RESUMEN

Almost 60 years after the assassination of President John F. Kennedy in 1963, the majority of Americans are still reluctant to believe official reports presented by the commissions gathered in 1964 and again in 1976 that determined the direction of the shot resulting in the fatal head injury. Long-withheld, confidential government files released in 2017 reignited the controversy. The present investigation computationally simulated projectile-skull-impacts from the direction specified in official reports and from three other directions. Detailed geometric models of the human head and ammunition, as well as known parameters from the assassination site served as the supportive base for analysis. Constitutive mathematical models for the impact of projectile material with skull tissues at supersonic speed were employed to analyze bone and bullet fragmentation mechanics. Simulated fracture characteristics of bone and bullet were compared with photographic and X-ray evidence. The most likely origin of the fatal shot was determined based on the degree of corresponding deformation and fragmentation between simulation and documented evidence. Computational corroboration could be established as physically consistent with high-speed impact from the rear, as established by the official commissions. Simulations of three other speculative shot origins did not correspond with the documented evidence.

Asunto(s)

Traumatismos Craneocerebrales , Heridas por Arma de Fuego , Simulación por Computador , Balística Forense/métodos , Homicidio , Humanos , Cráneo

RESUMEN

Hypovitaminosis D has been identified as a common risk factor for fragility fractures and poor fracture healing. Epidemiological data on vitamin D deficiency have been gathered in various populations, but the association between vertebral fragility fractures and hypovitaminosis D, especially in males, remains unclear. The purpose of this study was to evaluate serum levels of 25-hydroxyvitamin D (25-OH D) in patients presenting with vertebral fragility fractures and to determine whether patients with a vertebral fracture were at greater risk of hypovitaminosis D than a control population. Furthermore, we studied the seasonal variations in the serum vitamin D levels of tested patients in order to clarify the relationship between other known risk factors for osteoporosis and vitamin D levels. We measured the serum 25-OH D levels of 246 patients admitted with vertebral fractures (105 men, 141 female, mean age 69 years, sd 8.5), and in 392 orthopaedic patients with back pain and no fractures (219 men, 173 female, mean age 63 years, sd 11) to evaluate the prevalence of vitamin D insufficiency. Statistical analysis found a significant difference in vitamin D levels between patients with vertebral fragility fracture and the control group (p = 0.036). In addition, there was a significant main effect of the tested variables: obesity (p < 0.001), nicotine abuse (p = 0.002) and diabetes mellitus (p < 0.001). No statistical difference was found between vitamin D levels and gender (p = 0.34). Vitamin D insufficiency was shown to be a risk factor for vertebral fragility fractures in both men and women.

Asunto(s)

Fracturas Espontáneas/epidemiología , Fracturas Osteoporóticas/epidemiología , Fracturas de la Columna Vertebral/epidemiología , Deficiencia de Vitamina D/epidemiología , Vitamina D/análogos & derivados , Distribución por Edad , Anciano , Anciano de 80 o más Años , Densidad Ósea , Estudios de Cohortes , Comorbilidad , Femenino , Estudios de Seguimiento , Fijación de Fractura/métodos , Fracturas Espontáneas/diagnóstico por imagen , Fracturas Espontáneas/terapia , Humanos , Masculino , Persona de Mediana Edad , Fracturas Osteoporóticas/diagnóstico por imagen , Fracturas Osteoporóticas/terapia , Prevalencia , Estudios Prospectivos , Radiografía , Medición de Riesgo , Distribución por Sexo , Fracturas de la Columna Vertebral/diagnóstico por imagen , Resultado del Tratamiento , Vitamina D/sangre , Deficiencia de Vitamina D/diagnóstico

RESUMEN

PURPOSE: To investigate the possibility of increasing elution of fosfomycin, gentamicin, clindamycin, and vancomycin by the addition of dextran fluid during the cement-mixing phase. METHODS: In 12 test series, we produced standardized, antibiotic-loaded test specimens of cement, with and without addition of dextran, and determined their effectiveness against three reference pathogens in agar diffusion and elution tests. RESULTS: In the test series using combined agents, Refobacin(®)-Palacos(®)R plus fosfomycin continuously produced the largest zone of inhibition, both against methicillin-sensitive Staphylococcus aureus (p = 0.009) and against methicillin-resistant Staphylococcus aureus (p = 0.009). The addition of dextran to the various test series had no useful effect on the size of the zone of inhibition for any of the antibiotics tested. CONCLUSIONS: Dextran supplementation in Refobacin(®)-Palacos(®)R bone cement did not have the hope for positive effect on the elution rate of bound antibiotics.

