RESUMEN
Vascular endothelial cells (ECs) play a central role in the pathophysiology of many diseases. The use of targeted nanoparticles (NPs) to deliver therapeutics to ECs could dramatically improve efficacy by providing elevated and sustained intracellular drug levels. However, achieving sufficient levels of NP targeting in human settings remains elusive. Here, we overcome this barrier by engineering a monobody adapter that presents antibodies on the NP surface in a manner that fully preserves their antigen-binding function. This system improves targeting efficacy in cultured ECs under flow by >1000-fold over conventional antibody immobilization using amine coupling and enables robust delivery of NPs to the ECs of human kidneys undergoing ex vivo perfusion, a clinical setting used for organ transplant. Our monobody adapter also enables a simple plug-and-play capacity that facilitates the evaluation of a diverse array of targeted NPs. This technology has the potential to simplify and possibly accelerate both the development and clinical translation of EC-targeted nanomedicines.
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Células Endoteliales , Nanopartículas , Aminas , Anticuerpos , Sistemas de Liberación de Medicamentos , Humanos , Nanomedicina , OligonucleótidosRESUMEN
The stormwater runoff carries different pollutants that can reduce the quality of receiving waters due to diffuse pollutant loads. This research was aimed at evaluating the concentration of pollutants in stormwater and the application of SWMM (Storm Water Management Model) to an urban catchment in Lake Paranoá watershed to carry out the simulation of flow discharge with the hydraulic model, and subsequently to estimate the loads conveyed to the lake in ordinary events of precipitation. This study was carried out based on rainfall and runoff monitoring during events. It was confirmed that this model's results fit well in simulation of this type of watershed, leading to high value of the Nash-Sutcliffe coefficient after calibration but, as expected, precipitation distribution is a very important factor for calibration. Concerning water quality, it was observed that the event mean concentration values of suspended solids and chemical oxygen demand were high, indicating that the diffuse pollution is an important source of pollution of the receiving waters. The monitoring and modelling of stormwater are essential to identify diffuse pollution discharge, in searching for a sustainable solution.
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Monitoreo del Ambiente , Contaminación del Agua/análisis , Análisis de la Demanda Biológica de Oxígeno , Brasil , Lluvia , Calidad del AguaRESUMEN
Somatic inactivating mutations in epigenetic regulators are frequently found in combination in myelodysplastic syndrome (MDS). However, the mechanisms by which combinatory mutations in epigenetic regulators promote the development of MDS remain unknown. Here we performed epigenomic profiling of hematopoietic progenitors in MDS mice hypomorphic for Tet2 following the loss of the polycomb-group gene Ezh2 (Tet2KD/KDEzh2Δ/Δ). Aberrant DNA methylation propagated in a sequential manner from a Tet2-insufficient state to advanced MDS with deletion of Ezh2. Hyper-differentially methylated regions (hyper-DMRs) in Tet2KD/KDEzh2Δ/Δ MDS hematopoietic stem/progenitor cells were largely distinct from those in each single mutant and correlated with transcriptional repression. Although Tet2 hypomorph was responsible for enhancer hypermethylation, the loss of Ezh2 induced hyper-DMRs that were enriched for CpG islands of polycomb targets. Notably, Ezh2 targets largely lost the H3K27me3 mark while acquiring a significantly higher level of DNA methylation than Ezh1 targets that retained the mark. These findings indicate that Ezh2 targets are the major targets of the epigenetic switch in MDS with Ezh2 insufficiency. Our results provide a detailed trail for the epigenetic drift in a well-defined MDS model and demonstrate that the combined dysfunction of epigenetic regulators cooperatively remodels the epigenome in the pathogenesis of MDS.
