RESUMO
This year, 2019, marks the centennial of embryologist E. E. Just's discovery of what is known as the fast block to polyspermy. Just's observation of the subtle changes that occur at the egg's surface during fertilization (and in experimental parthenogenesis) led him to postulate that the egg, and indeed every cell, possesses a property he called independent irritability, which represents the cell's ability to respond in a physiologically-relevant way to a variety of signals or triggers. In this paper, I argue that Just's concept of independent irritability informed his contemporary Johannes Holtfreter as Holtfreter attempted to explain the phenomena of embryonic induction and competence and that Holtfreter, in turn, influenced Marc Kirschner and John Gerhart in their formulation of the theory of facilitated variation. Just's influence is especially evident in Gerhart and Kirschner's presentations of what they call weak linkage-a property of living systems that allows core processes and components to be mixed and matched in different ways to generate novel traits. Unfortunately, the connection between Holtfreter's work and Just's has remained hidden. This paper gives examples of phenomena that exhibit weak linkage, and it lays out the case that Just's concept of independent irritability, through Holtfreter, Gerhart, and Kirschner, has broadly infiltrated modern cell and developmental biology.
Assuntos
Biologia Celular/história , Biologia do Desenvolvimento/história , Poliploidia , Interações Espermatozoide-Óvulo/fisiologia , Animais , Indução Embrionária/fisiologia , Feminino , História do Século XX , História do Século XXI , Masculino , Oócitos/fisiologia , Espermatozoides/fisiologiaRESUMO
Recently, we showed that the fused chorismate-utilizing enzyme from the antibiotic-producing soil bacterium Streptomyces venezuelae is an anthranilate synthase (designated SvAS), not a 2-amino-2-deoxyisochorismate (ADIC) synthase, as was predicted based on its amino acid sequence similarity to the phenazine biosynthetic enzyme PhzE (an ADIC synthase). Here, we report the characterization of SvAS using steady-state kinetics, gel filtration chromatography, and laser light scattering. The recombinant His-tagged enzyme has Michaelis constants Km with respect to substrates chorismate and glutamine of 8.2 ± 0.2 µM and 0.84 ± 0.05 mM, respectively, and a catalytic rate constant k cat of 0.57 ± 0.02 s(-1) at 30 °C. Unlike most other anthranilate synthases, SvAS does not utilize ammonia as a substrate. The enzyme is competitively but non-cooperatively inhibited by tryptophan (K i = 11.1 ± 0.1 µM) and is active as a monomer. The finding that SvAS is a monomer jibes with the variety of association modes that have been observed for anthranilate synthases from different microorganisms, and it identifies the enzyme's minimal functional unit as a single TrpE-TrpG pair.
Assuntos
Antranilato Sintase/química , Catálise , Streptomyces/enzimologia , Sequência de Aminoácidos/genética , Antranilato Sintase/genética , Cinética , Estrutura Terciária de Proteína , Especificidade por Substrato , TriptofanoRESUMO
Ernest Everett Just (1883-1941) was an African American embryologist of international standing whose research interests lay in the area of fertilization and early development in marine invertebrates. Perhaps best known for his discovery of the dynamical and structural blocks to polyspermy that sweep over the egg upon fertilization, E. E. Just also was the first to associate cell surface changes with stages of embryonic development. He was deeply familiar with the natural history of the animals whose eggs he studied, and his knowledge of natural settings led him to emphasize the importance of using laboratory conditions that closely match those in nature. Based on more than 30 years of work, he came to believe that it was the cell surface that played the most critical role in development, heredity, and evolution. He promoted a holistic view of cells and organisms in opposition to the gene-centric view that was becoming more prevalent with the rise of genetics, but rejected the vitalism espoused by some biologists of his era, calling instead for "a physics and chemistry in a new dimension superimposed upon the now known physics and chemistry" to account for biological phenomena. Just's incisive critique of genetic reductionism finds echoes in contemporary multiscale, systems approaches in biology. His speculations on the relationship between developmental and evolutionary mechanisms resonate with today's evolutionary developmental biology. After a brief biographical sketch, this paper outlines and discusses some of Just's scientific contributions, and shows how his ideas remain relevant today.
