Pollen analysis of soil samples from the A.D. 79 level. Boscoreale, Oplontis, and Pompeii

Fifty samples of Roman time soil preserved under the thick ash layer of the A.D.79 eruption of Mt Vesuvius were studied by pollen analysis: 33 samples from a former vineyard surrounding a Villa Rustica at Boscoreale (excavation site 40 x 50 m), 13 samples taken along the 60 m long swimming pool in the sculpture garden of the Villa of Poppaea at Oplontis, and four samples from the formal garden (12.4 x 17.5 m) of the House of the Gold Bracelet in Pompeii. To avoid contamination with modern pollen all samples were taken immediately after uncovering a new portion of the A.D. 79 soil. For comparison also samples of modern Italian soils were studied. Using standard methods for pollen preparation the pollen content of 15 of the archaeological samples proved to be too little to reach a pollen sum of more than 100 grains. The pollen spectra of these samples are not shown in the pollen tables. (Flotation with a sodium tungstate solution, Na2WO4, D = 2.05, following treatment with HCl and NaOH would probably have given a somewhat better result. This method was, however, not available as too expensive at that time.) Although the archaeological samples were taken a few meters apart their pollen values differ very much from one sample to the other. E.g., at Boscoreale (SW quarter). the pollen values of Pinus range from 1.5 to 54.5% resp. from 1 to 244 pine pollen grains per 1 gram of soil, the extremes even found under pine trees. Vitis pollen was present in 7 of the 11 vineyard samples from Boscoreale (NE quarter) only. Although a maximum of 21.7% is reached, the values of Vitis are mostly below 1.5%. Even the values of common weeds differ very much, not only at Boscoreale, but also at the other two sites. The pollen concentration values show similar variations: 3 to 3053 grains and spores were found in 1 g of soil. The mean value (290) is much less than the number of pollen grains, which would fall on 1 cm2 of soil surface during one year. In contrast, the pollen and spore concentrations of the recent soil samples, treated in exactly the same manner, range from 9313 to almost 80000 grains per 1 g of soil. Evidently most of the Roman time pollen has disappeared since its deposition, the reasons not being clear. Not even species which are known to have been cultivated in the garden of Oplontis, like Citrus and Nerium, plant species with easily distinguishable pollen grains, could be traced by pollen analysis. The loss of most of the pollen grains originally contained in the soil prohibits any detailed interpretation of the Pompeian pollen data. The pollen counts merely name plant species which grew in the region, but not necessarily on the excavated plots.

Producción y gestión habitacional de los pobladores : articulación con la política de vivienda y barrio : trayectoria y problemática actual

