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Although the history of intraoperative neuromonitoring (IONM) dates back to the 19th century, the method did not evolve further than the mere differentiation of nerves until recently. Only the development of continuous IONM (cIONM) has allowed for non-stop analysis of excitation amplitude and latency during surgical procedures, which is nowadays integrated into the software of almost all commercially available neuromonitoring devices. The objective of cIONM is real-time monitoring of nerve status in order to recognize and prevent impending nerve injury and predict postoperative nerve function. Despite some drawbacks such as false-positive/negative alarms, technical artefacts, and rare adverse effects, cIONM remains a good instrument which is still under development. Active (acIONM) and passive (pcIONM) methods of cIONM are described in literature. The main fields of cIONM implementation are currently thyroid surgery (in which the vagal nerve is continuously stimulated) and surgery to the cerebellopontine angle (in which the facial nerve is either continuously stimulated or the discharge signal of the nerve is analyzed via pcIONM). In the latter surgery, continuous monitoring of the cochlear nerve is also established.
Assuntos
Complicações Intraoperatórias , Monitorização Intraoperatória , Ângulo Cerebelopontino , Nervo Facial , Humanos , Complicações Intraoperatórias/prevenção & controle , Tireoidectomia , Paralisia das Pregas VocaisRESUMO
Although the history of intraoperative neuromonitoring (IONM) dates back to the 19th century, the method did not evolve further than the mere differentiation of nerves until recently. Only the development of continuous IONM (cIONM) has allowed for non-stop analysis of excitation amplitude and latency during surgical procedures, which is nowadays integrated into the software of almost all commercially available neuromonitoring devices. The objective of cIONM is real-time monitoring of nerve status in order to recognize and prevent impending nerve injury and predict postoperative nerve function. Despite some drawbacks such as false-positive/negative alarms, technical artefacts, and rare adverse effects, cIONM remains a good instrument which is still under development. Active (acIONM) and passive (pcIONM) methods of cIONM are described in literature. The main fields of cIONM implementation are currently thyroid surgery (in which the vagal nerve is continuously stimulated) and surgery to the cerebellopontine angle (in which the facial nerve is either continuously stimulated or the discharge signal of the nerve is analyzed via pcIONM). In the latter surgery, continuous monitoring of the cochlear nerve is also established.
Assuntos
Monitorização Intraoperatória , Tireoidectomia , Paralisia das Pregas Vocais , Ângulo Cerebelopontino , Nervo Facial , Humanos , Complicações IntraoperatóriasRESUMO
BACKGROUND: Depression and diabetes commonly co-occur; however, the strength of the physiological effects of diabetes as mediating factors towards depression is uncertain. METHOD: We analyzed extensive clinical, epidemiological and laboratory data from n = 2081 Mexican Americans aged 35-64 years, recruited from the community as part of the Cameron County Hispanic Cohort (CCHC) divided into three groups: Diagnosed (self-reported) diabetes (DD, n = 335), Undiagnosed diabetes (UD, n = 227) and No diabetes (ND, n = 1519). UD participants denied being diagnosed with diabetes, but on testing met the 2010 American Diabetes Association and World Health Organization definitions of diabetes. Depression was measured using the Center for Epidemiological Studies - Depression (CES-D) scale. Weighted data were analyzed using dimensional and categorical outcomes using univariate and multivariate models. RESULTS: The DD group had significantly higher CES-D scores than both the ND and UD (p ⩽ 0.001) groups, whereas the ND and UD groups did not significantly differ from each other. The DD subjects were more likely to meet the CES-D cut-off score for depression compared to both the ND and UD groups (p = 0.001), respectively. The UD group was also less likely to meet the cut-off score for depression than the ND group (p = 0.003). Our main findings remained significant in models that controlled for socio-demographic and clinical confounders. CONCLUSIONS: Meeting clinical criteria for diabetes was not sufficient for increased depressive symptoms. Our findings suggest that the 'knowing that one is ill' is associated with depressive symptoms in diabetic subjects.