Assuntos
Invertebrados/embriologia , Invertebrados/genética , Óvulo/fisiologia , Interações Espermatozoide-Óvulo , Animais , Organismos Aquáticos , Evolução Biológica , Desenvolvimento Embrionário , História do Século XIX , História do Século XX , PartenogêneseRESUMO
As part of an overall project to characterize the streptomycin phosphotransferase enzyme APH(6)-Id, which confers bacterial resistance to streptomycin, we cloned, expressed, purified, and characterized the enzyme. When expressed in Escherichia coli, the recombinant enzyme increased by up to 70-fold the minimum inhibitory concentration needed to inhibit cell growth. Size-exclusion chromatography gave a molecular mass of 31.4 ± 1.3 kDa for the enzyme, showing that it functions as a monomer. Activity was assayed using three methods: (1) an HPLC-based method that measures the consumption of streptomycin over time; (2) a spectrophotometric method that utilizes a coupled assay; and (3) a radioenzymatic method that detects production of (32)P-labeled streptomycin phosphate. Altogether, the three methods demonstrated that streptomycin was consumed in the APH(6)-Id-catalyzed reaction, ATP was hydrolyzed, and streptomycin phosphate was produced in a substrate-dependent manner, demonstrating that APH(6)-Id is a streptomycin phosphotransferase. Steady-state kinetic analysis gave the following results: K(m)(streptomycin) of 0.38 ± 0.13 mM, K(m)(ATP) of 1.03 ± 0.1 mM, V(max) of 3.2 ± 1.1 µmol/min/mg, and k(cat) of 1.7 ± 0.6 s(-1). Our study demonstrates that APH(6)-Id is a bona fide streptomycin phosphotransferase, functions as a monomer, and confers resistance to streptomycin.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/química , Fosfotransferases (Aceptor do Grupo Álcool)/química , Estreptomicina/farmacologia , Antibacterianos/química , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Farmacorresistência Bacteriana , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Cinética , Testes de Sensibilidade Microbiana , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Streptomyces/enzimologia , Estreptomicina/químicaRESUMO
Ernest E. Just (1883-1941) is best known for his discovery of the "wave of negativity" that sweeps of the sea urchin egg during fertilization, and his elucidation of what are known as the fast and slow blocks to polyspermy. Just's contemporary Johannes Holtfreter (1901-1992) is known for his pioneering work in amphibian morphogenesis, which helped to lay the foundation for modern vertebrate developmental biology. This paper, after briefly describing the life and scientific contributions of Just, argues that his work and ideas strongly influenced two of the concepts for which Holtfreter is best known: tissue affinity and autoneuralization (or autoinduction). Specifically, this paper argues that, first, Just's experiments demonstrating developmental stage-specific changes in the adhesiveness of the blastomeres of cleavage embryos helped lay the foundation for Holtfreter's concept of tissue affinity and, second, Just's notion of the intrinsic irritability of the egg cell, which is evident in experimental parthenogenesis, strongly informed Holtfreter's concept of the nonspecific induction of neural tissue formation in amphibian gastrula ectoderm explants, a phenomenon known as autoinduction. Acknowledgment of these contributions by Just in no way diminishes the importance of Holtfreter's groundbreaking work. It does, however, extend the impact of Just's work into the area of embryo morphogenesis. It connects Just to Holtfreter and positions his work as an antecedent to embryo research that continues to this day.
Assuntos
Embriologia/história , Europa (Continente) , História do Século XX , Humanos , Estados UnidosRESUMO
The chloramphenicol producer Streptomyces venezuelae contains an enzyme, SvTrpEG, that has a high degree of amino acid sequence similarity to the phenazine biosynthetic enzyme PhzE of certain species of Pseudomonas. PhzE has the sequence signature of an anthranilate synthase, but recent evidence indicates that it catalyzes the production of 2-amino-2-deoxyisochorismate [corrected] (ADIC), an intermediate in the two-step anthranilate synthase reaction, not anthranilate. In order to determine if SvTrpEG is likewise an ADIC synthase, we have cloned the gene for SvTrpEG, expressed the recombinant enzyme in Escherichia coli, and purified the enzyme. Analysis of the SvTrpEG-catalyzed reaction mixture using UV-visible spectrophotometry, fluorescence spectrometry, and high-performance liquid chromatography shows that the product of the reaction is anthranilate, not ADIC. Our results therefore reveal that, despite its sequence similarity to PhzE, SvTrpEG is an anthranilate synthase, not an ADIC synthase.
