The shell effect on the room temperature photoluminescence from ZnO/MgO core/shell nanowires: exciton-phonon coupling and strain.

The room temperature photoluminescence from ZnO/MgO core/shell nanowires (NWs) grown by a simple two-step vapor transport method was studied for various MgO shell widths (w). Two distinct effects induced by the MgO shell were clearly identified. The first one, related to the ZnO/MgO interface formation, is evidenced by strong enhancements of the zero-phonon and first phonon replica of the excitonic emission, which are accompanied by a total suppression of its second phonon replica. This effect can be explained by the reduction of the band bending within the ZnO NW core that follows the removal of atmospheric adsorbates and associated surface traps during the MgO growth process on one hand, and a reduced exciton-phonon coupling as a result of the mechanical stabilization of the outermost ZnO NW monolayers by the MgO shell on the other hand. The second effect is the gradual increase of the excitonic emission and decrease in the defect related emission by up to two and one orders of magnitude, respectively, when w is increased in the ∼3-17 nm range. Uniaxial strain build-up within the ZnO NW core with increasing w, as detected by x-ray diffraction measurements, and photocarrier tunneling escape from the ZnO core through the MgO shell enabled by defect-states are proposed as possible mechanisms involved in this effect. These findings are expected to be of key significance for the efficient design and fabrication of ZnO/MgO NW heterostructures and devices.

Glucose biosensor based on functionalized ZnO nanowire/graphite films dispersed on a Pt electrode.

We present a glucose biosensor based on ZnO nanowire self-sustained films grown on compacted graphite flakes by the vapor transport method. Nanowire/graphite films were fragmented in water, filtered to form a colloidal suspension, subsequently functionalized with glucose oxidase and finally transferred to a metal electrode (Pt). The obtained devices were evaluated using scanning electron microscopy, energy-dispersive x-ray spectroscopy, cyclic voltammetry and chronoamperometry. The electrochemical responses of the devices were determined in buffer solutions with successive glucose aggregates using a tripolar electrode system. The nanostructured biosensors showed excellent analytical performance, with linear response to glucose concentrations, high sensitivity of up to ≈17 µA cm(-2) mM(-1) in the 0.03-1.52 mM glucose concentration range, relatively low Michaelis-Menten constant, excellent reproducibility and a fast response. The detection limits are more than an order of magnitude lower than those achievable in commercial biosensors for glucose control, which is promising for the development of glucose monitoring methods that do not require blood extraction from potentially diabetic patients. The strong detection enhancements provided by the functionalized nanostructures are much larger than the electrode surface-area increase and are discussed in terms of the physical and chemical mechanisms involved in the detection and transduction processes.

Management of postharvest grape withering to optimise the aroma of the final wine: A case study on Amarone.

Amarone wine is different from regular dry wine due to the postharvest withering of Corvina, Corvinone and Rondinella grapes. Grapes were withered in a commercial facility with variability in terms of temperature and relative humidity (R.H.). Sugar content reached 230-240gL(-1) and 280gL(-1) at 20% and 30% mass loss, respectively. Most of VOCs (volatile organic compounds) decreased during withering but few VOCs increased during withering and we considered as markers; in Corvinone they were methylhexanoate, dimethylsuccinate, nerol, nonanoic acid, and benzyl alcohol; in Corvina, benzyl alcohol, isoamyl alcohol, 1-hexanol, p-cymen-8-ol, 2,3 pinanediol, 3-oxo-ionol and 3-methyl-1-pentanol, coumaran and damascenone; in Rondinella, hexanol, nonanoic acid, methyl vanillate, damascenone, 3-oxo-ionol, eugenol, p-cymen-8-ol, 2,3 pinanediol, coumaran and raspberry keton. Olfactive descriptors of the wines and the potential aroma of the combination of Corvina wine with the wines of the other two varieties at different percentages of mass loss are reported.