Esta tesis analiza las acciones de los pobladores en la creación, consolidación y transformación de su hábitat y en su relación con la política pública de vivienda y barrio en Chile. A partir de la observación directa en terreno y de la revisión de material proveniente de diversas fuentes afirmamos que, aunque los pobladores han hecho un trabajo de producción del hábitat de gran magnitud y generalizado, las políticas públicas no han reconocido suficientemente su papel en la construcción de la ciudad, no han incorporado a cabalidad las potencialidades e innovaciones surgidas de sus prácticas y estrategias, y nunca les han abierto un espacio claro en la toma de decisiones y en la puesta en obra de los programas habitacionales. En el contexto latinoamericano, la política habitacional chilena de los últimos 20 años se ha considerado un éxito y un ejemplo a seguir, puesto que ha demostrado ser eficaz en la disminución del déficit habitacional. Sin embargo, ha tenido efectos urbanos y sociales nefastos, como la construcción de extensos bolsones periféricos de pobreza que se degradan aceleradamente, y la desintegración social que genera la expulsión de los sin casa a la periferia, donde pierden sus redes familiares y sociales. Desde una trinchera opuesta, los allegados, los sin casa que viven al alero de otras familias y representan la mayoría de la demanda por vivienda, exigen quedarse en barrios ya consolidados y evitan las periferias, en parte por mantener una red familiar y social que se sustenta en la proximidad física, en parte por los equipamientos y servicios con que cuentan estos barrios y la cercanía a las fuentes de empleo. Al mismo tiempo, los responsables de diseñar la política habitacional no han buscado establecer una forma de colaboración con los pobladores —principales receptores de la política— con el fin ajustar los programas públicos a las necesidades de las familias de bajos ingresos y a las realidades socioculturales de sus barrios. Por el contrario, han privilegiado una alianza con el sector privado, que conoce muy limitadamente las demandas de las familias. Así, en lugar de construir ciudades más justas, la política habitacional ha alimentado un mercado inmobiliario sustentado en la especulación del suelo y fomentado la industria de la construcción. La pregunta que guía esta investigación es cómo incorporar el conocimiento acumulado y los procedimientos probados por los pobladores al diseño y la implementación de programas habitacionales y urbanos que promuevan procesos de regeneración de las poblaciones y mejoren la distribución de la vivienda social en la ciudad. Sostenemos que los pobladores, a lo largo de una trayectoria de más de medio siglo, han adquirido y consolidado todas las competencias para construir vivienda, mejorar sus barrios e incorporarse a la discusión sobre ordenamiento territorial. Así, hoy están capacitados para asumir un papel protagónico en la definición de políticas públicas que apunte a la construcción de ciudades más sostenibles y equitativas. La producción social del hábitat vinculada al derecho a la ciudad y a la participación de los pobladores «desde abajo» está bastante documentada en la literatura latinoamericana. En Chile se han escrito numerosos trabajos y evaluaciones sobre la política habitacional, pero los estudios sobre el movimiento de pobladores, enfocados desde las ciencias sociales o multidisciplinares, tienen un auge primero, durante los años 60 y principios de los 70 y luego, en la segunda mitad de los 80, pero posteriormente dejan de publicarse, a excepción de algunas investigaciones de historia urbana o social. En cuanto a los estudios que abordan las acciones de los pobladores desde una mirada puesta en los resultados de la producción y la gestión habitacional y urbana, estos han sido especialmente escasos y ninguno abarca un período largo. La tesis aborda entonces las acciones específicas que emprenden los pobladores a distintas escalas territoriales —el conjunto, el barrio, la población, la ciudad y el país—, su relación con la política habitacional y su articulación con los demás actores que intervienen en la producción material del hábitat. Lo realizado por los pobladores se estudia a la luz del largo plazo, desde la promulgación de la primera ley de vivienda en 1906 hasta nuestros días, con el énfasis puesto entre los años 1990 y 2010, período de producción masiva y sostenida de vivienda social, financiada por el Estado y construida por el sector privado en la periferia urbana, y más detalladamente entre 2006 y 2010, cuando los pobladores irrumpen con la «gestión vecinal» y la «autogestión» como medios para implementar los programas habitacionales del gobierno. Para ello se recorre toda la trayectoria y se complementa con procesos particulares, a la manera de un lente de acercamiento con el cual se focalizan y amplifican trece casos de estudios, para ilustrar modos de producción y gestión concretos y mostrar cómo estos se inscriben en modos de hacer genéricos de los pobladores. Finalmente, con el lente centrado en el último ciclo de este proceso escribimos el capítulo inédito de los últimos veinte años de esta historia. Primero se realiza la reconstrucción de tres casos de estudio «en profundidad», que incluyen la génesis, la consolidación y las transformaciones del conjunto o barrio. Estos casos de estudio «en profundidad» se ponen en perspectiva reconstruyendo la trayectoria histórica de la producción y gestión realizada por los pobladores. Esta reconstrucción de largo período se profundiza con tres casos de estudio «específicos», de dimensión histórica, que tratan el conflicto del acceso a suelo. Finalmente se analizan las interrogantes que plantean estos procesos hoy en día para la producción y gestión de vivienda y barrio a futuro, a partir de entrevistas a actores claves y de la reconstrucción de siete casos de estudio «específicos» de acceso a suelo ilustrativos del período actual. La tesis sustenta que los pobladores, con las acciones de gestión y autogestión que realizan desde 2006, e interviniendo en la discusión sobre los instrumentos de planificación territorial a partir del mismo año, se sitúan actualmente en una nueva plataforma de acción y negociación desde la cual pueden incorporarse, con todas