ABSTRACT
OBJECTIVE: to study the relationship between thalamic metabolism and neurological outcome in patients who had sustained a traumatic brain injury (TBI). METHODS: nineteen patients who had sustained a severe TBI and ten control subjects were included in this study. Six of the 19 patients had a low level of consciousness (vegetative state or minimally conscious state), while thirteen showed normal consciousness. All patients underwent a PET with 18F-FDG, 459.4 +/- 470.9 days after the TBI. The FDG-PET images were normalized in intensity, with a metabolic template being created from data derived from all subjects. The thalamic trace was generated automatically with a mask of the region of interest in order to evaluate its metabolism. A comparison between the two groups was carried out by a two sample voxel-based T-test, under the General Linear Model (GLM) framework. RESULTS: patients with low consciousness had lower thalamic metabolism (MNI-Talairach coordinates: 12, -24, 18; T = 4.1) than patients with adequate awareness (14, -28, 6; T = 5.5). Control subjects showed the greatest thalamic metabolism compared to both patients groups. These differences in metabolism were more pronounced in the internal regions of the thalamus. CONCLUSIONS: the applied method may be a useful ancillary tool to assess neurological outcomes after a TBI, since it permits an objective quantitative assessment of metabolic function for groups of subjects. Our results confirm the vulnerability of the thalamus to suffering the effects of the acceleration-deceleration forces generated during a TBI. It is hypothesized that patients with low thalamic metabolism represent a subset of subjects highly vulnerable to neurological and functional disability after TBI.
Subject(s)
Brain Injuries , Thalamus/metabolism , Adolescent , Adult , Brain Injuries/metabolism , Brain Injuries/pathology , Brain Injuries/physiopathology , Coma/metabolism , Female , Fluorodeoxyglucose F18/metabolism , Humans , Male , Persistent Vegetative State/metabolism , Positron-Emission Tomography , Radiopharmaceuticals/metabolism , Thalamus/pathology , Young AdultABSTRACT
Objetivos: Estudiar la relación entre el metabolismo talámico y la situación neurológica en pacientes que han sufrido un traumatismo craneoencefálico (TCE). Material y métodos: Se incluyó a 19 pacientes que habían sufrido un TCE grave y 10 sujetos control. De los 19 pacientes, 6 presentaban un grado de alerta bajo (estado vegetativo o estado de mínima conciencia), mientras que 13 mostraban un grado de alerta normal. A todos los pacientes se les realizó una tomografía con emisión de positrones (PET) con 18-fluorodesoxiglucosa (18F-FDG) 459,4 ± 470,9 días después del TCE. Las imágenes de PET-FDG se normalizaron en intensidad, creándose posteriormente una plantilla metabólica del grupo entre todos los sujetos. El trazado talámico se generó automáticamente con una máscara de la región de interés. Se comparó el metabolismo talámico de los dos grupos de pacientes respecto al grupo control, para ello se utilizó un método de análisis basado en vóxel, con significación estadística, p < 0,05 corregido para múltiples comparaciones. Resultados: Los pacientes con grado de alerta bajo mostraron menor metabolismo talámico (coordenadas MNI-Talairach, 12, -24, 18; T = 4,1), con respecto a los sujetos control, que los pacientes con grado de alerta adecuado (14, -28, 6; T = 5,5). Estas diferencias en el metabolismo fueron más acentuadas en las regiones internas del tálamo. Conclusiones: La PET-FDG puede ser una herramienta útil para valorar la situación neurológica después de un TCE. El método utilizado permite una evaluación objetiva y cuantitativa de imágenes de PET-FDG para grupos de sujetos. Nuestros resultados confirman la vulnerabilidad del tálamo a sufrir los efectos de las fuerzas de aceleración-desaceleración generadas durante un TCE (AU)
