Flammability conditions for ultra-lean hydrogen premixed combustion based on flame-ball analyses

It has been reasoned that the structures of strongly cellular flames in very lean mixtures approach an array of flame balls, each burning as if it were isolated, thereby indicating a connection between the critical conditions required for existence of steady flame balls and those necessary for occurrence of self-sustained premixed combustion. This is the starting assumption of the present study, in which structures of near-limit steady sphericosym-metrical flame balls are investigated with the objective of providing analytic expressions for critical combustion conditions in ultra-lean hydrogen-oxygen mixtures diluted with N2 and water vapor. If attention were restricted to planar premixed flames, then the lean-limit mole fraction of H2 would be found to be roughly ten percent, more than twice the observed flammability limits, thereby emphasizing the relevance of the flame-ball phenomena. Numerical integrations using detailed models for chemistry and radiation show that a onestep chemical-kinetic reduced mechanism based on steady-state assumptions for all chemical intermediates, together with a simple, optically thin approximation for water-vapor radiation, can be used to compute near-limit fuel-lean flame balls with excellent accuracy. The previously developed one-step reaction rate includes a crossover temperature that determines in the first approximation a chemical-kinetic lean limit below which combustión cannot occur, with critical conditions achieved when the diffusion-controlled radiation-free peak temperature, computed with account taken of hydrogen Soret diffusion, is equal to the crossover temperature. First-order corrections are found by activation-energy asymptotics in a solution that involves a near-field radiation-free zone surrounding a spherical flame sheet, together with a far-field radiation-conduction balance for the temperature profile. Different scalings are found depending on whether or not the surrounding atmosphere contains wáter vapor, leading to different analytic expressions for the critical conditions for flame-ball existence, which give results in very good agreement with those obtained by detailed numerical computations.

Effects of radiation losses on very lean methane/air flames propagating upward in a vertical tube

The stationary upward propagation of a very lean methane/air flame in a long vertical tube open at the bottom and closed at the top is simulated numerically using a single overall chemical reaction to model combustion and assuming an optically thin gas and a transparent or non-reflecting tube wall to approximately account for radiation losses from CO2CO2 and H2OH2O. Buoyancy plays a dominant role in the propagation of these flames and causes a large region of low velocity of the burnt gas relative to the flame to appear below the flame front when the equivalence ratio is decreased. The size of this region scales with the radius of the tube, and its presence enhances the effect of radiation losses, which would be otherwise negligible for a standard flammability tube, given the small concentration of radiating species. Heat conduction is found to be important in the low velocity region and to lead to a conduction flux from the flame to the burnt gas that causes extinction at the flame tip for a value of the equivalence ratio near the flammability limit experimentally measured in the standard tube. The effect of radiation losses decreases with the radius of the tube. Numerical results and order-of-magnitude estimates show that, in the absence of radiation, a very lean flame front fails to propagate only after recirculation of the burnt gas extends to its reaction region and drastically changes its structure. This condition is not realized for the standard flammability tube, but it seems to account for the flammability limit measured in a tube of about half the radius of the standard tube.

The HOSHIN KANRI TREE. Cross-Plant Lean Shopfloor Management

Shopfloor Management (SM) empowerment methodologies have traditionally focused on two aspects: goal achievement following rigid structures, such as SQDCME, or evolutional aspects of empowerment factors away from strategic goal achievement. Furthermore, SM Methodologies have been organized almost solely around the hierarchical structure of the organization, failing systematically to cope with the challenges that Industry 4.0 is facing. The latter include the growing complexity of value-stream networks, sustainable empowerment of the workforce (Learning Factory), an autonomous and intelligent process management (Smart Factory), the need to cope with the increasing complexity of value-stream networks (VSN) and the leadership paradigm shift to strategic alignment. This paper presents a novel Lean SM Method (LSM) called ?HOSHIN KANRI Tree? (HKT), which is based on standardization of the communication patterns among process owners (POs) by PDCA. The standardization of communication patterns by HKT technology should bring enormous benefits in value stream (VS) performance, speed of standardization and learning rates to the Industry 4.0 generation of organizations. These potential advantages of HKT are being tested at present in worldwide research.

