Proton NMR studies of cytochrome c peroxidase mutant N82A: hyperfine resonance assignments, identification of two interconverting enzyme ofecies, quantitating the rate of interconversion, and determination of equilibrium constants

The cyanide-ligated form of the baker's yeast cytochrome c peroxidase mutant bearing the mutation Asn82-->Ala82 ([N82A]CcPCN) has been studied by proton NMR spectroscopy. This mutation alters an amino acid that forms a hydrogen bond to His52, the distal histidine residue that interacts in the heme pocket with heme-bound ligands. His52 is a residue critical to cytochrome c peroxidase's normal function. Proton hyperfine resonance assignments have been made for the cyanide-ligated form of the mutant by comparison with 1-D and NOESY spectra of the wild-type native enzyme. For [N82A]CcPCN, proton NMR spectra reveal two significant phenomena. First, similar to results published for the related mutant [N82D]CcPCN [Satterlee, J. D., et al. (1994) Eur. J. Biochem. 244, 81-87], for Ala82 mutation disrupts the hydrogen bond between His52 and the heme-ligated CN. Second, four of the 24 resolved hyperfine-shifted resonances are doubled in the mutant enzyme's proton spectrum, leading to the concept that the heme active site environment is dynamically microheterogeneous on a very localized scale. Two magnetically inequivalent enzyme forms are detected in a pure enzyme preparation. Varying temperature causes the two enzyme forms to interconvert. Magnetization transfer experiments further document this interconversion between enzyme forms and have been used to determine that the rate of interconversion is 250 (+/- 53) s-1. The equilibrium constant at 20 degrees C is 1.5. Equilibrium constants have been calculated at various temperatures between 5 and 29 degrees C leading to the following values: delta H = 60 kJ mol-1; delta S = 0.20 kJ K-1 mol-1.     

Neuronal migration disorders increase susceptibility to hyperthermia-induced seizures in developing rats

PURPOSE: Retrospective studies suggest that adult patients with intractable epilepsy may have a history of febrile seizures in childhood. Risk factors for a febrile seizure may include the rate of increase in the core temperature (T-core), its peak (Tmax), the duration of the temperature increase, or an underlying brain pathology. Recently, neuronal migration disorders (NMD) have been diagnosed with increasing frequency in patients with epilepsy, but the link between NMD, febrile seizures, and epilepsy is unclear. We studied rat pups rendered hyperthermic to ascertain the incidence of seizures, mortality, and extent of hippocampal cell loss in each group. METHODS: We exposed 14-day-old rat pups with experimentally induced NMD (n = 39) and age-matched controls (n = 30) to hyperthermia (core body temperature > 42 degrees C). RESULTS: The incidence of hyperthermia-induced behavioral seizures and mortality rate were significantly higher in rats with NMD than in controls (p < 0.05). The longer duration of hyperthermia resulted in a higher incidence of behavioral seizures and higher mortality rate (p < 0.05). In rats with NMD, hyperthermia resulted in hippocampal pyramidal cell loss independent of seizure activity; the extent of neuronal damage correlated positively with the duration of hyperthermia. In control rats, occasional neuronal loss and astrocytosis occurred only after prolonged hyperthermia. CONCLUSIONS: In immature rats, NMD lower the threshold to hyperthermia-induced behavioral seizures and hyperthermia in the presence of NMD may cause irreversible hippocampal neuronal damage.     

Extrahepatic metabolism of 4-methylumbelliferone and lidocaine in the anhepatic rabbit

OBJECTIVE: The initially well-fixed implants of total hip replacement (THR) are in the long-term subject to aseptic loosening. Many cytokines can contribute to osteolysis due to osteoclast recruitment and/or activation. However, in this respect tumor necrosis factor-alpha (TNF-alpha) plays a pivotal role, because it upregulates interleukin-1 and 6 and granulocyte-macrophage colony stimulating factor. The aim of this study was to assess the eventual presence, cellular localization and extent of expression of TNF-alpha in the synovial-like membrane at the implant or at the cement to bone interface compared to control synovial membrane. METHODS: Twenty samples from the synovial-like membrane of the periprosthetic tissues were compared to control samples. TNF-alpha containing cells were visualized using an avidin-biotin-peroxidase complex (ABC) method and analyzed by light microscopy, double labelling and image analysis. RESULTS: TNF-alpha was found in the periprosthetic tissues in fibroblasts and vascular endothelial cells, but mainly in the macrophages was it found to coincide with areas containing implant-derived debris. TNF-alpha containing cells were more numerous in the synovial-like membrane in the interface tissue from the proximal stem area (2816 +/- 318 cells) than in the control synovial membrane (565 +/- 93 cells, p < 0.01). Interestingly, similarly high TNF-alpha expression (3452 +/- 582 cells) was also seen in the synovial-like membrane of the pseudocapsule. CONCLUSION: These findings suggest that the foreign body-type host reaction caused by THR is characterized by the high expression of TNF-alpha. Because such expression occurred in the interface tissue between the implant and surrounding bone, TNF-alpha, due to its pivotal direct and indirect role in the activation and recruitment of osteoclasts, may contribute to periprosthetic osteolysis and to the loosening of THR.     

