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In 2015, the second cycle of the CONCORD programme established global surveillance of cancer survival as a metric of the effectiveness of health systems and to inform global policy on cancer control. CONCORD-3 updates the worldwide surveillance of cancer survival to 2014.

ABSTRACTThe gamma interferon (IFN-?) assay is widely used to measure cell-mediated immune (CMI) response for the early detection of tuberculosis infection. Processing whole-blood samples for CMI-based diagnostics is time sensitive and usually must occur within 8 h of collection to ensure optimal assay performance. In this study, we developed and tested a modified protocol, in which whole-blood samples from Mycobacterium bovis-infected cattle were diluted 1:1 in RPMI medium containing 0.3% fetal bovine serum (FBS) added or not to recombinant mouse interleukin-7 (rmIL-7) or rmIL-12, alone or in combination, and stored at 4°C. At 3 and 6 days postcollection, the diluted blood samples were adjusted to 10% FBS, dispensed into culture trays, stimulated with a bovine purified protein derivative from M. bovis, and incubated at 37°C in 5% CO2in air. Plasma was removed and assayed for an IFN-? response using bovine IFN-? enzyme-linked immunosorbent assay (ELISA) (Bovigam). The results were then compared with those obtained from the conventional procedure. The IFN-? responses of the samples stored up to 6 days postcollection in the supplemented RPMI medium were similar to those observed in the samples processed within 8 h after sampling, indicating that lymphocyte vitality and response were preserved. The addition of rmIL-7 and rmIL-12, alone or in combination, to culture medium can enhance lymphocyte survival and thus extends the time limit within which the IFN-? assay can be applied as a diagnostic tool in bovine tuberculosis surveillance and eradication.

Control of thyroid function in hyperthyroid women during pregnancy is based on antithyroid drugs (ATD) [propylthiouracil (PTU) and methimazole (MMI)]. While a teratogenic effect has been suggested for MMI and, more recently, for PTU, a clear demonstration is still lacking. Aim of this study was to assess the safety of ATD during pregnancy. A total of 379 pregnancies were retrospectively recruited in eight Italian Departments of Endocrinology and divided in five groups: (1) MMI-treated and euthyroid throughout pregnancy (n= 89); (2) MMI-treated and hyperthyroid on at least two occasions (n= 35); (3) PTU-treated women and euthyroid throughout pregnancy (n= 32); (4) PTU-treated women and hyperthyroid on at least two occasions (n= 20); and (5) non-ATD-treated (n= 203). Data on maternal thyroid function, miscarriages, type of delivery, neonatal weight, length and TSH, perinatal complications and congenital malformation were analyzed. The gestational age at delivery, the rate of vaginal delivery, neonatal weight, length and neonatal TSH did not significantly differ among groups. In all groups, the rates of spontaneous miscarriage and of major congenital malformations were not higher than in the general population. No newborns were born with a phenotype similar to those described in the “MMI embryopathy”. While a clear demonstration of a teratogenic effect of MMI is currently lacking, it seems reasonable to follow the current guidelines and advice for PTU treatment in hyperthyroid women during the first trimester of pregnancy. Further, large and prospective worldwide studies will be needed to fully clarify the issue of ATD safety during pregnancy.

Fine-needle aspiration (FNA) with cytologic evaluation is the most reliable tool for malignancy prediction in thyroid nodules, but cytologic diagnosis remains indeterminate for 12–18 % of nodules. BRAF V600E mutation has been reported to show a high specificity for malignant thyroid nodules and the use of this marker to refine indeterminate FNA cytology results may be a useful diagnostic adjunctive tool in the pre-operative evaluation of thyroid nodules. The aim of this study was to estimate the prevalence of BRAF exon 15 mutation (V600E) and its clinical value as a diagnostic tool in a series of thyroid nodules with indeterminate cytology from an area of borderline iodine deficiency. One hundred and fifty-three thyroid samples obtained by FNA of thyroid nodules from 151 patients were subjected to the analysis of BRAF V600E mutation by direct sequencing. In the study 54 nodules with indeterminate cytology, 56 benign and 43 malignant thyroid nodules were included. V600E BRAF gene mutation was demonstrated in 19/43 malignant nodules, in 0/56 benign nodules and in only 1/54 indeterminate nodules that, after histology, turned out to be at a papillary thyroid carcinoma. The application of BRAF exon 15 analysis showed limitations when applied to discriminate thyroid nodules with indeterminate cytology if wild-type BRAF is found, and there is no role for avoiding diagnostic thyroid surgery.

