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1.
Knee ; 34: 62-75, 2022 Jan.
Article in English | MEDLINE | ID: mdl-34883331

ABSTRACT

BACKGROUND: Extensive efforts have been made to understand joint kinematics and kinetics in total knee arthroplasty (TKA) in subjects with satisfactory outcomes during daily functional activities and clinical tests, but it remains unclear whether such movement characteristics hold the potential to indicate the underlying aetiology of unsatisfactory or bad TKA outcomes. PURPOSE: To investigate which kinematic and kinetic parameters assessed during passive clinical tests and functional activities of daily living are associated with poor functionality and underlying deficits after total knee replacement. METHODS: We focused on studies characterizing the kinematic or kinetic parameters of the knee joint that are associated with poor clinical outcome after TKA. Seventeen articles were included for the review, and kinematic and kinetic data from 719 patients with minimal follow up of 6 months were extracted and analyzed. RESULTS: Passive posterior translation at 90°flexionexhibited good potential for differentiating stable and unstable TKAs. Anterior-posterior (A-P) translation of the medial condyle at 0-30° and 30-60° flexion, A-P translation of the lateral condyle at 60-90°during closed chain exercises, as well asknee extension moment during stair ascent and descent, knee abduction moment during stair descent, knee internal rotation moment and plantar flexion moment during walking, 2ndpeak ground reaction force during stair ascent and walkingshowed the greatest promise as functional biomarkers for a dissatisfied/poor outcome knee after TKA. CONCLUSION: In this study, we systematically reviewed the state-of-the-art knowledge of kinematics and kinetics associated with functional deficits, and found 11 biomechanical parameters that showed promise for supportingdecision making in TKA.


Subject(s)
Arthroplasty, Replacement, Knee , Knee Prosthesis , Activities of Daily Living , Arthroplasty, Replacement, Knee/adverse effects , Biomechanical Phenomena , Humans , Kinetics , Knee Joint/surgery , Range of Motion, Articular
2.
Hautarzt ; 68(12): 959-967, 2017 Dec.
Article in German | MEDLINE | ID: mdl-29052749

ABSTRACT

BACKGROUND: More and more people worldwide and also in Germany are using botulinum toxin type A (BoNT-A) and hyaluronic acid injections for skin rejuvenation. OBJECTIVE: Study on body image and self-esteem of women with BoNT-A and/or hyaluronic acid filler treatment. MATERIAL AND METHODS: A total of 145 women who requested BoNT-A and/or hyaluronic acid injections completed a survey comprised of the body dysmorphic disorder questionnaire, the Rosenberg self-esteem scale and questionnaires on the attitudes and motives on measures for optimization of the body and demographic features. Using this instrument data on the body image and self-esteem as well as attitudes and motives for utilization of minimally invasive skin rejuvenation were collated. RESULTS: Female users of minimally invasive skin rejuvenation showed an overall higher socioeconomic status and an above average high monthly income. They lived in a partnership more often in comparison to women of equal age living in Berlin. The users of BoNT-A and/or hyaluronic acid fillers showed no conspicuous differences in body image and self-esteem. They showed a moderately positive attitude to body optimization procedures and 91% achieved their standard weight with a body mass index (BMI) of ≤25 kg/m2 in comparison to 56% of German women in the same age range (25 to ≥75 years old). CONCLUSION: In the first study of body image and self-esteem in users of BoNT­A and/or dermal fillers in German women, the users showed no signs of body dysmorphic patterns or disorders of self-esteem.


Subject(s)
Body Image , Botulinum Toxins, Type A/administration & dosage , Cosmetic Techniques/psychology , Hyaluronic Acid/administration & dosage , Rejuvenation/psychology , Self Concept , Skin Aging , Adult , Aged , Beauty , Body Dysmorphic Disorders/diagnosis , Body Dysmorphic Disorders/psychology , Female , Germany , Humans , Middle Aged , Motivation , Surveys and Questionnaires
3.
Case Rep Orthop ; 2016: 1498135, 2016.
Article in English | MEDLINE | ID: mdl-27504207

ABSTRACT

Articular cartilage defects at the knee joint are being identified and treated with increasing frequency. Chondrocytes may have strongest potential to generate high-quality repair tissue within the defective region, in particular when large diameter defects are present. Autologous chondrocyte implantation is not available in every country. We present a case where we spontaneously covered an acute cartilage defect, which was significantly larger than expected and loose during initial arthroscopic inspection after reading preoperative MRI, by mincing the separated fragment and directly implanting the autologous cartilage chips into the defective region.

