Andrea Branzi y la “Città senza architettura”: De la No-Stop City a los modelos de urbanización débil

El tema de estudio de esta tesis son las propuestas urbanas que Andrea Branzi ha desarrollado durante los últimos cincuenta años, centrándose especialmente en aquellas más elaboradas y completas: la No-Stop City (1970-71), que elabora como miembro del grupo radical Archizoom, y dos de sus modelos de urbanización débil, Agronica (1995) y el Master Plan para el Strijp Philips de Eindhoven (2000). Se trata de una parte de su obra que ha mantenido constante, a lo largo del tiempo, una propuesta de disolución de la arquitectura de notable consistencia que puede describirse con la fórmula “città senza architettura”, acuñada por él mismo. Una voluntad que ya se apunta en la muy variada producción de Archizoom previa a la No-Stop City, y que cristaliza y se hace explícita en este proyecto que aspiraba a: “liberar al hombre de la arquitectura”. A pesar de la continuidad de esta idea en el tiempo, la ciudad sin arquitectura de Branzi ha evolucionado claramente dando lugar a distintos tipos de disolución. Una disolución que, obviamente, no supone la efectiva desaparición de la disciplina, sino la formulación de una arquitectura “otra” basada en un replanteamiento radical de la naturaleza y el papel de la misma. Esta agenda contra la disciplina se ha desplegado a través de una serie de temas que socavan el objeto arquitectónico canónico (su vaciamiento expresivo, la pérdida de importancia de la envolvente y la forma acabada, el carácter anticompositivo, la independencia entre forma y función, la mutabilidad en el tiempo…), pero va más allá al poner en crisis el rol que la propia arquitectura ha tenido en la configuración material, política y simbólica del hábitat humano. Una pérdida de protagonismo y centralidad en la sociedad contemporánea que, en el discurso del arquitecto, implica necesariamente un papel subordinado. De este proceso de disolución surge un nuevo tipo de ciudad en la que la forma urbana o se ha perdido o se ha convertido en superflua, en la que se ha disuelto la zonificación funcional, cuyos espacios interiores se hallan en un proceso de permanente reprogramación que ignora las tipologías, que trasciende la división entre lo urbano y lo agrícola y que es, ante todo, un espacio de flujos y servicios. La crisis de la ciudad tradicional implica, en definitiva, un cambio en la naturaleza misma de lo urbano que pasa de considerarse un lugar físico y construido, a convertirse en una condición inmaterial y virtualmente omnipresente que se despliega independientemente de su soporte físico. En las investigaciones urbanas de Branzi convergen, además, muchas de las reflexiones que el arquitecto ha desarrollado sobre, y desde, las distintas “escalas” de la actividad profesional: diseño, arquitectura y urbanismo. Estas propuestas no sólo cuestionan las relaciones establecidas entre objetos, edificios, ciudades y territorios sino que ponen en cuestión estas mismas categorías. Unas ciudades sin arquitectura que se basan, en última instancia, en plantear preguntas que son muy sencillas y, por otra parte, eternas: ¿Qué es un edificio? ¿Qué es una ciudad? ABSTRACT The subject of study of this thesis are the urban proposals developed by Andrea Branzi over the last fifty years, with a special focus on the more developed and comprehensive ones: the No- Stop City (1970-1971), produced as a member of the architettura radicale group Archizoom, and two of his “weak urbanization models”: Agronica (1995) and the Master Plan for Philips Strijp in Eindhoven (2000). This area of his work has kept, over time, a remarkably consistent proposal for the dissolution of architecture that can be described with the motto città senza architettura (city without architecture), coined by himself. A determination, already latent in the very diverse production of Archizoom prior to No-Stop City, that crystallizes and becomes explicit in this project which was aimed to "liberate man from the architecture." Despite the continuity of this idea over time, Branzi’s city without architecture has clearly evolved leading to different types of dissolution. A dissolution that, obviously, does not mean the effective demise of the discipline, but rather, the formulation of an architecture autre based on a radical rethinking of its nature and role. This agenda against the discipline has been developed through a number of issues that undermine the canonical architectural object (its expressive emptying, the loss of importance of the envelope and the finished shape, the anticompositional character, the independence between form and function, the mutability over time...), but goes beyond it by putting into crisis the role that architecture itself has had in the material, political and symbolic configuration of the human habitat. A loss of prominence and centrality in contemporary society that, in the architect’s discourse, implies a subordinate role. From this dissolution process, a new type of city arises: a city where urban form has been lost or has become superfluous, in which functional zoning has dissolved, whose interiors are in a permanent process of reprogramming that ignores typologies, that transcends the division between urban and agricultural and becomes, above all, a space of flows and services. Ultimately, the crisis of the traditional city implies a change in the very nature of the urban that moves from being regarded as a physical and built place, to become an immaterial and virtually omnipresent condition that unfolds regardless of its physical medium. Many of the ideas Branzi has developed on, and from, the different "scales" of professional activity (design, architecture and urbanism) converge in his urban research. These proposals not only question the relations between objects, buildings, cities and territories but also these very categories. Cities without architecture that are based, ultimately, on raising simple questions that are, on the other hand, eternal: What is a building? What is a city?

