Proteasome inhibitors against amelanotic melanoma.

The incidence of malignant melanoma, the most aggressive skin cancer, is increasing constantly. Despite new targeted therapies, the prognosis for patients with metastatic disease remains poor. Thus, there is a need for new combinational treatments, and antineoplastic agents potentially valuable in this approach are inhibitors of the ubiquitin-proteasome system (UPS). In this work, we analyze the cytotoxicity mechanisms of proteasome inhibitors (MG-132, epoxomicin, and lactacystin) in a specific form of melanoma which does not synthesize melanin-the amelanotic melanoma (Ab cells). We found that the most cytotoxic of the compounds tested was epoxomicin. Caspase-9 activation as well as cytochrome C and AIF release from mitochondria indicated that exposure to epoxomicin induced the mitochondrial pathway of apoptosis. Epoxomicin treatment also resulted in accumulation of Bcl-2 family members-proapoptotic Noxa and antiapoptotic Mcl-1, which were postulated as the targets for bortezomib in melanoma. Inhibition of caspases by BAF revealed that cell death was partially caspase-independent. We observed no cell cycle arrest preceding the apoptosis of Ab cells, even though cdk inhibitors p21Cip1/Waf1 and p27Kip1 were up-regulated. The cell cycle was blocked only after inactivation of caspases by the pan-caspase inhibitor BAF. In summary, this is the first study exploring molecular mechanisms of cell death induced by epoxomicin in melanoma. We found that Ab cells died on the mitochondrial pathway of apoptosis and also partially by the caspase-independent way of death. Apoptosis induction was fast and efficient and was not preceded by cell cycle arrest.

A rare variations of the cephalic vein drainage: two cases report.

Throughout the years, anatomic studies have demonstrated numerous variations in the course of the cephalic vein (CV). There are, however, very rare cases of uncommon formation, course or termination of the vein to which our attention should be drawn. During a routine dissections conducted in the Department of Anatomy and Neurobiology, in two formalin-fixed cadavers, the very rare anatomical variants were found. In 80 year-old Caucasian female the right cephalic vein, after crossing the clavipectoral triangle, ascended anterior and superior to the clavicle and drained into the lateral branch of the right external jugular vein, which in turn opened to the right subclavian vein. In the second case, the dissection of 83 year-old Caucasian male cadaver revealed that after passing through the deltopectoral groove, the left cephalic vein run between clavicle and subclavius muscle to terminate in the left subclavian vein. Understanding of the topography, morphology and anatomical variations of the cephalic vein is important not only for the anatomists but for the clinicians and nurses as well. Such knowledge can prevent multiple complications during many invasive procedures including implantation of Cardiac Implantable Electronic Devices, central venous access, arteriovenous fistula creation or even iatrogenic injuries during clavicle or glenohumeral joint surgery.

The molecular mechanisms of cell death in the course of transient ischemia are differentiated in evolutionary distinguished brain structures.

There is large body of evidence suggesting distinct susceptibility to ischemia in various brain regions. However, the reason for this remains unexplained. Comparative studies of programmed cell death (PCD) pathways indicate their differentiated evolutional origin. The caspase-independent pathway is regarded as an older, whereas the caspase-dependent--as more advanced. In our study we address the question of whether there are any characteristic differences in the activation and course of PCD in phylogenetically and morphologically distinguished brain structures after transient focal ischemia. Using Western blot, we studied changes in expression of caspases: 3, 8, 9, and AIF in the frontoparietal neocortex, archicortex (CA1 and CA2 sectors of the hippocampus) and striatum, during reperfusion after 1 h occlusion of the middle cerebral artery. The caspase and AIF expression were differentiated between the studied structures. The activation of only the caspase-dependent pathway was observed in the neocortex. In the archicortex and striatum both caspase-dependent and caspase-independent pathways were activated, although in the latter the extrinsic apoptotic pathway was not activated. In summary, it is conceivable that structures of different evolutionary origin undergo cell-death processes with the participation of phylogenetically distinguished mechanisms. The previously reported unequal susceptibility to ischemia may co-exist with activation of different cell death pathways.

