Spatiotemporal variations in microcystin concentrations and in the proportions of microcystin-producing cells in several Microcystis aeruginosa populations.

With the aim of explaining the variations in microcystin (MC) concentrations during cyanobacterial blooms, we studied several Microcystis aeruginosa populations blooming in different freshwater ecosystems located in the same geographical area. As assessed by real-time PCR, it appeared that the potentially MC-producing cells (mcyB(+)) were predominant (70 to 100%) in all of these M. aeruginosa populations, with the exception of one population in which non-MC-producing cells always dominated. Apart from the population in the Grangent Reservoir, we found that the proportions of potentially MC-producing and non-MC-producing cells varied little over time, which was consistent with the fact that according to a previous study of the same populations, the intergenic transcribed spacer (ITS) genotype composition did not change (38). In the Grangent Reservoir, the MC-RR variant was the dominant microcystin variant throughout the bloom season, despite changes in the ITS composition and in the proportions of mcyB(+) cells. Finally, the variations in total MC concentrations (0.3 to 15 microg liter(-1)) and in the MC cellular quotas (0.01 to 3.4 pg cell(-1)) were high both between and within sites, and no correlation was found between the MC concentrations and the proportion of mcyB(+) cells. All of these findings demonstrate that very different results can be found for the proportions of potentially MC-producing and non-MC-producing cells and MC concentrations, even in M. aeruginosa populations living in more or less connected ecosystems, demonstrating the importance of the effect of very local environmental conditions on these parameters and also the difficulty of predicting the potential toxicity of Microcystis blooms.

Genetic diversity in Microcystis populations of a French storage reservoir assessed by sequencing of the 16S-23S rRNA intergenic spacer.

We compared the genetic diversity of the 16S-23S spacer of the rRNA gene (ITS1) in benthic and pelagic colonies of the Microcystis genus isolated from two different sampling stations with different depths and at two different sampling times (winter and summer) in the French storage reservoir of Grangent. In all, 66 ITS1 sequences were found in the different clone libraries. The nucleotide diversity of all the sampled isolates were in the same range (average number = 0.022) regardless of their origin, showing that several clones are involved in the summer bloom event and contribute to the high biomass production. Phylogenetic study and analysis of molecular variance (AMOVA) revealed no obvious genetic differentiation between the benthic and pelagic isolates. This finding confirms that the Microcystis genus in this lake is characterized by having both a benthic phase in winter and spring allowing this organism to survive in unfavorable environmental conditions, and a pelagic phase in summer and autumn when environmental conditions allow them to grow in the water column. Finally, comparing these sequences with those available in the GenBank database showed that some highly conserved genotypes are found throughout the world.

The centromeres of the Indian muntjac: evidence for the existence of multiple centromeres?

Unlike the centromeres of other species, the "compound' centromeres of the Indian muntjac span over exceptionally extended regions (Brinkley et al., 1984). We extend this concept and show that some of these centromeres are divisible into several chromomeres in which the light staining regions alternate with the dark staining C-band positive segments. Unlike the centromeres of other species where the centromere replicates as one unit, the replication of the sub-units constituting the centromere of the X-chromosome in the muntjac occurs at different times as at least three independent segments. The CREST staining of the centromere regions of even the smallest (Y2) chromosome is interrupted by non-staining segments. Electron microscopy shows similar interruptions in the continuity of the trilamellar kinetochore. Sister chromatid exchanges occur in the region of the centromeres and chromatid breaks within the centromere region occur in the non-fluorescent segments. We interpret these data to suggest that the centromere regions of the Indian muntjac are made up of independent multiple centromeres interrupted by non-centromeric chromatin. Relevance of these parameters in mutagenesis is briefly discussed.

Localization of anti-CENP antibodies and alphoid sequences in acentric heterochromatin in a breast cancer cell line.

Karyotype alterations are a hallmark of cancer cells. Of particular interest to our laboratory are the inactive centromeres and blocks of heterochromatin devoid of the accompanying centromere. When purified or monospecies anticentromere proteins (CENP) antibodies or the whole serum from scleroderma (crest) patients are applied to human chromosomes, the centromere region exhibits the label. When we treated MDA 435 cells with the anti-CENP-A, anti-CENP-B, or the whole serum, the label was apparent on heterochromatin pericentric to the active and inactive centromeres. Moreover, blocks of heterochromatin not associated with any centromere also exhibited the label. Anti-CENP-C, however, is more strictly confined to the centromere in discrete dots and is not detected on any region except the sites of active centromeres. Distribution of alpha sequences also shows a pattern compatible with its distribution in the heterochromatin. Apparently, the use of anti-CENP-A and anti-CENP-B antibodies or alphoid DNA may not detect either the centromere (primary constriction) or the kinetochore; CENP-C may be an exception.