Postharvest grape infection of Botrytis cinerea and its interactions with other moulds under withering conditions to produce noble-rotten grapes.

AIMS: To investigate the interactions between Botrytis cinerea and other moulds during grape withering and postharvest infection to obtain noble-rotten grapes. METHODS AND RESULTS: Strains of Botrytis cinerea, Penicillium expansum, Penicillium crustosum, Aspergillus niger, Fusarium verticilloides and Alternaria alternata, isolated from naturally withered grapes and identified by molecular tools, were used to infect Garganega and Corvina grapes. Individually sterilized berries were infected by a single inoculation of each strain or a simultaneous inoculation of B. cinerea together with one of each of the other moulds. Withering kinetics, glycerol, gluconic acid, total polyphenols, total anthocyanins and laccase activity greatly varied among each strain and also in respect to untreated berries. Successful noble rot settlement was ascertained by an additional infection assay carried out on nonsterilized berries. CONCLUSIONS: The suitability of inducing noble rot infection during grape withering and the improvement of the health of noble-rotten grapes have been demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides insights on the effects of mould interactions on withered grape quality. Implementing noble rot induction by postharvest infection in winery drying fruit rooms to standardize the level of grape botrytization is encouraged.

The inhibitory effects of wine phenolics on lysozyme activity against lactic acid bacteria.

The lysozyme of hen's egg white is used in winemaking to control spontaneous lactic acid bacteria (LAB). A total of eight LAB strains, isolated from grape must and wine, were used to assess the inhibitory effects of wine phenolics on lysozyme activity. The presence of phenolics, extracted from grape pomace, in growth medium reduced the mortality rate due to the lysozyme activity. This effect was especially clear in the case of strains belonging to Lactobacillus uvarum, Pediococcus parvulus and Oenococccus oeni, which are more sensitive to lysozyme than L. plantarum and L. hilgardii strains. Cell lysis assays carried out on four strains sensitive to lysozyme and Micrococcus lysodeikticus ATCC 4698, used as a reference strain, confirmed the inhibition of grape pomace phenolics on the muramidase. There was no interference from non-flavonoids, flavanols and flavonol compounds, when they were tested individually, on the lysozyme activity against the strains. Anthocyanins extracted from grape skins slightly inhibited the activity only against M. lysodeikticus. However, proanthocyanidins extracted from seed berries, strongly inhibited the lysozyme. In this extract, dimers were the predominant oligomers of flavan-3-ol. The study demonstrated that the effectiveness of lysozyme against LAB in red winemaking is related to the amount of low molecular weight proanthocyanidins that are released when the grapes are macerating.

On the role of arbitration of discordant double readings of screening mammography: experience from two Italian programmes.

PURPOSE: The authors sought to assess the role of arbitration by a third reader of discordant double readings to reduce the rate of recalls to diagnostic assessment. MATERIALS AND METHODS: A consecutive series of 7,660 double readings of screening examinations were considered. Discordant recalls were arbitrated by an expert reader (negative/positive). Diagnostic assessment was performed irrespective of arbitration results, and its outcome was used as reference standard for the study purpose. Assuming that negative arbitration would deny recall, its impact was assessed in terms of reduced recall rate and reduced cancer detection rate. Cost analysis of introducing arbitration was performed according to these results. RESULTS: Recalls at double reading were 528 (6.8%), of which 230 (43.5%) were concordant and 298 (56.5%) were discordant. The latter underwent arbitration, which was negative in 216 (72.4%) and positive in 82 (27.6%) cases, respectively. Overall, 49 cancers were detected (6.39 ‰ screened, 9.2% recalled): 43 cancers were detected among concordant (5.6 ‰ screened, 18.6% concordant) and six among discordant recalls (0.7 ‰ screened, 2.0% discordant). Six cancers were observed among arbitrated cases: five (6%) in positive and one (4.6 ‰) in negative arbitrations. Negative arbitration would have spared 216 assessment procedures (2.8% absolute, 40.9% relative reduction of recall rate) while missing one cancer case (0.13 ‰ absolute, 2.0% relative reduction of cancer detection rate). Arbitration cost was 74 euro, whereas 216 spared assessment procedures would have cost 14,558.4-23,346 euro. CONCLUSIONS: Arbitration is a cost-effective procedure that could be employed as a first measure to counterbalance excess recall rate observed in a double-reading scenario.

