RESUMEN
BACKGROUND.: It is inconclusive whether the perioperative administration of systemic lidocaine provides effective postoperative analgesia and enhances recovery in major orthopaedic surgery. We hypothesised that in adolescent and adult patients undergoing posterior spinal arthrodesis, a perioperative lidocaine infusion would reduce opioid requirements during the first 24 postoperative h. METHODS.: 70 patients undergoing posterior arthrodesis were enrolled in this prospective, randomised, double-blind, placebo-controlled clinical trial. Patients received total i.v. anaesthesia with propofol and remifentanil and were randomized to an adjuvant therapy with either lidocaine [i.v.-bolus injection of 1.5 mg kg -1 at induction of anaesthesia, followed by an infusion of 1.5 mg kg -1 h -1 which was continued until six h after arrival at the post-anaesthesia care unit] or placebo (equal volumes of saline). Postoperative pain was treated with patient-controlled i.v. morphine. Primary endpoints of this study were morphine requirements in the first postoperative 24 h. RESULTS.: Systemic lidocaine did not decrease morphine requirements in the first 24 postoperative h [lidocaine-group: 48 (23) mg (mean( sd )) vs placebo-group: 51(19) mg, P = 0.22]. Likewise, groups were not different with respect to the severity of postoperative pain, morphine consumption after 48 and 72 h, incidence of postoperative nausea and vomiting, perioperative inflammation, time to recovery of intestinal function, hospital length of stay, and quality of life (assessed preoperatively and one month postoperatively using the SF-12 physical and mental composite scores). CONCLUSIONS.: In our study, systemic lidocaine had no analgesic benefits in posterior arthrodesis when added to an opioid-based anaesthetic regimen. CLINICAL TRIAL REGISTRATION.: Eudra CT 2012-005264-98.
Asunto(s)
Analgésicos Opioides/uso terapéutico , Anestésicos Locales/uso terapéutico , Artrodesis/psicología , Lidocaína/uso terapéutico , Morfina/uso terapéutico , Dolor Postoperatorio/tratamiento farmacológico , Dolor Postoperatorio/prevención & control , Columna Vertebral/cirugía , Adolescente , Adulto , Analgesia Controlada por el Paciente , Anestésicos Locales/administración & dosificación , Anestésicos Locales/efectos adversos , Método Doble Ciego , Femenino , Humanos , Inyecciones Intravenosas , Tiempo de Internación , Lidocaína/administración & dosificación , Lidocaína/efectos adversos , Masculino , Persona de Mediana Edad , Resultados Negativos , Dolor Postoperatorio/psicología , Náusea y Vómito Posoperatorios/tratamiento farmacológico , Estudios Prospectivos , Calidad de Vida , Adulto JovenRESUMEN
The hormone leptin, which is thought to be primarily produced by adipose tissue, is a polypeptide that was initially characterized by its ability to regulate food intake and energy metabolism. Leptin appears to signal the status of body energy stores to the brain, resulting in the regulation of food intake and whole-body energy expenditure. Subsequently, it was recognized as a cytokine with a wide range of peripheral actions and is involved in the regulation of a number of physiological systems including reproduction. In the fed state, leptin circulates in the plasma in proportion to body adiposity in all species studied to date. However other factors such as sex, age, body mass index (BMI), sex steroids and pregnancy may also affect leptin levels in plasma. In pregnant mice and humans, the placenta is also a major site of leptin expression. Leptin circulates in biological fluids both as free protein and in a form that is bound to the soluble isoform of its receptor or other binding proteins such as one of the immunoglobulin superfamily members Siglec-6 (OB-BP1). Although the actions of leptin in the control of reproductive function are thought to be exerted mainly via the hypothalamic-pituitary-gonadal axis, there have also been reports of local direct effects of leptin at the peripheral level, however, these data appear contradictory. Therefore, there is a need to summarize the current status of research outcomes and analyze the possible reasons for differing results and thus provide researchers with new insight in designing experiments to investigate leptin effect on reproduction. Most importantly, our recent experimental data suggesting that reproductive performance is improved by decreasing concentrations of peripheral leptin was unexpected and cannot be explained by hypotheses drawn from the experiments of excessive exogenous leptin administration to normal animals or ob/ob mice.