las competencias necesarias, a la definición de las políticas públicas y así dotarlas de pertinencia y coherencia para contribuir a superar la pobreza con respuestas más acorde a sus realidades. ABSTRACT This thesis analyzes the actions of pobladores in the creation, consolidation and transformation of their habitat and their relationship with Chilean public housing and neighbourhood policy. Through direct observation in the field and the review of material from various sources we can affirm that although the pobladores have undertaken widespread work in the production of their environment, public policies have not sufficiently recognized their role in the construction of the city. Public policy has failed to fully incorporate the potential and innovation arising from practices and strategies employed by social housing recipients and has never opened a clear space for them in decision-making or the commissioning work of the housing programs. Within the Latin America context, the Chilean housing policy of the past 20 years has been considered a success and an example to follow given that it has proven effective in reducing the housing deficit. However it has had disastrous urban and social effects, such as construction of large peripheral pockets of poverty that degrade rapidly, and generates social disintegration through the expulsion of the homeless to the periphery, where they lose their family and social networks. On another front those homeless who live under the roof of relatives and who represent the majority of demand for social housing, request to stay in consolidated neighbourhoods avoiding the periphery, partly to maintain family and social networks based on physical proximity and partly because of the facilities and services available in these neighbourhoods and their adjacency to sources of employment. At the same time, those responsible for designing housing policy have not sought to establish a form of collaboration with the pobladores in order to adjust the public programs to the needs of low-income families and the socio-cultural realities of their neighbourhoods. On the contrary an alliance with the private sector has been favored, a sector which has very limited knowledge of the demands of the recipients. Therefore instead of building more equal cities, housing policy has fueled a housing market which supports land speculation and promotes the construction industry. The question leading this research is how to incorporate the accumulated knowledge and proven procedures of the pobladores in the design and implementation of programs that promote housing and urban regeneration processes and which could improve the distribution of social housing in the city. We maintain that social housing recipients over the course of half a century have acquired and consolidated all the skills to build housing, improve neighborhoods and join the discussion on city planning. These residents are now capable of assuming a leading role in defining public policies that aim to build more sustainable and equitable cities. The social production of the environment linked to the right to the city and resident participation from the «bottom-up» is well documented in Latin American literature. In Chile there are extensive written works and assessments on housing policy with multidisciplinary or social science studies on the movement of the pobladores peaking during the 60’s and early 70’s and then again in the second half of the 80’s but afterwards this stops, with the exception of some research on social or urban history. As for studies that address the actions of the pobladores looking at the results of production and housing and urban management these have been particularly scarce and none of which cover a long period of time. The thesis then addresses the specific actions undertaken by the pobladores at different territorial levels; the housing development, the neighbourhood, the community, the city and State, and their relation to housing policy and its coordination with other actors involved in the production process of the built environment. The accomplishments of the pobladores is studied over the long term, since the enactment of the first housing law in 1906 to the present, with an emphasis between 1990 and 2010, a period of mass production and sustained social housing which was State-funded and built by the private sector in the urban periphery, and in particular between 2006 and 2010, when the pobladores break with the «neighborhood management» and «self-management» as a means to implement the housing programs of the government. To this end the entire process is outlined and is complemented by specific processes which are placed under a lens in order to focus and amplify thirteen case studies illustrating actual ways of production and management and to show how these ways of doing things are generic to the pobladores. Finally, with the lens focused on the last cycle of this process we write the new chapter of the last twenty years of this history. First there is a reconstruction of three case studies «in depth», including their origins, consolidation and the transformation of the sector or neighborhood. These «in depth» case studies are put into perspective reconstructing the historical trajectory of the production and management by the pobladores. This reconstruction over a long period is given great depth by three «specific» case studies, of historical importance, dealing with the conflict of access to land. Finally we analyze the questions raised by these processes for the production and management of housing and neighborhood in the future, based on interviews with key players and the reconstruction of seven case studies specifically regarding access to land and which are illustrative of current practice. The thesis maintains that since 2006 the pobladores through actions of management and selfmanagement and their intervention in the debate on territorial planning has placed them on a new platform for action and negotiation from which they can incorporate themselves, with all the necessary capacities, in the definition of public policy and therefore provide it with a pertinence and coherence to help towards overcoming poverty with answers more according to their realities.