Detection and quantification of endolymphatic hydrops in the guinea pig cochlea by magnetic resonance microscopy

The experiments carried out on the model of immobilization stress permit establishing the antiulcer effect of preventive introduction of sodium succinate solution, milk serum solution and solution of milk serum with sodium succinate. A decrease in the degree and number of ulcers and in the integrative indices characterizing the ulcer process after introduction of the studied drugs was accompanied by a fall of the intensity of lipid peroxidation processes.     

Level and instability of day-to-day psychological well-being and risk for depression

Human alpha1-acid glycoprotein (AAG) is a mixture of at least two genetic variants: the A variant and the F1 and/or S variant or variants, which are encoded by two different genes. In a continuation of previous studies indicating specific drug transport roles for each AAG variant according to its separate genetic origin, this work was designed to (1) determine the affinities of the two main gene products of AAG (i.e., the A variant and a mixture of the F1 and S variants) for 35 chemically diverse drugs and (2) to obtain meaningful 3D-QSARs for each binding site. Affinities were obtained by displacement experiments, leading to qualitative indications about binding site characteristics. In particular, drugs binding selectively to the A variant displayed some common structural features, but this was not seen for the F1*S variants. Three-dimensional QSAR analyses using the CoMFA method yielded a steric model for binding to the A variant, from which a simplified haptophoric model was derived. In contrast, no statistically sound model was found for the F1*S variants, possibly due (among other reasons) to an insufficient number of high affinity ligands in the set.     

Meniscal regeneration in the long-term after total meniscectomy?

A cohort of 39 patients (28 male, 11 female) that had undergone total meniscectomy as adolescents (mean age 16 years) underwent FISP 3D Magnetic Resonance Imaging at a mean follow up of 30 years. The presence of meniscal tissue was assessed by two independent observers blinded to the operation details. The volume of any meniscal tissue present was calculated. A posterior horn remnant was seen in 57% of medial and 45% of lateral meniscectomy cases. The mean volume of an operated medial meniscal remnant was 0.29 mL compared with a mean volume of 1.15 mL for an intact medial meniscus. The mean volume of an operated lateral meniscal remnant was 0.30 mL compared with 1.07 mL for an intact lateral meniscus. We have shown that the incidence of incomplete excision of the posterior horn is more common after total medial meniscectomy, and that at a mean follow up of 30 years there is no convincing in vivo MRI evidence of long-term meniscal regeneration.     

Lipopolysaccharide-stimulated osteoclastogenesis is mediated by tumor necrosis factor via its P55 receptor

Chronic bone infection, as attends periodontitis, is often complicated by severe osteolysis. While LPS is believed to be central to the pathogenesis of the osteolytic lesion, the mechanisms by which this bacteria-derived molecule promotes bone resorption are unknown. We find that LPS induces bone marrow macrophages (BMMs) to express c-src, a protooncogene product that we demonstrate is a specific marker of commitment to the osteoclast phenotype. We next turned to possible soluble mediators of LPS-induced c-src. Of a number of osteoclastogenic cytokines tested, only TNF-alpha mirrors the c-src-enhancing effect of LPS. Suggesting that LPS augmentation of c-src is TNF-mediated, endotoxin sequentially induces BMM expression of TNF, followed by c-src. TNF and c-src expression, by cultured BMMs derived from LPS-injected mice, reflects duration of exposure to circulating endotoxin, intimating that endotoxin's effect in vivo is also mediated by TNF. Consistent with these findings, thalidomide (which antagonizes TNF action) attenuates c-src induction by LPS. An anti-TNF antibody blocks LPS enhancement of c-src mRNA, validating the cytokine's modulating role in vitro. Using BMMs of TNF receptor-deleted mice, we demonstrate that TNF induction of c-src is transmitted through the cytokine's p55, but not p75, receptor. Most importantly, LPS administered to wild-type mice prompts osteoclast precursor differentiation, manifest by profound osteoclastogenesis in marrow cultured ex vivo, and by a profusion of marrow-residing cells expressing the osteoclast marker tartrate resistant acid phosphatase, in vivo. In contrast, LPS does not substantially enhance osteoclast proliferation in mice lacking the p55TNF receptor, confirming that LPS-induced osteoclastogenesis is mediated by TNF in vivo via this receptor. Thus, therapy targeting TNF and/or its p55 receptor presents itself as a means of preventing the osteolysis of chronic bacterial infection.     

Up-regulation of CIR1/CROC1 expression upon cell immortalization and in tumor-derived human cell lines

Inactivation of 5-HT1A and [3H]5-HT binding sites by N-Ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline (EEDQ) was studied in regions of rat brain. After exposure to EEDQ (4 mg/kg body wt.) for 7 days, it is observed that the density of 5-HT1 receptor sites was decreased by nearly 20% in both cortex and hippocampus. The decrease, however, in 5-HT1A sites was more significant (70%) in both the regions. The affinity of [3H]5-HT to 5-HT1 sites was decreased significantly in both cortex and hippocampus after exposure to EEDQ, without affecting the Kd of 5-HT1A sites. Displacement studies suggested that EEDQ has high affinity to 5-HT1 sites with a Ki of 42.9+/-2.4 nM. After exposure neither basal nor 5-HT stimulated adenylyl cyclase activity was changed in cortex. The results of this study suggest that EEDQ decreases the density of 5-HT1 and 5-HT1A receptor sites but does not cause functional downregulation of these sites in rat brain.