Asunto(s)

Resinas Acrílicas/farmacocinética , Antibacterianos/farmacocinética , Cementos para Huesos/farmacocinética , Dextranos/farmacocinética , Gentamicinas/farmacocinética , Metilmetacrilatos/farmacocinética , Resinas Acrílicas/farmacología , Antibacterianos/farmacología , Bacillus subtilis/efectos de los fármacos , Clindamicina/farmacología , Dextranos/farmacología , Difusión , Pruebas Antimicrobianas de Difusión por Disco , Fosfomicina/farmacología , Gentamicinas/farmacología , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Metilmetacrilatos/farmacología , Vancomicina/farmacología

RESUMEN

BACKGROUND: The study presented here investigated the short-term effectiveness of one-off lumbar caudal epidural injection (EI) in sciatica in relationship to the reported duration of pain. MATERIALS AND METHODS: This retrospective analysis involved 106 consecutive in-patients who received either conservative treatment (Group I) or an additional EI on the first day of their treatment (Group II). Both groups were divided according to the duration of symptoms at the time of admission (less than three months, or more than six months). Propensity score matching was performed for the whole collective and the resulting subgroups. This incorporated gender, age and pain intensity at the time of admission. The target parameter were changes on a visual analogue scale (VAS) of pain intensity on days D1, D3, and D10 depending on the respective treatment. A routine evaluation of the mental variables anxiety, depression and somatisation was performed as part of the examination upon admission and their relationship to the success of treatment was later assessed. RESULTS: The mean age of the patients was 61.7 (± 11.6) in Group I and 63.6 (± 13.6) in Group II. 59 % of the patients were female (n = 63). The Lasègue sign was prevalent in 45 % of Group I and 51 % of Group II. The intensity of pain on the day of admission was similar in both groups (7.0 ± 1.0 for Group I, 6.7 ± 1.8 for Group II). The length of stay on the ward was also similar in both groups (10.2 ± 3.9 and 9.4 ± 3.7 d, respectively). It was found that, independent of the duration of symptoms, injection treatment was significantly more effective than conservative treatment only in the early stages (D1 and D3, p < 0.001). No differences could be found in the expression of these mental variables between treatment groups, as these factors showed no influence on the results of therapy. CONCLUSIONS: In the context of acute treatment a once only lumbar caudal epidural injection represents at most a short-term effectiveness for the therapy of sciatica. The results presented here indicate that neither the duration of symptoms nor the measured psychometric variables show any effect on the success of therapy.

Asunto(s)

Analgésicos/administración & dosificación , Bupivacaína/administración & dosificación , Dimensión del Dolor , Modalidades de Fisioterapia , Ciática/terapia , Administración Oral , Anestésicos Locales/administración & dosificación , Femenino , Humanos , Inyecciones Epidurales/métodos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Resultado del Tratamiento

