[Periarticular fractures of the upper and lower extremity. II: Therapeutic principle].
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Arm Injuries↗
Biomedical subjects
M Arand
Publications and source records attributed to M Arand.
Functional morphology of the spinal canal after endplate, wedge, and burst fractures.
Changes in the canal diameter during physiological motions are important considerations in the treatment of patients who have a burst fracture with the presence of bony fragments, but without neurologic deficit. In this in vitro study, the changes in the soft-canal diameter of the thoracolumbar region, when intact and after different fractures, was investigated under several different loading conditions. The soft-canal diameter was clearly identified on the lateral radiographs by attaching a series of steel balls to the posterior longitudinal ligament and ligamentum flavum in the midsagittal plane. Endplate, wedge, and burst fractures were produced incrementally in 19 three-vertebrae human cadaveric spine specimens by high-speed impacts. After each injury, a series of functional lateral radiographs were taken. The minimal canal diameter (MCD) was obtained by digitizing the images of the steel balls on radiographs using a custom-designed computer program. In the intact specimens, the MCD at the disc level changed significantly in flexion, extension, and compression, when compared with the MCD in the unloaded neutral position. However, the changes were small. The MCD after endplate and wedge fractures changed in a similar way. However, after the burst fracture, the MCD at the bone fragment level increased remarkably by a distraction force. It also significantly improved by an anterior shear force in comparison to the corresponding MCD in the neutral position. However, this change was smaller than the change due to the distraction force.
Biomechanical Phenomena↗
The catalytic activity of the endoplasmic reticulum-resident protein microsomal epoxide hydrolase towards carcinogens is retained on inversion of its membrane topology.
Diol epoxides formed by the sequential action of cytochrome P-450 and the microsomal epoxide hydrolase (mEH) in the endoplasmic reticulum (ER) represent an important class of ultimate carcinogenic metabolites of polycyclic aromatic hydrocarbons. The role of the membrane orientation of cytochrome P-450 and mEH relative to each other in this catalytic cascade is not known. Cytochrome P-450 is known to have a type I topology. According to the algorithm of Hartman, Rapoport and Lodish [(1989) Proc. Natl. Acad. Sci. U.S.A. 86, 5786-5790], which allows the prediction of the membrane topology of proteins, mEH should adopt a type II membrane topology. Experimentally, mEH membrane topology has been disputed. Here we demonstrate that, in contrast with the theoretical prediction, the rat mEH has exclusively a type I membrane topology. Moreover we show that this topology can be inverted without affecting the catalytic activity of mEH. Our conclusions are supported by the observation that two mEH constructs (mEHg1 and mEHg2), containing engineered potential glycosylation sites at two separate locations after the C-terminal site of the membrane anchor, were not glycosylated in fibroblasts. However, changing the net charge at the N-terminus of these engineered mEH proteins by +3 resulted in proteins (++mEHg1 and ++mEHg2) that became glycosylated and consequently had a type II topology. The sensitivity of these glycosylated proteins to endoglycosidase H indicated that, like the native mEH, they are still retained in the ER. The engineered mEH proteins were integrated into membranes as they were resistant to alkaline extraction. Interestingly, an insect mEH with a charge distribution in its N-terminus similar to ++mEHg1 has recently been isolated. This enzyme might well display a type II topology instead of the type I topology of the rat mEH. Importantly, mEHg1, having the natural cytosolic orientation, as well as ++mEHg1, having an artificial huminal orientation, displayed rather similar substrate turnovers for the mutagenic metabolite benzo[a]pyrene 4,5-oxide. To our knowledge this is the first report demonstrating that topological inversion of a protein within the membrane of the ER has only a moderate effect on its enzymic activity, despite differences in folding pathways and redox environments on each side of the membrane. This observation represents an important step in the evaluation of the influence of mEH membrane orientation in the cascade of events leading to the formation of ultimate carcinogenic metabolites, and for studying the general importance of metabolic channelling on the surface of membranes.
