Biomedical subjects

Barry Lai

Publications and source records attributed to Barry Lai.

At least 19 recordsLinked to original sources

PubMed2006

X-ray fluorescence microprobe imaging in biology and medicine.

Characteristic X-ray fluorescence is a technique that can be used to establish elemental concentrations for a large number of different chemical elements simultaneously in different locations in cell and tissue samples. Exposing the samples to an X-ray beam is the basis of X-ray fluorescence microscopy (XFM). This technique provides the excellent trace element sensitivity; and, due to the large penetration depth of hard X-rays, an opportunity to image whole cells and quantify elements on a per cell basis. Moreover, because specimens prepared for XFM do not require sectioning, they can be investigated close to their natural, hydrated state with cryogenic approaches. Until several years ago, XFM was not widely available to bio-medical communities, and rarely offered resolution better then several microns. This has changed drastically with the development of third-generation synchrotrons. Recent examples of elemental imaging of cells and tissues show the maturation of XFM imaging technique into an elegant and informative way to gain insight into cellular processes. Future developments of XFM-building of new XFM facilities with higher resolution, higher sensitivity or higher throughput will further advance studies of native elemental makeup of cells and provide the biological community including the budding area of bionanotechnology with a tool perfectly suited to monitor the distribution of metals including nanovectors and measure the results of interactions between the nanovectors and living cells and tissues.

Animals↗

PubMed2006

The endothelium-derived hyperpolarizing factor, H2O2, promotes metal-ion efflux in aortic endothelial cells: elemental mapping by a hard X-ray microprobe.

Hydrogen peroxide (H(2)O(2)) is a physiologic oxidant implicated in vascular cell signaling, although little is known about the biochemical consequences of its reaction with endothelial cells. Submicrometer-resolution hard X-ray elemental mapping of cultured porcine aortic endothelial cells (PAEC) has provided data on the global changes for intracellular elemental density within PAEC and indicates an efflux of metal ions and phosphorus from the cytoplasm after H(2)O(2) treatment. The synchrotron-radiation-induced X-ray emission experiments (SRIXE) show that H(2)O(2)-treated cells are irregularly shaped and exhibit blebbing indicative of increased permeability due to the damaged membrane. The SRIXE results suggest that H(2)O(2)-induced damage is largely restricted to the cell membrane as judged by the changes to membrane and cytoplasmic components rather than the cell nucleus. The SRIXE data also provide a mechanism for cell detoxification as the metal-ion efflux resulting from the initial H(2)O(2)-mediated changes to cell membrane potentially limits intracellular metal-mediated redox processes through Fenton-like chemistry. They may also explain the increased levels of these ions in atherosclerotic plaques, regardless of whether they are involved in plaque formation. Finally, the SRIXE data support the notion that cultured endothelial cells exposed to H(2)O(2) respond with enhanced cellular metal-ion efflux into the extracellular space.

Animals↗

PubMed2006

Melanosomal sequestration of cytotoxic drugs contributes to the intractability of malignant melanomas.

Multidrug resistance mechanisms underlying the intractability of malignant melanomas remain largely unknown. In this study, we demonstrate that the development of multidrug resistance in melanomas involves subcellular sequestration of intracellular cytotoxic drugs such as cis-diaminedichloroplatinum II (cisplatin; CDDP). CDDP is initially sequestered in subcellular organelles such as melanosomes, which significantly reduces its nuclear localization when compared with nonmelanoma/KB-3-1 epidermoid carcinoma cells. The melanosomal accumulation of CDDP remarkably modulates melanogenesis through a pronounced increase in tyrosinase activity. The altered melanogenesis manifested an approximately 8-fold increase in both intracellular pigmentation and extracellular transport of melanosomes containing CDDP. Thus, our experiments provide evidence that melanosomes contribute to the refractory properties of melanoma cells by sequestering cytotoxic drugs and increasing melanosome-mediated drug export. Preventing melanosomal sequestration of cytotoxic drugs by inhibiting the functions of melanosomes may have great potential as an approach to improving the chemosensitivity of melanoma cells.

Antineoplastic Agents↗

PubMed2006

The fate of platinum(II) and platinum(IV) anti-cancer agents in cancer cells and tumours.

