Leo Kesner

Copper(II)-catalyzed lipid peroxidation in liposomes and erythrocyte membranes

Cu++ was uniquely capable of catalyzing the peroxidation of rat erythrocyte membrane lipid in the presence of 10 mM H2O2, whereas several other transition metal ions were without significant effect. In contrast, peroxidation of soybean phospholipid liposomes could be catalyzed with decreasing efficiency by Co++, Cu++, Pb++, or Cr+++ also in the presence of H2O2. The effect of imidazole on Cu++-catalyzed lipid peroxidation was stimulatory in liposomes and inhibitory in membrane preparations, whereas EDTA, histidine, citrate and alanine inhibited peroxidation in both systems. EDTA could stop the peroxidation after initiation, but catalase could not, indicating that Cu++ alone was necessary for the propagation of the chain reaction. Competitive inhibition studies with various scavengers of hydroxyl radicals or singlet oxygen and the absence of significant reaction enhancement by D2O indicated that neither of these reactive oxygen species was a major mediator in the Cu++-H2O2 oxidative system. A copper-oxygen complex may be directly involved in the initiation of peroxidation. Normal erythrocyte membranes and phospholipid liposomes also differ in their sensitivities toward external oxidative stress. In the absence of H2O2, Cu++ (0.2 mM) was capable of catalyzing lipid peroxidation in liposomes, aged erythrocyte membranes and membranes from vitamin-E-deficient rats; however, freshly prepared membranes from control rats and liposomes containing α-tocopherol required H2O2 greater than 2 mM for the catalytic effect of Cu++ to be observed.

A novel calpain inhibitor for the treatment of acute experimental autoimmune encephalomyelitis

Aberrant activation of calpain plays a key role in the pathophysiology of several neurodegenerative disorders. Calpain is increasingly expressed in inflammatory cells in EAE and is significantly elevated in the white matter of patients with multiple sclerosis, thus calpain inhibition could be a target for therapeutic intervention. The experiments reported here employed a myelin oligodendrocyte glycoprotein-induced disease model in C57Bl/6 mice (EAE) and a novel calpain inhibitor, targeted to nervous tissue. CYLA was found to reduce clinical signs of EAE and prevent demyelination and inflammatory infiltration in a dose- and time-dependent manner. Oral administration of the diacetal prodrug was equally effective.

Prevention of Axonal Injury using Calpain Inhibitor in Chronic Progressive Experimental Autoimmune Encephalomyelitis

Axonal injury is the major correlate of permanent disability in neurodegenerative diseases such as multiple sclerosis (MS), especially in secondary-progressive MS which follows relapsing-remitting disease course. Proteolytic enzyme, calpain, is a potential candidate for causing axonal injury. Most current treatment options only target the inflammatory component of MS. Previous work using calpain inhibitor CYLA in our laboratory showed significant reduction in clinical sign, demyelination and tissue calpain content in acute experimental autoimmune encephalomyelitis (EAE). Here we evaluated markers of axonal injury (amyloid precursor protein, Na(v)1.6 channels), neuronal calpain content and the effect of CYLA on axonal protection using histological methods in chronic EAE [myelin oligodendrocyte glycoprotein (MOG)-induced disease model of MS]. Intraperitoneal application of CYLA (2 mg/mouse/day) significantly reduced the clinical signs, tissue calpain content, demyelination and inflammatory infiltration of EAE. Similarly, markers for axonal injury were barely detectable in the treated mice. Thus, this novel drug, which markedly suppresses the disease course, axonal injury and its progression, is a candidate for the treatment of a neurodegenerative disease such as multiple sclerosis.

Carnitine coupled pharmaceutical agents

Carnitine, which preferentially accumulates in cardiac and skeletal muscle, is coupled to a protease inhibitor, or any other pharmaceutically active compound for the purpose of site-specific drug delivery to these tissues. These products may be useful in a variety of muscle wasting diseases. They may also be useful in a variety of cardiac conditions including those produced by cardiac ischemia. They may also be useful as growth promoters for animals.

Degradation of articular cartilage by copper and hydrogen peroxide

When porcine articular cartilage particles were incubated in the presence of Cu2+ and H2O2 at pH 7.4, solubilization of collagen and proteoglycan was observed. Both agents were necessary and the rate of solubilization was concentration dependent. Other transition metal ions showed much lower catalytic activity. The solubilized polypeptides were polydispersed in size and the hydroxyproline content of the larger fragments was 13% by weight. Further incubation of the released material with Cu2+ and H2O2 resulted in further degradation and partial destruction of hydroxyproline residues. Competitive studies with scavengers of OH· and1O2 as well as the effect of D2O excluded these two species as major mediators in this system.