Assuntos
Antranilato Sintase/genética , Genes Bacterianos/genética , Streptomyces/enzimologia , Streptomyces/genética , Antranilato Sintase/metabolismo , Sequência de Bases , Catálise , Ácido Corísmico/metabolismo , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Dados de Sequência Molecular , Plasmídeos/genética , Reação em Cadeia da Polimerase , Pseudomonas/enzimologia , Pseudomonas/genética , Salmonella/enzimologia , Salmonella/genética , Termodinâmica , Transformação GenéticaRESUMO
First put forth in June 2005, the altered nuclear transfer-oocyte assisted reprogramming (ANT-OAR) proposal has been promoted as an ethically-acceptable alternative to the embryo-destructive methods now used to obtain embryonic stem cells. According to its proponents, the goal of ANT-OAR is to use the cloning process to create a pluripotent stem cell. This would be achieved through overexpression of the transcription factor Nanog (or a hypothetical substitute) both in the enucleated egg cell and in the somatic cell prior to transfer of its nucleus. Although the ethical acceptability of ANT-OAR has been publicly debated, its scientific feasibility has not. This paper aims to help rectify this situation. It argues that ANT-OAR, as currently conceived, cannot realistically work. It presents evidence from the scientific literature showing that Nanog cannot single-handedly establish pluripotency in cells, but rather works together with a network of other transcription factors to maintain pluripotency. It argues that ANT-OAR is based on a flawed understanding of stem cell biology, and emphasizes that, in this debate about embryonic stem cells, scientists must strive to accurately and realistically assess the feasibility of the embryo research strategies they propose.
Assuntos
Reprogramação Celular/fisiologia , Pesquisas com Embriões , Bases de Conhecimento , Técnicas de Transferência Nuclear , Oócitos/citologia , Projetos de Pesquisa , Animais , Fator de Transcrição CDX2 , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Pesquisas com Embriões/ética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Humanos , Proteína Homeobox Nanog , Células-Tronco Pluripotentes/citologiaRESUMO
Renowned biologist Ernest Everett Just (1883-1941) was an outspoken advocate for the classical embryologist's view of the cell; he believed that all the parts of the cell, but especially the cytoplasm, have important roles to play in the process of development, whereby a one-celled zygote becomes a many-celled animal. In opposition to geneticist Thomas Hunt Morgan, Just formulated a hypothesis for how the cell works in development, one that gave a more dominant role to cytoplasmic (instead of nuclear) factors. This paper argues that, in creating his hypothesis, Just applied insights from the African American intellectual community in which he was immersed, much as Charles Darwin applied insights from British political economist Thomas R. Malthus in formulating his theory of evolution by natural selection. This in no way diminishes the scientific validity of Just's (or Darwin's) hypothesis. Rather, it highlights Just's creativity and, as such, points to the importance of having diversity in science.
RESUMO
In the current study, we developed a HPLC method to quantitatively measure the permeability of the BpT-based chelators, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and 2-benzoylpyridine 4-allyl-3-thiosemicarbazone (Bp4aT), across human colorectal adenocarcinoma (Caco-2) monolayers as a model of gut absorption. In aqueous solution, Bp4eT and Bp4aT formed inter-convertible Z and E isomers that were resolved by HPLC. Peak area was linear with respect to chelator concentration. Acceptable within-day and between-day precision (<22%) and accuracy (85-115% of true values) were obtained over a range of 1.0-100µM for Bp4eT and 1.5-300µM for Bp4aT. Limits of detection were 0.3µM and 1µM for Bp4eT and Bp4aT, respectively, while corresponding limits of quantification were 1µM and 5µM. Both chelators showed significant ability to chelate iron in THP-1 cells using a calcein-based assay and no apparent cytotoxicity was observed within 24h. Ratios of the apical to basolateral and basolateral to apical transport for Bp4eT were 1.10 and 0.89 at 100µM and 300µM respectively, indicating equal bi-directional movement of the compounds. Similarly, ratios were 0.77 and 0.92 for Bp4aT, respectively. This study demonstrates that Bp4eT and Bp4aT can be efficiently transported through Caco-2 cells and can potentially be formulated for oral delivery.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Quelantes de Ferro/análise , Quelantes de Ferro/farmacocinética , Tiossemicarbazonas/análise , Tiossemicarbazonas/farmacocinética , Células CACO-2 , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Ferro/metabolismo , Quelantes de Ferro/química , Quelantes de Ferro/farmacologia , Isomerismo , Isoquinolinas/metabolismo , Limite de Detecção , Modelos Biológicos , Reprodutibilidade dos Testes , Tiossemicarbazonas/química , Tiossemicarbazonas/farmacologiaAssuntos
