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AIM: This study aimed to investigate and assess primary and secondary dental implant stability during the osseointegration period. METHODS: A total of 77 implants were placed in 42 patients with 26 males and 16 females. The study was conducted by comparing the resonance frequency analysis (RFA) values of the implants inserted in the lower jaw. RFA was done immediately after implant insertion and after 12 weeks. Results were statistically evaluated using SPSS Statistics for Windows, Version 7.1. Level of significance was set at P < 0.05. RESULTS: Significant differences were detected between the primary and secondary stability values, respectively. Maximum RFA value of 88 and the minimum value of 52 were observed. Stability values increased during the following three months, and all implants were successfully integrated without complication. CONCLUSION: Our results indicate and suggest that there is a strong linear correlation between implant stability and ISQ values that can be directly estimated by the RFA, especially in the posterior edentulous mandible. Osstell implant device could represent a useful tool which can be used to identify the risk for implant failure.

Purpose: The aim of this study was to analyze the implant stability quotient (ISQ) results of the resonance frequency (RF) during the first day of surgery and 3 months after dental implant placement in the maxilla. Material and methods: This study was approved by 40 patients with total or partial edentulous maxillary jaws underwent with 85 dental implants. The ISQ values were measured from these 85 implants at baseline and 3 months after placement. Osttell Mentor device was invented to measure the RF value of the implant fixture through the transducer or pin. Measurements were taken 4 times in each direction of the inserted oral dental implant. OPG X-ray images were also obtained to evaluate the bone and neighbouring anatomical structures. Results: The ISQ at baseline had a mean value of 58 (SD 5.91) for all implants placed in anterior and posterior sectors of maxillary ridges. The mean ISQ value on the 3rd month (secondary stability) after placement for all implants was 65 (SD 5.55). An ISQ value ≥65 was observed in 65 implants (78.82%) after 3 months of placement. Differences between primary and secondary mean ISQ values were statistically significant (p < 0.001). The one year cumulative success rate was 98.3% for tested and inserted oral implants. Conclusion: The results of presented study demonstrates and suggests that the influence and reliability of Osstell ISQ values, measured immediately and 3 months after placement it’s predictable for the oral implant outcomes, especially analyzing the sensibility of secondary RFA values.

BACKGROUND: The hyperparathyroidism (HPT) is a condition in which the parathyroid hormone (PTH) levels in the blood are increased. HPT is categorised into primary, secondary and tertiary. A rare entity that occurs in the lower jaw in association with HPT is the so-called brown tumour, which an osteolytic lesion is predominantly occurring in the lower jaw. It is usually a manifestation of the late stage of the disease. Osteosclerotic changes in other bones are almost always associated with renal osteodystrophy in secondary HPT and are extremely rare in primary HPT. This article reports a rare case of a brown tumour in the mandible as the first sign of a severe primary HPT, associated with osteosclerotic changes on the skull.CASE REPORT: A brown tumour in the mandible was diagnosed in 60 - year old female patient with no previous history of systemic disease. The x - rays showed radiolucent osteolytic lesion in the frontal area of the mandible affecting the lamina dura of the frontal teeth, and skull osteosclerosis in the form of salt and pepper sign. The blood analyses revealed increased values of PTH, calcitonin and β – cross-laps, indicating a primary HPT. The scintigraphy of the parathyroid glands showed a presence of adenoma in the left lower lobe. The tumour lesion was surgically removed together with the lower frontal teeth, and this was followed by total parathyroidectomy. The follow - up of one year did not reveal any signs of recurrence.CONCLUSION: It is critical to ensure that every osteolytic lesion in the maxillofacial region is examined thoroughly. Moreover, a proper and detailed systemic investigation should be performed. Patients should undergo regular check-ups to prevent late complications of HPT.

