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C Krebs

Publications and source records attributed to C Krebs.

At least 19 recordsLinked to original sources
PubMed1999

Biochemical and spectroscopic characterization of overexpressed fuscoredoxin from Escherichia coli.

Fuscoredoxin is a unique iron containing protein of yet unknown function originally discovered in the sulfate reducers of the genus Desulfovibrio. It contains two iron-sulfur clusters: a cubane [4Fe-4S] and a mixed oxo- and sulfido-bridged 4Fe cluster of unprecedented structure. The recent determination of the genomic sequence of Escherichia coli (E. coli) has revealed a homologue of fuscoredoxin in this facultative microbe. The presence of this gene in E. coli raises interesting questions regarding the function of fuscoredoxin and whether this gene represents a structural homologue of the better-characterized Desulfovibrio proteins. In order to explore the latter, an overexpression system for the E. coli fuscoredoxin gene was devised. The gene was cloned from genomic DNA by use of the polymerase chain reaction into the expression vector pT7-7 and overexpressed in E. coli BL21(DE3) cells. After two chromatographic steps a good yield of recombinant protein was obtained (approximately 4 mg of pure protein per liter of culture). The purified protein exhibits an optical spectrum characteristic of the homologue from D. desulfuricans, indicating that cofactor assembly was accomplished. Iron analysis indicated that the protein contains circa 8 iron atoms/molecule which were shown by EPR and Mössbauer spectroscopies to be present as two multinuclear clusters, albeit with slightly altered spectroscopic features. A comparison of the primary sequences of fuscoredoxins is presented and differences on cluster coordination modes are discussed on the light of the spectroscopic data.

Amino Acid Sequence↗
PubMed1999

The ferroxidase reaction of ferritin reveals a diferric mu-1,2 bridging peroxide intermediate in common with other O2-activating non-heme diiron proteins.

Ferritins are ubiquitous proteins that concentrate, store, and detoxify intracellular iron through oxidation of Fe2+ (ferroxidation), followed by translocation and hydrolysis to form a large inorganic mineral core. A series of mutagenesis, kinetics, and spectroscopic studies of ferritin led to the proposal that the oxidation/translocation path involves a diiron protein site. Recent stopped-flow absorption and rapid freeze-quench Mössbauer studies have identified a single peroxodiferric species as the initial transient intermediate formed in recombinant frog M ferritin during rapid ferroxidation [Pereira, S. A., Small, W., Krebs, C., Tavares, P., Edmondson, D. E., Theil, E. C., and Huynh, B. H. (1998) Biochemistry 37, 9871-9876]. To further characterize this transient intermediate and to establish unambiguously the peroxodiferric assignment, rapid freeze-quenching was used to trap the initial intermediate for resonance Raman investigation. Discrete vibrational modes are observed for this intermediate, indicating a single chromophore in a homogeneous state, in agreement with the Mössbauer conclusions. The frequency at 851 cm-1 is assigned as nu(O-O) of the bound peroxide, and the pair of frequencies at 485 and 499 cm-1 is attributed, respectively, to nus and nuas of Fe-O2-Fe. Identification of the chromophore as a micro-1,2 bridged diferric peroxide is provided by the isotope sensitivity of these Raman bands. Similar peroxodiferric intermediates have been detected in a mutant of the R2 subunit of ribonucleotide reductase from Escherichia coli and chemically reduced Delta9 stearoyl-acyl carrier protein desaturase (Delta9D), but in contrast, the ferritin intermediate is trapped from the true reaction pathway of the native protein. Differences in the Raman signatures of these peroxide species are assigned to variations in Fe-O-O-Fe angles and may relate to whether the iron is retained in the catalytic center or released as an oxidized product.

Animals↗
PubMed1998

Direct spectroscopic and kinetic evidence for the involvement of a peroxodiferric intermediate during the ferroxidase reaction in fast ferritin mineralization.

