The MAN antigens are non-lamin constituents of the nuclear lamina in vertebrate cells

The characterization of the human antiserum designated MAN has led to the identification of a subset of non-lamin proteins that are exclusively located at the nuclear periphery in all vertebrate cell types examined, from human to fish. Immunoreactive protein species were whown to comprise three major polypeptides of M _r 78000, 58000 and 40000. These antigens co-partitioned with the nuclear lamina during in situ isolation of nuclear matrices from lamin A/C-positive and-negative mammlian cells. Using double immunofluorescence, the spatial relationship of MAN antigens to type-A and type-B lamins was further examined throughout the cell cycle of lamin A/C-positive mammalian cells. In interphase HeLa and 3t3 cells, MAN antigens colocalized with both types of lamins at the periphery of the nucleus, but were absent from intranuclear foci of lamin B. As HeLa cells proceeded into mitosis, MAN antigens were seen to segregate from lamins A/C and coredistribute with lamin B. Lamins A/C disassembled during late prophase/early prometaphase and reassociated with chromatin in telophase/cytokinesis. In contrast, MAN antigens and lamin B dispersed late during prometaphase and reassembled on chromosomes in anaphase. Altogether, our data suggest that MAN antigens may play key functions in the maintenance of the structural integrity of the nuclear compartment in vertebrate cells.     

Paired box mutations in familial and sporadic aniridia predicts truncated aniridia proteins.

Aniridia, an autosomal dominant ocular disorder characterized by iris hypoplasia, results from mutations in the PAX6 gene, which encodes paired box and homeobox motifs. In this report we describe five new mutations in the paired box region of the human PAX6 gene that are associated with aniridia. The paired box mutations that we detected were in both familial (three) and sporadic (two) cases. All five mutations predict truncated PAX6 proteins. Our study indicates that early premature translational termination mutations in the PAX6 gene result in haploinsufficiency and generate the aniridia phenotype.     

Production and modifications of extracellular structures during development of chytridiomycetes

Summary In development of the primitive fungi, chytridiomycetes, unwalled zoospores bearing single, posterior flagella are transformed into walled, round-cells which elaborate the thallus. Production, structural modification, or release of extracellular material are involved with each transition of developmental stage. This article reviews the variety and developmental changes of extracellular materials found at the cell surface of chytridiomycetes. A cell coat, produced from Golgi-derived vesicles during zoosporogenesis, is visible around free swimming zoospores of some chytridiomycetes. How the zoospore surface receives and transduces signals is not widely explored, but it is known that fenestrated cisternae and simple cisternae, which are integrated into the microbody-lipid globule complex, are spatially and structurally associated with the plasma membrane and flagellar apparatus. This spatial association, as well as the cytochemical localization of calcium in fenestrated cisternae, suggest a mechanism for signal transduction and for regulation of zoospore motility. Zoospores become encased in a new layer of extracellular material as the zoospore encysts. Among some chytrids the source of this material is preexisting vesicles which fuse with the plasma membrane. Among other zoospores, a readily identifiable population of encystment vesicles is not apparent, demonstrating that there is no single pattern or mechanism for zoospore encystment in chytridiomycetes. Encysted zoospores developing into thalli, typically produce cell walls with a microfibrillar substructure. Ultrastructural analysis of walls reveals distinctive architecture and remarkable sculpturing which have been used in systematics of some members of chytridiomycetes. Nothing is known as to underlying controls of cytoskeletal elements and plasma membrane enzyme complexes in wall biogenesis. Many changes in cell surface structures accompany thallus maturation. Septa, many traversed with plasmodesmata, are produced in most chytrid thallus types. As sporangia and resting spores prepare for the production and release of zoospores, additional extracellular layers of material are frequently produced. Polarized deposits of extracellular material become discharge plugs, discharge vesicles, or endoopercula. Interstitial material is also released into cleavage furrows. Circumscissile or localized digestion of walls produce operculate or inoperculate exit ports for zoospore release. Cryofixation preserves more extensive extracellular material than does conventional chemical fixation, and broader application of cryofixation may radically alter our current view of cell surface structure. Thus chytridiomycetes exhibit a range in patterns for the occurrence and subsequent modifications of extracellular materials, even for members within the same order. The most universally recognized role for these extracellular materials is protection. Although there is a reasonable view of the types of extracellular material involved in chytridiomycete development, we have only limited understandings of their biogenesis or roles in regulation and communication, areas awaiting more investigations.Abbreviations DIC Nomarski-differential contrast optics - TEM transmission electron microscopy     

Effects of modeling and lineage on fishing behavior in the small-eared bushbaby (otolemur garnettii)

Thirty-eight bushbabies(Otolemur garnettii)were subjects in an observational learning study. We exposed them to one of three modeling conditions: (1) fishing model—one that actually performed fishing behavior; (2) nonfishing model—one that performed as a model in every way except performance of fishing behavior; and (3) no model. We assessed them with regard to latency to approach the fishbowl, latency to make an initial fishing attempt, duration of time spent in the vicinity of the fishbowls, and number of actual fishing attempts. Results indicate that subjects that were exposed to either fishing or nonfishing models were faster to approach the fishbowls and spent more time in the vicinity of the fishbowls than animals in the no-model condition Lineage, i.e., whether or not the animals’ parents fished, rather than modeling condition, was the best predictor of the latency to initial fishing attempt and the number of attempts made.     

