Kemal Baysal

Cation transport systems in mitochondria: Na+ and K+ uniports and exchangers

It is now well established that mitochondria contain three antiporters that transport monovalent cations. A latent, allosterically regulated K+/H+ antiport appears to serve as a cation-extruding device that helps maintain mitochondrial volume homeostasis. An apparently unregulated Na+/H+ antiport keeps matrix [Na+] low and the Na+-gradient equal to the H+-gradient. A Na+/Ca2+ antiport provides a Ca2+-extruding mechanism that permits the mitochondrion to regulate matrix [Ca2+] by balancing Ca2+ efflux against influx on the Ca2+-uniport. All three antiports have well-defined physiological roles and their molecular properties and regulatory features are now being determined. Mitochondria also contain monovalent cation uniports, such as the recently described ATP- and glibenclamide-sensitive K+ channel and ruthenium red-sensitive uniports for Na+ and K+. A physiological role of such uniports has not been established and their properties are just beginning to be defined.

Regulation of the mitochondrial Na+Ca2+ antiport by matrix pH☆

The effect of matrix pH (pHi) on the activity of the mitochondrial Na+/Ca2+ antiport has been studied using the fluorescence of SNARF-1 to monitor pHi and Na(+)-dependent efflux of accumulated Ca2+ to follow antiport activity. Heart mitochondria respiring in a KCl medium maintain a large delta pH (interior alkaline) and show optimal Na+/Ca2+ antiport only when the pH of the medium (pH0) is acid. Addition of nigericin to these mitochondria decreases delta pH and increases the membrane potential (delta psi). Nigericin strongly activates Na+/Ca2+ antiport at values of pH0 near 7.4 but inhibits antiport activity at acid pH0. When pHi is evaluated in these protocols, a sharp optimum in Na+/Ca2+ antiport activity is seen near pHi 7.6 in the presence or absence of nigericin. Activity falls off rapidly at more alkaline values of pHi. The effects of nigericin on Na+/Ca2+ antiport are duplicated by 20 mM acetate and by 3 mM phosphate. In each case the optimum rate of Na+/Ca2+ antiport is obtained at pHi 7.5 to 7.6 and changes in antiport activity do not correlate with changes in components of the driving force of the reaction (i.e., delta psi, delta pH, or the steady-state Na+ gradient). It is concluded that the Na+/Ca2+ antiport of heart mitochondria is very sensitive to matrix [H+] and that changes in pHi may contribute to the regulation of matrix Ca2+ levels.

MITOCHONDRIAL COMPLEX I, II/III, AND IV ACTIVITIES IN FAMILIAL AND SPORADIC PARKINSON'S DISEASE

A possible role of mitochondrial respiratory chain dysfunction in the pathogenesis of sporadic Parkinsons disease (PD) has been described. There are only a few reports concerning mitochondrial involvement in familial Parkinsons disease. The present study investigated mitochondrial complex I-IV activity in patients with sporadic and familial PD, compared to controls. Platelets were isolated from venous blood and platelet mitochondria were obtained through sonication and differential centrifugation. Complex I, II/III, and IV activities were measured in 17 patients with family history of Parkinsons disease (PDF), 15 patients with sporadic Parkinson disease (PDS), and 17 age-matched, healthy controls. The mitochondrial enzyme activities did not differ significantly between patient groups and controls. In addition, there was no correlation between mitochondrial complex activities and age, severity of disease, or age at onset of disease in the patient groups. In this study, the data indicate no significant differences in mitochondrial complex I-IV activities in PDF and PDS.

On the relationship between matrix free Mg2+ concentration and total Mg2+ in heart mitochondria