Objective: To study the relationship between thalamic metabolism and neurological outcome in patients who had sustained a traumatic brain injury (TBI). Methods: Nineteen patients who had sustained a severe TBI and ten control subjects were included in this study. Six of the 19 patients had a low level of consciousness (vegetative state or minimally conscious state), while thirteen showed normal consciousness. All patients underwent a PET with 18F-FDG, 459.4 ± 470.9 days after the TBI. The FDG-PET images were normalized in intensity, with a metabolic template being created from data derived from all subjects. The thalamic trace was generated automatically with a mask of the region of interest in order to evaluate its metabolism. A comparison between the two groups was carried out by a two sample voxel-based T-test, under the General Linear Model (GLM) framework. Results: Patients with low consciousness had lower thalamic metabolism (MNI-Talairach coordinates: 12, -24, 18; T = 4.1) than patients with adequate awareness (14, -28, 6; T = 5.5). Control subjects showed the greatest thalamic metabolism compared to both patients groups. These differences in metabolism were more pronounced in the internal regions of the thalamus. Conclusions: The applied method may be a useful ancillary tool to assess neurological outcomes after a TBI, since it permits an objective quantitative assessment of metabolic function for groups of subjects. Our results confirm the vulnerability of the thalamus to suffering the effects of the acceleration-deceleration forces generated during a TBI. It is hypothesized that patients with low thalamic metabolism represent a subset of subjects highly vulnerable to neurological and functional disability after TBI (AU)
Subject(s)
Humans , Thalamus/metabolism , Consciousness/classification , Craniocerebral Trauma/complications , Central Nervous System Diseases/epidemiology , Positron-Emission Tomography/methodsABSTRACT
Evidence for a key role of beta-amyloid (Abeta) in Alzheimer's disease has led to considerable interest in potential therapeutic strategies targeting enzymes involved in processing the amyloid precursor protein (APP). Beta-site APP Cleaving Enzyme (BACE or beta-secretase) is a membrane bound aspartyl protease that has been shown to be directly involved in Abeta production and, therefore, is at the forefront of therapeutic targets in the treatment of Alzheimer's disease. BACE-2, an enzyme closely related to BACE, regulates Abeta production in a manner antagonistic to BACE, suggesting that non-selective inhibition of BACE-2 by BACE inhibitors might impair the lowering of Abeta. The design of BACE inhibitors that do not inhibit BACE-2 would be enhanced by structural and kinetic studies, efforts that typically demand considerable amounts of both enzymes. A BACE-2 construct containing 19 residues of the BACE prosegment followed by the BACE-2 catalytic domain sequence, Asp36-Trp447, was produced in E. coli inclusion bodies (IB) at 110-140 mg/L cell culture. Exploration of a variety of refolding conditions resulted in an efficient method for refolding the resulting pro-BACE-2 construct, and this protein undergoes facile autocatalytic cleavage, optimal at pH 4, at the Leu40- downward arrow-Ala41 bond. Refolded BACE-2 was purified by anion exchange, molecular sieve, and affinity chromatographies, yielding 105 mg of homogeneous enzyme (kcat/ Km = 1.2 x 10(4) x M(-1) x sec(-1)) from 8 liters of E. coli cell culture.
Subject(s)
Amyloid Precursor Protein Secretases/chemistry , Aspartic Acid Endopeptidases/chemistry , Catalytic Domain/genetics , Amino Acid Sequence , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/metabolism , Chromatography, Affinity , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Inclusion Bodies/metabolism , Molecular Sequence Data , Protein Folding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Analysis, Protein , TemperatureABSTRACT
Suicidal attempts are relatively frequent and clinically relevant in patients with schizophrenia. Recent studies have found gray matter differences in suicidal and non-suicidal depressive patients. However, no previous neuroimaging study has investigated possible structural abnormalities associated to suicidal behaviors in patients with schizophrenia. A whole-brain magnetic resonance voxel-based morphometric examination was performed on 37 male patients meeting the DSM-IV criteria for schizophrenia. Thirteen (35.14%) patients had attempted suicide. A non-parametric permutation test was computed to perform the comparability between groups. An analysis of covariance (AnCova) model was constructed with a statistical threshold of p<0.05 corrected for multiple comparisons. After controlling for age and severity of illness, results showed significant gray matter density reduction in left superior temporal lobe (p=0.03) and left orbitofrontal cortex (p=0.04) in patients who had attempted suicide when comparing with non-suicidal patients. Although sample size limitations and potential clinical heterogeneity preclude definitive conclusions, these data point to structural differences in key cerebral areas. Neuroimaging studies are necessary to expand our knowledge of biological mechanisms underlying suicide in schizophrenia.
