RESUMO
Many organizations persist in working with others that engage in known, remediable structural discrimination. We name this practice interorganizational structural discrimination (ISD) and argue it is a pivotal contributor to inequities in science and medicine. We urge organizations to leverage their relationships and demand progress from collaborators.
RESUMO
Mentorship is vital for professional development in academic research and clinical practice, yet it faces challenges due to a limited number of experienced mentors and a lack of protected time for mentorship that may disproportionately affect women mentors in midcareer who are doing much of this "invisible work." The Push-Pull Mentoring Model offers a potential solution by emphasizing shared responsibility and active engagement between mentors and mentees; it fosters a flexible and collaborative approach that is mutually (though not necessarily equally) supportive of both individuals' career goals, with mentees pushing mentors up and facilitating opportunities in their realm of influence, including but not limited to sponsorship, while mentors are simultaneously pulling them up. The Push-Pull Mentoring Model provides a promising alternative to traditional mentoring models and may help institutions address the challenges associated with limited mentorship resources.
Assuntos
Tutoria , Mentores , Humanos , FemininoRESUMO
Sickle cell disease (SCD), an inherited blood disorder is due to a single amino acid substitution on the beta chain of hemoglobin, and is characterized by anemia, severe infections, acute and chronic pain, and multi-organ damage. The National Institutes of Health (NIH) is dedicated to support basic, translational and clinical science research to improve care and ultimately, to find a cure for SCD that causes such suffering. This report provides a detailed analysis of grants funded by the NIH for SCD research in Fiscal Years 2007 through 2013. During this period, the NIH supported 247 de novo grants totaling $272,210,367 that address various aspects of SCD. 83% of these funds supported research project grants investigating the following 5 scientific themes: Pathology of Sickle Red Blood Cells; Globin Gene Expression; Adhesion and Vascular Dysfunction; Neurological Complications and Organ-specific Dysfunction; and Pain Management and Intervention. The remaining 17% of total funds supported career development and training grants; Small Business Innovation Research (SBIR) and Small Business Technology Transfer (STTR) grants; large Center grants; and Conference grants. Further analysis showed that the National Heart, Lung, and Blood Institute (NHLBI) is the largest funder of SCD research within NIH with 67% of total grants, contributing 77% of total funds; followed by the National Institute for Digestive Diseases and Kidney (NIDDK) that is funding 19% of grants, contributing 13% of total funds. The remaining 14% of grants totaling 10% of the funds were supported by all other NIH Institutes/Centers (ICs) combined. In summary, the NIH is using multiple funding mechanisms to support a sickle cell disease research agenda that is intended to advance the detection, treatment, and cure of this debilitating genetic disease.
Assuntos
Anemia Falciforme/economia , Pesquisa Biomédica/economia , Organização do Financiamento/organização & administração , National Heart, Lung, and Blood Institute (U.S.)/economia , Anemia Falciforme/fisiopatologia , Anemia Falciforme/terapia , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/patologia , Adesão Celular , Eritrócitos/metabolismo , Eritrócitos/patologia , Expressão Gênica , Globinas/genética , Globinas/metabolismo , Humanos , Sistema Nervoso/fisiopatologia , Dor/tratamento farmacológico , Dor/fisiopatologia , Manejo da Dor/economia , Manejo da Dor/métodos , Transferência de Tecnologia , Estados UnidosRESUMO
ABSTRACT: Artificial Intelligence (AI) emerges as a transformative force, offering novel solutions to enhance medical education and mentorship in the specialty of Physical Medicine and Rehabilitation (PM&R). AI is a transformative technology that is being adopted in nearly every industry, In medicine, the use of AI in medical education is growing. AI may also assist with some of the challenges of mentorship, including the limited availability of experienced mentors, and the logistical difficulties of time and geography are some constraints of traditional mentorship. In this commentary, we discuss various models of AI in medical education and mentoring, including expert systems, conversational agents, and hybrid models. These models enable tailored guidance, broaden outreach within the PM&R community, and support continuous learning and development. Balancing AI's technical advantages with the essential human elements while addressing ethical considerations, AI integration into medical education and mentorship presents a paradigm shift towards a more accessible, responsive, and enriched experience in rehabilitation medicine.