The Regional Distribution of N-Acetylaspartylglutamate (NAAG) and Peptidase Activity Against NAAG in the Rat Nervous System

Abstract: N -Acetylaspartylglutamate (NAAG), a prevalent peptide in the vertebrate nervous system, may be hydrolyzed by extracellular peptidase activity to produce glutamate and N -acetylaspartate. Hydrolysis can be viewed as both inactivating the peptide after synaptic release and increasing synaptic levels of ambient glutamate. To test the hypothesis that NAAG and the peptidase activity that hydrolyzes it coexist as a unique, two-stage system of chemical neurotransmission, 50 discrete regions of the rat CNS were microdissected for assay. In each microregion, the concentration of NAAG was determined by radioimmunoassay and the peptidase activity was assayed using tritiated peptide as substrate. The NAAG concentration ranged from 2.4 nmol/mg of soluble protein in median eminence to 64 in thoracic spinal cord. Peptidase activity against NAAG ranged from 54 pmol of glutamate produced per milligram of membrane protein per minute in median eminence to 148 in superior colliculus. A linear relationship was observed between NAAG peptidase and NAAG concentration in 46 of the 50 areas, with a slope of 2.26 and a correlation coefficient of 0.45. These data support the hypothesis that hydrolysis of NAAG to glutamate and N -acetylaspartate is a consistent aspect of the physiology and metabolism of this peptide after synaptic release. The ratio of peptide concentration to peptidase activity was >0.3 in the following four areas: ventrolateral medulla and reticular formation where the peptide is concentrated in axons of passage, thoracic spinal cord, where NAAG is concentrated in ascending sensory tracts as well as motoneuron cell bodies, and ventroposterior thalamic nucleus.     

Development of a new transformant selection system for Penicillium chrysogenum: isolation and characterization of the P. chrysogenum acetyl-coenzyme A synthetase gene (facA) and its use as a homologous selection marker

A new transformation system for the filamentous fungus Penicillium chrysogenum is described, based on the use of the homologous acetyl-coenzyme A synthetase (facA) gene as a selection marker. Acetate-non-utilizing (Fac^–) strains of P. chrysogenum were obtained by positive selection for spontaneous resistance to fluoroacetate. Among these fac mutants putative facA strains were selected for a loss of acetyl-coenzyme A (CoA) synthetase activity. The facA gene, coding for the enzyme acetyl-CoA synthetase, was isolated from a P. chrysogenum genomic library using synthetic oligonucleotides derived from conserved regions from the corresponding genes of Aspergillus nidulans and Neurospora crassa. Vector pPC2-3, comprising a genomic 6.5 kb PstI fragment, was able to complement P. chrysogenum facA strains with frequencies up to 27 transformants·g^–1 DNA. Direct selection of transformants was accomplished using acetate and low amounts (0.001%) of glucose as carbon sources. About 50% of the transformants arose by integration of pPC2-3 DNA at the homologous facA locus and 50% by integration elsewhere in the genome. Determination of the nucleotide sequence of part of the cloned fragment showed the presence of an open reading frame of 2007 nucleotides, interrupted by five putative introns. Comparison of the nucleotide and the amino acid sequence of the facA gene of P. chrysogenum with the facA gene of A. nidulans reveals similarities of 80% and 89%, respectively. The putative introns present in the P. chrysogenum facA gene appear at identical positions as those in the A. nidulans facA gene, but show no significant sequence similarity. Correspondence to: R. F. M.van Gorcom     

Characterization of cell surface carbohydrates on asexual spores of the water moldSaprolegnia ferax

