Shaping of colony elements in Laomedea flexuosa Hinks (Hydrozoa, Thecaphora) includes a temporal and spatial control of skeleton hardening.

The colonies of thecate hydroids are covered with a chitinous tubelike outer skeleton, the perisarc. The perisarc shows a species-specific pattern of annuli, curvatures, and smooth parts. This pattern is exclusively formed at the growing tips at which the soft perisarc material is expelled by the underlying epithelium. Just behind the apex of the tip, this material hardens. We treated growing cultures of Laomedea flexuosa with substances we suspected would interfere with the hardening of the perisarc (L-cysteine, phenylthiourea) and those we expected would stimulate it (dopamine, N-acetyldopamine). We found that the former caused a widening of and the latter a reduction in the diameter of the perisarc tube. At the same time, the length of the structure elements changed so that the volume remained almost constant. We propose that normal development involves a spatial and temporal regulation of the hardening process. When the hardening occurs close to the apex, the diameter of the tube decreases. When it takes place farther from the apex, the innate tendency of the tip tissue to expand causes a widening of the skeleton tube. An oscillation of the position at which hardening takes place causes the formation of annuli.     

An H1-like protein from the macronucleus of Euplotes eurystomus

An H1-like protein has been purified from the macronucleus (MAC) of the hypotrichous ciliated protozoan, Euplotes eurystomus. It is present in amounts comparable to the inner histones and is extracted by treatment with 5% perchloric acid or 0.65 M NaCl, but not by 0.35 M NaCl. Treatment of soluble MAC chromatin with the ionic exchange resin AG 50W-X2 in 80 mM NaCl removes MAC H1 and yields H1-depleted chromatin. Mac H1 is lysine-rich and deficient in acidic amino acids. The stoichiometry of the H1 protein is reduced in mononucleosome preparations, consistent with its postulated interaction with linker DNA regions. Thermal denaturation and circular dichroism studies reveal that H1-depleted chromatin contains a larger portion of destabilized DNA than control chromatin. The molecular weight of Euplotes MAC H1 is significantly smaller than most reported H1 proteins. Comparisons are made with extracts of macronuclei from other hypotrichous ciliated protozoa and published reports of other lower eukaryotes.     

Initiation of primary cell cultures from human intracranial tumors on extracellular matrix from bovine corneal endothelial cells

Tissue specimens from 105 human gliomas and 57 human meningiomas were obtained at surgery, dissociated into single cells and small cell aggregates and then plated onto plain plastic tissue culture dishes and dishes which had been precoated with an extracellular matrix (ECM) derived from bovine corneal endothelium. In 80% of the glioma cases we observed a marked improvement in initial plating efficiency, colony formation and speed of attachment when cells were plated on ECM. In 5 cases cells attached only to the ECM-coated dishes but remained afloat in the untreated dishes. In addition it could be noted that over the first 2 days, those cells which had been initiated on ECM showed more signs of morphological differentiation, i.e., extension of cytoplasmic processes or formation of fiber networks between cell groups. If adaptation occurred and proliferation began in vitro, either immediately or after a several days' lag phase, both the ECM-cultured cells as well as those which slowly had adapted to culture on plastic could be passed on to untreated culture ware and perpetuated thereon. In the case of well-differentiated low-grade gliomas where no growth in culture took place, the cultures on ECM could at least be used for initial experiments in the primary cultures (P0). Meningiomas usually attached well to both, plastic or ECM. In 50% of our cases the plating efficiency was higher on ECM but after successful initial culture, the delay until the cells on plastic reached confluence in comparison with those on ECM was 1 or 2 days. Again there were 2 cases in which the cells would not plate on plastic. Here the cells which after 1 day were still afloat plated to more than 80% within the first 2 h after transfer to ECM. In all cases the cells from plastic and ECM cultures were indistinguishable and could be passed onto untreated dishes henceforth. In later culture stages ECM offers several advantages: It is easier to shift cells to serum-free defined culture conditions, the cells will grow at a faster rate on ECM when in higher passages and the maximal number of passages possible is higher on ECM.     

Isolation and characterization of an ipt gene from the Ti plasmid Bo542

Summary A 1.9 kb clone of the T-DNA region of the Agrobacterium tumefaciens Ti plasmid Bo542 which exhibited homology to the isopentenyl transferase (ipt) locus of pTiA6 was identified by low stringency DNA hybridization. Introduction of this segment of pTiBo542 DNA into cells of Nicotiana tabacum or N. glauca caused tumor formation in vivo, and allowed hormone independent growth in vitro. Furthermore, this DNA segment complemented ipt mutant strains of A. tumefaciens, restoring their ability to cause tumors on Kalanchöe leaves and tomato stems. The complete DNA sequence of this segment has been determined, revealing an open reading frame homologous to other known Agrobacterium ipt genes.     

Comparative biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons

Members of the Chenopodiaceae can accumulate high levels (>100 mol·(g DW)^-1) of glycine betaine (betaine) in leaves when salinized. Chenopodiaceae synthesize betaine by a two-step oxidation of choline (cholinebetaine aldehyde betaine), with the second step catalyzed by betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8). High betaine levels have also been reported in leaves of species from several distantly-related families of dicotyledons, raising the question of whether the same betaine-synthesis pathway is used in all cases.Fast atom bombardment mass spectrometry showed that betaine levels of >100 mol·(g DW)^-1 are present in Lycium ferocissimum Miers (Solanaceae), Helianthus annuus L. (Asteraceae), Convolvulus arvensis L. (Convolvulaceae), and Amaranthus caudatus L. (Amaranthaceae), that salinization promotes betaine accumulation in these plants, and that they can convert supplied choline to betaine aldehyde and betaine. Nicotiana tabacum L. and Lycopersicon lycopersicum (L.) Karst. ex Farw. (Solanaceae), Lactuca sativa L. (Asteraceae) and Ipomoea purpurea L. (Convolvulaceae) also contained betaine, but at a low level (0.1–0.5 mol·(g DW)^-1. Betaine aldehyde dehydrogenase activity assays, immunotitration and immunoblotting demonstrated that the betaine-accumulating species have a BADH enzyme recognized by antibodies raised against BADH from Spinacia oleracea L. (Chenopodiaceae), and that the M_r of the BADH monomer is in all cases close to 63 000. These data indicate that the cholinebetaine aldehydebetaine pathway may have evolved by vertical descent from an early angiosperm ancestor, and might be widespread (albeit not always strongly expressed) among flowering plants. Consistent with these suggestions, Magnolia x soulangiana was found to have a low level of betaine, and to express a protein of M_r 63 000 which cross-reacted with antibodies to BADH from Spinacia oleracea.Abbreviations BADH Betaine aldehyde dehydrogenase - DCIMS desorption chemical ionization mass spectrometry - FABMS fast atom bombardment mass spectrometry - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate - TLC thin-layer chromatography