Showing papers by "Thomas V. O'Halloran published in 2012"

Accumulation of cadmium in insulin-producing β cells

Abstract: Evidence suggests that chronic low level cadmium exposure impairs the function of insulin-producing β cells and may be associated with type-2 diabetes mellitus. Herein, we describe the cadmium content in primary human islets and define the uptake kinetics and effects of environmentally relevant cadmium concentrations in cultured β cells. The average cadmium content in islets from 10 non-diabetic human subjects was 29 ± 7 nmol/g protein (range 7 to 72 nmol/g protein). Exposure of the β-cell line MIN6 to CdCl 2 concentrations between 0.1 and 1.0 µmol/L resulted in a dose- and time-dependent uptake of cadmium over 72 h. This uptake resulted in an induction of metallthionein expression, likely enhancing cellular cadmium accumulation. Furthermore, cadmium accumulation resulted in an inhibition of glucose stimulated insulin secretion in MIN6 cells and primary mouse islets. Our results indicate that this impairment in β-cell function is not due to an increase in cell death or due to an increase in oxidative stress. We conclude that mouse β cells accumulate cadmium in a dose- and time-dependent manner over a prolonged time course at environmentally relevant concentrations. This uptake leads to a functional impairment of β-cell function without significant alterations in cell viability, expression of genes important for β-cell function or increase in oxidative stress.

A zinc-dependent mechanism regulates meiotic progression in mammalian oocytes.

Abstract: Precise coordination of meiotic progression is a critical determinant of an egg's capacity to be fertilized successfully, and zinc has emerged as a key regulatory element in this process. An early manifestation of a regulatory role for this transition metal is the significant increase in total intracellular zinc. This accumulation is essential for meiotic progression beyond telophase I and the establishment of meiotic arrest at metaphase II. The subsequent developmental event, fertilization, induces a rapid expulsion of labile zinc that is a hallmark event in meiotic resumption. In the present study, we show that the zinc fluxes work, in part, by altering the activity of the cytostatic factor (CSF), the cellular activity required for the establishment and maintenance of metaphase II arrest in the mature, unfertilized egg. We propose a model in which zinc exerts concentration-dependent regulation of meiosis through the CSF component EMI2, a zinc-binding protein. Together, the data support the conclusion that zinc itself, through its interaction with EMI2, is a central component of the CSF.

Zinc Maintains Prophase I Arrest in Mouse Oocytes Through Regulation of the MOS-MAPK Pathway

Abstract: Meiosis in mammalian females is marked by two arrest points, at prophase I and metaphase II, which must be tightly regulated in order to produce a haploid gamete at the time of fertilization. The transition metal zinc has emerged as a necessary and dynamic regulator of the establishment, maintenance, and exit from metaphase II arrest, but the roles of zinc during prophase I arrest are largely unknown. In this study, we investigate the mechanisms of zinc regulation during the first meiotic arrest. Disrupting zinc availability in the prophase I arrested oocyte by treatment with the heavy metal chelator N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine (TPEN) causes meiotic resumption even in the presence of pharmacological inhibitors of meiosis. We further show that the MOS-MAPK pathway mediates zinc-dependent prophase I arrest, as the pathway prematurely activates during TPEN-induced meiotic resumption. Conversely, inhibition of the MOS-MAPK pathway maintains prophase I arrest. While prolonged zinc insufficiency ultimately results in telophase I arrest, early and transient exposure of oocytes to TPEN is sufficient to induce meiotic resumption and bypass the telophase I block, allowing the formation of developmentally competent eggs upon parthenogenetic activation. These results establish zinc as a crucial regulator of meiosis throughout the entirety of oocyte maturation, including the maintenance of and release from the first and second meiotic arrest points.

Role of CTR4 in the Virulence of Cryptococcus neoformans

Abstract: While research has identified an important contribution for metals, such as iron, in microbial pathogenesis, the roles of other transition metals, such as copper, remain mostly unknown. Recent evidence points to a requirement for copper homeo- stasis in the virulence of Cryptococcus neoformans based on a role for a CUF1 copper regulatory factor in mouse models and in a human patient cohort. C. neoformans is an important fungal pathogen that results in an estimated 600,000 AIDS-related deaths yearly. In the present studies, we found that a C. neoformans mutant lacking the CUF1-dependent copper transporter, CTR4, grows normally in rich medium at 37°C but has reduced survival in macrophages and attenuated virulence in a mouse model. This reduced survival and virulence were traced to a growth defect under nutrient-restricted conditions. Expression studies us- ing a full-lengthCTR4-fluorescent fusion reporter construct demonstrated robust expression in macrophages, brain, and lung, the latter shown by ex vivo fluorescent imaging. Inductively coupled mass spectroscopy (ICP-MS) was used to probe the copper quota of fungal cells grown in defined medium and recovered from brain, which suggested a role for a copper-protective func- tion ofCTR4in combination with cell metallothioneins under copper-replete conditions. In summary, these data suggest a role forCTR4in copper-related homeostasis and subsequently in fungal virulence. IMPORTANCE Crytococcus neoformans is a significant global fungal pathogen, and copper homeostasis is a relatively unexplored aspect of microbial pathogenesis that could lead to novel therapeutics. Previous studies correlated expression levels of a Ctr4 copper transporter to development of meningoencephalitis in a patient cohort of solid-organ transplants, but a direct role for Ctr4 in mammalian pathogenesis has not been demonstrated. The present studies utilize a ctr4mutant strain which revealed an important role for CTR4 in C. neoformans infections in mice and relate the gene product to homeostatic control of copper and growth under nutrient-restricted conditions. Robust expression levels of CTR4during fungal infection were exploited to demonstrate expression and lung cryptococcal disease using ex vivo fluorescence imaging. In summary, these studies are the first to directly demonstrate a role for a copper transporter in fungal disease and provide an ex vivo imaging tool for further study of cryptococcal gene expression and pathogenesis.

Improved anti-proliferative effect of doxorubicin-containing polymer nanoparticles upon surface modification with cationic groups

Abstract: Polymer nanoparticles (PNPs) possessing a high density of drug payload have been successfully stabilized against aggregation in biological buffers after amine modification, which renders these PNPs positively charged. The resulting charge-stabilized PNPs retain their original narrow particle size distributions and well-defined spherical morphologies. This stabilization allows these PNPs to have an improved anti-proliferative effect on MDA-MB-231-Br human breast cancer cells compared to non-functionalized PNPs. As a non-cytotoxic control, similar surface-modified PNPs containing cholesterol in place of doxorubicin did not inhibit cell proliferation, indicating that the induced cytotoxic response was solely due to the doxorubicin release from the PNPs.

Modulation of oocyte meiotic progression and oocyte activation

Abstract: The present invention provides methods, compositions, and systems for reinitiating meiosis in cells in meiotic arrest and oocyte activation in fertilized, but un-activated, oocytes. In certain embodiments, Zn-binding moieties (e.g., zinc chelators) are used for reinitiating meiosis or oocyte activation.