Asunto(s)
Implantación del Embrión , Leptina/metabolismo , Reproducción , Transducción de Señal , Animales , Femenino , Fertilidad , Humanos , EmbarazoRESUMEN
Green fluorescent protein (GFP) is the most commonly used reporter of expression in cell biology despite evidence that it affects the cell physiology. The molecular mechanism of GFP-associated modifications has been largely unexplored. In this paper we investigated the proteome modifications following stable expression of GFP in breast cancer cells (MDA-MB-231). A combination of three different proteome analysis methods (2-DE, iTRAQ, label-free) was used to maximise proteome coverage. We found that GFP expression induces changes in expression of proteins that are associated with protein folding, cytoskeletal organisation and cellular immune response. In view of these findings, the use of GFP as a cell reporter should be carefully monitored.
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Artefactos , Neoplasias de la Mama/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteoma/metabolismo , Electroforesis en Gel Bidimensional , Femenino , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Células Tumorales CultivadasRESUMEN
Phosphatidylinositol (4,5) bisphosphate (PIP2) is an important signaling molecule located on the inner leaflet of the cell membrane. In order to perform its various signaling functions, it is suggested that PIP2 must be able to form localized clusters. In this study, we have used LAURDAN generalized polarization function (GP) with unlabeled PIP2 and single point fluorescence correlation spectroscopy and brightness analysis of various BODIPY labeled PIP2 to determine the presence of clusters in the membrane of giant unilamellar vesicles (GUVs) made of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin and cholesterol. We determined the number of freely diffusing fluorescent BODIPY molecules in the membrane and found that in GUVs containing various amounts of labeled PIP2, this number was significantly lower than in GUVs made with the control BODIPY labeled hexadecyl phosphatidylcholine (BODIPY-HPC). Also, we noted an increase in brightness of the labeled PIP2 particles with increasing labeled PIP2 molar fraction. Together with the observed change in LAURDAN GP with increasing molar fraction of unlabeled PIP2, these results demonstrate the presence of PIP2 enriched clusters that are smaller than the resolution limit of the fluorescent microscope. In addition, we report the presence of a hypsochromic shift of the fluorescence for the BODIPY labeled lipids that we attributed to clustering. This clustering result in a change in the partitioning of the lipids with the BODIPY labeled PIP2 lipids able to move between the liquid ordered and liquid disordered phase.
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Fosfatidilinositol 4,5-Difosfato/química , Liposomas Unilamelares/química , Compuestos de Boro/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/químicaRESUMEN
INTRODUCTION: The objective of this review is to give a state of affairs of meniscal transplantation, with the accent on preservation and surgical techniques. MATERIALS AND METHODS: All articles were selected by performing a search on the literature by using relevant keywords. The most relevant articles were selected with close attention to the publication date. RESULTS: When a meniscal tear is diagnosed, suture can be an option in the vascular zone, whereas the more frequently affected avascular zone heals poorly. A meniscectomy however is not without consequences, wherefore meniscal transplantation can be seen as a therapeutic option for pain reduction and improvement of function when the meniscus is lost. The meniscal scaffold, allograft and autograft can be currently withheld as possible grafts, where the meniscal scaffolds hold great promise as an alternative to the allograft. Various fixation techniques are therefore developed, where viable, deep frozen as well as cryopreservated allografts seem to give the most promising short term results. The transplantation can be performed using an open as well as an arthroscopic technique, using soft tissue fixation, bone plugs or blocks. De primacy of one technique can't be proven. In general meniscal transplantation can be considered as an acceptable procedure. DISCUSSION: Since the outcomes of different studies are difficult to compare, an attempt should be made to limit new studies to the comparison of one aspect. We can conclude that larger, more comparative randomised controlled long-term studies are necessary to resolve which techniques can give the best long-term results.
