Repeated blood instillation into the airway of the horse does not cause pulmonary fibrosis.

REASONS FOR PERFORMING THE STUDY: Exercise-induced pulmonary haemorrhage (EIPH) occurs in nearly all strenuously exercising horses. Recent studies have attempted to identify the role of free blood within the airspaces, in the lung fibrosis that develops within the lungs of EIPH horses. HYPOTHESIS: Repeated exposure of the equine lung to autologous blood results in lung fibrosis similar to that observed in spontaneous EIPH. METHODS: Forty ml of autologous blood from the jugular vein was instilled into preselected lung regions of 6 horses one, 2, 3, 4 or 5 times at 2 week intervals, with 40 ml of saline instilled into the contralateral lung serving as a control. The time interval between instillation of the first blood and euthanasia ranged from 2-10 weeks. The lung from each instillation site was harvested, and the histopathology was scored from each region based upon the presence and abundance of blood, haemosiderin and interstitial collagen. Consequently, at the time of euthanasia, the time since instillation of the first blood ranged from 2-10 weeks. RESULTS: Beyond retention of blood, and the accumulation of haemosiderin, there was no visible increase in perivascular and interstitial collagen within the blood-instilled lung sites. In a small number of regions, there were foci of bronchiolitis obliterans organising pneumonia with collagen accumulation within these foci, but no collagen accumulation with the characteristic perivascular and interstitial histological distribution seen in EIPH. CONCLUSIONS: Free blood within the airways of horses does not result in a qualitative increase in the amount of interstitial collagen within 8-10 weeks, and is therefore an unlikely aetiological factor in the lung collagen accumulation that occurs in EIPH. POTENTIAL RELEVANCE: This study emphasises the efficiency of the equine lung in clearing blood from the airspaces. Further, it suggests that the aetiopathogenesis of EIPH is not driven by events within the airspace lumen, but rather emanates from within the vasculature and lung interstitium.

Intrapericardial procedures for cardiac regeneration by stem cells: need for minimal invasive access (AttachLifter) to the normal pericardial cavity.

In view of the only modest functional and anatomical improvements achieved by bone marrow-derived cell transplantation in patients with heart disease, the question was addressed whether the intracoronary, transcoronary-venous, and intramyocardial delivery routes are adequate. It is hypothesized that an intrapericardial delivery of stem cells or activators of resident cardiac stem cells increases therapeutic benefits. From such an intrapericardial depot, cells or modulating factors, such as thymosin ß4 or Ac-SDKP, are expected to reach the myocardium with sustained kinetics. Novel tools which provide access to the pericardial space even in the absence of pericardial effusion are, therefore, described. When the pericardium becomes attached to the suction head (monitored by an increase in negative pressure), the pericardium is lifted from the epicardium ("AttachLifter"). The opening of the suction head ("Attacher") is narrowed by flexible clamps which grab the tissue and improve the vacuum seal in the case of uneven tissue. A ridge, i.e.,"needle guidance", on the suction head excludes injury to the epicardium, whereby the pericardium is punctured by a needle which resides outside the suction head. A fiberscope can be used to inspect the pericardium prior to puncture. Based on these procedures, the role of the pericardial space and the presence of pericardial effusion in cardiac regeneration can be assessed.

Strategies to mitigate diffuse phosphorus pollution during rewetting of fen peat soils.

The cultivation of fen peat soils (Eutric Histosols) for agricultural purposes, started in Europe about 250 years ago, resulting in decreased soil fertility, increased oxidation of peat and corresponding greenhouse gas fluxes to the atmosphere, nutrient transfer to aquatic ecosystems and losses in total area of the former native wetlands. To prevent these negative environmental effects set-aside programs and rewetting measures were promoted in recent years. Literature results and practical experiences showed that large scale rewetting of intensively used agricultural Histosols may result in mobilisation of phosphorus (P), its transport to adjacent surface waters and an accelerated eutrophication. The paper summarises results from an international European Community sponsored research project and demonstrates how results obtained at different scales and from different scientific disciplines were compiled to derive a strategy to carry out rewetting measures. Based on this findings a simple decision support system (DSS) for a hydrologically sensitive area in the Droemling catchment in north-eastern Germany was developed and since 2005 practically used to prevent freshwater resources from non point P pollution.