The matrix free magnesium ion concentration, [Mg2+]m, estimated using the fluorescent probe furaptra, averaged 0.67 mM in 15 preparations of beef heart mitochondria containing an average of 21 nmol total Mg2+ per mg protein. [Mg2+]m was compared with total Mg2+ during respiration-dependent uptake and efflux of Mg2+ and during osmotic swelling. In the absence of external Pi these mitochondria contain about 32 nmol/mg non-diffusible Mg-binding sites with an apparent Kd of 0.34 mM. [Mg2+]m depends on both the size of the total Mg2+ pool and the ability of matrix anions to provide Mg-ligands. Pi interacts strongly with Mg2+ to decrease [Mg2+]m and, in the absence of external Mg2+, promotes respiration-dependent Mg2+ efflux and a decrease in [Mg2+]m to very low levels. The uptake of Pi by respiring mitochondria converts delta pH to membrane potential (delta psi) and provides additional Mg-binding sites. This permits large accumulations of Mg2+ and Pi with little change in [Mg2+]m. Nigericin also converts delta pH to delta psi in respiring mitochondria and induces a large and rapid increase in both total Mg2+ and [Mg2+]m. Mersalyl increases the permeability of the mitochondrial membrane to cations and this also induces a marked increase in both total Mg2+ and [Mg2+]m. These results suggest that mitochondria take up Mg2+ by electrophoretic flux through membrane leak pathways, rather than via a specific Mg2+ transporter. Mitochondria swollen by respiration dependent uptake of potassium phosphate show decreased [Mg2+]m, whereas those swollen to the same extent in potassium acetate do not. This suggests that [Mg2+]m is well-buffered during osmotic volume changes unless there is also a change in ligand availability.

Novel short peptides isolated from phage display library inhibit vascular endothelial growth factor activity

Signal transduction through the vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) pathway has a pivotal importance in angiogenesis, and has therefore become a prime target in antitumor therapy. In search for peptides antagonizing VEGF binding to its receptors, we screened a random heptamer library displayed on phage for peptides that bind the whole VEGF165 molecule and inhibit VEGF dependent human umbilical vein endothelial cell (HUVEC) proliferation. Two selected peptides with sequences WHLPFKC and WHKPFRF were synthesized. Biacore and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis indicated that these peptides bind the VEGF homodimer in a concentration-dependent manner, with micromolar affinity, and with a 2:1 peptide: VEGF stoichiometry. They inhibited HUVEC proliferation in vitro by 77 and 55%, respectively. Taken together, our results indicate that these peptides could be potent inhibitors of angiogenesis. Furthermore, we show that the peptide-VEGF binding properties can be quantified, a prerequisite for the further optimization of binders.

Synthesis and characterization of poly(ethylene glycol)-poly(D,L-lactide-co-glycolide) poly(ethylene glycol) tri-block co-polymers modified with collagen : a model surface suitable for cell interaction

This study focused on the synthesis and characterization of poly(ethylene glycol)poly(D,L-lactide-co-glycolide)-poly(ethylene glycol) tri-block co-polymer (PEG-PDLLG-PEG), and its modification with type-I collagen. To this aim, a PEG-PDLLG-PEG tri-block co-polymer was synthesized in two steps by reacting poly(ethylene glycol)bis(carboxymethyl)ether with thionyl chloride to obtain an acyl-halide-terminated poly(ethylene glycol) and subsequently coupling this compound to hydroxyl-terminated poly(D,L-lactide-co-glycolide) (PDLLG). The new carboxyl end-groups of PEG-PDLLG-PEG were subsequently reacted with N-hydroxysuccinimide (NHS) in the presence of the hetero-bifunctional cross-linking agent dicyclohexylcarbodiimide (DCC) in order to activate the co-polymer for coupling with collagen. PEG-PDLLG-PEG and its activated form PEG-PDLLG-NHS were characterized by Fourier transform infrared (FT-IR) and 1H-NMR spectroscopy. Molecular weights of the polymeric products were determined by SEC. Type-I collagen in phosphate buffer was reacted with PEG-PDLLG-NHS. The resultant product, PEG-PDLLG-Col, was characterized by FT-IR. This biopolymer was used for preparation of a suitable surface for cell growth experiment. To measure the degree of cell proliferation, the films prepared with PDLLG, PEG-PDLLG-NHS and PEG-PDLLG-Col were seeded with L929 mouse fibroblasts. Cell growth was followed by SEM photography and quantitated by the neutral red uptake assay. It was shown that the attachment of collagen significantly increased the number of cells on the co-polymers.

Proteomic study of the microdissected aortic media in human thoracic aortic aneurysms.