Assuntos
Depressão/diagnóstico , Depressão/etnologia , Diabetes Mellitus/psicologia , Americanos Mexicanos/estatística & dados numéricos , Adulto , Estudos de Coortes , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Escalas de Graduação Psiquiátrica , Autorrelato , Fatores Socioeconômicos , Estados Unidos/etnologiaRESUMO
This study aimed to estimate the prevalence and risk factors for hepatitis C virus (HCV) infection in Mexican Americans living in South Texas. We tested plasma for the presence of HCV antibody from the Cameron County Hispanic Cohort (CCHC), a randomized, population-based cohort in an economically disadvantaged Mexican American community on the United States/Mexico border with high rates of chronic disease. A weighted prevalence of HCV antibody of 2·3% [n = 1131, 95% confidence interval (CI) 1·2-3·4] was found. Participants with diabetes had low rates of HCV antibody (0·4%, 95% CI 0·0-0·9) and logistic regression revealed a statistically significant negative association between HCV and diabetes (OR 0·20, 95% CI 0·05-0·77) after adjusting for sociodemographic and clinical factors. This conflicts with reported positive associations of diabetes and HCV infection. No classic risk factors were identified, but important differences between genders emerged in analysis. This population-based study of HCV in Mexican Americans suggests that national studies do not adequately describe the epidemiology of HCV in this border community and that unique risk factors may be involved.
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Coinfecção/epidemiologia , Diabetes Mellitus/epidemiologia , Hepacivirus/isolamento & purificação , Hepatite C/epidemiologia , Adulto , Coinfecção/etiologia , Estudos Transversais , Diabetes Mellitus/etiologia , Feminino , Hepatite C/virologia , Anticorpos Anti-Hepatite C/sangue , Humanos , Masculino , Americanos Mexicanos , Pessoa de Meia-Idade , Prevalência , Fatores de Risco , Texas/epidemiologiaRESUMO
In this Letter we study a system consisting of two nearly degenerate mechanical modes that couple to a single mode of an optical cavity. We show that this coupling leads to nearly complete (99.5%) hybridization of the two mechanical modes into a bright mode that experiences strong optomechanical interactions and a dark mode that experiences almost no optomechanical interactions. We use this hybridization to transfer energy between the mechanical modes with 40% efficiency.
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A photovoltaic tandem cell made of amorphous silicon (a-Si) and microcrystalline silicon (µc-Si) was investigated as a photocathode for hydrogen evolution in a photoelectrochemical device. The electronic and electrochemical properties of the samples were characterized using X-ray photoemission spectroscopy (XPS) and cyclic voltammetry (CV), whereas the morphology of the surface in contact with the electrolyte was investigated by scanning electron microscopy (SEM). The electric efficiency of the tandem cell was determined to be 5.2% in a photoelectrochemical (PEC) setup in acidic solution which is only about half of the photovoltaic efficiency of the tandem cell. A significant improvement in efficiency was achieved with platinum as a catalyst which was deposited by physical vapour deposition (PVD) under ultra-high vacuum (UHV) conditions.
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BACKGROUND: The number of elderly patients with head and neck cancer is increasing. However, there are few valid data on postoperative course after head and neck cancer surgery in elderly patients. The aim of this study was to evaluate the oncological outcome of elderly patients after surgical treatment for oro- and hypopharyngeal cancer. MATERIAL AND METHODS: The clinical data of 81 patients, separated into two age groups (62 < 65 years vs. 19 ≥ 65 years), were retrospectively analysed. The cohort comprised T1 and T2 oro- and hypopharyngeal cancer patients, who had undergone primary treatment with transoral laser surgery and neck dissection. Overall and disease-free survival times of the patients were analysed. Additionally, comorbidities and perioperative complications were compared between the two age groups. Median follow-up time was 5.9 years. RESULTS: Comparison of different clinical and histopathological data revealed no significant differences between the age groups. The Kaplan-Meier method revealed no significant difference in disease-free survival between the age groups (p = 0.52). Age had no effect on disease-free survival in uni- or multivariate analysis (p = 0.53 vs. 0.94). Surgery-related complications were observed in 13 patients (16 %), 11 cases of which concerned the younger group of patients. CONCLUSION: Transoral laser surgery and neck dissection can lead to satisfactory oncological and surgical outcomes in elderly patients with oro- and hypopharyngeal cancer.
