Membrane oxidation assay--a novel lipoxygenase-based evaluation of membrane oxidizability

A membrane oxidation assay is presented which uses isolated erythrocyte membranes ("ghosts") and lipoxygenase as a selective catalyst for the transfer of oxygen to cis - cis -1,4-pentadiene-moieties. The latter are, for instance, present in linoleic and arachidonic acids, both of which are integral parts of membranes. These non-conjugated double bonds represent energetically favorable sites for oxidative attack and therefore, may be rearranged and partially consumed during oxidative stress. Consequently, the measurement of oxygen consumption in the course of the lipoxygenase-mediated oxidation provides a tool for the quick and reliable determination of such double bonds. Significant inter-individual differences have been noted in 11 subjects, which also correlate to the total radical antioxidant parameter (TRAP) values obtained. This assay will be helpful in the assessment of oxidizable structures in erythrocyte membranes that may be diminished as a consequence of oxidative damage suffered by an individual. In conclusion, a simple and rapid assay for the assessment of the oxidizability of erythrocyte membranes is presented complementing the TRAP assay for plasma antioxidative status.     

Assignment of amide proton signals by combined evaluation of HN,NN and HNCA MAS-NMR correlation spectra

In this paper, we present a strategy for the ¹H^N resonance assignment in solid-state magic-angle spinning (MAS) NMR, using the -spectrin SH3 domain as an example. A novel 3D triple resonance experiment is presented that yields intraresidue H^N-N-C correlations, which was essential for the proton assignment. For the observable residues, 52 out of the 54 amide proton resonances were assigned from 2D (¹H-¹⁵N) and 3D (¹H-¹⁵N-¹³C) heteronuclear correlation spectra. It is demonstrated that proton-driven spin diffusion (PDSD) experiments recorded with long mixing times (4 s) are helpful for confirming the assignment of the protein backbone ¹⁵N resonances and as an aid in the amide proton assignment.     

A self-perpetuating vicious cycle of tissue damage in human hibernating myocardium

Recently, we proposed the hypothesis that a vicious cycle exists in human hibernating myocardium (HM) between the progression of myocyte degeneration and the development of fibrosis [1]. We now investigated the pathomechanism of this cycle in more detail and established a correlation between the severity of the morphological changes and the degree of postoperative functional recovery of HM.HM was diagnosed by dobutamine echocardiography, thallium-201 scintigraphy and radionuclide ventriculography. Functional recovery was present at 3 months after coronary bypass surgery but remained unchanged at 15 months. Forty patients were subdivided into 2 groups: A with complete and B with incomplete recovery. Biopsies taken during surgery and studied by electron microscopy, immunocytochemistry, rt-PCR, and morphometry revealed myocyte degeneration and inflammatory and fibrinogenic changes in a widened interstitial space. We report here for the first time an upregulation of TGF-₁ evident by a 5-fold increase of fibroblasts and macrophages exhibiting a TGF-₁ content 3-fold larger than in control, and a > 3-fold increase in TGF-₁ mRNA by rt-PCR. The number of angiotensin converting enzyme (ACE) containing structures was increased (n/mm²: control - 11.4, A - 17.6, B - 19.2, control vs. A and B, p < 0.05). Fibrosis was more severe in group B than A or control (%: C - 10.1; A - 21.2; B - 40.6; p < 0.05). Capillary density was significantly reduced (n/mm²: C - 1152; A - 782; B - 579, p < 0.05) and intercapillary distance was widened (m: C - 29.5, A - 36.1, B - 43.3, p < 0.05). The number of CD 3 (n/mm²: C - 5.0; A - 9.6; B - 9.4, ns) and CD 68 positive cells (n/mm²: C - 37.2; A - 80.7; B - 55.0, C vs. A p < 0.05) was elevated in HM as compared to control indicating an inflammatory reaction. Cut-off points for functional recovery are fibrosis > 32%, capillary density < 660/mm² and intercapillary distance > 39.0 m.In HM a self-perpetuating vicious cycle of tissue alterations leads to progressive replacement fibrosis and continuous intracellular degeneration which should be interrupted by early revascularization.     