4.
J Mater Chem B ; 4(46): 7423-7428, 2016 Dec 14.
Article in English | MEDLINE | ID: mdl-32263743

ABSTRACT

Superoxide radicals are associated with the development of many severe diseases, such as cancer. Under nonpathogenic conditions, the natural enzyme superoxide dismutase (SOD) regulates the intracellular superoxide concentrations, but nearly all tumor tissues show reduced SOD levels. Selective imaging in early progression stages remains a key requirement for efficient cancer diagnosis and treatment. Magnetic resonance imaging (MRI) as a noninvasive tool with high spatial resolution may offer advantages here, but MRI contrast agents exhibiting a redox-triggered change in the image contrast towards superoxide radicals have not been reported so far. Here we show that manganese oxide (MnO) nanoparticles (NPs) exhibit an intrinsic SOD-like activity, which is higher than that of the native Mn-dependent SOD. In addition, MnO NPs significantly enhance the MRI contrast when exposed to superoxide radicals, making them responsive MRI contrast agents for the treatment and imaging of cancer cells with reduced SOD levels.

6.
Eur J Phys Rehabil Med ; 51(6): 825-32, 2015 Dec.
Article in English | MEDLINE | ID: mdl-25779914

ABSTRACT

BACKGROUND: Impaired cervical joint position sense is a feature of chronic neck pain and is commonly argued to rely on abnormal cervical input. If true, muscle vibration, altering afferent input, but not mental interventions, should have an effect on head repositioning acuity and neck pain perception. AIM: The aim of the present study was to determine the short-term effects of neck muscle vibration, motor imagery, and action observation on cervical joint position sense and pressure pain threshold in people with chronic neck pain. DESIGN: Forty-five blinded participants with neck pain received concealed allocation and were randomized in three treatment groups. A blinded assessor performed pre- and post-test measurement. SETTING: Patients were recruited from secondary outpatient clinics in the southwest of Germany. POPULATION: Chronic, non specific neck pain patients without arm pain were recruited for this study. METHODS: A single intervention session of 5 minutes was delivered to each blinded participant. Patients were either allocated to one of the following three interventions: (1) neck muscle vibration; (2) motor imagery; (3) action observation. Primary outcomes were cervical joint position sense acuity and pressure pain threshold. Repeated measures ANOVAs were used to evaluate differences between groups and subjects. RESULTS: Repositioning acuity displayed significant time effects for vibration, motor imagery, and action observation (all P<0.05), but revealed no time*group effect. Pressure pain threshold demonstrated a time*group effect (P=0.042) as only vibration significantly increased pressure pain threshold (P=0.01). CONCLUSION: Although motor imagery and action observation did not modulate proprioceptive, afferent input, they nevertheless improved cervical joint position sense acuity. This indicates that, against the common opinion, changes in proprioceptive input are not prerequisite to improve joint repositioning performance. However, the short-term applications of these cognitive treatments had no effect on pressure pain thresholds, whereas vibration reduced pressure pain thresholds. This implies different underlying mechanisms after vibration and mental training. CLINICAL REHABILITATION IMPACT: Mental interventions were effective in improving cervical joint position sense and are easy to integrate in rehabilitation regimes. Neck muscle vibration is effective in improving cervical joint position sense and pressure pain thresholds within 5 minutes of application.


Subject(s)
Cervical Vertebrae/physiopathology , Imagery, Psychotherapy , Neck Pain/psychology , Neck Pain/rehabilitation , Pain Management/methods , Physical Therapy Modalities , Vibration/therapeutic use , Double-Blind Method , Female , Germany , Humans , Male , Middle Aged , Neck Pain/physiopathology , Pain Threshold , Proprioception/physiology , Treatment Outcome
7.
Infection ; 42(3): 511-9, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24474624

