Heterogeneous expression of immunotherapy candidate proteins gp100, MART-1, and tyrosinase in human melanoma cell lines and in human melanocytic lesions

In recent years, it has become evident that T cells can recognize peptides of melanocytic lineage antigens such as gp100, MART-1, and tyrosinase at the tumor cell surface and can subsequently destroy these cells. It is thus feasible to develop immunotherapeutic approaches based on the melanocytic marker profiles of melanoma cells. One of the predictors of the success rate of such a treatment is the extent of positive (target) tumor cells within the lesions of the patient. First, we investigated the presence of these three proteins in 18 human melanoma cell lines using RT-PCR and immunohistochemistry. In 11 cell lines, mRNA and protein of all three markers could be detected; in one cell line, only two markers were present, and six melanoma cell lines showed no evidence for these markers. Secondly, we stained frozen sections of 105 human melanocytic lesions, 13 common nevocellular nevi, 13 atypical nevi, 13 early primary melanomas (Breslow < 1.5 mm), 25 advanced primary melanomas (aPM; Breslow > or =1.5 mm), and 41 melanoma metastases (MM) with antibodies against glycoprotein 100, melanoma antigen recognized by T cells, and tyrosinase. In addition, we used the 3,4-dihydroxy-L-phenylalanine reaction to detect tyrosinase enzyme activity as a confirmation of the tyrosinase immunohistochemical results in a subset of the lesions. In the benign lesions, glycoprotein 100 was more prominently expressed in epidermal melanocytes, whereas melanoma antigen recognized by T cells was encountered in all or nearly all dermal melanocytes in all nevocellular nevi and atypical nevus lesions. Tyrosinase was found in a lower percentage of melanocytes, both in the epidermis and in the dermis within these lesions. With regard to heterogeneity of staining within the malignant lesions, we found that 54% (early primary melanomas), 48% (aPMs), and 56% (MM) of the lesions stained within the same staining category for all three proteins studied. Approximately 17% of the aPM and MM lesions did not show positive tumor cells for any of the three proteins. We conclude that a subgroup of patients with high expression should be selected for immunotherapeutic treatment approaches based on the presence of these proteins.     

A novel mtDNA deletion in an infant with Pearson syndrome

Pearson syndrome is a multisystem mitochondrial disorder of infancy that is associated with deletions in the mitochondrial DNA (mtDNA) genome. We report a study on a male infant with Pearson syndrome. Assessment of oxidative phosphorylation activity indicated combined respiratory-chain defects in muscle, liver and fibroblasts; in particular, activity of complex I was reduced. Analysis of the patient's mtDNA identified a novel heteroplasmic 2.461 kb deletion, present at levels greater than 50% of the total mtDNA in the tissues examined. The deletion spanned nucleotides 10368 to 12828 and was flanked by a 3 bp GCC direct repeat sequence. Gene sequences affected are subunits 3, 4, 4L and 5 of complex I, and tRNAs for arginine, histidine, serine and leucine. Our findings correlate with the multiorgan involvement observed in Pearson syndrome.     

Relationship of natural incidence and radiosensitivity for bone cancer in dogs

A comparison of the risk coefficients for 239Pu- or 226Ra-induced bone cancer in two canine breeds, one with a relatively low (beagle) and the other with a very high (St. Bernard) natural incidence, indicated only slightly higher risk in the giant breed. The differences in risk for skeletal malignancy in 239Pu and 226Ra dogs were nonsignificant (p > 0.05). Likewise, the values of the 239Pu:226Ra "toxicity ratios" for these respective breeds, using bone cancer as the endpoint, were not significantly different at the 0.05 level. The anatomical distribution of the radiation-induced bone tumors tended to be a function of both the bone mass and the skeletal distribution of the radionuclide, not the site of predilection for naturally occurring bone neoplasia. Although the etiology of the higher natural incidence of bone cancer in the St. Bernard was not determined, several possible factors, including a higher osteoblastic activity level in the St. Bernards, are presented. These data suggest that making extrapolations of radiation-induced bone cancer risk from animals to humans is valid.     

Several properties of low-molecular weight tetanus antitoxin

Following intravenous injection of tetanus antitoxin, obtained by tryptic digestion of the horse immunoglobulin "Diaferm-3", purification and concentration of active fragments, the antitoxin was eliminated from the rabbit organism three times more rapidly than after the injection of the original "Diaferm-3" antitoxin. After injection of the split antitoxin its urinary excretion lasted up to 6 days, whereas following injection of the "Diaferm-3" antitoxin it was excreted for up to 19 days; in the first case considerably less antitoxin was excreted than in the second one (2 and 3.5%, respectively). In both cases in the antitoxin excreted with urine represented monovalent. Fab'-fragments, producing a delay in precipitation in the cross reaction in agar gel between the tetanus toxoid and the tetanus antiserum. Fab'-fragment obtained by the mentioned method possessed anaphylactogenic properties.     