The educational learning lifecycle and the cloud computing

Currently, student dropout rates are a matter of concern among universities. Many research studies, aimed at discovering the causes, have been carried out. However, few solutions, that could serve all students and related problems, have been proposed so far. One such problem is caused by the lack of the "knowledge chain educational links" that occurs when students move onto higher studies without mastering their basic studies. Most regulated studies imparted at universities are designed so that some basic subjects serve as support for other, more complicated, subjects, thus forming a complicated knowledge network. When a link in this chain fails, student frustration occurs as it prevents him from fully understanding the following educational links. In this proposal we try to mitigate these disasters that stem, for the most part, the student?s frustration beyond his college stay. On one hand, we make a dissertation on the student?s learning process, which we divide into a series of phases that amount to what we call the "learning lifecycle." Also, we analyze at what stage the action by the stakeholders involved in this scenario: teachers and students; is most important. On the other hand, we consider that Information and Communication Technologies ICT, such as Cloud Computing, can help develop new ways, allowing for the teaching of higher education, while easing and facilitating the student?s learning process. But, methods and processes need to be defined as to direct the use of such technologies; in the teaching process in general, and within higher education in particular; in order to achieve optimum results. Our methodology integrates, as another actor, the ICT into the "Learning Lifecycle". We stimulate students to stop being merely spectators of their own education, and encourage them to take an active part in their training process. To do this, we offer a set of useful tools to determine not only academic failure causes, (for self assessment), but also to remedy these failures (with corrective actions); "discovered the causes it is easier to determine solutions?. We believe this study will be useful for both students and teachers. Students learn from their own experience and improve their learning process, while obtaining all of the "knowledge chain educational links? required in their studies. We stand by the motto "Studying to learn instead of studying to pass". Teachers will also be benefited by detecting where and how to strengthen their teaching proposals. All of this will also result in decreasing dropout rates.

Conditions for perfect focusing multiple point sources with the SMS design method

In this work, we demonstrate how it is possible to sharply image multiple object points. The Simultaneous Multiple Surface (SMS) design method has usually been presented as a method to couple N wave-front pairs with N surfaces, but recent findings show that when using N surfaces, we can obtain M image points when Nthe evolution of SMS method, from its basics, to imaging two object points through one surface, the transition from two to three objet points obtained by increasing the parallelism, and getting to the designs of six surfaces imaging up to eight object points. These designs are limited with the condition that the surfaces cannot be placed at the aperture stop. In the process of maximizing the object points to sharp image, we try to exhaust the degrees of freedom of aspherics and free-forms. We conjecture that maximal SMS designs are very close to a good solution, hence using them as a starting point for the optimization will lead us faster to a final optical system. We suggest here different optimization strategies which combined with the SMS method are proven to give the best solution. Through the example of imaging with the high aspect ratio, we compare the results obtained optimizing the rotational lens and using a combination of SMS method and optimization, showing that the second approach is giving significantly smaller value of overall RMS spot diameter.