Amino Acid Sequence↗
Cellular expression of human centromere protein C demonstrates a cyclic behavior with highest abundance in the G1 phase.
Centromere proteins are localized within the centromere-kinetochore complex, which can be proven by means of immunofluorescence microscopy and immunoelectron microscopy. In consequence, their putative functions seem to be related exclusively to mitosis, namely to the interaction of the chromosomal kinetochores with spindle microtubules. However, electron microscopy using immune sera enriched with specific antibodies against human centromere protein C (CENP-C) showed that it occurs not only in mitosis but during the whole cell cycle. Therefore, we investigated the cell cycle-specific expression of CENP-C systematically on protein and mRNA levels applying HeLa cells synchronized in all cell cycle phases. Immunoblotting confirmed protein expression during the whole cell cycle and revealed an increase of CENP-C from the S phase through the G2 phase and mitosis to highest abundance in the G1 phase. Since this was rather surprising, we verified it by quantifying phase-specific mRNA levels of CENP-C, paralleled by the amplification of suitable internal standards, using the polymerase chain reaction. The results were in excellent agreement with abundant protein amounts and confirmed the cyclic behavior of CENP-C during the cell cycle. In consequence, we postulate that in addition to its role in mitosis, CENP-C has a further role in the G1 phase that may be related to cell cycle control.
Cell Cycle↗
Granulocyte-macrophage-colony-stimulating factor enhances immune responses to melanoma-associated peptides in vivo.
Peptide epitopes derived from differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for MHC-restricted cytotoxic T lymphocytes (CTL). The characterization of multiple CTL-defined antigenic determinants has opened possibilities of development of antigen-targeted vaccines. In the present study, we determined CTL reactivity against melanoma-associated peptides derived from Melan A/MART-1, tyrosinase, and gp100/Pmel17 in 3 HLA-A2+ melanoma patients. Then, we assessed the immune responses to synthetic melanoma-associated peptides injected intradermally. After 3 cycles of immunization with peptide alone, we used systemic GM-CSF as an adjuvant during the fourth cycle of immunization. Enhanced DTH reactions and CD8+ CTL responses were observed after treatment with systemic GM-CSF. Immunohistochemical characterization of DTH-constituting elements revealed infiltrates of CD4+ and CD8+ T lymphocytes and strong expression of IL-2 and gammaIFN, suggesting the activation of CD4+ ThI and CD8+ CTL by peptides presented by MHC-class-I molecules of dermal APC. Objective tumor regression was documented in all patients. We conclude that systemic GM-CSF enhances immune responses to melanoma-associated peptides and supports CTL-mediated tumor rejection in vivo.
Adjuvants, Immunologic↗
Inverse relationship of melanocyte differentiation antigen expression in melanoma tissues and CD8+ cytotoxic-T-cell responses: evidence for immunoselection of antigen-loss variants in vivo.
Antigenic peptides derived from differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for MHC-restricted cytotoxic T lymphocytes (CTL). CTL directed against peptides derived from the Melan A/MART-1, tyrosinase and gp100/Pmel17 antigens can be detected in melanoma patients and in healthy controls. The presence of defined antigenic peptides and corresponding precursor CTL in patients with metastatic melanoma opens perspectives for the development of antigen-specific tumor vaccines. In this study, we examined the expression of Melan A/MART-1, tyrosinase and gp100lPmel17 in fresh melanoma tissues of HLA-A2+ patients and the spontaneous CTL reactivity against antigenic peptides derived from these antigens. Our results demonstrate an inverse correlation of antigen expression and CTL response to Melan A/MART-1 and tyrosinase in patients with metastatic melanoma. In 2 patients with advanced disease, CTL responses against Melan A/MART-1 and tyrosinase were induced by intradermal immunization with synthetic nona- or deca-peptides derived from these antigens. Metastases increasing in size over time showed a loss of Melan A/MART-1 expression in the presence of CTL in one patient. The regression of a metastasis with persistent tyrosinase expression was observed in the other patient after the induction of CTL, reactive against tyrosinase. We conclude that CTL responses against melanocyte differentiation antigens may mediate regression of antigen-positive tumors and select for antigen-loss variants in vivo.