RESUMO
The NifH (dimer) and ArsA proteins are structural homologs and share common motifs like nucleotide-binding domains, signal transduction domains and also possible similar metal center ligands. Given the similarity between two proteins, we investigated if the NifH protein from Azotobacter vinelandii could functionally substitute for the ArsA1 half of the ArsA protein of Escherichia coli. The chimeric NifH-ArsA2 protein was expressed and detected in the E. coli strain by Western blotting. Growth comparisons of E. coli strains containing plasmids encoding for complete ArsA, partial ArsA (ArsA2) or chimeric ArsA (NifH-ArsA2) in media with increasing sodium arsenite concentrations (0-5 mM) showed that the chimeric NifH-ArsA2 could substitute for the ArsA. This functional complementation demonstrated the strong conservation of essential domains that have been maintained in NifH and ArsA even after their divergence to perform varied functions.
Assuntos
Arsênio/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Bombas de Íon/metabolismo , Complexos Multienzimáticos/metabolismo , Oxirredutases/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Bombas de Íon/genética , Complexos Multienzimáticos/genética , Oxirredução , Oxirredutases/genética , Engenharia de Proteínas/métodosRESUMO
Four decades ago, U.S. life expectancy was within the same range as other high-income peer countries. However, during the past decades, the United States has fared worse in many key health domains resulting in shorter life expectancy and poorer health-a health disadvantage. The National Heart, Lung, and Blood Institute convened a panel of national and international health experts and stakeholders for a Think Tank meeting to explore the U.S. health disadvantage and to seek specific recommendations for implementation research opportunities for heart, lung, blood, and sleep disorders. Recommendations for National Heart, Lung, and Blood Institute consideration were made in several areas including understanding the drivers of the disadvantage, identifying potential solutions, creating strategic partnerships with common goals, and finally enhancing and fostering a research workforce for implementation research. Key recommendations included exploring why the United States is doing better for health indicators in a few areas compared with peer countries; targeting populations across the entire socioeconomic spectrum with interventions at all levels in order to prevent missing a substantial proportion of the disadvantage; assuring partnership have high-level goals that can create systemic change through collective impact; and finally, increasing opportunities for implementation research training to meet the current needs. Connecting with the research community at large and building on ongoing research efforts will be an important strategy. Broad partnerships and collaboration across the social, political, economic, and private sectors and all civil society will be critical-not only for implementation research but also for implementing the findings to have the desired population impact. Developing the relevant knowledge to tackle the U.S. health disadvantage is the necessary first step to improve U.S. health outcomes.
Assuntos
Pesquisa Biomédica , Doenças Cardiovasculares/prevenção & controle , Longevidade/fisiologia , National Heart, Lung, and Blood Institute (U.S.) , Guias de Prática Clínica como Assunto , Congressos como Assunto , Humanos , Estados UnidosRESUMO
CONTEXT: A review of interventions addressing obesity disparities could reveal gaps in the literature and provide guidance on future research, particularly for populations with a high prevalence of obesity and obesity-related cardiometabolic risk. EVIDENCE ACQUISITION: A systematic review of clinical trials in obesity disparities research that were published in 2011-2016 in PubMed/MEDLINE resulted in 328 peer-reviewed articles. Articles were excluded if they had no BMI, weight, or body composition measure as primary outcome or were foreign (n=201); were epidemiologic or secondary data analyses of clinical trials (n=12); design or protocol papers (n=54); systematic reviews (n=3); or retracted or duplicates (n=9). Forty-nine published trials were summarized and supplemented with a review of ongoing obesity disparities grants being funded by the National, Heart, Lung and Blood Institute. EVIDENCE SYNTHESIS: Of the 49 peer-reviewed trials, 27 targeted adults and 22 children only or parent-child dyads (5 of 22). Interventions were individually focused; mostly in single settings (e.g., school or community); of short duration (mostly ≤12 months); and primarily used behavioral modification (e.g., self-monitoring) strategies. Many of the trials had small sample sizes and moderate to high attrition rates. A meta-analysis of 13 adult trials obtained a pooled intervention effect of BMI -1.31 (95% CI=-2.11, -0.52, p=0.0012). Institutional review identified 140 ongoing obesity-related health disparities grants, but only 19% (n=27) were clinical trials. CONCLUSIONS: The reviews call for cardiovascular-related obesity disparities research that is long term and includes population research, and multilevel, policy, and environmental, or "whole of community," interventions.