Aggregation of hsp70 and hsc70 in vivo is distinct and temperature-dependent and their chaperone function is directly related to non-aggregated forms

We used non-denaturing gradient analysis of cell extracts before and after heat treatment of the cells and showed that hsp70 and hsc70 aggregate in vivo in a temperature-dependent fashion. Their aggregation profiles were found to be clearly distinguishable and sensitive to ATP depletion. Pore exclusion limit electrophoresis showed that these two proteins are mainly found in autoaggregated forms including dimers, trimers and oligomers. The addition of denatured luciferase to the cell extracts reversed the aggregation of both proteins towards their non-aggregated forms. Immunoprecipitation and Western-blot analysis showed that the non-aggregated form is the only one bound to denatured luciferase. Our results suggest that aggregated hsp70 and hsc70 represent predominantly self-associated molecules unable to exert chaperone activity. The cochaperone hsp40 was also found to be aggregated and, on addition of denatured luciferase, its aggregation was reversed to a non-aggregated state. Immunoprecipitation analysis indicated that hsp40 forms a complex with the non-aggregated form of hsc70 and denatured luciferase. These results confirm previous in vitro studies and support the suggestion that in vivo cytosolic hsp70 and hsc70 exist mainly in an oligomer-monomer equilibrium which is dependent on the environmental temperature, the levels of ATP and the presence of denatured proteins.     

Endoscopic features of the columnar-lined esophagus

Electrophysiological characterization of neurons within the rat subiculum was carried out with intracellular recordings in an in vitro slice preparation. Subicular neurons responded to threshold pulses of depolarizing current delivered at a resting membrane potential (RMP) of 45.7+/-5.8 mV (mean+/-SD, n=85) with an initial burst of three to five fast action potentials that rode on a depolarizing envelope and was terminated by an afterhyperpolarization (burst AHP) (duration 113+/-35 ms; peak amplitude 2.7+/-0.6 mV, n=10). Tonic firing replaced the bursting mode at membrane potential less negative than -55 mV. Suprathreshold depolarizing pulses evoked at RMP both an initial burst and successive tonic firing. Intracellular staining with biocytin showed morphological features typical of pyramidal cells (n=8). The relationship between frequency of repetitive firing and injected current (f-I) revealed that the burst firing frequency (250-300 Hz) was only slightly influenced by the amount of injected current. By contrast, the f-I curve of the tonic firing phase depended upon current intensity: it displayed an initial segment that increased at first linearly and then turned into a plateau for both the early and the late inter-spike intervals. The frequency of the tonic firing declined only slightly with time, thus suggesting a lack of adaptation. During tonic firing, each single action potential was followed by a fast AHP and a depolarizing afterpotential. Termination of repetitive firing was followed by an AHP (spike-train AHP; duration 223+/-101 ms, peak amplitude 5.6+/-2.4 mV, n=17). Fast spike-train and burst AHPs were reduced by bath application of the Ca2+-channel blockers Co2+ (2 mM) and Cd2+ (1 mM) (n=8), thus suggesting the participation of Ca2+-dependent K+ conductances in these AHPs. Subicular bursting neurons generated persistent, subthreshold voltage oscillations at 5.3+/-1 Hz (n=20) during steady depolarization positive to -60 mV; at values positive to -55 mV, the oscillatory activity could trigger clusters of single action potentials with a periodicity of 0.9-2 Hz. Oscillations were not prevented by application of excitatory amino acid receptor and GABA(A) receptor antagonists (n=5), Ca2+-channel blockers (n=5), or Cs+ (3 mM; n=4), but were abolished by the Na+-channel blocker tetrodotoxin (1 microM; n=6). Our findings demonstrate that pyramidal-like subicular neurons generate both bursting and non-adapting tonic firing, depending upon their membrane potential. These neurons also display oscillatory activity in the range of theta frequency that depends on the activation of a voltage-gated Na+ conductance. These electrophysiological properties may play a role in the process of signals arising from the hippocampal formation before being funnelled towards other limbic structures.     