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Traumatismos de la Rodilla/cirugía , Meniscos Tibiales/trasplante , Terapias en Investigación , Tendón Calcáneo/trasplante , Artroscopía , Criopreservación , Humanos , Preservación de Órganos/métodos , Ligamento Rotuliano/trasplante , Músculo Cuádriceps , Tendones/trasplante , Lesiones de Menisco Tibial , Trasplante Autólogo , Trasplante HomólogoRESUMEN
Some species display intersex variation in their rate of meiotic recombination, where recombination is usually suppressed in the heterogametic sex. Although no heteromorphic sex chromosomes have been detected in zebrafish (Danio rerio), genetic analysis has indicated a lower frequency of recombination in males relative to females. Our study of the meiotic recombination pattern in female zebrafish indicates that adult females have only a few meiotic oocytes that are found in groups in the ventral zone of the ovarian surface. We used antibody staining of human mutL homolog 1 (MLH1) protein to mark the sites of putative chiasmata to seek a physical basis for the pattern of recombination and its relative frequency in both sexes. We report that MLH1 foci are found mostly in distal regions of the synaptonemal complexes (SCs) in males, but tend to be more evenly distributed in females. Our cytological analysis yields a ratio of MLH1 foci per chromosome in males versus females of 1:1.55. This lower level of recombination in males is in general agreement with previously published results from linkage map analysis. However, the similar ratio of MLH1 foci per unit length of SCs in both sexes demonstrates a correlation between SC length and the frequency of recombination rather than a mechanism that suppresses recombination in males. Thus, chiasma interference seems to provide similar expression in males and females in agreement with the situation in humans, where oocytes with longer SCs display a higher level of recombination that is not a consequence of more closely spaced crossovers along the SCs.
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Intercambio Genético , Pez Cebra/genética , Animales , Femenino , Ligamiento Genético , Humanos , Cariotipificación , Masculino , Meiosis/genética , Recombinación GenéticaRESUMEN
During mammalian meiosis, transcriptional silencing of the XY bivalent is a necessary event where defects may lead to infertility in males. While not well understood, the mechanism of meiotic gene silencing is believed to be RNA-dependent. In this study, we investigated the types and localization of non-coding RNAs in the meiotic nucleus of the male mouse using a microarray screen with different cell isolates as well as FISH. We report that the dense body, a component of the murine spermatocyte sex body similar to that of a dense body in Chinese hamster spermatocytes, is DNA-negative but rich in proteins and RNA including miRNAs (micro RNAs) and piRNAs (PIWI associated small RNAs), or their precursors. Selective miRNAs and piRNAs localize to chromosome cores, telomeres and the sex body of spermatocytes. These RNAs have not previously been detected in meiotic nuclei. These RNAs appear to associate with the nucleolus of the Sertoli cells as well as with the dense body. While in MIWI-null male mice the nucleolar signal from miRNA and piRNA probes in Sertoli cells is largely diminished, a differential regulation must exist in meiotic nuclei since the localization of these two components appears to be unaffected in the null animal.
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Núcleo Celular/metabolismo , Meiosis/fisiología , MicroARNs/metabolismo , ARN Interferente Pequeño/metabolismo , Animales , Núcleo Celular/ultraestructura , Cromatina/metabolismo , Perfilación de la Expresión Génica , Humanos , Masculino , Mamíferos , Ratones , MicroARNs/genética , Modelos Biológicos , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Interferente Pequeño/genética , Células de Sertoli/metabolismo , Células de Sertoli/ultraestructura , Cromosomas Sexuales/ultraestructura , Espermatocitos/metabolismo , Espermatocitos/ultraestructura , Espermatogénesis/genética , Espermatogénesis/fisiologíaRESUMEN
We describe a 15-year-old girl who had winging of the right scapula develop after incorrect use of a thoracolumbar orthosis. The girl was treated for idiopathic scoliosis, but after 2 years of bracing, progressive scapular winging and diminished range of motion in the right shoulder was observed. The girl reported that the superior part of the brace frequently hooked under the tip of the right scapula. This resulted in complete neuropathy of the dorsal scapular nerve. When using a thoracolumbar orthosis in the treatment of children with scoliosis, physicians must consider potential compressive injuries to the dorsal scapular nerve.