Solitary plasmacytoma (SP) is an early-stage plasma cell malignancy that is in between monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM) along the spectrum of plasma cell disorders. SPs can be divided into 2 groups according to location: solitary bone plasmacytoma (SBP) – these occur most commonly in the vertebrae and secondarily in long bones, and extramedullary plasmacytoma (EMP) – these encompass all nonosseus SPs. The etiology is still unknown, and they generally display a destructive course with preponderance of males. Both lesions present a risk of progression to multiple myelom. Its presence in jaws is extremely rare and when it is seen, angulus and ramus mandible are most common sites of occurrence. Histopathological examination and systemic investigation are mandatory for confirmation but support of immunohistochemistry positivity for CD138 was also done for establishing the final diagnosis., Highlights • Solitary plasmacytoma of the jaw is a rare condition, especially in mandible. • Symptoms may be underestimated because they are considered nonspecific. • For jaw lesions it may be difficult, using routine hematoxylin and eosin stains, to determine whether the lesion is a neoplasm of plasma cells or an inflamatory lesion. • Immunoperoxidase techniques can be helpful in making the distinction between inflammatory and neoplastic lesions. • Plasma cell neoplasms demonstrate monoclonal light chains within the cytoplasm of plasma cells.

Odontogenic myxoma is rare benign neoplasm of jaw bones which is locally aggressive and non-metastasizing. А 23-year-old male sought medical attention with complaint of progressively increasing of asymptomatic swelling to the right side of maxilla causing serious facial asymmetry. Radiological examination, CT with 3D reconstruction was performed and it was shown well-defined unusual large radiolucent mass associated with impacted teeth within the right maxillary sinus extending into the nasal cavity with expansion of both buccal and palatal cortical plate of right maxilla. The treatment of choice was a surgical tumoral radical resection under general anaesthesia with surgical extraoral approach and preservation of the nearby structures. Odontogenic fibromyxoma was the definitive histopathological diagnosis. The postoperative period was uneventful, and the patient fully recovered. Long-term surveillance is required due to the high rate of local recurrence.

In this work, the micro-textural properties of CaO-based CO2 sorbents prepared in a thermo-gravimetric reactor were investigated by ex situ synchrotron-based Small Angle X-ray Scattering (SAXS), Ultra Small Angle X-ray Scattering (USAXS) and Wide Angle X-ray Scattering (WAXS). Six sorbent samples were produced by calcination in vacuum at different temperatures and durations of the heating step; additionally, four partially carbonated samples were produced, after calcination in vacuum, at different CaO conversions. The scattering intensity profiles were analyzed by means of the Unified Fit model. Bimodal pore size distributions (micro-pores and meso-pores) were identified and the micro-textural properties (porosity, specific surface and pore radius of gyration) of each of the two pore populations were quantified both for the calcined and for the carbonated samples. The micro-textural properties of a few calcined samples were compared with N2-adsorption measurements. For each pore population, the trends of porosity, specific surface and pore radius of gyration, in addition to the pore number density and sphericity factor, were obtained versus time during the calcination and versus the CaO conversion during the carbonation. The relevance of micro-pores with respect to macro-pores was assessed both as far as the porosity impact on the CO2 capture capacity and as far as the specific surface impact on the carbonation kinetics. For the calcined samples, the micro-textural properties of a partially calcined sample were compared with those of the completely calcined samples, and pore correlation distances were determined. For the carbonated samples, the specific surface per unit of particle volume results linearly dependent on the CaO conversion and, if expressed per unit of mass of unreacted CaO, does not change significantly for most of the CaO carbonation.