ABSTRACT

PURPOSE: Early prosthetic joint infection (PJI) can be treated with an intensive surgical debridement and implant retention (DAIR) of the prosthesis if (1) the prosthesis is stable, (2) the pathogen is not a difficult-to-treat microorganism, (3) symptoms have lasted for <3 weeks and (4) a sinus tract is absent. METHODS: We retrospectively evaluated the treatment outcome of early PJI in the hip and knee in a single orthopaedic centre. An early PJI was defined as a prosthesis infection within 3 months after primary implantation or revision surgery for a non-infectious cause. RESULTS: We identified 69 patients with confirmed early PJI, with a median age of 71 (range 33-84) years. Only 64 % presented with ≥2 acute signs of infection. The most commonly isolated bacteria were coagulase-negative staphylococci (38 %) and Staphylococcus aureus (25 %). Surgical procedures included DAIR (50 cases, 69 %) and two-stage exchange (19 cases, 31 %). At last follow-up, five of remaining living 67 patients (7.5 %) had a relapse of infection. The overall relapse-free survival of the prosthesis after 2 years was 92.3 % (95 % confidence interval 82-97 %) with no significant difference between DAIR and exchange of prosthesis. CONCLUSION: Our data suggest that an early PJI should be treated with DAIR as a less invasive procedure whenever possible according to the established treatment algorithm.


Subject(s)
Anti-Bacterial Agents/therapeutic use , Arthroplasty, Replacement/adverse effects , Debridement , Hip Joint/pathology , Knee Joint/pathology , Prosthesis-Related Infections/epidemiology , Prosthesis-Related Infections/therapy , Adult , Aged , Aged, 80 and over , Bacterial Infections/epidemiology , Bacterial Infections/pathology , Bacterial Infections/therapy , Female , Humans , Male , Middle Aged , Prosthesis-Related Infections/pathology , Retrospective Studies , Treatment Outcome
9.
Knee Surg Sports Traumatol Arthrosc ; 19(6): 967-72, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21085931

ABSTRACT

PURPOSE: The aim was to compare the patellar kinematics in the normal knee, fixed-bearing (FB) and mobile-bearing total knee replacement (MB-TKR). The hypothesis that a mobile-bearing TKR has a more natural patellar movement was tested. METHODS: Computer navigation was used to track the patella in nine whole lower extremities in the natural knee and in the same knee with a posterior stabilised FB-TKR and a posterior stabilised MB-TKR from 0° to 90° flexion. The form and position of the trochlea in the natural knee and the patellar groove of the TKR femoral component was also analysed. RESULTS: There were no differences between the FB and MB-TKRs. But the patella in the TKRs at flexion angles of more than 50° had a more medial tilt compared to the natural knee. The patella of the natural knee tended to rotate externally with flexion, this was not seen in both TKR types. There were no significant differences in absolute mediolateral translation nor in translation relative to the patellar groove. During flexion, the patella lost contact with its groove earlier in the TKRs. The radius of the patellar groove of the femoral component was larger. The groove extended more superiorly and less far posteriorly, it was also positioned further laterally compared to the natural knee. CONCLUSIONS: There are subtle kinematic differences in patellar tracking between the natural knee and a TKR presumably due to differences in the shape and position of the patellar groove. There are no kinematic differences in patellar movement between the FB- and MB-TKR.


Subject(s)
Arthroplasty, Replacement, Knee/methods , Image Interpretation, Computer-Assisted , Knee Joint/surgery , Knee Prosthesis , Range of Motion, Articular/physiology , Biomechanical Phenomena , Cadaver , Female , Humans , Male , Prosthesis Design , Reference Values , Sensitivity and Specificity , Weight-Bearing
10.
J Viral Hepat ; 18(12): 852-60, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21050341

ABSTRACT

Signalling activated by Toll-like receptors (TLRs) can result in the production of tumour necrosis factor alpha (TNF-α) which is implicated in hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infection. No study has examined or compared hepatic expression of TLRs in both HCV and HCV/HIV. Liver and peripheral blood mononuclear cells (PBMCs) were obtained from HCV & HCV/HIV-infected patients and PBMCs from HIV-infected patients. Liver RNA was analysed by microarray and reverse transcription quantitative PCR (RT-qPCR). PBMCs were analysed by flow cytometry. Associations with hepatic histology and infection type were sought. Forty-six HCV, 20 HIV and 27 HCV/HIV-infected patients were recruited. Increasing Metavir inflammatory activity score was associated with increased hepatic TLR mRNA by RT-qPCR: TLR2 (P ≤ 0.001), TLR4 (P = 0.008) and TNF-α (P ≤ 0.001). A high degree of correlation was seen between hepatic mRNA expression of TNF-αvs TLR2 (r(2) = 0.66, P < 0.0001) and TLR4 (r(2) = 0.60, P < 0.0001). No differences in TLR gene or protein expression was observed between HCV, HCV/HIV- or HIV-infected groups. Hepatic TLR2, TLR4 and TNF-α mRNA are associated with hepatic inflammation in both HCV and HCV/HIV infection. High correlation between TNF-α and TLR2/TLR4 suggests a role for the innate immune response in TNF-α production. Activation of the innate immune response appears to be independent of infection type.