Evidence of different fermentation behaviours of two indigenous strains of Saccharomyces cerevisiae and Saccharomyces uvarum isolated from Amarone wine.

AIMS: To explain the role of Saccharomyces cerevisiae and Saccharomyces uvarum strains (formerly Saccharomyces bayanus var. uvarum) in wine fermentation. METHODS AND RESULTS: Indigenous Saccharomyces spp. yeasts were isolated from Amarone wine (Italy) and analysed. Genotypes were correlated to phenotypes: Melibiose(-) and Melibiose(+) strains displayed a karyotype characterized by three and two bands between 225 and 365 kb, respectively. Two strains were identified by karyotype analysis (one as S. cerevisiae and the other as S. uvarum). The technological characterization of these two strains was conducted by microvinifications of Amarone wine. Wines differed by the contents of ethanol, residual sugars, acetic acid, glycerol, total polysaccharides, ethyl acetate, 2-phenylethanol and anthocyanins. Esterase and beta-glucosidase activities were assayed on whole cells during fermentation at 13 degrees and 20 degrees C. Saccharomyces uvarum displayed higher esterase activity at 13 degrees C, while S. cerevisiae displayed higher beta-glucosidase activity at both temperatures. CONCLUSIONS: The strains differed by important technological and qualitative traits affecting the fermentation kinetics and important aroma components of the wine. SIGNIFICANCE AND IMPACT OF THE STUDY: The contribution of indigenous strains of S. cerevisiae and S. uvarum to wine fermentation was ascertained under specific winemaking conditions. The use of these strains as starters in a winemaking process could differently modulate the wine sensory characteristics.

Notch signaling is involved in expression of thyrocyte differentiation markers and is down-regulated in thyroid tumors.

CONTEXT: Notch genes encode receptors for a signaling pathway that regulates cell growth and differentiation in various contexts, but the role of Notch signaling in thyroid follicular cells has never been fully published. OBJECTIVE: The objective of the study was to characterize the expression of Notch pathway components in thyroid follicular cells and Notch signaling activities in normal and transformed thyrocytes. DESIGN/SETTING AND PATIENTS: Expression of Notch pathway components and key markers of thyrocyte differentiation was analyzed in murine and human thyroid tissues (normal and tumoral) by quantitative RT-PCR and immunohistochemistry. The effects of Notch overexpression in human thyroid cancer cells and FTRL-5 cells were explored with analysis of gene expression, proliferation assays, and experiments involving transfection of a luciferase reporter construct containing human NIS promoter regions. RESULTS: Notch receptors are expressed during the development of murine thyrocytes, and their expression levels parallel those of thyroid differentiation markers. Notch signaling characterized also normal adult thyrocytes and is regulated by TSH. Notch pathway components are variably expressed in human normal thyroid tissue and thyroid tumors, but expression levels are clearly reduced in undifferentiated tumors. Overexpression of Notch-1 in thyroid cancer cells restores differentiation, reduces cell growth rates, and stimulates NIS expression via a direct action on the NIS promoter. CONCLUSION: Notch signaling is involved in the determination of thyroid cell fate and is a direct regulator of thyroid-specific gene expression. Its deregulation may contribute to the loss of differentiation associated with thyroid tumorigenesis.

Growth inhibition of medullary thyroid carcinoma cells by pyrazolo-pyrimidine derivates.

