Hyperpolarizing potentials in guinea pig hippocampal CA3 neurons

There is a bewildering variety of hyperpolarizing potentials which control activity in hippocampal pyramidal cells. These include an inhibitory postsynaptic potential (IPSP) with early and late components, voltage- and calcium-dependent potassium conductances, a voltage-dependent potassium conductance modulated by muscarinic agents (the M-current), and a complex and poorly understood afterhyperpolarization following epileptiform bursts. In hippocampal CA3 pyramidal cells, mossy fiber stimulation elicits an IPSP which is made up of two readily separable components. Using the in vitro slice preparation, we investigated the underlying ionic basis of these IPSP components and compared them to other hyperpolarizing potentials characteristic of the CA3 neurons. Intracellular recordings were obtained and then tissue was exposed to bathing medium low in chloride concentration or high in potassium concentration; the ion "blockers" EGTA (intracellular); tetraethylammonium (TEA) (intra- and extracellular), and barium and cobalt (extracellular); and the gamma-aminobutyric acid (GABA)/chloride antagonists penicillin, bicuculline and picrotoxin.     

Antibody localization of extensin in cell walls of carrot storage roots

The accumulation and cross-linking of hydroxyproline-rich glycoproteins (HRGPs) in cell walls of dicotyledonous plants has been correlated with a number of wall-strengthening phenomena. Polyclonal antibodies raised against glycosylated extensin-1, the most abundant HRGP in carrot (Daucus carota L.) cell walls, recognize this antigen on gel and dot blots and on thin sections of epoxy-embedded carrot-root cell walls. Since wall labeling can be largely reduced by preincubating the antibodies with purified extensin-1, most labeling can be attributed to recognition of this antigen. The remaining label may be the result of recognition of extensin-2, a second carrot HRGP, or other wall components (cellulose, hemicellulose and pectin are not recognized). Extensin-1 label was distributed quite uniformly across the cell wall but was absent from the expanded middle lamella at the intersection of three or more cells and was reduced in the narrow middle lamella between two cells. This distribution is essentially the same as that of cellulose. Because of limitations of this labeling technique, it is not possible to construct a complete model of the structure of the cross-linked extensin matrix. Nonetheless, short, linear arrays of gold particles may represent small portions of the extensin matrix or of individual extensin molecules as they are exposed on the surface of sections. These and other results presented here indicate that: a) newly synthesized extensin is added to the wall by intussusception; b) extensin cannot cross the middle lamella separating the walls of adjacent cells; and c) incorporation of extensin is a late event in the development of phloem-parenchyma cell walls in carrot.Abbreviations dE-1 antibodies antibodies raised against deglycosylated extensin 1 - ELISA enzyme-linked immunosorbant assay - gE-1 antibodies antibodies raised against glycosylated extensin 1 - HRGP hydroxyproline-rich glycoprotein - PAGE polyacrylamide gel electrophoresis - RG-1 rhamnogalacturonan I - SDS sodium dodecyl sulfate     

Cell cycle regulation during growth-dormancy cycles in pea axillary buds

Accumulation patterns of mRNAs corresponding to histones H2A and H4, ribosomal protein genes rpL27 and rpL34, MAP kinase, cdc2 kinase and cyclin B were analyzed during growth-dormancy cycles in pea (Pisum sativum cv. Alaska) axillary buds. The level of each of these mRNAs was low in dormant buds on intact plants, increased when buds were stimulated to grow by decapitating the terminal bud, decreased when buds ceased growing and became dormant, and then increased when buds began to grow again. Flow cytometry was used to determine nuclear DNA content during these developmental transitions. Dormant buds contain G₁ and G₂ nuclei (about 3:1 ratio), but only low levels of S phase nuclei. It is hypothesized that cells in dormant buds are arrested at three points in the cell cycle, in mid-G₁, at the G₁/S boundary and near the S/G₂ boundary. Based on the accumulation of histone H2A and H4 mRNAs, which are markers for S phase, cells arrested at the G₁/S boundary enter S within one hour of decaptitation. The presence of a cell population arrested in mid-G₁ is indicated by a second peak of histone mRNA accumulation 6 h after the first peak. Based on the accumulation of cyclin B mRNA, a marker for late G₂ and mitosis, cells arrested at G₁/S begin to divide between 12 and 18 h after decapitation. A small increase in the level of cyclin B mRNA at 6 h after decapitation may represent mitosis of the cells that had been arrested near the S/G₂ boundary. Accumulation of MAP kinase, cdc2 kinase, rpL27 and rpL34 mRNAs are correlated with cell proliferation but not with a particular phase of the cell cycle.     

