Porcine intestinal yeast species, Kazachstania slooffiae, a new potential protein source with favourable amino acid composition for animals.

There is little information about Kazachstania slooffiae which dominates among other yeasts in the pigs' intestine. Therefore, the aims of this study were to characterise the yeast cell contents and to investigate which nitrogen sources, organic acids and alcohols may be utilised or produced by this species. The results showed that, K. slooffiae could use urea, ammonium sulphate, peptides and single amino acids and produce thereby ethanol and formic acid. However, this yeast did not metabolise amino acids, lactic, butyric, propionic and acetic acids as sole carbon source. Using a global metabolite profiling approach employing gas chromatography and high-resolution liquid chromatography mass spectrometry, was found that the amount of peptides and dehydroascorbic acid considerably increased in the fermentation residues after yeast cultivation. It is noteworthy that the cells of K. slooffiae had higher contents of nitrogen and total amino acids (especially lysine) than the cells of nutritional yeast (Saccharomyces cerevisiae). This study indicates that due to potential production of peptides and formic acid in the intestinal tract, K. slooffiae might have an impact on the gut health. Moreover, from a nutritional standpoint, the cells of this yeast can be a good source of protein with useful amino acid composition for animal.

The outer membrane Omp85-like protein P39 influences metabolic homeostasis in mature Arabidopsis thaliana.

The Omp85 proteins form a large membrane protein family in bacteria and eukaryotes. Omp85 proteins are composed of a C-terminal ß-barrel-shaped membrane domain and one or more N-terminal polypeptide transport-associated (POTRA) domains. However, Arabidopsis thaliana contains two genes coding for Omp85 proteins without a POTRA domain. One gene is designated P39, according to the molecular weight of the encoded protein. The protein is targeted to plastids and it was established that p39 has electrophysiological properties similar to other Omp85 family members, particularly to that designated as Toc75V/Oep80. We analysed expression of the gene and characterised two T-DNA insertion mutants, focusing on alterations in photosynthetic activity, plastid ultrastructure, global expression profile and metabolome. We observed pronounced expression of P39, especially in veins. Mutants of P39 show growth aberrations, reduced photosynthetic activity and changes in plastid ultrastructure, particularly in the leaf tip. Further, they display global alteration of gene expression and metabolite content in leaves of mature plants. We conclude that the function of the plastid-localised and vein-specific Omp85 family protein p39 is important, but not essential, for maintenance of metabolic homeostasis of full-grown A. thaliana plants. Further, the function of p39 in veins influences the functionality of other plant tissues. The link connecting p39 function with metabolic regulation in mature A. thaliana is discussed.

VATS (video-assisted thoracic surgery) of undefined pulmonary nodules. Preoperative evaluation of videoendoscopic resectability.

Peripheral undefined pulmonary nodules have become a favorable indication for the videoendoscopic approach in thoracic surgery. In our latest experience, we also successfully applied this technique in centrally located lesions of the lung. In reviewing our first 29 cases, we looked for preoperative features of videoendoscopic resectability. From March 1992 to September 1993, 29 patients underwent videothoracoscopy for undefined pulmonary nodules at our hospital. This group consisted of 17 men and 12 women (aged 25 to 77 years). Pulmonary nodules of this group of patients were defined as centrally located when close attachment to the segmental or subsegmental bronchopulmonary unit was observed and/or the distance to the visceral pleura exceeded 10 mm. Nodules that did not meet any of these criteria were hence interpreted as peripheral lesions. In the course of 21 excisions of peripheral lesions, we had to convert to open thoracotomy only once for anatomic reasons. When using the video-assisted thoracic surgery (VATS) approach for centrally located lesions, we succeeded in removing four of six. We failed only if the lesions were located in the upper lobe but could easily apply the technique for centrally located lesions in the lower lobes. In conclusion, undefined peripheral pulmonary nodules are a favorite indication for VATS. Centrally located pulmonary nodules of the lower lobes can often be managed easily by VATS, especially if the interlobar fissure extends to the stem of the pulmonary artery. Centrally located pulmonary nodules in the upper lobes may not be suitable for the VATS approach due to the special anatomic arrangement of the upper lobe segmental arteries and bronchioles.

