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Biomedical subjects

S Spinelli

Publications and source records attributed to S Spinelli.

At least 19 recordsLinked to original sources
PubMed2006

[Sexual problems after allogenic stem cell transplantation].

Allogenic stem cell transplantation is an effective procedure for malignant haematological diseases, for these reasons the number of long-term survivors has increased as well as the concern for their quality of life. The late effects of transplantation can highly influence the patient's sexuality and relationships through psychological and physical factors. Psychological factors: regression due to sickness, staff-dependency, emotional and physical partner-dependence, distress related to sterility. Physical factors: hypo-oestrogenism, genital mucosae dryness and sometimes vulval or vaginal chronic graft-versus-host disease. Addressing the topic of intimacy may cause anxiety and embarrassment among nurses, haematological staff and practitioners with the risk of leaving patients in a sort of "conspiracy of silences".

Adult↗
PubMed2006

The management of severe vaginal obstruction from genital chronic graft-versus-host disease: diagnosis, surgical technique and follow-up.

AIM: Chronic graft-versus-host disease (GVHD) is one of the most important systemic late-onset complications of haematopoietic stem cell transplantation. Gynaecological manifestations are considered relatively rare, and involve lower genital tract skin and mucosa, causing vulvar scarring, vaginal stenosis, affecting the patients' sexual life, and leading to more serious complications as haematocolpos, haematometra and abscesses. Genital GVHD can be treated with topical therapy when mild to moderate disease is present. Surgery is indicated in advanced and complicated cases to restore normal anatomy. The aim of this study is to propose a standard approach for the management of such condition when medical therapy is not effective. METHODS: From May 2000 to January 2002, 8 patients suffering from genital chronic GVHD were operated in our institution. We describe clinical and sonographic presurgical assessment, simple or ultrasonographic guided surgical technique, postsurgical treatment and follow-up. RESULTS: Surgery was completely successful in restoring genital anatomy in all the cases. Mean follow-up was of 17 months (6 to 38 months). Two patients early discontinued the postsurgical treatment. At 1 month complete vaginal patency was found in 6 cases, weak partial adhesions in 1 case, while 1 patient refused follow-up. Eventually, complete vaginal patency was maintained in 2 cases, and partial adhesions were found in 5 cases. CONCLUSIONS: The combined use of clinical examination and endosonography provides a precise assessment of the level of the obstruction. The surgical technique here described is feasible and successful in restoring normal anatomy, while long term results seem related to the compliance towards the postsurgical treatment.

Adult↗
PubMed2002

Semen analysis following allogeneic bone marrow transplantation. Additional data for evidence-based counselling.

Knowledge of the impact of different conditioning regimens used in bone marrow transplantation on spermatogenesis is important in pre-BMT counselling for three reasons: (1) Most young patients who have not had children are concerned with their subsequent fertility; (2) For a number of diseases there are competing therapeutic options that may affect spermatogenesis more or less seriously; (3) Since spontaneous recovery of spermatogenesis is rare, it would be necessary to offer cryopreservation as soon as possible after diagnosis and prior to any treatment. This retrospective study evaluates 99 semen samples obtained in 64 patients who underwent BMT between 1982 and 1996. Recovery of spermatogenesis was observed in 90% of patients conditioned with cyclophosphamide (CY), in 50% of patients with CY plus busulphan (BU) or thiotepa and in 17% of patients with CY plus total body irradiation (TBI) or thoracoabdominal irradiation (TAI). Sperm quality following CY was within the normal range (WHO) in the majority of patients, whereas it was consistently severely impaired in patients who received irradiation or two alkylating agents. Following CY, spermatogenesis recovery was observed in 60% of patients tested 1 year post transplant and it was accomplished within the third year in 80% of cases. Following CY + TBI/TAI recovery of spermatogenesis never occurred before the 4th year post transplant and was demonstrated as late as 9 years in one patient who was azoospermic 1 year earlier. No statistical correlation between age and recovery of spermatogenesis could be demonstrated. The overall high incidence of azoospermia (70.3%) supports the indication for semen cryopreservation in young patients undergoing BMT. These results have implications for semen sample timing before and after BMT and underline a need to collect further data through prospective multi-center studies.