There is no effective treatment for recurrent or metastatic medullary thyroid carcinoma (MTC), a tumor arising from thyroid C-cells commonly presenting an inherited or acquired RET mutation. In this study we examined the sensitivity of two human MTC cell lines to novel pyrazolopyrimidine derivates, able to inhibit src-family tyrosine kinase activity. In TT cells [carrying the multiple endocrine neoplasia (MEN)2A Ret mutation Cys 634Trp] and MZ-CRC-1 cells (carrying the MEN2B RET mutation Met891Thr), one of these compounds, namely Si 34, determined a significant growth inhibitory effect (approximately 90% vs control for TT, 80% vs control for MZ-CRC-1) mainly due to enhanced cell mortality after a 6-day incubation. At concentrations that increased cell mortality, neither biochemical or morphological characteristics of apoptosis were detected in TT and MZCRC- 1 cells treated with Si 34. These results, when confirmed in other in vivo preclinical models, suggest that this novel tyrosine kinase inhibitor may be useful for the treatment of MTC.

BRAF mutations in papillary thyroid carcinomas inhibit genes involved in iodine metabolism.

CONTEXT: BRAF mutations are common in papillary thyroid carcinomas (PTCs). By affecting the expression of genes critically related to the development and differentiation of thyroid cancer, they may influence the prognosis of these tumors. OBJECTIVE: Our objective was to characterize the expression of thyroid-specific genes associated with BRAF mutation in PTCs. DESIGN/SETTING AND PATIENTS: We examined the expression of key markers of thyrocyte differentiation in 56 PTCs with BRAF mutations (BRAF-mut) and 37 with wild-type BRAF (BRAF-wt). Eight samples of normal thyroid tissue were analyzed as controls. Quantitative PCR was used to measure mRNA levels for the sodium/iodide symporter (NIS), apical iodide transporter (AIT-B), thyroglobulin (Tg), thyroperoxidase (TPO), TSH receptor (TSH-R), the transcription factor PAX8, and glucose transporter type 1 (Glut1). NIS protein expression and localization was also analyzed by immunohistochemistry. RESULTS: mRNA levels for all thyroid-specific genes were reduced in all PTCs vs. normal thyroid tissues. NIS, AIT-B, Tg, and TPO expression was significantly lower in BRAF-mut tumors than in the BRAF-wt group. Glut-1 transcript levels were increased in all PTCs, and additional increases were noted in BRAF-mut tumors. In both tumor subsets, the NIS protein that was expressed was abnormally retained in the cytoplasm. CONCLUSION: BRAF V600E mutation in PTCs is associated with reduced expression of key genes involved in iodine metabolism. This effect may alter the effectiveness of diagnostic and/or therapeutic use of radioiodine in BRAF-mut PTCs.

The vinification of partially dried grapes: a comparative fermentation study of Saccharomyces cerevisiae strains under high sugar stress.

AIMS: The study of the fermentation performance of Saccharomyces cerevisiae strains under high sugar stress during the vinification of partially dried grapes. METHODS AND RESULTS: Microvinification of partially dried grape must with sugar concentration of 35 degrees Brix was performed using four commercial strains to carry out alcoholic fermentation. A traditional red vinification without nutrients addition was applied. Yeasts displayed different efficiency to convert sugar in ethanol and varied in glycerol yield. Sugar consumption and ethanol level were attested at 80-87% and 143.5-158.0 g l(-1) respectively. High correlation between sugar and assimilable nitrogen consumption rate was observed. Statistical treatment of data by principal component analysis highlighted the different behaviours that strains exhibited in regard to the production of higher alcohols and other compounds important to wine quality. CONCLUSIONS: Saccharomyces cerevisiae strains displayed appreciable capability to overcome osmotic stress and to yield ethanol fermenting high sugar concentration grape must in winemaking condition. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provided insights on the strain contribution to wine quality subordinate to stress condition. This investigation is of applicative interest for winemaking and processing industry that use high sugar concentration musts.