Subject(s)
Frontal Lobe/pathology , Functional Laterality , Schizophrenia/pathology , Schizophrenic Psychology , Suicide/psychology , Temporal Lobe/pathology , Adult , Age Factors , Female , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Psychiatric Status Rating Scales , Statistics, NonparametricABSTRACT
BACE (beta-site APP cleaving enzyme) or beta-secretase, the enzyme responsible for processing APP to give the N-terminal portion of the Abeta peptide, is a membrane bound aspartyl protease consisting of an ectodomain catalytic unit, a C-terminal transmembrane segment and a cytoplasmic domain. Three BACE constructs, pET11a-BACE, pQE80L-BACE, and pQE70-BACE were designed to terminate at a position just before the transmembrane domain (Ser(432)) and are described schematically below. (1) pET11a-T7.Tag-G-S-M-(A-8GV......QTDES(432)), (2) pQE80L-Met-R-G-S-(His)(6)-G-S-I-E-T-D-(T(1)QH...QTDES(432)), and (3) pQE70-Met-BACE (R(36)GSFVEMG....PQTDES(432) (His) (6)) Each construct was over-expressed in Escherichia coli as inclusion bodies. The inclusion body proteins were solubilized in urea and refolded by dilution in water to yield active enzyme. Maximal activity for pET11a-BACE and pQE80L-BACE was usually reached at day 3 to 4, while construct pQE70-BACE required about 21 days. Active BACE was purified to homogeneity by anion-exchange chromatography and affinity chromatography over a column of immobilized peptide inhibitor. The process, easily scalable to 60 liters of cell culture, yielded in excess of 400 mg of active enzyme for crystallographic analysis. Highly purified pET11a-BACE and pQE70-BACE formed complexes with various inhibitors, the latter protein giving crystals diffracting up to 1.45 A resolution. In addition, a crystal form that does not require the presence of an inhibitor has been obtained for pQE70-BACE. This ligand-free crystal form has proven useful for the preparation of BACE-inhibitor complexes in soaking experiments.
Subject(s)
Amyloid Precursor Protein Secretases/isolation & purification , Aspartic Acid Endopeptidases/isolation & purification , Protein Folding , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Amino Acid Sequence , Amyloid Precursor Protein Secretases/chemistry , Amyloid Precursor Protein Secretases/genetics , Amyloid Precursor Protein Secretases/metabolism , Animals , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/genetics , Aspartic Acid Endopeptidases/metabolism , CHO Cells , Cricetinae , Cricetulus , Crystallization , Escherichia coli/genetics , Escherichia coli/metabolism , HIV Protease/metabolism , Humans , Molecular Sequence Data , Molecular Structure , Protein Conformation , Protein Precursors/chemistry , Protein Precursors/genetics , Protein Precursors/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , X-Ray DiffractionABSTRACT
Introducción. Desde la llegada de la neuroimagen numerosos estudios han tratado de analizar las diferencias en la respuesta emocional frente a la no emocional. La mayoría de estos estudios utilizan la modalidad visual (caras) y parten de los datos en sujetos normales. En el presente estudio se presenta un nuevo paradigma para el estudio de la respuesta emocional basado en la modalidad auditiva y diseñado específicamente para el estudio de la psicosis. Método. Se analizaron las palabras más frecuentes que oían los pacientes psicóticos con alucinaciones auditivas, se clasificaron según cinco categorías y a partir de las mismas se diseñó un tren de 13 palabras emocionales, comparándose con 13 palabras con la misma complejidad sintáctica y con una