Cardioselective sulfonylthiourea HMR 1098 blocks mitochondrial uncoupling induced by a KATP channel opener,P-1075, in beating rat hearts

We investigated effects of blockade of cardiac ATP-sensitive potassium channels (KATP) with a novel cardioselective sulfonylthiourea, HMR 1098, on metabolic uncoupling caused by a potent KATP opener, P-1075, in Langendorff-perfused rat hearts. We used (1) 87Rb-NMR to detect activation-deactivation of sarcolemmal KATP, (2) 31P-NMR to monitor high-energy phosphates, (3) oxygen uptake measurements to monitor cellular respiration, and (4) myocardial optical absorbance measurements at 603 nm to follow changes in cytochrome c oxidase redox state. Activation of sarcolemmal KATP by P-1075 (5 microM) and a mitochondrial uncoupler 2,4-dinitrophenol (DNP) (50 microM) stimulated Rb+ efflux from the hearts by 130% and 60%, respectively. HMR 1098 (5 and 30 microM) blocked activation of sarcolemmal KATP in situ. HMR 1098 also prevented cardiac arrest and mitochondrial uncoupling induced by P-1075, such as (a) depletion of phosphocreatine and ATP by 40%, (b) two-fold decrease in venous oxygen, and (c) reduction of cytochrome c oxidase (demonstrated by an increase in 603 nm optical absorbance). The metabolic effects of P-1075 can be readily explained by activation of putative mitochondrial KATP. We concluded that blockade of mitochondrial uncoupling by HMR 1098 included an inhibiting effect of HMR 1098 on sarcolemmal and mitochondrial KATP in beating rat hearts.     

Hyposmotic shock: effects on rubidium/potassium efflux in normal and ischemic rat hearts,assessed by 87Rb and 31P NMR

The study evaluated effects of hyposmotic shock on the rate of Rb(+)/K(+) efflux, intracellular pH and energetics in Langendorff-perfused rat hearts with the help of 87Rb- and 31P-NMR. Two models of hyposmotic shock were compared: (1) normosmotic hearts perfused with low [NaCl] (70 mM) buffer, (2) hyperosmotic hearts equilibrated with additional methyl alpha-D-glucopyranoside (Me-GPD, 90 or 33 mM) or urea (90 mM) perfused with normosmotic buffer. Four minutes after hyposmotic shock, Rb(+) efflux rate constant transiently increased approximately two-fold, while pH transiently decreased by 0.08 and 0.06 units, in the first and the second models, respectively, without significant changes in phosphocreatine and ATP. Hyposmotic shock (second model) did not change the rate of Rb(+)/K(+) uptake, indicating that the activity of Na(+)/K(+) ATPase was not affected. Dimethylamiloride (DMA) (10 microM) abolished activation of the Rb(+)/K(+) efflux in the second model; however, Na(+)/H(+) exchanger was not involved, because intracellular acidosis induced by the hyposmotic shock was not enhanced by DMA treatment. After 12 or 20 min of global ischemia, the rate of Rb(+)/K(+) efflux increased by 120%. Inhibitor of the ATP-sensitive potassium channels, glibenclamide (5 microM), partially (40%) decreased the rate constant; however, reperfusion with hyperosmolar buffer (90 mM Me-GPD) did not. We concluded that the shock-induced stimulation of Rb(+)/K(+) efflux occurred, at least partially, through the DMA-sensitive cation/H(+) exchanger and swelling-induced mechanisms did not considerably contribute to the ischemia-reperfusion-induced activation of Rb(+)/K(+) efflux.     

Evaluation of caspase 1 and sFas serum levels in patients with systemic sclerosis: correlation with lung dysfunction, joint and bone involvement

Recent studies point out at the role of apoptosis disturbances in the development of systemic sclerosis(SSc). The aim of our study was to examine caspase 1 and sFas serum levels in scleroderma patients and correlate the obtained results with skin involvement and internal organ changes. We studied 29 patients (14 with limited and 15 with diffuse SSc). The extension of skin involvement was measured using Total Skin Score (TSS). Internal organ involvement was assessed by specialist procedures. Serum caspase 1 and sFas levels were measured by enzyme-linked immunosorbent assay. We found correlation between sFas serum level and duration of Raynaud's phenomenon and TSS; caspase 1 serum level correlated only with TSS. Correlations between caspase 1 and lung dysfunction and sFas levels with joint and bone involvement in SSc patients were also observed. The obtained results revealed that disturbances of apoptosis might play a role in SSc pathogenesis. Caspase 1 and sFas serum levels correlate with the skin involvement severity, lung dysfunction, joint and bone changes.     

Biotransformation of waste lignin products by the soil-inhabiting yeast Trichosporon pullulans

In this study the biotransformation of lignin by-products of beechwood pulping with a soil-inhabiting yeast strain of Trichosporon pullulans was examined. The structural and molecular changes in the lignin during a cultivation process were determined by 13C NMR spectroscopy and gel permeation chromatography analysis, which confirmed the ability of the yeast strain tested to biodegrade lignin. Enzymatic analysis showed the presence of lignin peroxidase and Mn(II) peroxidase in the culture supernatant. The ligninolytic activity of both enzymes increased under carbon-depleted conditions. This observation is particularly important in the biodegradation of recalcitrant lignins in soil.     

Proteolytic mRNA expression in response to acute resistance exercise in human single skeletal muscle fibers.