Differential association of MUC5AC and CLCA1 expression in small cartilaginous airways of RAO-affected and control horses.

REASONS FOR PERFORMING STUDY: Airway mucus accumulation is associated with indoor irritant and allergen exposure in horses with recurrent airway obstruction (RAO). Epidermal growth factor receptor (EGFR) and a chloride channel (calcium activated, family member 1; CLCA1) are key signalling molecules involved in mucin gene expression. OBJECTIVES: We hypothesised that exposure to irritants and aeroallergens would lead to increased expression of the mucin gene eqMUC5AC and increased stored mucosubstance in the airways of RAO-affected horses, associated with increased neutrophils and CLCA1 and EGFR mRNA levels. METHODS: We performed quantitative RT-PCR of eqMUC5AC, CLCA1 and EGFR; volume density measurements of intraepithelial mucosubstances; and cytological differentiation of intraluminal inflammatory cells in small cartilaginous airways from cranial left and right and caudal left and right lung lobes of 5 clinically healthy and 5 RAO-affected horses that had been exposed to indoor stable environment for 5 days before euthanasia. RESULTS: Neutrophils were increased in RAO-affected horses compared to clinically healthy controls. EqMUC5AC mRNA levels were positively correlated with both CLCA1 and EGFR mRNA levels in RAO-affected horses but only with CLCA1 in controls. The relationship between eqMUC5AC and CLCA1 differed in the 2 groups of horses with RAO-affected animals overexpressing CLCA1 in relation to eqMUC5AC. CONCLUSIONS: These data implicate CLCA1 as a signalling molecule in the expression of eqMUC5AC in horses but also suggest differential regulation by CLCA1 and EGFR between horses with RAO and those with milder degrees of airway inflammation.

Regional distribution of collagen and haemosiderin in the lungs of horses with exercise-induced pulmonary haemorrhage.

REASONS FOR PERFORMING STUDY: Regional veno-occlusive remodelling of pulmonary veins in EIPH-affected horses, suggests that pulmonary veins may be central to pathogenesis. The current study quantified site-specific changes in vein walls, collagen and haemosiderin accumulation, and pleural vascular profiles in the lungs of horses suffering EIPH. HYPOTHESIS: In the caudodorsal lung regions of EIPH-affected horses, there is veno-occlusive remodelling with haemosiderosis, angiogenesis and fibrosis of the interstitium, interlobular septa and pleura. METHODS: Morphometric methods were used to analyse the distribution and accumulation of pulmonary collagen and haemosiderin, and to count pleural vascular profiles in the lungs of 5 EIPH-affected and 2 control horses. RESULTS: Vein wall thickness was greatest in the dorsocaudal lung and significantly correlated with haemosiderin accumulation. Increased venous, interstitial, pleural and septal collagen; lung haemosiderin; and pleural vascular profiles occurred together and changes were most pronounced in the dorsocaudal lung. Further, haemosiderin accumulation colocalised with decreased pulmonary vein lumen size. Vein wall thickening, haemosiderin accumulation and histological score were highly correlated and these changes occurred only in the caudodorsal part of the lung. CONCLUSION: The colocalisation of these changes suggests that regional (caudodorsal) venous remodelling plays an important role in the pathogenesis of EIPH. POTENTIAL RELEVANCE: The results support the hypothesis that repeated bouts of venous hypertension during strenuous exercise cause regional vein wall remodelling and collagen accumulation, venous occlusion and pulmonary capillary hypertension. Subjected to these high pressures, there is capillary stress failure, bleeding, haemosiderin accumulation and, subsequently, lung fibrosis.

