Effects of temperature on cytomorphogenesis and ultrastructure ofMicrasterias denticulata Bréb

Summary Exposure of growingMicrasterias cells to high (32°–36°C) and low (3°–10°C) temperatures produces changes in morphology that are accompanied by several ultrastructural alterations. Whereas low temperatures essentially cause simplification of cell ornamentation, a variety of cell malformations result from high temperature treatment. These are the loss of cell symmetry leading to markedly aberrant cell shapes and an increase of main lobes with reduced degree of differentiation. Preliminary studies indicate that a shift in the distribution of membrane-associated Ca²⁺ by elevated temperatures probably underlies these abnormal cytomorphogenetic events. Both, low and high temperature cause a reduction in size of the young half cell and affect cytoplasmic streaming. Moreover, nuclear migration is retarded and chloroplast arrangement is influenced by temperature treatment at both ranges. Growth velocity of primary wall responsible for cell shaping is increased at high and slowed down at low temperatures compared to cells grown at 20°C.The main ultrastructural alterations induced by high temperatures are an increase in amount and length of ER cisternae, the appearance of heat shock granule aggregations localized in the cytoplasm, a reduced number of ribosomes and polysomes, the presence of oil bodies in growing cells and a varying thickness of the primary wall. Influences of low temperatures on ultrastructure are less pronounced. They are manifested in the formation of large aggregations of ER cisternae slightly differing from those found in untreated cells, a disturbed arrangement of the microtubule system surrounding the nucleus and a decrease of the number of cell wall forming cytoplasmic vesicles.It is thought that most of the temperature effects are due to an influence on membranes probably an alteration of ionic currents and, in addition, a modulation of normal protein synthesis.Dedicated to my teacher Professor Oswald Kiermayer in deep gratitude     

Detachment of transformed cells

A parallel-plate flow chamber was used to quantify the detachment of normal cloned rat embryo fibroblasts (CREF) fibroblasts,ras-transformed CREF fibroblasts (CREF T24), and CREF T24 fibroblasts transfected with a Krev/RAP1A suppressor gene (HK B1) from a confluent monolayer of normal CREF fibroblasts to determine if the expression patterns of CD44 variants (mol wt 110 and 140 kDa) corresponded with detachment properties and metastatic potential. In the detachment assay, known shear stresses ranging from 20–24 dyn/cm² were applied to the adherent cells and the number of cells detached from the monolayer after 180 s was determined. Results showed that cellular expression of CD44 variants correlated with the metastatic potential of the cells and with the cells’ ability to detach from a monolayer of normal cells. Western blot analysis showed a low level of expression of the CD44 variants in the normal cell line, CREF, and the lowly metastatic cell line, HK B1. Detachment studies showed a low percentage of detachment of both of these cell lines from a normal cell monolayer. Tumor-derived (HK B1-T) and lung nodule-derived (HK B1-M) cell lines were established and both formed tumors and metastasis with reduced latency periods as compared to HK B1, but still showed a markedly delayed latency period compared to the highly metastatic cell line, CREF T24. Both of these cell lines showed a higher expression of the CD44 variants as compared to CREF and HK B1, and detached easier than CREF and HK B1. CREF T24 showed a much higher level of expression of the variants and had a higher percentage detachment than all other cell lines. To further test the role of the CD44 variants in the ability of the cells to detach from the normal monolayer, CREF cells were transfected with a DNA construct that constitutively expresses the CD44 variants and the detachment properties of three randomly selected clones were studied. Clones 2 and 3 showed a low level of expression of the CD44 variants after transfection and detached from the normal monolayer similar to CREF. Clone 1 showed a high level of expression of the CD44 variants and the detachment of these cells was significantly higher than CREF. From these results, it is concluded that in the five cell lines studied, expression of the CD44 variants play a significant role in the ability of the cells to detach from a monolayer of normal cells. It is hypothesized that this detachment may be an important component of a cell’s ability to metastasize.     