Adolescent↗
PubMed2001

Lateral recognition of a dye hapten by a llama VHH domain.

Camelids, camels and llamas, have a unique immune system able to produce heavy-chain only antibodies. Their VH domains (VHHs) are the smallest binding units produced by immune systems, and therefore suitable for biotechnological applications through heterologous expression. The recognition of protein antigens by these VHHs is rather well documented, while less is known about the VHH/hapten interactions. The recently reported X-ray structure of a VHH in complex with a copper-containing azo-dye settled the ability of VHH to recognize haptens by forming a cavity between the three complementarity-determining regions (CDR). Here we report the structures of a VHH (VHH A52) free or complexed with an azo-dye, RR1, without metal ion. The structure of the complex illustrates the involvement of CDR2, CDR3 and a framework residue in a lateral interaction with the hapten. Such a lateral combining site is comparable to that found in classical antibodies, although in the absence of the VL.

Amino Acid Sequence↗
PubMed2001

Selection, characterization and x-ray structure of anti-ampicillin single-chain Fv fragments from phage-displayed murine antibody libraries.

Single-chain Fv (scFv) antibody libraries were constructed from mice immunized with an ampicillin-bovine serum albumin conjugate. Several antibodies with specificity for intact ampicillin were selected by phage display and characterized. The antibody scFv fragment aL2 binds to intact ampicillin and shows no detectable cross-reactivity with hydrolyzed ampicillin. We determined the X-ray structures of two crystal forms of w.t. aL2, which differ mainly in the side-chain conformation of Trp H109 (according to a new consensus nomenclature Kabat residue number H95) in the extremely short (three residues) CDR H3 and the presence or absence of a well-resolved molecule of 2-methyl-pentane-2,4-diol in the bottom of the binding pocket. Attempts to co-crystallize aL2 with its antigen or to diffuse ampicillin into the wild-type aL2 crystals were unsuccessful, since crystal contacts obstruct the binding pocket. However, a mutant with two point mutations near the N terminus (Gln H6 replaced by Glu and Ala H10 (Kabat H9) replaced by Gly) crystallized in a form compatible with antigen-binding. Although the mutations affect the conformation of framework I, the conformations of the binding pocket of the uncomplexed wild-type aL2 and of the mutant complex were almost identical. The structure explains the specificity of the antibody for intact ampicillin and the degree of cross-reactivity of aL2 with a wide variety of ampicillin analogs. This antibody system will be very useful as a diagnostic reagent for antibiotics use and abuse, as a model for the effect of expression of antibiotic binding molecules in Escherichia coli, and for directed evolution towards high antibiotic resistance.

Amino Acid Sequence↗
PubMed2001

The importance of framework residues H6, H7 and H10 in antibody heavy chains: experimental evidence for a new structural subclassification of antibody V(H) domains.

The N-terminal segment (FR-H1) of the heavy chain (V(H)) of antibodies shows significant conformational variability correlating with the nature of the amino acids H6, H7 and H10 (Kabat H9). In this study, we have established a causal relationship between the local sequence and the structure of this framework region and linked this relationship to important biophysical properties such as affinity, folding yield and stability. We have generated six mutants of the scFv fragment aL2, covering some of the most abundant amino acid combinations in positions H6, H7 and H10 (according to a new consensus nomenclature, Kabat H9). For the aL2 wild-type (w.t.) with the sequence 6(Q)7(P)10(A) and for two of the mutants, the X-ray structures have been determined. The structure of the triple mutant aL2-6(E)7(S)10(G) shows the FR-H1 backbone conformations predicted for this amino acid combination, which is distinctly different from the structure of the w.t, thus supporting our hypothesis that these residues determine the conformation of this segment. The mutant aL2-6(E)7(P)10(G) represents a residue combination not occurring in natural antibody sequences. It shows a completely different, unique structure in the first beta-strand of V(H), not observed in natural Fv fragments and forms a novel type of diabody. Two V(H) domains of the mutant associate by swapping the first beta-strand. Concentration-dependent changes in Trp fluorescence indicate that this dimerization also occurs in solution. The mutations in amino acids H6, H7 and H10 (Kabat H9) influence the dimerization behavior of the scFv and its thermodynamic stability. All the observations reported here have practical implications for the cloning of Fv fragments with degenerate primers, as well as for the design of new antibodies by CDR grafting or synthetic libraries.

