Molecular cloning and sequence analysis of expansins--a highly conserved, multigene family of proteins that mediate cell wall extension in plants.

Expansins are unusual proteins discovered by virtue of their ability to mediate cell wall extension in plants. We identified cDNA clones for two cucumber expansins on the basis of peptide sequences of proteins purified from cucumber hypocotyls. The expansin cDNAs encode related proteins with signal peptides predicted to direct protein secretion to the cell wall. Northern blot analysis showed moderate transcript abundance in the growing region of the hypocotyl and no detectable transcripts in the nongrowing region. Rice and Arabidopsis expansin cDNAs were identified from collections of anonymous cDNAs (expressed sequence tags). Sequence comparisons indicate at least four distinct expansin cDNAs in rice and at least six in Arabidopsis. Expansins are highly conserved in size and sequence (60-87% amino acid sequence identity and 75-95% similarity between any pairwise comparison), and phylogenetic trees indicate that this multigene family formed before the evolutionary divergence of monocotyledons and dicotyledons. Sequence and motif analyses show no similarities to known functional domains that might account for expansin action on wall extension. A series of highly conserved tryptophans may function in expansin binding to cellulose or other glycans. The high conservation of this multigene family indicates that the mechanism by which expansins promote wall extensin tolerates little variation in protein structure.

Elongation factor 1 alpha concentration is highly correlated with the lysine content of maize endosperm.

Lysine is the most limiting essential amino acid in cereals, and for many years plant breeders have attempted to increase its concentration to improve the nutritional quality of these grains. The opaque2 mutation in maize doubles the lysine content in the endosperm, but the mechanism by which this occurs is unknown. We show that elongation factor 1 alpha (EF-1 alpha) is overexpressed in opaque2 endosperm compared with its normal counterpart and that there is a highly significant correlation between EF-1 alpha concentration and the total lysine content of the endosperm. This relationship is also true for two other cereals, sorghum and barley. It appears that genetic selection for genotypes with a high concentration of EF-1 alpha can significantly improve the nutritional quality of maize and other cereals.

Interaction of GroEL with a highly structured folding intermediate: iterative binding cycles do not involve unfolding.

The GroE proteins are molecular chaperones involved in protein folding. The general mechanism by which they facilitate folding is still enigmatic. One of the central open questions is the conformation of the GroEL-bound nonnative protein. Several suggestions have been made concerning the folding stage at which a protein can interact with GroEL. Furthermore, the possibility exists that binding of the nonnative protein to GroEL results in its unfolding. We have addressed these issues that are basic for understanding the GroE-mediated folding cycle by using folding intermediates of an Fab antibody fragment as molecular probes to define the binding properties of GroEL. We show that, in addition to binding to an early folding intermediate, GroEL is able to recognize and interact with a late quaternary-structured folding intermediate (Dc) without measurably unfolding it. Thus, the prerequisite for binding is not a certain folding stage of a nonnative protein. In contrast, general surface properties of nonnative proteins seem to be crucial for binding. Furthermore, unfolding of a highly structured intermediate does not necessarily occur upon binding to GroEL. Folding of Dc in the presence of GroEL and ATP involves cycles of binding and release. Because in this system no off-pathway reactions or kinetic traps are involved, a quantitative analysis of the reactivation kinetics observed is possible. Our results indicate that the association reaction of Dc and GroEL in the presence of ATP is rather slow, whereas in the absence of ATP association is several orders of magnitude more efficient. Therefore, it seems that ATP functions by inhibiting reassociation rather than promoting release of the bound substrate.

Immunohistochemical localization of adenylyl cyclase in rat brain indicates a highly selective concentration at synapses.