Rapid freeze-quench (RFQ) Mössbauer and stopped-flow absorption spectroscopy were used to monitor the ferritin ferroxidase reaction using recombinant (apo) frog M ferritin; the initial transient ferric species could be trapped by the RFQ method using low iron loading (36 Fe2+/ferritin molecule). Biphasic kinetics of ferroxidation were observed and measured directly by the Mössbauer method; a majority (85%) of the ferrous ions was oxidized at a fast rate of approximately 80 s-1 and the remainder at a much slower rate of approximately 1.7 s-1. In parallel with the fast phase oxidation of the Fe2+ ions, a single transient iron species is formed which exhibits magnetic properties (diamagnetic ground state) and Mössbauer parameters (DeltaEQ = 1.08 +/- 0.03 mm/s and delta = 0.62 +/- 0.02 mm/s) indicative of an antiferromagnetically coupled peroxodiferric complex. The formation and decay rates of this transient diiron species measured by the RFQ Mössbauer method match those of a transient blue species (lambdamax = 650 nm) determined by the stopped-flow absorbance measurement. Thus, the transient colored species is assigned to the same peroxodiferric intermediate. Similar transient colored species have been detected by other investigators in several other fast ferritins (H and M subunit types), such as the human H ferritin and the Escherichia coli ferritin, suggesting a similar mechanism for the ferritin ferroxidase step in all fast ferritins. Peroxodiferric complexes are also formed as early intermediates in the reaction of O2 with the catalytic diiron centers in the hydroxylase component of soluble methane monooxygenase (MMOH) and in the D84E mutant of the R2 subunit of E. coli ribonucleotide reductase. The proposal that a single protein site, with a structure homologous to the diiron centers in MMOH and R2, is involved in the ferritin ferroxidation step is confirmed by the observed kinetics, spectroscopic properties, and purity of the initial peroxodiferric species formed in the frog M ferritin.

Animals↗
PubMed1998

Differential expression of calbindin and calmodulin in motoneurons after hypoglossal axotomy.

Axotomy induces a profound modification of Ca2+ homeostasis in injured neurons which may lead to neuronal death. Remarkably, after axotomy and resection of the hypoglossal nerve, 65-75% of the hypoglossal motoneurons survive in the long term and this suggests some adaptive mechanisms compensating the massive calcium influx. As potential components of this adaptation, we have examined calmodulin and calbindin-D28k by in situ hybridisation and immunohistochemistry in motoneurons of the rat after hypoglossal nerve transection. Neuronal calbindin mRNA and protein content was low in normal state, transiently increased to 200% of the basal expression at 8 days post-operation (dpo), then declined to normal again until 28 dpo. Calmodulin mRNA was highly expressed in normal hypoglossal motoneurons and remained constant after axotomy. Calmodulin protein immunoreactivity, however, was transiently decreased in axotomised motoneurons suggesting post-transcriptional modification. The upregulation of calbindin expression may facilitate the survival of injured motoneurons.

Animals↗
PubMed1998

Spectroscopic characterization of a novel tetranuclear Fe cluster in an iron-sulfur protein isolated from Desulfovibrio desulfuricans.

Mossbauer and EPR spectroscopies were used to characterize the Fe clusters in an Fe-S protein isolated from Desulfovibrio desulfuricans (ATCC 27774). This protein was previously thought to contain hexanuclear Fe clusters, but a recent X-ray crystallographic measurement on a similar protein isolated from Desulfovibrio vulgaris showed that the protein contains two tetranuclear clusters, a cubane-type [4Fe-4S] cluster and a mixed-ligand cluster of novel structure [Lindley et al. (1997) Abstract, Chemistry of Metals in Biological Systems, European Research Conference, Tomar, Portugal]. Three protein samples poised at different redox potentials (as-purified, 40 and 320 mV) were investigated. In all three samples, the [4Fe-4S] cluster was found to be present in the diamagnetic 2+ oxidation state and exhibited typical Mossbauer spectra. The novel-structure cluster was found to be redox active. In the 320-mV and as-purified samples, the cluster is at a redox equilibrium between its fully oxidized and one-electron reduced states. In the 40-mV sample, the cluster is in a two-electron reduced state. Distinct spectral components associated with the four Fe sites of cluster 2 in the three oxidation states were identified. The spectroscopic parameters obtained for the Fe sites reflect different ligand environments, making it possible to assign the spectral components to individual Fe sites. In the fully oxidized state, all four iron ions are high-spin ferric and antiferromagnetically coupled to form a diamagnetic S = 0 state. In the one-electron and two-electron reduced states, the reducing electrons were found to localize, consecutively, onto two Fe sites that are rich in oxygen/nitrogen ligands. Based on the X-ray structure and the Mossbauer parameters, attempts could be made to identify the reduced Fe sites. For the two-electron reduced cluster, EPR and Mossbauer data indicate that the cluster is paramagnetic with a nonzero interger spin. For the one-electron reduced cluster, the data suggest a half-integer spin of 9/2. Characteristic fine and hyperfine parameters for all four Fe sites were obtained. Structural implications and the nature of the spin-coupling interactions are discussed.