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Tirantes/efectos adversos , Mononeuropatías/etiología , Escápula/inervación , Escoliosis/terapia , Adolescente , Diseño de Equipo , Femenino , HumanosRESUMEN
Studies using gene-linkage analysis have suggested that abnormal recombination during meiosis may lead to the production of aneuploid gametes; however, there is little direct evidence of a link between the two in human males. We analysed spermatocytes in the pachytene stage from a man with extremely high aneuploidy rates in his sperm. Testicular tissue specimens of the infertile man and two vasectomy reversals were processed with immuofluorescent techniques to visualize synaptonemal complex and recombination foci and fluorescent in situ hybridization on spermatocytes and sperm with probes for chromosomes 13, 21, 18, X and Y. We observed no recombination between sex chromosomes in the infertile man, while in two controls, we observed recombination rates of 79.3 and 81.0% between the sex chromosomes. This was associated with a total sex aneuploidy rate of 41.61% in testicular sperm of the infertile man (0.44 and 0.62% in two controls). Recombination on chromosome 21 was reduced in the infertile man, with 10.62% of spermatocytes showing no recombination (0 and 1.67% in two controls), as well as chromosome 13, with 53.98% having < or =1 recombination foci (22.05 and 21.67% in two controls). This was associated with increased aneuploidy for those chromosomes. Chromosome 18 aneuploidy was slightly increased, although there was no apparent decrease in recombination. These results provide the first evidence of both recombination and non-disjunction abnormalities in the same individual. This is also the only reported case of an infertile man who shows no recombination between the sex chromosomes, despite the formation of the sex body.
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Aneuploidia , Infertilidad Masculina/genética , Recombinación Genética , Espermatozoides/ultraestructura , Aborto Espontáneo/genética , Adulto , Cromosomas Humanos Par 18/ultraestructura , Cromosomas Humanos Par 21/ultraestructura , Humanos , Hibridación Fluorescente in Situ , Masculino , Fase Paquiteno , Cromosomas Sexuales/genética , Cromosomas Sexuales/ultraestructura , Testículo/patologíaRESUMEN
OBJECTIVE: The objective of the present study is to improve the accuracy and reliability of the spinal midline reconstructions in scoliosis from back shape data. Design. An active shape model which covers the variety of scoliotic curves with a minimum of adjustable parameters is designed for the reconstruction of the spinal midline based on rasterstereographic back surface measurements. BACKGROUND: To reduce the number of X-rays needed for patients with a scoliosis, an automatic method was developed for the reconstruction of spinal deformities from back shape data. METHODS: 264 digital X-rays of 264 patients with scoliosis were used as a training set. By examining the statistics of the vertebral body centres a point distribution model was derived. The model is used as a basis to reconstruct the spinal midline from back shape data (active shape model). RESULTS: 478 rasterstereographs of 114 scoliotic patients have been evaluated with this new procedure and with an existing procedure. Both procedures deliver a three-dimensional curve of the spinal midline. The frontal projections of these spinal midlines are compared with the vertebral centres of the corresponding 478 X-rays. The active shape model improved the results as compared to the existing procedure from sigma(x)=3.4mm to sigma(x)=3.0mm. CONCLUSION: The use of the active shape model improves the overall accuracy of the spinal midline reconstructions in scoliosis from back shape data.RELEVANCE. Improvements to the accuracy and the reliability of the three-dimensional spinal reconstruction based on back shape data, can lead to an additional reduction of X-rays for patients with a scoliosis.
