Cholinesterases in Single Nerve Cells Isolated from the Locus Ceruleus and from Nucleus of the Facial Nerve of the Rat: A Microgasometric Study

Cholinesterase activity in single nerve cell bodies isolated from the locus ceruleus and nucleus of the facial nerve of the rat was analyzed by the microgasometric method. Acetylcholinesterase activity is about the same in both types of cells. Nonspecific cholinesterase is present in noradrenergic cells of the locus ceruleus but not in the cholinergic cells of the nucleus of the facial nerve. The total activity of cholinesterases and the activity of acetylcholinesterase in nerve cell bodies isolated from the locus ceruleus remains practically unchanged from the tenth postnatal day until the age of 24 months. Depletion of noradrenaline by a high dose of reserpine does not influence the total activity of cholinesterases in nerve cell bodies of locus ceruleus.     

Distribution patterns of some subterranean Crustacea in the territory of the former Yugoslavia

A selection of the most representative distribution patterns of stygobiont animals is presented. The range of a genus may extend beyond the Dinaride karst, while some species exhibit a holo-Dinaric distribution. The mero-Dinaric distribution is organised in two vicarious centres (in the NW and SE) as well as an epilittoral and a paralittoral belt. The paralittoral distribution is a result of paleogeographic rather than present-day ecological conditions. Important differences between the interstitial faunas of Slovenia (and NE Italy) and Macedonia are probably the result of their different sources. Some other distribution patterns are discussed or summarized from the existing literature. The distribution patterns of this stygobiont fauna are extremely diverse, and are influenced by the geological complexity of the territory as well as by the richness of the fauna concerned. New distribution data for some Copepoda, Thermosbaenacea, and Amphipoda are appended.     

Comparison Between the Effects of Botulinum Toxin-Induced Paralysis and Denervation on Molecular Forms of Acetylcholinesterase in Muscles

Abstract: Velocity sedimentation analysis of acetylcholinesterase (AChE) molecular forms in the fast extensor digitorum longus muscle and in the slow soleus muscle of the rat was carried out on days 4, 8, and 14 after induction of muscle paralysis by botulinum toxin type A (BoTx). The results were compared with those observed after muscle denervation. In addition, the ability of BoTx-paralyzed muscles to resynthesize AChE was studied after irreversible inhibition of the preexistent enzyme by diisopropyl phosphorofluoridate. Major differences were observed between the effects of BoTx treatment and nerve section on AChE in the junctional region of the muscles. A precipitous drop in content of the asymmetric A12 AChE form was observed after denervation, whereas its decrease was much slower and less extensive in the BoTx-paralyzed muscles. Recovery of junctional AChE and of its A12 form after irreversible inhibition of the preexistent AChE in BoTx-paralyzed muscles was nevertheless very slow. It seems that a greater part of the junctional A12 AChE form pertains to a fraction with a very slow turnover that is rapidly degraded after denervation but not after BoTx-produced muscle paralysis. The postdenervation decrease in content of junctional A12 AChE is therefore not primarily due to muscle inactivity. The extrajunctional molecular forms of AChE seem to be regulated mostly by muscle activity because they undergo virtually identical changes both after denervation and BoTx paralysis. The differences observed in this respect between the fast and slow muscles after their inactivation must be intrinsic to muscles.     

Occurrence of Cladocera (Crustacea) in subterranean waters in Yugoslavia

Contrary to some localities in France and Spain, Cladocera occur in hypogean waters in Yugoslavia very sporadically. Beside the stygobitic species, Alona hercegovinae from caves and Alona smirnovi from interstitial waters, particularly some other Chydoridae seem to be suited for hypogean life. Chydorus sphaericus, reputed as one of the most euryoecious cladocerans, occurs also the deepest in interstitial waters as well as in caves. Other species, found in hypogean waters are Simocephalus vetulus, Ilyocryptus sordidus, Eurycercus lamellatus, Chydorus ovalis, Leydigia leydigi, Acroperus harpae and Biapertura affnis.     