El análisis de la construcción sin pérdidas (Lean Construction) y su relación con el Project & Construction Management : propuesta de regulación en España y su inclusión en la ley de la ordenación de la edificación

El presente trabajo se basa en la filosofía de la Construcción sin Pérdidas (“Lean Construction”), analizando la situación de esta filosofía en el sector de la edificación en el contexto internacional y español, respondiendo las siguientes preguntas: 1. ¿Cómo surge el “Lean Construction”? 2. ¿Cuáles son sus actividades, funciones y cometidos? 3. ¿Existe regulación del ¨Lean Construction” en otros países? 4. ¿Existe demanda del ¨Lean Construction” en España? 5. ¿Existe regulación del ¨Lean Construction” en España? 6. ¿Cómo debería ser la regulación ¨Lean Construction” en España? 7. ¿Cuál es la relación del “Lean Construction” con el “Project & Construction Management”? 8. ¿Cómo debería ser la regulación de “Lean Construction” en España considerando su relación con el “Project & Construction Management”? Las preguntas indicadas las hemos respondido detalladamente en el presente trabajo, a continuación se resume las respuestas a dichas preguntas: 1. El “Lean Construction” surge en agosto de 1992, cuando el investigador finlandés Lauri Koskela publicó en la Universidad de Stanford el reporte TECHNICAL REPORT N° 72 titulado “Application of the New Production Philosophy to Construction”. Un año más tarde el Dr. Koskela invitó a un grupo de especialistas en construcción al primer workshop de esta materia en Finlandia, dando origen al International Group for Lean Construction (IGLC) lo que ha permitido extender la filosofía a EEUU, Europa, América, Asia, Oceanía y África. “Lean Construction” es un sistema basado en el enfoque “Lean Production” desarrollado en Japón por Toyota Motors a partir de los años cincuenta, sistema que permitió a sus fábricas producir unidades con mayor eficiencia que las industrias americanas, con menores recursos, en menor tiempo, y con un número menor de errores de fabricación. 2. El sistema “Lean Construction” busca maximizar el valor y disminuir las pérdidas de los proyectos generando una coordinación eficiente entre los involucrados, manejando un proyecto como un sistema de producción, estrechando la colaboración entre los participantes de los proyectos, capacitándoles y empoderándoles, fomentando una cultura de cambio. Su propósito es desarrollar un proceso de construcción en el que no hayan accidentes, ni daños a equipos, instalaciones, entorno y comunidad, que se realice en conformidad con los requerimientos contractuales, sin defectos, en el plazo requerido, respetando los costes presupuestados y con un claro enfoque en la eliminación o reducción de las pérdidas, es decir, las actividades que no generen beneficios. El “Last Planner System”, o “Sistema del Último Planificador”, es un sistema del “Lean Construction” que por su propia naturaleza protege a la planificación y, por ende, ayuda a maximizar el valor y minimizar las pérdidas, optimizando de manera sustancial los sistemas de seguridad y salud. El “Lean Construction” se inició como un concepto enfocado a la ejecución de las obras, posteriormente se aplicó la filosofía a todas las etapas del proyecto. Actualmente considera el desarrollo total de un proyecto, desde que nace la idea hasta la culminación de la obra y puesta en marcha, considerando el ciclo de vida completo del proyecto. Es una filosofía de gestión, metodologías de trabajo y una cultura empresarial orientada a la eficiencia de los procesos y flujos. La filosofía “Lean Construction” se está expandiendo en todo el mundo, además está creciendo en su alcance, influyendo en la gestión contractual de los proyectos. Su primera evolución consistió en la creación del sistema “Lean Project Delivery System”, que es el concepto global de desarrollo de proyectos. Posteriormente, se proponen el “Target Value Design”, que consiste en diseñar de forma colaborativa para alcanzar los costes y el valor requerido, y el “Integrated Project Delivery”, en relación con sistemas de contratos relacionales (colaborativos) integrados, distintos a los contratos convencionales. 3. Se verificó que no existe regulación específica del ¨Lean Construction” en otros países, en otras palabras, no existe el agente con el nombre específico de “Especialista en Lean Construction” o similar, en consecuencia, es un agente adicional en el proyecto de la edificación, cuyas funciones y cometidos se pueden solapar con los del “Project Manager”, “Construction Manager”, “Contract Manager”, “Safety Manager”, entre otros. Sin embargo, se comprobó la existencia de formatos privados de contratos colaborativos de Integrated Project Delivery, los cuales podrían ser tomados como unas primeras referencias para futuras regulaciones. 4. Se verificó que sí existe demanda del ¨Lean Construction” en el desarrollo del presente trabajo, aunque aún su uso es incipiente, cada día existe más interesados en el tema. 5. No existe regulación del ¨Lean Construction” en España. 