Identification of molybdopterin guanine dinucleotide in formate dehydrogenase from Methanobacterium formicicum.

The pterin cofactor in formate dehydrogenase isolated from Methanobacterium formicium is identified as molybdopterin guanine dinucleotide. The pterin, stabilized as the alkylated, dicarboxamidomethyl derivative, is shown to have absorption and chromatographic properties identical to those of the previously characterized authentic compound. Treatment with nucleotide pyrophosphatase produced the expected degradation products GMP and carboxyamidomethyl molybdopterin. The molybdopterin guanine dinucleotide released from the enzyme by treatment with 95% dimethyl sulfoxide is shown to be functional in the in vitro reconstitution of the cofactor-deficient nitrate reductase in extracts of the Neurospora crassa nit-1 mutant.

Detection of myocardial involvement in systemic lupus erythematosus: mismatch between normal perfusion scans with 201Thallium and pathological 18FDG uptake.

BACKGROUND: Systemic lupus erythematosus (SLE) patients can frequently present cardiac symptoms, however its etiology is not well known. EXPERIMENTAL DESIGN: prospective study. SETTINGS: specialized out-patient unit for SLE patients at an university hospital. PATIENTS: 15 SLE patients (13 females, 2 males; age range 18-64 years). INTERVENTIONS: metabolic studies of the heart were done using 18F-deoxy-glucose (18FDG, 296-333 MBq on a 2-head hybrid system) as well as heart perfusion studies (111MBq 201Tl). Additional studies: resting ECG, echocardiography, stress ECG, immunological activity parameters, antibody analyses (ANA, ENA, anti-cardiolipin antibodies), CPK, troponin-T, and lipid profiles. MEASURES: degree of correlation between conventional diagnostics and the imaging techniques. RESULTS: Abnormal ECG in 10 cases, pericardial involvement in 11 cases, elevated CPK in 1 case. ANTIBODY PROFILES: anti-cardiolipin in 10/15, ENA in 9/15, ANA in 14/15. None of these changes were associated with parameters of immune activation. In the majority of cases (10/15) the 18FDG scan showed a speckled, inhomogeneous pattern of distribution, which contrasted sharply with a normal 201Tl scan. A similar pattern was observed in the patients with ocular mitochondrial myopathy, the anti-phospholipid syndrome as well as in dermatomyositis. CONCLUSIONS: Our preliminary results suggest that SLE patients with cardiac symptoms may have an abnormal glucose metabolism of the myocardium as shown by a pathological 18FDG scan, whereas perfusion appears to be normal (reversed mismatch). The lack of correlation with acute elevation of cardiac enzymes or with ECG changes suggest a chronic process.

A modified blow-gun syringe for remote injection of captive wildlife.

A modified syringe capable of automatic injection and suitable for use with a blow-gun is described. The syringe has been used successfully with white-tailed deer (Odocoileus virginianus) under confined conditions. Desirable characteristics for blow-gun syringes are discussed.

Expression of EAAT1 reflects a possible neuroprotective function of reactive astrocytes and activated microglia following human traumatic brain injury.

UNLABELLED: Glutamate-mediated excitotoxicity is known to cause secondary brain damage following stroke and traumatic brain injury (TBI). However, clinical trials using NMDA antagonists failed. Thus, glial excitatory amino acid transporters (EAATs) might be a promising target for therapeutic intervention. METHODS AND RESULTS: We examined expression of EAAT1 (GLAST) and EAAT2 (Glt-1) in 36 TBI cases by immunohistochemistry. Cortical expression of both EAATs decreased rapidly and widespread throughout the brain (in lesional, adjacent and remote areas) following TBI. In the white matter numbers of EAAT1+ parenchymal cells increased 39-fold within 24h (p<0.001) and remained markedly elevated till later stages in the lesion (90-fold, p<0.01) and in peri-lesional regions (86-fold, p<0.01). In contrast, EAAT2+ parenchymal cells and EAAT1+ or EAAT2+ perivascular cells did not increase significantly. Within the first days following TBI mainly activated microglia and thereafter mainly reactive astrocytes expressed EAAT1. Perivascular monocytes and foamy macrophages lacked EAAT1 immunoreactivity. We conclude that following TBI i) loss of cortical EAATs contributes to secondary brain damage, ii) glial EAAT1 expression reflects a potential neuroprotective function of microglia and astrocytes, iii) microglial EAAT1 expression is restricted to an early stage of activation, iv) blood-derived monocytes do not express EAAT1 and v) pharmacological modification of glial EAAT expression might further limit neuronal damage.