valencia emocional neutral. Se aplicó este paradigma para ver la activación cerebral mediante resonancia magnética funcional (RMf) en 10 varones sanos y diestros. Resultados. En los análisis preliminares se observa una clara diferenciación según el estímulo sea emocional o no emocional, tanto en la intensidad de la activación (corteza temporal derecha e izquierda) como en la activación de áreas específicas (precentral y supramarginal derecha). Conclusiones. El paradigma presentado permite observar una diferenciación en la activación cerebral de la respuesta a estímulos auditivos emocionales y podría ser de utilidad en pacientes psicóticos
Introduction. Since the arrival of neuroimaging numerous studies have tried to analyze the differences between emotional and non-emotional response. The majority of these studies use visual approach (faces) and begin with data in normal subjects. The present study introduces a new paradigm for the study of emotional response based on auditory approach and designed specifically for the study of psychoses. Method. The most frequent words heard by psychotic patients with auditory hallucinations were analyzed. They were classified according to five categories which were compared with 13 other words with the same structure but with a neutral emotional valency. This paradigm was applied to see the cerebral activation with functional magnetic resonance imaging (fMRI) in 10 right handed healthy males. Results. In the preliminary analysis a clear differentiation is observed depending on the type of stimulus applied (emotional or non-emotional), both in the intensity of activation (right and left temporal cortex) as in the activation of specific areas (right precentral and supramarginal gyrus) only with the emotional stimulus. Conclusions. The present paradigm allows the observation of a differentiation in the cerebral activation to emotional auditory stimulus and could be of utility in the study of psychotic patients
Subject(s)
Humans , Affect , Hallucinations/psychology , Magnetic Resonance Imaging , Psychotic Disorders/psychology , Vocabulary , Telencephalon/anatomy & histology , Telencephalon/metabolism , Hallucinations/complications , Hallucinations/diagnosis , Psychotic Disorders/diagnosisABSTRACT
INTRODUCTION: Since the arrival of neuroimaging numerous studies have tried to analyze the differences between emotional and non-emotional response. The majority of these studies use visual approach (faces) and begin with data in normal subjects. The present study introduces a new paradigm for the study of emotional response based on auditory approach and designed specifically for the study of psychoses. METHOD: The most frequent words heard by psychotic patients with auditory hallucinations were analyzed. They were classified according to five categories which were compared with 13 other words with the same structure but with a neutral emotional valency. This paradigm was applied to see the cerebral activation with functional magnetic resonance imaging (fMRI) in 10 right handed healthy males. RESULTS: In the preliminary analysis a clear differentiation is observed depending on the type of stimulus applied (emotional or non-emotional), both in the intensity of activation (right and left temporal cortex) as in the activation of specific areas (right precentral and supramarginal gyrus) only with the emotional stimulus. CONCLUSIONS: The present paradigm allows the observation of a differentiation in the cerebral activation to emotional auditory stimulus and could be of utility in the study of psychotic patients.