Ampicillin↗
PubMed2001

Crystal structure of aphrodisin, a sex pheromone from female hamster.

We have solved the crystal structure of aphrodisin, a pheromonal protein inducing a copulatory behaviour in male hamster, using MAD methods with selenium, at 1.63 A resolution. The monomeric protein belongs to the lipocalin family, and possesses a disulfide bridge in a loop between strands 2 and 3. This disulfide bridge is characteristic of a family of lipocalins mainly identified in rodents, and is analogous to the fifth disulfide bridge of the long neurotoxins, such as alpha cobratoxin. An elongated electron density was found inside the buried cavity, which might represent a serendipitous ligand of unknown origin. The analysis of the water accessible surfaces of the side-chains bordering the cavity indicates that Phe76 may be the door for the natural ligand to access the cavity. This residue defines the entry of the cavity as belonging to the consensus for lipocalins. The face bearing Phe76 might also serve for the interaction with the receptor.

Amino Acid Sequence↗
PubMed2001

Recombinant chemosensory protein (CSP2) from the moth Mamestra brassicae: crystallization and preliminary crystallographic study.

Chemosensory proteins (CSPs) are small proteins (13 kDa on average) present in several sensory organs from a wide range of insect species. They are believed to be involved in chemoperception (olfaction or taste) and to play a role in chemical transport from air or water to chemosensitive receptors. Here, the first crystals of a CSP originating from the moth Mamestra brassicae (Mbra) proboscis and expressed as recombinant protein in Escherichia coli periplasm are reported. Crystals of MbraCSP2 were obtained by the hanging-drop vapour-diffusion method under the following conditions: 1 microl of a 46 mg ml(-1) protein solution in 50 mM Tris pH 8.0 containing cetyl alcohol as ligand (1:5 molar ratio) was mixed with 1 microl of well solution containing 30% PEG 4000, 0.2 M sodium acetate in 100 mM Tris at pH 8.4. The protein-cetyl alcohol complex crystallizes in space group P2(1), with unit-cell parameters a = 47.9, b = 49.7, c = 50.3 A, beta = 110.1 degrees. With two molecules in the asymmetric unit, the V(M) is 2.15 A(3) Da(-1) and the solvent content is 42%. A complete data set has been collected at 1.6 A resolution on beamline ID14-2 (ESRF, Grenoble). Se-Met expression has been performed with a view to solving the CSP2 structure with MAD data collection using the Se absorption edge.

Animals↗
PubMed2001

DNA-interactive anticancer aza-anthrapyrazoles: biophysical and biochemical studies relevant to the mechanism of action.

The physicochemical and DNA-binding properties of anticancer 9-aza-anthrapyrazoles (9-aza-APs) were investigated and compared with the carbocyclic analogs losoxantrone (LX) and mitoxantrone (MX). Unlike their carbocyclic counterparts, the tested 9-aza-APs do not undergo self-aggregation phenomena. The pyridine nitrogen at position 9, missing in the carbocyclic derivatives, is involved in protonation equilibria at physiological pH. In addition, 9-aza-APs are electrochemically reduced at a potential intermediate between LX and MX. These data fully agree with quantum mechanical calculations. Binding to nucleic acids was examined by spectroscopic, chiroptical, and DNase I footprinting techniques as a function of ionic strength and base composition. The 9-aza-APs exhibit prominent affinity for DNA, with an important electrostatic contribution to the binding free energy. A very remarkable sequence preference pattern dramatically favors GC steps in double-helical DNA, whereas the carbocyclic reference compounds show a substantially lower selectivity for GC. A common DNA complexation geometry, considerably differing from that of MX, characterizes all anthrapyrazoles. Hence, bioisosteric substitution and ring-hydroxy deletion play an important role in defining the physicochemical properties and in modulating the affinity of anthrapyrazoles for the nucleic acid, the geometry of the intercalation complex, and the sequence specific contacts along the DNA chain. Drug stimulation of topoisomerase II-mediated DNA cleavage is remarkably attenuated in the aza-bioisosteric derivatives, suggesting that other non-enzyme-mediated cytotoxic mechanism(s), possibly connected with free radical production, are responsible for efficient cell killing. The biophysical and biochemical properties exhibited by 9-aza-APs contribute to clarifying the peculiar pharmacological profile of this family of compounds.