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Proteínas de Unión al ADN/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Epigénesis Genética , Regulación de la Expresión Génica , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Islas de CpG , Metilación de ADN , Proteínas de Unión al ADN/genética , Dioxigenasas , Modelos Animales de Enfermedad , Elementos de Facilitación Genéticos , Proteína Potenciadora del Homólogo Zeste 2/genética , Hematopoyesis/genética , Ratones , Ratones Noqueados , Ratones Transgénicos , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Motivos de Nucleótidos , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: The waiting time for deceased-donor kidney-only transplantations in Japan is long. Herein, we assessed the effect of length of dialysis on the outcomes of these patients. METHODS: We divided patients into 2 groups based on length of dialysis (Group A, <15 years, and Group B, ≥15 years), and compared the background and outcomes after kidney transplantation. RESULTS: Group A included 210 patients and Group B included 35 patients. In Group B, 20% of transplants were from living donors. Patient age (P = .017) and the hepatitis C infection rate (P = .018) were significantly higher in Group B, whereas hypertension (P = .011), diabetes (P = .041), and ABO-incompatibility rates (P = .015) were significantly higher in Group A. The 5- and 10-year survival rates were 97.0% and 95.4%, respectively, in Group A and 97.1% and 97.1%, respectively, in Group B. The 5- and 10-year graft survival rates were 95.4% and 84.8%, respectively, in Group A and 97.1% and 73.1%, respectively, in Group B. There were no significant differences between the groups in patient survival (P = .74) and graft survival (P = .72). The 5- and 10-year cardiovascular event-free survival rates were 95.9% and 92.4%, respectively, in Group A and 88.6% and 76.8%, respectively, in Group B. Cardiovascular event-free survival was significantly higher in Group A (P = .038). Cox stepwise multivariate analysis indicated that length of dialysis was a significant predictor of cardiovascular events (hazard risk, 1.007; range, 1.001-1.012; P = .012). CONCLUSION: The prognosis after kidney transplantation is promising even after a long length of dialysis, although evaluation of the cardiovascular risk is needed in these cases.
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Enfermedades Cardiovasculares/etiología , Fallo Renal Crónico/terapia , Trasplante de Riñón/mortalidad , Diálisis Renal/efectos adversos , Factores de Tiempo , Adulto , Incompatibilidad de Grupos Sanguíneos , Supervivencia sin Enfermedad , Femenino , Supervivencia de Injerto , Humanos , Japón , Trasplante de Riñón/métodos , Donadores Vivos , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Factores de Riesgo , Tasa de Supervivencia , Listas de EsperaRESUMEN
BACKGROUND: Barbed sutures have unidirectional circumferential shallow barbs, which distribute tension throughout the wound and close wound securely without the need to tie knots. OBJECTIVES: We compare two different methods of wound closure in elective plastic surgical cases: barbed 3/0 V-Loc™180 suture and smooth 3/0 Maxon™ sutures, both polyglyconate monofilament synthetic absorbable sutures. We assessed the aesthetic long-term results with a minimum two year follow up. METHODS: This is a prospective, randomized controlled study with internal control. A single surgeon performed all cases. Patients who underwent elective operations that involved long wound closure were enrolled in the study. Each patient acted as their own internal control with half their wound being sutured with 3/0 V-Loc™180 barbed suture and the other half with smooth 3/0 Maxon™ deep dermal sutures and then a subcuticular skin closure. In both groups, the superficial fascial system was closed with 1 Vicryl interrupted sutures on both sides. Long-term cosmesis was evaluated using the modified Hollander cosmesis score by review of standardized postoperative photographs by 9 blinded plastic surgeons and specialist registrars. RESULTS: The study reports on 33 female patients. The time taken for wound closure was significantly reduced using the barbed suture (p < 0.001). There was no difference in the complication ratio in either group. Two-year aesthetic outcome was significantly superior when using the barbed suture (p = 0.0075). CONCLUSION: Barbed sutures closure of long wounds is faster and produces a better long-term aesthetic outcome than smooth sutures.