Bacterial Proteins↗
PubMed1998

Altered expression of immune-related antigens by neuronophages does not improve neuronal survival after severe lesion of the facial nerve in rats.

Injection of Fluoro-Gold (FG) into the whiskerpad muscles of rats yields a permanent retrograde labeling of motoneurons in the facial nucleus. Following subsequent resection of 10 mm of the facial nerve, one-third of the facial motoneurons die and the microglia phagocytize the dead FG-labeled neurons, take up FG, and get labeled in vivo. The resulting identification of all FG-labeled cells allows long-term comparative investigations on the behavior of neuronophages. In this study, we used two groups of rats to test whether the quantified expression of five immune-related antigens by neuronophages was related to quantified decline in neuron number (counts after immunostaining for neuron-specific enolase) 3 to 224 days after resection of the facial nerve. Rats of the first group received standard food and those of the second group, pellets containing 1,000 ppm of the calcium channel blocker nimodipine. Image analysis of the number of FG-containing cells and the number and projection area of immunopositive neuronophages in serial sections for each antigen showed that nimodipine significantly attenuated the immunostaining for CR3, MHC class I, and class II antigens (monoclonal antibodies [MAbs] OX-42, OX-18, and OX-6); enhanced the expression of monocyte-macrophage-specific antigen (MAb ED1); and did not change the expression of rat macrophage differentiation antigen (MAb ED2). The altered expressions, however, had no effect on the loss of motoneurons in the lesioned facial nucleus. We conclude that the degree of expression of immune-related antigens by neuronophages has no influence on the delayed neuronal cell death induced by permanent target deprivation.

Animals↗
PubMed1997

Term ovine placental vasculature: comparison of sea level and high altitude conditions by corrosion cast and histomorphometry.

The placental vascular architecture differs significantly at high altitude from that at sea level in the human and guinea-pig. Four sheep between 137 and 140 days of gestation, kept near sea level throughout gestation, were used as a normoxic control group for comparison of the placental vasculature with 10 other ewes, kept at high altitude (3820 m above sea level; Barcroft Laboratory, White Mountain Research Station, CA, USA). Placentomes from both groups were prepared for histology and scanning electron microscopy of vascular corrosion casts. Singular perfusion of fetal placentae, as well as combined maternal/fetal injection was performed. The influence of long-term hypoxaemia was determined by qualitative and semi-quantitative evaluation of corrosion casts and histological sections. The fetal vessel casts show a distinct difference in the arrangement of vessels of all sizes in response to long-term hypoxaemia. In the control group, stem arteries and veins are straight and parallel. In contrast, this is much less evident in the hypoxaemic group because arterioles and venules branch off the stem vessels more frequently and in an irregular manner. This leads to a capillary bed that is much more dense due to increased branching and capillary coiling. These observations are confirmed by histomorphometry. In the fetal vessels of high altitude sheep placentomes, we observed a decreased number of vascular cross sections (21.6 +/- 4.7 SEM versus 27.7 +/- 4.0 SEM; P = 0.02). However, the average luminal size per cross section (77.9 +/- 10.5 microns2 SEM versus 59.4 +/- 7.4 microns2 SEM; P = 0.004) was increased at high altitude and the percentage of lumina of the total area (5.7 +/- 0.5 SEM versus 5.3 +/- 0.3 SEM; P = 0.09) indicated a trend towards an increase. In maternal vessels of high altitude placentomes, the number of vessel cross sections (6.5 +/- 0.7 SEM versus 6.0 +/- 0.5 SEM; P = 0.2) remained unchanged, whereas the average luminal size (1108 +/- 122 microns2 SEM versus 844 +/- 77 microns2 SEM; P < 0.001) and the percentage of lumina out of the total area (20.9 +/- 1.8 SEM versus 17.5 +/- 1.7 SEM; P < 0.001) were increased. The interhaemal distance appeared to be slightly but not significantly increased at high altitude. These findings indicate that at high altitude the sheep placenta develops an increased materno-fetal absorptive surface to help guarantee substance exchange.