Assuntos
Terapia Comportamental/métodos , Doenças Cardiovasculares/prevenção & controle , Disparidades nos Níveis de Saúde , Obesidade/terapia , Programas de Redução de Peso/métodos , Adulto , Doenças Cardiovasculares/epidemiologia , Doenças Cardiovasculares/etiologia , Criança , Ensaios Clínicos como Assunto , Humanos , Obesidade/complicações , Obesidade/epidemiologia , Prevalência , Projetos de Pesquisa/estatística & dados numéricos , Instituições Acadêmicas/estatística & dados numéricos , Programas de Redução de Peso/estatística & dados numéricosRESUMO
We have shown previously that the angiotensin II (Ang II) receptor AT2 reduces the intracellular levels of cGMP in Xenopus oocytes when activated by ligand binding, and the C-terminal cytoplasmic tail of the AT2 acts as a negative regulator of this function. Here we report the effects of mutations in the 2nd and 3rd intracellular loops of AT2 on AT2-mediated cGMP reduction. Mutating the highly conserved DRY motif (D141G-R142G-Y143A) of the 2nd ICL implicated in activating G(alpha) subunit of trimeric G-proteins did not affect AT2-mediated cGMP reduction. Moreover, anti-Gialpha antibody or phosphodiesterase inhibitor IBMX did not inhibit AT2-mediated cGMP reduction, suggesting that Gialpha activation and subsequent phosphodiesterase activation are not involved in this function. In contrast, mutations T250R-R251N and L255F-K256R located in the C-terminus of the 3rd ICL of AT2 retained ligand-binding properties of the wild-type AT2, and its ability to interact with the ErbB3 in yeast two-hybrid assay, but abolished AT2-mediated cGMP reduction. Similarities in the roles of ICLs of AT2 in AT2-mediated cGMP reduction in oocytes, and AT2-mediated SHP1 activation in COS-7 cells, (need of 3rd ICL for both functions and lack of involvement of DRY motif), suggest that the cascade of events in these two signaling mechanisms could be similar, and that an oocyte-specific SHP1-like protein may be involved in AT2-mediated cGMP reduction in these cells.
Assuntos
GMP Cíclico/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Feminino , Subunidades alfa de Proteínas de Ligação ao GTP/fisiologia , Técnicas In Vitro , Líquido Intracelular/metabolismo , Dados de Sequência Molecular , Mutação , Oócitos/metabolismo , Inibidores de Fosfodiesterase/farmacologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Receptor Tipo 2 de Angiotensina/química , Receptor Tipo 2 de Angiotensina/genética , Receptor ErbB-3/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo , Xenopus laevisRESUMO
Involvement of Angiotensin II (Ang II) in the regulation of sodium levels by modulating the Na+/H+ exchangers is demonstrated in many tissues. Screening of a mouse 17-day fetus cDNA library with the Angiotensin II receptor AT2 as the bait in yeast two-hybrid assay led us to identify an AT2-interacting mouse fetus peptide that shared 98% amino acid identity with the corresponding region of the human NHE6. NCBI Blast search showed that the clone 6430520C02 (GenBank Accession # AK032326) of the mouse genome project carried the complete sequence of this new mouse NHE6 isoform. The human and mouse NHE6 peptides share 97% overall homology. Further analysis showed that the region spanning the third intracellular loop and C-terminal cytoplasmic tail of the AT2 directly interacted with a 182 amino acid region that spans the predicted 5th intracellular loop and the initial part of the C-terminus of the mouse NHE6 in yeast two-hybrid assay. This 182-amino acid region that interacted with the AT2 also shares 98% homology with the corresponding region of rat NHE6 and therefore is highly conserved across species. We detected widespread expression of this NHE6 isoform in several rat tissues including 10-day fetus, 17-day fetus, and 30-day post-natal tissues of heart, brain, kidney and muscle. Moreover, the AT2 co-immunoiprecipitated with a hemagglutinin tagged NHE6 when expressed in human cell line MCF-7, and activated by AngII. This ligand-dependent complex formation between the AT2 and NHE6 suggests that the hormone Ang II may act as a regulator of NHE6, and Ang II-mediated direct protein-protein interaction between AT2 and NHE6 could be a mechanism for modulating the functions of the ubiquitously expressed NHE6 in different tissues.