Reactive astrocytes and activated microglial cells express EAAT1, but not EAAT2, reflecting a neuroprotective potential following ischaemia.

AIMS: Glutamate receptor antagonists have failed clinical stroke trials and it has been proposed that the action of N-methyl D-aspartate receptors is necessary for neuronal survival. Thus, excitatory amino acid transporters (EAATs) might be a promising therapeutic target. The aim of this study was to investigate glial expression of EAATs following ischaemia. METHODS AND RESULTS: Expression of EAAT1 (GLAST) and EAAT2 (Glt-1) in 24 cases of ischaemia was examined by immunohistochemistry. Cortical expression of both EAATs in the lesion decreased within 24 h (P < 0.01, each). Whereas EAAT1+ white matter cells increased 18-fold (P < 0.05) within 24 h in the lesion and remained elevated for months in adjacent (469-fold, P < 0.01) and remote areas (20-fold, P < 0.05), EAAT2+ white matter cells were equivalent in ischaemia and controls. In the first week after stroke mainly activated (ramified and amoeboid) microglia expressed EAAT1, whereas monocytic cells in perivascular spaces and foamy macrophages lacked EAAT1. After more than 1 week, predominantly reactive astrocytes expressed EAAT1. CONCLUSIONS: Microglial EAAT1 expression is restricted to the early/intermediate stage of activation and blood-derived (perivascular) monocytes do not contribute to EAAT1+ cells following ischaemia. Whether a pharmacological increase in glial EAAT expression may compensate for loss of cortical EAAT expression and reduce neuronal damage following stroke requires investigation by further functional studies.

Metabolism of formate in Methanobacterium formicicum.

Methanobacterium formicicum strain JF-1 was cultured with formate as the sole energy source in a pH-stat fermentor. Growth was exponential, and both methane production and formate consumption were linear functions of the growth rate. Hydrogen was produced in only trace amounts, and the dissolved H(2) concentration of the culture medium was below 1 muM. The effect of temperature or pH on the rate of methane formation was studied with a single fermentor culture in mid-log phase that was grown with formate under standard conditions at 37 degrees C and pH 7.6. Methane formation from formate occurred over the pH range from 6.5 to 8.6, with a maximum at pH 8.0. The maximum temperature of methanogenesis was 56 degrees C. H(2) production increased at higher temperatures. Hydrogen and formate were consumed throughout growth when both were present in saturating concentrations. The molar growth yields were 1.2 +/- 0.06 g (dry weight) per mol of formate and 4.8 +/- 0.24 g (dry weight) per mol of methane. Characteristics were compared for cultures grown with either formate or H(2)-CO(2) as the sole energy source at 37 degrees C and pH 7.6; the molar growth yield for methane of formate cultures was 4.8 g (dry weight) per mol, and that of H(2)-CO(2) cultures was 3.5 g (dry weight) per mol. Both formate and H(2)-CO(2) cultures had low efficiencies of electron transport phosphorylation; formate-cultured cells had greater specific activities of coenzyme F(420) than did H(2)-CO(2)-grown cultures. Hydrogenase, formate dehydrogenase, chromophoric factor F(342), and low levels of formyltetrahydrofolate synthetase were present in cells cultured with either substrate. Methyl viologen-dependent formate dehydrogenase was found in the soluble fraction from broken cells.

Composition of the coenzyme F420-dependent formate dehydrogenase from Methanobacterium formicicum.