One could argue that balneotherapy and mud therapy would have not lasted 2,000 years or so If they were not effective. No doubt a long history cannot be taken per seas scientific proof of efficacy. Some empiricism is still present in the field: the concept of spa itself is quite confounding, whereas spring waters are used for leisure purposes but also for non-acute patient therapy and late phases of clinical recovery. These confounding elements ultimately feed the opinion of those who aprioristically reject any potential beneficial effect of balneotherapy: instead, it should at least generate questions that deserve scientific answers. Clinical practices sequentially integrating pharmacological therapy with those natural principles for which a sufficient scientific demonstration is available, would probably cut the costs of public health, generating widespread advantages for the community. Recently, it has become evident that mineral waters may have intrinsic pharmacological properties. Of the numerous salts dissolved in thermal waters that might show pharmacological properties, for certain hydrogen sulfide (H2S) contained in sulfurous waters is the one that has obtained greater scientific attention, to which should be added the extensive scientific effort recently dedicated to H2S as a cellular gasotransmitter, independently from its natural sources. Dermatology and cosmetics are among the most studied applications of sulfurous waters, around which, however, some empiricism still confounds opinions: we therefore considered that a state-of-the-art focus on this topic might be timely and useful for future studies.

Hashimoto’s thyroiditis (HT) is an autoimmune disorder characterized by the presence of specific antibodies and by a lymphocytic infiltration of the thyroid secreting inflammatory cytokines. Macrophages, lymphocytes, and cytokines play a pivotal role in both development and progression of Th1-mediated autoimmune diseases, and a direct role in the destruction of thyroid follicles and follicular cell function in autoimmune thyroiditis. Integrins are integral membrane receptors involved in cell-extra-cellular matrix (ECM) interaction with both structural and signaling functions. The integrin-ECM interaction is necessary for the correct function and survival of thyroid follicular cells. The purpose of this study was to determine the effect of cytokine stimulation on integrin expression and signaling in the thyroid cell. Primary cultures from normal thyroids were treated with interferon-γ (IFN-γ), INF-α, tumor necrosis factor-α, interlukin 1a or these cytokines all together. Integrin expression, cell adhesion to fibronectin (FN) and FN-stimulated extracellular signal-regulated kinase (ERK) phosphorylation were determined after cytokine treatment. IFN-γ and IFN-α were the most effective, reducing the expression of the integrin αvβ3 and slightly increasing the α3β1. Cell treatment with IFN-γ strongly impaired cell adhesion to FN. At the same time, the treatment with IFN-γ dramatically inhibited the stimulation of ERK phosphorylation induced by cell adhesion to FN. In conclusion, IFN-γ inhibits the expression of the integrin αvβ3, reducing the cell adhesion to FN and the following intracellular signaling in thyroid cells in culture. These results suggest that integrins may be a target of the infiltrating lymphocytes and have a role in the pathogenesis of autoimmune thyroiditis.

The 3, 5, 3′-L-triiodothyronine (T3) partly derives by the deiodination of the prohormone 3, 5, 3′, 5′-L-tetraiodothyronine (T4) by the type 2 iodothyronine deiodinase (D2). The single-nucleotide polymorphism in the D2 gene at position 92 (Dio2T92A), generates an enzyme with a reduced T4to T3conversion velocity. Because thyroid hormones can modulate the immune response, we hypothesized a pathophysiological role for Dio2T92Apolymorphism in autoimmunity. The objective of this study was to investigate the Dio2T92Apolymorphism in relation to thyroid autoimmunity (TA). We compared the prevalence of Dio2T92Apolymorphism and serum thyroid hormone levels in healthy subjects and subjects with TA. A total of 110 subjects with TA and 106 controls were genotypized for Dio2T92Apolymorphism. Free T3 (FT3), free T4(FT4) and TSH were measured and compared with the Dio2T92Apolymorphism. Dio292T/A, Dio292A/A, and Dio292T/Thealthy subjects were 40.9%, 46.4%, and 12.7%, respectively. These prevalences were similar to those of some European countries whilst significantly different from that of Brazil. In the two groups of healthy subjects and TA subjects, Dio2T92Apolymorphism had a similar distribution with nonsignificant differences. Similarly, no significant differences were observed in the serum concentration of FT3, FT4, and TSH between subjects with different Dio2T92Apolymorphism. The FT4/FT3, and TSH/FT3ratios were higher in Dio292T/Tthan in Dio292T/Aand Dio292A/Asubjects in both TA and healthy groups, but these differences were not significant. In conclusion, the distribution of Dio2T92Apolymorphism may reflect geographical and ethnic differences, and it is not associated with TA.

