Synthesis and characterization of a perchlorotriphenylmethyl (trityl) triester radical: a potential sensor for superoxide and oxygen in biological systems

Synthesis and characterization of an inert perchlorotriphenylmethyl triester radical, PTM-TE, are reported. PTM-TE was prepared by a facile 3-step synthesis using Friedel-Crafts reaction of tetrachlorobenzene with chloroform followed by ethoxycarbonylation and subsequent oxidation. PTM-TE is paramagnetic and exhibits a single sharp EPR spectrum. In solution, the EPR linewidth of PTM-TE is highly sensitive to the dissolved oxygen content, thus enabling accurate measurement of oxygen concentration (oximetry). In addition, the radical also shows high reactivity towards superoxide. The ester radical has the potential for use as a high-sensitive probe for determination of oxygen concentration and superoxide in biological systems.     

Polynitroxyl-albumin (PNA) enhances myocardial infarction therapeutic effect of tempol in rat hearts subjected to regional ischemia-reperfusion

Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl, TPL), a low molecular weight stable nitroxyl radical (nitroxide), has been demonstrated in many in vitro and in vivo models to have protective effects against oxidative stress. The beneficial effect of TPL, however, is limited because of its short life-time in tissues. We have previously shown that polynitroxylated macromolecules such as polynitroxyl-human serum albumin (PNA) enable maintaining a sustained concentration of TPL for longer periods in tissues. PNA itself has previously been shown to inhibit ischemia-reperfusion (I/R) injury in the gut and to potentiate the activity of TPL. The aim of the present study was (i) to select an optimum formulation of PNA + TPL for therapeutic applications using in vivo EPR spectroscopy and (ii) to evaluate the efficacy of the PNA + TPL formulation in preventing I/R injury to heart, in an in vivo rat model. Rats were subjected to 45 min occlusion of the left anterior descending (LAD) coronary artery followed by 120 min reperfusion. PNA (100 mg/ml) + TPL (10 mg/ml), human serum albumin (HSA, 100 mg/ml) + TPL (10 mg/ml), or saline were injected 5 min before ischemia (3 ml/kg BW, i.v.) and 5 min before reperfusion (3 ml/kg BW, i.v.), followed by a 4 ml/kg BW infusion over 2 h reperfusion. Myocardial risk and infarct regions were then estimated. The results showed that the infarct volume, expressed as a percentage of the risk region, in the group treated with PNA + TPL was 39.7 +/- 3.1%, which was significantly smaller than for the saline (51.3 +/- 3.5%) or HSA + TPL (48.4 +/- 1.4%) groups. The results demonstrate that the PNA + TPL combination is very effective in reducing myocardial ischemia-reperfusion injury.     

Cellular basis of tissue regeneration by omentum

The omentum is a sheet-like tissue attached to the greater curvature of the stomach and contains secondary lymphoid organs called milky spots. The omentum has been used for its healing potential for over 100 years by transposing the omental pedicle to injured organs (omental transposition), but the mechanism by which omentum helps the healing process of damaged tissues is not well understood. Omental transposition promotes expansion of pancreatic islets, hepatocytes, embryonic kidney, and neurons. Omental cells (OCs) can be activated by foreign bodies in vivo. Once activated, they become a rich source for growth factors and express pluripotent stem cell markers. Moreover, OCs become engrafted in injured tissues suggesting that they might function as stem cells.Omentum consists of a variety of phenotypically and functionally distinctive cells. To understand the mechanism of tissue repair support by the omentum in more detail, we analyzed the cell subsets derived from the omentum on immune and inflammatory responses. Our data demonstrate that the omentum contains at least two groups of cells that support tissue repair, immunomodulatory myeloid derived suppressor cells and omnipotent stem cells that are indistinguishable from mesenchymal stem cells. Based on these data, we propose that the omentum is a designated organ for tissue repair and healing in response to foreign invasion and tissue damage.     

Characterization of the structural and functional changes in the myocardium following focal ischemia-reperfusion injury

High-resolution (11.7 T) cardiac magnetic resonance imaging (MRI) and histological approaches have been employed in tandem to characterize the secondary damage suffered by the murine myocardium following the initial insult caused by ischemia-reperfusion (I/R). I/R-induced changes in the myocardium were examined in five separate groups at the following time points after I/R: 1 h, day 1, day 3, day 7, and day 14. The infarct volume increased from 1 h to day 1 post-I/R. Over time, the loss of myocardial function was observed to be associated with increased infarct volume and worsened regional wall motion. In the infarct region, I/R caused a decrease in end-systolic thickness coupled with small changes in end-diastolic thickness, leading to massive wall thickening abnormalities. In addition, compromised wall thickening was also observed in left ventricular regions adjacent to the infarct region. A tight correlation (r2 = 0.85) between measured MRI and triphenyltetrazolium chloride (TTC) infarct volumes was noted. Our observation that until day 3 post-I/R the infarct size as measured by TTC staining and MRI was much larger than that of the myocyte-silent regions in trichrome- or hematoxylin-eosin-stained sections is consistent with the literature and leads to the conclusion that at such an early phase, the infarct site contains structurally intact myocytes that are functionally compromised. Over time, such affected myocytes were noted to structurally disappear, resulting in consistent infarct sizes obtained from MRI and TTC as well as trichrome and hematoxylin-eosin analyses on day 7 following I/R. Myocardial remodeling following I/R includes secondary myocyte death followed by the loss of cardiac function over time.     