The purpose of this study was to characterize changes in mRNA expression of select proteolytic markers in human slow-twitch [myosin heavy chain (MHC) I] and fast-twitch (MHC IIa) single skeletal muscle fibers following a bout of resistance exercise (RE). Muscle biopsies were obtained from the vastus lateralis of eight young healthy sedentary men [23 +/- 2 yr (mean +/- SD), 93 +/- 17 kg, 183 +/- 6 cm] before and 4 and 24 h after 3 x 10 repetitions of bilateral knee extensions at 65% of one repetition maximum. The mRNA levels of TNF-alpha, calpains 1 and 2, muscle RING (really interesting novel gene) finger-1 (MuRF-1), atrogin-1, caspase-3, B-cell leukemia/lymphoma (Bcl)-2, and Bcl-2-associated X protein (Bax) were quantified using real-time RT-PCR. Generally, MHC I fibers had higher (1.6- to 5.0-fold, P < 0.05) mRNA expression pre- and post-RE. One exception was a higher (1.6- to 3.9-fold, P < 0.05) Bax-to-Bcl-2 mRNA ratio in MHC IIa fibers pre- and post-RE. RE increased (1.4- to 4.8-fold, P < 0.05) MuRF-1 and caspase-3 mRNA levels 4-24 h post-RE in both fiber types, whereas Bax-to-Bcl-2 mRNA ratio increased 2.2-fold (P < 0.05) at 4 h post-RE only in MHC I fibers. These results suggest that MHC I fibers have a greater proteolytic mRNA expression pre- and post-RE compared with MHC IIa fibers. The greatest mRNA induction following RE was in MuRF-1 and caspase-3 in both fiber types. This altered and specific proteolytic mRNA expression among slow- and fast-twitch muscle fibers indicates that the ubiquitin/proteasomal and caspase pathways may play an important role in muscle remodeling with RE.     

Observation of Two Inorganic Phosphate NMR Resonances in the Perfused Hypothermic Rat Heart

The effect of hypothermia on isolated perfused rat hearts was studied with³¹P NMR. Hearts were continuously perfused with phosphate-free Krebs–Henseleit buffer while the perfusate temperature was adjusted. Perfusate pH was kept at 7.40 ± 0.02 throughout the experiments. Using the chemical shift difference between PCr and P_ithe intracellular pH was estimated. At 36, 20, and 10°C a cytosolic alkalinization at a pH of 7.05 ± 0.04, 7.21 ± 0.05, and 7.40 ± 0.03 was observed, respectively. At 10°C two P_iresonances were observed with a separation of 0.25 ppm. This resonance corresponded to a P_iresonance of a cellular compartment with a local pH of 7.78 ± 0.06, likely mitochondrial. This additional resonance disappeared upon warming of the hearts back to 36°C.     

Allergenic pollen and pollinosis in Warsaw

Summary Our study of fifty two hay fever patients included twenty six solely allergic to grass pollen and twenty six exhibiting allergy to various pollen species, such as hazel, birch, oak, poplar, andArtemisia. Their total and specific IgE response was evalutated by the immunoenzymatic method, while clinical reactivity was assessed by recording nasal and bronchial symptom scores between mid-March and mid-July. Simultaneously pollen counts were made. Polysensitized patients showed significantly higher levels of both total and specific IgE, which testifies to the enhanced quantitative and qualitative IgE. Multisensitized patients reacted earlier than patients sensitized to grass pollen only, which confirms that non-grass plants flowering only in the spring cause the priming effect on the nasal and bronchial mucosa. The early symptoms may be attributable to tree pollen sensitivity or may refletct higher grass pollen IgE levels in the polysensitized group. Characteristically, nasal symptoms preceded bronchial symptoms of several weeks.On comparing nasal washing from the polysensitized patients to washing from patients with grass pollen, we found much cytological material with the predominance of eosinophils.     

Protein a of quinolinate synthetase is the site of oxygen poisoning of pyridine nucleotide coenzyme synthesis in Escherichia coli

De novo biosynthesis of pyridine nucleotide coenzymes in Escherichia coli is initiated by an enzyme complex (quinolinate synthetase) containing protein B which converts l-aspartate into iminoaspartate protein A, which then generates quinolinate on the pathway to the coenzymes. This complex has been shown to be poisoned by hyperbaric oxygen. ^7,8 We performed assays made dependent on both proteins B and A versus only protein A, using cell-free extracts of hyperbaric-oxygen poisoned and aerobically grown cells. The specific activities were produced by a similar amounts of 68% and 60%, respectively, when measured in assays made dependent on enzymes B and A versus only protein A that was derived from oxygen-poisoned extract. Thus, protein A is the oxygen-sensitive component.     

Multiple forms of Pregnancy-Associated Glycoproteins released in vitro by porcine chorion or placentomal and interplacentomal explants of wild and domestic ruminants

Characterization of the Pregnancy-Associated Glycoproteins (PAG) is important for studies of reproduction of various eutherian domestic, wild and endangered mammals. Distinct chorionic PAG genes are expressed in embryo-origin cells: pre-placental trophoblast (TR) and in placental trophectoderm (TRD) of various entherians. This study demonstrates in vitro production of the PAG proteins during long-term cultures of various chorionic explants: porcine TR or TRD, cotyledonary (CT) of European bison (Eb), and CT or intercotyledonary (intCT)-TRD of the cattle. Chorionic proteins isolated from media were analyzed by homologous or heterologous Western immunoblotting with anti-PAG sera, raised against cellular bovine or secretory porcine antigens. Used anti-PAG sera identified diverse molecular forms of released PAG proteins: 43-69 kDa for EbPAG proteins, 40-85 kDa for bovine PAG (bPAG), and 43-73 kDa for porcine PAG (pPAG). Immunoblotting revealed also that both CT and intCT-TRD explants secreted equivalent amounts of bPAG proteins. This useful system of in vitro protein production can provide native chorionic PAG proteins with placental unique carbohydrate chains. The PAG proteins are required as standard markers for diagnostic tests of pregnancy in domestic and wild mammals, in which seasonal reproductive processes are relatively difficult to control.