Altitude↗
PubMed1997

Axotomy induces transient calbindin D28K immunoreactivity in hypoglossal motoneurons in vivo.

Calbindin D28K, an intracellular calcium-binding protein, acts as Ca2+ buffering system in the cytoplasm. By means of this property, calbindin may protect neurons against large fluctuations in free intracellular Ca2+ and, hence, may prevent cell death. Although axotomy causes a massive influx of calcium into the lesioned neurons, resection of the hypoglossal nerve does not induce extensive neuronal cell death in rats. Even several weeks after axotomy, about 70% of the motoneurons survive despite permanent target deprivation. The mechanisms responsible for this remarkable survival rate are unknown. In this study, we have looked at the modification of calbindin immunoreactivity in axotomized hypoglossal motoneurons. In non-axotomized motoneurons, no calbindin is detectable by immunocytochemistry. Axotomy induced an increase of calbindin immunoreactivity in lesioned motoneurons. This increase, visualised by the number of calbindin-immunoreactive neurons extended from 1 day to 28 days. At this time most, but not all, motoneurons located on the side of the lesion were calbindin-positive as shown by retrograde labeling and immunoquenching. From 14 days post operation, calbindin immunoreactivity decreased and reached its basal value after 35 days post operation. At that time, only fibres were still calbindin immunoreactive. Interestingly, calbindin-immunoreactivity was also increased in almost all cell nuclei, compatible with a nuclear regulation. These data are consistent with the hypothesis that, as a reaction to axotomy, motoneurons trigger an increase in calbindin expression which acts as a compensatory Ca(2+)-buffering system, enabling neurons to maintain Ca2+ homeostasis and the survival of many motoneurons after axotomy.

Animals↗
PubMed1997

Fetal villosity and microvasculature of the bovine placentome in the second half of gestation.

The architecture of the fetal villous tree and its vasculature in the bovine placentome were studied in the second half of gestation using both conventional histology and histology of ink-filled blood vessels. These were compared with corrosion casts of plastic fillings of the vasculature, prepared for scanning electron microscopy. This combination of morphological methods allows perception of the villous tree throughout gestation from broad-conical to tall-conical form where branch ramification occurs mainly at right angles to the stem. The stem villus typically contains a single central artery and several peripheral veins arranged in parallel. The proximal branches to the stem, the intermediate villi, contain a central arteriole and accompanying venules. The distal branches, the terminal villi, enclose capillary convolutions which consist of an afferent arterial capillary limb, capillary loops and efferent venous capillary limbs. Vascular interconnections exist within the terminal villi, as capillaries or venules between the capillary convolutions, serially bridging them in up to 5 places, and as capillary anastomoses between the capillary loops. Coiling and sinusoidal dilatations of these loops develop near the end of gestation. The intraplacentomal rearrangement of villous trees with progressive gestation and their morphological vascular adaptations are discussed in relation to placental function, including the ever increasing need for transplacental substance exchange. This adaptation allows the blood to traverse the shortest possible arterioarteriolar route to the periphery of the trees where exchange takes place. The need for an increasing blood flow stimulates capillary growth and at the same time optimises the blood flow reaching the placental barrier represented by the vessel cast surface. The capillaries also carry the blood back into the very voluminous system of venules and veins where back diffusion may occur. The total volume of terminal villi of bovine placentome, the 'working part' of villous trees, hence distinctly increases with respect to the stem and intermediate villi, the 'supplying part' of the villous tree. In morphological terms the efficiency of the bovine transplacental diffusional exchange is higher than in the closely related 'co-ruminants' sheep and goats and distinctly higher when compared with the human placenta.