Fractal analysis of lumbar vertebral cancellous bone architecture.

Osteoporosis is characterized by bone mineral density (BMD) decreasing and spongy bone rearrangement with consequent loss of elasticity and increased bone fragility. Quantitative computed tomography (QCT) quantifies bone mineral content but does not describe spongy architecture. Analysis of trabecular pattern may provide additional information to evaluate osteoporosis. The aim of this study was to determine whether the fractal analysis of the microradiography of lumbar vertebrae provides a reliable assessment of bone texture, which correlates with the BMD. The lumbar segment of the spine was removed from 22 cadavers with no history of back pain and examined with standard x-ray, traditional tomography, and quantitative computed tomography to measure BMD. The fractal dimension, which quantifies the image fractal complexity, was calculated on microradiographs of axial sections of the fourth lumbar vertebra to determine its characteristic spongy network. The relationship between the values of the BMD and those of the fractal dimension was evaluated by linear regression and a statistically significant correlation (R = 0.96) was found. These findings suggest that the application of fractal analysis to radiological analyses can provide valuable information on the trabecular pattern of vertebrae. Thus, fractal dimensions of trabecular bone structure should be considered as a supplement to BMD evaluation in the assessment of osteoporosis.

Distribution of S-layers on the surface of Bacillus cereus strains: phylogenetic origin and ecological pressure.

Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis have been described as members of the Bacillus cereus group but are, in fact, one species. B. anthracis is a mammal pathogen, B. thuringiensis an entomopathogen and B. cereus a ubiquitous soil bacterium and an occasional human pathogen. In two clinical isolates of B. cereus, in some B. thuringiensis strains and in B. anthracis, an S-layer has been described. We investigated how the S-layer is distributed in B. cereus, and whether phylogeny or ecology could explain its presence on the surface of some but not all strains. We first developed a simple biochemical assay to test for the presence of the S-layer. We then used the assay with 51 strains of known genetic relationship: 26 genetically diverse B. cereus and 25 non-B. anthracis of the B. anthracis cluster. When present, the genetic organization of the S-layer locus was analysed further. It was identical in B. cereus and B. anthracis. Nineteen strains harboured an S-layer, 16 of which belonged to the B. anthracis cluster. All 19 were B. cereus clinical isolates or B. thuringiensis, except for one soil and one dairy strain. These findings suggest a common phylogenetic origin for the S-layer at the surface of B. cereus strains and, presumably, ecological pressure on its maintenance.

Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments.

Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.

Anti-idiotypic response to antigrowth factor receptor monoclonal antibodies.

The immunogenicity of the idiotypic portions of two antigrowth factor receptor monoclonal antibodies (MAbs) was studied. Immunisation of allogeneic but not syngeneic mice with antihuman epidermal growth factor receptor (EGF-R) MAb MINT5 or anti-HER-2/neu MGR6 MAb elicited a detectable titre of circulating antibodies, particularly when the MAb was coupled with the keyhole limpet haemocyanin and administered together with Freund's adjuvant. The anti-Ab1 response to MAb MINT5 was slightly delayed as compared with the response obtained with MAb MGR6 and was mainly directed to the variable regions. In both cases, all anti-Ab1-positive sera specifically competed with the binding of homologous radiolabelled Ab1 to the relevant EGF-R+ or HER-2/neu+ target cells. Fusion of splenocytes from MINT5-immunised animals failed to produce MAb, whereas cell fusion was successful in generating a paratope-related MAb in the case of MGR6. The anti-MGR6 MAb-produced IdM6.4 inhibited the binding of MAb MGR6 on breast carcinoma cells, suggesting that it recognises an idiotope in or near the antigen combining site, and can be considered useful in the identification and purification of the Ab1 or its derivatives. We analysed whether a possible recognition of murine EGF-R by MAb MINT5 or a mimicry of EGF by the MAb idiotype prevented or delayed the development of an idiotypic cascade in mice. MINT5 inhibited human and murine EGF binding to the human EGF-R, whereas the anti-Ab1 response competed with MINT5 but not with murine EGF binding to A431 human epidermoid carcinoma cells. Moreover, MINT5 did not recognise the murine EGF-R. In a phase I clinical study, no detectable levels of human antimouse antibody response were observed in 5 of the 6 treated cancer patients. The ability of MAb MINT5 to block human EGF-R function, together with its low immunogenicity in patients, raise the possibility of its application in carcinoma immunotherapy.