Subject(s)
Affect , Brain/anatomy & histology , Brain/metabolism , Hallucinations/psychology , Magnetic Resonance Imaging , Psychotic Disorders/psychology , Vocabulary , Hallucinations/complications , Hallucinations/diagnosis , Humans , Psychotic Disorders/complications , Psychotic Disorders/diagnosisABSTRACT
BACKGROUND: The Multiple Sclerosis Functional Composite (MSFC) is a multidimensional clinical outcome measure that includes quantitative tests of leg function/ambulation (Timed 25-Foot Walk), arm function (9-Hole Peg Test), and cognitive function (Paced Auditory Serial Addition Test). The MSFC is the primary outcome measure in the ongoing multinational phase 3 trial of interferon beta-1a (Avonex) in patients with secondary progressive MS. OBJECTIVE: To assess the practice effects, reliability, and validity of the MSFC clinical outcome measure. DESIGN: Examining technicians underwent formal training using standardized materials. The MSFC was performed according to a standardized protocol. The 436 patients enrolled in the International Multiple Sclerosis Secondary Progressive Avonex Controlled Trial underwent 3 prebaseline MSFC testing sessions before randomization. RESULTS: Practice effects were evident initially for the MSFC but stabilized by the fourth administration. The Paced Auditory Serial Addition Test demonstrated the most prominent practice effects. The reliability of the MSFC was excellent, with an intraclass correlation coefficient for session 3 (final prebaseline session) vs session 4 (baseline) of 0.90. The MSFC at baseline correlated moderately strongly with the Kurtzke Expanded Disability Status Scale. Among the MSFC components, the Timed 25-Foot Walk correlated most closely. Correlations among the 3 MSFC components were weak, suggesting they assess distinct aspects of neurologic function in patients with MS. CONCLUSIONS: The MSFC demonstrated excellent intrarater reliability in this multinational phase 3 trial. Three prebaseline testing sessions were sufficient to compensate for practice effects. The pattern of correlations among the MSFC, its components, and the Kurtzke Expanded Disability Status Scale supported the validity of the MSFC.
Subject(s)
Health Personnel/education , Multiple Sclerosis/diagnosis , Neurologic Examination/methods , Outcome Assessment, Health Care , Sickness Impact Profile , Adult , Female , Humans , Male , Middle Aged , Multiple Sclerosis/classification , Quality of Life , Reproducibility of Results , Statistics, NonparametricABSTRACT
BACKGROUND: T1 hypointense lesions (T1 black holes) are focal areas of relatively severe CNS tissue damage detected by MRI in patients with MS. OBJECTIVE: To determine the natural history of T1 hypointense lesions in relapsing MS and the utility of T1 hypointense lesions as outcome measures in MS clinical trials. METHODS: MR studies were from the Multiple Sclerosis Collaborative Research Group trial. Longitudinal results are reported in 80 placebo- and 80 interferon beta-1a (IFNbeta-1a)-treated patients with mild to moderate disability relapsing-remitting MS. RESULTS: There was a small but significant correlation between T1 hypointense lesion volume and disability at baseline and on trial (r = 0.22, r = 0.28). In placebo patients there was a 29.2% increase in the mean volume of T1 hypointense lesions (median 124.5 mm3) over 2 years (p < 0.001 for change from baseline), as compared to an 11.8% increase (median 40 mm3) in the IFNbeta-1a-treated patients (change from baseline not significant). These treatment group comparisons did not quite reach significance. The most significant contributor to change in T1 hypointense lesions was the baseline number of enhancing lesions (model r2 = 0.554). Placebo patients with more active disease, defined by enhancing lesions at baseline, were the only group to show a significant increase in T1 hypointense lesion volume from baseline. CONCLUSION: The development of T1 hypointense lesions is strongly influenced by prior inflammatory disease activity, as indicated by enhancing lesions. These results suggest that treatment with once weekly IM IFNbeta-1a (30 mcg) slows the 2-year accumulation of these lesions in the brain.