Sequence and diversity of T-cell receptor alpha V, J, and C genes of the owl monkey Aotus nancymaae

 We cloned and sequenced TcR alpha chain cDNA of three healthy Aotus nancymaae monkeys. Fifteen different TRAJ segments and 9 different TRAV genes were identified in the 29 rearrangements analyzed. As expected from the greater phylogenetic distance, A. nancymaae TRA gene sequences diverged more from the human sequences than those of the chimpanzee or the rhesus macaque. However, no Aotus TRAJ segment or TRAV gene was found which lacked a human counterpart. These counterparts were AJ02, AJ05, AJ09, AJ15, AJ22, AJ23, AJ28, AJ30, AJ32, AJ34, AJ37, AJ40, AJ42, AJ45, AJ52 and AV2S1, AV2S3, AV3S1, AV8S1, AV12S1, AV15S1, ADV21S1/DV5, AV22S1S and AV23S1, respectively. In most cases the identity of amino acid sequences between corresponding Aotus and human genes was greater than 80%. This marked conservation of TRA gene sequences indicates a close structural relationship of Aotus and human TcR and demonstrates that the TcR repertoire in primates is remarkably stable. The results support the concept of using Aotus monkeys, which are susceptible to infection with the human malaria parasite Plasmodium falciparum, as an animal model for the evaluation of molecularly defined malaria vaccine candidates. Received: 15 January 1998 / Revised: 23 March 1998     

Left ventricular assist device for end-stage heart failure: results of the first LVAD destination program in the Netherlands

PurposeMechanical circulatory support with a continuous-flow left ventricular assist device (LVAD) may be a valuable treatment in end-stage heart failure patients for an extended period of time. The purpose of this study was to evaluate the safety and efficacy of implantation of a continuous-flow LVAD in end-stage heart failure patients within the first destination program in the Netherlands.MethodsA third-generation LVAD was implanted in 16 heart failure patients (age 61 ± 8; 81 % male; left ventricular ejection fraction 20 ± 6 %) as destination therapy. All patients were ineligible for heart transplant. At baseline, 3 and 6 months, New York Heart Association (NYHA) functional class, quality-of-life and exercise capacity were assessed. Clinical adverse events were registered.ResultsSurvival at 30 days and 6 months was 88 and 75 %, respectively. In the postoperative phase, 6 (38 %) patients required continuous veno-venous haemofiltration for renal failure and 2 (13 %) patients required extracorporeal membrane oxygenation because of severe right ventricular failure. During follow-up, NYHA functional class and quality-of-life improved from 3.7 ± 0.1 to 2.3 ± 0.1 and 57 ± 5 to 23 ± 3 at 6 months (P < 0.001), respectively. The 6 min walking distance improved from 168 ± 42 m to 291 ± 29 m at 6 months (P = 0.001).ConclusionContinuous-flow LVAD therapy is a promising treatment for patients with end-stage heart failure ineligible for heart transplant.     

Multiparametric MRI of Epiphyseal Cartilage Necrosis (Osteochondrosis) with Histological Validation in a Goat Model

Purpose

To evaluate multiple MRI parameters in a surgical model of osteochondrosis (OC) in goats.

Methods

Focal ischemic lesions of two different sizes were induced in the epiphyseal cartilage of the medial femoral condyles of goats at 4 days of age by surgical transection of cartilage canal blood vessels. Goats were euthanized and specimens harvested 3, 4, 5, 6, 9 and 10 weeks post-op. Ex vivo MRI scans were conducted at 9.4 Tesla for mapping the T₁, T₂, T_1ρ, adiabatic T_1ρ and T_RAFF relaxation times of articular cartilage, unaffected epiphyseal cartilage, and epiphyseal cartilage within the area of the induced lesion. After MRI scans, safranin O staining was conducted to validate areas of ischemic necrosis induced in the medial femoral condyles of six goats, and to allow comparison of MRI findings with the semi-quantitative proteoglycan assessment in corresponding safranin O-stained histological sections.

Results

All relaxation time constants differentiated normal epiphyseal cartilage from lesions of ischemic cartilage necrosis, and the histological staining results confirmed the proteoglycan (PG) loss in the areas of ischemia. In the scanned specimens, all of the measured relaxation time constants were higher in the articular than in the normal epiphyseal cartilage, consistently allowing differentiation between these two tissues.

Conclusions

Multiparametric MRI provided a sensitive approach to discriminate between necrotic and viable epiphyseal cartilage and between articular and epiphyseal cartilage, which may be useful for diagnosing and monitoring OC lesions and, potentially, for assessing effectiveness of treatment interventions.