Structure of hemocyanin subunit CaeSS2 of the crustacean Mediterranean crab Carcinus aestuarii

Arthropodan hemocyanins are giant respiratory proteins responsible for oxygen transport. They exhibit unusual assemblies of up to 48 structural subunits. Hemocyanin from Carcinus aestuarii contains three major and two minor structural subunits. Here, we reveal the primary structure of the gamma-type 75 kDa subunit of Carcinus aestuarii hemocyanin, CaeSS2, and combine structure-based sequence alignments, tryptophan fluorescence, and glycosylation analyses to provide insights into the structural and functional organisation of CaeSS2. We identify three functional domains and three conserved histidine residues that most likely participate in the formation of the copper active site in domain 2. Oxygen-binding ability of Carcinus aestuarii Hc and its structural subunit 2 was studied using CD and fluorescence spectroscopy. Removing the copper dioxygen system from the active site led to a decrease of the melting temperature, which can be explained by a stabilizing effect of the binding metal ion. To study the quenching effect of the active site copper ions in hemocyanins, the copper complex Cu(II)(PuPhPy)2+ was used, which appears as a very strong quencher of the tryptophan emission. Furthermore, the structural localization was clarified and found to explain the observed fluorescence behavior of the protein. Sugar analysis reveals that CaeSS2 is glycosylated, and oligosaccharide chains connected to three O-glycosylated and one N-glycosylated sites were found.     

Reconstitution of Carcinus maenas (crustacea) hemocyanin,in the presence of nonionic detergent

Carcinus maenas hemocyanin was reconstituted from the apo-protein using copper(I) chloride in presence of Triton X-100 at physiological pH. In these conditions Cu⁺ is stable and permitted a kinetic study of the reconstitution reaction to be made.     

Conformational behavior of α,α-dialkylated peptides

The preferred conformations of N-acetyl-N′-methyl amides of some dialkylglycines have been determined by empirical conformational-energy calculations; minimum-energy conformations were located by minimizing the energy with respect to all the dihedral angles of the molecules. The conformational space of these compounds is sterically restricted, and low-energy conformations are found only in the regions of fully extended and helical structures. Increasing the bulkiness of the substituents on the C^α, the fully extended conformation becomes gradually more stable than the helical structure preferred in the cases of dimethylglycine. This trend is, however, strongly dependent on the bond angles between the substituents on the C^α atom: In particular, helical structures are favored by standard values (111°) of the N-C^α-C′ angle, while fully extended conformations are favored by smaller values of the same angle, as experimentally observed, for instance, in the case of α,α-di-n-propylglycine.     

Characterization of a murine monoclonal antibody specific for an allotypic determinant on T cell antigen receptor

Repeated immunization of normal C57L/J (H-2b) mice with peripheral T cells from BALB.B (H-2b) mice results in the production of antibodies which react with the T cell receptor. A monoclonal antibody-producing hybridoma, F23.1, was isolated from immunized C57L/J mice showing this property. This monoclonal antibody recognizes approximately 25% of peripheral T cells in BALB mice. It stains approximately the same fraction of T cells and precipitates the same heterodimer as the rat monoclonal antibody described previously that was made against isolated receptor material. The allotypic determinant recognized by this monoclonal antibody is present in most common laboratory strains (BALB, C57BL, CBA, A, DBA, C3H) and is absent in C57L, C57BR, and SJL mice. Sorting peripheral T cells from BALB.B or (SJL X BALB/c)F1 mice for the F23.1+ and F23.1- subsets revealed that both populations contain approximately the same CTL precursor frequency for alloantigen. Thus, the T cell receptor allotype defined by F23.1 is present on CTL. Furthermore, cytotoxicity mediated by an F23.1+ CTL line could be blocked specifically by the F23.1 monoclonal antibody. Under appropriate conditions, the monoclonal antibody F23.1 bound to Sepharose 4B beads can induce resting peripheral T lymphocytes of allotype-positive strains to proliferate.     

Radiations and female fertility

Hundreds of thousands of young women are diagnosed with cancer each year, and due to recent advances in screening programs, diagnostic methods and treatment options, survival rates have significantly improved. Radiation therapy plays an important role in cancer treatment and in some cases it constitutes the first therapy proposed to the patient. However, ionizing radiations have a gonadotoxic action with long-term effects that include ovarian insufficiency, pubertal arrest and subsequent infertility. Cranial irradiation may lead to disruption of the hypothalamic-pituitary-gonadal axis, with consequent dysregulation of the normal hormonal secretion. The uterus might be damaged by radiotherapy, as well. In fact, exposure to radiation during childhood leads to altered uterine vascularization, decreased uterine volume and elasticity, myometrial fibrosis and necrosis, endometrial atrophy and insufficiency. As radiations have a relevant impact on reproductive potential, fertility preservation procedures should be carried out before and/or during anticancer treatments. Fertility preservation strategies have been employed for some years now and have recently been diversified thanks to advances in reproductive biology. Aim of this paper is to give an overview of the various effects of radiotherapy on female reproductive function and to describe the current fertility preservation options.     