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Dorso/patología , Dorso/fisiopatología , Interpretación de Imagen Asistida por Computador/métodos , Imagenología Tridimensional/métodos , Modelos Biológicos , Fotogrametría/métodos , Escoliosis/diagnóstico , Escoliosis/fisiopatología , Inteligencia Artificial , Simulación por Computador , Humanos , Reconocimiento de Normas Patrones Automatizadas/métodos , Interpretación de Imagen Radiográfica Asistida por Computador/métodosRESUMEN
The targeted deletion of the meiotic chromosome core component MmSYCP3 results in chromosome synaptic failure at male meiotic prophase, extended meiotic chromosomes, male sterility, oocyte aneuploidy and absence of the MmSYCP2 chromosome core component. To test the functions of SYCP2 and SYCP3 proteins in the cores, we determined the effect of their deletion on homology recognition by whole chromosome painting and the effect on chromatin loop attachment to the cores with endogenous and exogenous sequences. Because we observed that the alignment of cores is between homologs, it suggested that alignment is not a function of the chromosome core components but might be mediated by chromatin-chromatin interactions. The alignment function therefore appears to be separate from intimate synapsis function of homologous cores that is observed to be defective in the SYCP3-/- males. To examine the functions of the SYCP2 and 3 core proteins in chromatin loop attachment, we measured the loop sizes of the centromeric major satellite chromatin and the organization of an exogenous transgene in SYCP3+/+ and SYCP3-/- males. We observed that these satellite chromatin loops have a normal appearance in SYCP3-/- males, but the loop regulation of a 2-Mb exogenous lambda phage insert appears to be altered. Normally the insert fails to attach to the core except by flanking endogenous sequences, but in the absence of SYCP2 and SYCP3, there appears to be multiple attachments to the core. This suggests that the selective preference for the attachment of mouse sequences to the chromosome core in the wild-type male is impaired in the SYCP3-/- male. Apparently the SYCP2 and SYCP3 proteins function in the specificity of chromatin attachment to the chromosome core.
Asunto(s)
Proteínas Cromosómicas no Histona/fisiología , Emparejamiento Cromosómico , Proteínas Nucleares/fisiología , Espermatocitos/citología , Animales , Bacteriófago lambda/genética , Proteínas de Ciclo Celular , Cromatina/fisiología , Proteínas Cromosómicas no Histona/genética , Pintura Cromosómica , ADN Satélite/química , Proteínas de Unión al ADN , Femenino , Eliminación de Gen , Masculino , Ratones , Ratones Transgénicos , Proteínas Nucleares/genética , Conformación de Ácido Nucleico , Alineación de SecuenciaRESUMEN
Infertile men have an increased frequency of aneuploid sperm. We have determined that decreased recombination is associated with the production of aneuploid sperm in humans. The aim of this study was to determine whether some cases of infertility are associated with decreased meiotic recombination. Analysis of the early stages of meiosis was performed in a 33-year-old man with non-obstructive azoospermia. Newly developed immunocytogenetic techniques were used to identify the synaptonemal complex (SC) in various stages of prophase. Antibodies to meiotic proteins identified the SC (SYN1/SCP3), the centromere (CREST) and recombination sites (MLH1). Only 36 meiotic spreads were recovered from the infertile man, compared with hundreds available from controls. One-third of the cells were in zygotene compared with 4% in controls, demonstrating an inability of bivalents to synapse and progress to pachytene. The infertile man had a greatly reduced frequency of recombination, with a mean of only 32.7 MLH1 foci/cell (range 1-60) compared with 46.0 (range 21-62) in control donors. A high proportion of cells (73%) contained at least one autosomal bivalent with zero MLH1 foci, compared with only 4.5% in control donors. Discontinuities in the SC were also more prevalent (68% of cells versus 26% in controls). This is the first demonstration of dramatic pachytene-stage abnormalities in an infertile man using these powerful new immunocytogenetic techniques.