Assuntos
Neoplasias Hipofaríngeas/cirurgia , Terapia a Laser/métodos , Esvaziamento Cervical/métodos , Neoplasias Orofaríngeas/cirurgia , Idoso , Idoso de 80 Anos ou mais , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Hipofaríngeas/diagnóstico , Masculino , Pessoa de Meia-Idade , Neoplasias Orofaríngeas/diagnóstico , Procedimentos Cirúrgicos Otorrinolaringológicos/métodos , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Transmission of hepatitis C (HCV) in Pakistan is a continuing public health problem; 15 years ago it was linked to the practice of reusing therapeutic instruments in healthcare settings. We sought to examine current risk factors for HCV transmission in a hospital population in Karachi, Pakistan. We enrolled 300 laboratory-confirmed HCV-positive participants and 300 laboratory confirmed HCV-negative participants from clinics at Indus Hospital. Independent and significant risk factors for both men and women were: receiving o12 injections in the past year, blood transfusions, having had dental work performed, and delivery in hospital or transfusion for women. Interestingly, being of Mohajir origin or born in Sindh province were protective.Encouragingly, a strong protective effect was observed for those that reported bringing their own needle for injections (59%). The widespread reuse of therapeutic needles in healthcare settings in Karachi remains a major driver of the HCV epidemic.
Assuntos
Infecção Hospitalar/epidemiologia , Infecção Hospitalar/transmissão , Instalações de Saúde , Hepatite C/epidemiologia , Hepatite C/transmissão , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Paquistão/epidemiologia , Fatores de RiscoRESUMO
The design of structurally diverse enzymes is constrained by long-range interactions that are necessary for accurate folding. We introduce an atomistic and machine learning strategy for the combinatorial assembly and design of enzymes (CADENZ) to design fragments that combine with one another to generate diverse, low-energy structures with stable catalytic constellations. We applied CADENZ to endoxylanases and used activity-based protein profiling to recover thousands of structurally diverse enzymes. Functional designs exhibit high active-site preorganization and more stable and compact packing outside the active site. Implementing these lessons into CADENZ led to a 10-fold improved hit rate and more than 10,000 recovered enzymes. This design-test-learn loop can be applied, in principle, to any modular protein family, yielding huge diversity and general lessons on protein design principles.
Assuntos
Técnicas de Química Combinatória , Endo-1,4-beta-Xilanases , Engenharia de Proteínas , Catálise , Domínio Catalítico , Engenharia de Proteínas/métodos , Endo-1,4-beta-Xilanases/químicaRESUMO
AIMS: Cervical cancer incidence and mortality rates are approximately 55% higher in the Rio Grande Valley (RGV) along the Texas-Mexico border compared with the average rates in the US. Our aim was to improve cervical cancer prevention efforts in the RGV through a comprehensive multilevel intervention initiative focused on community education, patient navigation, and training of local providers. METHODS: We initiated a program in the RGV which consisted of (1) community education, (2) patient navigation, and (3) a training/mentoring program for local medical providers including hands-on training courses coupled with telementoring using Project ECHO® (Extension for Community Health Outcomes). We assessed the number of women undergoing cervical cancer screening, diagnosis, and treatment at three participating clinics caring for underserved women in the region. RESULTS: From November 2014 to October 2018, 14,846 women underwent cervical cancer screening. A total of 2030 (13.7%) women underwent colposcopy for abnormal results (179% increase over baseline) and 453 women underwent loop electrosurgical excision procedures (LEEPs) for treatment of cervical dysplasia. Invasive cancer was diagnosed in 39 women who were navigated to a gynecologic oncologist for treatment. Seven local medical providers were trained to perform colposcopy and/or LEEP. Project ECHO telementoring videoconferences were held every 2 weeks for a total 101 sessions with an average of 22 participants per session and a total of 180 patient cases presented and discussed. CONCLUSIONS: Our program led to a large number of women undergoing diagnosis and treatment of cervical dysplasia in the RGV. If sustained, we anticipate these efforts will decrease cervical cancer rates in the region. The program is currently being expanded to additional underserved areas of Texas and globally to low- and middle-income countries.