Subject(s)
Adjuvants, Immunologic/therapeutic use , Brain/pathology , Interferon-beta/therapeutic use , Magnetic Resonance Imaging , Multiple Sclerosis, Relapsing-Remitting/diagnosis , Adjuvants, Immunologic/adverse effects , Adult , Brain/drug effects , Disease Progression , Female , Humans , Injections, Intramuscular , Interferon beta-1a , Interferon-beta/adverse effects , Longitudinal Studies , Male , Multiple Sclerosis, Relapsing-Remitting/drug therapyABSTRACT
The skeletal extracellular matrix produced by osteoblasts contains the glycoprotein fibronectin, which regulates the adhesion, differentiation and function of various adherent cells. Interactions with fibronectin are required for osteoblast differentiation in vitro, since fibronectin antagonists added to cultures of immature fetal calvarial osteoblasts inhibit their progressive differentiation. To determine if fibronectin plays a unique role in fully differentiated osteoblasts, cultures that had already formed mineralized nodules in vitro were treated with fibronectin antagonists. Fibronectin antibodies caused >95% of the cells in the mature cultures to display characteristic features of apoptosis (nuclear condensation, apoptotic body formation, DNA laddering) within 24 hours. Cells appeared to acquire sensitivity to fibronectin antibody-induced apoptosis as a consequence of differentiation, since antibodies failed to kill immature cells and the first cells killed were those associated with mature nodules. Intact plasma fibronectin, as well as fragments corresponding to the amino-terminal, cell-binding, and carboxy-terminal domains of fibronectin, independently induced apoptosis of mature (day-13), but not immature (day-4), osteoblasts. Finally, transforming growth factor-beta1 partially protected cells from the apoptotic effects of fibronectin antagonists. Thus, in the course of maturation cultured osteoblasts switch from depending on fibronectin for differentiation to depending on fibronectin for survival. These data suggest that fibronectin, together with transforming growth factor-beta1, may affect bone formation, in part by regulating the survival of osteoblasts.
Subject(s)
Apoptosis/physiology , Fibronectins/metabolism , Osteoblasts/cytology , Animals , Antibodies/isolation & purification , Antibodies/pharmacology , Apoptosis/drug effects , Cell Differentiation/physiology , Cells, Cultured , Cellular Senescence/physiology , Fibronectins/immunology , Immunoglobulin G/isolation & purification , Immunoglobulin G/pharmacology , Microscopy, Electron , Osteoblasts/chemistry , Osteoblasts/ultrastructure , Rats , Skull/cytology , Staphylococcal Protein A , Transforming Growth Factor beta/pharmacologyABSTRACT
Human cytomegalovirus protease (CMV PR) is a target for the development of antiviral therapeutics. To obtain large amounts of native protease, a 268-amino-acid polypeptide with a hexahistidinyl tag at the C terminus was expressed in Escherichia coli. The first 262 amino acids of the recombinant protein were identical to the amino acid sequence of native CMV PR, except for mutations introduced at the internal cleavage site to eliminate autoproteolysis at that site. The hexahistidinyl tag was placed downstream of amino acid 262 of the native CMV PR sequence. In this design, the Ala-Ser bond at amino acids 256-257 constitutes a site naturally cleaved by the protease during capsid maturation. The 268-amino-acid polypeptide with the (His)6 tag was expressed at high levels in E. coli as inclusion bodies. After solubilization of the inclusion bodies, the protease was purified to homogeneity by a single step using Ni2+ affinity chromatography. The protease was refolded to an active enzyme using dialysis which leads to effective autocleavage of the Ala-Ser bond at amino acids 256-257 to remove 12 amino acids including the (His)6 tag from the C terminus of the protein. This strategy yielded large amounts of highly purified CMV PR with the native N terminus and C terminus. Approximately 40 mg of purified CMV PR was obtained per liter of cell culture using this strategy. The enzymatic activity of CMV PR purified from inclusion bodies and refolded to an active enzyme was similar to the enzymatic activity of CMV PR expressed as a soluble protein in E. coli. In addition, the refolded CMV PR could be crystallized for X-ray diffraction.
