained >50% ferroxidase activity (which was not as affected by azide). Modelling of mouse ceruloplasmin against the known X-ray structure of human ceruloplasmin indicated subtle but potentially significant changes in the pPD- and azide-binding sites. Purification and in-gel assays after native PAGE confirmed that mouse ceruloplasmin had ferroxidase activity but revealed an additional ferroxidase in ceruloplasmin knockout mouse plasma, which is also seen in size-exclusion chromatography. In the wild-type mouse, the 'ceruloplasmin' peak contained approximately 55% of the total copper, but ceruloplasmin knockout plasma exposed a major additional peak (180 kDa) which co-eluted with ferroxidase activity. Two other ferroxidases (700 and 2000 Da) were also detected in mouse and human plasma. Mammalian blood thus contains copper components and ferroxidases not reported previously.

Copper binding components of blood plasma and organs, and their responses to influx of large doses of (65)Cu, in the mouse.

To establish for the first time how mice might differ from rats and humans in terms of copper transport, excretion, and copper binding proteins, plasma and organ cytosols from adult female C57CL6 mice were fractionated and analyzed by directly coupled size exclusion HPLC-ICP-MS, before and after i.p. injection of large doses of (65)Cu. Plasma from untreated mice had different proportions of Cu associated with transcuprein/macroglobulin, ceruloplasmin and albumin than in humans and rats, and two previously undetected copper peaks (Mr 700 k and 15 k) were observed. Cytosols had Cu peaks seen previously in rat liver (Mr > 1,000 k, 45 k and 11 k) plus one of 110 kDa. (65)Cu (141 microg) administered over 14 h, initially loaded plasma albumin and mainly entered liver and kidney (especially 28 kDa and 11 kDa components). Components of other organs were less (but still significantly) enriched. (63)Cu/(65)Cu ratios returned almost to normal by 14 days, indicating a robust system for excreting excess copper. We conclude that there are significant differences but also strong similarities in copper metabolism between mice, rats and humans; that the liver is able to buffer enormous changes in copper status; and that a large number of mammalian copper proteins remain to be identified.

Use of stable isotopically enriched proteins and directly coupled high-performance liquid chromatography inductively coupled plasma mass spectrometry for quantitatively monitoring the transfer of metals between proteins.

Studies have shown that metallothionein (MT) may play an important role in modulating the activity of certain Zn-regulated enzymes under various oxidoreductive conditions by either donating or removing Zn. To better determine the role of MT in interprotein metal transfer, we describe a procedure that uses stable isotopically enriched (67)Zn(7) metallothionein 2 ((67)Zn(7)-MT-2) to quantitatively determine the stoichiometry of transfer of Zn from the protein to a recipient apo-metalloenzyme, apo-carbonic anhydrase (apo-CA) by directly coupled ion exchange high-performance liquid chromatography inductively coupled plasma mass spectrometry. Quantitatively, the transfer of (67)Zn was consistent with the enzymatic activation of the apo-enzyme as judged by its esterase activity and ability to cleave p-nitrophenyl acetate. Maximum enzyme activation occurred at an MT-2:apo-CA molar ratio of 1, implying the release of a single atom of Zn from MT-2. Preincubation of (67)Zn(7)-MT-2 with an excess of oxidized glutathione (GSSG) increased metal donation fourfold, whereas reduced glutathione (GSH) inhibited donation by approximately 50%. By using multiple recipient and donor proteins having different stable isotopic signatures, the technique has the potential for quantitatively studying the kinetic and thermodynamic aspects of Zn transfer between numerous competing ligands in vitro, an important first step toward understanding the regulatory role of this metal in protein functioning and cellular metabolism in vivo.

Concentration of contaminants in breeding bird eggs from the Colorado River Delta, Mexico.

Organic contaminants (organochlorine [OC], organophosphorus [OP] pesticides and polychlorinated biphenyls [PCBs]), and metals (As, Cd, Hg, Pb, and Se) are a concern to avian health in the Colorado River delta, Mexico. We determined concentrations of contaminants in eggs of three breeding species of birds from the delta (mourning doves [Zenaida macroura], burrowing owls [Athene cunicularia], and marsh wrens [Cistothorus palustris]). We collected 27 eggs of mourning doves, eight eggs of burrowing owls, and 18 eggs of marsh wrens for analyses. Polychlorinated biphenyls, OC, and OP pesticides were analyzed by gas chromatography equipped with an electron capture detector, and metals were analyzed by inductively coupled plasma mass spectrometry. The non-ortho PCB congeners (PCB 77 and 126) were found in mourning dove and burrowing owl eggs at concentrations in which hatchability can be affected. Mean selenium concentration found in marsh wren eggs (5.6 microg/g dry wt) exceeded the level of concern. Arsenic and Cd were found at higher than normal concentrations, Hg concentrations did not exceed the level of concern in any of the species, and Pb concentrations were higher in eggs of species subject to hunting. With the exception of lead, marsh wren eggs contained the highest metal concentrations.

Metal donation and apo-metalloenzyme activation by stable isotopically labeled metallothionein.

Coupled HPLC-ICP-MS has been used to quantitatively study the effects of GSSG and GSH on the ability of metallothionein (MTII) to donate essential and non-essential metals to apo-carbonic anhydrase. Stable isotopically labeled (67)Zn(3)Cd(4) MTII was used to enable Zn donated from MTII to be differentiated from extraneous sources of Zn. Transfer of both (67)Zn and Cd from MTII to apo-carbonic anhydrase was noted in the absence of either GSSG or GSH. GSSG increased the initial transfer of both Zn and Cd. Thereafter, a gradual increase in the (67)Zn content at the expense of Cd was noted over 24-h indicating continued interaction and exchange between MTII and the enzyme commensurate with the relative preferences shown by the proteins for these two metals. Although GSH also increased transfer of (67)Zn from MT it reduced the simultaneous transfer of Cd to the enzyme thereby conferring protection against Cd induced activation.

Anodic stripping voltammetry combined on-line with inductively coupled plasma-MS via a direct-injection high-efficiency nebulizer.

A direct-injection high-efficiency nebulizer (DIHEN) is used to couple a thin-layer electrochemical flow cell on-line with an ICP-mass spectrometer to perform anodic stripping voltammetry (ASV) at a thin mercury film followed by subsequent ICPMS measurements for the stripped metal analytes. The resultant hyphenated technique (ASV-DIHEN-ICPMS) is capable of analyzing select heavy metals present at ultratrace levels (down to low-ppt to sub-ppt levels) that are lower than the detection limits obtained by conventional ICPMS. In addition to its good analytical performance, the technique offers other attractive features such as the ability to eliminate detrimental matrix effects that can compromise ICPMS analyses and the possibility of probing electrode reactions involving trace amounts metal species with ICPMS. For conducting ASV on-line with ICPMS, the DIHEN was found to be more advantageous than the microconcentric nebulizer in terms of minimizing memory effects and potential artifacts caused by the erosion of the Hg film into the flowing solution stream. Compared to a direct injection nebulizer (DIN), the DIHEN was easier to operate. Moreover, its simpler design and the lack of back pressure from the DIHEN capillary made it more compatible with coupling to the thin-layer electrochemical cell than a DIN system.