Effect of magnitude and direction of force on laryngeal abduction: implications for the nerve-muscle pedicle graft technique.

REASONS FOR PERFORMING STUDY: The nerve-muscle pedicle graft technique is a treatment for recurrent laryngeal neuropathy (RLN), but the optimal placement of the pedicles within the cricoarytenoideus dorsalis (CAD) muscle is unknown. HYPOTHESIS: The magnitude and direction of force placed on the muscular process of the left arytenoid cartilage affects the magnitude of laryngeal abduction. METHODS: Five larynges were harvested from cadavers. Using increments of 0.98 N, a dead-weight force generator applied a force of 0-14.7 N for 1 min each to the left muscular process at 0, 10, 20, 30, 40, 50, 60 and 70 degrees angles. The rima glottis was photographed digitally 1 min after each force had been applied. Distances between biomarkers (Lines 1-4) and right to left angle quotient (RLQ) were used to assess the degree of left arytenoid abduction. RESULTS: Increasing force from 0-14.7 N progressively and significantly increased the length of all lines and RLQ, indicating abduction. Furthermore, there was a significant interaction between force and angles. Applying forces of 7.84 N or greater (Lines 2-4 and RLQ) or 11.76 N or greater (Line 1) at angles 0, 10, 20 and 30 degrees resulted in significantly greater abduction than applying the same forces at 40, 50, 60 and 70 degrees. Angles of 0-30 degrees correspond with the direction of pull exerted by the lateral compartment of the CAD muscle. CONCLUSION: In RLN, nerve-muscle pedicle grafts should be placed preferentially in the lateral rather than in the medial compartment of the CAD muscle. POTENTIAL RELEVANCE: The information presented can be used to assist surgeons in the planning and application of the nerve-muscle pedicle graft procedure.

Spatial and temporal variability of sediment deposition on artificial-lawn traps in a floodplain of the River Elbe.

Artificial-lawn mats were used as sediment traps in floodplains to measure sediment input and composition during flood events. To estimate the natural variability, 10 traps were installed during two flood waves at three different morphological units in a meander loop of the River Elbe. The geochemical composition of deposited and suspended matter was compared. The sediment input showed weak correlations with concentration and composition of river water. It also correlated poorly with flood duration and level as well as distance of trap position from the main river. This is due to the high variability of the inundation, different morphological conditions and the variability of sources. The composition of the deposits and the suspended matter in the river water was comparable. Hence, for the investigated river reach, the expected pollution of the floodplain sediments can be derived from the pollution of the suspended matter in the river during the flood wave.

In situ soil water extraction: a review.

The knowledge of the composition and fluxes of vadose zone water is essential for a wide range of scientific and practical fields, including water-use management, pesticide registration, fate of xenobiotics, monitoring of disposal from mining and industries, nutrient management of agricultural and forest ecosystems, ecology, and environmental protection. Nowadays, water and solute flow can be monitored using either in situ methods or minimally invasive geophysical measurements. In situ information, however, is necessary to interpret most geophysical data sets and to determine the chemical composition of seepage water. Therefore, we present a comprehensive review of in situ soil water extraction methods to monitor solute concentration, solute transport, and to calculate mass balances in natural soils. We distinguished six different sampling devices: porous cups, porous plates, capillary wicks, pan lysimeters, resin boxes, and lysimeters. For each of the six sampling devices we discuss the basic principles, the advantages and disadvantages, and limits of data acquisition. We also give decision guidance for the selection of the appropriate sampling system. The choice of material is addressed in terms of potential contamination, filtering, and sorption of the target substances. The information provided in this review will support scientists and professionals in optimizing their experimental set-up for meeting their specific goals.

Histological evaluation of the equine larynx after unilateral laser-assisted ventriculocordectomy.

