Mucins and molluscan calcification. Molecular characterization of mucoperlin, a novel mucin-like protein from the nacreous shell layer of the fan mussel Pinna nobilis (Bivalvia, pteriomorphia)

A cDNA expression library constructed from mantle tissue mRNA of the Mediterranean fan mussel Pinna nobilis was screened with antibodies raised against the acetic acid-soluble shell matrix of the same species. This resulted in the isolation of a 2138-base pair cDNA, containing 13 tandem repeats of 93 base pairs. The deduced protein has a molecular mass of 66.7 kDa and a isoelectric point of 4.8. This protein, which is enriched in serine and proline residues, was overexpressed, purified, and used for producing polyclonal antibodies. Immunological in situ and in vitro tests showed that the protein is localized in the nacreous aragonitic layer of P. nobilis, but not in the calcitic prisms. Because this protein of the nacre of P. nobilis exhibits some mucin-like characteristics, we propose the name mucoperlin. This is the first paper reporting the cloning of a molluscan mucin and the first molecular evidence for the involvement of a mucin in molluscan calcification. This finding corroborates our previous hypothesis that some of the proteinaceous constituents of the molluscan shell matrix would derive from mucins, common to many metazoan lineages of the late Precambrian (Marin, F., Smith, M., Isa, Y., Muyzer, G. and Westbroek, P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1554-1559). The adaptation of an ancestral mucin to a new function, the regulation of the mineralization process, may be one of the molecular events, among others, that would explain the simultaneous emergence of organized calcification in many metazoan lineages during the Cambrian explosion.     

Role of thyroglobulin endocytic pathways in the control of thyroid hormone release

Thyroglobulin (Tg), the thyroid hormoneprecursor, is synthesized by thyrocytes and secreted into the colloid.Hormone release requires uptake of Tg by thyrocytes and degradation inlysosomes. This process must be precisely regulated. Tg uptake occursmainly by micropinocytosis, which can result from both fluid-phasepinocytosis and receptor-mediated endocytosis. Because Tg is highlyconcentrated in the colloid, fluid-phase pinocytosis or low-affinityreceptors should provide sufficient Tg uptake for hormone release;high-affinity receptors may serve to target Tg away from lysosomes,through recycling into the colloid or by transcytosis into thebloodstream. Several apical receptors have been suggested toplay roles in Tg uptake and intracellular trafficking. A thyroidasialoglycoprotein receptor may internalize and recycle immature formsof Tg back to the colloid, a function also attributed to an as yetunidentified N-acetylglucosamine receptor. Megalin mediatesTg uptake by thyrocytes, especially under intense thyroid-stimulatinghormone stimulation, resulting in transcytosis of Tg from the colloidto the bloodstream, a function that prevents excessive hormone release.

     

Bile acid secretion during rat liver carcinogenesis

Retro-differentiation of liver parenchyma during neoplastic processes is characterized by the expression of tumor antigens, such as alpha-fetoprotein and the placental isoenzyme of glutathione-S-transferase (GST-P). To investigate whether this may also affect a typical liver function such as bile acid secretion was the aim of this work. Rat hepatocarcinogenesis was induced by diethylnitrosamine (i.p., 200 mg/Kg body weight at day 0) and promoted by two-thirds partial hepatectomy (at day 21) plus 2-acetamidofluorene administration (50 mg/Kg body weight, subcutaneously, twice a week from day 14 to day 35). In order to carry out planimetric measurements of neoplastic tissue after immunohistochemical staining, a novel monoclonal antibody (MAb 14.1.3) against GST-P with no cross-reactivity against the major liver isoform of GST (GST-H) was raised. Analysis of total biliary bile acid output using the 3alpha-hydroxysteroid dehydrogenase method indicated that a significant reduction (-26%) occurred during the formation of GST-P-positive foci (12 wk). This was restored to normal values during adenoma formation (16-20 wk), but decreased again during carcinoma transformation (32 wk). These changes were not parallel to that observed in bile flow, which was progressively but slightly decreased throughout the whole period under study. HPLC analysis of bile samples collected for 1 h at different time points during hepatocarcinogenesis revealed that in contrast to what happens during cholestatic disease, a continuous and progressive increase in the cholic acid-to-chenodeoxycholic acid ratio (from 4.4+/-0.5 in control animals to 15.1+/-1.9 in rats with hepatocellular carcinoma) occurs. A significant and transient increase at 16 wk (+120%) in the proportion of bile acids amidated with glycine as compared to those conjugated with taurine was also observed. These results indicate that the mechanisms accounting for the secretion of major bile acids are modified differently at various steps of rat liver tumor development.     