Identification and quantification of endogenous gibberellins in apical buds and the cambial region of Eucalyptus

Endogenous gibberellins (GAs) were extracted and purified from apical buds of Eucalyptus nitens (Deane and Maid.) Maid. and the cambial region of E. globulus (Labill.). then analysed by capillary gas chromatography-mass spectrometry. GA₁ GA₁₉ GA₂₀ and GA₂₉ were identified by full scan mass spectra. Kovats retention indices and high resolution selected ion monitoring. Using deuterated internal standards. GA₁. GA₁₉. GA₂₀ and putative GA₂₉ and GA₅₃ were quantified in the apical buds, while GA₄. GA₈. GA₉ and GA₄₄ were shown to be either absent or present at very low levels. From the cambial region. GA₁ and GA₂₀ were quantified at levels of 0.30 ng (g fresh weight)-¹ and 8.8 ng (g fresh weight)-¹ respectively. These data suggest that the early 13-hydroxylation pathway is the dominant pathway for GA biosynthesis in Eucalyptus .     

Determination of the structure of the DNA binding domain of gamma delta resolvase in solution.

The DNA binding domain (DBD) of gamma delta resolvase (residues 141-183) is responsible for the interaction of this site-specific DNA recombinase with consensus site DNA within the gamma delta transposable element in Escherichia coli. Based on chemical-shift comparisons, the proteolytically isolated DBD displays side-chain interactions within a hydrophobic core that are highly similar to those of this domain when part of the intact enzyme (Liu T, Liu DJ, DeRose EF, Mullen GP, 1993, J Biol Chem 268:16309-16315). The structure of the DBD in solution has been determined using restraints obtained from 2-dimensional proton NMR data and is represented by 17 conformers. Experimental restraints included 458 distances based on analysis of nuclear Overhauser effect connectivities, 17 phi and chi 1 torsion angles based on analysis of couplings, and 17 backbone hydrogen bonds determined from NH exchange data. With respect to the computed average structure, these conformers display an RMS deviation of 0.67 A for the heavy backbone atoms and 1.49 A for all heavy atoms within residues 149-180. The DBD consists of 3 alpha-helices comprising residues D149-Q157, S162-T167, and R172-N183. Helix-2 and helix-3 form a backbone fold, which is similar to the canonical helix-turn-helix motif. The conformation of the NH2-terminal residues, G141-R148, appears flexible in solution. A hydrophobic core is formed by side chains donated by essentially all hydrophobic residues within the helices and turns. Helix-1 and helix-3 cross with a right-handed folding topology. The structure is consistent with a mechanism of DNA binding in which contacts are made by the hydrophilic face of helix-3 in the major groove and the amino-terminal arm in the minor groove. This structure represents an important step toward analysis of the mechanism of DNA interaction by gamma delta resolvase and provides initial structure-function comparisons among the divergent DBDs of related resolvases and invertases.     

Selectively 13C-enriched DNA: 13C and 1H assignments of a triple helix by two-dimensional relayed HMQC experiments

Summary We present NMR studies of an intramolecular triple helix, the three strands of which have been linked by a hexaethylene glycol chain. To overcome the generally encountered difficulties of assignment in the homopyrimidine strands, the carbon C1 of the pyrimidines were selectively ¹³C-enriched. Assignments of the aromatic and sugar protons were obtained from NOESY-HMQC and TOCSY-HMQC spectra. We show that the recognition of a DNA duplex by a third strand via triplex formation is easily carried out in solution by observing the changes of the ¹H1–¹³C1 connectivities as a function of pH. Furthermore, the conformation of the sugars has been found to be C2-endo, on the basis of the coupling constant values directly measured in an HSQC spectrum.     

Expression of CD44 Splice Variants During Lymphocyte Activation and Tumor Progression

Recently, splice variants of CD44 have been described that confer metastatic potential to non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies against variant CD44 (CD44v) sequences, we have examined the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and in colorectal neoplasia. Lymphohematopoietic cells express low levels of CD44v glycoproteins. During the process of lymphocyte activation in vitro and in vivo, expression of CD44v glycoproteins is transiently upregulated. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that confers metastatic capability. In human colorectal neoplasia we observed overexpression of CD44 splice variants in all invasive carcinomas. Already at early stages of colorectal tumor progression exon v5 epitopes were overexpressed. Tumor progression was strongly related to expression of CD44 isoforms containing exon v6 encoded domains. The findings establish CD44 variants as tumor progression markers in colorectal cancer.     