Animals↗
PubMed1997

Calretinin is transiently expressed in tendon fibroblasts of the paravertebral region of the chick embryo.

Although calretinin is an intracellular calcium acceptor protein essentially located in neurons, we have previously shown that calretinin was also expressed in the mesenchymal cells forming the intervertebral disc. Here, we describe, using immunohistochemistry, the transient expression of calretinin in fibroblasts responsible for the tendon formation in the paravertebral region. We have looked at chick embryos from embryonic day 4 to day 18. At embryonic day 6, calretinin immunoreactivity was detected in chick embryo cells located laterally to the spinal cord between two groups of developing muscular cells. At embryonic day 8, calretinin immunoreactivity intensity was the highest in cells showing a fibroblast morphology. After embryonic day 8, when fibroblast proliferation decreased and differentiation increased, calretinin immunoreactivity progressively disappeared. Interestingly, calretinin could not be detected in fibroblasts of the anterior and posterior limbs at any investigated age. Because calretinin expression appeared selectively and transiently in the fibroblasts of the paravertebral region, we may conclude that the phenotype of those fibroblasts is different from the limbs fibroblasts.

Animals↗
PubMed1996

Intrauterine growth restriction with absent end-diastolic flow velocity in the umbilical artery is associated with maldevelopment of the placental terminal villous tree.

OBJECTIVE: Our purpose was to evaluate the structure of placental terminal villi and their capillaries in pregnancies complicated by intrauterine growth restriction with absent end-diastolic flow velocity in the umbilical artery. STUDY DESIGN: Glutaraldehyde-perfusion-fixed villous tissue and a plastic cast of the vessels in at least two cotyledons were prepared from 10 cases with intrauterine growth restriction and 9 gestational age-matched control placentas. The structure and dimensions of 20 terminal capillary loops per cast were determined by scanning electron microscopic examination, and their appearances were correlated with the peripheral villi of the perfusion-fixed villous tissue. RESULTS: Capillary loops in the growth-restricted cases were sparse in number and significantly longer than in the control cases (218 microns [72] vs 137 microns [30], mean and SD, p < 0.05). They exhibited fewer branches (4.0 [1.9] per loop vs 6.1 [2.2], p < 0.05) and a majority of loops were uncoiled (79% vs 18%, p < 0.05). The villous tissues from the growth-restricted cases demonstrated elongated villi, consistent with the cast findings. The trophoblast surface was wrinkled and in some areas covered by fibrin plaques. CONCLUSIONS: The terminal villous compartment of the placenta appears to be maldeveloped in preterm intrauterine growth restriction pregnancies where absent end-diastolic flow velocity is demonstrated in the umbilical artery before delivery. These findings are consistent with an increase in fetoplacental vascular impedance at the capillary level and may account for the impaired gas and nutrient transfer in this disorder.

Blood Flow Velocity↗
PubMed1996

[Architecture and fine structure of the terminal blood vessels in the marginal papillae of the tongue in newborn piglets].

The vascular architecture of the marginal papillae of the tongue was examined in one to five day old piglets by means of light and transmission electron microscopy and scanning microscopy of vascular corrosion casts. The marginal papillae of the tongue exhibit a "rope ladder-like" principle of blood supply. Every papilla contains one ascending arteriole, which branches into single capillary loops each supplying a protrusion of the multiply branched base of the capillary body. The transition of the arteriole takes place on the tip of the papilla. Subsequently the capillary loops converge onto this venule. Precapillary sphincters are observed at the origin of the capillary loops. This type of vessel arrangement offers the opportunity to use the capillary system at maximum efficiency, thus allowing an erection of the papillae marginales during the process of suckling.

Animals↗
PubMed1994

Transanal internal sphincter myomectomy: indications, operative procedure and results.