Reducing the impact of EV charging on the electric grid

One of the main objectives of European Commission related to climate and energy is the well-known 20-20-20 targets to be achieved in 2020: Europe has to reduce greenhouse gas emissions of at least 20% below 1990 levels, 20% of EU energy consumption has to come from renewable resources and, finally, a 20% reduction in primary energy use compared with projected levels, has to be achieved by improving energy efficiency. In order to reach these objectives, it is necessary to reduce the overall emissions, mainly in transport (reducing CO2, NOx and other pollutants), and to increase the penetration of the intermittent renewable energy. A high deployment of battery electric (BEVs) and plug-in hybrid electric vehicles (PHEVs), with a low-cost source of energy storage, could help to achieve both targets. Hybrid electric vehicles (HEVs) use a combination of a conventional internal combustion engine (ICE) with one (or more) electric motor. There are different grades of hybridation from micro-hybrids with start-stop capability, mild hybrids (with kinetic energy recovery), medium hybrids (mild hybrids plus energy assist) and full hybrids (medium hybrids plus electric launch capability). These last types of vehicles use a typical battery capacity around 1-2 kWh. Plug in hybrid electric vehicles (PHEVs) use larger battery capacities to achieve limited electric-only driving range. These vehicles are charged by on-board electricity generation or either plugging into electric outlets. Typical battery capacity is around 10 kWh. Battery Electric Vehicles (BEVs) are only driven by electric power and their typical battery capacity is around 15-20 kWh. One type of PHEV, the Extended Range Electric Vehicle (EREV), operates as a BEV until its plug-in battery capacity is depleted; at which point its gasoline engine powers an electric generator to extend the vehicle's range. The charging of PHEVs (including EREVs) and BEVs will have different impacts to the electric grid, depending on the number of vehicles and the start time for charging. Initially, the lecture will start analyzing the electrical power requirements for charging PHEVs-BEVs in Flanders region (Belgium) under different charging scenarios. Secondly and based on an activity-based microsimulation mobility model, an efficient method to reduce this impact will be presented.

50 Years Ago We Marched: What began on the grounds of Lincoln University, ended on the steps of the Alabama State Capitol