Bright sources of high energy electromagnetic radiation are widely employed in fundamental research as well as in industry and medicine. This steadily growing interest motivated the construction of several facilities aiming at the realisation of sources of intense X- and gamma-ray pulses. To date, free electron lasers and synchrotrons provide intense sources of photons with energies up to 10-100 keV. Facilities under construction based on incoherent Compton back scattering of an optical laser pulse off an electron beam are expected to yield photon beams with energy up to 19.5 MeV and peak brilliance in the range 10$^{20}$-10$^{23}$ photons s$^{-1}$ mrad$^{-2}$ mm$^{-2}$ per 0.1% bandwidth. Here, we demonstrate a novel mechanism based on the strongly amplified synchrotron emission which occurs when a sufficiently dense electron beam interacts with a millimetre thickness solid target. For electron beam densities exceeding approximately $3\times10^{19}\text{ cm$^{-3}$}$ filamentation instability occurs with the self-generation of 10$^{7}$-10$^{8}$ gauss magnetic fields where the electrons of the beam are trapped. This results into a giant amplification of synchrotron emission with the production of collimated gamma-ray pulses with peak brilliance above $10^{25}$ photons s$^{-1}$ mrad$^{-2}$ mm$^{-2}$ per 0.1% bandwidth and photon energies ranging from 200 keV up to several hundreds MeV. These findings pave the way to compact, high-repetition-rate (kHz) sources of short (30 fs), collimated (mrad) and high flux ($>10^{12}$ photons/s) gamma-ray pulses., Full-text access to a view-only version of the published paper by the following SharedIt link: https://rdcu.be/LGtC This is part of the Springer Nature Content Sharing Initiative (https://www.springernature.com/gp/researchers/sharedit). Enhanced PDF features such as annotation tools, one-click supplements, citation file exports and article metrics are freely available

Background: The patients that are subjects to oral-surgical interventions produce large amounts of steroids in comparison with healthy patients which are not a subject to any dental intervention. The aim of research was to determine the level of stress hormone cortisol in serum, arterial blood pressure and arterial pulse, and to compare the effectiveness of the usage of lidocaine with adrenalin in comparison with lidocaine without adrenalin during the tooth extraction. Patients and methods: This clinical research includes patients with indication of tooth extraction divided in hypertensive and normotensive patients. Results: There is no important statistical distinction between groups, for the cortisol levels before, during and after tooth extraction regardless of the type of anesthetic used, while we registered higher values of systolic and diastolic values at hypertensive patients, regardless of the type of anesthetic Conclusion: There is significant systolic and diastolic blood pressure rise in both groups of patients hypertensive and normotensive patients, (regardless of anesthetic used with or without vasoconstrictor), who underwent tooth extraction. The special emphasize is attributed to hypertensive patients where these changes are more significant. As per cortisol level and pulse rate, our results indicate no significant statistical difference in between groups.

BACKGROUND: Oncocytoma or oxyphilic adenoma is uncommon salivary gland tumour, occurs predominantly in the in patients older than 60 years of age. Clinically oncocytoma resemble other salivary tumours while histology is typically consisting of oncocytes with many hyperplastic mitochondria. It usually occurs in the parotid gland. Because the features of oncocytoma are similar to those of other benign and low-grade malignant salivary tumours, clinical diagnosis is often challenging.CASE PRESENTATION: This report presents the pathologic and imaging findings of an oncocytoma arising in the deep lobe of the right parotid gland in a 74-year-old male. Oncocytoma was diagnosed on the basis of histological, magnetic resonance imaging (MRI), scintigraphic findings and fine needle aspiration cytology (FNAC).CONCLUSION: This case was unique because in the literature there are few articles about the rare presentation and deep lobe location of this type of parotid oncocytoma.

The advancements in epigenetics of oral squamous cell carcinoma (OSCC), are made in regard to DNA hypermethylation of MGMT, DAPK, ECAD (E-cadherin) and p16, as an important component of oral carcinogenesis and new potential biomarkers in molecular diagnostic strategies. The objective of the study was to evaluate the methylation status of the proposed genes and their possible role in the tumor genesis and diagnosis of OSCC. Materials and methods: From sixty surgically treated and molecularly analyzed patients, we obtained three groups of bioptical materials: tumor, normal contralateral and healthy tissues. Comparison of the frequencies of DNA methylation for all transcripts was utilized to validated their potential role in the cancerogenesis and detection of OSCC. Results: The most often methylated genes in the tumor samples were ECAD, MGMT, DAPK followed by p16 genes (90% vs 75% vs 75% vs 52,5%), respectively. We observed frequent methylated genes in contralateral mucosa and consistently unmethylated- 0% in healthy samples. ECAD methylated genes showed the highest sensitivity for diagnosing OSCC in tumor and contralateral tissues (90% and 89,7% respectively, with a specificity of 100%). Conclusion: ECAD and MGMT have tumor-specific signatures and can be considered as potential noninvasive diagnostic biomarkers in OSCC.