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Meiosis , Oligospermia/genética , Oligospermia/fisiopatología , Adulto , Emparejamiento Cromosómico , Análisis Citogenético/métodos , Humanos , Masculino , ProfaseRESUMEN
We present our experience of treatment by physiotherapy, continuous passive motion and strapping in a series of 100 clubfeet classified on a scale of severity according to Dimeglio. Twenty-five percent were good after conservative treatment, and 75% required an operation. There were no recurrences or additional procedures. Before the introduction of the functional treatment in our department, 100% required some sort of surgical intervention and 51% required an additional procedure. In comparison with the results published by Dimeglio et al., the greatest discordance is observed in grade 2 clubfeet.
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Pie Equinovaro/terapia , Pie Equinovaro/cirugía , Femenino , Humanos , Lactante , Masculino , Terapia Pasiva Continua de Movimiento , Resultado del TratamientoRESUMEN
Spectral karyotyping (SKY) represents an effective tool to detect individual chromosomes and analyze major karyotype abnormalities within an entire genome. We have tested the feasibility of combining SKY and FISH/protein detection in order to combine SKY's unique abilities with specific loci detection. Our experimental results demonstrate that various combined protocols involving SKY, FISH and immunostaining work well when proper procedures are used. This combined approach allows the tracking of key genes or targeted chromosome regions while monitoring changes throughout the whole genome. It is particularly useful when simultaneously monitoring the behavior of both protein complexes and DNA loci within the genome. The details of this methodology are described and systematically tested in this communication.
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Cromosomas/metabolismo , Proteínas de Unión al ADN/metabolismo , Genoma , Inmunohistoquímica/métodos , Hibridación Fluorescente in Situ/métodos , Cariotipificación/métodos , Animales , Cromosomas/genética , ADN/genética , ADN/metabolismo , Sondas de ADN , Proteínas de Unión al ADN/análisis , Colorantes Fluorescentes , Genes , Humanos , Meiosis/genética , Ratones , Mapeo Físico de Cromosoma/métodos , Unión Proteica , Células Tumorales CultivadasRESUMEN
The behavior of meiotic chromosomes differs in several respects from that of their mitotic counterparts, resulting in the generation of genetically distinct haploid cells. This has been attributed in part to a meiosis-specific chromatin-associated protein structure, the synaptonemal complex. This complex consist of two parallel axial elements, each one associated with a pair of sister chromatids, and a transverse filament located between the synapsed homologous chromosomes. Recently, a different protein structure, the cohesin complex, was shown to be associated with meiotic chromosomes and to be required for chromosome segregation. To explore the functions of the two different protein structures, the synaptonemal complex and the cohesin complex, in mammalian male meiotic cells, we have analyzed how absence of the axial element affects early meiotic chromosome behavior. We find that the synaptonemal complex protein 3 (SCP3) is a main determinant of axial-element assembly and is required for attachment of this structure to meiotic chromosomes, whereas SCP2 helps shape the in vivo structure of the axial element. We also show that formation of a cohesin-containing chromosomal core in meiotic nuclei does not require SCP3 or SCP2. Our results also suggest that the cohesin core recruits recombination proteins and promotes synapsis between homologous chromosomes in the absence of an axial element. A model for early meiotic chromosome pairing and synapsis is proposed.