6. Uno de los objetivos fundamentales de esta tesis es el de regular esta figura cuando actúe en un proyecto, definir y realizar una estructura de Agente de la Edificación, según la Ley de Ordenación de la Edificación (LOE), y de esta manera poder introducirla dentro de la Legislación Española, protegiéndola de eventuales responsabilidades civiles. En España existe jurisprudencia (sentencias de los tribunales de justicia españoles) con jurisdicción civil basada en la LOE para absolver o condenar a agentes de la edificación que son definidos en los tribunales como “gestores constructivos” o similares. Por este motivo, en un futuro los tribunales podrían dictaminar responsabilidades solidarias entre el especialista “Lean Construction” y otros agentes del proyecto, dependiendo de sus actuaciones, y según se implemente el “Lean Project Delivery System”, el “Target Value Design” y el “Integrated Project Delivery”. Por otro lado, es posible que el nivel de actuación del especialista “Lean Construcción” pueda abarcar la gestión del diseño, la gestión de la ejecución material (construcción), la gestión de contratos, o la gestión integral de todo el proyecto de edificación, esto último, en concordancia con la última Norma ISO 21500:2012 o UNE-ISO 21500:2013 Directrices para la dirección y gestión de proyectos. En consecuencia, se debería incorporar adecuadamente a uno o más agentes de la edificación en la LOE de acuerdo a sus funciones y responsabilidades según los niveles de actuación del “Especialista en Lean Construction”. Se propone la creación de los siguientes agentes: Gestor del Diseño, Gestor Constructivo y Gestor de Contratos, cuyas definiciones están desarrolladas en este trabajo. Estas figuras son definidas de manera general, puesto que cualquier “Project Manager” o “DIPE”, gestor BIM (Building Information Modeling), o similar, puede actuar como uno o varios de ellos. También se propone la creación del agente “Gestor de la Construcción sin Pérdidas”, como aquel agente que asume las actuaciones del “gestor de diseño”, “gestor constructivo” y “gestor de contratos” con un enfoque en los principios del Lean Production. 7. En la tesis se demuestra, por medio del uso de la ISO 21500, que ambos sistemas son complementarios, de manera que los proyectos pueden tener ambos enfoques y ser compatibilizados. Un proyecto que use el “Project & Construction Management” puede perfectamente apoyarse en las herramientas y técnicas del “Lean Construction” para asegurar la eliminación o reducción de las pérdidas, es decir, las actividades que no generen valor, diseñando el sistema de producción, el sistema de diseño o el sistema de contratos. 8. Se debería incorporar adecuadamente al agente de la edificación “Especialista en Lean Construction” o similar y al agente ¨Especialista en Project & Construction Management” o DIPE en la Ley de Ordenación de la Edificación (LOE) de acuerdo a sus funciones y responsabilidades, puesto que la jurisprudencia se ha basado para absolver o condenar en la referida Ley. Uno de los objetivos fundamentales de esta tesis es el de regular la figura del “Especialista en Lean Construction” cuando actúa simultáneamente con el DIPE, y realizar una estructura de Agente de la Edificación según la LOE, y de esta manera protegerlo de eventuales responsabilidades solidarias. Esta investigación comprueba que la propuesta de definición del agente de edificación DIPE, según la LOE, presentada en la tesis doctoral del Doctor Manuel Soler Severino es compatible con las nuevas definiciones propuestas. El agente DIPE puede asumir los roles de los diferentes gestores propuestos en esta tesis si es que se especializa en dichas materias, o, si lo estima pertinente, recomendar sus contrataciones. ABSTRACT This work is based on the Lean Construction philosophy; an analysis is made herein with regard to the situation of this philosophy in the building sector within the international and Spanish context, replying to the following questions: 1. How did the concept of Lean Construction emerge? 2. Which are the activities, functions and objectives of Lean Construction? 3. Are there regulations on Lean Construction in other countries? 4. Is there a demand for Lean Construction in Spain? 5. Are there regulations on Lean Construction in Spain? 6. How should regulations on Lean Construction be developed in Spain? 7. What is the relationship between Lean Construction and the Project & Construction Management? 8. How should regulations on Lean Construction be developed in Spain considering its relationship with the Project & Construction Management? We have answered these questions in detail here and the replies are summarized as follows: 1. The concept of Lean Construction emerged in august of 1992, when Finnish researcher Lauri Koskela published in Stanford University TECHNICAL REPORT N° 72 entitled “Application of the New Production Philosophy to Construction”. A year later, Professor Koskela invited a group of construction specialists to Finland to the first workshop conducted on this matter; thus, the International Group for Lean Construction (IGLC) was established, which has contributed to extending the philosophy to the United States, Europe, the Americas, Asia, Oceania, and Africa. Lean Construction is a system based on the Lean Production approach, which was developed in Japan by Toyota Motors in the 1950s. Thanks to this system, the Toyota plants were able to produce more units, with greater efficiency than the American industry, less resources, in less time, and with fewer manufacturing errors. 