Patterns of protein synthesis in dormant and growing vegetative buds of pea

Lateral buds on intact pea plants (Pisum sativum L. cv. Alaska) remain dormant until they are stimulated to develop by decapitating the terminal bud. Using two-dimensional gel electrophoresis, we have examined the protein content of terminal and lateral buds from intact plants and from plants at various times after decapitation. Silver-staining and in-vivo-labeling demonstrated very different sets of proteins. The level of expression of 18 stained and 25 labeled proteins was altered when growth was stimulated; this represents 3.4% and 9.1% of the total proteins detected by each method, respectively. Within 24 h of being stimulated, lateral buds doubled in length and their protein content was qualitatively nearly the same as that of terminal buds. Six hours after decapitation, before the onset of detectable growth, the overall pattern of protein synthesis in lateral buds was more like that of growing lateral buds or of terminal buds than that of dormant lateral buds. Direct application of N⁶-furfurylaminopurine (kinetin) to buds on intact plants stimulated their growth and resulted in the same pattern of protein synthesis as did decapitation. Inhibition of bud growth by addition of indole-3-acetic acid to the stumps of decapitated plants resulted in the synthesis of dormancy-related proteins. Lateral buds at all stages of development incorporated labeled amino acids at similar rates, indicating that metabolic activity is not a component of dormancy in these buds.Abbreviations IAA indole-3-acetic acid - IEF isoelectric focusing - KIN kinetin (N⁶-furfurylaminopurine) - SDS sodium dodecylsulfate - TCA trichloroacetic acid - 2D-PAGE two-dimensional polyacrylamide gel electrophoresis     

A second extensin-like hydroxyproline-rich glycoprotein from carrot cell walls

The insoluble extensin matrix of dicot cell walls has been studied most fruitfully by examining the salt-extractable precursors to this matrix. Multiple extensin-like hydroxyproline-rich glycoproteins (HRGPs) have been isolated, or their existence inferred, from tomato, potato, bean, soybean, melon, carrot, and other plants. We and others previously have studied a carrot extensin which we call extensin-1. Here we report on the properties of extensin-2, a second salt-extractable hydroxyproline-rich glycoprotein from carrot. Like extensin-1, extensin-2 contains large amounts of hydroxyproline, serine, histidine, and lysine. In contrast, its tyrosine content is only about one-third that of extensin-1. Arabinose and galactose are the most abundant neutral sugars in both proteins, and nearly identical buoyant densities in CsCl suggest a similar proportion of carbohydrate in each. The size of extensin-2 is about half the size of extensin-1 based on: (a) the measured lengths of shadowed molecules (about 40 versus 84 nanometers); (b) the migration of extensin-2 in acid-urea gels relative to monomers, dimers, and trimers of extensin-1; and (c) the Stokes' radii of these molecules as determined by gel filtration chromatography. Electron microscopy of shadowed extensin-2 molecules indicates that they contain kinks, which may indicate the presence of intramolecular isodityrosine cross-links, but intermolecular cross-links, either with other extensin-2 molecules or extensin-1 molecules, are observed rarely if ever.     

Are annulate lamellae in the Drosophila embryo the result of overproduction of nuclear pore components?

Annulate lamellae are cytoplasmic organelles composed of stacked sheets of membrane containing pores that are structurally indistinguishable from nuclear pores. The functions of annulate lamellae are not well understood. Although they may be found in virtually any eucaryotic cell, they occur most commonly in transformed and embryonic tissues. In Drosophila, annulate lamellae are found in the syncytial blastoderm embryo as it is cleaved to form the cellular blastoderm. The cytological events of the cellularization process are well documented, and may be used as temporal landmarks when studying changes in annulate lamellae. By using morphometric techniques to analyze electron micrographs of embryos, we are able to calculate the number of pores per nucleus in nuclear envelopes and annulate lamellae during progressive stages of cellularization. We find that annulate lamellae pores remain at a low level while nuclear envelopes are expanding and acquiring pores in early interphase. Once nuclear envelopes are saturated with pores, however, the number of annulate lamellae pores increases more than 10-fold in 9 min. Over the next 30 min it gradually declines to the initial low level. On the basis of these results, we propose (a) that pore synthesis and assembly continues after nuclear envelopes have been saturated with pores; (b) that these supernumerary pores accumulate transiently in cytoplasmic annulate lamellae; and (c) that because these pores are not needed by the embryo they are subsequently degraded.     