Asunto(s)
Meiosis/fisiología , Proteínas Nucleares/fisiología , Sinapsis/fisiología , Animales , Células COS , Proteínas de Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona , Cromosomas/fisiología , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas , Proteínas Recombinantes/genética , CohesinasRESUMEN
The spermatid nucleus and cytoplasm undergo dramatic morphological modifications during spermatid differentiation into mature sperm. Some of the external force causing this nuclear shaping is generated by a microtubular structure termed the manchette, which attaches to the perinuclear ring of the spermatid. Here, we report the isolation and characterization of a protein component of this perinuclear ring in an immunological screening of a mouse testis cDNA library. We termed this protein CLIP-50 because of its high similarity at the amino acid level to the C-terminal region of the microtubule-binding protein CLIP-170/restin. CLIP-50 lacks the characteristic microtubule-binding motif, but retains a portion of the predicted coiled-coiled domain and the metal-binding motif. The CLIP-50 transcript and protein are abundant in testis. The protein is also expressed in heart, lung, kidney, and skin. A distinct size variant exists in brain. In the spermatids, CLIP-50 protein localizes specifically to the centriolar region where the sperm tail originates and to the perinuclear ring from which the manchette emerges. CLIP-50 staining is retained in the ring throughout its migration over the surface of the nucleus which accompanies the nuclear shaping into its characteristic sperm configuration. This localization pattern indicates a very specific function for this novel CLIP derivative during mouse spermiogenesis.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Espermatogénesis , Espermatozoides/citología , Espermatozoides/metabolismo , Testículo/metabolismo , Animales , Western Blotting , Clonación Molecular , Perfilación de la Expresión Génica , Inmunohistoquímica , Proteínas de Filamentos Intermediarios/química , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Proteínas de Neoplasias/química , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/genética , Ratas , Espermátides/citología , Espermátides/metabolismo , Testículo/citologíaRESUMEN
The structural maintenance of chromosomes (SMC) protein encoded by the fission yeast rad18 gene is involved in several DNA repair processes and has an essential function in DNA replication and mitotic control. It has a heterodimeric partner SMC protein, Spr18, with which it forms the core of a multiprotein complex. We have now isolated the human orthologues of rad18 and spr18 and designated them hSMC6 and hSMC5. Both proteins are about 1100 amino acids in length and are 27-28% identical to their fission yeast orthologues, with much greater identity within their N- and C-terminal globular domains. The hSMC6 and hSMC5 proteins interact to form a tight complex analogous to the yeast Rad18/Spr18 heterodimer. In proliferating human cells the proteins are bound to both chromatin and the nucleoskeleton. In addition, we have detected a phosphorylated form of hSMC6 that localizes to interchromatin granule clusters. Both the total level of hSMC6 and its phosphorylated form remain constant through the cell cycle. Both hSMC5 and hSMC6 proteins are expressed at extremely high levels in the testis and associate with the sex chromosomes in the late stages of meiotic prophase, suggesting a possible role for these proteins in meiosis.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/química , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/química , Secuencia de Aminoácidos , Animales , Northern Blotting , Western Blotting , Proteínas de Ciclo Celular/metabolismo , División Celular , Línea Celular , Línea Celular Transformada , Núcleo Celular/metabolismo , Células Cultivadas , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Clonación Molecular , ADN Complementario/metabolismo , Proteínas de Unión al ADN/química , Dimerización , Fibroblastos/metabolismo , Humanos , Meiosis , Ratones , Microscopía Fluorescente , Datos de Secuencia Molecular , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Distribución Tisular , Ubiquitina-Proteína LigasasRESUMEN
The spectral karyotyping procedure of in situ hybridization with chromosome-specific probes assigns a unique colour code to each of the 21 mouse mitotic chromosomes. We have adapted this procedure to meiotic prophase chromosomes, and the results show that each of the pachytene or metaphase I bivalents can be identified. This technique has the potential to recognize synaptic anomalies and chromosome-specific structural and behavioural characteristics. We confirm these potentials by the recognition of the heterologous synapsis of the X and Y chromosomes and by the variances of synaptonemal complex lengths for each of the colour-coded bivalents in eight prophase nuclei.
Asunto(s)
Pintura Cromosómica/métodos , Cariotipificación/métodos , Meiosis/genética , Animales , Masculino , Ratones , Profase/genética , Testículo/citología , Cromosoma X/genética , Cromosoma Y/genéticaRESUMEN
SYCP3 localizes to the lateral elements of the synaptonemal complex and is essential for male meiosis. The genomic structure of SYCP3 consists of nine exons spanning approximately 14 kb. In mouse and rat, but not in hamster, the putative translation start of SYCP3 is present in the first exon. The putative promoter of SYCP3 was also cloned and shown to drive transcription of a reporter gene in somatic cells.