Analysis of the role of the COL1 domain and its adjacent cysteine-containing sequence in the chain assembly of type IX collagen

The mechanisms of chain selection and assembly of type IX collagen, a heterotrimer 1(IX)2(IX)3(IX), must differ from that of fibrillar collagens since it lacks the characteristic C-propeptide of these latter molecules. We have tested the hypothesis that the information required for this process is contained within the C-terminal triple helical disulfide-bonded region (LMW). The reassociations of the purified LMW fragments of pepsinized bovine type IX collagen were followed by the formation of disulfide-bonded multimers. Our data demonstrate that only three triple helical assemblies form readily, (1)₃, (2)₃, and 123. The information required for chain selection and assembly is thus, at least in part, contained in the studied fragments. Molecular stoichiometries different from the classical heterotrimer may thus also form under certain conditions.     

A Glycoprotein Expressed by Human Fibrous Astrocytes is a Hyaluronate-Binding Protein and a Member of the CD44 Family

We have isolated and characterized an antigen from normal human brain called p80, so called because it migrated with an Mr of 80 kDa on SDS PAGE. The Mr of 80 kDa consists of a protein of about 55-60 kDa and carbohydrate (20-25 kDa). The carbohydrate is almost entirely of the N-linked type, although a small amount of O-linked carbohydrate was detected. Cross-reactivity with monoclonal antibodies A3D8 and A1G3 showed that p80 could therefore be considered an isoform of the CD44 adhesion molecules. In addition, specific binding to hyaluronate which was not competed for by proteoglycan demonstrated that it involved different sites than the proteoglycan binding sites. We also observed that fucoidan and dextran sulphate increased the binding by 200-250% while chondroitin sulphate C also increased the binding but to a lesser extent. Heparin, heparan sulphate and chondroitin sulphates A and B did not have such an effect. The binding of p80 to hyaluronate was pH dependent with a maximum at pH 6.4. We concluded that p80 was an astrocyte specific adhesion molecule.     

Dominant role of local dipoles in stabilizing uncompensated charges on a sulfate sequestered in a periplasmic active transport protein.

Electrostatic interactions are among the key factors determining the structure and function of proteins. Here we report experimental results that illuminate the functional importance of local dipoles to these interactions. The refined 1.7-A X-ray structure of the liganded form of the sulfate-binding protein, a primary sulfate active transport receptor of Salmonella typhimurium, shows that the sulfate dianion is completely buried and bound by hydrogen bonds (mostly main-chain peptide NH groups) and van der Waals forces. The sulfate is also closely linked, via one of these peptide units, to a His residue. It is also adjacent to the N-termini of three alpha-helices, of which the two shortest have their C-termini "capped" by Arg residues. Site-directed mutagenesis of the recombinant Escherichia coli sulfate receptor had no effect on sulfate-binding activity when an Asn residue was substituted for the positively charged His and the two Arg (changed singly and together) residues. These results, combined with other observations, further solidify the idea that stabilization of uncompensated charges in a protein is a highly localized process that involves a collection of local dipoles, including those of peptide units confined to the first turns of helices. The contribution of helix macrodipoles appears insignificant.     

Behavioural responses of the vine weevil, Otiorhynchus sulcatus, to semiochemicals from conspecifics, Otiorhynchus salicicola, and host plants

The vine weevil Otiorhynchus sulcatus is a parthenogenetic reproducing species which forages for suitable host plants at night, but is found congregated in dark places during the day. Frass of this weevil species is suspected to contain attractive compounds that are host‐plant related. Using a still‐air olfactometer, we tested adult vine weevils at night for their behavioural response to odours from conspecifics, feeding on a mixture of spindle tree (Euonymus fortunei) and yew (Taxus baccata), and to a sexually reproducing related species (Otiorhynchus salicicola), feeding on a mixture of ivy (Hedera helix) and cherry laurel (Prunus laurocerasus). Their attraction to conspecifics and O. salicicola appeared to be related to frass production. Freshly collected frass from O. sulcatus and from O. salicicola males and females was attractive. Prunus laurocerasus and H. helix have not been observed to be hosts of the vine weevil in the field. However, our tests showed that the vine weevil was attracted to mechanically damaged leaves of both plant species, whereas undamaged leaves were not attractive. Only undamaged young unfolding leaves of H. helix were also attractive. The attraction to odours from mechanically damaged host and non‐host plants suggested the involvement of compounds that are commonly found in many plant species. The involvement of plant compounds and/or aggregation pheromones in attraction to frass of the vine weevil and frass of the related weevil species O. salicicola is discussed.