In vitro and in vivo stability and anti-tumour efficacy of an anti-EGFR/anti-CD3 F(ab')2 bispecific monoclonal antibody.

The in vitro and in vivo stability and anti-tumour efficacy of the anti-EGFR/anti-CD3 bispecific monoclonal antibody (biMAb), M26.1, were analysed. The interaction of the intact biMAb with Fc receptor I (Fc gamma RI) present on human leucocytes was not observed when the antibody was used as an F(ab')2 fragment. A CD8+ T-cell clone coated with M26.1 F(ab')2 was as effective as the intact biMAb in inducing IGROV1 target cell lysis when tested in a 51Cr-release assay. Variable levels of reduction of F(ab')2 to monovalent F(ab') were observed upon incubation with human ovarian cancer ascitic fluid (OCAF) or with human glioblastoma cavity fluid (GCF), but not with mouse or human sera. Activated lymphocytes coated with F(ab')2 and incubated in vitro with GCF or OCAF for 24 and 48 h respectively maintained their targeting. Thus, the F(ab')2, when present as a soluble molecule, but not when bound to T cells, might lose some functional activity as a consequence of partial reduction to F(ab'). In normal mice, M26.1 F(ab')2 retained full cytotoxic activity in the circulation, and clearance values were similar to those obtained with parental and other MAb F(ab')2. Treatment of IGROV1 tumour-bearing mice with activated human lymphocytes coated with the M26.1 F(ab')2 significantly prolonged survival of the animals compared with tumour-bearing untreated and control mice treated with lymphocytes or F(ab')2 alone. Together, these results suggest the clinical usefulness of bispecific M26.1 F(ab')2 as a targeting agent for local treatment of tumours such as glioma and ovarian cancers that express variable levels of epidermal growth factor receptor (EGFR).

Anti-tumor efficacy of an anti-epidermal-growth-factor-receptor monoclonal antibody and its F(ab')2 fragment against high- and low-EGFR-expressing carcinomas in nude mice.

Monoclonal antibody (MAb) MINT5 specifically detects the epidermal-growth-factor receptor (EGFR). In vitro analyses of intact MINT5 (IgG1) and its F(ab')2 fragment indicated that both forms of the MAb inhibited binding of 125I-mEGF to EGFR, induced receptor internalization and blocked EGF-induced EGFR tyrosine-kinase activation in A431 cells. Both forms of the MAb also inhibited to the same extent the proliferation of the carcinoma cell lines A431 and IGROVI, despite the difference in EGFR levels on the cells. The detection of TGF alpha mRNA and the inhibition of cell growth in EGF-free conditions by anti-EGFR MAb indicated the involvement of an EGFR/TGF alpha autocrine/paracrine pathway in the in vitro growth of both cell lines. Analysis of mice xenotransplanted s.c. with A431 cells and treated with MINT5 revealed a block in A431 tumor take in 6 of 10 animals when intact MAb was administered from day 0 to day 11. On a molar basis, F(ab')2 at the same dose was ineffective, although at a 7-fold higher dose F(ab')2 reduced s.c. tumor growth by 80%. At the same dose, intact MINT5 MAb reduced s.c. growth of the EGFR-negative MeWo cell line by 5%. Survival of mice bearing IGROVI i.p. xenotransplants and treated locally with either form of MAb was significantly prolonged even when treatment was initiated on day 3. Corrected doses of intact and F(ab')2 fragment, which accounted for the difference in serum half-lives of the MAb forms, resulted in similar survival rates in the tumor-bearing mice. These pre-clinical results suggest that MINT5 MAb might be safely used for systemic therapy of EGFR-over-expressing tumors. Loco-regional therapy might be contemplated in the case of tumors with moderate/low EGFR expression.