Assuntos
Receptor Tipo 2 de Angiotensina/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Sequência de Aminoácidos , Angiotensina II/farmacologia , Animais , Sequência de Bases , Células Cultivadas , Sequência Conservada , Expressão Gênica , Humanos , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Ratos , Trocadores de Sódio-Hidrogênio/química , Trocadores de Sódio-Hidrogênio/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
A stop codon at position 322 was introduced to generate a truncated, C-terminal-deleted AT2 receptor. Expression studies in Xenopus oocytes showed that C-terminal-deleted AT2 had reduced affinity to [(125)I]angiotensin II (K(d)=1.7 nM) and enhanced binding of the AT2-specific peptidic ligand [(125)I]CGP42112A (K(d)=0.097 nM). AT2 activation by angiotensin II resulted in reduction of cGMP levels in oocytes and this reduction was further enhanced by C-terminal deletion, implying that the C-terminus may have a negative effect on the AT2-mediated cGMP reduction. Moreover, interaction of the AT2 with the ATP-binding domain of the human ErbB3 receptor in yeast two-hybrid assay was abolished by C-terminal deletion. In summary, the C-terminal cytoplasmic tail of AT2 modulates its ligand binding and signaling properties.
Assuntos
Angiotensina II/metabolismo , Receptores de Angiotensina/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Sequência de Bases , Códon de Terminação , GMP Cíclico/metabolismo , Primers do DNA , Humanos , Ligantes , Dados de Sequência Molecular , Ligação Proteica , Receptores de Angiotensina/química , Receptores de Angiotensina/genética , Homologia de Sequência de Aminoácidos , XenopusAssuntos
Anti-Hipertensivos/uso terapêutico , Pesquisa Biomédica , Negro ou Afro-Americano/genética , Disparidades em Assistência à Saúde , Hipertensão/tratamento farmacológico , Adulto , Negro ou Afro-Americano/estatística & dados numéricos , Distribuição por Idade , Idoso , Determinação da Pressão Arterial/métodos , Feminino , Humanos , Hipertensão/etnologia , Hipertensão/genética , Hipertensão/fisiopatologia , Masculino , Pessoa de Meia-Idade , Avaliação das Necessidades , Prevalência , Índice de Gravidade de Doença , Distribuição por Sexo , Estados Unidos/epidemiologia , População Branca/genética , População Branca/estatística & dados numéricosRESUMO
Peptidyl-prolyl cis/trans isomerases (PPIases) play a pivotal role in catalyzing the correct folding of many prokaryotic and eukaryotic proteins that are implicated in a variety of biological functions, ranging from cell cycle regulation to bacterial infection. The nif accessory protein NifM, which is essential for the biogenesis of a functional NifH component of nitrogenase, is a PPIase. To understand the nature of the molecular signature that defines the NifM dependence of NifH, we screened a library of nifH mutants in the nitrogen-fixing bacterium Azotobacter vinelandii for mutants that acquired NifM independence. Here, we report that NifH can acquire NifM independence when the conserved Pro258 located in the C-terminal region of NifH, which wraps around the other subunit in the NifH dimer, is replaced by serine.
Assuntos
Azotobacter vinelandii/enzimologia , Oxirredutases/genética , Oxirredutases/metabolismo , Peptidilprolil Isomerase/metabolismo , Sequência de Aminoácidos , Azotobacter vinelandii/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação ProteicaRESUMO
Azotobacter vinelandii belongs to the y subdivision of eubacteria and has one of the highest respiratory rates. It is considered to be among the probable progenitors of mitochondria. Group II introns were originally identified on organelle genomes. Analysis of the A. vinelandii genome for the presence of group II introns using a deduced group II intron consensus sequence identified two putative introns. The first intron (AVI) which was found to be inserted in the groEL, an essential gene, was already characterized. Our study identified another group II intron (AV2) in A. vinelandii genome. This intron is inserted in a mobile genetic element, similar to most of the group II introns in bacteria, which in this case is a transposase like gene, tnpAl. This putative TnpAl protein is 52% identical to TnpA, the transposase of bacteriophage Lambda, and 85% identical to TnpAl of Pseudomonas stutzeri. Sequence analysis showed that this intron encodes a reverse transcriptase (RT) like motif in domain IV, similar to other group II introns. The RT of this intron open reading frame (ORF) is 53% homologous with that of AVI intron and 66% homologous with that of Pseudomonas putida (Tn5041c) intron. Secondary structure analysis showed that this intron has the typical sub-group IIB1 structure, but the EBS2-IBS2 interaction appears to be missing. Using the RNA generated by in vitro transcription of the intron sequence with its flanking exons, in vitro splicing experiments were performed. It was found that the AV2 intron is functional, despite of lacking the EBS2-IBS2 interaction that plays a role in exon recognition.