REASONS FOR PERFORMING STUDY: Trans-endoscopic laser surgery, such as unilateral laser-assisted ventriculocordectomy (LVC), has gained popularity in the treatment of RLN because a laryngotomy incision or general anaesthesia are not required. However, removal of the vocal fold and ventricle takes considerable laser energy and could cause collateral tissue damage, including injury to the adjacent laryngeal cartilages. OBJECTIVES: To document the histological effects of laser surgery on laryngeal tissues in horses that have undergone LVC for the treatment of laryngeal hemiplegia (LH). METHODS: Six horses were used: 4 with experimentally induced LH that had subsequently undergone LVC 6 months prior to euthanasia; and, 2 horses were used as controls. One of the control horses with naturally occurring LH was used to study the effect of neuropathy alone, whereas the other was subjected to euthanasia immediately following LVC to evaluate the acute effect of laser surgery. Using a band saw, each larynx was sectioned transversely at 5 mm intervals and evaluated histologically. RESULTS: Acutely, LVC caused thermal damage to adjacent soft tissues but did not affect the histology of the laryngeal cartilages. Six months after LVC, laryngeal cartilages were histologically normal and there was squamous metaplasia of the repaired laryngeal mucosa, resulting in restitution of the mucosal integrity. CONCLUSIONS: Using a diode laser in contact fashion at 20 W, LVC can be used to remove the laryngeal vocal fold and ventricle without causing laryngeal cartilage damage. POTENTIAL RELEVANCE: Laryngeal chondritis is an unlikely consequence of LVC.

Pulmonary response to airway instillation of autologous blood in horses.

REASONS FOR PERFORMING STUDY: Exercise-induced pulmonary haemorrhage (EIPH) occurs in the majority of horses performing strenuous exercise. Associated pulmonary lesions include alveolar and airway wall fibrosis, which may enhance the severity of EIPH. Further work is required to understand the pulmonary response to blood in the equine airways. OBJECTIVES: To confirm that a single instillation of autologous blood into horse airways is associated with alveolar wall fibrosis, and to determine if blood in the airways is also associated with peribronchiolar fibrosis. METHODS: Paired regions of each lung were inoculated with blood or saline at 14 and 7 days, and 48, 24 and 6 h before euthanasia. Resulting lesions were described histologically and alveolar and airway wall collagen was quantified. RESULTS: The main lesion observed on histology was hypertrophy and hyperplasia of type II pneumocytes at 7 days after blood instillation. This lesion was no longer present at 14 days. There were no significant effects of lung region, treatment (saline or autologous blood instillation), nor significant treatment-time interactions in the amount of collagen in the interstitium or in the peribronchial regions. CONCLUSION: A single instillation of autologous blood in lung regions is not associated with pulmonary fibrosis. POTENTIAL RELEVANCE: Pulmonary fibrosis and lung remodelling, characteristic of EIPH, are important because these lesions may enhance the severity of bleeding during exercise. A single instillation of autologous blood in the airspaces of the lung is not associated with pulmonary fibrosis. Therefore the pulmonary fibrosis described in EIPH must have other causes, such as repetitive bleeds, or the presence of blood in the pulmonary interstitium in addition to the airspaces. Prevention of pulmonary fibrosis through therapeutic intervention requires a better understanding of these mechanisms.

Effect of tracheal mucus and tracheal cytology on racing performance in Thoroughbred racehorses.