Copper (II) complex-catalyzed oxidation of NADH by hydrogen peroxide

Among various metal ions of physiological interest, Cu2+ is uniquely capable of catalyzing the oxidation of NADH by H2O2. This oxidation is stimulated about fivefold in the presence of imidazole. A similar activating effect is found for some imidazole derivatives (1-methyl imidazole, 2-methyl imidazole, andN-acetyl-L-histidine). Some other imidazole-containing compounds (L-histidine,L-histidine methyl ester, andL-carnosine), however, inhibit the Cu2+-catalyzed peroxidation of NADH. Other chelating agents such as EDTA andL-alanine are also inhibitory. Stoichiometry for NADH oxidation per mole of H2O2 utilized is 1, which excludes the possibility of a two-step oxidation mechanism with a nucleotide free-radical intermediate. About 92% of the NADH oxidation product can be identified as enzymatically active NAD+. D2O, 2,5-dimethylfuran, and 1,4-diazabicyclo [2.2.2]-octane have no significant effect on the oxidation, thus excluding1O2 as a mediator. Similarly, OH· is also not a likely intermediate, since the system is not affected by various scavengers of this radical. The results suggest that a copper-hydrogen peroxide intermediate, when complexed with suitable ligands, can generate still another oxygen species much more reactive than its parent compound, H2O2.

Amino acid content of muscle and plasma with altered pH.

The free amino acid contents of plasma and skeletal muscle were determined in rats with K + -deficiency, acute respiratory acidosis or acute respiratory alkalosis. K + -deficiency caused a reduction in intracellular pH (pH i ) and an elevation of extracellular pH (pH e ). The acute respiratory acidosis reduced skeletal muscle pH 1 to the same extent K + -deficiency did; the respiratory alkalosis imposed elevated pH e to a level comparable with K + -deficiency. This made it possible to determine whether or not altered pH 1 or pH e might directly influence the movement of amino acids into (or out of) skeletal muscle cells. Low-K + muscle contained increased amounts of basic amino acids, primarily lysine. These increases accounted for about 30 per cent of the alkali metal cation deficit found in K + -deficient muscle. Aside from changes in basic amino acids, low-K + skeletal muscle had increased amounts of threonine, serine, glutamine and citrulline; there were decreased concentrations of isoleucine plus leucine, valine, alanine and aspartic acid. No comparable pattern of amino acid changes were observed in normal rats with comparably reduced pH 1 or elevated pH e induced by changing pCO 2 . These findings indicate that altered pH i or pH e have no direct influence on free amino acid movements across rat skeletal muscle cells in vivo.

Studies on the hydrolytic action of pronase on derivatized and non-derivatized proteins

1. 1. The products of pronase hydrolysis of native, oxidized, dinitrophenylated, and dinitrophenylated-oxiodized RNAase (polyribonucleotide 2-oligonucleotidotransferase (cyclizing), EC 2.7.7.16) were fractionated by means of silica-gel-column chromatography of their 2,4-dinitrophenyl derivatives. 2. 2. With the exception of glycine and proline, most of the amino acids, including asparagine, glutamine and methionine sulfone, were found to be well liberated. 3. 3. Pronase action on a dinitrophenylated protein yielded ϵ-DNP-lysine but not O-DNP-tyrosine or imidazole-DNP-histidine. 4. 4. It was pointed out that the introduction of toluene into the solvent system eliminates the need for preliminary extraction procedures commonly employed for the silica gel column chromatography of DNP derivatives.

A novel calpain inhibitor for treatment of transient retinal ischemia in the rat.

After an acute ischemia/reperfusion of the rat retina, the activation of cytotoxic proteases, including calpain, results in necrosis and apoptosis of retinal ganglion cells resulting in their degeneration. Using a systemically administered calpain inhibitor that crosses the blood–retinal barrier would provide for novel systemic intervention that protects the retina from acute injury and loss of function. Herein, we study a novel calpain peptide inhibitor, cysteic–leucyl–argininal (CYLA), in an in-vivo rat model of retinal ischemia to determine functional protection using electroretinography. The CYLA prodrug was administered intraperitoneally before and/or after ischemia–reperfusion at concentrations of 20–40 mg/kg. We found that administering 20 mg/kg of CYLA only after ischemia provides significant preservation of retinal function.

[9] Determination of amino acids as DNP derivatives by automatic partition chromatography

Publisher Summary This chapter describes the automatic partition chromatography method for the determination of amino acids as DNP derivatives. A gradient elution system composed of n -heptane, tert -amyl alcohol, and methylethyl ketene is deaerated, water-saturated, and pumped through a hydrated silica gel column at a uniform flow rate. The effluent is passed through a photometer from which absorbance is continuously measured. By passing a small volume of toluene through the column prior to gradient elution, unreacted dinitrofluorobenzene, dinitrophenol, and dinitroaniline are eluted before the amino acids, thus eliminating the necessity for preliminary extraction of the reaction mixture. Reproducibility and quantitation depend upon the precise control of a variety of factors. These include the uniform preparation of hydrated silica gel columns, use of deaerated water-saturated solvents in a consistent manner, and the stability of the photometric recording system. The chapter focuses on the used reagents, apparatus, column preparation, dinitrophenylation, column operation, and calibration procedures. Continuous counting of radioactive materials from the column effluent is also discussed.