Dissociation of minor satellite from the centromere in mouse.

The minor satellite DNA of mouse is believed to constitute the centromere. We report that centromeres of some chromosomes in the Cl1D cells of mouse are not associated with this DNA even though the latter is present on these chromosomes. The satellite DNA was detected distally from the centromere and could not be mistaken as a component of the centromere. We also report that the site of the primary constriction may not always coincide with the site of the anti-kinetochore antibody reaction. Whereas the regions containing the major satellite decondense upon treatment with bisbenzimidazole (Hoechst 33258), the sites carrying minor satellite resist decondensing.

Three overlapping lct genes involved in L-lactate utilization by Escherichia coli.

In Escherichia coli, the lct locus at min 80 on the chromosome map is associated with ability to grow on L-lactate and to synthesize a substrate-inducible flavin-linked dehydrogenase. Similar to that of the glpD-encoded aerobic glycerol-3-phosphate dehydrogenase, the level of induced enzyme activity is elevated by aerobiosis. Both of these controls are mediated by the two-component signal transduction system ArcB/ArcA, although sensitivity to the control is much more striking for L-lactate dehydrogenase. This study disclosed that the lct locus contained three overlapping genes in the clockwise order of lctD (encoding a flavin mononucleotide-dependent dehydrogenase), lctR (encoding a putative regulator), and lctP (encoding a permease) on the chromosomal map. These genes, however, are transcribed in the counterclockwise direction. No homology in amino acid sequence was found between aerobic glycerol-3-phosphate dehydrogenase and L-lactate dehydrogenase. A phi (lctD-lac) mutant was inducible by L-lactate but not D-lactate. Although the mutant lost the ability to grow on L-lactate, growth on D-lactate, known to depend on a different enzyme, remained normal.

Signal transduction by the epidermal growth factor receptor is attenuated by a COOH-terminal domain serine phosphorylation site.

It has been proposed that the acute desensitization of epidermal growth factor receptor (EGF-R) function can be accounted for, in part, by the effect of EGF to increase phosphorylation of the receptor at Ser1046/7 (Countaway, J.L., Nairn, A.C., and Davis, R.J. (1992) J. Biol. Chem. 267, 1129-1140). Here, we show that the mutational removal of this phosphorylation site causes an activation of EGF-R function and a potentiation of signal transduction. The mechanism of potentiation results from 1) defective down-regulation of the EGF-R when cells are incubated with high concentrations of EGF; and 2) increased EGF-stimulated tyrosine phosphorylation. The increased EGF-stimulated phosphorylation is associated with an alteration of the apparent specificity of tyrosine phosphorylation and is independent of the down-regulation defect. Together, these data strongly support the hypothesis that Ser1046/7 is a biologically significant site of regulatory phosphorylation of the EGF-R.

Mutational analysis of the cytoplasmic tail of the human transferrin receptor. Identification of a sub-domain that is required for rapid endocytosis.

It has been reported that the sequence Tyr20-X-Arg-Phe23 present within the cytoplasmic tail of the transferrin receptor may represent a tyrosine internalization signal (Collawn, J.F., Stangel, M., Kuhn, L.A., Esekogwu, V., Jing, S., Trowbridge, I.S., and Tainer, J. A. (1990) Cell 63, 1061-1072). However, as Tyr20 is not conserved between species (Alvarez, E., Gironès, N., and Davis, R. J. (1990) Biochem. J. 267, 31-35), the functional role of the putative tyrosine internalization signal is not clear. To address this question, we constructed a series of 32 deletions and point mutations within the cytoplasmic tail of the human transferrin receptor. The effect of these mutations on the apparent first order rate constant for receptor endocytosis was examined. It was found that the region of the cytoplasmic tail that is proximal to the transmembrane domain (residues 28-58) is dispensable for rapid endocytosis. In contrast, the distal region of the cytoplasmic tail (residues 1-27) was found to be both necessary and sufficient for the rapid internalization of the transferrin receptor. The region identified includes Tyr20-X-Arg-Phe23, but is significantly larger than this tetrapeptide. It is therefore likely that structural information in addition to the proposed tyrosine internalization signal is required for endocytosis. To test this hypothesis, we investigated whether a heterologous tyrosine internalization signal (from the low density lipoprotein receptor) could function to cause the rapid endocytosis of the transferrin receptor. It was observed that this heterologous tyrosine internalization signal did not allow rapid endocytosis. We conclude that the putative tyrosine internalization signal (Tyr20-Thr-Arg-Phe23) is not sufficient to determine rapid endocytosis of the transferrin receptor. The data reported here indicate that the transferrin receptor internalization signal is formed by a larger cytoplasmic tail structure located at the amino terminus of the receptor.

Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase.

A growth factor-stimulated (MAP2-related) protein kinase, ERT, that phosphorylates the epidermal growth factor receptor at Thr669 has been purified from KB human tumor cells by Northwood and co-workers (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). The ERT protein kinase has a restricted substrate specificity, and the structural determinants employed for substrate recognition by this enzyme have not been defined. As an approach toward understanding the specificity of substrate phosphorylation, we have used an in vitro assay to identify additional substrates for the ERT protein kinase. In this report we describe two novel substrates: (a) the human c-myc protein at Ser62 and (b) the rat c-jun protein at Ser246. Alignment of the primary sequences surrounding the phosphorylation sites located within the epidermal growth factor receptor (Thr669), Myc (Ser62), and Jun (Ser246) demonstrated a marked similarity. The observed consensus sequence was Pro-Leu-Ser/Thr-Pro. We propose that this sequence forms part of a substrate structure that is recognized by the ERT protein kinase.

Inhibitory insulin-like growth factor-binding protein: cloning, complete sequence, and physiological regulation.

In this study we report the preparation of a human osteosarcoma cell cDNA library and describe the isolation and sequence determination of a clone encoding the complete sequence of a novel human insulin-like growth factor (IGF)-binding protein (hIGFBP-4). Previous work indicated that hIGFBP-4 is the predominant IGFBP expressed by human osteoblast-like cells, and that IGFBP-4 binds and inhibits the mitogenic activities of IGF-I and IGF-II. Sequence determination revealed that hIGFBP-4 is a unique gene product with significant amino- and carboxy-terminal sequence similarity to three other known IGFBPs. Identical alignment of 18 cysteines in IGFBP-4 and the three other IGFBPs is a key structural feature of this protein family. In vitro studies of human osteoblast-like cells suggest that PTH regulates the expression of hIGFBP-4 and that the PTH effect is mediated through a cAMP mechanism. hIGFBP-4 mRNA was also expressed in skin fibroblasts, and thus, this inhibitory IGFBP could be an important physiological regulator of IGF actions in bone cells and other cell types as well.

Aromatase activity in human adipose tissue stromal cells: effect of growth factors.

Adipose tissue is a major, nonglandular site for the aromatization of androgens to estrogens. In this tissue, the aromatase activity resides primarily in the stromal cells, and we have used cultures of stromal cells to study the effects of insulin and insulin-like growth factor I (IGF-I) on aromatase activity. Adipose tissue, obtained during indicated surgery, was digested with collagenase, and the stromal cells were isolated and cultured. Aromatase activity was determined by measuring the tritiated water (3H2O) in the medium after incubating stromal cells with [1 beta-3H]androstenedione. Insulin and IGF-I had no effect on the aromatase activity in cultured adipose stromal cells at concentrations of 10 to 1,000 microU/ml. However, insulin (100 to 1,000 microU/ml) and IGF-I (500 ng/ml) markedly attenuated the stimulatory effect of (Bu)2cAMP, but significantly augmented the dexamethasone-stimulated aromatase activity. The greater effects of IGF-I compared with the effect of insulin are compatible with both effects being mediated through the IGF-I compared with the effect of insulin are compatible with both effects being mediated through the IGF-I receptor. In addition, the effects of insulin in attenuating the aromatase activity in adipose tissue could potentiate its role in hyperandrogenic syndromes in women.

Chronic urticaria: a model questionnaire for patient screening.