Inflammatory tenosynovitis is an inflammation that involves the tendons and synovial sheaths caused by minor trauma repeated for a long period of time. This inflammatory disease may be involved in the onset of tunnel carpal syndrome (CTS), because of the thickening of the tendon sheath that may produce an increase in the carpal canal pressure and damage of the median nerve in the wrist. Recent studies suggest that in patients with CTS pathological changes occur in the subsynovial connective tissue, such as vascular proliferation and non-inflammatory synovial fibrosis. However, little is known about the pathological mechanism of tenosynovial thickening. The aim of this study is to evaluate the potential role of vascular endothelial growth factor (VEGF), prostaglandin E2 (PGE2) and trasforming growth factor-beta (TGF-β) in the modifications of connective synovial tissue of CTS specimens in order to determine whether these factors play a role in the development of this disease. Ten specimens from patients with CTS and four control tissues (cadavers) were analyzed by immunohistochemistry using specific antibodies against these growth factors. A temporary increase in the production of these molecules was found in cells within the vessels and synovial lining during the intermediate phase of the syndrome, when the histology of the tenosynovium changes from oedematous to fibrotic. Our data confirm a close correlation between the expression of PGE2and VEGF. Recent histological examinations have shown a marked increase in vascular proliferation and reduction of fibroblast density in specimens from CTS patients during the intermediate phase. Our study indicates that the expression of TGF-β in fibroblasts and vascular endothelial cells of synovial connective tissues of CTS patients was significantly higher than in those of controls. These findings suggest that angiogenesis appears to take place as a part of a regenerative reaction that results in fibrosis. We believe that VEGF, TGF-β and PGE2may be potential therapeutic targets in the treatment of this disease although proof of this evidence requires further studies.

The aim of this retrospective multicentre study was to report the continued occurrence of compartment syndrome secondary to paediatric supracondylar humeral fractures in the period 1995 to 2005. The inclusion criteria were children with a closed, low-energy supracondylar fracture with no associated fractures or vascular compromise, who subsequently developed compartment syndrome. There were 11 patients (seven girls and four boys) identified from eight hospitals in three countries. Ten patients with severe elbow swelling documented at presentation had a mean delay before surgery of 22 hours (6 to 64). One patient without severe swelling documented at presentation suffered arterial entrapment following reduction, with a subsequent compartment syndrome requiring fasciotomy 25 hours after the index procedure.This series is noteworthy, as all patients had low-energy injuries and presented with an intact radial pulse. Significant swelling at presentation and delay in fracture reduction may be important warning signs for the development of a compartment syndrome in children with supracondylar fractures of the humerus.

Inter-comparisons in the gas exchange patterns and root characteristics under both well-watered and drought conditions were done in three-years-old seedlings of three oak species (Quercus cerrisL., Q. frainettoTen., and Q. ilexL.) growing in controlled environment. Well-watered Q. cerrishad greater physiological performances than other oaks, but under drought it was not able to face the water stress showing also structural modifications such as reduction of root length and average diameter. On the other hand, Q. ilexmaintained root growth both in drought or well-watered soils. Moreover, it was able to keep open stomata also under water stress, although stomatal conductance (gs) was low. Q. frainettohad an intermediate position in regard to its physiological and root structural characteristics between Q. cerrisand Q. ilexunder drought stress. For all oaks the relationship between gsand the ratio of sub-stomatal and ambient CO2concentration (Ci/Ca) highlighted the dynamic adaptation of gsto the increase of hydraulic resistances of leaf, stem, and roots portions, more evident during the air humidity change and progressive soil dehydration. This suggests a well-triggered above-and under-ground mechanism to endure the drought stress.Inter-comparisons in the gas exchange patterns and root characteristics under both well-watered and drought conditions were done in three-years-old seedlings of three oak species (Quercus cerrisL., Q. frainettoTen., and Q. ilexL.) growing in controlled environment. Well-watered Q. cerrishad greater physiological performances than other oaks, but under drought it was not able to face the water stress showing also structural modifications such as reduction of root length and average diameter. On the other hand, Q. ilexmaintained root growth both in drought or well-watered soils. Moreover, it was able to keep open stomata also under water stress, although stomatal conductance (gs) was low. Q. frainettohad an intermediate position in regard to its physiological and root structural characteristics between Q. cerrisand Q. ilexunder drought stress. For all oaks the relationship between gsand the ratio of sub-stomatal and ambient CO2concentration (Ci/Ca) highlighted the dynamic adaptation of gsto the increase of hydraulic resistances of leaf, stem, and roots portions, more evident during the air humidity change and progressive soil dehydration. This suggests a well-triggered above-and under-ground mechanism to endure the drought stress.

In the frame of the foreseen climate global changes we analysed the physiological responses of Arbutus unedo L. to the variations of carbon dioxide concentration, leaf temperature, and irradiance by measurements of leaf gas exchange and leaf water potential performed both in field and in the laboratory. Stomatal conductance was not affected by increase of leaf temperature. The growth conditions of potted plants likely made stomata more sensitive to the variation of external parameters than naturally growing plants. The interaction between high CO2 concentration and temperature involved important down-regulation mechanisms in the metabolic pathway of the carbon fixation. From an ecological point of view, the ability of A. unedo to adapt to the field stress makes it highly competitive in the Mediterranean plant community.