Genotypic and phenotypic characterization of a large,diverse population of maize near‐isogenic lines

Genome‐wide association (GWA) studies can identify quantitative trait loci (QTL) putatively underlying traits of interest, and nested association mapping (NAM) can further assess allelic series. Near‐isogenic lines (NILs) can be used to characterize, dissect and validate QTL, but the development of NILs is costly. Previous studies have utilized limited numbers of NILs and introgression donors. We characterized a panel of 1270 maize NILs derived from crosses between 18 diverse inbred lines and the recurrent inbred parent B73, referred to as the nested NILs (nNILs). The nNILs were phenotyped for flowering time, height and resistance to three foliar diseases, and genotyped with genotyping‐by‐sequencing. Across traits, broad‐sense heritability (0.4–0.8) was relatively high. The 896 genotyped nNILs contain 2638 introgressions, which span the entire genome with substantial overlap within and among allele donors. GWA with the whole panel identified 29 QTL for height and disease resistance with allelic variation across donors. To date, this is the largest and most diverse publicly available panel of maize NILs to be phenotypically and genotypically characterized. The nNILs are a valuable resource for the maize community, providing an extensive collection of introgressions from the founders of the maize NAM population in a B73 background combined with data on six agronomically important traits and from genotyping‐by‐sequencing. We demonstrate that the nNILs can be used for QTL mapping and allelic testing. The majority of nNILs had four or fewer introgressions, and could readily be used for future fine mapping studies.     

Characterization of DRGs,developmentally regulated GTP-binding proteins,from pea and Arabidopsis

Developmentally regulated GTP-binding proteins (DRGs) from animals and fungi are highly conserved but have no known function. Here we characterize DRGs from pea (PsDRG) and Arabidopsis (AtDRG). Amino acid sequences of AtDRG and PsDRG were 90% identical to each other and about 65% identical to human DRG. Genomic Southern blotting indicated that AtDRG and PsDRG probably are single-copy genes. PsDRG mRNA accumulated preferentially in growing organs (root apices, growing axillary buds and elongating stems) compared with their non-growing counterparts. At DRG mRNA was relatively abundant in Arabidopsis leaves, stems and siliques, less abundant in flowers and flower buds, and barely detectable in roots. Histone mRNAs are known to accumulate predominantly during S phase of the cell cycle and are markers for proliferating cells. The patterns of histone H2A mRNA accumulation in pea and Arabidopsis organs were very similar to those of DRG mRNAs. An antiserum raised against a PsDRG N-terminal fusion protein recognized 43 and 45 kDa proteins. PsDRG proteins were more abundant in growing pea roots and stems than in non-growing organs, but they were equally abundant in growing and dormant axillary buds. After differential centrifugation, PsDRG proteins were found primarily in the microsomal (150 000×g pellet) and soluble (150 000×g supernatant) cell fractions.     

Cross-linking patterns in salt-extractable extensin from carrot cell walls

Extensins are hydroxyproline-rich glycoproteins (HRGPs) found in the primary cell walls of dicots. Extensin monomers are secreted into the wall and covalently bound to each other, presumably by isodityrosine (IDT) cross-links, to form a rigid matrix. Expression of the extensin matrix is correlated with inhibition of cell elongation during normal development and with increased resistance to virulent pathogens. We have isolated extensin from carrot root tissue (Daucus carota L.) by published techniques and have used gel filtration chromatography to purify fractions enriched in monomers and oligomers. We refer to this protein as “extensin-1” to distinguish it from “extensin-2,” a second extensin-like HRGP from carrot which we will describe later. We prepared extensin-1 for electron microscopy by shadowing it with platinum. Monomers are highly elongated (84 nanometers) and kinked at several sites. Kinks occur at all sites on molecules with nearly equal probability, but do not appear to occur at their ends. The distribution of kinks is similar to that of tyrosine-lysine-tyrosine sequences, which have been shown to be capable of forming intramolecular IDT cross-links, so we suggest that kinks are visible manifestations of intramolecular IDTs. Oligomers likely result from IDT cross-links between monomers, and may be regarded as transient precursors of the fully cross-linked matrix. Nearly 60% of cross-links involve the ends of molecules while the rest are scattered among internal sites. We discuss how the relative positions and proportions of intra- and intermolecular cross-links in extensin-1 may affect the structure, and in turn the function, of the extensin matrix.