Evaluating patients with chronic urticaria is frequently a time-consuming, costly, and frustrating undertaking. Taking a thorough, detailed history should always be the first step in the diagnostic process as associations that may not be apparent to the patient are important to the physician. We developed a questionnaire to be used as an initial historical database in evaluating chronic urticaria and to help efficiently establish the diagnosis of underlying causes.

Assembly of Escherichia coli fumarate reductase holoenzyme.

The production and assembly of the four fumarate reductase polypeptides into holoenzyme was studied in vivo in a T7-promoter-conditional expression system. No posttranslational modification of any of the subunits was detected, although the ratio of polypeptides produced varied with the temperature at which expression occurred. FrdC and FrdD, the membrane anchor polypeptides, assembled rapidly into the membrane and then were capped with FrdA and FrdB in separate events. Truncation of the C-terminal domain of FrdD by insertion of transposon Tn5 into the frdD cistron interfered with membrane insertion of the anchor polypeptides and assembly of the holoenzyme. Proteolytic degradation of truncated FrdD was implicated in the production of a soluble FrdABC trimer.

Regulation of in vitro expression of the Escherichia coli frd operon: alanine and Fnr represent positive and negative control elements.

The frdABCD operon of Escherichia coli encodes the anaerobically expressed terminal electron transport enzyme, fumarate reductase. Two mutually exclusive hairpin loop structures can occur in frdmRNA just downstream of the start of the frdA cistron. The mRNA sequence involved encodes a stretch of sequence rich in Ala and uses all four of the codons for this amino acid. In vitro expression of the frdABCD operon showed that as the level of plasmid DNA was increased from 150 fmol to 225 fmol, transcription of mRNA was suddenly elevated 6.5-fold, consistent with the concept of titrating out a repressor protein. Further studies showed that the concomitant 10.9-fold increase in translation of protein was heavily biased towards the proximal end of the operon, with little or no expression of FrdC or FrdD and a ratio of FrdA:FrdB of 2.6:1. Addition of Ala to the S-30 extract caused a 6.1-fold amplification of frd messenger transcription, a 17.6-fold increase in Frd protein translation, and a balancing of the subunit ratios to 1:1:1:1. The expression of the bla gene carried on the plasmid was not affected by DNA titration or the addition of Ala. When fnr DNA was added in equimolar ratio to frdDNA the amplification of fumarate reductase expression by Ala was abolished and the ratio of subunits produced showed a high degree of polarity with or without Ala.

Investigation of Escherichia coli fumarate reductase subunit function using transposon Tn5.

Seventy two Tn5 transposon insertions were isolated in the frd operon carried on the multicopy plasmid pFRD79. The polar nature of these mutations permitted examination of the expression and localization of the frd polypeptides in novel subunit combinations. The minimal catalytic unit is the FRDA plus B dimer. A transposon within frdB (frdB::Tn5) produces inactive, soluble FRDA polypeptide which has covalently attached 8 alpha(N3-histidyl)flavin adenine dinucleotide cofactor. A transposon mutation within frdC (frdC::Tn5) produces soluble, catalytically active dimer. An insertion in frdD (frdD::Tn5) produces both a soluble trimer composed of FRDABC, and a tetramer of FRDABC and truncated FRDD bound to the inner membrane. Eighty percent of the activity is in the soluble form. Using this mutant, the requirement for FRDD both for optimal activity of the catalytic domain and for proper anchorage in the cytoplasmic membrane was demonstrated.

Dapsone in the treatment of erosive lichen planus.

A patient with erosive lichen planus responded to therapy with dapsone after multiple therapeutic modalities had failed. The potential usefulness of dapsone therapy for lymphocyte-mediated inflammatory diseases such as erosive lichen planus is suggested.

Self-healing juvenile cutaneous mucinosis.

A case of self-healing juvenile cutaneous mucinosis is presented. It demonstrates typical features of the disease: early age of onset, infiltrated plaque lesions of the head and torso, nodules of the face and periarticular regions, rapid onset accompanied by inflammatory phenomena, and spontaneous resolution over a few months. Skin biopsy showed the deposition of the mucin, hyaluronic acid, mainly in the upper reticular dermis, and a mild increase in fibroblasts and mast cells. Results of studies of B and T lymphocytes were normal. Although three cases have been reported in Europe, to our knowledge this is the first reported case in the English-language literature.