SRIXE mapping has been used to gain insight into the fate of platinum(II) and platinum(IV) complexes in cells and tumours treated with anticancer active complexes to facilitate the development of improved drugs. SRIXE maps were collected of thin sections of human ovarian (A2780) cancer cells treated with bromine containing platinum complexes, cis-[PtCl(2)(3-Brpyr)(NH(3))] (3-Brpyr=3-bromopyridine) and cis,trans,cis-[PtCl(2)(OAcBr)(2)(NH(3))(2)] (OAcBr=bromoacetate), or a platinum complex with an intercalator attached cis-[PtCl(2)(2-[(3-aminopropyl)amino]-9,10-anthracenedione)(NH(3))]. After 24h the complexes appear to be localised in the cell nucleus with a lower concentration in the surrounding cytoplasm. In cells treated with cis-[PtCl(2)(3-Brpyr)(NH(3))] the concentration of bromine was substantially higher than in control cells and the bromine was co-localised with the platinum consistent with the 3-bromopyridine ligand remaining bound to the platinum. The cells treated with cis,trans,cis-[PtCl(2)(OAcBr)(2)(NH(3))(2)] also showed an increased level of bromine, but to a much lesser extent than for those treated with cis-[PtCl(2)(3-Brpyr)(NH(3))] suggestive of substantial reduction of the platinum(IV) complex. Maps were also collected from thin sections of a 4T1.2 neo 1 mammary tumour xenograft removed from a mouse 3h after treatment with cis,trans,cis-[PtCl(2)(OH)(2)(NH(3))(2)] and revealed selective uptake of platinum by one cell.

Animals↗

PubMed2006

Metal exposure and Alzheimer's pathogenesis.

With the growing aging population in Western countries, Alzheimer's disease (AD) has become a major public health concern. No preventive measure and effective treatment for this burdensome disease is currently available. Genetic, biochemical, and neuropathological data strongly suggest that Abeta amyloidosis, which originates from the amyloidogenic processing of a metalloprotein-amyloid precursor protein (APP), is the key event in AD pathology. However, neurochemical factors that impact upon the age-dependent cerebral Abeta amyloidogenesis are not well recognized. Growing data indicate that cerebral dysregulation of biometals, environmental metal exposure, and oxidative stress contribute to AD pathology. Herein we provided further evidence that both metals (such as Cu) and H(2)O(2) promote formation of neurotoxic Abeta oligomers. Moreover, we first demonstrated that laser capture microdissection coupled with X-ray fluorescence microscopy can be applied to determine elemental profiles (S, Fe, Cu, and Zn) in Abeta amyloid plaques. Clearly the fundamental biochemical mechanisms linking brain biometal metabolism, environmental metal exposure, and AD pathophysiology warrant further investigation. Nevertheless, the study of APP and Abeta metallobiology may identify potential targets for therapeutic intervention and/or provide diagnostic methods for AD.

Alzheimer Disease↗

PubMed2006

Correlative microXRF and optical immunofluorescence microscopy of adherent cells labeled with ultrasmall gold particles.

Synchrotron-based X-ray fluorescence microscopy (microXRF) is a powerful tool to study the two-dimensional distribution of a wide range of biologically relevant elements in tissues and cells. By growing mouse fibroblast cells directly on formvar-carbon coated electron microscopy grids, microXRF elemental maps with well-defined subcellular resolution were obtained. In order to colocalize the elemental distribution with the location of specific cellular structures and organelles, we explored the application of a commercially available secondary antibody conjugated to FluoroNanogold, a dual-label that combines a regular organic fluorophore with a 1.4 nm Au-cluster as xenobiotic label for microXRF imaging. Adherent mouse fibroblast cells were grown on silicon nitride windows serving as biocompatible XRF support substrate, and labeled with FluoroNanogold in combination with primary antibodies specific for mitochondria or the Golgi apparatus, respectively. Raster scanning of the in-air dried cells with an incident X-ray energy of 11.95 keV, sufficient to ensure excitation of the Au Lalpha line, provided two-dimensional maps with submicron resolution for Au as well as for most biologically relevant elements. MicroXRF proved to be sufficiently sensitive to image the location and structural details of the Au-labeled organelles, which correlated well with the subcellular distribution visualized by means of optical fluorescence microscopy.