Subject(s)
HIV Infections/pathology , Hepatitis C/pathology , Inflammation/pathology , Liver/pathology , Toll-Like Receptor 2/biosynthesis , Toll-Like Receptor 4/biosynthesis , Tumor Necrosis Factor-alpha/metabolism , Adult , Coinfection/immunology , Coinfection/pathology , Female , Gene Expression Profiling , HIV Infections/immunology , Hepatitis C/immunology , Humans , Leukocytes, Mononuclear/immunology , Liver/immunology , Male , Middle Aged , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction
11.
Clin Oral Investig ; 13(2): 203-8, 2009 Jun.
Article in English | MEDLINE | ID: mdl-18853203

ABSTRACT

Professional recommendations for individual oral hygiene mostly include tooth brushing at least twice daily for 2-3 min with gentle force using the Bass technique or modifications of it. This study evaluated whether habitual tooth brushing actually meets these standards. Uninstructed adults (n = 103; mean age 31 +/- 6 years; 61 female, 42 male) with habitual manual tooth brushing were given a self-administered questionnaire about the frequency of brushing and a computer system recorded their brushing technique, duration and force. The majority (79.6%) of participants brushed twice daily. The mean brushing duration was 96.6 +/- 36.0 s, the mean brushing force was 2.3 +/- 0.7 N (max. 4.1 N), and no significant differences were found for quadrants. Most subjects (73.8%) brushed with circling, 8.7% with horizontal/scrubbing, 13.6% with horizontal/circling and 3.9% with vertical/sweeping movements. Modified Bass technique was not observed. When appropriate brushing habits were defined as brushing at least twice daily for 120 s with a brushing force of less than 3 N and with circling or vertical sweeping movements, only 25.2% of the participants fulfilled all criteria, emphasising the ongoing need for oral hygiene education.


Subject(s)
Toothbrushing/methods , Toothbrushing/standards , Adult , Dental Stress Analysis , Female , Health Education, Dental , Humans , Male , Oral Hygiene/education , Reproducibility of Results , Surveys and Questionnaires , Young Adult
12.
J Viral Hepat ; 15(12): 888-900, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18673429

ABSTRACT

Hepatitis B virus (HBV) infection is a major cause of liver-related morbidity and mortality. Toll-like receptors (TLRs) have recently been recognized to play an important role in the pathogenesis of chronic hepatitis B (CH-B). Furthermore, manipulation of TLR signalling pathways shows potential as an antiviral therapeutic strategy. Whether hepatocytes themselves possess intact TLR signalling pathways remains controversial. It is critical that cell culture models be developed to allow investigation of the interaction between HBV and the TLR signalling pathways. We have screened three hepatocyte cell lines for the integrity of pro-inflammatory responses and antiviral cytokines following stimulation with interleukin-1 (IL-1) and different TLR ligands. We observed that Huh-7, HepG2 and PH5CH8 cells selectively responded to IL-1 and TLR2 ligands, leading to the activation of NF-kappaB. In addition, the PH5CH8 cell lines were able to induce type 1 interferon (IFN) via both TLR3 and RIG-I following stimulation with poly I:C, HepG2 cells mounted an IFN response via RIG-I only, whereas Huh-7 cells were unresponsive. We conclude that the hepatocyte cell lines investigated display a repertoire of TLR signalling, albeit limited, suggesting that hepatocytes may themselves play an active role in innate immune responses to viruses such as HBV. Furthermore, particular hepatoma cell lines are suitable for investigating the interaction between HBV and hepatocyte-expressed pattern recognition receptors.