A New HPLC Analytical Method to Study Fungal Caffeine Metabolism

A simple and rapid HPLC method has been developed to analyse all the methylxanthines that can be produced by N-demethylation of 1,3,7-trimethylxanthine (caffeine). This method is particularly suitable to study caffeine metabolism of a filamentous fungus (Aspergillus sp V12A25) cultivated in a synthetic liquid medium containing caffeine as the sole source of nitrogen.     

Viral Oncoproteins Discriminate between p53 and the p53 Homolog p73

p73 is a recently identified member of the p53 family. Previously it was shown that p73 can, when overproduced in p53-defective tumor cells, activate p53-responsive promoters and induce apoptosis. In this report we describe the generation of anti-p73 monoclonal antibodies and confirm that two previously described p73 isoforms are produced in mammalian cells. Furthermore, we show that these two isoforms can bind to canonical p53 DNA-binding sites in electrophoretic mobility shift assays. Despite the high degree of similarity between p53 and p73, we found that adenovirus E1B 55K, simian virus 40 T, and human papillomavirus E6 do not physically interact with p73. The observation that viral oncoproteins discriminate between p53 and p73 suggests that the functions of these two proteins may differ under physiological conditions. Furthermore, they suggest that inactivation of p73 may not be required for transformation.     

Speciation and phylogeography of giant petrels Macronectes

We examine global phylogeography of the two forms of giant petrel Macronectes spp. Although previously considered to be a single taxon, and despite debate over the status of some populations and the existence of minimal genetic data (one mitochondrial cytochrome b sequence per form), the current consensus based on morphology is that there are two species, Northern Giant Petrel M. halli and Southern Giant Petrel M. giganteus. This study examined genetic variation at cytochrome b as well as six microsatellite loci in giant petrels from 22 islands, representing most island groups at which the two species breed. Both markers support separate species status, although sequence divergence in cytochrome b was only 0.42% (corrected). Divergence was estimated to have occurred approximately 0.2 mya, but with some colonies apparently separated for longer (up to 0.5 my). Three clades were found within giant petrels, which separated approximately 0.7 mya, with the Southern Giant Petrel paraphyletic to a monophyletic Northern Giant Petrel. There was evidence of past fragmentation during the Pleistocene, with subsequent secondary contact within Southern Giant Petrels. The analysis also suggested a period of past population expansion that corresponded roughly to the timing of speciation and the separation of an ancestral giant petrel population from the fulmar Fulmarus clade.     

First cytochemical study of haemocytes from the crab Carcinus aestuarii (Crustacea,Decapoda)