Animals↗
PubMed2001

A novel 9-aza-anthrapyrazole effective against human prostatic carcinoma xenografts.

OBJECTIVES: Systematic investigation of a novel series of intercalating agents, 9-aza-anthrapyrazoles, has led to the identification of a promising analogue, BBR 3438. This study describes the antitumour efficacy of the novel compound in human prostate carcinoma models and the molecular/cellular basis of its activity. METHODS AND RESULTS: The novel 9-aza-anthrapyrazole BBR 3438 was significantly more effective than doxorubicin and losoxantrone (DuP-941) in two of the three tested prostate carcinoma models. The superior activity was more evident in PC3 tumour, since BBR 3438 produced an appreciable rate of complete tumour regressions. Under these conditions, the drug-induced antiproliferative activity paralleled delayed apoptosis. Tumour response to in vivo drug treatment was associated with an early down-regulation of Bcl-2, which was somewhat more marked for the aza compound. In fact, the 9-aza-anthrapyrazole induced DNA cleavage in vitro with isolated DNA topoisomerase II (isoform alpha) and DNA strand breaks in prostatic carcinoma cells. Although the molecular effects of losoxantrone and the 9-aza analogue on the enzyme target were comparable, the cytotoxic effects of BBR 3438 could be enhanced by long-term exposure as a consequence of favourable cellular accumulation and prominent DNA-binding affinity. In addition, a lower reduction potential of the 9-aza-anthrapyrazole in comparison with classical anthrapyrazoles suggests an increased ability of the drug to induce oxidative stress following free radical production, which may be a contributing factor in determining the long-term response (i.e. delayed cell death) to genotoxic damage. CONCLUSIONS: BBR 3438 exhibited a unique profile of preclinical activity with a superior efficacy against prostatic carcinoma models compared to reference compounds (doxorubicin and losoxantrone). The antitumour efficacy of BBR 3438 against prostatic carcinoma could be the result of a combination of favourable events, including enhanced intracellular accumulation and an increased DNA-binding affinity favouring the accumulation of multiple sublethal or lethal damage. In spite of its enhanced cytotoxic potency, the 9-aza compound was better tolerated in vivo than losoxantrone, thus improving the therapeutic index. The preclinical profile of efficacy against prostatic carcinoma, a tumour resistant to conventional antitumour drugs, makes the novel 9-aza-anthrapyrazole BBR 3438 a promising candidate for clinical evaluation.

Adenocarcinoma↗
PubMed2000

N4-(omega-Aminoalkyl)-1-[(omega-aminoalkyl)amino]-4- acridinecarboxamides: novel, potent, cytotoxic, and DNA-binding agents.

A series of DNA-binding potential antitumor agents, (omega-aminoalkyl)-4-acridinecarboxamides, has been prepared either by reduction of the corresponding (omega-aminoalkyl)-9-oxo-9, 10-dihydro-4-acridinecarboxamides with aluminum amalgam or by aminolysis of the corresponding (omega-aminoalkyl)-1-chloro-4-acridinecarboxamides with the suitable amine. The noncovalent DNA-binding properties of these compounds have been examined using a fluorometric technique. In vitro cytotoxic potencies of these derivatives toward six tumor cell lines, including human colon adenocarcinoma (HT29) and human ovarian carcinoma (A2780-sensitive, A2780cisR cisplatin-resistant, CH1-sensitive, CH1cisR cisplatin-resistant, and SKOV-3) cells, are described and compared to that of reference drugs. One highly DNA affinic analogue (3a) has been identified with a useful broad spectrum of cytotoxic activity in the 4-7 nM range (mean IC(50) of 6 nM).