REASON FOR PERFORMING STUDY: Accumulations of mucus within the trachea are often found during endoscopic examinations of the airways of poorly performing racehorses, but the clinical importance of this finding is unknown. OBJECTIVES: To determine the effect of tracheal mucus, pharyngeal lymphoid hyperplasia (PLH) and cytological indices of tracheal aspirate on racing performance in Thoroughbred horses assessed by race place and whether the horse was raced. METHODS: Endoscopic examination of the nasopharynx, larynx and trachea was performed, and a tracheal aspirate obtained monthly at Thistledown racetrack from April to December, 2002 and 2003. Horses received a score of 0-4 for the degree of PLH and 0-4 for the amount of mucus visible in the trachea. The tracheal aspirate was assessed for turbidity, and total and differential cell counts. Generalised estimating equations models were used as repeated measures models for each risk factor and the level of association assessed through the risk factor's P value in the model. RESULTS: Moderate to severe tracheal mucus (2-4) was a risk factor for poor racing performance. There was no association between degree of PLH, cell counts or turbidity of tracheal wash fluid and racing performance. However, horses that raced had higher total neutrophil counts in tracheal wash aspirates than horses that did not race. CONCLUSIONS: Grades 2-4 tracheal mucus should be considered a potential cause of poor racing performance in Thoroughbred horses. CLINICAL RELEVANCE: Because moderate to severe tracheal mucus accumulation, and not increased tracheal neutrophils, was a risk factor for poor racing performance, functionally significant airway inflammation may best be confirmed by the presence of mucus rather than increased number of neutrophils in the trachea.

Copper(I) and copper(II) in complexes of biochemical significance studied by X-ray photoelectron spectroscopy.

X-ray photoelectron spectroscopic measurements of copper complexes of biochemical significance were carried out to permit the conclusion whether or not copper is present in the Cu(I) or Cu(II) state. Only one single homogeneous signal in the X-ray photoelectron spectra of the Cu(I) 2p1/2 and 2p3/2 levels was seen regardless whatever Cu(I) complex was used. By contrast one more or less split satellite in addition to the main 2p copper signal appeared when Cu(II) complexes were studied. The extent of satellite splitting was dependent on the nature of the ligands coordinated with Cu(II). Thus, a strong splitting was observed in the spectra of Cu-(trifluoroacetylacetonate)2 and Cu-(biuret)2Cl2 were Cu(II) is exclusively bound to oxygen having a formal double bond. No such splitting was seen in Cu(II) chelates where the metal was bound to single bonded oxygen and/or nitrogen. It excited great interest to see that in the antiferromagnetically coupled Cu(II) complexes Cu2-(succinate)2-4H2O, Cu-(HCCO)2, CuO and in the completely diamagnetic Cu2-(u,3-diphenyltriazene)4 complex Cu(II) could be detected. The reaction of Cu(I) and Cu(II) with the thiol sulphur of either cysteine, penicillamine or alpha-mercaptopropionylglycine yielded Cu(I) complexes. During the X-ray exposure of the different samples photoreduction of Cu(II) was not observed. For example, the satellite structure of the copper 2p levels using Cu(II)-cystine remained unchanged during the measurement.

Circular dichroism of metallothioneins. A structural approach.

A comprehensive study on circular dichroism of metallothioneins containing Zn, Cd and Cu was carried out. The contributions of the metals, the sulphur and the polypeptide chain to the observed Cotton effects was shown. From the pH dependency of the extrinsic Cotton effects which are due to the metal-thiolate chromophore the stability of the metal clusters was found to decrease in the order Cu greater than Cd greater than Zn. The pH values corresponding to the dissociation of half of the bound metal ions are 0.44 for Cu-thionein, 3.05 for Cd-thionein and 4.6 for Zn-thionein. The extrinsic Cotton effects of Cd, Zn-thioneins of varying Cd to Zn ratio could be simulated using the difference circular dichroic spectra of Cd-thionein (bands at 227, 242.5 and 262 nm), Zn-thionein (bands at 225 and 244 nm) and the circular dichroic spectrum of cysteine-thionein (band at 200 nm, shoulder at 225 nm). Since during the dissociation of the metals the circular dichroic spectra exhibited changes only in amplitude and not in shape we can conclude that the dissociation of the metal ions involves the complete sequential degradation of metal clusters. In the near-ultraviolet region the metal-free proteins show only Cotton effects attributable to a disulphide chromophore. Thus Cotton bands are observed for cystine-thionein at 282.5 and 260 nm. From the intrinsic circular dichroism of Cd- and Zn-thionein (negative Cotton effect at 200 nm, shoulder at 225 nm) it follows that the protein conformation consists of less than 5% helical or pleated sheet structure and therefore has to be classified as unordered structure or "fixed" random coil

Characterization of iron-sulphur centres of plant mitochondria by microwave power saturation.

