Direct fluorescence localization of an endogenous N-acetyl-glucosamine-specific lectin in the thyroid gland

A sugar-binding protein, or endogenous lectin, was localized on fixed and paraffin-embedded thyroid sections by means of fluorescein-labelled neoglycoproteins. Fluorescence microscopy showed the binding of this lectin to be dependent on calcium ions and acidic pH and indicated sugar specificity for GlcNAc moieties only. In human, porcine and murine thyrocytes, specific binding was observed mainly on subcellular organelles. Conversely, in rabbit thyrocytes, specific labelling was seen mostly at the apical cell surface facing the follicular lumen. The possibility that this novel endogenous lectin is involved in the Tg metabolism is considered.     

Protein-disulfide isomerase (PDI) in FRTL5 cells. pH-dependent thyroglobulin/PDI interactions determine a novel PDI function in the post-endoplasmic reticulum of thyrocytes

Thyroglobulin (TG) is secreted by the thyrocytes into the follicular lumen of the thyroid. After maturation and hormone formation, TG is endocytosed and delivered to lysosomes. Quality control mechanisms may occur during this bidirectional traffic since 1) several molecular chaperones are cosecreted with TG in vivo, and 2) lysosomal targeting of immature TG is thought to be prevented via the interaction, in acidic conditions, between the Ser(789)-Met(1172) TG hormonogenic domain (BD) and an unidentified membrane receptor. We investigated the secretion and cell surface expression of PDI and other chaperones in the FRTL5 thyroid cell line, and then studied the characteristics of the interaction between TG and PDI. We demonstrated that PDI, but also other chaperones such as calnexin and KDEL-containing proteins are exposed at the cell surface. We observed on living cells or membrane preparations that PDI specifically binds TG in acidic conditions, and that only BD is involved in binding. Surface plasmon resonance analysis of TG/PDI interactions indicated: 1) that PDI bound TG but only in acidic conditions, and that it preferentially recognized immature molecules, and 2) BD is involved in binding even if cysteine-rich modules are deleted. The notion that PDI acts as an "escort" for immature TG in acidic post-endoplasmic reticulum compartments is discussed.     

Performance of 18S rDNA helix E23 for phylogenetic relationships within and between the Rotifera-Acanthocephala clades

The species diversity of the phylum Rotifera has been largely studied on the basis of morphological characters. However, cladistic relationships within this group are poorly resolved due to extensive homoplasy in morphological traits, substantial phenotypic plasticity and a poor fossil record. We undertook this study to determine if a phylogeny based on partial 18S rDNA, which included the helix E23 of 18S rDNA sequence, was concordant with established taxonomic relationships within the order Ploimida (class: Monogononta). We also estimated the level of polymorphism within clones and populations of Ploimida 'species'. Finally, we included the Cycliophora Symbion pandora as outgroup and the variable helix E23 region to examine the influence of their signal on the evolutionary relationships among Acanthocephala, Bdelloidea and Ploimida. Phylogenetic reconstruction was performed using maximum parsimony, neighbour joining and maximum likelihood methods. We found 1) that morphologically similar Ploimida 'species' show vastly different 18S E23 rDNA sequences; 2) inclusion of the helix E23 of 18S rDNA and its secondary structure analysis results in better resolution of family level relationships within the Ploimida; 3) an impact of Symbion pandora as an outgroup with inclusion of the helix E23 on the relationships between the Rotifera and the Acanthocephala; and 4) partial incongruence and differential substitution rate between conserved region and helix E23 region of the 18S rDNA gene depending on the taxomic group studied.     

Origin and development of river zooplankton: example of the Marne

Zooplankton composition and growth in the river Marne (France)were studied on a space scale of 300 km in July 1991. There werethree distinct areas along the river: the immediate reservoir outlet(pK 652), the natural river called middle Marne (from pK 652to pK 799 downstream) and the channeled river (from pK 799 topK 975 downstream). A typical lake community, characterized both byan abundance of microcrustaceans and a high zooplanktonconcentration was found immediately downstream of the reservoir Marne(Der-Chantecocq Lake). Here, large microcrustaceans (copepods,daphnids), and large rotifers (Keratella cochlearisrobusta and Polyarthra dolichoptera-vulgaris) rapidlydisappeared, and small rotifer species (<120 m) dominated theplankton. Their populations (specially Keratella c. cochlearis)proliferated in the middle Marne as far as 100 km downstream(up to 288 ind. l-1) but were considerably reduced (20 to35 ind. l-1) where the river is channeled, algal resourcesdecline and turbidity increases.The dominance of small organisms such as rotifers, in riverplankton is assumed to be the result of fish predation on largezooplankton as well as of a short generation time which allowstheir in situ reproduction, in spite of a short residencetime of the water.     

Preparation and partial characterization of polyclonal antibodies raised against a preselected sequence of calcium dependent lectins

The synthetic-peptide strategy was used to generate antibodies raised against calcium-dependent lectins of vertebrates. We demonstrate that a synthetic peptide predicted from the amino acid sequence of the carbohydrate recognition domain can induce blocking antibodies which would react with, or in close vicinity of, the binding site of the parent molecule. As the preselected sequence was chosen in a consensus sequence region, we also present preliminary investigations of the use of specific antisera as a common biological probe against calcium dependent lectins. The availability of monospecific polyclonal sera opens new possibilities in biochemical and structural studies as well as immunodection of calcium dependent lectins.     