Amino Acid Sequence↗
Generation of cytotoxic T-cell responses with synthetic melanoma-associated peptides in vivo: implications for tumor vaccines with melanoma-associated antigens.
Peptide epitopes derived from differentiation antigens of the melanocyte lineage have been identified in human melanomas and normal cultured melanocytes as targets for MHC-restricted cytotoxic T lymphocytes (CTL). Characterization of multiple CTL-defined antigenic determinants and the presence of corresponding precursor CTL open perspectives for the development of antigen-based vaccines. In the present study, we determined the CTL reactivity against melanoma-associated peptides derived from Melan A/MART-1, tyrosinase and gp100/Pmel17 in 10 HLA-A2+ melanoma patients and 10 healthy individuals. Then, we examined the immunological effects and toxicity of intradermal inoculation of synthetic melanoma-associated peptides. Six patients with advanced melanoma received weekly intradermal injections of 6 melanoma-associated peptides and the influenza matrix peptide as a control for 4 consecutive weeks. DTH reactions were observed in 5/6 patients at the injections sites of the tyrosinase signal peptide and of the influenza matrix peptide. No toxic side effects were observed. Changes in CTL reactivity after peptide vaccination were assessed by an MLPC assay for each peptide. Generation of peptide-specific CTL was documented against Melan A/MART-1-derived peptide epitopes, the tyrosinase signal peptide and the influenza matrix peptide after vaccination. A decreasing CTL response against the internal tyrosinase peptide was documented in 1 patient through the course of vaccination and a decrease in DTH reactions. No major tumor regressions were observed. Two patients with rapidly progressive disease before vaccination have shown disease stabilization since vaccinations started. In conclusion, our results demonstrate that peptide alone injected intradermally may generate antigen-specific DTH reactions and an increase of antigen-specific CTL reactivity.
Adult↗
A multiplex polymerase chain reaction protocol for the simultaneous analysis of the glutathione S-transferase GSTM1 and GSTT1 polymorphisms.
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Base Sequence↗
Asp333, Asp495, and His523 form the catalytic triad of rat soluble epoxide hydrolase.
On the basis of the sequence similarity between mammalian epoxide hydrolases and bacterial haloalkane dehalogenase reported earlier (Arand, M., Grant, D. F., Beetham, J. K., Friedberg, T., Oesch, F., and Hammock, B. D. (1994) FEBS Lett. 338, 251-256; Beetham, J. K., Grant, D., Arand, M., Garbarino, J., Kiyosue, T., Pinot, F., Oesch, F., Belknap, W. R., Shinozaki, K., and hammock, B. D. (1995) DNA Cell. Biol. 14, 61-71) we selected candidate amino acid residues for the putative catalytic triad of the rat soluble epoxide hydrolase. The predicted amino acid residues were exchanged by site-directed mutagenesis of the epoxide hydrolase cDNA, followed by the expression of the respective mutant enzymes in Escherichia coli. A total of 25 different mutants were analyzed for their epoxide hydrolase activity toward the model substrate trans-stilbene oxide. In case of impaired catalytic activity of a given mutant, the structural integrity of the recombinant enzyme protein was assessed either by its ability to covalently bind the substrate trans-stilbene oxide or by affinity purification on benzyl thio-Sepharose, using the soluble epoxide hydrolase-specific competitive inhibitor 4-fluorochalcone oxide to release the bound enzyme from the affinity matrix. Of the mutants under investigation, only those with changes in the positions Asp333, Asp495, and His523 were completely inactive toward the model substrate trans-stilbene oxide while retaining the proper protein fold. These amino acids were exactly those previously predicted by sequence alignment. Exchange of the amino acid residues flanking the catalytic nucleophile Asp333 significantly changed the kinetic properties of the enzyme. Mutation of His332 to Gln had no apparent effect on the Km but led to a heavily reduced Vmax (5% that of the wild type) of the mutant enzyme, while the exchange of Trp334 against Phe strongly increased the Km (7-fold) and also moderately enhanced the Vmax (2-fold) of the corresponding mutant. Mutation of Trp540 apparently had a strong effect on the protein conformation.