LINCOLN UNIVERSITY - On March 25, 1965, a bus loaded with Lincoln University students and staff arrived in Montgomery, Ala. to join the Selma march for racial and voting equality. Although the Civil Rights Act of 1964 was in force, African-Americans continued to feel the effects of segregation. The 1960s was a decade of social unrest and change. In the Deep South, specifically Alabama, racial segregation was a cultural norm resistant to change. Governor George Wallace never concealed his personal viewpoints and political stance of the white majority, declaring “Segregation now, segregation tomorrow, segregation forever.” The march was aimed at obtaining African-Americans their constitutionally protected right to vote. However, Alabama’s deep-rooted culture of racial bias began to be challenged by a shift in American attitudes towards equality. Both black and whites wanted to end discrimination by using passive resistance, a movement utilized by Dr. Martin Luther King Jr. That passive resistance was often met with violence, sometimes at the hands of law enforcement and local citizens. The Selma to Montgomery march was a result of a protest for voting equality. The Student Nonviolent Coordinating Committee (SNCC) and the Southern Christian Leadership Counsel (SCLC) among other students marched along the streets to bring awareness to the voter registration campaign, which was organized to end discrimination in voting based on race. Violent acts of police officers and others were some of the everyday challenges protesters were facing. Forty-one participants from Lincoln University arrived in Montgomery to take part in the 1965 march for equality. Students from Lincoln University’s Journalism 383 class spent part of their 2015 spring semester researching the historical event. Here are their stories: Peter Kellogg “We’ve been watching the television, reading about it in the newspapers,” said Peter Kellogg during a February 2015 telephone interview. “Everyone knew the civil rights movement was going on, and it was important that we give him (Robert Newton) some assistance … and Newton said we needed to get involve and do something,” Kellogg, a lecturer in the 1960s at Lincoln University, discussed how the bus trip originated. “That’s why the bus happened,” Kellogg said. “Because of what he (Newton) did - that’s why Lincoln students went and participated.” “People were excited and the people along the sidewalk were supportive,” Kellogg said. However, the mood flipped from excited to scared and feeling intimidated. “It seems though every office building there was a guy in a blue uniform with binoculars standing in the crowd with troops and police. And if looks could kill me, we could have all been dead.” He says the hatred and intimidation was intense. Kellogg, being white, was an immediate target among many white people. He didn’t realize how dangerous the event in Alabama was until he and the others in the bus heard about the death of Viola Liuzzo. The married mother of five from Detroit was shot and killed by members of the Ku Klux Klan while shuttling activists to the Montgomery airport. “We found out about her death on the ride back,” Kellogg recalled. “Because it was a loss of life, and it shows the violence … we could have been exposed to that danger!” After returning to LU, Kellogg’s outlook on life took a dramatic turn. Kellogg noted King’s belief that a person should be willing to die for important causes. “The idea is that life is about something larger and more important than your own immediate gratification, and career success or personal achievements,” Kellogg said. “The civil rights movement … it made me, it made my life more significant because it was about something important.” The civil rights movement influenced Kellogg to change his career path and to become a black history lecturer. Until this day, he has no regrets and believes that his choices made him as a better individual. The bus ride to Alabama, he says, began with the actions of just one student. Robert Newton Robert Newton was the initiator, recruiter and leader of the Lincoln University movement to join Dr. Martin Luther King’s march in Selma. “In the 60s much of the civil rights activists came out of college,” said Newton during a recent phone interview. Many of the events that involved segregation compelled college students to fight for equality. “We had selected boycotts of merchants, when blacks were not allowed to try on clothes,” Newton said. “You could buy clothes at department stores, but no blacks could work at the department stores as sales people. If you bought clothes there you couldn’t try them on, you had to buy them first and take them home and try them on.” Newton said the students risked their lives to be a part of history and influence change. He not only recognized the historic event of his fellow Lincolnites, but also recognized other college students and historical black colleges and universities who played a vital role in history. “You had the S.N.C.C organization, in terms of voting rights and other things, including a lot of participation and working off the bureau,” Newton said. Other schools and places such as UNT, Greenville and Howard University and other historically black schools had groups that came out as leaders. Newton believes that much has changed from 50 years ago. “I think we’ve certainly come a long way from what I’ve seen from the standpoint of growing up outside of Birmingham, Alabama,” Newton said. He believes that college campuses today are more organized in their approach to social causes. “The campus appears to be some more integrated amongst students in terms of organizations and friendships.” Barbara Flint Dr. Barbara Flint grew up in the southern part of Arkansas and came to Lincoln University in 1961. She describes her experience at Lincoln as “being at Lincoln when the world was changing.“ She was an active member of Lincoln’s History Club, which focused on current events and issues and influenced her decision to join the Selma march. “The first idea was to raise some money and then we started talking about ‘why can’t we go?’ I very much wanted to be a living witness in history.” Reflecting on the march and journey to Montgomery, Flint describes it as being filled with tension. “We were very conscious of the fact that once we got on the road past Tennessee we didn’t know what was going to happen,” said Flint during a February 2015 phone interview. “Many of the students had not been beyond Missouri, so they didn’t have that sense of what happens in the South. Having lived there you knew the balance as well as what is likely to happen and what is not likely to happen. As my father use to say, ‘you have to know how to stay on that line of balance.’” Upon arriving in Alabama she remembers the feeling of excitement and relief from everyone on the bus. “We were tired and very happy to be there and we were trying to figure out where we were going to join and get into the march,” Flint said. “There were so many people coming in and then we were also trying to stay together; that was one of the things that really stuck out for me, not just for us but the people who were coming in. You didn’t want to lose sight of the people you came with.” Flint says she was keenly aware of her surroundings. For her, it was more than just marching forward. “I can still hear those helicopters now,” Flint recalled. “Every time the helicopters would come over the sound would make people jump and look up - I think that demonstrated the extent of the tenseness that was there at the time because the helicopters kept coming over every few minutes.” She said that the marchers sang “we are not afraid,” but that fear remained with every step. “Just having been there and being a witness and marching you realize that I’m one of those drops that’s going to make up this flood and with this flood things will move,” said Flint. As a student at Lincoln in 1965, Flint says the Selma experience undoubtedly changed her life. “You can’t expect to do exactly what you came to Lincoln to do,” Flint says. “That march - along with all the other marchers and the action that was taking place - directly changed the paths that I and many other people at Lincoln would take.” She says current students and new generations need to reflect on their personal role in society. “Decide what needs to be done and ask yourself ‘how can I best contribute to it?’” Flint said. She notes technology and social media can be used to reach audiences in ways unavailable to her generation in 1965. “So you don’t always have to wait for someone else to step out there and say ‘let’s march,’ you can express your vision and your views and you have the means to do so (so) others can follow you. Jaci Newsom Jaci Newsom came to Lincoln in 1965 from Atlanta. She came to Lincoln to major in sociology and being in Jefferson City was largely different from what she had grown up with. “To be able to come into a restaurant, sit down and be served a nice meal was eye-opening to me,” said Newsom during a recent interview. She eventually became accustomed to the relaxed attitude of Missouri and was shocked by the situation she encountered on an out-of-town trip. “I took a bus trip from Atlanta to Pensacola and I encountered the worse racism that I have ever seen. I was at bus stop, I went in to be served and they would not serve me. There was a policeman sitting there at the table and he told me that privately owned places could select not to serve you.” Newsom describes her experience of marching in Montgomery as being one with a purpose. “We felt as though we achieved something - we felt a sense of unity,” Newsom said. “We were very excited (because) we were going to hear from Martin Luther King. To actually be in the presence of him and the other civil rights workers there was just such enthusiasm and excitement yet there was also some apprehension of what we might encounter.” Many of the marchers showed their inspiration and determination while pressing forward towards the grounds of the Alabama Capitol building. Newsom recalled that the marchers were singing the lyrics “ain’t gonna let nobody turn me around” and “we shall overcome.” “ I started seeing people just like me,” Newsom said. “I don’t recall any of the scowling, the hitting, the things I would see on TV later. I just saw a sea of humanity marching towards the Capitol. I don’t remember what Martin Luther King said but it was always the same message: keep the faith; we’re going to get where we’re going and let us remember what our purpose is.” Newsom offers advice on what individuals can do to make their society a more productive and peaceful place. “We have come a long way and we have ways to change things that we did not have before,” Newsom said. “You need to work in positive ways to change.” Referencing the recent unrest in Ferguson, Mo., she believes that people become destructive as a way to show and vent anger. Her generation, she says, was raised to react in lawful ways – and believe in hope. “We have faith to do things in a way that was lawful and it makes me sad what people do when they feel without hope, and there is hope,” Newsom says. “Non-violence does work - we need to include everyone to make this world a better place.” Newsom graduated from Lincoln in 1969 and describes her experience at Lincoln as, “I grew up and did more growing at Lincoln than I think I did for the rest of my life.”