SPC3, an anti-HIV peptide construct derived from the viral envelope,binds and enters HIV target cells

SPC₃ is a peptide construct (eight branches of the GPGRAF motif) derived from the consensus sequence present at the apex of the third variable domain of the human immunodeficiency virus (HIV) envelope (Env). It presents a potent anti-HIV activity and is currently tested in phase II clinical trials (FDA protocol 257A). Its mode of action remains unclear. It was thought that SPC₃ exerts its effect both during HIV interaction with CD4⁺ cells but also through interference either with a post-binding event or with Env processing. Accordingly, SPC₃ was supposed to be able to bind and to enter CD4⁺ cells. In this work, we addressed these points. SPC₃ was found to interact with CD4⁺ cell membrane with a K_0.5 value in the range of 500 nm . The binding of SPC₃ to CD4⁺ cells involves its interaction with a cell membrane associated protein which is pronase sensitive and different from CD4. This interaction was similar from 2 to 37°C. The maximum binding occurred at acidic pH whereas the interaction was inhibited in alkaline conditions. We observed also that SPC₃ was internalized rapidly into the cells—the maximal intracell amount was reached within 30 min—where it remained stable for at least 24 h. Altogether, these data suggest that SPC₃ can exert its antiviral activity via interference with events occurring at the cell surface but also into the target cell. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Mammalian peptidylglycine alpha-amidating monooxygenase mRNA expression can be modulated by the La autoantigen

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal alpha-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3' untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3' UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3' UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3' UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules.     

Impact of turbulence and turbidity on the grazing rate of the rotifer Brachionus calyciflorus (Pallas)

The impact of turbulence and turbidity on Brachionus calyciflorus grazing rate was determined in short feeding periods (10 min), using labelled Chlorella pyrenoïdosa. The response to water motion of B. calyciflorus depends on it physiological state: the grazing rate of recently fed amictic females stomach green (with one or two eggs) is significantly reduced in agitated environments compared with non-agitated environments. In contrast, the grazing rate of starved amictic females is not reduced by water motion, whatever its velocity (V1=0.18 m s?1 and V2=0.22 m s?1). In the presence of suspended particles (3–6-μm silica beads), a larger reduction in grazing rate is observed in agitated water at any water velocity (V1=0.18 m s?1 or V2=0.22 m s?1), than in stagnant water. A synergy between turbulence and turbidity is unfavourable to feeding of rotifers.     

Intracellular degradation of the HIV-1 envelope glycoprotein. Evidence for, and some characteristics of, an endoplasmic reticulum degradation pathway.

Analysis of the fate of HIV-1 envelope protein gp160 (Env) has shown that newly synthesized proteins may be degraded within the biosynthetic pathway and that this degradation may take place in compartments other than the lysosomes. The fate of newly synthesized Env was studied in living BHK-21 cells with the recombinant vaccinia virus expression system. We found that gp160 not only undergoes physiological endoproteolytic cleavage, producing gp120, but is also degraded, producing proteolytic fragments of 120 kDa to 26 kDa in size, as determined by SDS/PAGE in non reducing conditions. Analysis of the 120-kDa proteolytic fragment, and comparison with gp120, showed that it is composed of peptides linked by disulfides bonds and lacks the V3-loop epitope and the C-terminal domain of gp120 (amino acids 506-516). A permeabilized cell system, with impaired transport of labeled Env from the endoplasmic reticulum (ER) to Golgi compartments, was developed to determine the site of degradation and to define some biochemical characteristics of the intracellular degradation process. In the semipermeable BHK-21 cells, there was: (a) no gp120 production (b), a progressive decrease in the amount of newly synthesized gp160 and a concomitant increase in the amount of a 120-kDa proteolytic fragment. This fragment had the same biochemical characteristics as the 120-kDa proteolytic fragment found in living nonpermeabilized cells, and (c) susceptibility of the V3 loop. This degradation process occurred in the ER, as shown by both biochemical and indirect immunofluorescence analysis. Furthermore, there was evidence that changes in redox state are involved in the ER-dependent envelope degradation pathway because adding reducing agents to permeabilized cells caused dose-dependent degradation of the 120-kDa proteolytic fragment and of the remaining gp160 glycoprotein. Thus our results provide direct evidence that regulated degradation of the HIV-1 envelope glycoprotein may take place in the ER of infected cells.     

The N-acetylglucosamine-specific receptor of the thyroid. Binding characteristics, partial characterization, and potential role

The binding characteristics of the GlcNAc binding protein present in thyroid membranes (Consiglio, E., Shifrin, S., Yavin, Z., Ambesi-Impiombato, F.S., Rall, J.E., Salvatore, G., and Kohn, L.D. (1981) J. Biol. Chem. 256, 10592-10599) were reinvestigated using neoglycoproteins as probes. Plasma membrane preparations from porcine thyroid specifically bound 125I-GlcNAc35-bovine serum albumin. Binding was dependent on the presence of calcium. Binding of ligand to receptor was minimal at neutral pH and maximal at pH 5.0. Equilibrium binding studies indicated positive cooperativity of binding and a site capacity of about 60 pmol/mg of protein. Competition studies were compatible with a specificity hierarchy of GlcNAc much greater than Gal; no recognition of mannose, fucose, or glucose moieties was noted. The receptor was detergent-solubilized from plasma membrane preparations and on the basis of the defined binding properties, purified by chromatography on a GlcNAc-Sepharose affinity column. The purified GlcNAc thyroid receptor has a subunit molecular size of about 45 kDa and appears to be an oligomer composed of three subunits. The receptor was identified as a component of thyrocytes by in situ cytochemical localization with fluorescent neoglycoproteins. In certain cases it was mainly present on, or near, the apical cell surface. It is suggested that this GlcNAc receptor functions in thyroglobulin metabolism, possibly involved in recycling of internalized thyroglobulin molecules back into the follicular lumen.