Inhibition of thymidylate synthase by hydroxyurea in rapidly proliferating P815 mastocytoma cells

The incorporation of [¹⁴C]deoxycytidine, [³H]deoxyuridine, and [³H]thymidine, respectively into pyrimidine bases of DNA has been measured in rapidly proliferating P815 mouse mastocytoma cells in the presence of hydroxyurea. The incorporation of [¹⁴C]deoxycytidine-derived radioactivity into DNA cytosines is increased when compared to the incorporation into DNA thymines. The [³H]deoxyuridine-derived radioactivity is incorporated solely into DNA thymines and this incorporation is inhibited by hydroxyurea in a dose-dependent manner. This suggests an inhibitory effect of hydroxyurea on the thymidylate synthase which was proved in experiments in which the conversion of deoxyuridine monophosphate into deoxythymidine monophosphate catalysed by a crude enzyme preparation from P815 cells was inhibited in the presence of hydroxyurea. Enzymatic DNA methylation as measured by the conversion of incorporated [¹⁴C]deoxycytidine into 5-methylcytosines was not affected by hydroxyurea.     

Reconstruction of the absorption spectrum of Synechocystis sp. PCC 6803 optical mutants from the in vivo signature of individual pigments

Photosynthesis Research - In this work, we reconstructed the absorption spectrum of different Synechocystis sp. PCC 6803 optical strains by summing the computed signature of all pigments present in...     

Inhibition of both BRAF and MEK in BRAFV600E mutant melanoma restores compromised dendritic cell (DC) function while having differential direct effects on DC properties

Purpose

Dendritic cells (DCs) can induce strong tumor-specific T-cell immune responses. Constitutive upregulation of the mitogen-activated protein kinase (MAPK) pathway by a BRAF^V600 mutation, which is present in about 50 % of metastatic melanomas, may be linked to compromised function of DCs in the tumor microenvironment. Targeting both MEK and BRAF has shown efficacy in BRAF^V600 mutant melanoma.

Methods

We co-cultured monocyte-derived human DCs with melanoma cell lines pretreated with the MEK inhibitor U0126 or the BRAF inhibitor vemurafenib. Cytokine production (IL-12 and TNF-α) and surface marker expression (CD80, CD83, and CD86) in DCs matured with the Toll-like receptor 3/Melanoma Differentiation-Associated protein 5 agonist polyI:C was examined. Additionally, DC function, viability, and T-cell priming capacity were assessed upon direct exposure to U0126 and vemurafenib.

Results

Cytokine production and co-stimulation marker expression were suppressed in polyI:C-matured DCs exposed to melanoma cells in co-cultures. This suppression was reversed by MAPK blockade with U0126 and/or vemurafenib only in melanoma cell lines carrying a BRAF^V600E mutation. Furthermore, when testing the effect of U0126 directly on DCs, marked inhibition of function, viability, and DC priming capacity was observed. In contrast, vemurafenib had no effect on DC function across a wide range of dose concentrations.

Conclusions

BRAF^V600E mutant melanoma cells modulate DC through the MAPK pathway as its blockade can reverse suppression of DC function. MEK inhibition negatively impacts DC function and viability if applied directly. In contrast, vemurafenib does not have detrimental effects on important functions of DCs and may therefore be a superior candidate for combination immunotherapy approaches in melanoma patients.     

Molecular simulation of protein aggregation

Computer simulation offers unique possibilities for investigating molecular-level phenomena difficult to probe experimentally. Drawing from a wealth of studies concerning protein folding, computational studies of protein aggregation are emerging. These studies have been successful in capturing aspects of aggregation known from experiment and are being used to refine experimental methods aimed at abating aggregation. Here we review molecular-simulation studies of protein aggregation conducted in our laboratory. Specific attention is devoted to issues with implications for biotechnology.