The coenzyme F420-dependent formate dehydrogenase from Methanobacterium formicicum was purified to electrophoretic homogeneity by anoxic procedures which included the addition of azide, flavin adenine dinucleotide (FAD), glycerol, and 2-mercaptoethanol to all buffer solutions to stabilize activity. The enzyme contains, in approximate molar ratios, 1 FAD molecule and 1 molybdenum, 2 zinc, 21 to 24 iron, and 25 to 29 inorganic sulfur atoms. Denaturation of the enzyme released a molybdopterin cofactor. The enzyme has a molecular weight of 177,000 and consists of one each of two different subunits, giving the composition alpha 1 beta 1. The molecular weight of the alpha-subunit is 85,000, and that of the beta-subunit is 53,000. The UV-visible spectrum is typical of nonheme iron-sulfur flavoprotein. Reduction of the enzyme facilitated dissociation of FAD, and the FAD-depleted enzyme was unable to reduce coenzyme F420. Preincubation of the FAD-depleted enzyme with FAD restored coenzyme F420-dependent activity.

Properties of formate dehydrogenase in Methanobacterium formicicum.

Soluble formate dehydrogenase from Methanobacterium formicicum was purified 71-fold with a yield of 35%. Purification was performed anaerobically in the presence of 10 mM sodium azide which stabilized the enzyme. The purified enzyme reduced, with formate, 50 mumol of methyl viologen per min per mg of protein and 8.2 mumol of coenzyme F420 per min per mg of protein. The apparent Km for 7,8-didemethyl-8-hydroxy-5-deazariboflavin, a hydrolytic derivative of coenzyme F420, was 10-fold greater (63 microM) than for coenzyme F420 (6 microM). The purified enzyme also reduced flavin mononucleotide (Km = 13 microM) and flavin adenine dinucleotide (Km = 25 microM) with formate, but did not reduce NAD+ or NADP+. The reduction of NADP+ with formate required formate dehydrogenase, coenzyme F420, and coenzyme F420:NADP+ oxidoreductase. The formate dehydrogenase had an optimal pH of 7.9 when assayed with the physiological electron acceptor coenzyme F420. The optimal reaction rate occurred at 55 degrees C. The molecular weight was 288,000 as determined by gel filtration. The purified formate dehydrogenase was strongly inhibited by cyanide (Ki = 6 microM), azide (Ki = 39 microM), alpha,alpha-dipyridyl, and 1,10-phenanthroline. Denaturation of the purified formate dehydrogenase with sodium dodecyl sulfate under aerobic conditions revealed a fluorescent compound. Maximal excitation occurred at 385 nm, with minor peaks at 277 and 302 nm. Maximal fluorescence emission occurred at 455 nm.

FAD requirement for the reduction of coenzyme F420 by formate dehydrogenase from Methanobacterium formicicum.

The partial purification of the formate dehydrogenase from cell-free extracts of Methanobacterium formicicum decreased the rate of coenzyme F420 reduction 175-fold relative to the rate of methyl viologen reduction. FAD, isolated from this organism, reactivated the coenzyme F420-dependent activity of purified formate dehydrogenase and restored the activity ratio (coenzyme F420/methyl viologen) to near that in cell-free extracts. Neither flavin mononucleotide nor FADH2 replaced FAD. The reduced form of FAD inhibited the reactivation of coenzyme F420-dependent formate dehydrogenase activity by the oxidized form. The results suggest that native formate dehydrogenase from Methanobacterium formicicum contains noncovalently bound FAD that is required for coenzyme F420-dependent activity.

[Several years observation of a Catergen (cyanidanol-3) induced immunohemolysis]. / Mehrjährige Verlaufsbeobachtung einer durch Catergen (Cyanidanol-3) induzierten Immunhämolyse.

A report is presented on repeated severe episodes of cyanidanol-induced immune hemolysis in a 75-year-old woman suffering from chronic active hepatitis. One year after the first hemolytic episode, high titers of anticyanidanol antibodies were still demonstrated in the patient's serum (as well as sensitization of the patient's lymphocytes by the drug). 4 years later virtually no decrease of the antibody titer was found. Further investigations of the patient's serum revealed antibodies against flavonoids with free hydroxyl groups (cyanidanol and rutin). However, no antibodies against flavonoids with derivatized hydroxyl groups (troxerutin, mixture of hydroxy-ethyl-rutin and silibinin) could be demonstrated. Sensitization with flavonoids other than cyanidanol preceding cyanidanol-induced hemolysis has been proposed by others. In this case storage of approximately 75 flavonoid containing preparations in a chemist's shop in the patient's house may have contributed to the astonishing persistence of dry dependent antibodies.

Molybdopterin cofactor from Methanobacterium formicicum formate dehydrogenase.