Assuntos
Azotobacter vinelandii/genética , Genoma Bacteriano , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Bacterianos/genética , DNA Bacteriano/genética , Íntrons , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Splicing de RNA , RNA Bacteriano/química , RNA Bacteriano/genética , Homologia de Sequência de AminoácidosRESUMO
The MoFe protein of the complex metalloenzyme nitrogenase folds as a heterotetramer containing two copies each of the homologous alpha and beta subunits, encoded by the nifD and the nifK genes respectively. Recently, the functional expression of a fusion NifD-K protein of nitrogenase was demonstrated in Azotobacter vinelandii, strongly implying that the MoFe protein is flexible as it could accommodate major structural changes, yet remain functional [M.H. Suh, L. Pulakat, N. Gavini, J. Biol. Chem. 278 (2003) 5353-5360]. This finding led us to further explore the type of interaction between the fused MoFe protein units. We aimed to determine whether an interaction exists between the two fusion MoFe proteins to form a homodimer that is equivalent to native heterotetrameric MoFe protein. Using the Bacteriomatch Two-Hybrid System, translationally fused constructs of NifD-K (fusion) with the full-length lambdaCI of the pBT bait vector and also NifD-K (fusion) with the N-terminal alpha-RNAP of the pTRG target vector were made. To compare the extent of interaction between the fused NifD-K proteins to that of the beta-beta interactions in the native MoFe protein, we proceeded to generate translationally fused constructs of NifK with the alpha-RNAP of the pTRG vector and lambdaCI protein of the pBT vector. The strength of the interaction between the proteins in study was determined by measuring the beta-galactosidase activity and extent of ampicillin resistance of the colonies expressing these proteins. This analysis demonstrated that direct protein-protein interaction exists between NifD-K fusion proteins, suggesting that they exist as homodimers. As the interaction takes place at the beta-interfaces of the NifD-K fusion proteins, we propose that these homodimers of NifD-K fusion protein may function in a similar manner as that of the heterotetrameric native MoFe protein. The observation that the extent of protein-protein interaction between the beta-subunits of the native MoFe protein in BacterioMatch Two-Hybrid System is comparable to the extent of protein-protein interaction observed between the NifD-K fusion proteins in the same system further supports this idea.
Assuntos
Azotobacter vinelandii/enzimologia , Molibdoferredoxina/metabolismo , Azotobacter vinelandii/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Dimerização , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Estrutura Molecular , Molibdoferredoxina/química , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias , beta-Galactosidase/metabolismoRESUMO
Studies on Angiotensin II (Ang II) receptor type AT1 have suggested that interaction between the two highly conserved residues, Tyr292 in the 7th transmembrane domain (TMD) and the Asp74 in the 2nd TMD, is critical for linking the Ang II binding and AT1 receptor-Gq protein coupling. In the Ang II receptor type AT2, the Asp is conserved (Asp90 in 2nd TMD), however, there is no Tyr residue in the 7th TMD and Phe308 occupies the analogous position to Tyr292 of the AT1. Replacing this Phe308 with Ala reduced receptor affinity to peptidic ligands (125)I-Ang II (K(d) = 0.37 nM) and (125)I-CGP42112A (K(d) = 0.56 nM), but retained the ability of the AT2 to reduce cGMP levels in Xenopus oocytes. Thus, the Phe308 of the AT2 does not mimic the role of Tyr292 of the AT1 in the receptor activation upon Ang II binding. We have also shown that the M8 mutant of the AT2 with the 7th TMD similar to that of wild type AT2 can couple to PLC like the AT1 and bind the AT2-specific ligands with high affinity. Since the Ang II is shown to bind to both the AT1 and the AT2 in an identical manner, we propose that the absence of Tyr in the 7th TMD of the AT2 does not prevent the receptor from coupling to Gq-protein, rather may contribute to the freedom of the AT2 to couple to trimeric G-proteins in both G- betagamma dependent and independent manners upon Ang II binding.