Animals↗

PubMed2006

c-Type cytochrome-dependent formation of U(IV) nanoparticles by Shewanella oneidensis.

Modern approaches for bioremediation of radionuclide contaminated environments are based on the ability of microorganisms to effectively catalyze changes in the oxidation states of metals that in turn influence their solubility. Although microbial metal reduction has been identified as an effective means for immobilizing highly-soluble uranium(VI) complexes in situ, the biomolecular mechanisms of U(VI) reduction are not well understood. Here, we show that c-type cytochromes of a dissimilatory metal-reducing bacterium, Shewanella oneidensis MR-1, are essential for the reduction of U(VI) and formation of extracellular UO(2) nanoparticles. In particular, the outer membrane (OM) decaheme cytochrome MtrC (metal reduction), previously implicated in Mn(IV) and Fe(III) reduction, directly transferred electrons to U(VI). Additionally, deletions of mtrC and/or omcA significantly affected the in vivo U(VI) reduction rate relative to wild-type MR-1. Similar to the wild-type, the mutants accumulated UO(2) nanoparticles extracellularly to high densities in association with an extracellular polymeric substance (EPS). In wild-type cells, this UO(2)-EPS matrix exhibited glycocalyx-like properties and contained multiple elements of the OM, polysaccharide, and heme-containing proteins. Using a novel combination of methods including synchrotron-based X-ray fluorescence microscopy and high-resolution immune-electron microscopy, we demonstrate a close association of the extracellular UO(2) nanoparticles with MtrC and OmcA (outer membrane cytochrome). This is the first study to our knowledge to directly localize the OM-associated cytochromes with EPS, which contains biogenic UO(2) nanoparticles. In the environment, such association of UO(2) nanoparticles with biopolymers may exert a strong influence on subsequent behavior including susceptibility to oxidation by O(2) or transport in soils and sediments.

Bacterial Outer Membrane Proteins↗

PubMed2005

Intracellular mapping of the distribution of metals derived from the antitumor metallocenes.

The intracellular distribution of transition metals in V79 Chinese hamster lung cells treated with subtoxic doses of the organometallic anticancer complexes Cp(2)MCl(2), where Cp is eta (5) -cyclopentadienyl and M is Mo, Nb, Ti, or V, has been studied by synchrotron-based X-ray fluorescence (XRF). While significantly higher concentrations of Mo and Nb were found in treated cells compared with control cells, distinct differences in the cellular distribution of each metal were observed. Analysis of thin sections of cells was consistent with some localization of Mo in the nucleus. Studies with a noncytotoxic thiol derivative of molybdocene dichloride showed an uneven distribution of Mo in the cells. For comparison, the low levels of Ti and V in cells treated with the more toxic titanocene and vanadocene complexes, respectively, resulted in metal concentrations at the detection limit of XRF. The results agree with independent chemical studies that have concluded that the biological chemistry of each of the metallocene dihalides is unique.

Animals↗

PubMed2005

Engineering metal-impurity nanodefects for low-cost solar cells.

As the demand for high-quality solar-cell feedstock exceeds supply and drives prices upwards, cheaper but dirtier alternative feedstock materials are being developed. Successful use of these alternative feedstocks requires that one rigorously control the deleterious effects of the more abundant metallic impurities. In this study, we demonstrate how metal nanodefect engineering can be used to reduce the electrical activity of metallic impurities, resulting in dramatic enhancements of performance even in heavily contaminated solar-cell material. Highly sensitive synchrotron-based measurements directly confirm that the spatial and size distributions of metal nanodefects regulate the minority-carrier diffusion length, a key parameter for determining the actual performance of solar-cell devices. By engineering the distributions of metal-impurity nanodefects in a controlled fashion, the minority-carrier diffusion length can be increased by up to a factor of four, indicating that the use of lower-quality feedstocks with proper controls may be a viable alternative to producing cost-effective solar cells.

Crystallization↗

PubMed2005

Imaging of the intracellular topography of copper with a fluorescent sensor and by synchrotron x-ray fluorescence microscopy.