Assuntos
Navegação de Pacientes , Displasia do Colo do Útero , Neoplasias do Colo do Útero , Feminino , Humanos , Masculino , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/prevenção & controle , Texas/epidemiologia , México/epidemiologia , Detecção Precoce de CâncerRESUMO
Ebola virus is very pathogenic in humans. It induces an acute hemorrhagic fever that leads to death in about 70% of patients. We compared the immune responses of patients who died from Ebola virus disease with those who survived during two large outbreaks in 1996 in Gabon. In survivors, early and increasing levels of IgG, directed mainly against the nucleoprotein and the 40-kDa viral protein, were followed by clearance of circulating viral antigen and activation of cytotoxic T cells, which was indicated by the upregulation of FasL, perforin, CD28 and gamma interferon mRNA in peripheral blood mononuclear cells. In contrast, fatal infection was characterized by impaired humoral responses, with absent specific IgG and barely detectable IgM. Early activation of T cells, indicated by mRNA patterns in peripheral blood mononuclear cells and considerable release of gamma interferon in plasma, was followed in the days preceding death by the disappearance of T cell-related mRNA (including CD3 and CD8). DNA fragmentation in blood leukocytes and release of 41/7 nuclear matrix protein in plasma indicated that massive intravascular apoptosis proceeded relentlessly during the last 5 days of life. Thus, events very early in Ebola virus infection determine the control of viral replication and recovery or catastrophic illness and death.
Assuntos
Anticorpos Antivirais/sangue , Apoptose , Surtos de Doenças , Doença pelo Vírus Ebola/mortalidade , Leucócitos/patologia , Antígenos CD28/biossíntese , Proteína Ligante Fas , Gabão/epidemiologia , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Interferon gama/biossíntese , Glicoproteínas de Membrana/biossíntese , Nucleoproteínas/imunologia , Perforina , Proteínas Citotóxicas Formadoras de Poros , Linfócitos T Citotóxicos/imunologia , Regulação para Cima , Proteínas do Core Viral/imunologiaRESUMO
BACKGROUND: The Rio Grande Valley (RGV) and Laredo regions located along the Texas-Mexico border consist of seven counties with a population of approximately 1.5 million people and a high uninsured rate (33.5%). Cervical cancer mortality in these border counties is approximately 30% higher than the rest of Texas. The RGV and Laredo areas were studied to better understand the state of access to cervical cancer prevention services along the Texas-Mexico border. METHODS: Data on the population served and the services provided were analyzed to determine the gap between cervical cancer screenings recommended versus those received. Through interviews, we gathered the perspectives of 16 local stakeholders regarding cervical cancer screening for underserved individuals in the region. FINDINGS: It is estimated that 69,139 uninsured women aged 21-64 years in the RGV/Laredo per year are recommended to undergo cervical cancer screening with Papanicolaou (Pap) and/or human papillomavirus (HPV) testing, but only 8941 (12.9%) Pap tests are being performed by the Federally Qualified Health Center (FQHC) serving uninsured women in these regions. Systemic barriers identified include insufficient provider clinical capacity, the high cost of healthcare, and uncertainty about government funding sources. Patient barriers identified include inadequate knowledge on navigating the local healthcare system, low health literacy, lack of money and childcare, an inability to miss work, limited transportation, and fear of deportation. CONCLUSION: Decreasing the disparity between cervical cancer screening services provided and those recommended requires addressing the barriers, identified by local experts, which prevent uninsured women from accessing care. These challenges are being addressed through ongoing programs and collaborations.