In vivo EPR measurement of glutathione in tumor-bearing mice using improved disulfide biradical probe

Disulfide nitroxide biradicals, DNB, have been used for glutathione, GSH, measurements by X-band electron paramagnetic resonance, EPR, in various cells and tissues. In the present paper, the postulated potential use of DNB for EPR detection of GSH in vivo was explored. Isotopic substitution in the structure of the DNB was performed for the enhancement of its EPR spectral properties. (15)N substitution in the NO fragment of the DNB decreased the number of EPR spectral lines and resulted in an approximately two-fold increase in the signal-to-noise ratio, SNR. An additional two-fold increase in the SNR was achieved by substitution of the hydrogen atoms with deuterium resulting in narrowing the EPR lines from 1.35 G to 0.95 G. The spectral changes of DNB upon reaction with GSH and cysteine were studied in vitro in a wide range of pHs at room temperature and "body" temperature, 37 degrees C, and the corresponding bimolecular rate constants were calculated. In in vivo experiments the kinetics of the L-band EPR spectral changes after injection of DNB into ovarian xenograft tumors grown in nude mice were measured by L-band EPR spectroscopy, and analyzed in terms of the two main contributing reactions, splitting of the disulfide bond and reduction of the NO fragment. The initial exponential increase of the "monoradical" peak intensity has been used for the calculation of the GSH concentration using the value of the observed rate constant for the reaction of DNB with GSH, k(obs) (pH 7.1, 37 degrees C)=2.6 M(-1)s(-1). The concentrations of GSH in cisplatin-resistant and cisplatin-sensitive tumors were found to be 3.3 mM and 1.8 mM, respectively, in quantitative agreement with the in vitro data.     

Assessment of wound-site redox environment and the significance of Rac2 in cutaneous healing

We have previously reported that H(2)O(2) is actively generated by cells at the wound site and that H(2)O(2)-driven redox signaling supports wound angiogenesis and healing. In this study, we have standardized a novel and effective electron paramagnetic resonance spectroscopy-based approach to assess the redox environment of the dermal wound site in vivo. Rac2 regulates inducible NADPH oxidase activation and other functional responses in neutrophils. Using Rac2-deficient mice we sought to investigate the significance of Rac2 in the wound-site redox environment and healing responses. Noninvasive measurements of metabolism of topically applied nitroxide (15)N-perdeuterated tempone in murine excisional dermal wounds demonstrated that the wound site is rich in oxidants, the levels of which peak 2 days postwounding in the inflammatory phase. Rac2-deficient mice had threefold lower production of superoxide compared to controls with similar wounds. In these mice, a lower wound-site superoxide level was associated with compromised wound closure. Immunostaining of wound edges harvested during the inflammatory phase showed that the numbers of phagocytic cells recruited to the wound site in Rac2-deficient and control mice were similar, but the amount of lipid peroxidation was significantly lower in Rac2-deficient mice, indicating compromised NADPH oxidase activity. Taken together, the findings of this study support that the wound site is rich in oxidants. Rac2 significantly contributes to oxidant production at the wound site and supports the healing process.     

Origination of the split structure of spliceosomal genes from random genetic sequences

The mechanism by which protein-coding portions of eukaryotic genes came to be separated by long non-coding stretches of DNA, and the purpose for this perplexing arrangement, have remained unresolved fundamental biological problems for three decades. We report here a plausible solution to this problem based on analysis of open reading frame (ORF) length constraints in the genomes of nine diverse species. If primordial nucleic acid sequences were random in sequence, functional proteins that are innately long would not be encoded due to the frequent occurrence of stop codons. The best possible way that a long protein-coding sequence could have been derived was by evolving a split-structure from the random DNA (or RNA) sequence. Results of the systematic analyses of nine complete genome sequences presented here suggests that perhaps the major underlying structural features of split-genes have evolved due to the indigenous occurrence of split protein-coding genes in primordial random nucleotide sequence. The results also suggest that intron-rich genes containing short exons may have been the original form of genes intrinsically occurring in random DNA, and that intron-poor genes containing long exons were perhaps derived from the original intron-rich genes.     

A naphthalocyanine-based EPR probe for localized measurements of tissue oxygenation.

A new electron paramagnetic resonance (EPR) oximetry probe, based on a naphthalocyanine macrocycle, is reported to exhibit high oxygen sensitivity and favorable EPR characteristics for biological applications. The free radical probe, lithium naphthalocyanine (LiNc), is synthesized as fine microcrystalline powder with particle size less than 1 microm and high spin density. It exhibits a single sharp EPR peak, whose width varies linearly with oxygen partial pressure (pO2). The EPR spectrum is nonsaturable at typical microwave power levels (< 25 mW at X-band). These unique characteristics make this probe ideal for measuring oxygen concentration in biological tissues, in vivo. The peak-to-peak width under anoxic conditions is 0.51 G (at X-band), and it increases linearly with increase in oxygen partial pressure and reaches 26.0 G for 100% oxygen (760 mmHg), showing an oxygen sensitivity of 34 mG/mmHg. The probe responds to changes in pO2 quickly and reproducibly, thus enabling dynamic measurements of regional oxygenation in real time. The application of this probe for oximetry is demonstrated in an in vivo biological system. The changes in pO2 were monitored in the leg muscle tissue of a living mouse breathing room air and carbogen (95% oxygen + 5% CO2), alternatively. The mean pO2 measured with this probe in muscle tissues was consistent with values reported previously using other methods. Overall, the probe shows very desirable characteristics for localized measurements of tissue oxygenation.