2. The Lean Construction system aims at maximizing the value of projects while reducing waste, producing an effective coordination among those involved; it manages projects as a production system, enhancing collaboration between the parties that participate in the projects while building their capacities, empowering them, and promoting a culture of change. Its purpose is to develop a construction process free of accidents, without damages to the equipment, facilities, environment and community, flawless, in accordance with contractual requirements, within the terms established, respecting budgeted costs, and with a clear approach to eliminating or reducing waste, that is, activities that do not generate benefits. The Last Planner System is a Lean Construction system, which by its own nature protects planning and, therefore, helps to maximize the value and minimize waste, optimizing substantially the safety and health systems. Lean Construction started as a concept focused on the execution of works, and subsequently the philosophy was applied to all the stages of the project. At present it considers the project’s total development, since the time ideas are born until the completion and start-up of the work, taking into account the entire life cycle of the project. It is a philosophy of management, work methodologies, and entrepreneurial culture aimed at the effectiveness of processes and flows. The Lean Construction philosophy is extending all over the world and its scope is becoming broader, having greater influence on the contractual management of projects. It evolved initially through the creation of the Lean Project Delivery System, a global project development concept. Later on, the Target Value Design was developed, based on collaborative design to achieve the costs and value required, as well as the Integrated Project Delivery, in connection with integrated relational (collaborative) contract systems, as opposed to conventional contracts. 3. It was verified that no specific regulations on Lean Construction exist in other countries, in other words, there are no agents with the specific name of “Lean Construction Specialist” or other similar names; therefore, it is an additional agent in building projects, which functions and objectives can overlap those of the Project Manager, Construction Manager, Contract Manager, or Safety Manager, among others. However, the existence of private collaborative contracts of Integrated Project Delivery was confirmed, which could be considered as first references for future regulations. 4. There is a demand for Lean Construction in the development of this work; even though it is still emerging, there is a growing interest in this topic. 5. There are no regulations on Lean Construction in Spain. 6. One of the main objectives of this thesis is to regulate this role when acting in a project, and to define and develop a Building Agent structure, according to the Building Standards Law (LOE by its acronym in Spanish), in order to be able to incorporate it into the Spanish law, protecting it from civil liabilities. In Spain there is jurisprudence in civil jurisdiction based on the LOE to acquit or convict building agents, which are defined in the courts as “construction managers” or similar. For this reason, courts could establish in the future joint and several liabilities between the Lean Construction Specialist and other agents of the project, depending on their actions and based on the implementation of the Lean Project Delivery System, the Target Value Design, and the Integrated Project Delivery. On the other hand, it is possible that the level of action of the Lean Construction Specialist may comprise design management, construction management and contract management, or the integral management of the entire building project in accordance with the last ISO 21500:2012 or UNE-ISO 21500:2013, guidelines for the management of projects. Accordingly, one or more building agents should be appropriately incorporated into the LOE according to their functions and responsibilities and based on the levels of action of the Lean Construction Specialist. The creation of the following agents is proposed: Design Manager, Construction Manager, and Contract Manager, which definitions are developed in this work. These agents are defined in general, since any Project Manager or DIPE, Building Information Modeling (BIM) Manager or similar, may act as one or as many of them. The creation of the Lean Construction Manager is also proposed, as the agent that takes on the role of the Design Manager, Construction Manager and Contract Manager with a focus on the Lean Production principles. 7. In the thesis it is demonstrated that through the implementation of the ISO 21500, both systems are supplementary, so projects may have both approaches and be compatible. A project that applies the Project & Construction Management may perfectly have the support of the tools, techniques and practices of Lean Construction to ensure the elimination or reduction of losses, that is, those activities that do not generate value, thus designing the production system, the design system, or the contract system. 8. The Lean Construction Specialist or similar and the Specialist in Project & Construction Management should be incorporated appropriately into the LOE according to their functions and responsibilities, since jurisprudence has been based on such Law to acquit or convict. One of the main objectives of this thesis is the regulate the role of the Lean Construction Specialist when acting simultaneously with the DIPE, and to develop a structure of the building agent, according to the LOE, and in this way protect such agent from joint and several liabilities. This research proves that the proposal to define the DIPE building agent, according to the LOE, and presented in the doctoral dissertation of Manuel Soler Severino, Ph.D. is compatible with the new definitions proposed. The DIPE agent may assume the roles of the different managers proposed in this thesis if he specializes in those topics or, if deemed pertinent, recommends that they be engaged.