The electron spin relaxation of iron-sulphur centres and ubisemiquinones of plant mitochondria was studied by microwave power saturation of the respective EPR signals. In the microwave power saturation technique, the experimental saturation data were fitted by a least-squares procedure to a saturation function which is characterized by the power for half-saturation (P1/2) and the inhomogeneity parameter (b). Since the theoretical saturation curves were based on a one-electron spin system, it became possible to differentiate between EPR signals of iron-sulphur centres which have similar g values but different P1/2 values. If the difference in the P1/2 values of the overlapped components was small, no significant deviation from these theoretical saturation curves was observed, as shown for the overlapped signals of centre S-3 and the Ruzicka centre of mung bean mitochondria. By contrast, the microwave power saturation data for the g = 1.93 signal (17--26 K) of Arum maculatum submitochondrial particles reduced by succinate could not be fitted using one-electron saturation curves. Reduction by NADH resulted in a stronger deviation. Since the iron-sulphur centres of Complex I were present only in an unusually low concentration in A. maculatum mitochondria, it was proposed that an iron-sulphur centre of the external NADH dehydrogenase contributes to the spectrum of centre S-1. For mung bean mitochondria, the g = 1.93 signal below 20 K could be attributed mainly to centre N-2. The microwave power saturation technique was also suitable for detecting magnetic interactions between paramagnetic centres. From the saturation data of the complex spectrum attributable to centre S-3 and an interacting ubisemiquinone pair in mung bean mitochondria (oxidized state) followed that centre S-3 has a faster electron spin relaxation than the ubisemiquinone molecules. It is noteworthy that the differences in the relaxation rates were maintained despite the interaction between centre S-3 and the ubisemiquinones. Furthermore, a relaxation enhancement was observed for centre S-1 of A. maculatum submitochondrial particles upon reduction of centre S-2 by dithionite. This indicated a magnetic interaction between centres S-1 and S-2.

Electron spin relaxation of iron-sulphur proteins studied by microwave power saturation.

The electron-spin relaxation of iron-sulphur centres in a range of simple proteins (ferredoxin, high-potential iron-sulphur protein and rubredoxin) was investigated by means of the temperature dependence and microwave power saturation of the EPR signal. The proteins containing [2Fe-2S] centres all showed temperature optima higher than those for [4Fe-4S] centres, but the difference between the slowest-relaxing [4Fe-4S] protein (Chromatium high-potential iron-sulphur protein) and the fastest-relaxing [2Fe-2S] protein (Halobacterium halobium ferredoxin) was small. A greater distinction was seen in the power saturation behaviour at low temperature (10--20 K). The behaviour of the signal intensity as a function of microwave power was analyzed in terms of the power for half saturation P 1/2 and the degree of homogeneous/inhomogeneous broadening. The effect of distorting the protein structure by salts, organic solvents and urea was to decrease the electron-spin relaxation rate as shown by a decreased value of P 1/2. The addition of Ni2+ as a paramagnetic perturbing agent caused an increase in the electron-spin relaxation rate of all the proteins, with the exception of adrenal ferredoxin, as shown by an increased P 1/2 and, in a few cases, broadening of the linewidth. Ferricyanide, a commonly used oxidizing agent, has similar effects. These results are discussed in relation to the use of paramagnetic probes to determine whether iron-sulphur centres are near to a membrane surface. Spin-spin interactions between two paramagnetic centres in a protein molecule such as a 2[4Fe-4S] ferredoxin, lead to more rapid electron-spin relaxation. This method was used to detect a spin-spin interaction between molybdenum V and centre Fe-SI in xanthine oxidase.