Acridines↗
PubMed2000

The insect attractant 1-octen-3-ol is the natural ligand of bovine odorant-binding protein.

Bovine odorant-binding protein (bOBP) is a dimeric lipocalin present in large amounts in the respiratory and olfactory nasal mucosa. The structure of bOBP refined at 2.0-A resolution revealed an elongated volume of electron density inside each buried cavity, indicating the presence of one (or several) naturally occurring copurified ligand(s) (Tegoni et al. (1996) Nat. Struct. Biol. 3, 863-867; Bianchet et al. (1996) Nat. Struct. Biol. 3, 934-939). In the present work, by combining mass spectrometry, x-ray crystallography (1.8-A resolution), and fluorescence, it has been unambiguously established that natural bOBP contains the racemic form of 1-octen-3-ol. This volatile substance is a typical component of bovine breath and in general of odorous body emanations of humans and animals. The compound 1-octen-3-ol is also an extremely potent olfactory attractant for many insect species, including some parasite vectors like Anopheles (Plasmodium) or Glossina (Trypanosoma). For the first time, a function can be assigned to an OBP, with a possible role of bOBP in the ecological relationships between bovine and insect species.

Animals↗
PubMed2000

Crystal structure of a cohesin module from Clostridium cellulolyticum: implications for dockerin recognition.

In the assembly of the Clostridium cellulolyticum cellulosome, the multiple cohesin modules of the scaffolding protein CipC serve as receptors for cellulolytic enzymes which bear a dockerin module. The X-ray structure of a type I C. cellulolyticum cohesin module (Cc-cohesin) has been solved using molecular replacement, and refined at 2.0 A resolution. Despite a rather low sequence identity of 32 %, this module has a fold close to those of the two Clostridium thermocellum cohesin (Ct-cohesin) modules whose 3D structures have been determined previously. Cc-cohesin forms a dimer in the crystal, as do the two Ct-cohesins. We show here that the dimer exists in solution and that addition of dockerin-containing proteins dissociates the dimer. This suggests that the dimerization interface and the cohesin/dockerin interface may overlap. The nature of the overall surface and of the dimer interface of Cc-cohesin differ notably from those of the Ct-cohesin modules, being much less polar, and this may explain the species specificity observed in the cohesin/dockerin interaction of C. cellulolyticum and C. thermocellum. We have produced a topology model of a C. cellulolyticum dockerin and of a Cc-cohesin/dockerin complex using homology modeling and available biochemical data. Our model suggests that a special residue pair, already identified in dockerin sequences, is located at the center of the cohesin surface putatively interacting with the dockerin.

Amino Acid Sequence↗
PubMed2000

In vivo cytoreduction studies and cell sorting--enhanced tumor-cell detection in high-risk neuroblastoma patients: implications for leukapheresis strategies.

PURPOSE: To improve autologous leukapheresis strategies in high-risk neuroblastoma (NB) patients with extensive bone marrow involvement at diagnosis. PATIENTS AND METHODS: Anti-G(D2) immunocytochemistry (sensitivity, 1 in 10(5) to 10(6) leukocytes) was used to evaluate blood and bone marrow disease at diagnosis and during the recovery phase of the first six chemotherapy cycles in 57 patients with stage 4 NB and bone marrow disease at diagnosis. A total of 42 leukapheresis samples from the same patients were evaluated with immunocytology, and in 24 of these patients, an anti-G(D2) immunomagnetic enrichment step was used to enhance tumor-cell detection. RESULTS: Tumor cytoreduction was much faster in blood compared with bone marrow (3.2 logs after the first cycle and 2.1 logs after the first two cycles, respectively). Bone marrow disease was often detectable throughout induction, with a trend to plateau after the fourth cycle. By direct anti-G(D2) immunocytology, a positive leukapheresis sample was obtained in 7% of patients after either the fifth or sixth cycle; when NB cell immunomagnetic enrichment was applied, 25% of patients had a positive leukapheresis sample (sensitivity, 1 in 10(7) to 10(8) leukocytes). CONCLUSION: Standard chemotherapy seems to deliver most of its in vivo purging effect within the first four cycles. In patients with overt marrow disease at diagnosis, postponing hematopoietic stem-cell collection beyond this point may not be justified. Tumor-cell clearance in blood seems to be quite rapid, and earlier collections via peripheral-blood leukapheresis might be feasible. Immunomagnetically enhanced NB cell detection can be highly sensitive and can indicate whether ex vivo purging should be considered.

