Preparation and characterization of geodin. A betagamma-crystallin-type protein from a sponge

Geodin is a protein encoded by a sponge gene homologous to genes from the betagamma-crystallins superfamily. The interest for this crystallin-type protein stems from the phylogenesis of porifera, commonly called sponges, the earliest divergence event in the history of metazoans. Here we report the preparation of geodin as a recombinant protein from Escherichia coli, its characterization through physico-chemical analyses, and a model of its 3D structure based on homology modelling. Geodin is a monomeric protein of about 18 kDa, with an all-beta structure, as all other crystallins in the superfamily, but more prone to unfold in the presence of chemical denaturants, when compared with other homologues from the superfamily. Its thermal unfolding, studied by far- and near-CD, and by calorimetry, is described by a two-state model. Geodin appears to be structurally similar in many respects to the bacterial protein S crystallin, with which it also shares a significant, albeit more modest stabilizing effect exerted by calcium ions. These results suggest that the crystallin-type structural scaffold, employed in the evolution of bacteria and moulds, was successfully recruited very early in the evolution of metazoa.     

Tyrosine-sulfated peptides functionally reconstitute a CCR5 variant lacking a critical amino-terminal region

Entry of most primary human immunodeficiency virus, type 1 (HIV-1) isolates into their target cells requires the cellular receptor CD4 and the G protein-coupled chemokine coreceptor CCR5. An acidic, tyrosine-rich, and tyrosine-sulfated domain of the CCR5 amino terminus plays a critical role in the ability of CCR5 to serve as an HIV-1 coreceptor, and tyrosine-sulfated peptides based on this region physically associate with the HIV-1 envelope glycoprotein gp120 and slow HIV-1 entry into CCR5-expressing cells. Here we show that the same tyrosine-sulfated peptides, but not their unsulfated analogs, can restore the HIV-1 coreceptor activity of a CCR5 variant lacking residues 2-17 of its amino terminus. Additionally, these sulfated peptides restored the ability of this CCR5 variant to mobilize calcium in response to the chemokines macrophage inflammatory factors 1alpha and 1beta. These observations show that a tyrosine-sulfated region of the CCR5 amino terminus can function independently to mediate association of chemokines and the HIV-1 envelope glycoprotein with the remaining domains of CCR5.     

An antibody against a sequence of human topoisomerase II gives different immunofluorescence patterns in quiescent and proliferating nuclei of Pisum sativum L.

Immunofluorescence with an antibody against a C-terminal sequenceof human topoisomerase II has been performed on nuclei releasedfrom different tissues of Pisum sativum L. All the nuclei labelledand preincubation of the antibody with the corresponding immunogenpeptide strongly decreased the fluorescence. The labelling pattern(particularly nucleolar) was different in quiescent and proliferatingnuclei and changed during germination. In prophase nuclei, thelabelling was at the periphery of the condensing chromosomes,and in metaphase chromosomes, a characteristic labelling atpericentromeric regions was found. A computer search indicatedthat, apart from mammalian topoisomerase II, the immunogen peptidedid not match any other sequenced protein which could reasonablybe present in plant nuclei. The possible relation between theantigen recognized and topoisomerase II is discussed. Key words: Topoisomerase II, seed germination, Pisum sativum, immunofluorescence, nuclear proteins     

Host‐ and stage‐dependent secretome of the arbuscular mycorrhizal fungus Rhizophagus irregularis

Arbuscular mycorrhizal fungi form the most wide‐spread endosymbiosis with plants. There is very little host specificity in this interaction, however host preferences as well as varying symbiotic efficiencies have been observed. We hypothesize that secreted proteins (SPs) may act as fungal effectors to control symbiotic efficiency in a host‐dependent manner. Therefore, we studied whether arbuscular mycorrhizal (AM) fungi adjust their secretome in a host‐ and stage‐dependent manner to contribute to their extremely wide host range. We investigated the expression of SP‐encoding genes of Rhizophagus irregularis in three evolutionary distantly related plant species, Medicago truncatula, Nicotiana benthamiana and Allium schoenoprasum. In addition we used laser microdissection in combination with RNA‐seq to study SP expression at different stages of the interaction in Medicago. Our data indicate that most expressed SPs show roughly equal expression levels in the interaction with all three host plants. In addition, a subset shows significant differential expression depending on the host plant. Furthermore, SP expression is controlled locally in the hyphal network in response to host‐dependent cues. Overall, this study presents a comprehensive analysis of the R. irregularis secretome, which now offers a solid basis to direct functional studies on the role of fungal SPs in AM symbiosis.     