Computational methods were applied to the Pd-catalyzed intramolecular allylations of resonance-stabilized carbanions obtained from amide and ketone substrates, with the aim of rationalizing the endo vs exo selectivity in the cyclizations. In addition, ester substrates were prepared and subjected to the Pd-catalyzed cyclization conditions, and were found to form lactones via exo attack. DFT calculations with the BP86/LACVP*+ level of theory with a CH2Cl2 solvation correction reproduce the relative transition state energies. The preference for exo-cyclization of the nitrogen-containing starting material appears to result from the preference for near-planarity of the amide N. Both the oxygen and nitrogen tethers are too short to allow efficient endo cyclization, whereas the carbon tether is long enough to allow favorable endo cyclization. The carbon tether also disfavors the exo cyclization transition states slightly from eclipsing interaction, leading to almost isoenergetic exo and endo transition states, and thus accounting for the experimentally observed mixture of five- and seven-membered-ring products.

Abstract -- A maximum-likelihood (ML) estimator of the correlation dimension d₂ of fractal sets of points not affected by the left-hand truncation of their inter-distances is defined. Such truncation might produce significant biases of the ML estimates of d₂ when the observed scale range of the phenomenon is very narrow, as often occurs in seismological studies. A second very simple algorithm based on the determination of the first two moments of the inter-distances distribution (SOM) is also proposed, itself not biased by the left-hand truncation effect. The asymptotic variance of the ML estimates is given. Statistical tests carried out on data samples with different sizes extracted from populations of inter-distances following a power law, suggested that the sample variance of the estimates obtained by the proposed methods are not significantly different, and are well estimated by the asymptotic variance also for samples containing a few hundred inter-distances. To examine the effects of different sources of systematic errors, the two estimators were also applied to sets of inter-distances between points belonging to statistical fractal distributions, baker's maps and experimental distributions of earthquake epicentres. For a full evaluation of the results achieved by the methods proposed here, these were compared with those obtained by the ML estimator for untruncated samples or by the least-squares algorithm.

OBJECTIVE: The 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, lovastatin, induces apoptosis in the thyroid cell line TAD-2 and in proliferating normal human thyroid cells in culture, through a p53-independent mechanism involving caspase-3-like proteases. The combination of lovastatin with other anti-neoplastic drugs potentiates chemotherapy of tumors. This drug has been suggested for the chemotherapy of tumors and is potentially useful in the treatment of thyroid proliferative diseases. Based on this premise, we analyzed in more detail the role of some molecular effectors and the role of the caspase family proteases in the lovastatin-induced apoptotic pathway in TAD-2 cells. METHODS: TAD-2 cells were treated with lovastatin to induce apoptosis, and expression of p53, Bc1-2, Bcl-XL and Bax was analyzed by Western blot. Caspase activation was evaluated by the assay of enzymatic activity with chromogenic peptides and Western blot. Nuclear, cytosolic and mitochondrial fractions were prepared by differential centrifugation and the presence of cytochrome c and lamin B was evaluated by Western blot. RESULTS: p53, Bc1-2, Bcl-XL and Bax protein expression were unchanged during apoptosis. Cytochrome c was released from mitochondria into the cytosol, a pivotal event in the activation of caspase-3. Caspase-3 and -6 but not caspase-2 were activated, and proteolysis of PARP and lamin B, a caspase-6 substrate located in the inner nuclear membrane, was demonstrated by Western blot. The nuclear localization of lamin B was also inhibited by lovastatin. CONCLUSIONS: These data demonstrate that, in TAD-2 thyroid cells, lovastatin induces lamin B proteolysis and inhibits its nuclear localization and induces cytochrome c release from mitochondria into the cytosol.

OBJECTIVE: To evaluate the molecular mechanisms of the inhibitory effects of amiodarone and its active metabolite, desethylamiodarone (DEA) on thyroid hormone action. MATERIALS AND METHODS: The reporter construct ME-TRE-TK-CAT or TSHbeta-TRE-TK-CAT, containing the nucleotide sequence of the thyroid hormone response element (TRE) of either malic enzyme (ME) or TSHbeta genes, thymidine kinase (TK) and chloramphenicol acetyltransferase (CAT) was transiently transfected with RSV-TRbeta into NIH3T3 cells. Gel mobility shift assay (EMSA) was performed using labelled synthetic oligonucleotides containing the ME-TRE and in vitro translated thyroid hormone receptor (TR)beta. RESULTS: Addition of 1 micromol/l T4 or T3 to the culture medium increased the basal level of ME-TRE-TK-CAT by 4.5- and 12.5-fold respectively. Amiodarone or DEA (1 micromol/l) increased CAT activity by 1.4- and 3.4-fold respectively. Combination of DEA with T4 or T3 increased CAT activity by 9.4- and 18.9-fold respectively. These data suggested that DEA, but not amiodarone, had a synergistic effect with thyroid hormone on ME-TRE, rather than the postulated inhibitory action; we supposed that this was due to overexpression of the transfected TR into the cells. When the amount of RSV-TRbeta was reduced until it was present in a limited amount, allowing competition between thyroid hormone and the drug, addition of 1 micromol/l DEA decreased the T3-dependent expression of the reporter gene by 50%. The inhibitory effect of DEA was partially due to a reduced binding of TR to ME-TRE, as assessed by EMSA. DEA activated the TR-dependent down-regulation by the negative TSH-TRE, although at low level (35% of the down-regulation produced by T3), whereas amiodarone was ineffective. Addition of 1 micromol/l DEA to T3-containing medium reduced the T3-TR-mediated down-regulation of TSH-TRE to 55%. CONCLUSIONS: Our results demonstrate that DEA, but not amiodarone, exerts a direct, although weak, effect on genes that are regulated by thyroid hormone. High concentrations of DEA antagonize the action of T3 at the molecular level, interacting with TR and reducing its binding to TREs. This effect may contribute to the hypothyroid-like effect observed in peripheral tissues of patients receiving amiodarone treatment.