From October 1, 1981, until December 31, 1991, we performed clinical, radiological, electromanometrical, histochemical and histological diagnostic procedures in 906 patients under 15 years of age, because of several distinct colonic and anorectal disorders. 739 were clinically constipated and in 272 of them hypertonicity of the internal anal sphincter with or without achalasia could be demonstrated by manometry. 121 of these patients were submitted to a posterior, transanal, partially resective internal sphincteromyomectomy, according to a slightly modified classical procedure. Follow-up of our operated patients ranged from 1 month to 9 years, with a mean of 3 years and 2 months and overall satisfactory results in 87.6% based on clinical evaluation. Additionally, 55.4% of the operated patients had early postoperative manometric controls (n = 67) and 77.6% of these (n = 52) accepted a third manometry, in order to evaluate late results of our procedure. We could demonstrate that early postoperative anorectal electromanometry shows a highly significant decrease of the internal anal sphincter pressure correlating with a clinical improvement in 98.5% of these patients (n = 67). Most late postoperative examinations showed recurrent elevation of the manometric internal sphincter pressure parameters, exceeding the mean values of our own normal controls. Nevertheless, clinically 98.1% of these patients had a persistent satisfactory remission of their symptoms, thus not correlating with the manometric findings in these cases. We conclude, that transanal internal sphincter myomectomy is a safe surgical procedure, which leads to satisfactory results in approximately 90% of chronically constipated children with anal sphincter hypertonicity, if very precise indications for operation are considered.

Adolescent↗
PubMed1991

Anorectal electromanometry in the diagnosis of neuronal intestinal dysplasia in childhood.

The diagnosis of neuronal intestinal dysplasia (NID) is currently established by histochemical procedures. From October 1, 1981, until July 31, 1990, we submitted a total number of 737 patients under 15 years of age with several distinct colonic and anorectal disorders, to clinical, radiological, electromanometrical, histochemical and histological evaluation. 573 had a clinical diagnosis of chronic constipation, and in 38 of these NID could be demonstrated by histochemical examination. Motivated by our own findings with the anorectal electromanometry in these patients we performed a double-blind prospective study to find out if there are any particular and pathognomonic manometric parameters of NID in childhood. 80% of our patients with NID diagnosed prospectively since April 1st, 1986, had a relaxation of the internal anal sphincter which was not proportional to the volume of rectal distention. Anorectal hyperexcitability was also present in these patients, whereas a statistically highly significant (p less than 0.01) increase of the amplitude of anorectal fluctuations (7.27 +/- 1.12 mmHg) as compared to the values measured in our own patients with functional chronic constipation (2.87 +/- 0.33 mmHg) could be demonstrated. Considering only those patients who simultaneously presented all of the above mentioned electromanometric criteria (e.g. non-proportional relaxation of the internal anal sphincter, anorectal hyperexcitability, increased amplitude of anorectal fluctuations) without an increase of the anorectal pressure profile, we could demonstrate that the correlation between the electromanometric diagnosis of NID and the final histochemical diagnosis was 100%. Nevertheless, under these conditions, 30% (9 out of 30) of patients with NID were not recognized electromanometrically because they had been excluded as false negative cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1991

Effect of growth factors on collagen metabolism in cultured human heart fibroblasts.

Using human heart fibroblasts (HHF), we studied the effect of basic fibroblast growth factor (bFGF) and transforming growth factor-beta (TGF-beta) on the gene expression of type I collagen, collagenase and tissue inhibitor of metalloproteinases (TIMP). Initially, treatment of HHF with bFGF alone (10 ng/ml) resulted in elevated secretion of collagenase into the culture medium. Subsequent treatment of HHF with TGF-beta in combination with bFGF suppressed collagenase secretion. Northern blot analysis reinforced this observation by revealing an enhancement of the steady-state mRNA level of collagenase in response to bFGF. In order to examine if the collagenase gene was affected by bFGF at the transcriptional level, transfection experiments were carried out with a plasmid containing collagenase promoter linked to chloramphenicol acetyltransferase gene (CAT). Basic FGF stimulated CAT activity by four-fold, indicating increased promoter activity whereas the combination of TGF-beta and bFGF resulted in decreased CAT activity. TGF-beta was shown to increase type I collagen and TIMP mRNA levels by 2.5- and 2.1-fold, respectively. These results suggest that TGF-beta and bFGF may play a pivotal role in regulating collagen metabolism in HHF.

Cells, Cultured↗