Evidence for a role of a perferryl-oxygen complex, FeO3+, in the N-oxygenation of amines by cytochrome P450 enzymes

Two epithelial tumour lines, HeLa and KB, were treated with okadaic acid and calyculin A, specific inhibitors of Ser/Thr phosphatases (PP), esp. PP1 and PP2A. Morphological criteria, analysis of DNA fragmentation and studies of membrane integrity revealed that both agents concentration- and time-dependently induced apoptosis at nanomolar concentrations which in these cells was associated with the stimulation of a transglutaminase activity. Since a non-functional derivative of okadaic acid did not affect cell viability apoptosis was apparently related to the inhibition of PP1 and PP2A. Membrane damage marker activity was delayed by at least 24 h when compared to nuclear alterations.     

Design,characterisation and drug release study of polymeric,drug‐eluting single layer thin films on the surface of intraocular lenses

To design, develop and study a novel drug delivery system for intraocular applications. The spin coating technique was applied to develop a polymeric, drug‐eluting thin film consisting of a blend of organic polymers [poly (D, L lactide coglycolide) lactide: glycolide 75: 25, PLGA and polycaprolactone, PCL] and dexamethasone on the surface of intraocular lenses (IOLs). The initial durability of the IOLs during spinning was assessed. Information about the structural and optical properties of the modified IOLs was extracted using atomic force microscopy, scanning electron microscopy and spectroscopic ellipsometry. A drug release study was conducted for 8 weeks. The IOLs were durable in spinning speeds higher than the ones used to develop thin films. Single‐layer thin films were successfully developed on the optics and the haptics of the lenses. The films formed nanopores with encapsulated aggregates of dexamethasone. The spectroscopic ellipsometry showed an acceptable optical transparency of the lenses regardless of the deposition of the drug‐eluting films on their surface. The drug release study demonstrated gradual dexamethasone release over the selected period. In conclusion, the novel drug‐eluting IOL system exhibited desired properties regarding its transparency and drug release rate. Further research is necessary to assess their suitability as an intraocular drug delivery system.Inspec keywords: ellipsometry, encapsulation, nanoporous materials, spin coating, polymer blends, biodegradable materials, surface treatment, polymer films, atomic force microscopy, transparency, nanomedicine, durability, scanning electron microscopy, drug delivery systemsOther keywords: drug release, intraocular lenses, intraocular applications, spin coating technique, modified IOLs, spectroscopic ellipsometry, dexamethasone release, transparency, drug release rate, intraocular drug delivery system, drug‐eluting IOL system, polymeric drug‐eluting single‐layer thin films, optical properties, structural properties     

The synchronization principle in modelling of binding and attention

Recent atomic 3-D reconstructions of the acto-myosin interface suggest that electrostatic interactions are important in the initial phase of cross-bridge formation. Earlier biochemical studies had also given strong evidence for the ionic strength dependence of this step in the cross-bridge cycle. We have probed these interactions by altering the ionic strength (Gamma/2) of the medium mainly with K+, imidazole+ and EGTA2- to vary charge shielding. We examined the effect of ionic strength on the kinetics of rigor development at low Ca2+ (experimental temperature 18-22 degrees C) in chemically skinned single fast-twitch fibres of mouse extensor digitorum longus (EDL) muscle. On average the delay before rigor onset was 10 times longer, the maximum rate of rigor tension development was 10 times slower, the steady-state rigor tension was 3 times lower and the in-phase stiffness was 2 times lower at high (230 mM) compared to low (60 mM) ionic strength. These results were modelled by calculating ATP depletion in the fibre due to diffusional loss of ATP and acto-myosin Mg.ATPase activity. The difference in delay before rigor onset at low and high ionic strength could be explained in our model by assuming a 15 times higher Mg.ATPase activity and a threefold increase in Km in relaxing conditions at low ionic strength. Activation by Ca2+ induced at different time points before and during onset of rigor confirmed the calculated time course of ATP depletion. We have also investigated ionic strength effects on rigor development with the activated troponin/tropomyosin complex. ATP withdrawal at maximum activation by Ca2+ induced force transients which led into a "high rigor" state. The peak forces of these force transients were very similar at low and high ionic strength. The subsequent decrease in tension was only 10% slower and steady-state "high rigor" tension was reduced by only 27% at high compared to low ionic strength. Addition of 10 mM phosphate to lower cross-bridge attachment strongly suppressed the transient increases in force at high ionic strength and reduced the steady-state rigor tension by 17%. A qualitatively similar but smaller effect of phosphate was observed at low ionic strength where steady-state rigor force was reduced by 10%. The data presented in this study show a very strong effect of ionic strength on rigor development in relaxed fibres whereas the ionic strength dependence of rigor development after thin filament activation was much less. The data confirm the importance of electrostatic interactions in cross-bridge attachment and cross-bridge-attachment-induced activation of thin filaments.