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Cicatriz/prevención & control , Cirugía Plástica/instrumentación , Suturas/clasificación , Implantes Absorbibles , Adulto , Cicatriz/etiología , Procedimientos Quirúrgicos Electivos/métodos , Diseño de Equipo , Estética , Femenino , Estudios de Seguimiento , Humanos , Mamoplastia/instrumentación , Mamoplastia/métodos , Persona de Mediana Edad , Estudios Prospectivos , Método Simple Ciego , Trasplante de Piel/efectos adversos , Cirugía Plástica/métodos , Colgajos Quirúrgicos , Resultado del TratamientoRESUMEN
This paper considers moments due to friction forces on the human fingertip. A computational technique called the friction moment arc method is presented. The method computes the static and/or dynamic friction moment independent of a friction force calculation. In addition, a new finger holder to display friction moment is presented. This device incorporates a small brushless motor and disk, and connects the human's finger to an interface finger of the five-fingered haptic interface robot HIRO II. Subjects' perception of friction moment while wearing the finger holder, as well as perceptions during object manipulation in a virtual reality environment, were evaluated experimentally.
RESUMEN
BACKGROUND: Apoptosis is important for regulating spermatogenesis. The protein mRHBDD1 (mouse homolog of human RHBDD1)/rRHBDD1 (rat homolog of human RHBDD1) is highly expressed in the testis and is involved in apoptosis of spermatogonia. GC-1, a spermatogonia cell line, has the capacity to differentiate into spermatids within the seminiferous tubules. We constructed mRHBDD1 knockdown GC-1 cells and evaluated their capacity to differentiate into spermatids in mouse seminiferous tubules. RESULTS: Stable mRHBDD1 knockdown GC-1 cells were sensitive to apoptotic stimuli, PS341 and UV irradiation. In vitro, they survived and proliferated normally. However, they lost the ability to survive and differentiate in mouse seminiferous tubules. CONCLUSION: Our findings suggest that mRHBDD1 may be associated with mammalian spermatogenesis.
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Receptores ErbB/fisiología , Túbulos Seminíferos/fisiología , Espermatogénesis/fisiología , Espermatogonias/citología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Western Blotting , Ácidos Borónicos/farmacología , Bortezomib , Línea Celular , Proliferación Celular , Receptores ErbB/genética , Receptores ErbB/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos BALB C , Pirazinas/farmacología , ARN Interferente Pequeño/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Regeneración , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Túbulos Seminíferos/citología , Túbulos Seminíferos/cirugía , Espermatogénesis/genética , Espermatogonias/metabolismo , Espermatogonias/trasplante , Testículo/citología , Testículo/metabolismo , Transfección , Rayos UltravioletaRESUMEN
OBJECTIVE: To examine the relationship between an antibody against GAPDH-2, a sperm-specific protein, and infertility of female mice. DESIGN: Basic research. SETTING: National Research Institute for Family Planning Beijing, World Health Organization Collaboration Center of Human Reproduction. ANIMAL(S): New Zealand rabbit, NIH and ICR mice. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Enzyme-linked immunoabsorbent assay, Western blot and indirect immunostaining assays, standard fertility assay, and sperm agglutination assay. RESULT(S): Antibodies against the full-length GAPDH-2 were raised. Its specificity was assessed by immunoblotting and indirect immunostaining assays. The antibody immunoreacted with human sperm GAPDH-2 and the mouse homolog GAPDS but did not cross-react with GAPDH. Treatment of female mice with IP injection of anti-GAPDH-2 serum significantly reduced their fertility. Anti-GAPDH-2 serum caused the agglutination of normal mice sperm in vitro. The anti-GAPDH-2 antibody was detectable in the sera and uterine fluid of the mice immunized with GAPDH-2. CONCLUSION(S): These results show that GAPDH-2 should be further evaluated as a promising candidate in the development of an antifertility immunogen. Detecting anti-GAPDH-2 antibodies in the bodily fluid of subjects afflicted with indeterminate infertility may be a new diagnostic index.