Characteristics of the turbulent/nonturbulent interface in boundary layers, jets and shear-free turbulence

The characteristics of turbulent/nonturbulent interfaces (TNTI) from boundary layers, jets and shear-free turbulence are compared using direct numerical simulations. The TNTI location is detected by assessing the volume of turbulent flow as function of the vorticity magnitude and is shown to be equivalent to other procedures using a scalar field. Vorticity maps show that the boundary layer contains a larger range of scales at the interface than in jets and shear-free turbulence where the change in vorticity characteristics across the TNTI is much more dramatic. The intermittency parameter shows that the extent of the intermittency region for jets and boundary layers is similar and is much bigger than in shear-free turbulence, and can be used to compute the vorticity threshold defining the TNTI location. The statistics of the vorticity jump across the TNTI exhibit the imprint of a large range of scales, from the Kolmogorov micro-scale to scales much bigger than the Taylor scale. Finally, it is shown that contrary to the classical view, the low-vorticity spots inside the jet are statistically similar to isotropic turbulence, suggesting that engulfing pockets simply do not exist in jets

Typing of urinary JC virus DNA offers a novel means of tracing human migrations

Although polyomavirus JC (JCV) is the proven pathogen of progressive multifocal leukoencephalopathy, the fatal demyelinating disease, this virus is ubiquitous as a usually harmless symbiote among human beings. JCV propagates in the adult kidney and excretes its progeny in urine, from which JCV DNA can readily be recovered. The main mode of transmission of JCV is from parents to children through long cohabitation. In this study, we collected a substantial number of urine samples from native inhabitants of 34 countries in Europe, Africa, and Asia. A 610-bp segment of JCV DNA was amplified from each urine sample, and its DNA sequence was determined. A worldwide phylogenetic tree subsequently constructed revealed the presence of nine subtypes including minor ones. Five subtypes (EU, Af2, B1, SC, and CY) occupied rather large territories that overlapped with each other at their boundaries. The entire Europe, northern Africa, and western Asia were the domain of EU, whereas the domain of Af2 included nearly all of Africa and southwestern Asia all the way to the northeastern edge of India. Partially overlapping domains in Asia were occupied by subtypes B1, SC, and CY. Of particular interest was the recovery of JCV subtypes in a pocket or pockets that were separated by great geographic distances from the main domains of those subtypes. Certain of these pockets can readily be explained by recent migrations of human populations carrying these subtypes. Overall, it appears that JCV genotyping promises to reveal previously unknown human migration routes: ancient as well as recent.

(4-Aminomethyl)phenylguanidine derivatives as nonpeptidic highly selective inhibitors of human urokinase

Increased expression of the serine protease urokinase-type plasminogen activator (uPA) in tumor tissues is highly correlated with tumor cell migration, invasion, proliferation, progression, and metastasis. Thus inhibition of uPA activity represents a promising target for antimetastatic therapy. So far, only the x-ray crystal structure of uPA inactivated by H-Glu-Gly-Arg-chloromethylketone has been reported, thus limited data are available for a rational structure-based design of uPA inhibitors. Taking into account the trypsin-like arginine specificity of uPA, (4-aminomethyl)phenylguanidine was selected as a potential P1 residue and iterative derivatization of its amino group with various hydrophobic residues, and structure–activity relationship-based optimization of the spacer in terms of hydrogen bond acceptor/donor properties led to N-(1-adamantyl)-N′-(4-guanidinobenzyl)urea as a highly selective nonpeptidic uPA inhibitor. The x-ray crystal structure of the uPA B-chain complexed with this inhibitor revealed a surprising binding mode consisting of the expected insertion of the phenylguanidine moiety into the S1 pocket, but with the adamantyl residue protruding toward the hydrophobic S1′ enzyme subsite, thus exposing the ureido group to hydrogen-bonding interactions. Although in this enzyme-bound state the inhibitor is crossing the active site, interactions with the catalytic residues Ser-195 and His-57 are not observed, but their side chains are spatially displaced for steric reasons. Compared with other trypsin-like serine proteases, the S2 and S3/S4 pockets of uPA are reduced in size because of the 99-insertion loop. Therefore, the peculiar binding mode of the new type of uPA inhibitors offers the possibility of exploiting optimized interactions at the S1′/S2′ subsites to further enhance selectivity and potency. Because crystals of the uPA/benzamidine complex allow inhibitor exchange by soaking procedures, the structure-based design of new generations of uPA inhibitors can rely on the assistance of x-ray analysis.