Developmental expression of SNAP-25 protein in the rat striatum and cerebral cortex.

The developmental changes of 25-kDa synaptosomal-associated protein (SNAP-25) expression in the rat striatum and cerebral cortex were examined using Western- blotting and densitometric scanning of immunoblots. Analysis of the striatum extracts from postnatal day 0 (P0) to postnatal day 120 (P120) demonstrated that SNAP-25 is poorly expressed until P14. From this point the expression level gradually increases to reach a maximum on P60 and then decreases. The pattern of SNAP-25 expression in the rat cerebral cortex is different. Two peaks are observed, the first on P10 and the second on P60, after which the expression level decreases. These results appear to confirm the role of SNAP-25 protein in axon outgrowth and synaptogenesis in the nervous system.

Hippocampal interleukin-1beta in the juvenile and middle-aged rat: response to chronic forced swim or high-light open-field stress stimulation.

It is postulated that stress differentially affects interleukin-1beta (IL-1beta) during ontogenetic life. This study examined the influence of chronic exposure to forced swim (FS) stress or high-light open-field (HL-OF) stress on interleukin-1beta (IL-1beta). The total level of IL-1beta protein was assessed by Western blot analysis of hippocampal extracts. Double immunofluorescence staining was used to reveal the percentage of IL-1beta/NeuN (NeuN - neuronal marker) cells in the CA1, CA3 and dentate gyrus (DG) hippocampal subfields. Juvenile (P28; P - postnatal day) and middle-aged (P360) rats were used in the experiment. The research showed no significant differences in IL-1beta protein levels between P28 and P360 non-stress rats. However, a substantial increase in the percentage of IL-1beta-ir neurons in the CA1, CA3 and DG in P360 rats was observed. Chronic FS had no significant influence on IL-1beta expression in the hippocampus or on the percentage of IL-1beta-ir neurons in CA1, CA3 and DG hippocampal subfields in either age group. During HL-OF, the IL-1beta level was significantly increased in the hippocampus of P28 and P360 rats, whereas a marked increase in the percentage of IL-1beta-ir neurons in the CA1, CA3 and DG hippocampal areas occurred only in P360 animals. These results indicate that chronic HL-OF stimulation was the factor inducing changes in the IL-1beta protein levels in P28 and P360 rats and in the percentage of IL-1beta/NeuN-ir cells in the hippocampus of P360 animals.

NPY-, SOM- and VIP-containing interneurons in postnatal development of the rat claustrum.

A growing body of evidence indicates the common origin of the claustrum, endopiriform nucleus, and the basolateral nuclear complex of amygdala from the lateral and ventral parts of the pallium, as the claustroamygdaloid complex. It seems very probable that at least some of the claustral interneurons derive from subcortical sources. The postnatal development of neuropeptide Y-, somatostatin- and vasoactive intestinal polypeptide-containing interneurons was studied during the 4 postnatal months (P0-P120; P, postnatal day). The study was conducted on 45 Wistar rats of both sexes. Our results indicate that neuropeptide-containing interneurons are not morphologically mature at the moment of birth. The characteristic features of neuronal bodies and the relatively long period of postnatal development may indicate their migration from the subcortical neurogenetic centers. Morphological changes in the neuropil are also reported. Although developmental patterns differ between various neuropeptide-containing neuronal subpopulations, two phases of development can be distinguished in each of them: the early phase (P0-P4) during which undifferentiated neurons and neuropil dominate, and the late phase (P7-P28) during which the characteristic features of an adult-like structure gradually appear. Later these observed developmental changes are terminated. The postnatal development of neuropeptide-containing interneurons is completed after 4 weeks of life. This period, which is important for the structural and functional development of the claustrum, must be taken into account in future studies on this structure.