Adolescent↗
PubMed2000

Mammalian odorant binding proteins.

Odorant binding proteins (OBPs) pertain to one of the most abundant classes of proteins found in the olfactory apparatus. OBPs are a sub-class of lipocalins, defined by their property of reversibly binding volatile chemicals, that we call 'odorants'. Numerous sequences of OBPs are now available, derived from protein sequencing from nasal mucus material, or from DNA sequences. The structural knowledge of OBPs has been improved too in recent years, with the availability of two X-ray structures. The physiological role of OBPs remains, however, essentially hypothetical, and most probably, not linked to a function of odor transport. The present knowledge on OBP biochemistry, sequence and structure will be examined here in relation to the different functional hypotheses proposed for OBPs.

Animals↗
PubMed2000

Complexes of porcine odorant binding protein with odorant molecules belonging to different chemical classes.

Porcine odorant binding protein (pOBP) is a monomer of 157 amino acid residues, purified in abundance from pig nasal mucosa. In contrast to the observation on lipocalins as retinol binding protein (RBP), major urinary protein (MUP) or bovine odorant binding protein (bOBP), no naturally occurring ligand was found in the beta-barrel cavity of pOBP. Porcine OBP was therefore chosen as a simple model for structure/function studies with odorant molecules. In competition experiments with tritiated pyrazine, the affinity of pOBP towards several odorant molecules belonging to different chemical classes has been found to be of the micromolar order, with a 1:1 stoichiometry. The X-ray structures of pOBP complexed to these molecules were determined at resolution between 2.15 and 1.4 A. As expected, the electron density of the odorant molecules was observed into the hydrophobic beta-barrel of the lipocalin. Inside this cavity, very few specific interactions were established between the odorant molecule and the amino acid side-chains, which did not undergo significant conformational change. The high B-factors observed for the odorant molecules as well as the existence of alternative conformations reveal a non-specific mode of binding of the odorant molecules in the cavity.

Animals↗
PubMed2000

Camelid heavy-chain variable domains provide efficient combining sites to haptens.

Camelids can produce antibodies devoid of light chains and CH1 domains (Hamers-Casterman, C. et al. (1993) Nature 363, 446-448). Camelid heavy-chain variable domains (VHH) have high affinities for protein antigens and the structures of two of these complexes have been determined (Desmyter, A. et al. (1996) Nature Struc. Biol. 3, 803-811; Decanniere, K. et al. (1999) Structure 7, 361-370). However, the small size of these VHHs and their monomeric nature bring into question their capacity to bind haptens. Here, we have successfully raised llama antibodies against the hapten azo-dye Reactive Red (RR6) and determined the crystal structure of the complex between a dimer of this hapten and a VHH fragment. The surface of interaction between the VHH and the dimeric hapten is large, with an area of ca. 300 A(2); this correlates well with the low-dissociation constant of 22 nM measured for the monomer. The VHH fragment provides an efficient combining site to the RR6, using its three CDR loops. In particular, CDR1 provides a strong interaction to the hapten through two histidine residues bound to its copper atoms. VHH fragments might, therefore, prove to be valuable tools for selecting, removing, or capturing haptens. They are likely to play a role in biotechnology extending beyond protein recognition alone.

Amino Acid Sequence↗