Control of pre-mRNA accumulation by the essential yeast protein Nrd1 requires high-affinity transcript binding and a domain implicated in RNA polymerase II association

Nrd1 is an essential yeast protein of unknown function that has an RNA recognition motif (RRM) in its carboxyl half and a putative RNA polymerase II-binding domain, the CTD-binding motif, at its amino terminus. Nrd1 mediates a severe reduction in pre-mRNA production from a reporter gene bearing an exogenous sequence element in its intron. The effect of the inserted element is highly sequence-specific and is accompanied by the appearance of 3′-truncated transcripts. We have proposed that Nrd1 binds to the exogenous sequence element in the nascent pre-mRNA during transcription, aided by the CTD-binding motif, and directs 3′-end formation a short distance downstream. Here we show that highly purified Nrd1 carboxyl half binds tightly to the RNA element in vitro with sequence specificity that correlates with the efficiency of cis-element-directed down-regulation in vivo. A large deletion in the CTD-binding motif blocks down-regulation but does not affect the essential function of Nrd1. Furthermore, a nonsense mutant allele that produces truncated Nrd1 protein lacking the RRM has a dominant-negative effect on down-regulation but not on cell growth. Viability of this and several other nonsense alleles of Nrd1 appears to require translational readthrough, which in one case is extremely efficient. Thus the CTD-binding motif of Nrd1 is important for pre-mRNA down-regulation but is not required for the essential function of Nrd1. In contrast, the RNA-binding activity of Nrd1 appears to be required both for down-regulation and for its essential function.