Copper is an essential micronutrient that plays a central role for a broad range of biological processes. Although there is compelling evidence that the intracellular milieu does not contain any free copper ions, the rapid kinetics of copper uptake and release suggests the presence of a labile intracellular copper pool. To elucidate the subcellular localization of this pool, we have synthesized and characterized a membrane-permeable, copper-selective fluorescent sensor (CTAP-1). Upon addition of Cu(I), the sensor exhibits a 4.6-fold emission enhancement and reaches a quantum yield of 14%. The sensor exhibits excellent selectivity toward Cu(I), and its emission response is not compromised by the presence of millimolar concentrations of Ca(II) or Mg(II) ions. Variable temperature dynamic NMR studies revealed a rapid Cu(I) self-exchange equilibrium with a low activation barrier of deltaG++ = 44 kJ.mol(-1) and k(obs) approximately 10(5) s(-1) at room temperature. Mouse fibroblast cells (3T3) incubated with the sensor produced a copper-dependent perinuclear staining pattern, which colocalizes with the subcellular locations of mitochondria and the Golgi apparatus. To evaluate and confirm the sensor's copper-selectivity, we determined the subcellular topography of copper by synchrotron-based x-ray fluorescence microscopy. Furthermore, microprobe x-ray absorption measurements at various subcellular locations showed a near-edge feature that is characteristic for low-coordinate monovalent copper but does not resemble the published spectra for metallothionein or glutathione. The presented data provide a coherent picture with strong evidence for a kinetically labile copper pool, which is predominantly localized in the mitochondria and the Golgi apparatus.

Animals↗

PubMed2005

Time-dependent uptake, distribution and biotransformation of chromium(VI) in individual and bulk human lung cells: application of synchrotron radiation techniques.

Chromium(VI) is a human carcinogen, primarily affecting the respiratory tract probably via active transport into cells, followed by the reduction to Cr(III) with the formation of DNA-damaging intermediates. Distribution of Cr and endogenous elements within A549 human lung adenocarcinoma epithelial cells, following treatment with Cr(VI) (100 microM, 20 min or 4 h) were studied by synchrotron-radiation-induced X-ray emission (SRIXE) of single freeze-dried cells. After the 20-min treatment, Cr was confined to a small area of the cytoplasm and strongly co-localized with S, Cl, K, and Ca. After the 4-h treatment, Cr was distributed throughout the cell, with higher concentrations in the nucleus and the cytoplasmic membrane. This time-dependence corresponded to approximately 100% or 0% clonogenic survival of the cells following the 20-min or 4-h treatments, respectively, and could potentially be explained by a new cellular protective mechanism. Such processes may also be important in reducing the potential hazards of Cr(III) dietary supplements, for which there is emerging evidence that they exert their anti-diabetic effects via biological oxidation to Cr(VI). The predominance of Cr(III) was confirmed by micro-XANES spectroscopy of intracellular Cr hotspots. X-ray absorption spectroscopy (XANES and EXAFS, using freeze-dried cells after the 0-4-h treatments) was used to gain insight into the chemical structures of Cr(III) complexes formed during the intracellular reduction of Cr(VI). The polynuclear nature of such complexes (probably with a combination of carboxylato and hydroxo bridging groups and O-donor atoms of small peptides or proteins) was established by XAFS data analyses.

Biotransformation↗

PubMed2005

Zinc concentration in esophageal biopsy specimens measured by x-ray fluorescence and esophageal cancer risk.