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Anticuerpos/farmacología , Anticonceptivos/inmunología , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/inmunología , Gliceraldehído-3-Fosfato Deshidrogenasas/inmunología , Infertilidad Femenina/inmunología , Espermatozoides/inmunología , Animales , Anticuerpos/sangre , Especificidad de Anticuerpos , Líquidos Corporales/inmunología , Anticonceptivos/farmacología , Reacciones Cruzadas , Femenino , Fertilidad/inmunología , Regulación Enzimológica de la Expresión Génica , Gliceraldehído-3-Fosfato Deshidrogenasa (Fosforilante)/genética , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Humanos , Inmunización , Masculino , Ratones , Ratones Endogámicos ICR , Conejos , Aglutinación Espermática/inmunología , Testículo/citología , Testículo/inmunología , Útero/inmunologíaRESUMEN
Rhomboid family members are widely conserved and found in all three kingdoms of life. They are serine proteases and serve important regulatory functions. In the present study, a novel gene highly expressed in the testis, RHBDD1, is shown to be a new member of the Rhomboid family, participating in the cleavage of BIK, a proapoptotic member of the Bcl-2 family. The RHBDD1-involved proteolytic modification is upstream of the BIK protein degradation pathway. Mutagenesis studies show that the amino acid residues glycine142 and serine144 of RHBDD1 are crucial for its activity in cleaving BIK at a site located in the transmembrane region. Overexpression or knock-down of RHBDD1 in HEK 293T cells can reduce or enhance BIK-mediated apoptosis, respectively. The present findings suggest that, by acting as a serine protease, RHBDD1 modulates BIK-mediated apoptotic activity.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis/genética , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Testículo/metabolismo , Northern Blotting , Western Blotting , Línea Celular , Cartilla de ADN/genética , Humanos , Masculino , Proteínas Mitocondriales , Modelos Biológicos , Mutagénesis , Interferencia de ARN , Análisis de Secuencia de ADNRESUMEN
To uncover novel genes potentially involved in embryo development, especially at the midblastula transition (MBT) phase in the developing embryo of zebrafish, Affymetrix zebrafish GeneChip microarray analysis was carried out on the expression of 14,900 gene transcripts. The results of the analysis showed that 360 genes were clearly up-regulated and 119 genes were markedly down-regulated. Many of these genes were involved in transcription factor activity, nucleic acid binding, and cell growth. The present study showed that significant changes in transcript abundance occurred during the MBT phase. The expression of eight of these 479 genes was identified by reverse transcription-polymerase chain reaction analysis, confirming the microarray results. The WSB1 gene, found to be down-regulated by the microarray and reverse transcription-polymerase chain reaction analyses, was selected for further study. Sequence analysis of the WSB1 gene showed that it encodes a protein with 75% identity to the corresponding active human orthologs. In addition, WSB1 gene expression was detected at a higher level at 2 h post fertilization and at a lower level at 4 h post fertilization, consistent with the chip results. Overexpression of the WSB1 gene can result in the formation of abnormalities in embryos, as determined by fluorescence-activated cell sorting. The present study showed unequivocally that the occurrence of WSB1 expression is an important event during the MBT phase in the development of zebrafish embryos.
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Blástula/crecimiento & desarrollo , Blástula/metabolismo , Desarrollo Embrionario/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Pez Cebra/fisiología , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas de Pez Cebra/genéticaAsunto(s)
Conductos Pancreáticos/anomalías , Dolor Abdominal/diagnóstico , Dolor Abdominal/cirugía , Colangiopancreatografia Retrógrada Endoscópica , Pancreatocolangiografía por Resonancia Magnética , Diagnóstico Diferencial , Femenino , Humanos , Persona de Mediana Edad , Conductos Pancreáticos/cirugía , Tomografía Computarizada por Rayos XRESUMEN
The determination of bile acid concentration in urine is useful for the screening and diagnosis of various hepatobiliary diseases. Currently, there is no concise method to determine bile acid concentration in urine. This study describes a bile acid biosensor fabricated by electrochemical technique for urinalysis. The micro-planar electrodes employed for the study consisted of a working electrode (platinum), a counter electrode (platinum) and a reference electrode (silver/silver chloride (Ag/AgCl)). The sensor chip was coated with Nafion using a spin-coater in order to both eliminate many interference species in urine and achieve long-term stability of the reference electrode. Nafion coating allowed the sensor chip to prevent the electrode reaction from interference species in urine, because it is charged negative strongly (Nafion contains sulfonic acid group). Three enzymes (bile acid sulfate sulfatase: BSS, beta-hydroxysteroid dehydrogenase: beta-HSD, and NADH oxidase: NHO) were immobilized by glutaraldehyde (GA: cross-linker) onto the sensor chip, because the immobilization of enzymes by GA is simple and commonly carried out. The sensor chip was able to detect bile acid in buffer solution. The optimum enzyme ratio immobilized onto the sensor chip was BSS:beta-HSD:NHO=4:4:20 U/1 chip. There was a relationship between the concentration of bile acid and the response current value. The dynamic range of the sensor chip was 2-100 microM for bile acid. Additionally, bile acid in the urine specimen could be detected using this bile acid biosensor. We present a simple and rapid bile acid biosensor with high sensitivity and high reproducibility.