Início da Vida Humana , Clonagem de Organismos/ética , Embrião de Mamíferos , Técnicas de Transferência Nuclear , Criação de Embriões para Pesquisa/ética , Células-Tronco , Embrião de Mamíferos/citologia , Desenvolvimento Embrionário , Determinismo Genético , Engenharia Genética/ética , Humanos , Biologia de SistemasRESUMO
Ecological developmental biology (Eco-Devo) involves the study of development in its natural environmental context as opposed to the laboratory setting. Ernest E. Just was an early 20th century African-American embryologist who devoted his career to studying the early development of marine invertebrates in the United States and abroad. Through detailed study of the fertilization process, he came to see the cell cortex as playing a central role in development, inheritance, and evolution. This paper, after presenting some of Just's scientific and philosophical contributions, argues that Just was an Eco-Devo biologist. Three lines of evidence are given. First, Just believed that intimate knowledge of the natural history of the marine animal under study--hence, the natural setting in which fertilization occurs--was essential. Second, he stressed the importance of the egg's "normality"--how well its condition in the laboratory corresponds to the natural, fertilizable state. Finally, Just was an organicist, believing that organisms are holistic systems with emergent properties that arise from their organization and complexity. Although other scientists may stand out more clearly as founding architects of Eco-Devo, E. E. Just, with his unwavering insistence on the normality and holistic integrity of the egg cell, was one of its purest adherents.
Assuntos
Biologia do Desenvolvimento/história , Ecologia/história , Embriologia/história , Animais , História do Século XIX , História do Século XX , Estados UnidosRESUMO
Thermostable anthranilate synthase from the marine sulfate-reducing hyperthermophile Archaeoglobus fulgidus has been expressed in Escherichia coli, purified, and characterized. The functional enzyme is an alpha2beta2 heterotetrameric complex of molecular mass 150+/-15 kDa. It is composed of two TrpE (50 kDa) and two TrpG (18 kDa) subunits. The extrinsic factors glycerol (25%) and potassium chloride (2 M) stabilized the recombinant enzyme against thermal inactivation. In the presence of these extrinsic factors, the enzyme was highly thermostable, exhibiting a half-life of thermal inactivation of about 1 h at 85 degrees C. The kinetic constants for the enzyme under these conditions were: Km (chorismate) 84 microM, Km (glutamine) 7.0 mM, kcat 0.25 s(-1), and pH optimum 8.0. The enzyme was competitively, though non-cooperatively, inhibited by tryptophan.
Assuntos
Antranilato Sintase/metabolismo , Archaeoglobus fulgidus/enzimologia , Antranilato Sintase/antagonistas & inibidores , Antranilato Sintase/química , Antranilato Sintase/genética , Archaeoglobus fulgidus/genética , Estabilidade Enzimática/efeitos dos fármacos , Genes Arqueais , Glicerol/farmacologia , Cinética , Peso Molecular , Cloreto de Potássio/farmacologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , Triptofano/farmacologiaRESUMO
The DNA polymerase I from Thermus aquaticus (Taq polymerase) performs lagging-strand DNA synthesis and DNA repair. Taq polymerase contains a polymerase domain for synthesizing a new DNA strand and a 5'-nuclease domain for cleaving RNA primers or damaged DNA strands. The extended crystal structure of Taq polymerase poses a puzzle on how this enzyme coordinates its polymerase and the nuclease activities to generate only a nick. Using contrast variation solution small angle neutron scattering, we have examined the conformational changes that occur in Taq polymerase upon binding "overlap flap" DNA, a structure-specific DNA substrate that mimics the substrate in strand replacement reactions. In solution, apoTaq polymerase has an overall expanded equilibrium conformation similar to that in the crystal structure. Upon binding to the DNA substrate, both the polymerase and the nuclease domains adopt more compact overall conformations, but these changes are not enough to bring the two active sites close enough to generate a nick. Reconstruction of the three-dimensional molecular envelope from small angle neutron scattering data shows that in the DNA-bound form, the nuclease domain is lifted up relative to its position in the non-DNA-bound form so as to be in closer contact with the thumb and palm subdomains of the polymerase domain. The results suggest that a form of structure sensing is responsible for the coordination of the polymerase and nuclease activities in nick generation. However, interactions between the polymerase and the nuclease domains can assist in the transfer of the DNA substrate from one active site to the other.