Subject(s)
Hepatocytes/immunology , Hepatocytes/metabolism , Immunity, Innate , Signal Transduction , Cell Line , Gene Expression , Humans , Interferons/genetics , Interferons/immunology , NF-kappa B/genetics , NF-kappa B/immunology , Toll-Like Receptors/genetics , Toll-Like Receptors/immunology
13.
Unfallchirurg ; 108(3): 239-40, 2005 Mar.
Article in German | MEDLINE | ID: mdl-15645199

ABSTRACT

Pertrochanteric femur fracture is rare in patients with lower leg amputation. Using supracondylar traction takes full advantage of the extension table. Intraoperative insertion of a Steinmann pin for traction is a well-known low-risk procedure. In our opinion, this kind of extension is a simple procedure that carries no risks for postoperative prosthetic management in patients with lower leg amputation.


Subject(s)
Amputees/rehabilitation , Bone Nails , Femoral Fractures/therapy , Fracture Fixation, Intramedullary/methods , Knee Joint/surgery , Traction/instrumentation , Traction/methods , Aged , Humans , Male , Treatment Outcome
14.
J Exp Med ; 194(11): 1549-59, 2001 Dec 03.
Article in English | MEDLINE | ID: mdl-11733570

ABSTRACT

The stroma of solid tumors is a complex network of different cell types. We analyzed stroma cell interactions in two tumor models during cyclophosphamide (Cy)-induced tumor rejection. In growing tumors, tumor infiltrating macrophages (TIMs) produced interleukin (IL)-10. Beginning 6 h after Cy-treatment T cells in the tumor were inactivated and TIMs switched to interferon (IFN)-gamma production. Both, IL-10 production before and IFN-gamma production after Cy-treatment by TIMs required T cells. With the same kinetics as TIMs started to produce IFN-gamma the tumor vasculature was destroyed which required IFN-gamma receptor expression on host but not tumor cells. These events preceded hemorrhagic necrosis and residual tumor cell elimination by T cells. Together, T cells regulate the function of TIMs and tumor rejection can be induced by disturbing the stroma network.


Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Cyclophosphamide/pharmacology , Fibrosarcoma/immunology , Plasmacytoma/immunology , Stromal Cells/drug effects , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Fibrosarcoma/drug therapy , Interferon-gamma/biosynthesis , Interferon-gamma/immunology , Interleukin-10/biosynthesis , Lymphocytes, Tumor-Infiltrating , Macrophages/immunology , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Nude , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/immunology , Neovascularization, Pathologic/immunology , Plasmacytoma/drug therapy , Receptors, Interferon/genetics , Receptors, Interferon/immunology , Stromal Cells/immunology , Time Factors , Interferon gamma Receptor
15.
J Exp Med ; 194(12): 1767-75, 2001 Dec 17.
Article in English | MEDLINE | ID: mdl-11748278

ABSTRACT

Activation of tumor-associated CD8(+) cytotoxic T lymphocytes (CTLs) often requires antigen representation, e.g., by dendritic cells (DCs), and CD4(+) T cell help. Previously, we showed that CTL-mediated tumor immunity required interleukin 4 (IL-4) during the immunization but not effector phase. To determine the source and target cells of IL-4, we performed adoptive T cell transfers using CD4(+) and CD8(+) T cells from IL-4(-/-) and IL-4R(-/-) mice and analyzed CTL generation. Even though necessary for CTL generation, CD4(+) T cells did not need to express IL-4 or IL-4R. Surprisingly, CTL generation required IL-4 but not IL-4R expression by CD8(+) T cells. As IL-4 (a) was expressed by naive CD8(+) T cells within 24 h after antigen encounter, (b) IL-4 induced DC maturation, and (c) CTL development was impaired in T cell-reconstituted IL-4R(-/-) mice, CD8(+) T cell-derived IL-4 appears to act on DCs. We conclude that CD4(+) and CD8(+) T cells provide different signals for DC activation during CTL generation.


Subject(s)
Carcinoma, Renal Cell/immunology , Cytotoxicity, Immunologic , Interleukin-4/immunology , Kidney Neoplasms/immunology , T-Lymphocytes, Cytotoxic/immunology , Adoptive Transfer , Animals , CD8 Antigens/immunology , Carcinoma, Renal Cell/pathology , Kidney Neoplasms/pathology , Mice , Mice, Inbred BALB C , Mice, SCID , Neoplasm Transplantation , Receptors, Interleukin-4/immunology
16.
J Virol ; 75(23): 11930-4, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11689679

ABSTRACT

Delivering attenuated lentivirus vaccines as proviral DNA would be simple and inexpensive. Inoculation of macaques with wild-type simian immunodeficiency virus strain mac239 (SIV(mac239)) DNA or SIV(mac239) DNA containing a single deletion in the 3' nef-long terminal repeat overlap region (nef/LTR) led to sustained SIV infections and AIDS. Injection of SIV(mac239) DNA containing identical deletions in both the 5' LTR and 3' nef/LTR resulted in attenuated SIV infections and substantial protection against subsequent mucosal SIV(mac251) challenge.