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Ácidos y Sales Biliares/análisis , Ácidos y Sales Biliares/orina , Técnicas Biosensibles/instrumentación , Polímeros de Fluorocarbono , HumanosRESUMEN
YWK-II protein is a sperm membrane component, structurally related to human placenta amyloid precursor protein homolog (APPH) and rat amyloid precursor-like protein 2 (APLP2). Its transmembrane-cytoplasmic domain has high homology (70.6%) with that of betaA4-amyloid precursor protein (APP) found in brain plaques of subjects with Alzheimer's disease. The gene encoding the YWK-II protein is expressed in various mammalian cells and tissues. In the present study, splicing patterns of YWK-II mRNA and the content of YWK-II mRNA in mouse testes, eggs, and cumulus cells were determined. Three different YWK-II transcripts were found in testes and eggs, while cumulus cells contained an additional transcript. In mouse eggs, the content of YWK-II transcript exceeded that of APP. An alternative splicing region was located in the vicinity of the kunitz protease inhibitor (KPI) domain, which may be the basis for the formation of multiple transcripts. YWK-II protein was immunolocated in male and female gametes. It was localized in the plasma membrane of mouse eggs and spermatozoa. In the male reproductive system of the mouse, the YWK-II gene was expressed in germ cells at various stages of differentiation. In mature spermatozoa, the YWK-II protein occurred in the plasma membrane enveloping the acrosome. Triggering the acrosome reaction incited a release of the YWK-II protein attached to the liberated membrane vesicles. The occurrence of the YWK-II protein in the plasma membranes of mouse gametes suggests its involvement in sperm-egg interaction.
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Precursor de Proteína beta-Amiloide/fisiología , Fertilización/fisiología , Proteínas del Tejido Nervioso/fisiología , Óvulo/fisiología , Espermatozoides/fisiología , Empalme Alternativo/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Femenino , Masculino , Ratones , Microscopía Inmunoelectrónica , Proteínas del Tejido Nervioso/genética , Estructura Terciaria de Proteína/genética , Testículo/fisiologíaRESUMEN
HSD-3.8 cDNA (accession number AF311312) encodes a human sperm component. A 0.7 kb fragment (HSD-0.7) containing three immunological epitopes of HSD-3.8 cDNA was prepared and expressed in E. coli. Immunization of female rats with the recombinant HSD-0.7 proteins induced infertility. A cDNA fragment encoding the C-terminal 144 amino acids of human G-protein beta l subunit (Gbeta1-C144) was screened by yeast two-hybrid, when HSD-0.7 segment was used as a bait. Recombinant His6-tagged-Gbeta1-C144 protein was expressed in E. coli BL21 and Anti-Gbeta1 serum was raised with purified Gbeta1-C144. HA-tagged HSD-0.7 and FLAG-tagged Gbeta1 plasmids were constructed and co-transfected into human embryonal kidney 293 cells. Two proteins were localized at superimposable sites in the cytoplasm, and they formed a complex when 500 micromol/L GDP existed. Overexpression of HSD-0.7 activated the G-protein-mediated extracellular signal-regulated kinases (ERK1/2); however, the truncated fragments of HSD-0.7, which lacked either TPR domain or P-loop, lost the ability to activate the ERK1/2 pathway. Further study revealed that the activation of ERK1/2 was protein kinase C (PKC) rather than Ras dependent. These results provide evidence that HSD-3.8 present in spermatocytes and sperm may participate in spermatogenesis and fertilization process by activating the PKC-dependent ERK1/2 signal transduction pathway.