Bioactive peptides: Conformational studies of [TYR4] cyclolinopeptide A

The solid state conformational analysis of [Tyr⁴] cyclolinopeptide A has been carried out by x-ray diffraction studies. The crystal structure of the monoclinic form, grown from a dioxane-water mixture [a = 9.849 (5) Å, b = 20.752 (4) Å, c = 16.728 (5) Å, β = 98.83 (3)°, space group P2₁, Z = 2], shows the presence of five intramolecular N-H? O?C hydrogen bonds, with formation of one C₁₇ ring structure, one α-turn (C₁₃), one inverse γ-turn (C₇), and two β-turns (C₁₀, one of type III and one of type 1). The Pro¹-Pro² peptide unit is cis (ω = 5°) all others are trans. The structure is almost superimposable with that of cyclolinopeptide A. The rms deviation for the atoms of the backbones is on the average 0.33 Å. © 1995 John Wiley & Sons, Inc.     

Solid-state geometry and conformation of linear,diastereoisomeric oligoprolines

We have carried out a systematic analysis of the solid-state conformational preferences of a number of linear homo-oligoprolines (to the tetramer) by ir absorption and x-ray diffraction. The peptides present different chiral sequences (tacticities), various types (urethane and amide) of N-protecting groups, and free and blocked C-termini (which imply different capabilities of forming H-bonds). The following conclusions can be drawn: (i) values for the geometry of the prolyl residue and the peptide bond in the cis and in the trans conformations are proposed; (ii) in general the conformational angles φ and ψ in the linear homo-oligoprolines have values appropriate for the polyproline II structure (conformation F); (iii) the pyrrolidine ring shows various types of puckering with no apparent relation to the backbone conformation; (iv) Pro-Pro peptide bonds generally take the trans conformation, the few cases of cis conformation being formed by Pro residues of different chirality; (v) the single H-bond donor — OH, when present, is always bonded to H-acceptors, which can be either the urethane or the amide or the peptide carbonyl but never the carbonyl group of the — COOH moiety.     

Quantitative comparison of carcinogenicity, mutagenicity and electrophilicity of 10 direct-acting alkylating agents and of the initial O: 7-alkylguanine ratio in DNA with carcinogenic potency in rodents

The quantitative relationship between carcinogenicity in rodents and mutagenicity in Salmonella typhimurium was examined, by using 10 monofunctional alkylating agents, including N-nitrosamides, alkyl methanesulfonates, epoxides, β-propiolactone and 1,3-propane sultone. The compounds were assayed for mutagenicity in two S. typhimurium strains (TA1535 and TA100) and in plate and liquid assays. The mutagenic activity of the agents was compared with their alkylating activity towards 4-(4′-nitrobenzyl)pyridine and with their half-lives (solvolysis constants) in an aqueous medium. No correlations between these variables were found, nor was mutagenic activity correlated with estimates of carcinogenicity in rodents.There was a positive relationship between carcinogenicity and the initial ratios of 7-: O⁶-alkylguanine formed or expected after their reaction with double-stranded DNA in vitro. The results suggest that alkylation of guanine at position O⁶ (or at other O atoms of DNA bases) may be a critical DNA-base modification that determines the overall carcinogenicity of these alkylating agents in rodents.     

Construction and properties of new Lyt-congenic strains and anti-Lyt-2.2 and anti-Lyt-3.1 monoclonal antibodies

Hybridomas producing mouse monoclonal IgM antibodies specific for Lyt-2.2 and Lyt-3.1 T-cell surface alloantigens have been constructed. Cytotoxic titers of ascites fluids were found to be 10(-6) or greater and no lysis of thymocytes of congenic strains bearing the alternative allele was observed at the lowest dilutions tested (1:2). The anti-Lyt-2.2 monoclonal antibody (HO-2.2) specifically precipiated from extracts of Lyt-2.2-positive thymocytes molecular species indistinguishable from those precipitated by conventional anti-Lyt-2.2 sera. However, by immunoprecipitation criteria (though not by cytotoxicity), the anti-Lyt-3.1 antibody (HO-3.1) demonstrated some cross-reactivity with similar molecular species from Lyt-3.1-negative thymocytes. In addition, three new strains of mice differing from existing strains in the region of the Lyt-2 and Lyt-3 loci have been constructed. They are: C.C58-Lyt-2a, Lyt-3a and C.AKR-Lyt-2a, Lyt-3a, congenic with Balb/cAn and bearing Lyt-2a and Lyt-3a alleles of C58/J and AKR/J, respectively; and AKR.C-Lyt-2b, Lyt-3b congenic with AKR/J and bearing the Lyt-2b and Lyt-3b alleles of Balb/cJ.