The research was aimed at obtaining copper-enriched cells of Saccharomyces cerevisiae , developing an efficient metal delivery system for human nutrition and therapeutic treatments. To produce glutathione (GSH)–copper conjugates, four samples of commercial baker's yeasts ( S. cerevisiae ) were employed in biotransformation trials with copper acetate. GSH production was found variable, ranging from 0.15 to 1.2% cell dw. Trials carried out with copper-adapted cells resulted in GSH and conjugate limited biosynthesis. The highest copper conjugate level (1.7% dw) was instead obtained in experiments set up by employing not adapted cells. Cell disruption by sonication and subsequent analysis by atomic absorption evidenced the presence of very interesting amount of intracellular copper, 1.8 ± 0.2 mg/g cell dw (i.e. 28 μmol copper/g cell dw). The proposed procedure can be considered an interesting opportunity to further the range of application of yeast cells culture, for the set up of a copper delivery system for therapeutic treatments.

The construction of the map of the landscape in the environmental hinterland of Expo 2015: new general plan of the Groane - The Groane: the only narrow passage that is free between Milan, Varese and Como, one of the most congested places in the country and nevertheless one of those that is most supported with Gdp and the productivity of all Lombardy, its commuters and Vat numbers. It is a sort of 15 km tunnel buffeted between the exceptional processes of urbanisation which have inevitably (and irreparably) impaired its valuable land resources, except of course for the protected area of the Groane Park, which has recently adopted our new general plan for the provincial ‘General Co-ordination Plan’. Its contiguity with the cumbersome attraction of the Expo 2015 - which could have generated dangerous settlement repercussions even for the park - has not, however, caused a murmur to be raised, either because of the economic crisis or, more probably, because the environmental importance of the Groane Park is considered today as value added to be preserved in all ways. In fact there has been absolutely no assault on the land of the park for residential purposes at all. On the contrary, 60% of the areas subject to the new plan increase the perimeter of protection (11% more than at present) on the basis of an initial landscape map which is presented in the article with the construction procedures and the relative methodology.

Rapamycin induces chromosome malsegregation in mammalian cell lines and yeast. Previous studies indicate that the function impaired in ataxia-telangiectasia (A-T) patients is necessary for both the growth inhibition and the chromosome malsegregation induced by rapamycin, and that treating the non-tumorigenic Chinese hamster cell line CHEF/18 with rapamycin results in supernumerary centrosomes and multipolar spindles. In this paper we report that lymphoblastoid cell lines established from A-T patients as well as hamster A-T-like cells are more resistant to rapamycin than the respective normal cell lines. Two cell lines derived from Nijmegen Breakage Syndrome (NBS) patients, who have clinical symptoms similar to those of A-T but a different molecular defect, were not resistant to rapamycin. Both A-T lymphoblastoid cells and A-T-like fibroblasts had giant centrosomes formed by more than two areas of γ-tubulin-reacting material. Such giant centrosomes were also observed in CHEF/18 cells after prolonged treatment with rapamycin. Formation of giant centrosomes, possibly due to the coalescence of supernumerary centrosomes, was associated with increased aneuploidy in treated cells. Expression analysis of cell-cycle regulatory genes in rapamycin-treated human lymphoblastoid cells indicated that rapamycin decreased the expression of the tumor suppressor gene GADD45. The levels of RB, p21 and p53 mRNA were also decreased, although to a lesser extent. As rapamycin is often used as an immunosuppressant in pediatric transplant patients, these data indicate that caution should be taken, especially when the drug is given for prolonged periods of time. Environ. Mol. Mutagen., 2005. © 2005 Wiley-Liss, Inc.