RESUMEN

INTRODUCTION: The present study investigated the incidence and risk factors of heterotopic ossification (HO) after implantation of knee prosthesis. MATERIALS AND METHODS: We undertook a retrospective cohort study in 434 cases (363 patients) treated with a total knee replant using a Press-Fit-Condylar (P.F.C.(®)Sigma(®)) prosthesis. The occurrence of HO in radiograph after a follow-up period of 11.2 ± 2.4 months was correlated in a regression model with a variety of influencing factors. RESULTS: 21 patients (4.8 %) developed heterotopic ossifications, all located in the area of the distal femur. The only risk factor found concerning the development of HO was osteoarthritis when compared to rheumatoid arthritis (OR = 4.07, 95 % CI 1.18-14.05; p = 0.0201) and postoperative wound healing problems (OR = 11.32, 95 % CI 3.26-39.33; p = 0.0001). Notching (OR = 2.22, 95 % CI 0.92-5.36; p = 0.0765) and osteophyte forming (hypertrophic) arthrosis (OR = 2.40, 95 % CI 0.97-5.95; p = 0.0596), however, were associated with the development of a bony spur in the contact area of the femoral component of the prosthesis. CONCLUSIONS: Our study has revealed that patients with rheumatoid arthritis are at lower risk of HO than patients with osteoarthritis. An impairment of wound healing would appear to promote the development of a HO. Notching and hypertrophic arthrosis are highly likely to be associated with the development of a bony spur in the ventral contact area of the prosthesis.

Asunto(s)

Artroplastia de Reemplazo de Rodilla/efectos adversos , Fémur , Prótesis de la Rodilla/efectos adversos , Osificación Heterotópica/etiología , Osteofito/etiología , Adulto , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/complicaciones , Artritis Reumatoide/cirugía , Femenino , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Osteoartritis de la Rodilla/complicaciones , Osteoartritis de la Rodilla/cirugía , Implantación de Prótesis/efectos adversos , Estudios Retrospectivos , Factores de Riesgo

RESUMEN

AIM: Lateral release is a substantial component of the distal soft tissue procedure, which plays a major role in the framework of the lapidus-fusion and the near-to-basis osteotomy in the hallux valgus surgery. The extent of release which is reached during this procedure is controversially discussed. The goal of this anatomic study was the evaluation of the effect of the sequential disconnection of lateral contraction structures on the alignment of the large toe. METHOD: On 8 anatomical hallux valgus preparations in which each foot was fastened in a holding device under defined continuous lateral pull, the soft tissue at the lateral aspect of the large toe was surgically split in the following sequential order: the lateral joint capsule, the tendon of the adductor hallucis muscle and afterwards the transverse metatarsal ligament. Before each procedure an X-ray was taken. We documented the different changes of the hallux valgus angle, the intermetatarsal 1/2 angle as well as the proximal and distal articulation angles. RESULTS: The hallux valgus angle was predominantly and significantly improved by capsule splitting and tenotomy of the tendon of the adductor hallucis muscle. A significant correction of the intermetatarsal angle did not take place however. CONCLUSION: It was shown that the substantial element of an effective distal soft tissue procedure for hallux valgus is the splitting of the capsule. The disconnection of the transverse metatarsal ligament did not lead to a further correction and can therefore be neglected.

Asunto(s)

Tejido Conectivo/patología , Tejido Conectivo/fisiopatología , Hallux Valgus/cirugía , Modelos Anatómicos , Modelos Biológicos , Dedos del Pie/patología , Dedos del Pie/fisiopatología , Humanos , Cápsula Articular/cirugía , Rango del Movimiento Articular , Dedos del Pie/cirugía

RESUMEN

Provision of sufficient post-operative pain therapy is an obligation in the clinical management of patients. A wide range of medical, technical and organizational options is used to improve post-operative pain management in orthopaedic surgery. Measurement of pain is as important as the correct use of analgesics and application techniques. Standardized pain therapy algorithms should facilitate autonomous treatment of patients. Additional procedures like patient-controlled analgesia or local catheter for pain are necessary for individualized or operation-specific pain therapy. The balanced combination in postoperative pain therapy could reduce side effects and complication rates, increase mobility and enhance patient satisfaction.