Subject(s)
Cytomegalovirus/enzymology , Histidine , Serine Endopeptidases/isolation & purification , Amino Acid Sequence , Bacteriophage lambda/genetics , Catalysis , Crystallization , Cytomegalovirus/genetics , Escherichia coli/genetics , Genetic Vectors/genetics , Humans , Inclusion Bodies/enzymology , Molecular Sequence Data , Peptides/chemistry , Protein Folding , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Sequence Alignment , Serine Endopeptidases/biosynthesis , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Solubility , Substrate SpecificityABSTRACT
The secretion of fibronectin by differentiating osteoblasts and its accumulation at sites of osteogenesis suggest that fibronectin participates in bone formation. To test this directly, we determined whether fibronectin-cell interactions regulate progressive differentiation of cultured fetal rat calvarial osteoblasts. Spatial distributions of alpha 5 integrin subunit, fibronectin, osteopontin (bone sialoprotein I) and osteocalcin (bone Gla-protein) were similar in fetal rat calvaria and mineralized, bone-like nodules formed by cultured osteoblasts. Addition of anti-fibronectin antibodies to cultures at confluence reduced subsequent formation of nodules to less than 10% of control values, showing that fibronectin is required for normal nodule morphogenesis. Anti-fibronectin antibodies selectively inhibited steady-state expression of mRNA for genes associated with osteoblast differentiation; mRNA levels for alkaline phosphatase and osteocalcin were suppressed, whereas fibronectin, type I collagen and osteopontin were unaffected. To identify functionally relevant domains of fibronectin, we treated cells with soluble fibronectin fragments and peptides. Cell-binding fibronectin fragments (type III repeats 6-10) containing the Arg-Gly-Asp (RGD) sequence blocked both nodule initiation and maturation, whether or not they contained a functional synergy site. In contrast, addition of the RGD-containing peptide GRGDSPK alone did not inhibit nodule initiation, although it did block nodule maturation. Thus, in addition to the RGD sequence, other features of the large cell-binding fragments contribute to the full osteogenic effects of fibronectin. Nodule formation and osteoblast differentiation resumed after anti-fibronectin antibodies or GRGDSPK peptides were omitted from the media, showing that the inhibition was reversible and the treatments were not cytotoxic. Outside the central cell-binding domain, peptides from the IIICS region and antibodies to the N terminus did not inhibit nodule formation. We conclude that osteoblasts interact with the central cell-binding domain of endogenously produced fibronectin during early stages of differentiation, and that these interactions regulate both normal morphogenesis and gene expression.
Subject(s)
Fibronectins/metabolism , Osteoblasts/cytology , Osteoblasts/metabolism , Amino Acid Sequence , Animals , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Differentiation/physiology , Cells, Cultured , Extracellular Matrix/metabolism , Fibronectins/chemistry , Fibronectins/pharmacology , Gene Expression , Models, Biological , Molecular Sequence Data , Oligopeptides/chemistry , Oligopeptides/pharmacology , Osteoblasts/drug effects , Osteogenesis/physiology , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Rats , Skull/cytology , Skull/metabolismABSTRACT
The differentiaton of bone cells is a complex multistep process. Bone is somewhat unusual in that it is very actively and continually remodeled in the adult and that maintenance of its mass in the mature organism is exquisitely sensitive to mechanical as well as chemical signals. Bone is also unique because it consists of a very large amount of extracellular matrix (ECM) that is mineralized. The integrin family of ECM receptors has been shown to play an important role in tissue morphogenesis in several systems. Our studies on the regulation of matrix remodeling enzymes by integrins in rabbit synovial fibroblasts show that two b1 integrin fibronectin (FN) receptor complexes (alpha 5 beta 1 and alpha 4 beta 1) cooperate in detecting subtle changes in the composition of the ECM. As a result of signal transduction by these integrins, the levels of mRNA and protein for several members of the metalloproteinase family are regulated in these cells. We have also used antibody and RGD peptide perturbation studies to determine the significance of cell/ECM interactions to normal osteogenesis. We found that interactions between the cell binding domain of FN and integrins are required for both normal morphogenesis and gene expression in cultured osteoblasts that differentiate to form bone-like tissue in culture. These data lead us to propose that beta 1 integrins play an important role in osteoblast differentiation as well as in bone remodeling.