Crucial residues in the carboxy-terminal end of C1 inhibitor revealed by pathogenic mutants impaired in secretion or function.

The last exon of the C1-1NH gene was screened for point mutations in 36 unrelated hereditary angioedema patients. Mutations were found in eight patients, predicting changes in the short COOH-terminal region which anchors the reactive site loop on its COOH-terminal side. The effects of each of these mutations were examined in transiently transfected Cos-7 cells. Complete intracellular retention or degradation was observed with substitutions in the COOH-terminal strands 4B or 5B: Leu459-->Pro, Leu459-->Arg, and Pro467-->Arg were all blocked at early stages of intracellular transport, but differences in the immunofluorescence patterns indicated that a significant fraction of the Leu459-->Pro and of the Pro467-->Arg proteins reached a compartment distinct from the endoplasmic reticulum. In line with previous findings with alpha 1-antitrypsin, chain termination within strand 5B resulted in rapid degradation. Mutant Val451-->Met, in strand 1C, and mutant Pro476-->Ser, replacing the invariant proline near the COOH terminus, yielded reduced secretion, but these extracellular proteins were unable to bind the target protease C1s. Presence of low levels of both dysfunctional proteins in patient plasmas defies the conventional classification of C1 inhibitor deficiencies as type I or type II. These data point to a key role of certain residues in the conserved COOH-terminal region of serpins in determining the protein foldings compatible with transport and proper exposure of the reactive site loop.

C1 inhibitor mutations which affect intracellular transport and secretion in type I hereditary angioedema.

A cluster of point mutations was found in the region of exon 8 of the C1 INH gene which codes for the 28 C-terminal amino acids. Seven of these mutations introduce amino acid changes and one results in a stop codon. Upon transient expression in monkey kidney Cos-7 cells all of these C1 inhibitor mutants showed an impaired intracellular transport and most of them failed to be secreted. Biochemical and immunofluorescence studies indicated that the defective proteins accumulate or are degraded mainly in the endoplasmic reticulum. The product of deletions of exons 4, which has an internal in frame deletion of 45 amino acids, also fails to be secreted.

A critical comparison of three internalization assays applied to the evaluation of a given mAb as a toxin-carrier candidate.

In the attempt to define a strategy for screening new monoclonal antibodies (mAb) that could be appropriate for clinical application in oncology, we evaluated the suitability of three methods: a direct internalization assay (DIA), an indirect internalization assay (IIA) and an indirect cytotoxicity assay (ICA), by applying them to already selected mAb. The latter were directed against three antigenic systems [38-kDa glycoprotein (gp38), epidermal growth factor receptor, and the neu oncogene product], which, according to their tumor selectivity, could be considered suitable for mAb-guided therapy. The dose-dependent and time-dependent binding, as well as the low intra-assay variability, demonstrated the reliability of the three tests. However, a certain degree of inter-assay variability was observed in each one, the highest value being that found when IIA was applied. Furthermore, the degree of variability, as well as the predictability, seemed to be more related to the mAb/antigen (Ag) combination used rather than to the test applied. From the overall data we suggest a procedure to be applied for screening purposes. As a first approach applied to the raw material, ICA is only suitable for screening in the case of an already selected toxin whereas IIA may be helpful to eliminate the true negative mAb. After purification of the relevant mAb a repeated analysis using DIA could allow the selection of true internalizing mAb. However, this second screening should be followed by a further analysis of the fate of the Ag-Ab complex after internalization.