Asunto(s)

Analgésicos Opioides/uso terapéutico , Analgésicos/uso terapéutico , Procedimientos Ortopédicos , Dolor Postoperatorio/tratamiento farmacológico , Heridas y Lesiones/cirugía , Analgesia Epidural , Analgésicos/efectos adversos , Analgésicos Opioides/efectos adversos , Anestesia de Conducción , Humanos , Bombas de Infusión , Cuidados a Largo Plazo , Dimensión del Dolor

RESUMEN

Inadequate levels of all-trans-retinol in the blood cause retinal dysfunction; hence, genes implicated in retinal vitamin-A metabolism represent candidates for inherited retinal degenerations. In the current study, molecular genetic analysis of a consanguineous pedigree segregating for non-syndromic autosomal recessive retinitis pigmentosa (arRP) indicated that the affected siblings were homozygous by descent for a G4763A nucleotide substitution in RLBP1, the gene encoding cellular retinaldehyde-binding protein (CRALBP). This substitution is predicted to replace an arginine with glutamine at residue 150. CRALBP is not expressed in photoreceptors but is abundant in the retinal pigment epithelium (RPE) and Müller cells of the neuroretina, where it carries 11-cis-retinol and 11-cis-retinaldehyde. When expressed in bacteria, recombinant CRALBP (rCRALBP) containing the R150Q substitution was less soluble than wild-type rCRALBP. Mutant rCRALBP was purified from the soluble cell lysate and the protein structure was verified by mass spectrometry. The mutant protein lacked the ability to bind 11-cis-retinaldehyde. These findings suggest that arRP in the current pedigree results from a lack of functional CRALBP, presumably leading to disruption of retinal vitamin-A metabolism.

Asunto(s)

Proteínas Portadoras/genética , Mutación , Retinitis Pigmentosa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/química , Consanguinidad , Secuencia Conservada , Análisis Mutacional de ADN , Cartilla de ADN/genética , Femenino , Genes Recesivos , Humanos , Técnicas In Vitro , Masculino , Datos de Secuencia Molecular , Linaje , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Retinaldehído/metabolismo

RESUMEN

Follicle-stimulating hormone (FSH) is in the family of pituitary/placental glycoprotein hormones which also includes luteinizing hormone (LH), chorionic gonadotropin (hCG), and thyroid-stimulating hormone. These hormones are heterodimers composed of common alpha- and similar but unique beta-subunits. The 21 amino acid loop between Y33 and F53 of the FSH beta-subunit (L2 beta) can be switched into L2 beta of hCG beta without a loss of receptor binding, yet mutation of hFSH beta 37LVY39 to 37AAA39 was antecendent to a 20-fold reduction in receptor binding (based on ID50). A mutation in the LH beta gene, which causes Q54 to be R, causes hypogonadism. This residue is conserved in the glycoprotein hormones and corresponds to Q48 in hFSH beta. Mutation of hFSH beta 48QKTCT52 to 48AAACA52 resulted in a failure of heterodimer formation. In the current study single mutations were made to pinpoint which of the seven hFSH beta residues in the 37LVY39 to 37AAA39 and the 48QKTCT52 to 48AAACA52 mutants were responsible for the observed phenotypes. A single mutation of T52 to alanine was sufficient to cause a reduction in expression of heterodimeric hormone. Single mutants Q48A, T50A, V38A, Y39A, and, to a lesser extent, T52A formed heterodimer. However, these hFSH mutants were markedly unstable at pH 2.0. Thus, acid dissociation can be used to reveal metastable forms of this protein. Mutant hFSH beta Q48A was also 8-fold less active than wild-type hFSH when assayed for binding to hFSH receptors. hFSH beta V38A and Y39A mutants affected receptor binding; however, neither mutation alone caused greater than a 2-fold decrease in receptor binding activity. In summary, these results identify single important residues in the long loop (between Y33 and F53) of the hFSH beta-subunit which are required for proper subunit interactions that provide conformational stability which in turn is necessary for FSH-receptor interaction.