The molybdopterin cofactor from the formate dehydrogenase of Methanobacterium formicicum was studied. The cofactor was released by guanidine denaturation of homogeneous enzyme, which also released greater than 80% of the molybdenum present in the enzyme. The anoxically isolated cofactor was nonfluorescent, but after exposure to air it fluoresced with spectra similar to those of described molybdopterin cofactors. Aerobic release from acid-denatured formate dehydrogenase in the presence of I2 and potassium iodide produced a mixture of fluorescent products. Alkaline permanganate oxidation of the mixture yielded pterin-6-carboxylic acid as the only detectable fluorescent product. The results showed that the cofactor from formate dehydrogenase contained a pterin nucleus with a 6-alkyl side chain of unknown structure. Covalently bound phosphate was also present. The isolated cofactor was unable to complement the cofactor-deficient nitrate reductase of the Neurospora crassa nit-1 mutant.

Kinetics of Formate Metabolism in Methanobacterium formicicum and Methanospirillum hungatei.

The kinetics of formate metabolism in Methanobacterium formicicum and Methanospirillum hungatei were studied with log-phase formate-grown cultures. The progress of formate degradation was followed by the formyltetrahydrofolate synthetase assay for formate and fitted to the integrated form of the Michaelis-Menten equation. The K(m) and V(max) values for Methanobacterium formicicum were 0.58 mM formate and 0.037 mol of formate h g (dry weight), respectively. The lowest concentration of formate metabolized by Methanobacterium formicicum was 26 muM. The K(m) and V(max) values for Methanospirillum hungatei were 0.22 mM and 0.044 mol of formate h g (dry weight), respectively. The lowest concentration of formate metabolized by Methanospirillum hungatei was 15 muM. The apparent K(m) for formate by formate dehydrogenase in cell-free extracts of Methanospirillum hungatei was 0.11 mM. The K(m) for H(2) uptake by cultures of Methanobacterium formicicum was 6 muM dissolved H(2). Formate and H(2) were equivalent electron donors for methanogenesis when both substrates were above saturation; however, H(2) uptake was severely depressed when formate was above saturation and the dissolved H(2) was below 6 muM. Formate-grown cultures of Methanobacterium formicicum that were substrate limited for 57 h showed an immediate increase in growth and methanogenesis when formate was added to above saturation.

Renaturation of Escherichia coli-derived recombinant human macrophage colony-stimulating factor.

Recombinant human macrophage colony-stimulating factor (rhM-CSF), a homodimeric, disulfide bonded protein, was expressed in Escherichia coli in the form of inclusion bodies. Reduced and denatured rhM-CSF monomers were refolded in the presence of a thiol mixture (reduced and oxidized glutathione) and a low concentration of denaturing agent (urea or guanidinium chloride). Refolding was monitored by nonreducing gel electrophoresis and recovery of bioactivity. The effects of denaturant type and concentration, protein concentration, concentration of thiol/disulfide reagents, temperature, and presence of impurities on the kinetics of rhM-CSF renaturation were investigated. Low denaturant concentrations (<0.5 M urea) and high protein concentrations (>0.4 mg/ml) in the refolding mixture resulted in increased formation of aggregates, although aggregation was never significant even when refolding was carried out at room temperature. Higher protein concentration resulted in higher rates but did not lead to increased yields, due to the formation of unwanted aggregates. Experiments conducted at room temperature resulted in slightly higher rates than those conducted at 4 degrees C. Although the initial renaturation rate for solubilized inclusion body protein without purification was higher than that of the reversed-phase purified reduced denatured rhM-CSF, the final renaturation yield was much higher for the purified material. A maximum refolding yield of 95% was obtained for the purified material at the following refolding conditions: 0.5 M urea, 50 mM Tris, 1.25 mM DTT, 2 mM GSH, 2 mM GSSG, 22 degrees C, pH 8, [protein] = 0.13 mg/ml.

Clinical presentation of rupture of the left-ventricular free wall after myocardial infarction: report of five cases with successful surgical repair.