From ab initio quantum mechanics to molecular neurobiology: A cation–π binding site in the nicotinic receptor

The nicotinic acetylcholine receptor is the prototype ligand-gated ion channel. A number of aromatic amino acids have been identified as contributing to the agonist binding site, suggesting that cation–π interactions may be involved in binding the quaternary ammonium group of the agonist, acetylcholine. Here we show a compelling correlation between: (i) ab initio quantum mechanical predictions of cation–π binding abilities and (ii) EC50 values for acetylcholine at the receptor for a series of tryptophan derivatives that were incorporated into the receptor by using the in vivo nonsense-suppression method for unnatural amino acid incorporation. Such a correlation is seen at one, and only one, of the aromatic residues—tryptophan-149 of the α subunit. This finding indicates that, on binding, the cationic, quaternary ammonium group of acetylcholine makes van der Waals contact with the indole side chain of α tryptophan-149, providing the most precise structural information to date on this receptor. Consistent with this model, a tethered quaternary ammonium group emanating from position α149 produces a constitutively active receptor.

Human ATP-binding cassette transporter 1 (ABC1): Genomic organization and identification of the genetic defect in the original Tangier disease kindred

Tangier disease is characterized by low serum high density lipoproteins and a biochemical defect in the cellular efflux of lipids to high density lipoproteins. ABC1, a member of the ATP-binding cassette family, recently has been identified as the defective gene in Tangier disease. We report here the organization of the human ABC1 gene and the identification of a mutation in the ABC1 gene from the original Tangier disease kindred. The organization of the human ABC1 gene is similar to that of the mouse ABC1 gene and other related ABC genes. The ABC1 gene contains 49 exons that range in size from 33 to 249 bp and is over 70 kb in length. Sequence analysis of the ABC1 gene revealed that the proband for Tangier disease was homozygous for a deletion of nucleotides 3283 and 3284 (TC) in exon 22. The deletion results in a frameshift mutation and a premature stop codon starting at nucleotide 3375. The product is predicted to encode a nonfunctional protein of 1,084 aa, which is approximately half the size of the full-length ABC1 protein. The loss of a Mnl1 restriction site, which results from the deletion, was used to establish the genotype of the rest of the kindred. In summary, we report on the genomic organization of the human ABC1 gene and identify a frameshift mutation in the ABC1 gene of the index case of Tangier disease. These results will be useful in the future characterization of the structure and function of the ABC1 gene and the analysis of additional ABC1 mutations in patients with Tangier disease.

Translational coupling by modulation of feedback repression in the IF3 operon of Escherichia coli

A pseudoknot formed by a long-range interaction in the mRNA of the initiation factor 3 (IF3) operon is involved in the translational repression of the gene encoding ribosomal protein L35 by another ribosomal protein, L20. The nucleotides forming the 5′ strand of the key stem of the pseudoknot are located within the gene for IF3, whereas those forming the 3′ strand are located 280 nt downstream, immediately upstream of the Shine–Dalgarno sequence of the gene for L35. Here we show that premature termination of IF3 translation at a nonsense codon introduced upstream of the pseudoknot results in a substantial enhancement of L20-mediated repression of L35 expression. Conversely, an increase of IF3 translation decreases repression. These results, in addition to an analysis of the effect of mutations in sequences forming the pseudoknot, indicate that IF3 translation decreases L20-mediated repression of L35 expression. We propose that ribosomes translating IF3 disrupt the pseudoknot and thereby attenuate repression. The result is a novel type of translational coupling, where unfolding of the pseudoknot by ribosomes translating IF3 does not increase expression of L35 directly, but alleviates its repression by L20.