Assuntos
Angiotensina II/metabolismo , Fenilalanina/metabolismo , Receptor Tipo 2 de Angiotensina/genética , Receptor Tipo 2 de Angiotensina/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , CMP Cíclico/metabolismo , Humanos , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oócitos/fisiologia , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Receptor Tipo 2 de Angiotensina/química , Tirosina/metabolismo , XenopusRESUMO
The nifEN encodes an E2N2 tetrameric metalloprotein complex that serves as scaffold for assembly of the FeMo cofactor of nitrogenase. In most diazotrophs, the NifE and NifN are translated as separate polypeptides and then assembled into tetrameric E2N2 complex. However, in Anabaena variabilis which has two nif clusters that encode two different NifEN complexes, the NifEN2 is encoded by a single nifE-N like gene, which has high homology to the NifE at amino-terminus and to the NifN at the carboxy-terminus. These observations implied that a metalloprotein like NifEN can accommodate large variations in their amino acid composition and also in the way they are synthesized (as two separate proteins or as a single protein) and yet remain functional. In Azotobacter vinelandii NifE and NifN are synthesized separately. To test whether NifEN could retain its functionality when encoded by a single gene, we generated a translational fusion of the nifE and nifN genes of A. vinelandii that could encode a large NifE-N fusion protein. When expressed in the nifEN-minus strain of A. vinelandii, the nifE-N gene fusion could complement the NifEN function. Western blot analysis by using polyclonal NifEN antibodies revealed that the complementing nifEN product is a large NifE-N fusion protein unit. The fact that the gene fusion of nifE-N specifies a functional NifE-N fusion protein reflects that these metalloproteins can accommodate a wide range of flexibility in their gene organization, structure, and assembly.
Assuntos
Azotobacter vinelandii/metabolismo , Genes Bacterianos , Metaloproteínas/genética , Metaloproteínas/fisiologia , Molibdoferredoxina/metabolismo , Sequência de Aminoácidos , Fusão Gênica Artificial , Azotobacter vinelandii/citologia , Azotobacter vinelandii/genética , Sequência de Bases , Divisão Celular , Substâncias Macromoleculares , Metaloproteínas/biossíntese , Dados de Sequência Molecular , Plasmídeos , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Alinhamento de SequênciaRESUMO
Studies utilizing several physical, biochemical and spectroscopic methods have suggested that Azotobacter vinelandii contains multiple copies (40-80) of its chromosome per cell, whereas genetic analysis indicated that these cells function like haploid cells. To further verify if A. vinelandii indeed contains 40-80 copies of its chromosome per cell, we have developed an 'in vivo chromosome counting' technique. The basic principle of this technique is to introduce the same genetic marker on the chromosome and on an extrachromosomal element of known copy number into the bacterium. The copy number of the chromosome can be determined by comparing the intensity of the hybridization signal generated by the DNA fragment carrying the chromosomal marker with that of the extrachromosomal marker when the total DNA isolated from this strain is hybridized with a probe made of the same genetic marker DNA. To do this we used an A. vinelandii BG102 strain which carries a kanamycin resistance marker gene integrated into the nifY locus on its chromosome(s). The plasmids pRK293 and pKT230, which can replicate in A. vinelandii and carry the kanamycin resistance gene (similar to the one present on the chromosome of A. vinelandii BG102), served as the extrachromosomal elements with known copy number. Southern blotting and hybridization analysis of the total DNA, isolated from A. vinelandii BG102 containing these plasmids, with a probe made of the kanamycin resistance gene clearly indicated that the copy number of A. vinelandii chromosome is slightly lower than the copy number of the low-copy plasmid pRK293 and about 21-fold lower than the copy number of the high copy plasmid pKT230. We believe that this 'in vivo chromosome counting' technique can be used for determination of the copy number of the chromosome in other cells with appropriate modifications in the nature of the extrachromosomal element and the genetic marker.
Assuntos
Azotobacter vinelandii/genética , Cromossomos Bacterianos , Análise Citogenética , Resistência a Canamicina/genética , Marcadores Genéticos , Plasmídeos/genética , PoliploidiaRESUMO
The MoFe protein component of the complex metalloenzyme nitrogenase is an alpha2beta2 tetramer encoded by the nifD and the nifK genes. In nitrogen fixing organisms, the alpha and beta subunits are translated as separate polypeptides and then assembled into tetrameric MoFe protein complex that includes two types of metal centers, the P cluster and the FeMo cofactor. In Azotobacter vinelandii, the NifEN complex, the site for biosynthesis of the FeMo cofactor, is an alpha2beta2 tetramer that is structurally similar to the MoFe protein and encoded as two separate polypeptides by the nifE and the nifN genes. In Anabaena variabilis it was shown that a NifE-N fusion protein encoded by translationally fused nifE and nifN genes can support biological nitrogen fixation. The structural similarity between the MoFe protein and the NifEN complex prompted us to test whether the MoFe protein could also be functional when synthesized as a single protein encoded by nifD-K translational fusion. Here we report that the NifD-K fusion protein encoded by nifD-K translational fusion in A. vinelandii is a large protein (as determined by Western blot analysis) and is capable of supporting biological nitrogen fixation. These results imply that the MoFe protein is flexible in that it can accommodate major structural changes and remain functional.