Asunto(s)

Hormona Folículo Estimulante/química , Hormona Folículo Estimulante/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Receptores de HFE/química , Receptores de HFE/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calorimetría , Línea Celular , Dicroismo Circular , ADN Complementario , Hormona Folículo Estimulante de Subunidad beta , Humanos , Cinética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos , Mutación Puntual , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Spodoptera , Termodinámica , Transfección

RESUMEN

Follicle stimulating hormone (FSH) is a gonadotropin and member of the pituitary/placental glycoprotein hormone family which bind to G-protein-coupled receptors. These hormones are heterodimers composed of a common alpha and distinct beta -subunits. Previous experimental evidence suggested that the FSH beta -subunit long loop comprised of amino acids Tyr33 to Phe53 is involved in receptor binding and activation and in subunit interaction. According to recently reported crystal structures of human chorionic gonadotropin (hCG), the homologous long loop of the beta -subunit of hCG associates with the alpha -subunit and is partially exposed to solvent. This report describes the results of scanning alanine mutagenesis used to determine if amino acid side chains in this region of the molecule are required for receptor binding and/or subunit contact. Five mutations were made which spanned this loop and the mutant FSH beta-subunits were co-expressed with alpha-subunit in a Baculovirus-infected insect-cell expression system. Mutation of 48QKTCT52 to 48AAACA52 produced a FSH beta-subunit that failed to form heterodimer, consistent with the crystal structure of hCG which shows these amino acids are buried at the subunit interface. The four remaining mutants produced heterodimer and were assayed for binding to and activation of human FSH receptors. Mutation of 37LVY39 to 37AAA39 caused a 20-fold reduction binding (ID50 of 7.0 nM compared with 0.3 nM for wildtype). Mutation of 34TRDL37 to 34AAAA37 or 44RPKI47 to 44APAA47 caused lesser but measurable effects with ID50 values of 1.1 nM and 1.9 nM, respectively. The (40)KDPA(43) to 40KDPA43 to 40AAPA43 mutation had little effect on receptor binding (ID50 = 0.5 nM).

Asunto(s)

Hormona Folículo Estimulante Humana , Hormona Folículo Estimulante de Subunidad beta , Hormona Folículo Estimulante/genética , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/genética , Alanina , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , ADN Complementario/genética , Hormona Folículo Estimulante/química , Humanos , Datos de Secuencia Molecular , Nucleopoliedrovirus/genética , Fragmentos de Péptidos/química , Ratas , Receptores de HFE/metabolismo , Transducción de Señal , Relación Estructura-Actividad

RESUMEN

Recent analyses of human FSH (hFSH) using antipeptide antibodies, monoclonal antibodies, and chimeric constructions of hCG/hFSH strongly suggest that the C-terminal region, including residues 81-100 of the hFSH beta-subunit, is involved in subunit association as well as hFSH heterodimer binding and/or activation of receptor. To test this hypothesis, site-directed mutagenesis was used to generate five triple alanine mutants of the C-terminal region of hFSH beta: Q81, H83, G85; K86, D88, S89; D90, S91, T92; D93, T95, V96; and R97, G98, L99. The baculovirus-infected insect cell system was used for expression. High Five cells were infected with virus harboring either delta hFSH beta complementary DNA (cDNA) or wild-type hFSH beta (hFSH beta wt) cDNA and coinfected with virus containing hFSH alpha cDNA. After infections, media were assayed for FSH using a heterodimer-specific enzyme-linked immunosorbent capture assay. All delta hFSH beta subunits formed heterodimers with hFSH alpha wt subunit and were secreted in the medium. These results suggest, for all five mutants, that side chains of amino acids substituted with alanine had no significant role in subunit association. The FSHs delta hFSH and hFSHwt were tested in a RRA, using cell lines that express the hFSH receptor, to determine if there were any changes in binding activity. Similarly, delta hFSH and hFSHwt were compared for receptor activation by measuring the levels of progesterone production in an in vitro FSH bioassay. delta hFSH-(93-96) exhibited minimal binding activity and no detectable steroidogenic activity. delta hFSH-(97-99) showed reduced binding affinity compared with that of hFSHwt, whereas the binding potency and bioactivity of the remaining delta hFSH were comparable to those of hFSHwt. These data demonstrate that within the hFSH beta-(81-99) region, FSH receptor-binding sites are contained within the sequence 93-99.