The maturity-onset diabetes of the young (MODY1) transcription factor HNF4α regulates expression of genes required for glucose transport and metabolism

Hepatocyte nuclear factor 4α (HNF4α) plays a critical role in regulating the expression of many genes essential for normal functioning of liver, gut, kidney, and pancreatic islets. A nonsense mutation (Q268X) in exon 7 of the HNF4α gene is responsible for an autosomal dominant, early-onset form of non-insulin-dependent diabetes mellitus (maturity-onset diabetes of the young; gene named MODY1). Although this mutation is predicted to delete 187 C-terminal amino acids of the HNF4α protein the molecular mechanism by which it causes diabetes is unknown. To address this, we first studied the functional properties of the MODY1 mutant protein. We show that it has lost its transcriptional transactivation activity, fails to dimerize and bind DNA, implying that the MODY1 phenotype is because of a loss of HNF4α function. The effect of loss of function on HNF4α target gene expression was investigated further in embryonic stem cells, which are amenable to genetic manipulation and can be induced to form visceral endoderm. Because the visceral endoderm shares many properties with the liver and pancreatic β-cells, including expression of genes for glucose transport and metabolism, it offers an ideal system to investigate HNF4-dependent gene regulation in glucose homeostasis. By exploiting this system we have identified several genes encoding components of the glucose-dependent insulin secretion pathway whose expression is dependent upon HNF4α. These include glucose transporter 2, and the glycolytic enzymes aldolase B and glyceraldehyde-3-phosphate dehydrogenase, and liver pyruvate kinase. In addition we have found that expression of the fatty acid binding proteins and cellular retinol binding protein also are down-regulated in the absence of HNF4α. These data provide direct evidence that HNF4α is critical for regulating glucose transport and glycolysis and in doing so is crucial for maintaining glucose homeostasis.

Sequence dependent hypermutation of the immunoglobulin heavy chain in cultured B cells

The variable (V) regions of immunoglobulin heavy and light chains undergo high rates of somatic mutation during the immune response. Although point mutations accumulate throughout the V regions and their immediate flanking sequences, analysis of large numbers of mutations that have arisen in vivo reveal that the triplet AGC appears to be most susceptible to mutation. We have stably transfected B cell lines with γ2a heavy chain constructs containing TAG nonsense codons in their V regions that are part of either a putative (T)AGC hot spot or a (T)AGA non-hot spot motif. Using an ELISA spot assay to detect revertants and fluctuation analysis to determine rates of mutation, the rate of reversion of the TAG nonsense codon has been determined for different motifs in different parts of the V region. In the NSO plasma cell line, the (T)AGC hot spot motif mutates at rates of ≈6 × 10−4/bp per generation and ≈3 × 10−5/bp per generation at residues 38 and 94 in the V region. At each of these locations, the (T)AGC hot spot motif is 20–30 times more likely to undergo mutation than the (T)AGA non-hot spot motif. Moreover, the AGA non-hot spot motif mutates at as high a rate as the hot spot motif when it is located adjacent to hot spot motifs, suggesting that more extended sequences influence susceptibility to mutation.

Infrequent Translation of a Nonsense Codon Is Sufficient to Decrease mRNA Level

In many organisms nonsense mutations decrease the level of mRNA. In the case of mammalian cells, it is still controversial whether translation is required for this nonsense-mediated RNA decrease (NMD). Although previous analyzes have shown that conditions that impede translation termination at nonsense codons also prevent NMD, the residual level of termination was unknown in these experiments. Moreover, the conditions used to impede termination might also have interfered with NMD in other ways. Because of these uncertainties, we have tested the effects of limiting translation of a nonsense codon in a different way, using two mutations in the immunoglobulin μ heavy chain gene. For this purpose we exploited an exceptional nonsense mutation at codon 3, which efficiently terminates translation but nonetheless maintains a high level of μ mRNA. We have shown 1) that translation of Ter462 in the double mutant occurs at only ∼4% the normal frequency, and 2) that Ter462 in cis with Ter3 can induce NMD. That is, translation of Ter462 at this low (4%) frequency is sufficient to induce NMD.