Subject(s)
Bone Remodeling/physiology , Connective Tissue/metabolism , Extracellular Matrix/physiology , Integrins/physiology , Osteoblasts/physiology , Alkaline Phosphatase/analysis , Alkaline Phosphatase/genetics , Amino Acid Sequence , Animals , Cell Differentiation/physiology , Cells, Cultured , Collagen/analysis , Collagen/physiology , Connective Tissue/physiology , Connective Tissue Cells , Extracellular Matrix/chemistry , Extracellular Matrix/metabolism , Gene Expression Regulation, Developmental , Integrins/analysis , Integrins/metabolism , Molecular Sequence Data , Morphogenesis/physiology , Osteoblasts/cytology , RNA, Messenger/analysis , RNA, Messenger/genetics , Rats , Receptors, Fibronectin/analysis , Receptors, Fibronectin/physiology , Signal Transduction/physiologySubject(s)
HIV Protease/chemistry , HIV Protease/metabolism , Point Mutation , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Amino Acid Sequence , Autolysis , Enzyme Stability , HIV Protease/biosynthesis , Humans , Kinetics , Molecular Sequence Data , Recombinant Proteins/biosynthesis , Substrate SpecificitySubject(s)
HIV Protease/chemistry , HIV Protease/metabolism , HIV-1/enzymology , Mutagenesis, Site-Directed , Amino Acid Sequence , Catalysis , Cloning, Molecular/methods , Enzyme Stability , Escherichia coli , HIV Protease/isolation & purification , Kinetics , Molecular Sequence Data , Oligopeptides/chemistry , Oligopeptides/metabolism , Peptide Fragments/chemistry , Peptide Fragments/isolation & purification , Point Mutation , Polymerase Chain Reaction , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Restriction Mapping , Substrate SpecificityABSTRACT
Site-directed mutagenesis of autolysis sites in the human immunodeficiency virus type 1 (HIV-1) protease was applied in an analysis of enzyme specificity; the protease served, therefore, as both enzyme and substrate in this study. Inspection of natural substrates of all retroviral proteases revealed the absence of beta-branched amino acids at the P1 site and of Lys anywhere from P2 through P2'. Accordingly, several mutants of the HIV-1 protease were engineered in which these excluded amino acids were substituted at their respective P positions at the three major sites of autolysis in the wild-type protease (Leu5-Trp6, Leu33-Glu34, and Leu63-Ile64), and the mutant enzymes were evaluated in terms of their resistance to autodegradation. All of the mutant HIV-1 proteases, expressed as inclusion bodies in Escherichia coli, were enzymatically active after refolding, and all showed greatly diminished rates of cleavage at the altered autolysis sites. Some, however, were not viable enzymatically because of poor physical characteristics. This was the case for mutants having Lys replacements of Glu residues at P2' and for another in which all three P1 leucines were replaced by Ile. However, one of the mutant proteases, Q7K/L33I/L63I, was highly resistant to autolysis, while retaining the physical properties, specificity, and susceptibility to inhibition of the wild-type enzyme. Q7K/L33I/L63I should find useful application as a stable surrogate of the HIV-1 protease. Overall, our results can be interpreted relative to a model in which the active HIV-1 protease dimer is in equilibrium with monomeric, disordered species which serve as the substrates for autolysis.
Subject(s)
HIV Protease/metabolism , HIV-1/enzymology , Protein Processing, Post-Translational , Amino Acid Sequence , HIV Protease/genetics , HIV Protease Inhibitors/pharmacology , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligopeptides/metabolism , Protein Conformation , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The protease encoded by the human immunodeficiency virus type 1 (HIV-1) was engineered in Escherichia coli as a construct in which the natural 99-residue polypeptide was preceded by an NH2-terminal methionine initiator. Inclusion bodies harboring the recombinant HIV-1 protease were dissolved in 50% acetic acid and the solution was subjected to gel filtration on a column of Sephadex G-75. The protein, eluted in the second of two peaks, migrated in SDS-PAGE as a single sharp band of M(r) approximately 10,000. The purified HIV-1 protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer at pH 5.5. This method of purification, which has also been applied to the purification of HIV-2 protease, provides a single-step procedure to produce 100 mg quantities of fully active enzyme.