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Antígenos de Superficie/fisiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Proteínas de Unión al GTP/fisiología , Espermatozoides/metabolismo , Animales , Antígenos de Superficie/metabolismo , Western Blotting , Células COS , Línea Celular , Chlorocebus aethiops , Citoplasma/metabolismo , ADN Complementario/metabolismo , Escherichia coli/metabolismo , Femenino , Proteínas de Unión al GTP/metabolismo , Humanos , Inmunoprecipitación , Sistema de Señalización de MAP Quinasas , Masculino , Microscopía Fluorescente , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Ovario/metabolismo , Plásmidos/metabolismo , Proteína Quinasa C/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Transducción de Señal , Espermatogénesis , Testículo/metabolismo , Distribución Tisular , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
Gamma-aminobutyric acid type A (GABA(A)) receptors are the major sites of inhibitory action of fast synaptic neurotransmission in the brain. Their receptors are also widely distributed in peripheral and endocrine tissues. A full-length cDNA encoding a novel splice variant of beta3 subunit of GABA(A) receptor, designated as beta3t, was identified in rat testis. This isoform contains a segment, having identical amino acid sequence as the beta3 subunit of neuronal GABA(A) receptors except for a section composed of 25 different amino acid sequence in the N-terminus. Northern blot shows that this isoform is found in rat testis. The beta3t isoform mRNA was detected in germ cells in the late step of spermatogenesis by in situ hybridization assay. Results of immunohistochemical and immunocytochemical assays indicate that the beta3t isoform is expressed in rat testis and spermatozoa. To determine a possible function of the N-terminal 25 amino acid segment, a recombinant plasmid of beta3t-EGFPC was constructed by fusing green fluorescent protein to the C-terminus of the beta3t isoform. The chimera product failed to be translocated unto the cell surface when expressed in HEK 293 cells; whereas, the beta3 subunit of rat brain is incorporated into the plasma membrane. In conclusion, the present results show that one variant of beta3 subunit of GABA(A) receptor, designated as beta3t, is found in germ cells of rat testis and sperm. The inability of the beta3t variant to target into the plasma membrane maybe a consequence of the unique 25 amino acid segment in the N-terminus.
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Empalme Alternativo/fisiología , Receptores de GABA-A/biosíntesis , Espermatozoides/metabolismo , Testículo/metabolismo , Empalme Alternativo/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Membrana Celular/metabolismo , Humanos , Masculino , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Especificidad de Órganos/fisiología , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Ratas , Receptores de GABA-A/genética , Espermatozoides/citología , Testículo/citologíaRESUMEN
The method of laser capture microdissection (LCM) combined with suppressive subtractive hybridization (SSH) was developed to isolate specific germ cells from human testis sections and to identify the genes expressed during differentiation and development. In the present study, over 10,000 primary spermatocytes and round spermatid cells were successfully isolated by LCM. Using the cDNAs from primary spermatocytes and round spermatids, SSH cDNAs library of primary spermatocyte-specific was constructed. The average insert size of the cDNA isolated from 75 randomly picked white clones was 500 bp, ranging from 250 bp to 1.7 kb. Using the dot-blot method, a total of 421 clones were examined, resulting in the identification of 390 positive clones emitting strong signals. Partial sequence of cDNAs prepared from each clone was determined with an overall success rate of 84.4%. Genes encoding cytochrome c oxidase II and the rescue factor-humanin were most frequently expressed in primary spermatocytes, suggesting their roles involved in meiosis.