Subject(s)
Simian Immunodeficiency Virus/immunology , Vaccines, DNA/administration & dosage , Viral Vaccines/administration & dosage , Amino Acid Sequence , Animals , Antibodies, Viral/biosynthesis , Base Sequence , CD4 Lymphocyte Count , DNA Primers , Interferon-gamma/biosynthesis , Macaca nemestrina , Molecular Sequence Data , Mutagenesis , Plasmids , RNA, Viral/blood , Repetitive Sequences, Nucleic Acid , Vaccines, DNA/chemistry , Vaccines, DNA/immunology , Viral Vaccines/chemistry , Viral Vaccines/immunology
17.
Eur J Biochem ; 268(16): 4562-9, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11502218

ABSTRACT

The D1 protein constitutes one of the reaction center subunits of photosystem II and turns over rapidly due to photooxidative damage. Here, we studied the degradation of a truncated D1 protein. A plasmid with a precise deletion in the reading frame of the psbA gene encoding D1 was introduced into the chloroplast of Chlamydomonas reinhardtii. A homoplasmic mutant containing the desired gene was able to synthesize the truncated form of the polypeptide, but could not accumulate significant levels of it. As a consequence, other central photosystem II subunits did not assemble within the thylakoid membrane. In vivo pulse-chase experiments showed that the abnormal D1 protein is rapidly degraded in the light. Degradation was delayed in the light in the presence of an uncoupler, or when cells were incubated in the dark. Pulse-chase experiments performed in vitro indicate that an ATP and metal-dependent protease is responsible for the breakdown process. The paper describes the first in vivo and in vitro functional test for ATP-dependent degradation of a defect polypeptide in chloroplasts. The possible involvement of proteases similar to those removing abnormal proteins in prokaryotic organisms is discussed on the basis of proteases recently identified in chloroplasts.


Subject(s)
Adenosine Triphosphate/physiology , Chloroplasts/metabolism , Photosynthetic Reaction Center Complex Proteins/metabolism , Photosystem II Protein Complex , Plant Proteins/analysis , RNA, Plant/analysis , Thylakoids/chemistry
18.
Mol Microbiol ; 40(2): 498-507, 2001 Apr.
Article in English | MEDLINE | ID: mdl-11309131

ABSTRACT

We have developed a simple three-step method for transferring oriC mutations from plasmids to the Escherichia coli chromosome. Ten oriC mutations were used to replace the wild-type chromosomal origin of a recBCsbcB host by recombination. The mutations were subsequently transferred to a wild-type host by transduction. oriC mutants with a mutated DnaA box R1 were not obtained, suggesting that R1 is essential for chromosomal origin function. The other mutant strains showed the same growth rates, DNA contents and cell mass as wild-type cells. Mutations in the left half of oriC, in DnaA boxes M, R2 or R3 or in the Fis or IHF binding sites caused moderate asynchrony of the initiation of chromosome replication, as measured by flow cytometry. In mutants with a scrambled DnaA box R4 or with a modified distance between DnaA boxes R3 and R4, initiations were severely asynchronous. Except for oriC14 and oriC21, mutated oriCs could not, or could only poorly, support minichromosome replication, whereas most of them supported chromosome replication, showing that the classical definition of a minimal oriC is not valid for chromosome replication. We present evidence that the functionality of certain mutated oriCs is far better on the chromosome than on a minichromosome.