Basolateral Localization of the Caenorhabditis elegans Epidermal Growth Factor Receptor in Epithelial Cells by the PDZ Protein LIN-10

In Caenorhabditis elegans, the EGF receptor (encoded by let-23) is localized to the basolateral membrane domain of the epithelial vulval precursor cells, where it acts through a conserved Ras/MAP kinase signaling pathway to induce vulval differentiation. lin-10 acts in LET-23 receptor tyrosine kinase basolateral localization, because lin-10 mutations result in mislocalization of LET-23 to the apical membrane domain and cause a signaling defective (vulvaless) phenotype. We demonstrate that the previous molecular identification of lin-10 was incorrect, and we identify a new gene corresponding to the lin-10 genetic locus. lin-10 encodes a protein with regions of similarity to mammalian X11/mint proteins, containing a phosphotyrosine-binding and two PDZ domains. A nonsense lin-10 allele that truncates both PDZ domains only partially reduces lin-10 gene activity, suggesting that these protein interaction domains are not essential for LIN-10 function in vulval induction. Immunocytochemical experiments show that LIN-10 is expressed in vulval epithelial cells and in neurons. LIN-10 is present at low levels in the cytoplasm and at the plasma membrane and at high levels at or near the Golgi. LIN-10 may function in secretion of LET-23 to the basolateral membrane domain, or it may be involved in tethering LET-23 at the basolateral plasma membrane once it is secreted.

Coupled Translocation Events Generate Topological Heterogeneity at the Endoplasmic Reticulum Membrane

Topogenic determinants that direct protein topology at the endoplasmic reticulum membrane usually function with high fidelity to establish a uniform topological orientation for any given polypeptide. Here we show, however, that through the coupling of sequential translocation events, native topogenic determinants are capable of generating two alternate transmembrane structures at the endoplasmic reticulum membrane. Using defined chimeric and epitope-tagged full-length proteins, we found that topogenic activities of two C-trans (type II) signal anchor sequences, encoded within the seventh and eighth transmembrane (TM) segments of human P-glycoprotein were directly coupled by an inefficient stop transfer (ST) sequence (TM7b) contained within the C-terminus half of TM7. Remarkably, these activities enabled TM7 to achieve both a single- and a double-spanning TM topology with nearly equal efficiency. In addition, ST and C-trans signal anchor activities encoded by TM8 were tightly linked to the weak ST activity, and hence topological fate, of TM7b. This interaction enabled TM8 to span the membrane in either a type I or a type II orientation. Pleiotropic structural features contributing to this unusual topogenic behavior included 1) a short, flexible peptide loop connecting TM7a and TM7b, 2) hydrophobic residues within TM7b, and 3) hydrophilic residues between TM7b and TM8.

Mutations in the organic cation/carnitine transporter OCTN2 in primary carnitine deficiency

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation caused by defective carnitine transport. This disease presents early in life with hypoketotic hypoglycemia or later in life with skeletal myopathy or cardiomyopathy. The gene for this condition maps to 5q31.2–32 and OCTN2, an organic cation/carnitine transporter, also maps to the same chromosomal region. Here we test the causative role of OCTN2 in primary carnitine deficiency by searching for mutations in this gene in affected patients. Fibroblasts from patients with primary carnitine deficiency lacked mediated carnitine transport. Transfection of patient’s fibroblasts with the OCTN2 cDNA partially restored carnitine transport. Sequencing of the OCTN2 gene revealed different mutations in two unrelated patients. The first patient was homozygous (and both parents heterozygous) for a single base pair substitution converting the codon for Arg-282 to a STOP codon (R282X). The second patient was a compound heterozygote for a paternal 1-bp insertion producing a STOP codon (Y401X) and a maternal 1-bp deletion that produced a frameshift creating a subsequent STOP codon (458X). These mutations decreased the levels of mature OCTN2 mRNA and resulted in nonfunctional transporters, confirming that defects in the organic cation/carnitine transporter OCTN2 are responsible for primary carnitine deficiency.