Only three isoforms of adenylyl cyclase (EC 4.6.1.1) mRNAs (AC1, -2, and -5) are expressed at high levels in rat brain. AC1 occurs predominantly in hippocampus and cerebellum, AC5 is restricted to the basal ganglia, whereas AC2 is more widely expressed, but at much lower levels. The distribution and abundance of adenylyl cyclase protein were examined by immunohistochemistry with an antiserum that recognizes a peptide sequence shared by all known mammalian adenylyl cyclase isoforms. The immunoreactivity in striatum and hippocampus could be readily interpreted within the context of previous in situ hybridization studies. However, extending the information that could be gathered by comparisons with in situ hybridization analysis, it was apparent that staining was confined to the neuropil--corresponding to immunoreactive dendrites and axon terminals. Electron microscopy indicated a remarkably selective subcellular distribution of adenylyl cyclase protein. In the CA1 area of the hippocampus, the densest immunoreactivity was seen in postsynaptic densities in dendritic spine heads. Labeled presynaptic axon terminals were also observed, indicating the participation of adenylyl cyclase in the regulation of neurotransmitter release. The selective concentration of adenylyl cyclases at synaptic sites provides morphological data for understanding the pre- and postsynaptic roles of adenylyl cyclase in discrete neuronal circuits in rat brain. The apparent clustering of adenylyl cyclases, coupled with other data that suggest higher-order associations of regulatory elements including G proteins, N-methyl-D-aspartate receptors, and cAMP-dependent protein kinases, suggests not only that the primary structural information has been encoded to render the cAMP system responsive to the Ca(2+)-signaling system but also that higher-order strictures are in place to ensure that Ca2+ signals are economically delivered and propagated.

Restriction fragment length polymorphism-coupled domain-directed differential display: a highly efficient technique for expression analysis of multigene families.

In this paper, a reverse-transcriptase PCR-based protocol suitable for efficient expression analysis of multigene families is presented. The method combines restriction fragment length polymorphism (RFLP) technology with a gene family-specific version of mRNA differential display and hence is called "RFLP-coupled domain-directed differential display. "With this method, expression of all members of a multigene family at many different developmental stages, in diverse tissues and even in different organisms, can be displayed on one gel. Moreover, bands of interest, representing gene family members, are directly accessible to sequence analysis, without the need for subcloning. The method thus enables a detailed, high-resolution expression analysis of known gene family members as well as the identification and characterization of new ones. Here the technique was used to analyze differential expression of MADS-box genes in male and female inflorescences of maize (Zea mays ssp. mays). Six different MADS-box genes could be identified, being either specifically expressed in the female sex or preferentially expressed in male or female inflorescences, respectively. Other possible applications of the method are discussed.

Construction and characterization of a highly complex retroviral library for lineage analysis.

Replication-incompetent retroviral vectors encoding histochemical reporter genes have been used for studying lineal relationships in a variety of species. A crucial element in the interpretation of data generated by this method is the identification of sibling relationships, or clonal boundaries. The use of a library of viruses in which each member is unique can greatly facilitate this aspect of the analysis. A previously reported murine retroviral library containing about 80 members demonstrated the utility of the library approach. However, the relatively low number of tags in the murine library necessitated using low infection rates in order to give confidence in clonal assignments. To obviate the need for low infection rates, a far more complex library was created and characterized. The CHAPOL library was constructed such that each member encodes a histochemical reporter gene and has a DNA tag derived from a degenerate oligonucleotide pool synthesized to have a complexity of > 1 x 10(7). The library was tested after infection of cells in vitro or in vivo. The DNA tag from each histochemically labeled cell or clone of cells was recovered by PCR and sequenced for unambiguous identification. Three hundred and twenty tags have been identified after infection, and so far no tag has been seen to result from more than one independent infection. Thus, an equal distribution of inserts is suggested, and Monte Carlo analysis predicts a complexity of > 10(4) members.

The alpha and gamma 1 isoforms of protein phosphatase 1 are highly and specifically concentrated in dendritic spines.

Protein phosphatase 1 (PP1) is a highly conserved enzyme that has been implicated in diverse biological processes in the brain as well as in nonneuronal tissues. The present study used light and electron microscopic immunocytochemistry to characterize the distribution of two PP1 isoforms, PP1 alpha and PP1 gamma 1, in the rat neostriatum. Both isoforms are heterogeneously distributed in brain with the highest immunoreactivity being found in the neostriatum and hippocampal formation. Further, both isoforms are highly and specifically concentrated in dendritic spines. Weak immunoreactivity is present in dendrites, axons, and some axon terminals. Immunoreactivity for PP1 alpha is also present in the perikaryal cytoplasm and nuclei of most medium- and large-sized neostriatal neurons. The specific localization of PP1 in dendritic spines is consistent with a central role for this enzyme in signal transduction. The data support the concept that, in the course of evolution, spines developed as specialized signal transduction organelles enabling neurons to integrate diverse inputs from multiple afferent nerve terminals.

A cyclophilin from the polycentric anaerobic rumen fungus Orpinomyces sp. strain PC-2 is highly homologous to vertebrate cyclophilin B.