In order to compare oral and high-dose iv corticosteroid therapy for Graves’ disease, 25 patients with Graves’ ophthalmopathy were treated with two weekly iv injections of 1g of methylprednisolone diluted in 250–500 ml of physiological solution for 6 weeks, and were compared to a group of 26 patients treated with oral prednisone at a dose of 60–80 mg/day progressively reduced every 2 weeks for a total duration of 4–6 months. The efficacy of treatment was evaluated using the ophthalmopathy index score. Patients were followed at 3, 6, 12 months, and afterwards yearly. All patients showed a significant improvement in signs and symptoms of orbital inflammation and a slight improvement in proptosis and diplopia. Relevant side-effects were reported from patients receiving oral therapy, but no significant side-effects were observed in patients treated with high iv doses; a few cases presented with gastric pain (highly sensitive to aluminium oxide or ranitidine), while most of the patients referred to cutaneous rashes and a metal taste that disappeared some hours after the infusion. Improvements observed after treatment have been stable in both groups. In conclusion, in addition to a lower incidence of side-effects compared to the classic oral therapy, the high-dose iv steroid therapy provides efficient and stable improvement in Graves’ ophthalmopathy.

Edible plants can be contaminated by air diffused pollutants. Research on stress caused by pollutants, which involves the biomonitoringof ornamental trees in the city of Palermo, including Citrusfruits,which are of considerable importance in Sicily's agricultural economy andagroindustrial programmes, has been carried out.Morphological observations of ornamental Citrusrevealed anevergreen foliage and excellent leaf characteristics, even in environmentalcontexts where other plant species were found to have undergone morpho-anatomical changes.Stress symptoms related to the presence of lead which, supposedly, couldhave been absorbed by the pericarp or by the pulp of the navel orangebrasiliano N.L. 92 and 2 grapefruit cultivars were observed. Fruits, sections of pulp and leaves of brasiliano oranges collected from thefield in Lascari (Palermo, 38° N) of the “Centro di studio per ilmiglioramento genetico degli agrumi” (C.N.R. Palermo) and from theexperimental sites of the Faculty of Agriculture in the Parco d'Orleans inPalermo were compared. Fruits of the cultivars Ruby Red Star and Marshpicked from the field located in the Parco d'Orleans were also objects ofobservation.Lead, iron, manganese and copper were measured by atomic absorptionspectrophotometry and we conclude that, in these particular cultivationconditions, the brasiliano orange would not appear to be a suitable markerspecies and seems to be safe for agro-alimentary use.

Treatment of dopaminergic rat PC12 cells with human immunodeficiency virus, type 1 (HIV-1) Tat protein or tat cDNA inhibited the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for the dopamine biosynthetic pathway, as well as the production and release of dopamine into the culture medium. Moreover, the Tat addition to PC12 cells up-regulated the expression of the inducible cAMP early repressor (ICER), a specific member of the cAMP-responsive element modulator transcription factor family, in a cAMP-dependent manner. In turn, ICER overexpression abrogated the transcription activity of the TH promoter in PC12 cells, strongly suggesting ICER involvement in Tat-mediated inhibition of TH gene expression. In vivo injection of synthetic HIV-1 Tat protein into the striatum of healthy rats induced a subclinical Parkinson's-like disease that became manifested only when the animals were treated with amphetamine. As early as one week postinjection, the histochemical examination of the rat substantia nigra showed a reduced staining of neurons expressing TH followed by a loss of TH(+) neurons at later time points. As Tat protein can be locally released into the central nervous system by HIV-1-infected microglial cells, our findings may contribute to the explanation of the pathogenesis of the motorial abnormalities often reported in HIV-1 seropositive individuals.