Asunto(s)

Aminoácidos/análisis , Hormona Folículo Estimulante/química , Hormona Folículo Estimulante/metabolismo , Receptores de HFE/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Hormona Folículo Estimulante/genética , Humanos , Insectos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Sondas de Oligonucleótidos/genética

RESUMEN

Residues Phe33 and Arg35, individually, and a composite mutation of residues Arg42, Ser43, and Lys44 were changed to alanine in the human glycoprotein hormone common alpha-subunit using site-directed mutagenesis. These specific residues are highly conserved across species and have by chemical modification and synthetic peptide approaches been implicated in the binding of human chorionic gonadotropin (hCG) to leutinizing hormone (LH) receptor. In the present study we tested the hypothesis that specific alpha-subunit amino acid residues which stabilize the hormone receptor interaction for hCG have the same function in human follicle-stimulating hormone (hFSH). Wild type or mutant alpha-subunit cDNAs were coexpressed with wild type hFSH or hCG beta cDNA in sialylation defective Chinese hamster ovary cells. Recombinant hormones were tested in a radioligand receptor competition assay, using rat testis membranes as a source of FSH and LH receptors. Mutant hFSH heterodimers F33A-FSH, R35A-FSH, Arg42-Ser43-Lys44/Ala42-Ala43-Ala44- FSH all displaced 125I-hFSH in a similar fashion, indicating that these residues are not important for binding of hFSH to the rat FSH receptor. On the other hand, F33A-CG evidenced a 5-fold decrease in binding, while R35A-CG had over a 100-fold decrease in binding to the rat LH receptor when compared to the wild type recombinant hCG. These data demonstrate that a receptor-binding site on the common alpha-subunit which is very important for hCG binding to LH receptor is not important for the binding of hFSH to FSH receptor. Our interpretation of these findings is that there are fundamental structural differences in the receptor interface contacts of the common alpha-subunit, which stabilize receptor binding among members of the glycoprotein hormone family.

Asunto(s)

Aminoácidos/genética , Hormonas Glicoproteicas de Subunidad alfa/genética , Mutagénesis Sitio-Dirigida , Secuencia de Aminoácidos , Animales , Células CHO , Gonadotropina Coriónica/metabolismo , Cricetinae , Hormona Folículo Estimulante/metabolismo , Hormonas Glicoproteicas de Subunidad alfa/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Ratas , Ratas Wistar , Receptores de HFE/metabolismo , Proteínas Recombinantes/genética

RESUMEN

Previous analyses of the topology of human follitropin (hFSH) with monoclonal antibodies and antipeptide antibodies have led to a current operating hypothesis that some amino acids within the hFSH beta 33-53 region are surface oriented, and others participate in subunit contact. Protein structural analysis predicts beta-turns within this region, and the immunochemical studies indicate that the ends may be involved in subunit contact. In this study, hFSH beta was mutagenized to change 34TRDL37 to 34AAAA37 or 48QKTCT52 to 48AAACA52, allowing us to study the ends of the hFSH beta 33-53 sequence contiguous with the hFSH beta sequence. Wild-type and mutant cDNAs were coexpressed with alpha-subunit cDNA in CHOPro-5 cells. Wild-type hFSH was secreted from cells cotransfected with wild-type hFSH alpha and hFSH beta cDNAs, as expected. However, heterodimeric hFSH was minimally detected in the medium from cells transfected with the 34TRDL37 mutant and was not detected in the case of the 48QKTCT52 mutant. Analysis of cell lysates (intracellular FSH) by immunoprecipitation and polyacrylamide gel electrophoresis showed that wild-type and mutant beta-subunits were indistinguishable and recoverable intact from each cell line. Additionally, analysis of lysates with a conformation-specific monoclonal antibody 3G3 revealed that similar levels of properly folded beta-subunit were produced in cells expressing wild-type or either mutated beta-subunit. These data indicate that the flanking amino acids of the hFSH beta 33-53 region, in particular 48QKTCT52, are critical for assembly of hFSH heterodimer.