The electron spin relaxation of the electron acceptors of photosystem I reaction centre studied by microwave power saturation.

Photosystem I particles from spinach were reduced by illumination at 77 K. Under these conditions the one-electrom transfer from P-700 resulted in a reduction of only one acceptor molecule of the reaction centre. The EPR signals at g=2.05, 1.94 and 1.86 were attributed to reduced centre A and the smaller signals at g=2.07, 1.92 and 1.89 to reduced centre B. Reduction of both centres by dithionite in the dark lead to signals at g=2.05, 1.99, 1.96, 1.94, 1.92 and 1.89. Thus, the features at g=2.07 and 1.86 disappeared and new signals at g=1.99 and 1.96 were observed. From the spectral changes it followed that the iron-sulphur centres A and B interact magnetically. Temperature dependent EPR spectra demonstrated a faster electron spin relaxation of centre A than of centre B. These conclusions were corroborated using microwave power saturation of the respective EPR signals. The saturation data of the fully reduced centres A and B could not be fitted using the saturation equation for a one-electron spin system. The magnetic interaction between the (4Fe-4S) CENTRes of the electron acceptors A and B resulted in saturation properties which are simular to those of the 2(4Fe-4S) ferredoxin from Clostridium pasteurianum. For centre X a high proportion of homogeneous broadening of the EPR lines was inferred from the inhomogeneity parameter (b=1.83). It was, therefore, concluded that centre X is most probably an anion radical of chlorophyll. From the low temperature necessary for observing the EPR signal of centre X followed that the drastic relaxation enhancement has to be attributed to a magnetic interaction of the anion radical with iron.

Oxidation-reduction reactions of copper-thiolate centres in Cu-thionein.

Cu-thionein from yeast was investigated by EPR spectroscopy to probe the oxidation state of copper, and the effects on it of oxidizing and reducing agents. At pH 0.2 the copper was released, but no EPR signal from Cu(II) was observed, unless air was present. Optical experiments did not detect any disulphide groups which might have been formed during anaerobic release of copper. The mercurial, p-hydroxymercuribenzoate caused the release of EPR-detectable copper only under aerobic conditions, and EDTA caused release of Cu(II) on heating. No reduction of the copper-thiolate units in Cu-thionein by ascorbate was detected. Potentiometric titrations with hexachloroiridate(IV) or hexacyanoferrate(III) produced several different Cu(II) EPR signals at various stages of oxidation. The former oxidizing agent required a lower oxidation-reduction potential (+350 mV) to oxidize the copper, than the latter (+410 mV) and neither titration was fully reversible. The EPR signal from Cu(II) oxidized by hexachloroiridate(IV) resembled that produced by p-hydroxy-mercuribenzoate in air, suggesting that the copper was released from its thiolate ligands. It is concluded that the EPR non-detectable copper in the native protein is Cu(I). Oxidation-reduction of the copper-thiolate clusters of Cu-thionein is proposed to be decisive for controlling storage and transport of cellular copper.

Lisinopril-mediated regression of myocardial fibrosis in patients with hypertensive heart disease.

