Rebecca A. Bozym

Free zinc ions outside a narrow concentration range are toxic to a variety of cells in vitro.

The zinc(II) ion has recently been implicated in a number of novel functions and pathologies in loci as diverse as the brain, retina, small intestine, prostate, heart, pancreas, and immune system. Zinc ions are a required nutrient but elevated concentrations are known to kill cells in vitro. Paradoxical observations regarding zincs effects have appeared frequently in the literature, and often their physiological relevance is unclear. We found that for PC-12, HeLa and HT-29 cell lines as well as primary cultures of cardiac myocytes and neurons in vitro in differing media, approximately 5 nmol/L free zinc (pZn = 8.3, where pZn is defined as – log10 [free Zn2+]) produced apparently healthy cells, but 20-fold higher or (in one case) lower concentrations were usually harmful as judged by multiple criteria. These results indicate that (1) the free zinc ion levels of media should be controlled with a metal ion buffer; (2) adding zinc or strong zinc ligands to an insufficiently buffered medium may lead to unpredictably low or high free zinc levels that are often harmful to cells; and (3) it is generally desirable to measure free zinc ion levels due to the presence of contaminating zinc in many biochemicals and unknown buffering capacity of many media.

Release of Intracellular Calcium Stores Facilitates Coxsackievirus Entry into Polarized Endothelial Cells

Group B coxsackieviruses (CVB) are associated with viral-induced heart disease and are among the leading causes of aseptic meningitis worldwide. Here we show that CVB entry into polarized brain microvasculature and aortic endothelial cells triggers a depletion of intracellular calcium stores initiated through viral attachment to the apical attachment factor decay-accelerating factor. Calcium release was dependent upon a signaling cascade that required the activity of the Src family of tyrosine kinases, phospholipase C, and the inositol 1,4,5-trisphosphate receptor isoform 3. CVB-mediated calcium release was required for the activation of calpain-2, a calcium-dependent cysteine protease, which controlled the vesicular trafficking of internalized CVB particles. These data point to a specific role for calcium signaling in CVB entry into polarized endothelial monolayers and highlight the unique signaling mechanisms used by these viruses to cross endothelial barriers.

Excitation ratiometric fluorescent biosensor for zinc ion at picomolar levels

Zinc is a metal ion of increasing significance in several biomedical fields, including neuroscience, immunology, reproductive biology, and cancer. Fluorescent indicators have added greatly to our understanding of the biology of several metal ions, most notably calcium. Despite substantial efforts, only recently have zinc indicators been developed which are sufficiently selective for use in the complex intra- and extracellular milieus, and which are capable of quantifying the free zinc levels with some degree of reliability. However, these indicators (such as FuraZin-1 and Newport Green DCF) have only modest sensitivity, and there is growing evidence that significantly lower levels of free zinc may be biologically relevant in some instances. We have adapted the peerless selectivity and sensitivity of a carbonic anhydrase-based indicator system to an excitation ratiometric format based on resonance energy transfer: i.e., where the zinc ion level is transduced as the ratio of fluorescence intensities excited at two different excitation wavelengths, which is preferred for fluorescence microscopy. The system exhibits more than a 60% increase in the ratio of intensity excited at 365 nm to that excited at 546 nm (emission observed at 617 nm). The detection limit is about 10 pM in zinc buffered systems, a 10-1000-fold improvement on the Fura indicators (which respond to Ca and Mg as well), and a 10 000-fold improvement on the recently described FuraZin-1.

Chapter 14 Determination of Zinc Using Carbonic Anhydrase-Based Fluorescence Biosensors

This chapter summarizes the use of carbonic anhydrase (CA)-based fluorescent indicators to determine free zinc in solution, in cells, and in subcellular organelles. Expression (both in situ and in vitro) and preparation of CA-based indicators are described, together with techniques of their use, and procedures to minimize contamination. Recipes for zinc buffers are supplied.