BACKGROUND: In rodents, zinc deficiency potentiates the effects of certain nitrosamines that act as esophageal carcinogens. Studies of the association between zinc and esophageal squamous cell carcinoma in humans have been hampered by plasma zinc homeostasis, which obscures individual differences in total zinc stores, and by the uncertainty regarding zinc bioavailability when estimating dietary zinc intake because phytate from whole grains effectively prohibits zinc absorption. By using baseline tissue biopsy specimens collected in a prospective observational study, we determined the association between incident esophageal squamous cell carcinoma and baseline element concentrations in tissue sections from residents of Linzhou, China, participating in a nutrition intervention trial. METHODS: We used x-ray fluorescence spectroscopy to measure zinc, copper, iron, nickel, and sulfur concentrations in single 5-microm-thick sections from formalin-fixed, paraffin-embedded esophageal biopsy specimens collected in 1985 from 60 eventual case and 72 control subjects. Subjects were matched on baseline histology and followed for 16 years. We used Cox proportional hazards models to estimate the hazard ratios (HRs) and 95% confidence intervals (CIs) for the association between each element and risk of incident esophageal cancer. All statistical tests were two-sided. RESULTS: The risk of developing esophageal cancer was much lower for subjects in the highest quartile of esophageal tissue zinc concentration compared with those in the lowest quartile (HR = 0.21, 95% CI = 0.065 to 0.68). The association was statistically significant across quartiles (P(trend) = .015). Individuals in the highest quartile of sulfur concentration had a lower risk of esophageal cancer than individuals in the lowest quartile (HR = 0.29, 95% CI = 0.095 to 0.85), but the association across quartiles was not statistically significant (P(trend) = .081). There was no association between copper, iron, or nickel concentrations and risk of esophageal cancer. CONCLUSION: High tissue zinc concentration was strongly associated with a reduced risk of developing esophageal squamous cell carcinoma. X-ray fluorescence spectroscopy can be used to assess relationships among concentrations of both nutritional and toxic elements and disease risk in banked tissue specimens.

Biopsy↗

PubMed2005

Elemental analysis of Mycobacterium avium-, Mycobacterium tuberculosis-, and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system.

Mycobacterium avium and Mycobacterium tuberculosis are human pathogens that infect and replicate within macrophages. Both organisms live in phagosomes that fail to fuse with lysosomes and have adapted their lifestyle to accommodate the changing environment within the endosomal system. Among the many environmental factors that could influence expression of bacterial genes are the concentrations of single elements within the phagosomes. We used a novel hard x-ray microprobe with suboptical spatial resolution to analyze characteristic x-ray fluorescence of 10 single elements inside phagosomes of macrophages infected with M. tuberculosis and M. avium or with avirulent M. smegmatis. The iron concentration decreased over time in phagosomes of macrophages infected with Mycobacterium smegmatis but increased in those infected with pathogenic mycobacteria. Autoradiography of infected macrophages incubated with (59)Fe-loaded transferrin demonstrated that the bacteria could acquire iron delivered via the endocytic route, confirming the results obtained in the x-ray microscopy. In addition, the concentrations of chlorine, calcium, potassium, manganese, copper, and zinc were shown to differ between the vacuole of pathogenic mycobacteria and M. smegmatis. Differences in the concentration of several elements between M. avium and M. tuberculosis vacuoles were also observed. Activation of macrophages with recombinant IFN-gamma or TNF-alpha before infection altered the concentrations of elements in the phagosome, which was not observed in cells activated following infection. Siderophore knockout M. tuberculosis vacuoles exhibited retarded acquisition of iron compared with phagosomes with wild-type M. tuberculosis. This is a unique approach to define the environmental conditions within the pathogen-containing compartment.

Animals↗

PubMed2005

Changes of the phagosomal elemental concentrations by Mycobacterium tuberculosis Mramp.

Pathogenic mycobacteria survive within phagosomes which are thought to represent a nutrient-restricted environment. Divalent cation transporters of the Nramp family in phagosomes and mycobacteria (Mramp) may compete for metals that are crucial for bacterial survival. The elemental concentrations in phagosomes of macrophages infected with wild-type Mycobacterium tuberculosis (M. tuberculosis strain H37Rv) and a M. tuberculosis Mramp knockout mutant (Mramp-KO), derived from a clinical isolate isogenic to the strain MT103, were compared. Time points of 1 and 24 h after infection of mouse peritoneal macrophages (bcg(S)) were compared in both cases. Increased concentrations of P, Ni and Zn and reduced Cl concentration in Mramp-KO after 1 h of infection were observed, compared to M. tuberculosis vacuoles. After 24 h of infection, significant differences in the P, Cl and Zn concentrations were still present. The Mramp-KO phagosome showed a significant increase of P, Ca, Mn, Fe and Zn concentrations between 1 and 24 h after infection, while the concentrations of K and Ni decreased. In the M. tuberculosis vacuole, the Fe concentration showed a similar increase, while the Cl concentration decreased. The fact that the concentration of several divalent cations increased in the Mramp-KO strain suggests that Mramp may have no impact on the import of these divalent cations into the mycobacterium, but may function as a cation efflux pump. The concordant increase of Fe concentrations within M. tuberculosis, as well as within the Mramp-KO vacuoles, implies that Mramp, in contrast to siderophores, might not be important for the attraction of Fe and its retention in phagosomes of unstimulated macrophages.