Physiological response to long-term peripheral and central leptin infusion in lean and obese mice

Recent data have identified leptin as an afferent signal in a negative-feedback loop regulating the mass of the adipose tissue. High leptin levels are observed in obese humans and rodents, suggesting that, in some cases, obesity is the result of leptin insensitivity. This hypothesis was tested by comparing the response to peripherally and centrally administered leptin among lean and three obese strains of mice: diet-induced obese AKR/J, New Zealand Obese (NZO), and Ay. Subcutaneous leptin infusion to lean mice resulted in a dose-dependent loss of body weight at physiologic plasma levels. Chronic infusions of leptin intracerebroventricularly (i.c.v.) at doses of 3 ng/hr or greater resulted in complete depletion of visible adipose tissue, which was maintained throughout 30 days of continuous i.c.v. infusion. Direct measurement of energy balance indicated that leptin treatment did not increase total energy expenditure but prevented the decrease that follows reduced food intake. Diet-induced obese mice lost weight in response to peripheral leptin but were less sensitive than lean mice. NZO mice were unresponsive to peripheral leptin but were responsive to i.c.v. leptin. Ay mice did not respond to subcutaneous leptin and were 1/100 as sensitive to i.c.v. leptin. The decreased response to leptin in diet-induced obese, NZO, and Ay mice suggests that obesity in these strains is the result of leptin resistance. In NZO mice, leptin resistance may be the result of decreased transport of leptin into the cerebrospinal fluid, whereas in Ay mice, leptin resistance probably results from defects downstream of the leptin receptor in the hypothalamus.

Multiple pathways to bypass the enhancer requirement of sigma 54 RNA polymerase: Roles for DNA and protein determinants

Sigma 54 is a required factor for bacterial RNA polymerase to respond to enhancers and directs a mechanism that is a hybrid between bacterial and eukaryotic transcription. Three pathways were found that bypass the enhancer requirement in vitro. These rely on either deletion of the sigma 54 N terminus or destruction of the DNA consensus −12 promoter recognition element or altering solution conditions to favor transient DNA melting. Each of these allows unstable heparin-sensitive pre-initiation complexes to form that can be driven to transcribe in the absence of both enhancer protein and ATP β–γ hydrolysis. These disparate pathways are proposed to have a common basis in that multiple N-terminal contacts may mediate the interactions between the polymerase and the DNA region where melting originates. The results raise possibilities for common features of open complex formation by different RNA polymerases.

