Fate of micelles and quantum dots in cells.

Micelles and quantum dots have been used as experimental drug delivery systems and imaging tools both in vitro and in vivo. Investigations of their fate at the subcellular level require different surface-core modifications. Among the most common modifications are those with fluorescent probes, dense-core metals or radionucleids. Cellular fate of several fluorescent probes incorporated into poly(caprolactone)-b-copolymer micelles (PCL-b-PEO) was followed by confocal microscopy, and colloidal gold incorporated in poly 4-vinyl pyridine-PEO micelles were developed to explore micelle fate by electron microscopy. More recently, we have examined quantum dots (QDs) as the next-generation-labels for cells and nanoparticulate drug carriers amenable both to confocal and electron microscopic analyses. Effects of QDs at the cellular and subcellular levels and their integrity were studied. Results from different studies suggest that size, charge and surface manipulations of QDs may play a role in their subcellular distribution. Examples of pharmacological agents incorporated into block copolymer micelles, administered or attached to QD surfaces show how the final biological outcome (e.g. cell death, proliferation or differentiation) depends on physical properties of these nanoparticles.     

Gamma-tubulin in chicken erythrocytes: changes in localization during cell differentiation and characterization of cytoplasmic complexes.

The mechanism of marginal band (MB) formation in differentiating erythroid cells is not fully understood, and the proteins involved in nucleation of MB microtubules are largely unknown. To gain insights into the function of gamma-tubulin in MB formation, we have followed its distribution in developing chicken erythrocytes and characterized soluble forms of the protein. In early stages of erythroid cells differentiation, gamma-tubulin was present in microtubule-organizing centers, mitotic spindles, as well as on MB. Its subcellular localization changed in the course of differentiation, and in postnatal peripheral erythrocytes gamma-tubulin was found only in soluble forms. After cold-induced depolymerization gamma-tubulin in erythroid cells formed large clusters that were not observed in matured cells, and re-growth experiments demonstrated that gamma-tubulin was not present in distinct nucleation structures at the cell periphery. Soluble gamma-tubulin formed complexes of various size and large complexes were prone to dissociation in the presence of high salt concentration. Interaction of gamma-tubulin with tubulin dimers was revealed by precipitation experiments. gamma-Tubulin occurred in multiple charge variants whose number increased in the course of erythrocyte differentiation and corresponded with decreased binding to MB. The presented data demonstrate for the first time that gamma-tubulin is a substrate for developmentally regulated posttranslational modifications and that the binding properties of gamma-tubulin or its complexes change during differentiation events.     

Immunological consequence of renal transplantation and immunosuppression. I. Alterations in human natural killer-cell activity

The role and activity of natural killer (NK) cells following renal transplantation remain unknown. To monitor NK activity, a⁵¹Cr release of K-562 targets in prednisone-and azathioprine-treated patients receiving renal allografts was utilized. In 18 patients in whom NK activity was measured prior to and after transplantation, a significant diminution in NK activity within 3 weeks following transplantation was demonstrated compared to pretransplant values (34.71 vs 12.20%, respectively;P<0.001). In 11 subjects who had NK activity assayed at various intervals after transplantation but not prior to allografting, mean NK values were markedly lower (mean, 14.2%) than those of normal volunteers or patients maintained on hemodialysis (P<0.001). The latter two control groups demonstrated no difference (P = NS) in mean NK activity (39.46 vs 35.82%, respectively). In 5 of the 29 patients evaluated with good long-term graft function (mean, 2.7 years), restitution of normal NK activity was demonstrated. In two patients with bacterial infections, NK activity increased from 39.29 to 51.7% and from 13.54 to 20.00%. After infection, these values were 35.3% in the former and 3.39% in the latter. Viral infection did not appear to affect NK activity significantly. NK activity was increased in only one of seven patients with documented rejection episodes. In three of such patients, NK activity declined significantly following pulse methylprednisolone therapy. These results indicate that (1) NK-cell activity significantly decreases immediately after transplantation, probably as a result of immunosuppressive therapy; (2) NK activity does not appear to be stimulated by the alloreactive rejection process; (3) NK activity may be augmented in the course of bacterial but not viral infections; and (4) long-term allograft survival may be associated with a restoration of NK-cell levels in certain recipients.     

Partial purification and characterization of anhydrotetracycline oxygenase of Streptomyces aureofaciens

Anhydrotetracycline oxygenase was purified both by affinity chromatography and by hydrophobic interaction chromatography. Molecular weight of anhydrotetracycline oxygenase was determined to be 115,000 by Sephadex G-200 gel filtration. Using preparative isoelectric focusing the isoelectric point of the enzyme was estimated to be 5.3. The enzyme showed a sensitivity to thiol-specific inhibitors. During the hydrophobic interaction purification step, the activity dropped considerably. Reactivation occurred when a heat treated crude extract was added to the reaction mixture.     