To facilitate the estimation of acro-megalic activity a prospective study was done comparing, against a clinical score, the effectiveness of serum IGF-I, IGFBP3 and the IGF-I/IGFBP3 molar ratio. Sixty nine observations were distributed in three groups: Group I=patients before surgery; group II=patients improved but still clinically active; group III=patients clinically inactive. Suppression of serum GH levels one hour after an oral glucose load was in agreement with the clinical score in 21/22 observations. Increases in serum IGF-I and IGFBP3 levels were similarly frequent: both 100% in group I, 80% and 95% in group II, 9% and 36% in group III, respectively. The frequency of abnormal molar ratios was 95%, 40% and 0% in the same groups. Log IGF-I, log IGFBP3, and log molar ratio correlated significantly with the clinical scores (r=0.873, r=0.692, and r=0.829, respectively). Conclusions: The IGF-I/IGF-BP3 molar ratio was not better than either IGF-I or IGFBP3 in detecting activity in the three groups of patients studied. Both IGF-I and IGFBP3 appear comparably useful for the diagnosis and follow-up of acromegalic patients. Since IGF- I is a more biologically meaningful parameter it might be preferable.

Leukocyte Ig-like receptor (LIR)-1 is a member of the Ig superfamily which has been shown to bind the human cytomegalovirus MHC class I homologue UL-18 protein. In this study, we have analyzed the expression and function of LIR-1 in human NK cells. We show that LIR-1 is expressed by a subset of NK cells variable in size among different donors. When compared to the known HLA class I-specific NK receptors, the expression of LIR-1 was found to be partially overlapped with that of CD94-NKG2A or with that of killer inhibitory receptors (KIR) belonging to the Ig superfamily. The use of the soluble form of UL-18 molecule revealed, in double fluorescence analysis, a selective binding to LIR-1 + cells while no correlation was observed between expression of either KIR or CD94-NKG2A molecules and ability to bind UL18. We further determined whether LIR-1 could also function as receptor for HLA class I molecules. To this end, we assessed the capability of LIR-1 + NK cell clones of lysing HLA class I- target cells transfected with different class I alleles, including HLA-A, -B, -C and -G alleles. Data revealed that LIR-1 functions as a broad HLA class I-specific inhibitory receptor recognizing different alleles coded for by different HLA loci.

In this paper are shown the results obtained on Citrus sinensis(L.) Osbeck during the sampling campaigns performed in Burriana (Spain) in the frame of B.E.M.A. project. The daily trends of photosynthesis (A) showed higher values at morning and a decrement during the hottest hours of the day; for leaf transpiration (E) we observed a slight increment during the day. During different months (May '96, June '97 and July '95) we noted a progressive reduction in gas exchange of the leaves in relation to build-up of summer stress condition (−49% and −60% for June and July respect to May). In particular, net photosynthesis and leaf transpiration measured on Citrusleaves were, in general, characterised by low average values (2.7 ± 0.06 (s.e.) μmol CO2m−2s−1and 0.665 ± 0.021 (s.e.) mmol H2O m−2s−1for current leaves, respectively). The relationship between biogenic volatile organic compound (BVOC) emission rates normalised by assimilation (A) and gas exchange parameters as E/A ratio is reported. This latter is considered as an useful functional index to determine different vegetation types on a functional basis. We also found a good correlation between BVOC normalised emissions with air temperature. This behaviour has been reported for other species having monoterpene compounds in storage structures as Pinusspecies. Moreover, it is evident an uncoupling between BVOC emissions and photosynthesis.

The pore structure of zeolite Y consists of 13 Å supercages connected through 7 Å windows. This study deals with the intrazeolitic photoelectron transfer from trisbipyridyl ruthenium (II) (Ru(bpy)3²⁺) synthesized within zeolite Y supercages to ion-exchanged bipyridinium molecules in neighboring supercages. Three N,N‘-dialkyl-2,2‘-bipyridinium ions and a 4,4‘-bipyridinium ion with reduction potentials varying from −0.37 to −0.65 V have been studied. For the 2,2‘-bipyridinium salts (members of the diquat family), two, three, and four CH2 bridging units abbreviated as 2DQ²⁺, 3DQ²⁺, and 4DQ²⁺ , respectively, have been examined. The fourth viologen is 1,1‘-dimethyl-4,4‘-bipyridinium, commonly known as methyl viologen and abbreviated here as MV²⁺. Because of the limitations of the time-resolved diffuse reflectance instrument, only a lower limit of the forward electron transfer rate constant from Ru(bpy)3²⁺* to the bipyridinium ion was obtained and is >10⁷ s^-1. The back electron transfer from the photogenerated bipyridinium radical ions to Ru(bpy)3³⁺ was monitored at different intrazeolitic bipyridinium concentrations. At low loadings of bipyridinium ions (1 per 10−15 supercages), the transient signal between 360 and 390 nm has contributions from both Ru(bpy)3²⁺* and the bipyridinium radical ions, since the bipyridinium ion concentration was not high enough to quench all of the Ru(bpy)3²⁺*. The decay of this signal (360−390 nm) could be fitted to the sum of two exponentials, representing the disappearance of unquenched Ru(bpy)3²⁺* and the back electron transfer from the bipyridinium radical ions to Ru(bpy)3³⁺. The rate constants for the back electron transfer for 2DQ²⁺, MV²⁺, 3DQ²⁺, and 4DQ²⁺ were found to be 4.0 × 10⁴, 1.7 × 10⁴, 1.1 × 10⁴, and 7.3 × 10³ s^-1. The decrease in the electron transfer rates with increasing driving force for the reaction indicates that the electron transfer is occurring in the Marcus inverted region. At high loadings of the bipyridinium ions (1.2−1.7 molecules/supercage), the bipyridinium radical ions were considerably longer lived, and a simple exponential decay no longer described the loss of the bipyridinium radical signal. A model that allows for electron exchange processes between bipyridinium ions to compete with the back electron transfer was necessary. This kinetic model allowed us to extract the back electron transfer rate at high loadings along with the rate constants for electron hopping and a second-order electron (bipyridinium radical)/hole (Ru(bpy)3³⁺) recombination process. For the series 2DQ²⁺, MV²⁺, 3DQ²⁺, and 4DQ²⁺ with high loading, the back electron transfer rate constants were 2.5 × 10⁵, 9 × 10⁴, 1.8 × 10⁵, and 1.2 × 10⁵ s^-1, higher than the low loading samples. In the high loading case, longer lived charge separation was observed because of the presence of a route for charge propagation by electron hopping via the densely packed viologen molecules.