Synergistic interaction between leptin and cholecystokinin to reduce short-term food intake in lean mice

Leptin is a circulating protein involved in the long-term regulation of food intake and body weight. Cholecystokinin (CCK) is released postprandially and elicits satiety signals. We investigated the interaction between leptin and CCK-8 in the short-term regulation of food intake induced by 24-hr fasting in lean mice. Leptin, injected intraperitoneally (i.p.) at low doses (4–120 μg/kg), which did not influence feeding behavior for the first 3 hr postinjection, decreased food intake dose dependently by 47–83% during the first hour when coinjected with a subthreshold dose of CCK. Such an interaction was not observed between leptin and bombesin. The food-reducing effect of leptin injected with CCK was not associated with alterations in gastric emptying or locomotor behavior. Leptin–CCK action was blocked by systemic capsaicin at a dose inducing functional ablation of sensory afferent fibers and by devazepide, a CCK-A receptor antagonist but not by the CCK-B receptor antagonist, L-365,260. The decrease in food intake which occurs 5 hr after i.p. injection of leptin alone was also blunted by devazepide. Coinjection of leptin and CCK enhanced the number of Fos-positive cells in the hypothalamic paraventricular nucleus by 60%, whereas leptin or CCK alone did not modify Fos expression. These results indicate the existence of a functional synergistic interaction between leptin and CCK leading to early suppression of food intake which involves CCK-A receptors and capsaicin-sensitive afferent fibers.

Insulin depletion leads to adipose-specific cell death in obese but not lean mice

Mutation of the obese gene produces obesity, hyperinsulinemia, and compensatory “overexpression” of the defective gene. As insulin activates obese gene expression, it seemed possible that hyperinsulinemia might be responsible for overexpression of the gene. To address this question we rapidly neutralized circulating insulin by injection of an insulin antibody. Unexpectedly, insulin depletion in obese (ob/ob or db/db) mice caused massive adipose RNA degradation confirmed by histological analysis to result from adipocyte cell death by a largely necrotic mechanism. This effect was not observed in lean littermates and was completely corrected by coadministration of insulin. Comparison of multiple tissues demonstrated that the effect was restricted to adipose tissue. Insulin depletion in obese mice by administration of streptozotocin also led to cell death, but this death was less extensive and appeared to be apoptotic in mechanism. Thus insulin may promote the survival side of the physiological balance between adipocyte survival and death.

RNA polymerase sigma factor determines start-site selection but is not required for upstream promoter element activation on heteroduplex (bubble) templates

Sequence-selective transcription by bacterial RNA polymerase (RNAP) requires σ factor that participates in both promoter recognition and DNA melting. RNAP lacking σ (core enzyme) will initiate RNA synthesis from duplex ends, nicks, gaps, and single-stranded regions. We have used DNA templates containing short regions of heteroduplex (bubbles) to compare initiation in the presence and absence of various σ factors. Using bubble templates containing the σD-dependent flagellin promoter, with or without its associated upstream promoter (UP) element, we demonstrate that UP element stimulation occurs efficiently even in the absence of σ. This supports a model in which the UP element acts primarily through the α subunit of core enzyme to increase the initial association of RNAP with the promoter. Core and holoenzyme do differ substantially in the template positions chosen for initiation: σD restricts initiation to sites 8–9 nucleotides downstream of the conserved −10 element. Remarkably, σA also has a dramatic effect on start-site selection even though the σA holoenzyme is inactive on the corresponding homoduplexes. The start sites chosen by the σA holoenzyme are located 8 nucleotides downstream of sequences on the nontemplate strand that resemble the conserved −10 hexamer recognized by σA. Thus, σA appears to recognize the −10 region even in a single-stranded state. We propose that in addition to its described roles in promoter recognition and start-site melting, σ also localizes the transcription start site.

Regulation of toxin synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor

Clostridium difficile, a causative agent of antibiotic-associated diarrhea and its potentially lethal form, pseudomembranous colitis, produces two large protein toxins that are responsible for the cellular damage associated with the disease. The level of toxin production appears to be critical for determining the severity of the disease, but the mechanism by which toxin synthesis is regulated is unknown. The product of a gene, txeR, that lies just upstream of the tox gene cluster was shown to be needed for tox gene expression in vivo and to activate promoter-specific transcription of the tox genes in vitro in conjunction with RNA polymerases from C. difficile, Bacillus subtilis, or Escherichia coli. TxeR was shown to function as an alternative sigma factor for RNA polymerase. Because homologs of TxeR regulate synthesis of toxins and a bacteriocin in other Clostridium species, TxeR appears to be a prototype for a novel mode of regulation of toxin genes.

Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity

Perilipin coats the lipid droplets of adipocytes and is thought to have a role in regulating triacylglycerol hydrolysis. To study the role of perilipin in vivo, we have created a perilipin knockout mouse. Perilipin null (peri−/−) and wild-type (peri+/+) mice consume equal amounts of food, but the adipose tissue mass in the null animals is reduced to ≈30% of that in wild-type animals. Isolated adipocytes of perilipin null mice exhibit elevated basal lipolysis because of the loss of the protective function of perilipin. They also exhibit dramatically attenuated stimulated lipolytic activity, indicating that perilipin is required for maximal lipolytic activity. Plasma leptin concentrations in null animals were greater than expected for the reduced adipose mass. The peri−/− animals have a greater lean body mass and increased metabolic rate but they also show an increased tendency to develop glucose intolerance and peripheral insulin resistance. When fed a high-fat diet, the perilipin null animals are resistant to diet-induced obesity but not to glucose intolerance. The data reveal a major role for perilipin in adipose lipid metabolism and suggest perilipin as a potential target for attacking problems associated with obesity.

Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting.

Escherichia coli RNA polymerase (RNAP) alpha subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2 alpha-->alpha 2-->alpha 2 beta-->alpha 2 beta beta'-->alpha 2 beta beta' sigma. In this work, we have used hydroxyl-radical protein footprinting to define determinants of alpha for interaction with beta, beta', and sigma. Our results indicate that amino acids 30-75 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta (i.e., in alpha 2 beta, alpha 2 beta beta', and alpha 2 beta beta' sigma), and amino acids 175-210 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta' (i.e., in alpha 2 beta beta' and alpha 2 beta beta' sigma). The protected regions are conserved in the alpha homologs of prokaryotic, eukaryotic, archaeal, and chloroplast RNAPs and contain sites of substitutions that affect RNAP assembly. We conclude that the protected regions define determinants of alpha for direct functional interaction with beta and beta'. The observed maximal magnitude of protection upon interaction with beta and the observed maximal magnitude of protection upon interaction with beta' both correspond to the expected value for complete protection of one of the two alpha protomers of RNAP (i.e., 50% protection). We propose that only one of the two alpha protomers of RNAP interacts with beta and that only one of the two alpha protomers of RNAP interacts with beta'.

The sigma subunit of Escherichia coli RNA polymerase senses promoter spacing.

The promoters recognized by sigma 70, the primary sigma of Escherichia coli, consist of two highly conserved hexamers located at -10 and -35 bp from the start point of transcription, separated by a preferred spacing of 17 bp. sigma factors have two distinct DNA binding domains that recognize the two hexamer sequences. However, the component of RNA polymerase recognizing the length of the spacing between hexamers has not been determined. Using an equilibrium DNA binding competition assay, we demonstrate that a polypeptide of sigma 70 carrying both DNA binding domains is very sensitive to promoter spacing, whereas a sigma 70 polypeptide with only one DNA binding domain is not. Furthermore, a mutant sigma, selected for increasing transcription of the minimal lac promoter (18-bp spacer), has an altered response to promoter spacing in vivo and in vitro. Our data support the idea that sigma makes simultaneous, productive contacts at both the -10 and the -35 regions of the promoter and discerns the spacing between these conserved regions.

Location of a potential transport binding site in a sigma class glutathione transferase by x-ray crystallography.

The crystal structure of the sigma class glutathione transferase from squid digestive gland in complex with S-(3-iodobenzyl)glutathione reveals a third binding site for the glutathione conjugate besides the two in the active sites of the dimer. The additional binding site is near the crystallographic two-fold axis between the two alpha 4-turn-alpha 5 motifs. The principal binding interactions with the conjugate include specific electrostatic interactions between the peptide and the two subunits and a hydrophobic cavity found across the two-fold axis that accommodates the 3-iodobenzyl group. Thus, two identical, symmetry-related but mutually exclusive binding modes for the third conjugate are observed. The hydrophobic pocket is about 14 A from the hydroxyl group of Tyr-7 in the active site. This site is a potential transport binding site for hydrophobic molecules or their glutathione conjugates.