Aortic aneurysm is a complex multifactorial disease, and its molecular mechanism is not understood. In thoracic aortic aneurysm (TAA), the expansion of the aortic wall is lead by extracellular matrix (ECM) degeneration in the medial layer, which leads to weakening of the aortic wall. This dilatation may end in rupture and-if untreated-death. The aortic media is composed of vascular smooth muscle cells (VSMCs) and proteins involved in aortic elasticity and distensibility. Delineating their functional and quantitative decrease is critical in elucidating the disease causing mechanisms as well as the development of new preventive therapies. Laser microdissection (LMD) is an advanced technology that enables the isolation of the desired portion of tissue or cells for proteomics analysis, while preserving their integrity. In our study, the aortic media layers of 36 TAA patients and 8 controls were dissected using LMD technology. The proteins isolated from these tissue samples were subjected to comparative proteomic analysis by nano-LC-MS/MS, which enabled the identification of 352 proteins in aortic media. Among these, 41 proteins were differentially expressed in the TAA group with respect to control group, and all were downregulated in the patients. Of these medial proteins, 25 are novel, and their association with TAA is reported for the first time in our study. Subsequent analysis of the data by ingenuity pathway analysis (IPA) shows that the majority of differentially expressed proteins were found to be cytoskeletal-associated proteins and components of the ECM which are critical in maintaining aortic integrity. Our results indicate that the protein expression profile in the aortic media from TAA patients differs significantly from controls. Further analysis of the mechanism points to markers of pathological ECM remodeling, which, in turn, affect VSMC cytosolic structure and architecture. In the future, the detailed investigation of the differentially expressed proteins may provide insight into the elucidation of the pathological processes underlying aneurysms.

Synthesis and characterization of glycolide, L-lactide, and PDMS-based terpolymers as a support for cell cultures

Poly(lactic acid)/poly(glycolic acid)/poly(dimethylsiloxane) (PLGA/TEGOMER) terpolymers have been synthesized by the ring-opening polymerization of L-lactide and glycolide with α,ω-amine-terminated poly(dimethylsiloxane) prepolymer, using stannous octoate as a catalyst. The resulting terpolymers were characterized by various analytical techniques including size exclusion chromatography, 1H-nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectroscopy, and differential scanning calorimetry. The data showed that the terpolymers presented an amorphous structure. The glass transition temperature decreased with increasing TEGOMER unit content. For in vitro degradation studies, porous films were fabricated using a solvent-casting, particulate leaching technique. Degradation of the PLGA/TEGOMER terpolymer was studied in phosphate-buffered saline at pH 7.4 and 37°C. The degradation was followed by intrinsic viscosity, mass loss, and molecular weight measurements, and 1H-NMR spectroscopy. The mass loss after 55 days was 76% for the PLGA/TEGOMER (71/24/5) sample. Cell growth experiments using Swiss 3T3 fibroblasts demonstrated that PLGA/TEGOMER terpolymer matrices allow the attachment and growth of cells.

Abstract B48: Inhibition of recurrence of craniopharyngiomas with imatinib mesylate-loaded poly(lactide-co-glycolide) (PLGA) microspheres

Background: Recurrence is highly seen in craniopharyngiomas. Due to their close proximity to the vital structures, gross-total resection, which is thought to be the major determinant of the recurrence, is not always possible for craniopharyngiomas. So, post-operative long-term effective therapy approaches are needed to prevent the recurrence. Besides, our recent studies showed that there is a positive correlation between the recurrence and angiogenic potential of craniopharyngioma through over activation of PDGF (platelet-derived growth factor) signaling pathway. Objective: In this study, we aimed to investigate the inhibitory potential of local, long-term delivery of imatinib mesylate (PDGFR-B blocker) on recurrence of adult craniopharyngiomas via PLGA-type biodegradable microspheres. Methods: Imatinib mesylate containing PLGA polymers composed of different lactic/glycolic acid concentrations, molecular weights and drug compositions were synthesized by modified double emulsion/solvent evaporation technique. After in vitro characterization of protein holding, surface morphology, entrapment efficiency and drug release kinetics, inhibitory potential of microspheres on neovascularization was tested on craniopharyngioma tumor samples implanted in rat cornea. Results: Imatinib containing PLGA microspheres in different LA:GA ratios (LA:GA, 50:50 (M38); 75:25(M70); 85:15(M71)) considerably reduced the neovascularization induced by recurrent tumor samples in an in vivo rat cornea angiogenesis model, p Conclusion: Long-term, local delivery of imatinib mesylate to the post-surgical tumoral cavity using biodegradable microspheres is promising approach to prevent the recurrence of craniopharyngiomas. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B48.