We report on the implementation of a confocal microscopy system based on a 2.9 THz quantum cascade laser source. Lateral and axial resolutions better than 70 mu m and 400 mu m, respectively, are achieved, with a large contrast enhancement compared to the non-confocal arrangement. The capability of resolving overlapping objects lying on different longitudinal planes is also clearly demonstrated.

The aim of this study is to determine the prognostic value of available clinical and histological parameters to predict subclinical nodal metastases and survival. For that purpose we used a modification of the Jakobsson [9] grading system proposed by Richard W. Nason [12] and applied it to a series of patients with oral carcinoma. Correlation between histological parameters and cervical lymph node metastasis were made and the chi-square test showed a strong significant association between mode of invasion, depth of tumor invasion, vascular invasion and cervical metastases. Analysis of overall survival showed that overall survival strongly correlates with cervical node metastases (p=0.0004). We found that overall survival correlates with vascular invasion (p=0.005) and also overall survival correlates with tumour depth (p=0.001). There is a strong relationship between malignancy score and survival (p=0.00001) and a high malignancy score is associated with poor prognoses.a total 32 with over 14 points of malignancy score, 25 (78.1%) died and 7 (21.9%) are alive. Analysis of the risk factors for subclinical cervical metastases in patients with oral cancer is important for predicting prognosis and achieving a high survival rate.

The assignment of admissible degrees of change to the building stock of an historic centre is one of the most important factors to be taken into account when developing an urban plan, because it establishes the possibilities of making changes to an existing urban fabric, or not; and since urban cores formed in ancient times contain the most significant concentrations of high-quality historic/architectural heritage, it is necessary to identify a way of specifically breaking down the changes to each building that are possible. In the case of Como, analysis of the morphological and socioeconomic characteristics of the fabric of the walled town and its extramural historic villages, and then the application of multivariate geostatistics, produced one such classification of this context in the form of six possible scenarios for change, of which the most significant was selected as the most effective for setting in train a profound upgrading of Como's built fabric, which is currently in a state of advanced degradation.

Myotonic dystrophy is a multisystemic disorder, due to a CTG triplet expansion at the 3'UTR of the DM1 gene encoding for myotonic dystrophy protein kinase. Recent studies indicate that decreased DMPK levels could account for part of the symptoms suggesting a role of this protein in skeletal muscle differentiation. To investigate this aspect, polyclonal antibodies were raised against two peptides of the catalytic domain and against the human full-length DMPK (DMFL). In western blots, anti-hDMFL antibody was able to detect low amounts of purified human recombinant protein and recognized the splicing isoforms in heart and stomach of overexpressing mice. In human muscle extracts, this antibody specifically recognized a protein of apparent molecular weight of 85 kDa and it specifically stained neuromuscular junctions in skeletal muscle sections. In contrast, both anti-peptide antibodies demonstrated low specificity for either denatured or native DMPK, suggesting that these two epitopes are probably cryptic sites. Using anti-hDMFL, the expression and localization of DMPK was studied in human skeletal muscle cells (SkMC). Western blot analysis indicated that the antibody recognizes a main protein of apparent MW of 75 kDa, which appears to be expressed during differentiation into myotubes. Immunolocalization showed low levels of DMPK in the cytoplasm of undifferentiated cells; during differentiation the staining became more intense and was localized to the terminal part of the cells, suggesting that DMPK might have a role in cell elongation and fusion.

The toxicity of two conjugates containing ribosome-inactivating proteins (RIPs, i.e. saporin and ricin-A chain x-linked to transferrin) has been measured on a prostatic cancer line (PC3) naturally overexpressing the transferrin receptor, in the presence of monensin and chloroquine. This paper investigates whether the increased toxicity of Tf-RIPs induced by monensin and chloroquine may be due to alterations of the normal endocytotic pathway of the complexes mediated by the transferrin receptor. Monensin, besides inducing alkalinization of normally acid intracellular compartments, causes an accumulation of the receptor-bound Tf-RIP in a perinuclear region contiguous to the cisternae of the trans-Golgi network. Chloroquine, though increasing the intracellular pH, seems not to modify the endocytotic pathway of these chimeric molecules. We believe that the enhanced toxicity of the Tf-RIPs may be related to intracellular alkalinization (i.e., endosomal or lysosomal pH) rather than to the effects on the recycling of transferrin receptor-bound toxins. We conclude that the efficacy of chimeric toxins may be modulated not only by the carrier used for their engineering but also by addition of drugs able to influence the stability and activation of the toxins inside the cell.