Scienderin is a Ca+-dependent actin filament severing protein present in chromaffin cells, platelets and a variety of secretory cells. It has been suggested that scinderin is involved in chromaffin cell F-actin dynamics and that this actin network controls the delivery of secretory vesicles to plasma membrane exocytotic sites. Moreover, scinderin redistribution and activity may be regulated by pH and Ca2+ in resting and stimulated cells. Here we describe the molecular cloning, the nucleotide sequence and the expression of bovine chromaffin cell scinderin cDNA. The fusion protein obtained cross-reacts with native scinderin antibodies and binds phosphatidylserine (PS), phosphatidylinositol 4,5-bisphosphate (PIP2) and actin in a Ca+-dependent manner. Antibodies raised against the fusion protein produced the same cellular staining patterns for scinderin as anti-native scinderin. Nucleotide and amino acid sequence analysis indicate that scinderin has six domains each containing three internal sequence motifs, two actin and two PIP2 binding sites and has 63 and 53% homology with gelsolin and villin. These data indicate that scinderin is a novel member of the family of Ca2+-dependent F-actin severing proteins which includes gelsolin and villin.

We investigated the expression of CD4 antigen in normal bone marrow (BM) samples, enriched in CD34+ hematopoietic progenitor cells. At flow cytometry, a significant fraction (ranging from 25% to 65%) of CD34+ cells also showed low levels of CD4 antigen on their surface. The CD4 receptor densities on the surface of hematopoietic progenitors was approximately 50% that of peripheral blood monocytes and 5% of peripheral blood T lymphocytes. In immunoprecipitation experiments, the CD4 antigen expressed by BM hematopoietic progenitors appeared to be the same form expressed by mature peripheral blood CD4+ cells and appeared to be a potentially functional receptor for human immunodeficiency virus-type 1, because it specifically bound recombinant envelope gp120. Moreover, BM samples, highly enriched in CD34+ cells, showed the presence of CD4 mRNA at reverse transcription- polymerase chain reaction examination. In experiments of complement- mediated cytotoxicity with Leu3-a+Leu3b anti-CD4 monoclonal antibody, a significant reduction in the number of both classes of megakaryocyte (burst-forming unit-meg [BFU-meg] and colony-forming unit-meg [CFU- meg]) and granulocyte/macrophage (CFU-GM) progenitors was observed, whereas erythroid (BFU-E) progenitors were only slightly affected. Moreover, purified CD4+ BM cells obtained by immunomagnetic selection, using high concentrations of Leu3a+Leu3b, showed a colony-forming ability of megakaryocyte and granulocyte/macrophage progenitors comparable with that of CD4- BM cells. In conclusion, the present data show that immature hematopoietic progenitor cells express low levels of CD4, the high-affinity receptor of human immunodeficiency virus-type 1.

Programmed cell death is activated, by different stimuli and in many cell types, to regulate cell population balance during tissue proliferation and embryogenesis. Its initial event seems to be, in most cases, the activation of a Ca2+-dependent endonuclease, causing DNA cleavage into nucleosomic fragments. Its morphological expression is characterized by deep nuclear changes, consisting of typical cap-shaped chromatin marginations, followed by nuclear fragmentation and final formation of numerous micronuclei. Cytoplasmic damage appears in a very late stage of the process and the greatest part of the phenomenon appears to take place despite good preservation of the plasma membrane and organellar component. In the present study we analyzed apoptosis in camptothecin-treated HL60 leukaemia cells, and in freshly isolated mouse thymocytes treated with dexamethasone. The process was first quantified and time monitored by flow cytometry. Subsequently the specimens were processed for morphological examination in order to investigate the behaviour of the different nuclear domains. To follow DNA and RNA localization, we utilized osmium ammine and DNase-colloidal gold cytochemical reactions. The concentration of most DNA in the cap-shaped structures was demonstrated by these reactions. Confocal microscopy of cells processed by in situ nick-translation suggested that DNA was firstly cleaved and subsequently condensed in cup-shaped structures. Despite the strong nuclear modifications, nucleoli could be clearly recognized until the late apoptotic stages.