Assuntos
Acessibilidade aos Serviços de Saúde/economia , Pessoas sem Cobertura de Seguro de Saúde/psicologia , Teste de Papanicolaou/economia , Neoplasias do Colo do Útero/diagnóstico , Adulto , Detecção Precoce de Câncer/economia , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , México , Pessoa de Meia-Idade , Texas , Neoplasias do Colo do Útero/economia , Neoplasias do Colo do Útero/prevenção & controle , Esfregaço Vaginal/economia , Adulto JovemRESUMO
OBJECTIVE: To find the prevalence of HIV infection and risk behaviors among injecting drug users (IDUs) in Karachi, Pakistan. DESIGN: A cross-sectional study of IDUs conducted in Karachi, Pakistan from February through June 1996. RESULTS: Of the 242 IDUs, 11 (4%) refused HIV testing. One (0.4%; 95% confidence interval (CI) = 0.37-0.48%) was HIV positive. All subjects were male. Over the past 6 months 47% had engaged in receptive needle sharing, 38% had perceived a change in their social network, 22% had had sexual intercourse, of whom only 7% always used condoms, and none had washed their needles with bleach. Younger age (28 vs. 31 years; p = 0.01), younger age at first injection (25 vs. 28 years; p = 0.001), fewer years of schooling (3 vs. 5 years; p = 0.001), lower monthly income (70 dollars vs. 80 dollars; p = 0.03), inhaling fumes of heroin from a foil in the year before injecting (OR = 4.8; CI = 2.2-10.3), injecting first time with heroin (OR = 3.6; CI = 1.2-12.6), having a temporary job (OR = 2.5; CI = 1.2-5.2), and a perceived change in one's social network (OR = 4.4; CI = 2.4-7.9) were all associated with receptive needle sharing. IDUs who knew about HIV spread through contaminated needles were less likely to share (OR = 0.4; CI 0.2-0.8). In the final logistic regression model receptive needle sharing was associated with inhaling of fumes of heroin on a foil in the year prior to injecting (adjusted OR = 5.6; CI = 2.6-12.0), a perceived change in one's social network (adjusted OR = 4.0; CI = 2.2-7.4), and inversely associated with age at first time of injection (beta = -0.07; p = 0.002). CONCLUSION: Background HIV prevalence was low among IDUs in Karachi despite high-risk behavior in 1996. In order to control HIV transmission among IDUs in Pakistan, continual HIV surveillance with well-coordinated and effective HIV risk reduction, and drug demand reduction programs need to be implemented among drug users.
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Assunção de Riscos , Abuso de Substâncias por Via Intravenosa , Adulto , Estudos Transversais , HIV , Infecções por HIV/imunologia , Infecções por HIV/transmissão , Humanos , Masculino , Uso Comum de Agulhas e Seringas , PaquistãoRESUMO
A human serum DNA-binding protein (C3DP) derived from complement component C3 has been found in elevated concentrations in the sera of individuals with malignant diseases. An assay system has been devised which reveals quantitative values of serum C3DP levels. Results obtained using this system indicate that normal human sera have an average C3DP level of 242 mug/ml (range, 40 to 146), whereas sera from individuals with active carcinomas have an average C3DP level of 242 mug/ml (range, 146 to 400). Sera from individuals with active leukemias, lymphomas, and melanomas all had elevated levels of C3DP, whereas sera from individuals with polycythemia vera or other nonmalignant diseases had normal or only slightly elevated C3DP concentrations. No tissue specificity seems to be required for malignant growths to result in elevated C3DP serum concentrations. The levels of C3DP in 79% of the individuals who experienced disease remission were found to decline to normal values, concurrent with the disease regression. Patients who did not respond to therapy regimens retained high C3DP levels.