Amino Acid Sequence↗
Investigating the role of the microsomal epoxide hydrolase membrane topology and its implication for drug metabolism pathways.
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Animals↗
Influence of varying muscle forces on lumbar intradiscal pressure: an in vitro study.
The purposes of this study were to determine the effect of including muscle forces in the experimental loading of the spine on the intradiscal pressure and to determine whether this effect correlates with previously established in vivo data. We modeled the spine muscles as of five distinct groups and isolated the effect of each group on the intradiscal pressure (L4-L5). Seven human lumbosacral spines were tested in pure flexion/extension, right/left lateral bending, and left/right axial rotation moments. Stimulated muscle activity strongly influenced load-pressure characteristics, especially for the multifidus. Without muscle forces active, pressure increased proportionately with increasing moment. With five pairs of symmetrical constant muscle forces active (80 N per pair) the pressure increased more than 200% in neutral position and did not increase with increasing moment. The pressure without muscle forces and without axial preload was 0.12 MPa, which is about the same found by earlier in vivo studies of anesthetized subjects in prone position. With simulated muscle forces, the pressure was 0.39 MPa and in the range found for non-anesthetized subjects. We conclude that simulating muscle forces substantially affects intradiscal pressure.
Adult↗
Inducing properties of rifampicin and rifabutin for selected enzyme activities of the cytochrome P-450 and UDP-glucuronosyltransferase superfamilies in female rat liver.
Important species differences have been reported concerning the induction properties of rifampicin towards enzymes of the P-450 superfamily. Mice, rabbits and humans are far more responsive than rats and guinea pigs. In the present study a strong induction of cytochrome P-450 3A-dependent enzyme activities was observed in female rat liver microsomes after high dose treatment (> or = 250 mg/kg/day for 9 days) with rifampicin, resulting in an up to 30-fold enhanced hydroxylation rate of testosterone in the 2 beta-, 6 beta- and 15 beta-position in vitro. Other cytochrome P-450 isozyme-selective reactions were not, or only marginally, affected. A steep increase in cytochrome P-450 3A activity on a moderate elevation of the dose administered, together with the previously observed lack of efficient induction with doses below 200 mg/kg/day demonstrated that there is a threshold in enzyme induction by rifampicin. For rifabutin such a threshold was not apparent. Induction by rifabutin showed an isoenzyme-selectivity profile similar to that produced by rifampicin, but the maximally achievable induction of cytochrome P-450 3A by rifabutin was about two-fold lower compared with rifampicin. Rifampicin and rifabutin enhanced the glucuronidation of 1-naphthol, 4-hydroxybiphenyl and beta-estradiol by a factor of two to three. The potential implications of the enzyme induction by rifampicin derivatives in terms of possible drug-drug interactions are discussed.
Animals↗
Cytolytic T cell reactivity against melanoma-associated differentiation antigens in peripheral blood of melanoma patients and healthy individuals.