Rupture of the left-ventricular free wall may not always result in immediate irreversible hemodynamic collapse. We report a series of five patients (4 male, 1 female; age 59-79 years) successfully operated for postinfarction free-wall rupture with good long-term results. Two patients presented with syncopy and acute tamponade three days after an acute myocardial infarction. In two patients with atypical chest pain and congestive heart failure, a large pericardial effusion and an extreme localized thinning of a myocardial scar region was seen several weeks after an uncomplicated myocardial infarct. In one patient a pseudoaneurysm was detected, which developed asymptomatically within three weeks after a posterior myocardial infarct. In all cases myocardial rupture was suspected after an echocardiographic examination. At surgery a hemopericardium and a localized rupture site were found. The surgical procedure included closure of the defect by direct suture or patch, CABG in 3 cases, and mitral valve replacement in one patient. The postoperative course was uneventful, only one patient needed IABP for 24 hours. Three patients returned to NYHA functional class I, one patient to class II, and one patient to class III. The latter patient died of heart failure 17 months postoperatively, and the other patients are still alive 4,18,24, and 26 months postoperatively. Thus clinical representation of left-ventricular free-wall rupture after myocardial infarction can be highly variable. But close cooperation between experienced echocardiographers and surgeons may allow successful corrections with good long term-results.

Mechanistic studies of the coenzyme F420 reducing formate dehydrogenase from Methanobacterium formicicum.

Mechanistic studies have been undertaken on the coenzyme F420 dependent formate dehydrogenase from Methanobacterium formicicum. The enzyme was specific for the si face hydride transfer to C5 of F420 and joins three other F420-recognizing methanogen enzymes in this stereospecificity, consistent perhaps with a common type of binding site for this 8-hydroxy-5-deazariboflavin. While catalysis probably occurs by hydride transfer from formate to the enzyme to generate an EH2 species and then by hydride transfer back out to F420, the formate-derived hydrogen exchanged with solvent protons before transfer back out to F420. The kinetics of hydride transfer from formate revealed that this step is not rate determining, which suggests that the rate-determining step is an internal electron transfer. The deflavo formate dehydrogenase was amenable to reconstitution with flavin analogues. The enzyme was sensitive to alterations in FAD structure in the 6-, 7-, and 8-loci of the benzenoid moiety in the isoalloxazine ring.

Formate dehydrogenase from Methanobacterium formicicum. Electron paramagnetic resonance spectroscopy of the molybdenum and iron-sulfur centers.

Formate dehydrogenase from Methanobacterium formicicum was examined by electron paramagnetic resonance spectroscopy. Although oxidized enzyme yielded no EPR signals over the temperature range 8-200 K, dithionite reduction resulted in generation of two paramagnetic components. The first, a nearly isotropic signal visible at temperatures below 200 K with g1 = 2.018, g2 = 2.003, and g3 = 1.994, exhibited nuclear hyperfine interaction with two equivalent protons (A1 = 0.45, A2 = 0.6, and A3 = 0.55 milliTeslas). EPR spectra of partially reduced 95Mo-enriched formate dehydrogenase exhibited additional 3-4 milliTeslas splittings, due to spin interaction with the 95Mo nucleus. Thus, this signal is due to a Mo center. This is the first reported example of a Mo center with gav greater than 2.0 in a biological system. The second species, a rhombic signal visible below 40 K with g values of g1 = 2.0465, g2 = 1.9482, and g3 = 1.9111 showed no hyperfine coupling and was assigned to reduced Fe/S. Both paramagnetic species could be detected in samples of M. formicicum whole cells anaerobically reduced with sodium formate. The Mo(V) signal was altered following addition of cyanide (g1 = 1.996, g2 = 1.988, and g3 = 1.980). Growth of bacteria in the presence of 1 mM WO4(2-) resulted in abolition of the Mo(V) EPR signal and formate dehydrogenase activity. Em, 7.7 was -330 mV for Mo(VI)/Mo(V) and -470 mV for Mo(V)/Mo(IV).

Heavy metal levels in feathers of wild turkeys from Virginia.

Levels of lead, cadmium, nickel, and zinc were determined in feathers of 175 wild turkeys (Meleagris gallopava) shot by hunters in 19 Virginia counties in 2 physiographic regions. Lead and nickel levels did not vary by county, region, sex, or age. Zinc and cadmium levels were significantly (P less than 0.01) higher in adult turkeys.