Calcium signals and calpain-dependent necrosis are essential for release of coxsackievirus B from polarized intestinal epithelial cells

In contrast to nonpolarized cells, coxsackievirus B (CVB)–infected polarized intestinal Caco-2 cells undergo necrotic cell death triggered by inositol 1,4,5-trisphosphate receptor–dependent calcium release. This CVB-induced necrosis depends on Ca2+-activated calpain-2, which is required for disruption of the apical tight junction complex.

In situ Measurement of Free Zinc in an Ischemia Model and Cell Culture Using a Ratiometric Fluorescence-Based Biosensor

Zinc ion is of growing interest in medicine and biology generally, and especially in the ischemic brain and other tissues. We have developed ratiometric fluorescence-based biosensors for the study of zinc in these systems; the biosensors use apocarbonic anhydrase variants as recognition elements that offer high sensitivity and selectivity. We report continuous in situ, in vivo measurement of nanomolar extracellular zinc in the brain of an animal model of ischemia using a ratiometric fiber optic biosensor. We also report the development of an expressible excitation ratiometric indicator of zinc ion suitable for use in cells that exhibits picomolar sensitivity. Finally, we also report the discovery that the Zn complex of the chelator TPEN seems to be comparably apoptogenic to the free chelator itself.

In vivo and intracellular sensing and imaging of free zinc ion

We describe new methods for the study of zinc in biological specimens. Intracellular free zinc was determined at levels down to picomolar using an excitation ratiometric fluorescence-based biosensing approach using a carbonic anhydrase variant as transducer. A new fiber optic sensor suitable for in vivo use is also described using laser excitation and an emission ratiometric approach; the zinc concentration range of sensor response can be selected to fit the application.

Imaging Intracellular and Intra-Mitochondrial Free Zinc Ion Concentrations Following Hypoxia/Hypoglycemia with an Expressible Fluorescence Biosensor

Zinc is a “trace” metal necessary for cellular function, but excess free zinc ion is toxic to most cells (1,2,9) We and others have observed increased intra- and extra-cellular free zinc concentrations following cerebral ischemia (3,4). Substantial evidence indicates that mitochondrial dysfunction plays a significant role in neuronal death following ischemia (5), and both mitochondrial dysfunction and increased intracellular zinc have been associated with increased production of reactive oxygen species (ROS) and ultimately apoptosis (6,7). Zinc, potently, inhibits mitochondrial enzymes involved in energy production and destruction of ROS, potentially promoting mitochondrial dysfunction (8). We targeted our expressible fluorescent zinc biosensor (9) to mitochondria of PC12 cells, to ratiometrically image the intra-mitochondrial zinc concentration at resting (pM) levels. We imaged cytoplasmic and mitochondrial zinc levels in cells deprived of oxygen and glucose (OGD), a widely used model of ischemia. Our data indicate that both intra-mitochondrial and cytoplasmic zinc concentrations increase substantially following OGD. Further experiments indicate that the zinc is released from intracellular sites rather than entering the cell from external sources. Supported by NIH EB03924.1. Canzoniero, L.M., et al. (1999) Journal of Neuroscience 19:RC31, 1–62. Zodl, B, et al. (2003) Journal of Inorganic Biochemistry 97, 324–3303. Tonder, N., et al. (1990) Neuroscience Letters 109, 247–2524. Frederickson, C.J., et al (2006) Experimental Neurology 198, 285–2935. Fiskum G., et al. (2008) Mitochondria and Oxidative Stress in Neurodegenerative Disorders: Annals of the New York Academy of Science. 1147, 129–1386. Weiss, J.H., et al. (2000) Trends in Pharmacological Science 21, 395–4017. Jiang, D., et al. (2001) Journal of Biological Chemistry 276, 47524–475298. Gazaryan, I.G., et al. (2007) Journal of Biological Chemistry 282, 24373–243809. Bozym, R.A., et al. (2006) ACS Chemical Biology 1, 103–111.