A cyclophilin (CyP) purified to homogeneity from the polycentric anaerobic rumen fungus Orpinomyces sp. strain PC-2 had a molecular mass of 20.5 kDa and a pI of 8.1. The protein catalyzed the isomerization of the prolyl peptide bond of N-succinyl-Ala-Ala-(cis,trans)-Pro-Phe p-nitroanilide with a kcat/Km value of 9.3 x 10(6) M-1.s-1 at 10 degrees C and pH 7.8. Cyclosporin A strongly inhibited this peptidylprolyl cis-trans isomerase activity with an IC50 of 19.6 nM. The sequence of the first 30 N-terminal amino acids of this CyP had high homology with the N-terminal sequences of other eukaryotic CyPs. By use of a DNA hybridization probe amplified by PCR with degenerate oligonucleotide primers designed based on the amino acid sequences of the N terminus of this CyP and highly conserved internal regions of other CyPs, a full-length cDNA clone was isolated. It possessed an open reading frame encoding a polypeptide of 203 amino acids with a calculated molecular weight of 21,969, containing a putative hydrophobic signal peptide sequence of 22 amino acids preceding the N terminus of the mature enzyme and a C-terminal sequence, Lys-Ala-Glu-Leu, characteristic of an endoplasmic reticulum retention signal. The Orpinomyces PC-2 CyP is a typical type B CyP. The amino acid sequence of the Orpinomyces CyP exhibits striking degrees of identity with the corresponding human (70%), bovine (69%), mouse (68%), chicken (66%), maize (61%), and yeast (54%) proteins. Phylogenetic analysis based on the CyP sequences indicated that the evolutionary origin of the Orpinomyces CyP was closely related with CyPs of animals.

The ionized gas in the CALIFA early-type galaxies I. Mapping two representative cases: NGC 6762 and NGC 5966

As part of the ongoing CALIFA survey, we have conducted a thorough bidimensional analysis of the ionized gas in two E/S0 galaxies, NGC 6762 and NGC 5966, aiming to shed light on the nature of their warm ionized ISM. Specifically, we present optical (3745–7300 Å) integral field spectroscopy obtained with the PMAS/PPAK integral field spectrophotometer. Its wide field-of-view (1′ × 1′) covers the entire optical extent of each galaxy down to faint continuum surface brightnesses. To recover the nebular lines, we modeled and subtracted the underlying stellar continuum from the observed spectra using the STARLIGHT spectral synthesis code. The pure emission-line spectra were used to investigate the gas properties and determine the possible sources of ionization. We show the advantages of IFU data in interpreting the complex nature of the ionized gas in NGC 6762 and NGC 5966. In NGC 6762, the ionized gas and stellar emission display similar morphologies, while the emission line morphology is elongated in NGC 5966, spanning ~6 kpc, and is oriented roughly orthogonal to the major axis of the stellar continuum ellipsoid. Whereas gas and stars are kinematically aligned in NGC 6762, the gas is kinematically decoupled from the stars in NGC 5966. A decoupled rotating disk or an “ionization cone” are two possible interpretations of the elongated ionized gas structure in NGC 5966. The latter would be the first “ionization cone” of such a dimension detected within a weak emission-line galaxy. Both galaxies have weak emission-lines relative to the continuum[EW(Hα) ≲ 3 Å] and have very low excitation, log([OIII]λ5007/Hβ) ≲ 0.5. Based on optical diagnostic ratios ([OIII]λ5007/Hβ, [NII]λ6584/Hα, [SII]λ6717, 6731/Hα, [OI]λ6300/Hα), both objects contain a LINER nucleus and an extended LINER-like gas emission. The emission line ratios do not vary significantly with radius or aperture, which indicates that the nebular properties are spatially homogeneous. The gas emission in NGC 6762 can be best explained by photoionization by pAGB stars without the need of invoking any other excitation mechanism. In the case of NGC 5966, the presence of a nuclear ionizing source seems to be required to shape the elongated gas emission feature in the “ionization cone” scenario, although ionization by pAGB stars cannot be ruled out. Further study of this object is needed to clarify the nature of its elongated gas structure.

Separation and fractionation of order and disorder in highly polydisperse systems

We study a polydisperse soft-spheres model for colloids by means of microcanonical Monte Carlo simulations. We consider a polydispersity as high as 24%. Although solidification occurs, neither a crystal nor an amorphous state are thermodynamically stable. A finite size scaling analysis reveals that in the thermodynamic limit: a the fluid-solid transition is rather a crystal-amorphous phase-separation, b such phase-separation is preceded by the dynamic glass transition, and c small and big particles arrange themselves in the two phases according to a complex pattern not predicted by any fractionation scenario.