Structure-intestinal transport and structure-metabolism correlations of some potential cancerostatic pyrimidine nucleosides in isolated rat jejunum

Summary Both transport and biotransformation processes for a series of pyrimidine nucleobases, ribonucleosides, 2-deoxyribonucleosides, and acetyl and 5-substituted derivatives of the cancerostatic agent araC were studied in the isolated everted rat jejunum with a continuous perfusion technique. Metabolic alterations during penetration were assessed by HPLC. 5-Halogeno and 5-deoxy derivatives of cytosine nucleosides exhibited higher transport rates and higher stability towards the deamination reaction than did unsubstituted derivatives. Octanol-buffer partition coefficients were estimated for the study compounds, and fragmental constants for the sugar moieties of nucleosides were assessed. With the present study compounds there was no correlation between lipophilicity and transport rate, as previously reported, but there was a correlation between lipophilicity and metabolic alteration of araC derivatives (r=0.99, n=5).     

Problems associated with sterilization using ethylene oxide. Residues in treated materials

The paper deals with problems associated with reduction of undesirable effects of ethylene oxide in polymers in medical devices on the patient's health. The authors explain the need of careful elaboration and validation of the sterilization and aeration process incl assessment of ethylene oxide (EO) residues. The authors investigated the effect of the type of material and conditions of sterilization and aeration on the assessed EO concentration. For research of the behaviour of different polymers in the sterilization process model sterilizations of actual items of medical devices with a known composition proved more suitable than assessment in medical devices from medical institutions. The main conclusions of the investigation were a classification of polymers into those suitable and unsuitable for sterilization or resterilization, and attention was also drawn to poor reproducibility of results in old sterilizers, in particular those lacking effective aeration in aerators.     

Mutation analysis of RyR2 gene in patients after arrhythmic storm

IntroductionMutations of RyR2 gene encoding calcium channel of sarcoplazmatic reticulum are the cause of congenital catecholaminergic polymorphic ventricular tachycardia. The aim of this study was to test the hypothesis that RyR2 variants can increase occurrence of malignant arrhythmias in patients with structural heart diseases.MethodsThe investigated group consisted of 36 patients with structural heart diseases with ICD implanted who suffered arrhythmic storm. In the control group there were 141 patients with coronary artery disease who were hospitalized at our department owing to an acute coronary event and they were alive at least 3 years after the index event. Thus, they could be considered as a group with a low risk of sudden cardiac death. In all of them mutation analysis of RyR2 gene was performed.ResultsWe detected 16 different sequence changes of RyR2 gene in both groups. None of the found nucleotide polymorphisms led to amino acid changes, were located close to splice sites or had any similarity to known splicing enhancer motifs. The occurrence of these variants was not different in both groups.ConclusionsThe prevalence of RyR2 gene variants was not different in cases versus controls suggesting a limited role of this gene in the arrhythmogenesis in structural heart disease patients.     

Tumoricidal properties of rat peritoneal macrophages activated with various activators depend on nitrogen oxid synthesis.

Cytolytic activity of mineral oil elicited rat peritoneal macrophages activated by lipopolysaccharide (LPS) and/or rIFN-gamma, rIL-2, Zymosan and PMA (4-beta-phorbol-12-beta-myristate 13-alpha-acetate) was detected in the presence of various concentrations of L-arginine. This paralleled the NO2- production in the presence, but not in the absence, of L-arginine. Significant amount of NO2- was detected in the peritoneal macrophages cultured with 0.4 mmol of L-arginine 8 days and the last 24 h with LPS at a concentration of 1 microgram/ml. No significant differences were found between activated peritoneal macrophages obtained from normal (healthy) and/or from tumor bearing rats to induce tumoricidal activity and NO2- production under the same experimental conditions. The results showed that the major cytolytic mechanism against BP6-Tu2 and U 937 tumor cell lines is L-arginine-dependent nitrogen oxide synthesis of activated rat peritoneal macrophages.     

Radiosurgery with the Leksell gamma knife in the treatment of solitary brain metastasis--5-year results

During five years (1992-1997) 215 patients with solitary brain metastases were treated in the Department of Stereotactic and Radiation Neurosurgery (117 men and 98 women). The most frequent first neurological symptoms were: headache, hemiparesis, seizure, mental and behavioural disturbances. Location of lesion was supratentorial in 163 patients (75.8%) and infratentorial in 52 cases (24.2%). Treatment results: complete and partial regression in 175 patients (81.4%), stabilisation in 30 of patients (14.0%), local progression in 10 patients (4.6%). Local recurrence after initially effective treatment was observed in 11 pts (5.1%). The severity of neurological symptoms was measured by RTOG 4 grades scoring system. The acute and late toxicity was evaluated by SOMA scale (RTOG--Radiation Therapy Oncology Group): grading 0-5. We observed acute toxicity (score 3 and 4) in 20 patients (9.3%) and late (score 3 and 4) in 9 patients (4.2%). Minimal follow up for all patients was 10 months. Median survival for 159 dead patients was 7 months (ranged from 2 to 35 months), median survival for surviving 56 pts was 15.5 months (ranged from 10 to 56 months). One-year survival was 39.5% of all patients.