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Complemento C3 , Proteínas do Sistema Complemento , Neoplasias/sangue , Proteínas de Transporte/sangue , DNA de Neoplasias/sangue , Feminino , Humanos , Masculino , Neoplasias/terapia , Ligação Proteica , Radioimunoensaio , Remissão EspontâneaRESUMO
The treatment of lymph node metastases involving the carotid artery is controversial. The aim of the present study was to determine the outcomes of head and neck cancer patients with radiographic carotid artery involvement in neck metastases. A total of 27 patients with head and neck cancer and radiologically diagnosed advanced metastases involving the common carotid artery or internal carotid artery were enrolled. All patients underwent a primary or salvage neck dissection and surgical carotid peeling. The oncological outcome and survival of all patients were analyzed. Loco-regional control was observed in 13 of the 27 patients (48.1%). During follow-up, five patients (18.5%) developed second primaries and 11 (40.7%) developed distant metastases. The survival time was poor independent of regional control. The median overall survival was 1.55 years and disease-free survival was 0.71 year. Radiographic carotid artery involvement in neck metastases in head and neck cancer appears to correlate with a poor long-term prognosis, with a high rate of distant metastases despite loco-regional control.
Assuntos
Artérias Carótidas/diagnóstico por imagem , Artérias Carótidas/patologia , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Neoplasias de Cabeça e Pescoço/patologia , Tomografia Computadorizada por Raios X , Adulto , Idoso , Artérias Carótidas/cirurgia , Feminino , Neoplasias de Cabeça e Pescoço/cirurgia , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Esvaziamento Cervical , Prognóstico , Taxa de Sobrevida , Resultado do TratamentoAssuntos
Artéria Carótida Interna/anormalidades , Obstrução Nasal/etiologia , Nasofaringe/irrigação sanguínea , Orofaringe/irrigação sanguínea , Seios Paranasais/cirurgia , Complicações Pós-Operatórias/etiologia , Pulso Arterial , Apneia Obstrutiva do Sono/etiologia , Ronco/etiologia , Conchas Nasais/cirurgia , Idoso , Angiografia , Artéria Carótida Interna/diagnóstico por imagem , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Obstrução Nasal/diagnóstico por imagem , Complicações Pós-Operatórias/diagnóstico por imagem , Apneia Obstrutiva do Sono/diagnóstico por imagem , Tomografia Computadorizada por Raios XRESUMO
Transcription of the gene elements that form the variable region of immunoglobulin heavy chains has been proposed to represent the process that controls access for the recombination enzymes in their sequential steps of catalysis. Evidence for germline transcription of VH gene elements, as part of VH to DJH recombination, has been limited to transcripts of only a few gene elements. We have examined normal fetal liver mRNA by Northern blotting and present evidence for germline transcripts from six human VH gene families. The candidate VH4 transcripts have been confirmed as germline transcripts by hybridization with 3' flanking sequences that would have been removed by recombination from mature VHDJH genes. The candidate transcripts for VH1, VH3, VH4 and VH6 have been confirmed by polymerase chain reaction amplification with primers from the 3' flanking sequences of these gene families and determination of the sequence of these products. Determination of sequence from two clones of VH1, VH3 and VH4 indicates that more than one gene from each of these families is transcribed. PCR amplification of VH4 and VH6 with primers specific for the leader sequence (exon 1) and 3' flanking sequence indicate that these transcripts are spliced, representing RNA processing. Germline transcripts from these families are also present in normal human bone marrow. These results indicate that transcriptional activation of germline VH gene elements is a general phenomenon in tissues undergoing V to DJ recombination.