Summary In asexual reproduction of the water mold,Saprolegnia ferax, four distinct and sequentially produced spores are involved in dispersal, two of which are motile and two of which are nonmotile. Composition of cell surface glycoproteins may be important in dispersal strategies for each of these stages. Binding patterns of fluorescently labelled lectins were investigated to identify differences in glycoproteins of asexually produced dispersal stages. The pattern of lectin binding to zoospores was diverse. FITC-Con A bound to surfaces of zoospores and membranes of the water expulsion vacuole system, indicating the prescence of mannosyl and glucosyl residues. In zoospores incubated for more than 30 min in FITC-WGA and FITC-GS II. which bind N-acetyl glucosamine, fluorescence was sometimes localized in peripheral, intracellular patches. In shorter incubations, secondary zoospores bound these lectins along the groove region where K-bodies were located. Surfaces of cystospores typically bound FITC-WGA, but not FITC-GS II. FITC-GS II, however, bound to empty cystospore walls, probably because reactive sugars were available at the inner surface of the wall. Germ tubes emerging from cystospores bound labelled WGA and GS II, but not Con A. The same lectin binding pattern was found along discharge papilla of primary cystospores, indicating that modifications in cystospore walls associated with direct germination and zoospore discharge were similar. Thus, glycoproteins involved in early establishment of the hyphal system differ from those forming the cell surface of cystospores. Differences in the binding pattern of lectins to zoospores and cystospores highlight differences between cell surface carbohydrates of motile and nonmotile asexual stages.Abbreviations BPA lectin fromBauhinia purpurea - C₁ primary cystospore - C₂ secondary cystospore - Con A concanavalin A, lectin fromCanavalia ensiformis - DBA lectin fromDolichos biflorus - DIC Nomarski differential interference contrast optics - DS dilute salts - FITC fluorescein isothiocyanate - FUC fucose - Gal galactose - GalNAc N-acetyl galactosamine - Glc glucose - GlcNAc N-acetyl glucosamine - GS I Griffonia simplicifolia lectin I - GS II G. simplicifolia lectin II - Man mannose - MPA lectin fromMaclura pomifera - PC phase contrast optics - PNA lectin fromArachis hypogaea - SBA soybean agglutinin, lectin fromGlycine max - UEA-1 lectin fromUlex europaeus - WGA wheat germ agglutinin fromTriticum vulgare - WV water expulsion vacuole     

Bacteria Associated with Cysts of the Soybean Cyst Nematode (Heterodera glycines)

The soybean cyst nematode (SCN), Heterodera glycines, causes economically significant damage to soybeans (Glycine max) in many parts of the world. The cysts of this nematode can remain quiescent in soils for many years as a reservoir of infection for future crops. To investigate bacterial communities associated with SCN cysts, cysts were obtained from eight SCN-infested farms in southern Ontario, Canada, and analyzed by culture-dependent and -independent means. Confocal laser scanning microscopy observations of cyst contents revealed a microbial flora located on the cyst exterior, within a polymer plug region and within the cyst. Microscopic counts using 5-(4,6-dichlorotriazine-2-yl)aminofluorescein staining and in situ hybridization (EUB 338) indicated that the cysts contained (2.6 ± 0.5) × 10⁵ bacteria (mean ± standard deviation) with various cellular morphologies. Filamentous fungi were also observed. Live-dead staining indicated that the majority of cyst bacteria were viable. The probe Nile red also bound to the interior polymer, indicating that it is lipid rich in nature. Bacterial community profiles determined by denaturing gradient gel electrophoresis analysis were simple in composition. Bands shared by all eight samples included the actinobacterium genera Actinomadura and Streptomyces. A collection of 290 bacteria were obtained by plating macerated surface-sterilized cysts onto nutrient broth yeast extract agar or on actinomycete medium. These were clustered into groups of siblings by repetitive extragenic palindromic PCR fingerprinting, and representative isolates were tentatively identified on the basis of 16S rRNA gene sequence. Thirty phylotypes were detected, with the collection dominated by Lysobacter and Variovorax spp. This study has revealed the cysts of this important plant pathogen to be rich in a variety of bacteria, some of which could presumably play a role in the ecology of SCN or have potential as biocontrol agents.     

Low‐dose linearity: The rule or the exception?

In 1976, Crump, Hoel, Langley, and Peto described how almost any dose‐response relationship for carcinogens becomes linear at low doses when background cancers are taken into account. This has been used, by the U.S. Environmental Protection Agency, USEPA, as partial justification for a regulatory posture that assumes low‐dose linearity, as is illustrated by a discussion of regulation of benzene as a carcinogen. The argument depends critically on the assumption that the pollutant and the background proceed by the same biological mechanism. In this paper we show that the same argument applies to noncancer end points also. We discuss the application to a number of situations: reduction in lung function and consequent increase in death rate due to (particulate) air pollution; reduction in IQ and hence (in extreme cases) mental deficiency due to radiation in utero; reduction of sperm count and hence increase in male infertility due to DBCP exposure. We conclude that, although the biological basis for the health effect response is different, in each case low‐dose linearity might arise from the same mathematical effect discussed by Crump et al. (1976). We then examine other situations and toxic end points where low‐dose linearity might apply by the same argument. We urge that biologists and chemists should concentrate efforts on comparing the biological and pharmacokinetic processes that apply to the pollutant and the background. Finally, we discuss some public policy implications of the possibility that low dose linearity may be the rule rather than the exception for environmental exposures.