Search for ultrahigh energy neutrinos in highly inclined events at the Pierre Auger Observatory

The Surface Detector of the Pierre Auger Observatory is sensitive to neutrinos of all flavors above 0.1 EeV. These interact through charged and neutral currents in the atmosphere giving rise to extensive air showers. When interacting deeply in the atmosphere at nearly horizontal incidence, neutrinos can be distinguished from regular hadronic cosmic rays by the broad time structure of their shower signals in the water-Cherenkov detectors. In this paper we present for the first time an analysis based on down-going neutrinos. We describe the search procedure, the possible sources of background, the method to compute the exposure and the associated systematic uncertainties. No candidate neutrinos have been found in data collected from 1 January 2004 to 31 May 2010. Assuming an E-2 differential energy spectrum the limit on the single-flavor neutrino is E(2)dN/dE < 1.74 x 10(-7)GeVcm(-2)s(-1)sr(-1) at 90% C.L. in the energy range 1 x 10(17) eV < E < 1 x 10(20)eV.

Molecular signature of highly conductive metal-molecule-metal junctions

The simplicity of single-molecule junctions based on direct bonding of a small molecule between two metallic electrodes makes them an ideal system for the study of fundamental questions related to molecular electronics. Here we study the conductance properties of six different types of molecules by suspending individual molecules between Pt electrodes. All the molecular junctions show a typical conductance of about 1G0 which is ascribed to the dominant role of the Pt contacts. However, despite the metalliclike conductivity, the individual molecular signature is well expressed by the effect of molecular vibrations in the inelastic contribution to the conductance.

A highly resistive layer within the crust of X-ray pulsars limits their spin periods

The lack of isolated X-ray pulsars with spin periods longer than 12 s raises the question of where the population of evolved high-magnetic-field neutron stars has gone. Unlike canonical radiopulsars, X-ray pulsars are not subject to physical limits to the emission mechanism nor observational biases against the detection of sources with longer periods. Here we show that a highly resistive layer in the innermost part of the crust of neutron stars naturally limits the spin period to a maximum value of about 10–20 s. This highly resistive layer is expected if the inner crust is amorphous and heterogeneous in nuclear charge, possibly owing to the existence of a nuclear ‘pasta’ phase. Our findings suggest that the maximum period of isolated X-ray pulsars may be the first observational evidence for an amorphous inner crust, whose properties can be further constrained by future X-ray timing missions combined with more detailed models.

A Highly Efficient Solvent-Free Asymmetric Direct Aldol Reaction Organocatalyzed by Recoverable (s)-Binam-L-Prolinamides. ESI-MS Evidence of the Enamine-Iminium Formation

Recoverable (Sa)-binam-l-prolinamide in combination with benzoic acid is used as catalysts in the direct aldol reaction between cycloalkyl, alkyl, and α-functionalized ketones and aldehydes under solvent-free reaction conditions. Three different methods are assayed: simple conventional magnetic stirring, magnetic stirring after previous dissolution in THF and evaporation, and ball mill technique. These procedures allow one to reduce not only the amount of required ketone to 2 equiv but also the reaction time to give the aldol products with regio-, diastereo-, and enantioselectivities comparable to those in organic or aqueous solvents. Generally anti-isomers are mainly obtained with enantioselectivities up to 97%. The reaction can be carried out under these conditions also using aldehydes as nucleophiles, yielding after in situ reduction of the aldol products the corresponding chiral 1,3-diols with moderate to high enantioselectivities mainly as anti-isomers. The aldol reaction has been studied by the use of positive ESI-MS technique, providing the evidence of the formation of the corresponding enamine−iminium intermediates.

Colloidal gold immobilized on mesoporous silica as a highly active and selective catalyst for styrene epoxidation with H2O2

Colloidal gold nanoparticles were synthesized by different procedures affording suspensions with two different mean sizes (2 and 5 nm). Au catalysts were prepared by sol immobilization onto several silica frameworks with different 2D and 3D mesoporosities. The catalysts were tested in styrene oxidation reactions showing excellent efficiency and selectivity. The effect of nanoparticle size and mesoporous framework on the physical and catalytic properties of the final materials was studied. The most selective catalyst was prepared from the 5 nm Au nanoparticles and the more interconnected silica framework (3D mesoporosity).