Protein engineering of Bacillus thuringiensis δ-endotoxin: Mutations at domain II of CryIAb enhance receptor affinity and toxicity toward gypsy moth larvae

Substitutions or deletions of domain II loop residues of Bacillus thuringiensis δ-endotoxin CryIAb were constructed using site-directed mutagenesis techniques to investigate their functional roles in receptor binding and toxicity toward gypsy moth (Lymantria dispar). Substitution of loop 2 residue N372 with Ala or Gly (N372A, N372G) increased the toxicity against gypsy moth larvae 8-fold and enhanced binding affinity to gypsy moth midgut brush border membrane vesicles (BBMV) ≈4-fold. Deletion of N372 (D3), however, substantially reduced toxicity (>21 times) as well as binding affinity, suggesting that residue N372 is involved in receptor binding. Interestingly, a triple mutant, DF-1 (N372A, A282G and L283S), has a 36-fold increase in toxicity to gypsy moth neonates compared with wild-type toxin. The enhanced activity of DF-1 was correlated with higher binding affinity (18-fold) and binding site concentrations. Dissociation binding assays suggested that the off-rate of the BBMV-bound mutant toxins was similar to that of the wild type. However, DF-1 toxin bound 4 times more than the wild-type and N372A toxins, and it was directly correlated with binding affinity and potency. Protein blots of gypsy moth BBMV probed with labeled N372A, DF-1, and CryIAb toxins recognized a common 210-kDa protein, indicating that the increased activity of the mutants was not caused by binding to additional receptor(s). The improved binding affinity of N372A and DF-1 suggest that a shorter side chain at these loops may fit the toxin more efficiently to the binding pockets. These results offer an excellent model system for engineering δ-endotoxins with higher potency and wider spectra of target pests by improving receptor binding interactions.

The crystal structure of modified bovine fibrinogen

Here we report the crystal structure at ≈4-Å resolution of a selectively proteolyzed bovine fibrinogen. This key component in hemostasis is an elongated 340-kDa glycoprotein in the plasma that upon activation by thrombin self-assembles to form the fibrin clot. The crystals are unusual because they are made up of end-to-end bonded molecules that form flexible filaments. We have visualized the entire coiled-coil region of the molecule, which has a planar sigmoidal shape. The primary polymerization receptor pockets at the ends of the molecule face the same way throughout the end-to-end bonded filaments, and based on this conformation, we have developed an improved model of the two-stranded protofibril that is the basic building block in fibrin. Near the middle of the coiled-coil region, the plasmin-sensitive segment is a hinge about which the molecule adopts different conformations. This segment also includes the boundary between the three- and four-stranded portions of the coiled coil, indicating the location on the backbone that anchors the extended flexible Aα arm. We suggest that a flexible branch point in the molecule may help accommodate variability in the structure of the fibrin clot.

Small antibody-like proteins with prescribed ligand specificities derived from the lipocalin fold

We demonstrate that the ligand pocket of a lipocalin from Pieris brassicae, the bilin-binding protein (BBP), can be reshaped by combinatorial protein design such that it recognizes fluorescein, an established immunological hapten. For this purpose 16 residues at the center of the binding site, which is formed by four loops on top of an eight-stranded β-barrel, were subjected to random mutagenesis. Fluorescein-binding BBP variants were then selected from the mutant library by bacterial phage display. Three variants were identified that complex fluorescein with high affinity, exhibiting dissociation constants as low as 35.2 nM. Notably, one of these variants effects almost complete quenching of the ligand fluorescence, similarly as an anti-fluorescein antibody. Detailed ligand-binding studies and site-directed mutagenesis experiments indicated (i) that the molecular recognition of fluorescein is specific and (ii) that charged residues at the center of the pocket are responsible for tight complex formation. Sequence comparison of the BBP variants directed against fluorescein with the wild-type protein and with further variants that were selected against several other ligands revealed that all of the randomized amino acid positions are variable. Hence, a lipocalin can be used for generating molecular pockets with a diversity of shapes. We term this class of engineered proteins “anticalins.” Their one-domain scaffold makes them a promising alternative to antibodies to create a stable receptor protein for a ligand of choice.