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ADN Complementario/genética , Regulación del Desarrollo de la Expresión Génica , Microdisección/métodos , Espermátides/química , Espermatocitos/química , Testículo/citología , Clonación Molecular , ADN Complementario/análisis , ADN Complementario/aislamiento & purificación , Complejo IV de Transporte de Electrones/metabolismo , Biblioteca de Genes , Técnicas Histológicas , Humanos , Hibridación in Situ/métodos , Rayos Láser , Masculino , Microdisección/instrumentación , Reacción en Cadena de la Polimerasa , ARN/genética , Análisis de Secuencia de ADN/métodos , Espermátides/citología , Espermátides/metabolismo , Espermatocitos/citología , Espermatocitos/metabolismo , Espermatogénesis/genéticaRESUMEN
hSMP-1 is a human sperm membrane protein expressed during development. It is a testis-specific component produced during male germ cell differentiation. Proteins that interact with hSMP-1 were identified by the application of the yeast two-hybrid system. One of the components, RanBPM, was found to be associated with hSMP-1 under both in vitro and in vivo conditions. In the human testis, RanBPM is produced in spermatogonia and primary spermatocytes, suggesting expression during the early stages of spermatogenesis; whereas in the rat testis, it is located in round and elongated spermatids, similar to hSMP-1, suggesting expression of both components during spermiogenesis. Images obtained by immunofluorescence and confocal scanning microscopy of CHO-K1 cells co-transfected with pEGFP-C1-hSMP-1 and pDsRed1-Nl-RanBPM revealed that RanBPM and hSMP-1 are distributed in discrete loci throughout the cytoplasm. When superimposed, the stained spots appeared as congruent yellow areas, indicative of co-localization and probable complex formation of these two components. This interaction between hSMP-1 and RanBPM may be involved in the process of male germ cell differentiation. In CHO-Kl cells transfected with pEGFP-Cl-hSMP-1, the exogenously expressed hSMP-1 was found to co-localize with alpha-tubulin. Depolymerization of microtubules can be induced in CHO-Kl cells by cold treatment. In cells transfected with the pEGFP-Cl vector, the dispersed tubulins promptly reassembled upon warming. However, in cells transfected with pEGFP-Cl-hSMP-1, reassembly of the dispersed tubulins was blocked even upon rewarming of the cells. These findings suggest that hSMP-1 interacts with tubulins and thereby may modulate microtubule assembly and/or activity.
Asunto(s)
Proteínas de la Membrana/metabolismo , Centro Organizador de los Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Espermatozoides/metabolismo , Testículo/metabolismo , Proteína de Unión al GTP ran/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Antígenos de Superficie , Células CHO , Clonación Molecular , Cricetinae , Proteínas del Citoesqueleto , Regulación del Desarrollo de la Expresión Génica , Biblioteca de Genes , Células HeLa , Humanos , Inmunohistoquímica , Masculino , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/genética , Pruebas de Precipitina , Ratas , Espermatogénesis , Proteína de Unión al GTP ran/biosíntesis , Proteína de Unión al GTP ran/genéticaRESUMEN
PROBLEM: The 80 kDa human sperm antigen (HSA) is a sperm-specific and conserved antigen, capable of inducing immunological infertility. Partial N-terminal amino acid sequences of 80 kDa HSA (Peptide NT) and its peptides obtained by digestion with endoproteinase Lys-C (peptides 1-4) and endoproteinase Glu-C (peptides 5-6) did not show any sequence homology with reported known proteins deposited in the Gen-Bank. These sequenced peptides were synthesized and conjugated to key hole limpet haemocyanin (KLH) and evaluated for its antifertility effects. The present communication describes the characterization of these peptides and their antibodies. METHOD OF STUDY: Peptides NT, 1, 2, 3 and 4 were synthesized and conjugated to KLH. Antibodies to KLH conjugated peptides were raised in rabbits by active immunization and the antibody titer was determined by enzyme-linked immunosorbent assay (ELISA) using sperm extract coated wells. The binding specificity of the synthetic peptides or purified 80 kDa HSA to their antibodies was assessed in the presence of various doses of respective synthetic peptides or 80 kDa HSA. The binding specificity was further confirmed by Western blot analysis. Antipeptide antibodies were also checked for sperm agglutinating activity, in-vitro. RESULTS: Active immunization of rabbits elicited significant antibody titers against the synthetic peptides, except for peptide 3. Antipeptide antibodies specifically recognized the native protein in an ELISA and induced in-vitro agglutination of human, rat and monkey sperm. In addition, Western blot analysis showed that these antipeptide antibodies specifically bind to the 80 kDa HSA band of the sperm extract. CONCLUSION: Synthetic peptides of 80 kDa HSA are immunogenic and antibodies raised against these peptides recognize the native protein detected by ELISA, Western blot analysis. In addition, they possess sperm agglutinating activity. These findings suggest that they are promising candidates in the development of immunocontraceptives.