A leftward deletional α+ thalassemia found in East Sicily in conjunction with heterozygous β-thalassemia

Summary Two types of ⁺-antitrypsin thalassemia (-/) have been described, respectively termed leftward and rightward, which correspond to nonhomologous crossing-over in different homology zones X and Z within the -globin gene cluster. Up to now the leftward type has been described only in Asiatic populations, whereas the rightward type is universally distributed. We report here a first case of leftward deletion observed in a Sicilian male. This raises the question of an identical or not crossing-over event.     

Studies on the nephron of an elasmobranch fish Scyliorhinus stellaris (L.)

Summary The nephron of the elasmobranch Scyliorhinus stellaris was studied by macerating kidneys of newly hatched specimens in a solution of arsenic anhydride and by sectioning material of the same age and of late embryos. It was found that each nephron consists of (a) a dorsal conical portion containing a skein of canals, loosely intertwined and immersed in blood sinuses, (b) a Malpighian corpuscle, (c) a ventral portion formed by a tightly packed bundle of five tubules. It was not possible to disentangle the ventral tubules so as to recognize their sequence. It may be stated that the neck, originating from the corpuscle and coming back to it after a bend, forms two of such tubules; two more are similarly connected at the distal end by another loop; one forms the collecting duct. The dorsal part of the nephron contains two canals, greatly folded and interlaced. When completely dissociated they appeared to consist of (a) a larger and longer one, corresponding to the brush border portion of all vertebrates (it is thick for most its length, but becomes thin at one end), and (b) a smaller and shorter one, lined by low cells throughout, with no brush border. Although it is not possible to decide so far which of these canals precedes the other, it was ascertained that they are not continuous with each other inside the dorsal portion, but are connected by means of a loop situated in the ventral bundle. The renal tubule passes by, and adheres to the corpuscle four times. The small dorsal canal, although it does not correspond, either in topography or in structure, to the features of the alleged special segments, may be something peculiar to elasmobranchs.Dedicated to Professor Wolfgang Bargmann on his 60th birthday. — Research carried out under contracts with the U. S. Atomic Energy Commission (NYO-3355-3) and Euratom (043-65-1 BIOI), and supported by the Italian C. N. R. and Ministry of Education. — Thanks are due to Dr. P. Dohrn and the staff of the Zoological Station for their help in securing the material and to Dr. Elena Vivori who revised the English.     

Changes in dopamine uptake and developmental effects of dopamine receptor inactivation in the sea urchin

[³H]-dopamine ([³H]-DA) uptake was measured in the presence or absence of the catecholamine uptake inhibitor nomifensine in both unfertilized and fertilized eggs. Specific [³H]-DA uptake depended on time and [³H]-DA concentration; it was high in unfertilized eggs, declined 20–30 min after fertilization, and rose again during cleavage. Irreversible inactivation of dopamine receptors by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) resulted in a complete loss of sensitivity of egg adenylate cyclase to dopamine stimulation. In fertilized eggs treated with EEDQ for 1 hr, restoration of adenylate cyclase activity sensitive to dopamine stimulation could be observed 4 hr after the end of treatment, thus suggesting the appearance of new dopamine receptors in cleaving eggs. Short-term EEDQ treatment on unfertilized eggs, although not impairing fertilization, resulted in cleavage inhibition; the same treatment carried out soon after fertilization, on the other hand, elicited no effect on development. On the contrary, in embryos subjected to continuous treatment with EEDQ, development was impaired independent of the stage at which the treatment was started. © 1995 Wiley-Liss, Inc.     

The preparation of high specific activity [3H]chloramphenicol base and chloramphenicol labeled in the propanediol side chain

Methods are described for the synthesis of [³H]chloramphenicol and derivatives labeled on carbon 1 of the propanediol side chain, with a specific activity of about 2 mCi/μmol. The labeling step involves the reduction of the I-oxo derivative of N-acetyl chloramphenicol base by KB³H₄ to produce a mixture of the d (?) threo- and d (?) erythro-diastereoisomers, since carbon 1 is an asymmetric carbon atom. The two isomers were separated by thin-layer chromatography after acetylation of the two free hydroxyls. After hydrolysis of the three acetyl groups, the biologically active d (?) threo-[1 ? ³H]chloramphenicol base was converted to chloramphenicol. Modification of the above procedures allows the rapid and simple preparation of the mixed d (?) threo- and d (?) erythro-isomers of [1 ? ³H]chloramphenicol. This mixture can be used where the presence of the inactive d (?) erythro-isomer of chloramphenicol is not important. The modified procedure also allows the preparation of the mixed isomers of [1 ? ³H]chloramphenicol base and of chloramphenicol analogs. Attempts to prepare a 3-aldehyde derivative of chloramphenicol were not successful. If this could be done, reduction of this derivative with KB³H₄ would produce the correct isomer of chloramphenicol since carbon atom 3 on the propanediol side chain is not an asymmetric carbon atom.