A number of techniques are currently employed for the fractionation of heterogeneous cell populations or for the separation of cells in different phases of their cycle. With the development of osmotically inert colloidal silica particles media, density gradient centrifugation became an established method for the separation and purification of cells and subcellular particles. We have applied this technique to the separation of cycling from resting Friend erythroleukemia cells, to obtain purified populations for further biological assays. The flow cytometric analysis of DNA content of the different fractions obtained by the gradient and stained with Propidium lodide (PI), showed the S compartment highly concentrated in the 1.073/77g/ml interface, while the upper levels of the gradient were highly enriched of cells in G1 phase. Moreover, the dual parameter analysis of DNA content by means of Bromodeoxyuridine (BrdUrd) incorporation and PI staining, showed that part of the cells in the 1.067/73 fraction represented the early S phase even if their DNA level, measured on the basis of PI fluorescence was within the diploid cell cluster. This method seems to be suitable to obtain pure cell fractions even when dealing with numerically large populations.

HeLa metaphase chromosomes were exammed by means of “in lens” field emission scanning electron microscopy, which permits high resolution detection of uncoated biological samples. By using uncoated chromosomes as a model for comparison we report evidence of how traditional scanning electron microscopy techniques such as metal coating and conductive methods can generate errors in chromosome structure evaluation, since both give rise to morphological artifacts. By comparing the morphology of uncoated chromosomes obtained by two different isolation procedures, such as that utilized in standard cytogenetics and the polyamine method, we have drawn the following conclusions: (a) the standard cytogenetic method gives rise to a chromosome structure consisting of a flattened network of 10 nm fibers, in which higher order chromatin organization is absent. (b) Chromosomes obtained by the polyamine method show both three-dimensional profile and higher level folding of chromatin fibers, supporting the loop chromosome organization previously suggested by scanning electron microscopy observation of hexylene glycol isolated chromosomes.

Scinderin is a calcium-dependent actin filament-severing protein recently discovered in the chromaffin cells of adrenal medulla. In view of the wide tissue distribution of gelsolin, another actin filament-severing protein, experiments were performed to determine the tissue expression of scinderin. Extracts prepared from different bovine tissues were tested by actin-DNase I Sepharose 4B-binding procedure and immunoprecipitation followed by immunoblotting with scinderin and gelsolin antibodies. Among the tissues tested, scinderin was found to be present in the adrenal medulla, brain, anterior and posterior pituitaries, kidney, salivary gland and testis. Scinderin was not found in liver, plasma, skeletal and heart muscles. Gelsolin was expressed in all of the above tissues. The results suggest that scinderin seems to be restricted to tissues with high secretory activity.

Natural killer cells express clonally distributed receptors specific for major histocompatibility complex class I molecules. The human leukocyte antigen (HLA)-C-specific receptors have been molecularly identified and cloned. They exist not only as inhibitory (p58) but also as activatory (p50) receptors. Here we show that p50 and p58 are highly homologous in their extracellular regions formed by two Ig-like domains. In contrast, major differences exist in their transmembrane and cytoplasmic portions. Whereas p 58 displays a 76-84-amino acid cytoplasmic tail containing an unusual antigen receptor activation motif, p50 is characterized by a shorter 39-amino acid tail. In addition, whereas p58 has a nonpolar transmembrane portion, p50 contains the charged amino acid Lys. These data strongly suggest that receptors with identical HLA-C allele specificity can mediate functions of opposite sign owing to their different transmembrane/cytoplasmic portions.

On the basis of the consideration that cell-free models cannot precisely mimic the complexity of the intracellular environment, we used a system to investigate the mechanisms that enable antigen-presenting cells (APC) to bind exogenous peptides through their human leukocyte antigen (HLA) molecules. We evaluated the uptake of the radiolabeled peptide 17-29-Tyr of influenza virus matrix protein by B-EBV cell lines, under various conditions. The results can be summarized as follows: a)the kinetics of peptide binding and release are very fast in living, fully competent cells; b)the peptide-HLA complexes are short-living and the DR molecules continuously undergo peptidic exchange; c)using glutaraldehyde-fixed cells, the kinetics of the two phenomena are slow, closely resembling those observed with the same peptide and purified, immobilized DR molecules.The data suggest that in APC, cellular mechanisms are operative that increase the efficiency of both loading and unloading of Class II HLA with exogenous peptides. This is likely to be related to the recycling of Class II molecules to intracellular compartments, were binding takes place.The observation that the HLA-peptide complex is a dynamic structure, suggests the possibility of replacing natural peptides with synthetic ones at this level, in order to regulate the immune response.