Subject(s)
Chromosomes, Bacterial , Escherichia coli/genetics , Escherichia coli/physiology , Mutation , Replication Origin/genetics , Blotting, Southern , DNA Replication , Flow Cytometry , Plasmids/genetics , Polymerase Chain Reaction , Recombination, Genetic , Replication Origin/physiology , Sequence Analysis, DNA , Transduction, Genetic
19.
J Biol Chem ; 276(30): 27864-72, 2001 Jul 27.
Article in English | MEDLINE | ID: mdl-11323423

ABSTRACT

Human mutations in the transcription factor SOX9 cause campomelic dysplasia/autosomal sex reversal. Here we identify and characterize two novel heterozygous mutations, F154L and A158T, that substitute conserved "hydrophobic core" amino acids of the high mobility group domain at positions thought to stabilize SOX9 conformation. Circular dichroism studies indicated that both mutations disrupt alpha-helicity within their high mobility group domain, whereas tertiary structure is essentially maintained as judged by fluorescence spectroscopy. In cultured cells, strictly nuclear localization was observed for wild type SOX9 and the F154L mutant; however, the A158T mutant showed a 2-fold reduction in nuclear import efficiency. Importin-beta was demonstrated to be the nuclear transport receptor recognized by SOX9, with both mutant proteins binding importin-beta with wild type affinity. Whereas DNA bending was unaffected, DNA binding was drastically reduced in both mutants (to 5% of wild type activity in F154L, 17% in A158T). Despite this large effect, transcriptional activation in cultured cells was only reduced to 26% in F154L and 62% in A158T of wild type activity, suggesting that a small loss of SOX9 transactivation activity could be sufficient to disrupt proper regulation of target genes during bone and testis formation. Thus, clinically relevant mutations of SOX9 affect protein structure leading to compound effects of reduced nuclear import and reduced DNA binding, the net effect being loss of transcriptional activation.


Subject(s)
Abnormalities, Multiple/genetics , Active Transport, Cell Nucleus/genetics , Bone and Bones/abnormalities , DNA/metabolism , Disorders of Sex Development , High Mobility Group Proteins/genetics , High Mobility Group Proteins/metabolism , Mutation , Point Mutation , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation , Adult , Amino Acid Sequence , Animals , COS Cells , Cell Nucleus/metabolism , Cells, Cultured , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Female , Genes, Dominant , Heterozygote , High Mobility Group Proteins/chemistry , Humans , Immunohistochemistry , Infant, Newborn , Karyopherins , Karyotyping , Kinetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Nuclear Proteins/metabolism , Phenotype , Polymorphism, Single-Stranded Conformational , Protein Binding , Protein Conformation , Protein Denaturation , Protein Structure, Tertiary , SOX9 Transcription Factor , Sequence Analysis, DNA , Spectrometry, Fluorescence , Structure-Activity Relationship , Temperature , Transcription Factors/chemistry , Transfection , Tryptophan/metabolism
20.
Arch Biochem Biophys ; 374(2): 229-40, 2000 Feb 15.
Article in English | MEDLINE | ID: mdl-10666302

ABSTRACT

A new DNA polymerase activity, distinct from DNA polymerase gamma, has been identified in bovine heart mitochondria. First detected among proteins isolated in a complex with mitochondrial DNA, the DNA polymerase activity has been partially purified 47,000-fold. Enzyme activity separates from DNA polymerase gamma on several chromatographic columns and appears to copurify with a 38 +/- 2-kDa polypeptide. Unlike DNA polymerase gamma, this enzyme is relatively resistant to inhibition by N-ethylmaleimide and dideoxynucleotides, has moderately low monovalent and high divalent cation requirements, and possesses 20-fold-higher apparent K(m) values for deoxynucleotides. The enzyme polymerizes deoxynucleotides onto a primed template DNA in a relatively nonprocessive fashion and lacks a detectable 3' to 5' exonuclease activity. Many of these characteristics resemble a beta-like mitochondrial DNA polymerase previously identified in, and considered unique to, trypanosomes. We propose that the bovine and trypanosomal enzymes are related and represent a new class of ubiquitous mitochondrial DNA polymerases.


Subject(s)
DNA Polymerase beta/metabolism , Mitochondria, Heart/enzymology , Animals , Cations, Divalent/pharmacology , Cations, Monovalent/pharmacology , Cattle , Chromatography, Affinity , Chromatography, Ion Exchange , DNA Polymerase beta/isolation & purification , DNA Polymerase gamma , DNA, Mitochondrial/isolation & purification , DNA, Mitochondrial/metabolism , DNA-Directed DNA Polymerase/isolation & purification , Enzyme Stability , Ethylmaleimide/pharmacology , Kinetics , Molecular Weight , Substrate Specificity , Templates, Genetic
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