For the first time, a morphological study of haemocytes from the crab Carcinus aestuarii was carried out by means of light microscopy and differing cytochemical assays. Analysis of haemocyte size frequency distribution (performed by means of a Coulter Counter) revealed the presence of two distinct haemocyte fractions in C. aestuarii haemolymph, depending on cell size. The first fraction was of about 3–5 µm in diameter and 30–50 fL in volume, the second was of about 6–12 µm in diameter and over 200 fL in volume. Mean cell diameter and volume were 8.20±1.7 µm and 272.30±143.5 fL, respectively. Haemocytes observed under light microscope were distinguished in three cell types: granulocytes (28%; 11.94±1.43 µm in diameter) with evident cytoplasmic granules, semigranulocytes (27%; 12.38±1.76 µm in diameter) with less granules than granulocytes, and hyalinocytes (44%; 7.88±1.6 µm in diameter) without granules. In addition, a peculiar cell type was occasionally found (about 1%): it was 25–30 µm in diameter and had a great vacuole and a peripheral cytoplasm with granules. Granulocyte and semigranulocyte granules stained in vivo with Neutral Red, indicating that they were lysosomes. Giemsa’s dye confirmed that granulocytes and semigranulocytes were larger than hyalinocytes. Pappenheim’s panoptical staining and Ehrlich’s triacid mixture allowed to distinguish granule-containing cells (including semigranulocytes) in acidophils (64%), basophils (35%) and neutrophils (1%). Hyalinocytes showed always a basophilic cytoplasm. Haemocytes were positive to the PAS reaction for carbohydrates, even if cytoplasm carbohydrate distribution varied among cell types. Lastly, lipids were found on cell membrane and in cytoplasm of all haemocyte types in the form of black spots produced after Sudan Black B staining. The morphological characterisation of C. aestuarii haemocytes by light microscopy was necessary before performing both ultrastructural and functional studies of circulating cells.Key words: Carcinus aestuarii, crab, haemocytes, light microscopy, cytochemical assays, morphological characterisation.     

Discovery of a novel series of potent S1P1 agonists

The discovery of a novel series of S1P1 agonists is described. Starting from a micromolar HTS positive, iterative optimization gave rise to several single-digit nanomolar S1P1 agonists. The compounds were able to induce internalization of the S1P1 receptor, and a selected compound was shown to be able to induce lymphopenia in mice after oral dosing.     

The properties and contribution of the Corynebacterium glutamicum MscS variant to fine-tuning of osmotic adaptation

Based on sequence similarity, the mscCG gene product of Corynebacterium glutamicum belongs to the family of MscS-type mechanosensitive channels. In order to investigate the physiological significance of MscCG in response to osmotic shifts in detail, we studied its properties using both patch-clamp techniques and betaine efflux kinetics. After heterologous expression in an Escherichiacoli strain devoid of mechanosensitive channels, in patch-clamp analysis of giant E. coli spheroplasts MscCG showed the typical pressure dependent gating behavior of a stretch-activated channel with a current/voltage dependence indicating a strongly rectifying behavior. Apart from that, MscCG is characterized by significant functional differences with respect to conductance, ion selectivity and desensitation behavior as compared to MscS from E. coli. Deletion and complementation studies in C. glutamicum showed a significant contribution of MscCG to betaine efflux in response to hypoosmotic conditions. A detailed analysis of concomitant betaine uptake (by the betaine transporter BetP) and efflux (by MscCG) under hyperosmotic conditions indicates that MscCG may act in osmoregulation in C. glutamicum by fine-tuning the steady state concentration of compatible solutes in the cytoplasm which are accumulated in response to hyperosmotic stress.     

Energy transfer pathways in the CP24 and CP26 antenna complexes of higher plant photosystem II: a comparative study

Antenna complexes are key components of plant photosynthesis, the process that converts sunlight, CO₂, and water into oxygen and sugars. We report the first (to our knowledge) femtosecond transient absorption study on the light-harvesting pigment-protein complexes CP26 (Lhcb5) and CP24 (Lhcb6) of Photosystem II. The complexes are excited at three different wavelengths in the chlorophyll (Chl) Qy region. Both complexes show a single subpicosecond Chl b to Chl a transfer process. In addition, a reduction in the population of the intermediate states (in the 660-670 nm range) as compared to light-harvesting complex II is correlated in CP26 to the absence of both Chls a604 and b605. However, Chl forms around 670 nm are still present in the Chl a Qy range, which undergoes relaxation with slow rates (10-15 ps). This reduction in intermediate-state amplitude CP24 shows a distinctive narrow band at 670 nm connected with Chls b and decaying to the low-energy Chl a states in 3-5 ps. This 670 nm band, which is fully populated in 0.6 ps together with the Chl a low-energy states, is proposed to originate from Chl 602 or 603. In this study, we monitored the energy flow within two minor complexes, and our results may help elucidate these structures in the future.