Antigenic peptides derived from several differentiation antigens of the melanocyte lineage were recently identified in human melanomas as targets for HLA-A2.1-restricted cytotoxic T lymphocytes (CTLs). To examine their potential role in tumour-directed immune responses in vivo, we determined CTL reactivity against seven antigenic peptides derived from the Melan A/MART-1, tyrosinase and gp100/Pmel17 antigens in the peripheral blood of 10 HLA-A2+ healthy controls and 26 HLA-A2+ melanoma patients. The influenza matrix peptide (GILGFVFTL) presented by HLA-A2.1 was used as a control peptide. CTL reactivity was assessed in a mixed lymphocyte 'peptide' culture assay. Reactivity against Melan A/MART-1-derived peptide antigens was readily detectable in both melanoma patients and controls. Reactivity directed against tyrosinase-derived peptide antigens was also detected in both melanoma patients and healthy individuals, but less frequently. A measurable response against gp100/Pmel17-derived antigens was found in 1/10 controls and in 1/26 of the melanoma patients. Reactivity against the influenza matrix peptide was common in both melanoma patients and controls. Our findings show that precursor CTLs against melanocyte differentiation antigens can be detected in peripheral blood of melanoma patients and healthy individuals. The pattern of CTL reactivity directed against melanoma-associated antigens does not seem to be altered in melanoma patients. Despite antigen-specific CTL reactivity, tumour growth was not prevented in melanoma patients and autoimmune phenomena were not detected in healthy individuals. It remains to be determined whether precursor CTLs recognizing melanocyte differentiation antigens can be activated by immunization and lead to effective tumour rejection in vivo.
Amino Acid Sequence↗
[Polytrauma and economics].
Treatment of polytrauma patients requires a large number of well trained staff and expensive medical equipment. The high medical standard in polytrauma care resulted in a reduction of a 40% lethality in the 70's to 10 to 15% today. The changes in german health systems (GSG) cut down the financial resources and call for careful evaluation of costs in trauma centers. Our analysis of the costs of 1 multiple injured patient in 1996 showed, that the reimbursements of hospital charges ended in a deficit of DM 30,000,-. Our department treats approximately 100 polytraumas a year which implies a budget deficit of 3 million DM per year. Today the aim of trauma centers is the maintenance of a high surgical standard in trauma care together with adequate reimbursement of hospital expenses. Trauma care and life saving interventions must not be shortened by financial restrictions. The trauma surgeon has to take care for his multiple injured patients and balanced financial reimbursements as well.
Adult↗
Validity of the three-column theory of thoracolumbar fractures. A biomechanic investigation.
STUDY DESIGN: This study validated the three-column theory of fractures by correlating the multidirectional instabilities and the vertebral injuries to each of the three columns, using a biomechanic trauma model. OBJECTIVES: The objective was to validate the three-column theory as applied to the thoracolumbar fractures. SUMMARY OF BACKGROUND DATA: The widely used three-column theory of fractures for classification and evaluation was based on retrospective analysis of radiographs. No biomechanic study, using realistic spinal fractures and multidirectional instability measurements, was available. METHODS: Using 16 fresh cadaveric thoracolumbar human spine specimens, two groups of burst fractures were produced by either simple axial compression or flexion-compression, using a high-speed trauma model. Multidirectional flexibility was measured before and after the trauma, thus quantifying the instability of the burst fracture. Computed tomography scans were taken after the fracture, and a newly developed injury scoring scheme quantified the injuries to the anterior, middle, and posterior columns. Statistical correlations were obtained between the flexibility parameters and injuries to each of the three columns. RESULTS: In the axial compression group, the middle column injury, compared with the other two columns, showed the highest correlations to eight of the nine flexibility parameters (average R2 = 0.77). In the flexion-compression group, again the middle column injury showed the highest correlations to eight of the nine flexibility parameters (average R2 = 0.85). CONCLUSIONS: The results of this study supported the three-column theory of the thoracolumbar fractures and bolstered the concept of the middle column being the primary determinant of mechanical stability of this region of the spine.
Adult↗
Stability increase of the lumbar spine with different muscle groups. A biomechanical in vitro study.