Vasopressin is the key regulator of water homeostasis in vertebrates. Central to its antidiuretic action in mammals is the redistribution of the water channel aquaporin 2 (AQP2) from intracellular vesicles to the apical membrane of kidney epithelial cells, an event initiated by an increase in cAMP and activation of protein kinase A. The subsequent steps of the signaling cascade are not known. To identify proteins involved in the AQP2 shuttle we exploited a recently developed cell line (CD8) derived from the rabbit cortical collecting duct and stably transfected with rat AQP2 cDNA. Treatment of CD8 cells with pertussis toxin (PTX) inhibited both the vasopressin-induced increase in water permeability and the redistribution of AQP2 from an intracellular compartment to the apical membrane. ADP-ribosylation studies revealed the presence of at least two major PTX substrates. Correspondingly, two alpha subunits of PTX-sensitive G proteins, Galphai2 and Galphai3, were identified by Western blotting. Introduction of a synthetic peptide corresponding to the C terminus of the Gi3 alpha subunit into permeabilized CD8 cells efficiently inhibited the cAMP-induced AQP2 translocation; a peptide corresponding to the alpha subunits of Gi1/2 was much less potent. Thus a member of the Gi family, most likely Gi3, is involved in the cAMP-triggered targeting of AQP2-bearing vesicles to the apical membrane of kidney epithelial cells.

The intracellular dynamics of fluorescent conjugates of the toxic lectin ricin was followed by video fluorescence microscopy on living CHO cells, demonstrating that the ricin heterodimer and its isolated B chain, after binding to the plasma membrane receptors, migrate to and accumulate in the Golgi apparatus following internalization. A ricin derivative labelled with fluorescein on the A chain and rhodamine on the B chain did not display significant splitting of the A-B heterodimer during translocation of the toxin to the Golgi; this novel finding provides support for the hypothesis that further processing of ricin takes place in this cellular compartment.

State of the art in optical microscopyOptical microscopy is still and increasingly one of the most valuable tools in biological investigation. In particular, confocal microscopies are capable of achieving best performances in the study of three-dimensional fluorescent and reflecting specimens. Nevertheless, current techniques adopted in confocal microscopy present some drawbacks and limitations that stimulate to devise and set-up further techniques, suited to a wider range of applications.Advantages of confocal microscopes mainly correspond to an improved spatial resolution, especially in the axial direction. Depending on the narrow-field scanning approach used, there are two main forms of confocal microscopes: single-point (SP) and multi-point (MP) ones. Unfortunately, SP confocal microscopes require the use of lasers as illumination sources with consequent high costs and scarce spectral flexibility. Moreover, specimen photo-damage due to relatively high instantaneous irradiation doses involved, can often limit their investigative capabilities. On the other hand, proposed MP confocal microscopes still rely on the revolving-disk approach and exhibit a relatively low luminous efficiency, substantial constructional complexity, and limited contrast in the study of thick fluorescent objects.

Using fluorescence microscopy a green fluorescent spot was observed in both green and bleached Euglena gracilis cells. Shape, dimensions and location indicate this fluorescent organelle to be the paraflagellar body (PFB). The spectroscopic characteristics strongly suggest the flavin nature of the luminescent centers, while their ordered arrangement can be deduced from the polarization of the emitted light.

The spectroscopic properties of stigma inside green and dark-grown cells and of isolated stigma globules have been studied by means of a microspectrophotometer build in the Laboratory. On the base of these results and of the analysis of the absorption spectra of a stigma suspension, cell suspension and the cell methanolic extracts, it can be inferred that pigments localized in the stigma are free carotenoids which are not closely packed and do not show an ordered arrangement. Furthermore, the efficiency of the stigma as shading device is duscussed.