A quantitative method for evaluating the stabilities of nucleic acids

We report a general method for screening, in solution, the impact of deviations from canonical Watson-Crick composition on the thermodynamic stability of nucleic acid duplexes. We demonstrate how fluorescence resonance energy transfer (FRET) can be used to detect directly free energy differences between an initially formed “reference” duplex (usually a Watson-Crick duplex) and a related “test” duplex containing a lesion/alteration of interest (e.g., a mismatch, a modified, a deleted, or a bulged base, etc.). In one application, one titrates into a solution containing a fluorescently labeled, FRET-active, reference duplex, an unlabeled, single-stranded nucleic acid (test strand), which may or may not compete successfully to form a new duplex. When a new duplex forms by strand displacement, it will not exhibit FRET. The resultant titration curve (normalized fluorescence intensity vs. logarithm of test strand concentration) yields a value for the difference in stability (free energy) between the newly formed, test strand-containing duplex and the initial reference duplex. The use of competitive equilibria in this assay allows the measurement of equilibrium association constants that far exceed the magnitudes accessible by conventional titrimetric techniques. Additionally, because of the sensitivity of fluorescence, the method requires several orders of magnitude less material than most other solution methods. We discuss the advantages of this method for detecting and characterizing any modification that alters duplex stability, including, but not limited to, mutagenic lesions. We underscore the wide range of accessible free energy values that can be defined by this method, the applicability of the method in probing for a myriad of nucleic acid variations, such as single nucleotide polymorphisms, and the potential of the method for high throughput screening.

Mutations of γ-aminobutyric acid and glycine receptors change alcohol cutoff: Evidence for an alcohol receptor?

Alcohols in the homologous series of n-alcohols increase in central nervous system depressant potency with increasing chain length until a “cutoff” is reached, after which further increases in molecular size no longer increase alcohol potency. A similar phenomenon has been observed in the regulation of ligand-gated ion channels by alcohols. Different ligand-gated ion channels exhibit radically different cutoff points, suggesting the existence of discrete alcohol binding pockets of variable size on these membrane proteins. The identification of amino acid residues that determine the alcohol cutoff may, therefore, provide information about the location of alcohol binding sites. Alcohol regulation of the glycine receptor is critically dependent on specific amino acid residues in transmembrane domains 2 and 3 of the α subunit. We now demonstrate that these residues in the glycine α1 and the γ-aminobutyric acid ρ1 receptors also control alcohol cutoff. By mutation of Ser-267 to Gln, it was possible to decrease the cutoff in the glycine α1 receptor, whereas mutation of Ile-307 and/or Trp-328 in the γ-aminobutyric acid ρ1 receptor to smaller residues increased the cutoff. These results support the existence of alcohol binding pockets in these membrane proteins and suggest that the amino acid residues present at these positions can control the size of the alcohol binding cavity.

Catalytic mechanism of the adenylyl and guanylyl cyclases: Modeling and mutational analysis

The adenylyl and guanylyl cyclases catalyze the formation of 3′,5′-cyclic adenosine or guanosine monophosphate from the corresponding nucleoside 5′-triphosphate. The guanylyl cyclases, the mammalian adenylyl cyclases, and their microbial homologues function as pairs of homologous catalytic domains. The crystal structure of the rat type II adenylyl cyclase C2 catalytic domain was used to model by homology a mammalian adenylyl cyclase C1-C2 domain pair, a homodimeric adenylyl cyclase of Dictyostelium discoideum, a heterodimeric soluble guanylyl cyclase, and a homodimeric membrane guanylyl cyclase. Mg2+ATP or Mg2+GTP were docked into the active sites based on known stereochemical constraints on their conformation. The models are consistent with the activities of seven active-site mutants. Asp-310 and Glu-432 of type I adenylyl cyclase coordinate a Mg2+ ion. The D310S and D310A mutants have 10-fold reduced Vmax and altered [Mg2+] dependence. The NTP purine moieties bind in mostly hydrophobic pockets. Specificity is conferred by a Lys and an Asp in adenylyl cyclase, and a Glu, an Arg, and a Cys in guanylyl cyclase. The models predict that an Asp from one domain is a general base in the reaction, and that the transition state is stabilized by a conserved Asn-Arg pair on the other domain.