Asunto(s)

Hormona Folículo Estimulante/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Hormona Folículo Estimulante/química , Hormona Folículo Estimulante/genética , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Sondas de Oligonucleótidos/genética , Pliegue de Proteína , Proteínas Recombinantes

RESUMEN

Foci, nodules of cellular overgrowth, that appear after confluence are an in vitro characteristic of malignant transformation. A well-studied in vitro model of estrogen-dependent tumors is the MCF-7 cell line, derived from a pleural metastasis of a human breast adenocarcinoma. We report that cultivation of MCF-7 cells, using routine methods, results in extensive estrogen-stimulated postconfluent cell accumulation characterized by discrete three-dimensional arrays. Side view Nomarski optical sections revealed these to be principally multicellular foci with occasional domes and pseudoacinar vacuoles. This effect on MCF-7 cell growth occurs in media containing fetal bovine serum but not with calf serum or charcoal-dextran-treated fetal bovine serum unless supplemented with estrogens. Foci formation starts 5-6 days after confluence, and the number of foci generated is a function of the concentration of added estrogens. Foci formation is suppressed by the antiestrogens Tamoxifen and LY 156758. Addition of progesterone, testosterone, or dexamethasone had little or no effect, while various estrogens (ethinyl estradiol, diethylstilbestrol, and moxestrol) induced foci development. Clones derived from single cells of the initial MCF-7 population revealed a wide variance in estrogen-induced foci formation, demonstrating heterogeneity of this tumor cell line. The postconfluent cell growth of the estrogen receptor-deficient cell line, MDA-MB-231, contrasted with MCF-7 by developing an extensive multilayer morphology devoid of discrete structures. The tumorigenic potential of the MCF-7 cells used in our experiments was confirmed by their estrogen-dependent growth in immunosuppressed male BDF1 mice. These data suggest an estrogen receptor-based mechanism for the development of multicellular foci during postconfluent growth of MCF-7 cells. After confluence, foci, in contrast to the quiescent surrounding monolayer, retain proliferating cells. Focus formation, therefore, reflects the heterogeneous responsiveness of these cells to estrogens and should provide a model permitting in vitro comparisons between the progenitor cells of multicellular foci and the monolayer population.

Asunto(s)

Adenocarcinoma/patología , Neoplasias de la Mama/patología , Estradiol/farmacología , Estrógenos/farmacología , Neoplasias Hormono-Dependientes/patología , Animales , Bovinos , División Celular , Inhibición de Contacto/efectos de los fármacos , Medios de Cultivo , Humanos , Masculino , Ratones , Ratones Endogámicos , Trasplante de Neoplasias , Fenotipo , Piperidinas/farmacología , Clorhidrato de Raloxifeno , Receptores de Estrógenos/fisiología , Tamoxifeno/farmacología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/patología

RESUMEN

Cells cultured on thin plastic (e.g. Formvar, Teflon, polycarbonate) membranes can be clearly imaged from the side in vivo by video microscopy. We have used this flexible-substratum technique to examine the behaviour and properties of primary cilia in confluent cultures of the kidney epithelial cell lines PtK1, PtK2, LLC-PK1, MDCK and BSC-40. In these cells primary cilia appear as rigid rods, up to 55 micron long, which project at various angles from the dorsal cell surface. The length distribution of primary cilia in confluent cultures is a distinct characteristic of each established kidney cell line examined, with LLC-PK1 exhibiting three distinct length populations. Primary cilia of kidney cell lines bend passively in response to flow but do not display propagated bending or vortical motions. Up to 26% of the cilia in the cell types examined possess one or more conspicuous swellings along the ciliary shaft. Treatment with 0.05% trypsin, which is sufficient to cause cell rounding, does not induce the resorption or shedding of the cilium. These direct observations demonstrate that kidney epithelial-cell primary cilia are non-motile and longer than previously thought, and suggest that their length represents a phenotypic marker for each cell line.

Asunto(s)

Riñón/citología , Animales , Células Cultivadas , Cilios/fisiología , Técnicas Citológicas , Riñón/efectos de los fármacos , Tripsina/farmacología