BACKGROUND: In arterial hypertension, left ventricular hypertrophy (LVH) includes myocyte hypertrophy and fibrosis, which leads to LV diastolic dysfunction and, finally, heart failure. In spontaneously hypertensive rats, myocardial fibrosis was regressed and LV diastolic function was improved by treatment with the angiotensin-converting enzyme inhibitor lisinopril. Whether this holds true for patients with hypertensive heart disease was addressed in this prospective, randomized, double-blind trial. METHODS AND RESULTS: A total of 35 patients with primary hypertension, LVH, and LV diastolic dysfunction were treated with either lisinopril (n=18) or hydrochlorothiazide (HCTZ; n=17). At baseline and after 6 months, LV catheterization with endomyocardial biopsy, Doppler echocardiography with measurements of LV peak flow velocities during early filling and atrial contraction and isovolumic relaxation time, and 24-hour blood pressure monitoring were performed. Myocardial fibrosis was measured by LV collagen volume fraction and myocardial hydroxyproline concentration. With lisinopril, collagen volume fraction decreased from 6.9+/-0.6% to 6. 3+/-0.6% (P:<0.05 versus HCTZ) and myocardial hydroxyproline concentration from 9.9+/-0.3 to 8.3+/-0.4 microg/mg of LV dry weight (P:<0.00001 versus HCTZ); this was associated with an increase in the early filling and atrial contraction LV peak flow velocity ratio from 0.72+/-0.04 to 0.91+/-0.06 (P:<0.05 versus HCTZ) and a decrease in isovolumic relaxation time from 123+/-9 to 81+/-5 ms (P:<0.00002 versus HCTZ). Normalized blood pressure did not significantly change in either group. No LVH regression occurred in lisinopril-treated patients, whereas with HCTZ, myocyte diameter was reduced from 22. 1+/-0.6 to 20.7+/-0.7 microm (P:<0.01 versus lisinopril). CONCLUSIONS: In patients with hypertensive heart disease, angiotensin-converting enzyme inhibition with lisinopril can regress myocardial fibrosis, irrespective of LVH regression, and it is accompanied by improved LV diastolic function.

Subacute and long term effect of swimming training on blood pressure in young and old spontaneously hypertensive rats.

The effects of swimming training (three weeks' training with the duration increasing up to a maximum of 180 min per day, at a water temperature of 36 degrees C) on arterial blood pressure were studied in 4, 11, and 18 month old spontaneously hypertensive rats. In addition, in the 11 month old rats the change in blood pressure after individual exercise was determined. The significance of a training induced loss of body weight in lowering blood pressure was assessed by pair feeding of sedentary age matched spontaneously hypertensive rats. Blood pressure was reduced by approximately 50 mmHg within 8-10 days, except in the oldest rats, which tolerated the physical activity poorly and had, if any, only a moderate fall in blood pressure. It was possible to distinguish between subacute transient effects lasting for not more than one day and long term effects. Blood pressures were 20-25 mmHg lower after individual swimming routines than those before exercise when measured on the ninth day of the training programme. On cessation of training, blood pressures approached those of sedentary rats within two weeks. It seems that the loss of body weight was of minor importance in lowering blood pressure under these experimental conditions.

Transcriptional modulators targeted at fuel metabolism of hypertrophied heart.

The transition of nonfailing to failing cardiac hypertrophy cannot be prevented by current drug regimens. This investigation examined whether possible drug targets have remained unexplored because they do not result in acute improvement of heart function. Of major importance, in this respect, is an inadequate performance of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2). In the present approach, binding sequences within the proximal promoter of SERCA2 are described which may be useful in the development of drugs (i.e., transcriptional modulators) that interfere selectively with the transcription of genes of the cardiomyocyte. The proximal promoter region of the SERCA2 genes has a thyroid response element, 9 potential Sp1-binding sites (5'-GGGCGG-3', 5'-CCGCCC-3' and 5'-GGGAGG-3'), and an E-box motif (5'-CACATG-3'), which may function as glucose response elements. This region also has 2 putative fatty-acid response elements (5'-GGGGGA-3'). It is proposed that the beneficial effects of the camitine palmitoyltransferase-1 inhibitor etomoxir arise from a shift in fuel metabolism involving glucose response elements and/or peroxisomal proliferator-activated receptors. Although the relative contribution of these DNA regulatory elements remains to be defined, it appears that they provide the driving force that prevents the decrease in transcriptional activity of the SERCA2 gene in the hypertrophic heart. It is further concluded that etomoxir represents a member of a novel class of transcriptional modulators that improve function of hypertrophied hearts with unimpeded blood flow by modulating gene expression of the cardiomyocyte.