Red blood cells from patients homozygotes for hemoglobin S (HbS) have been studied using a computer-controlled microspectrophotometer, which allows measurements of spectra and dynamics to be undertaken in a single erythrocyte. Complete photodissociation of HbCO results in polymerization of intracellular deoxyhemoglobin S and deformation of the cell. This is associated with a delayed optical change, which, for the same cell, was found to be highly reproducible between repeated cycles of sickling. Comparison of photographic records and absorbance time courses indicates that an erythrocyte, once having undergone a photochemically induced sickling event, always deforms along the same axis during subsequent cycles. This behaviour implies that the cell retains a ‘memory’ of its previous cycle(s) possibly via slow relaxations of the membrane. In addition, rebinding of CO to intracellular hemoglobin was found to be slower if measured after deformation of the cell, with possible important implications for the pathological mechanism of sickling.

An experimental setup that provides the measurement of the spatial variation of the responsiveness of a single photodiode belonging to a linear metal-oxide-semiconductor (MOS) photoarray is described. An optical microscanning technique has been employed to sequentially illuminate a small area of a single photoelement in order to achieve the mapping of its spatial responsiveness. Experimental results for a single pixel are graphically presented.

Microspectrophotometry has been applied to single red blood cells to reinvestigate the linked processes of diffusion of gases inside the erythrocyte and their combination with hemoglobin. The experiments took advantage of the photosensitivity of the cabron monoxide derivative of hemoglobin, which allows ligand release from the CO-saturated red cells under strong illumination and recombination when the light is switched off. The photochemical method was also used to study the kinetics of sickling on ligand removal in single erythrocytes of Hb S carriers. The results give new information on the mechanism of the sickling process.

A fluorescent derivative of a chimeric toxin between human pro-urokinase and the plant ribosome-inactivating protein saporin (p-uPA-Sap(TRITC)), has been prepared in order to study the endocytosis of this potentially antimetastatic conjugate in the murine model cell line LB6 clone19 (Cl19) transfected with the human urokinase receptor gene. The physiological internalization of urokinase-inhibitor complexes is triggered by the interaction of plasminogen inhibitors (PAIs) with receptors belonging to the low density lipoprotein-related receptor protein (LRP) family, and involves a macro-quaternary structure including uPAR, LRP, and PAIs. However, in contrast to this mechanism, we observed a two-step process: first, the urokinase receptor (uPAR) acts as the anchoring factor on the plasma membrane; subsequently, LRP acts as the endocytic trigger. Once the chimera is bound to the plasma membrane by interaction with uPAR, we suggest that a possible exchange may occur to transfer the toxin to LRP via the saporin moiety and begin the internalization. So an unusual endocytic process is described, where the toxin enters the cell via a receptor different from that used to bind the plasma membrane.

Human transferrin (Tf) and saporin-6 (Sap), a ribosome inactivating protein from Saponaria officinalis, were chemically conjugated: the reaction generated two chimeras (called Tf-Sap) that proved to be cytotoxic to HepG2 cells. Electrophoretic and chromatographic analysis revealed that the two conjugates contained saporin and Tf in a 2:1 or 1:1 molar ratio (140 and 110 KDa, respectively). Free saporin is essentially nontoxic, whereas Tf-Sap efficiently kills HepG2 cells, although its ID50 (= 6 nM) is 1000-fold greater than that of ricin. Intracellular transport of these toxins was followed by in vivo fluorescence video microscopy, preparing the conjugates starting from rhodamine isothiocyanate-labeled saporin. Image analysis of living HepG2 cells exposed to fluorescent Tf-Sap revealed that the endocytotic pathway involving passage through secondary endosomes is dictated by Tf and is different from that of ricin (the dimeric toxin from Ricinus communis), which is delivered to the Golgi apparatus, the probable site of activation. We discuss whether differences in toxicity between ricin and Tf-Sap can be attributed to the different mechanisms of transport and activation.