Animals↗

PubMed2004

Elemental and redox analysis of single bacterial cells by x-ray microbeam analysis.

High-energy x-ray fluorescence measurements were used to make elemental maps and qualitative chemical analyses of individual Pseudomonas fluorescens strain NCIMB 11764 cells. Marked differences between planktonic and adhered cells were seen in the morphology, elemental composition, and sensitivity to Cr(VI) of hydrated cells at spatial scales of 150 nm. This technology can be applied to natural geomicrobiological systems.

Bacterial Adhesion↗

PubMed2003

The cellular distribution and oxidation state of platinum(II) and platinum(IV) antitumour complexes in cancer cells.

The cellular distribution of platinum in A2780 ovarian cancer cells treated with cisplatin and platinum(IV) complexes with a range of reduction potentials has been examined using elemental analysis (synchrotron radiation-induced X-ray emission). The cellular distribution of platinum(IV) drugs after 24 h is similar to that of cisplatin, consistent with the majority of administered platinum(IV) drugs being reduced. Micro-X-ray absorption near-edge spectra of cells treated with cisplatin and platinum(IV) complexes confirmed the reduction of platinum(IV) to platinum(II). In cells treated, the most difficult to reduce complex, cis, trans, cis-[PtCl(2)(OH)(2)(NH(3))(2)], platinum(IV) was detected in the cells along with platinum(II). The observations are in accordance with the relative ease of reduction of the platinum(IV) complexes used and support the requirement of reduction for activation of platinum(IV) complexes.

Antineoplastic Agents↗

PubMed2003

The direct mapping of the uptake of platinum anticancer agents in individual human ovarian adenocarcinoma cells using a hard X-ray microprobe.

Uptake of platinum-based anticancer compounds into individual human ovarian andenocarcinoma cells was measured using an X-ray microprobe. The uptake of cisplatin, a platinum-based compound, in drug-resistant cells is decreased by approximately 50% after 24 h, compared with the uptake of the drug in nonresistant cells over the same time period. The Pt103 derivative of the drug, in contrast, showed an increased uptake by an order of magnitude in resistant cells over the same time period. Increased uptake appears to allow Pt103 to overcome the resistance mechanism developed by the cell. This work additionally shows that the X-ray microprobe is able to directly quantify Pt drug uptake on a subcellular level and can measure the mass of Pt down to a detectable limit of 20 attograms of Pt (2 x 10(-17) grams or 6 x 10(4) Pt atoms) in 1 s. Such exquisite elemental sensitivity combined with high spatial resolution paves the way for quantitative submicron three-dimensional mapping of elemental distributions within individual cells.

Adenocarcinoma↗

PubMed2003

Biology of TiO2-oligonucleotide nanocomposites.

Emerging areas of nanotechnology hold the promise of overcoming the limitations of existing technologies for intracellular manipulation. These new developments provide approaches for the creation of chemical-biological hybrid nanocomposites that can be introduced into cells and subsequently used to initiate intracellular processes or biochemical reactions. Such nanocomposites would advance medical biotechnology, just as they are improving microarray technology and imaging in biology and medicine, and introducing new possibilities in chemistry and material sciences. Here we describe the behaviour of 45-A nanoparticles of titanium dioxide semiconductor combined with oligonucleotide DNA into nanocomposites in vivo and in vitro. These nanocomposites not only retain the intrinsic photocatalytic capacity of TiO2 and the bioactivity of the oligonucleotide DNA (covalently attached to the TiO2 nanoparticle), but also possess the chemically and biologically unique new property of a light-inducible nucleic acid endonuclease, which could become a new tool for gene therapy.

Animals↗