Asunto(s)
Antígenos/inmunología , Péptidos/inmunología , Espermatozoides/inmunología , Pruebas de Aglutinación , Secuencia de Aminoácidos , Animales , Anticuerpos , Especificidad de Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo/inmunología , Antígenos/química , Anticoncepción Inmunológica , Ensayo de Inmunoadsorción Enzimática , Hemocianinas/inmunología , Humanos , Inmunización , Masculino , Péptidos/química , ConejosRESUMEN
Mouse sp56 is considered as one of the candidates for mouse zona pellucida 3 (mZP3) receptor. Up to date, its homologue has only been cloned from guinea pig, namely AM67. Based on the cDNA sequence of mouse sp56, we designed a pair of primer to amplify its homologue from rat testis cDNA. Using RT-PCR, two fragments of 743 bp and 938 bp were amplified. The PCR products show very high homology to mouse sp56. However, the 743 bp product completely lacks one of the seven Sushi domains of mouse sp56. Using the 743 bp product as the probe to detect the expression profile of sp56 in rat tissues, Northern blot shows that a approximately 2.0 kb mRNA expresses specifically in testis. Employed the RACE method, two full cDNA sequences of rat sp56 were obtained. A Mr approximately 42 KD band was detected in denatured and non-reducing protein sample of rat testis and sperm with anti-mouse sp56 monoclonal antibody by Western blot method. Rat sp56 was localized on rat sperm head by the indirect immunofluorescence method. Rat sp56 immunoreactivity was detected from the early pachytene spermatocytes and throughout the spermatogenesis. Its cloning will further our understanding of the mechanism of the sperm-egg recognition and binding.
Asunto(s)
Proteínas del Huevo/metabolismo , Fertilización/fisiología , Glicoproteínas de Membrana/metabolismo , Receptores de Superficie Celular/aislamiento & purificación , Espermatozoides/metabolismo , Testículo/metabolismo , Animales , Anticuerpos , Clonación Molecular , ADN Complementario/análisis , ADN Complementario/genética , Técnica del Anticuerpo Fluorescente , Masculino , Ratones , Datos de Secuencia Molecular , Ratas , Ratas Wistar , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Espermatozoides/citología , Testículo/citología , Glicoproteínas de la Zona PelúcidaRESUMEN
The expression of stage-specific genes during spermatogenesis was determined by isolating two segments of rat seminiferous tubule at different stages of the germinal epithelium cycle delineated by transillumination-delineated microdissection, combined with differential display polymerase chain reaction to identify the differential transcripts formed. A total of 22 cDNAs were identified and accepted by GenBank as new expressed sequence tags. One of the expressed sequence tags was radiolabeled and used as a probe to screen a rat testis cDNA library. A novel full-length cDNA composed of 2228 bp, designated as RSD-3 (rat sperm DNA no.3, GenBank accession no. AF094609) was isolated and characterized. The reading frame encodes a polypeptide consisting of 526 amino acid residues, containing a number of DNA binding motifs and phosphorylation sites for PKC, CK-II, and p34cdc2. Northern blot of mRNA prepared from various tissues of adult rats showed that RSD-3 is expressed only in the testis. The initial expression of the RSD-3 gene was detected in the testis on the 30th postnatal day and attained adult level on the 60th postnatal day. Immunolocalization of RSD-3 in germ cells of rat testis showed that its expression is restricted to primary spermatocytes, undergoing meiosis division I. A human testis homologue of RSD-3 cDNA, designated as HSD-3.1 (GenBank accession no. AF144487) was isolated by screening the Human Testis Rapid-Screen arrayed cDNA library panels by RT-PCR. The exon-intron boundaries of HSD-3.1 gene were determined by aligning the cDNA sequence with the corresponding genome sequence. The cDNA consisted of 12 exons that span approximately 52.8 kb of the genome sequence and was mapped to chromosome 14q31.3.