Assuntos
Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Fígado/imunologia , Fígado/metabolismo , Transcrição Gênica/imunologia , Adulto , Sequência de Bases , Northern Blotting , Medula Óssea/imunologia , Medula Óssea/metabolismo , Feto , Células Germinativas/imunologia , Humanos , Dados de Sequência Molecular , Família Multigênica/imunologia , Reação em Cadeia da PolimeraseRESUMO
Cavity optomechanics offers powerful methods for controlling optical fields and mechanical motion. A number of proposals have predicted that this control can be extended considerably in devices where multiple cavity modes couple to each other via the motion of a single mechanical oscillator. Here we study the dynamic properties of such a multimode optomechanical device, in which the coupling between cavity modes results from mechanically induced avoided crossings in the cavity's spectrum. Near the avoided crossings we find that the optical spring shows distinct features that arise from the interaction between cavity modes. Precisely at an avoided crossing, we show that the particular form of the optical spring provides a classical analogue of a quantum non-demolition measurement of the intracavity photon number. The mechanical oscillator's Brownian motion, an important source of noise in these measurements, is minimized by operating the device at cryogenic temperature (500 mK).
RESUMO
The Sm core proteins of U1, U2, U4/U6 and U5 snRNPs include B(B1), B'(B2), N(B3), D1, D2, D3, E, F and G polypeptides. We have isolated genomic clones encoding the Sm-D1 protein using the Sm-D1 cDNA as probe. Southern blotting and DNA sequencing analysis of these clones revealed the presence of an Sm-D1 multigene family in the human genome. Three gene members have been identified. Two of the genes are without introns and contain mutations compared to the cDNA sequence. They appear to be processed pseudogenes. The third gene, termed SNRPD1, shares 100% identity to the cDNA sequence including both 5'- and 3'-untranslated regions (UTR); it contains three introns. Analysis of the 5'-flanking region of the SNRPD1 gene revealed promoter activity, suggesting this is the functional gene that encodes the Sm-D1 protein. The promoter activity was localized in a 0.38 kb PstI fragment using CAT reporter gene fusion assays. Addition of an SV40 enhancer element did not enhance the transcription directed by that fragment. Sequence comparison of the 0.38 kb promoter sequence with the promoters of the Sm-E gene and U1 snRNA genes revealed several homologous motifs, suggesting that genes encoding the snRNP components may be coordinately regulated.
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Autoantígenos/genética , Genes , Regiões Promotoras Genéticas , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U1/genética , Ribonucleoproteínas Nucleares Pequenas , Sequência de Aminoácidos , Autoantígenos/química , Autoantígenos/isolamento & purificação , Sequência de Bases , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ribonucleoproteína Nuclear Pequena U1/química , Ribonucleoproteína Nuclear Pequena U1/isolamento & purificação , Análise de Sequência de DNA , Homologia de Sequência do Ácido Nucleico , Proteínas Centrais de snRNPRESUMO
To conduct functional and autoimmunity studies, we overproduced human Sm-D1 (hSm-D1), a small nuclear ribonucleoprotein 'core' protein and autoantigen, in Escherichia coli and Saccharomyces cerevisiae. Optimal expression in these organisms was achieved by designing vectors that synthesized abundant hSm-D1 mRNA under the control of the strong, regulatable promoters: T7 phi 10 (E. coli) and GAL1 (yeast). In addition, efficient translation initiation of the hSm-D1 coding sequence was effected in E. coli by utilizing a two-cistron approach; for expression in yeast, we created a 5' untranslated leader whose sequence was based on the consensus of highly expressed genes in S. cerevisiae. The hSm-D1 protein accumulated at high levels in both bacteria and yeast, representing, respectively, approx. 10% and 7% of the total protein. However, in comparison with the authentic protein, the recombinant hSm-D1 displayed different immunoreactive determinants as assessed by Western blot. We thus conclude that certain hSm-D1 immunologic properties are most likely dependent on posttranslational modifications that take place in the cells of higher eukaryotes.