Stepwise Photoinhibition of Photosystem II1 : Studies with Synechocystis Species PCC 6803 Mutants with a Modified D-E Loop of the Reaction Center Polypeptide D1

Several mutant strains of Synechocystis sp. PCC 6803 with large deletions in the D-E loop of the photosystem II (PSII) reaction center polypeptide D1 were subjected to high light to investigate the role of this hydrophilic loop in the photoinhibition cascade of PSII. The tolerance of PSII to photoinhibition in the autotrophic mutant ΔR225-F239 (PD), when oxygen evolution was monitored with 2,6-dichloro-p-benzoquinone and the equal susceptibility compared with control when monitored with bicarbonate, suggested an inactivation of the QB-binding niche as the first event in the photoinhibition cascade in vivo. This step in PD was largely reversible at low light without the need for protein synthesis. Only the next event, inactivation of QA reduction, was irreversible and gave a signal for D1 polypeptide degradation. The heterotrophic deletion mutants ΔG240-V249 and ΔR225-V249 had severely modified QB pockets, yet exhibited high rates of 2,6-dichloro-p-benzoquinone-mediated oxygen evolution and less tolerance to photoinhibition than PD. Moreover, the protein-synthesis-dependent recovery of PSII from photoinhibition was impaired in the ΔG240-V249 and ΔR225-V249 mutants because of the effects of the mutations on the expression of the psbA-2 gene. No specific sequences in the D-E loop were found to be essential for high rates of D1 polypeptide degradation.

Defining the roles of individual residues in the single-stranded DNA binding site of PcrA helicase

Crystal structures and biochemical analyses of PcrA helicase provide evidence for a model for processive DNA unwinding that involves coupling of single-stranded DNA (ssDNA) tracking to a duplex destabilization activity. The DNA tracking model invokes ATP-dependent flipping of bases between several pockets on the enzyme formed by conserved aromatic amino acid residues. We have used site-directed mutagenesis to confirm the requirement of all of these residues for helicase activity. We also demonstrate that the duplex unwinding defects correlate with an inability of certain mutant proteins to translocate effectively on ssDNA. Moreover, the results define an essential triad of residues within the ssDNA binding site that comprise the ATP-driven DNA motor itself.

The structure of bovine F1-ATPase complexed with the antibiotic inhibitor aurovertin B.

In the structure of bovine mitochondrial F1-ATPase that was previously determined with crystals grown in the presence of adenylyl-imidodiphosphate (AMP-PNP) and ADP, the three catalytic beta-subunits have different conformations and nucleotide occupancies. Adenylyl-imidodiphosphate is bound to one beta-subunit (betaTP), ADP is bound to the second (betaDP), and no nucleotide is bound to the third (betaE). Here we show that the uncompetitive inhibitor aurovertin B binds to bovine F1 at two equivalent sites in betaTP and betaE, in a cleft between the nucleotide binding and C-terminal domains. In betaDP, the aurovertin B pocket is incomplete and is inaccessible to the inhibitor. The aurovertin B bound to betaTP interacts with alpha-Glu399 in the adjacent alphaTP subunit, whereas the aurovertin B bound to betaE is too distant from alphaE to make an equivalent interaction. Both sites encompass betaArg-412, which was shown by mutational studies to be involved in binding aurovertin. Except for minor changes around the aurovertin pockets, the structure of bovine F1-ATPase is the same as determined previously. Aurovertin B appears to act by preventing closure of the catalytic interfaces, which is essential for a catalytic mechanism involving cyclic interconversion of catalytic sites.

Crystallographic analysis of endogenous peptides associated with HLA-DR1 suggests a common, polyproline II-like conformation for bound peptides.

The structure of the human major histocompatibility complex (MHC) class II molecule HLA-DR1 derived from the human lymphoblastoid cell line LG-2 has been determined in a complex with the Staphylococcus aureus enterotoxin B superantigen. The HLA-DR1 molecule contains a mixture of endogenous peptides derived from cellular or serum proteins bound in the antigen-binding site, which copurify with the class II molecule. Continuous electron density for 13 amino acid residues is observed in the MHC peptide-binding site, suggesting that this is the core length of peptide that forms common interactions with the MHC molecule. Electron density is also observed for side chains of the endogenous peptides. The electron density corresponding to peptide side chains that interact with the DR1-binding site is more clearly defined than the electron density that extends out of the binding site. The regions of the endogenous peptides that interact with DRI are therefore either more restricted in conformation or sequence than the peptide side chains or amino acids that project out of the peptide-binding site. The hydrogen-bond interactions and conformation of a peptide model built into the electron density are similar to other HLA-DR-peptide structures. The bound peptides assume a regular conformation that is similar to a polyproline type II helix. The side-chain pockets and conserved asparagine residues of the DR1 molecule are well-positioned to interact with peptides in the polyproline type II conformation and may restrict the range of acceptable peptide conformations.