STUDY DESIGN: This study investigated the influence of five different muscle groups on the monosegmental motion (L4-L5) during pure flexion/extension, lateral bending, and axial rotation moments. OBJECTIVES: The results showed and compared the effect of different muscle groups acting in different directions on the stability of a single motion segment to find loading conditions for in vitro experiments that simulate more physiologically reasonable loads. SUMMARY OF BACKGROUND DATA: In spine biomechanics research, most in vitro experiments have been carried out without applying muscle forces, even though these forces stabilize the spinal column in vivo. METHODS: Seven human lumbosacral spines were tested in a spine tester that allows simulation of up to five symmetrical muscle forces. Changing pure flexion/extension, lateral bending, and axial rotation moments up to +/- 3.75 Nm were applied without muscle forces, with different muscle groups and combinations. The three-dimensional monosegmental motion was determined using an instrumented spatial linkage system. RESULTS: Simulated muscle forces were found to strongly influence load-deformation characteristics. Muscle action generally decreased the range of motion and the neutral zone of the motion segments. This was most evident for flexion and extension. After five pairs of symmetrical, constant muscle forces were applied (80 N per pair), the range of motion decreased about 93% in flexion and 85% in extension. The total neutral zone for flexion and extension was decreased by 83% muscle action. The multifidus muscle group had the strongest influence. CONCLUSION: This experiment showed the importance of including at least some of the most important muscle groups in in vitro experiments on lumbar spine specimens.
Adult↗
Xenobiotic metabolizing enzyme activities and viability are well preserved in EDTA-isolated rat liver parenchymal cells after cryopreservation.
Rat liver parenchymal cells (PC) were isolated by EDTA perfusion and were purified by a subsequent Percoll centrifugation. The isolated PC had a viability of 95%, as judged by trypan blue exclusion. Freshly isolated PC were cryopreserved with an optimized protocol in a computer-controlled freezer. After thawing, the PC still retained a viability of 89%. The activities of representative xenobiotic metabolizing enzymes were compared between freshly isolated and cryopreserved PC after thawing. The cytochrome P450 content and the cytochrome P450 2C11 isoenzyme activity, determined by hydroxylation of testosterone in intact cells, were not affected by the cryopreservation. The following phase II enzyme activities were also well maintained after cryopreservation: Phenol sulfotransferase (92%), 1-naphthol UDP-glucuronosyl transferase (95%), soluble epoxide hydrolase (87%), and glutathione S-transferase (88%), determined with broad spectrum substrate 1-chloro-2,4-dinitrobenzene. However, there was a significant decrease in plating efficiency between freshly isolated (86%) and cryopreserved (57%) PC when they were cultured. The initial quality of the freshly isolated PC is decisive for the success of cryopreservation. These results support the use of cryopreserved PC in pharmacology and toxicology with the aim to reduce the number of experimental animals used.
Animal Testing Alternatives↗
Fracture pattern and instability of thoracolumbar injuries.
Spinal fractures are common in the thoracolumbar region. Assessment of fracture instability is often made from fracture pattern seen on plain radiographs or CT scans. The purpose of this in vitro study was to correlate three-dimensional flexibility to each fracture type, i.e., endplate, wedge, and burst. Ten fresh cadaveric human spine specimens (T11-L1) were incrementally impacted in a high-speed trauma apparatus until a fracture occurred. All fractures were produced by the same mechanism (axial compression/flexion load). The occurrence of a fracture was monitored by lateral radiographs of the specimen, whose canal was lined with 1.6-mm steel balls. After each impact, the specimen was studied for its flexibility in flexion, extension, left and right lateral bindings, and left and right axial rotations. The flexibility was determined in response to the application of maximum pure moments of 7.5 Nm. Each moment was applied individually and in three load cycles. Parameters of neutral zone (NZ) and range of motion (ROM) were computed. Average flexion-extension ROM (and NZ) for intact, endplate, wedge, and burst fracture were respectively, 12.7 degrees (1.3 degrees), 13.9 degrees (1.7 degrees), 19.2 degrees (3.2 degrees), 22.0 degrees (6.0 degrees). The average lateral bending ROM (NZ) were 12.6 degrees (1.2 degrees), 13.6 degrees (1.9 degrees), 19.1 degrees (3.7 degrees), 27.2 degrees (9.8 degrees). The average axial rotation ROM (NZ) were 4.7 degrees (0.4 degree), 6.1 degrees (0.7 degrees), 7.1 degrees (1.0 degree), 12.9 degrees (3.1 degrees).(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