Melanocortin system in cancer-related cachexia

The melanocortin system plays a pivotal role in the regulation of appetite and energy balance. It was recognized to play an important role in the development of cancer-related cachexia, a debilitating condition characterized by progressive body wasting associated with anorexia, increased resting energy expediture and loss of fat as well as lean body mass that cannot be simply prevented or treated by adequate nutritional support.     

Efficient total synthesis of (+)-dihydropinidine, (-)-epidihydropinidine, and (-)-pinidinone

The 2,6-disubstituted piperidine alkaloids (+)-dihydropinidine (1), (-)-epidihydropinidine (2) (as HCl salts), and (-)-pinidinone (3) were efficiently synthesized from (S)-epichlorohydrin (7) as common substrate using regioselective Wacker-Tsuji oxidation of alkenylazides 10 and 14 as well as a highly diastereoselective reduction of cyclic imine 11 as key steps. The protecting group free total syntheses represent the up to date shortest routes with highest overall yields for all three naturally occurring alkaloids (1-3). The first single-crystal X-ray analysis of (-)-epidihydropinidine hydrochloride (2·HCl) confirmed its proposed absolute configuration to be (2S,6S), corresponding to that of the isolated natural product.     

Azithromycin inhibits macrophage interleukin-1β production through inhibition of activator protein-1 in lipopolysaccharide-induced murine pulmonary neutrophilia

Macrolide antibiotics, including azithromycin, also possess anti-inflammatory properties. However, the molecular mechanism(s) of activity as well as the target cells for their action have not been unambiguously identified as yet. In this study, the effects of azithromycin on lipopolysaccharide (LPS)-induced pulmonary neutrophilia were investigated in mice. Using immunohistochemistry, mRNA and specific protein assays, we confirmed that azithromycin ameliorates LPS-induced pulmonary neutrophilia by inhibiting interleukin-1β (IL-1β) expression and production selectively in alveolar macrophages as well as in LPS-stimulated J774.2 macrophage-derived cells in vitro. Inhibition by azithromycin of neutrophilia and IL-1β was accompanied by prevention of nuclear expression of activator protein-1 (AP-1) in both alveolar macrophages and J774.2 cells. The macrolide did not alter nuclear factor kappa B (NF-κB) or extracellular signal-regulated kinase 1/2 (ERK1/2) expression, activation or localization in LPS-stimulated lungs or in J774.2 cells. In conclusion, we have shown that inhibition of LPS-induced pulmonary neutrophilia and IL-1β concentrations in lung tissue following azithromycin treatment is mediated through effects on alveolar macrophages. In addition, we have shown for the first time, in an in vivo model, that azithromycin inhibits AP-1 activation in alveolar macrophages, an action confirmed on J774.2 cells in vitro.     

Cytotoxicity and effects on inflammatory response of modified types of cellulose in macrophage-like THP-1 cells

The cytotoxicity and in vitro effects of six variously modified types of cellulose (OC--oxidized cellulose, NaOC--oxidized cellulose sodium salt, DAC--dialdehyde cellulose, CMC--carboxymethyl cellulose, MFC--microfibrilated cellulose, and MCC--microcrystalline cellulose) on the inflammatory response in macrophage-like THP-1 cells were examined, with special focus on their ability to influence gene expression and the production of TNF-α. The study provides evidence that DAC exerts a marked effect on the induction of TNF-α gene expression and its subsequent production in human macrophages. Thus, the use of DAC for anti-hemorrhagic or wound-healing therapy should be considered carefully with regard to its pro-inflammatory activity. On the contrary, MCC showed significant anti-inflammatory effects in the LPS-induced conditions, which might be beneficial for the treatment of non-healing chronic wounds, e.g., diabetic or venous ulcers.     

Multitarget antithrombotic drugs

Thromboembolic disorders are still the leading causes of morbidity and mortality in developed societies. Therefore, prophylaxis and treatment of arterial and venous thrombosis are among the main therapeutic challenges nowadays. Simultaneous action on several targets involved in pathology of thrombosis offers potential advantages compared to existing drugs which were developed as selective modulators of single targets. The review focuses on dual inhibitors of coagulation enzymes, dual antiaggregatory compounds exerting their action on different combinations of platelet targets, as well as on anticoagulant/antiaggregatory compounds which interfere with at least one target involved in blood coagulation and at least one target engaged in the process leading to platelet aggregation.     

Treating patients with HIV and Hepatitis B and C infections: Croatian dental students' knowledge, attitudes, and risk perceptions

Dentists and dental students can be exposed to the human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) during routine work. The aims of this study were to assess a group of dental students' knowledge about HIV, HBV, and HCV infections; assess their attitudes and risk perceptions about the treatment of patients with HIV, HBV, and HCV; and identify factors associated with their knowledge and willingness to treat these patients. An anonymous survey was administered to 534 predoctoral students at the School of Dental Medicine, University of Zagreb, Croatia. The response rate was 71.9 percent. Students' knowledge increased with each year of study. Senior students (in their third, fourth, and fifth years) had more professional attitudes and were significantly more positive about dentists' professional obligation to treat patients who are HIV-positive than were junior students (in their first and second years; p=0.0002). Senior students also expressed significantly more willingness to treat intravenous drug users and patients with hepatitis (p=0.016 and p=0.033, respectively). Female students were significantly more convinced than male students that routine dental treatment carried a significant risk of HIV and hepatitis infection (p=0.025). These students' knowledge negatively correlated with the lack of willingness to treat intravenous drug users and patients with hepatitis, and they expressed their willingness to receive further theoretical and practical education on this topic.     

Chimeric CYP21A1P/CYP21A2 genes identified in Czech patients with congenital adrenal hyperplasia

Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive disorders caused by an enzymatic deficiency which impairs the biosynthesis of cortisol and, in the majority of severe cases, also the biosynthesis of aldosterone. Approximately 95% of all CAH cases are caused by mutations in the steroid 21-hydroxylase gene (CYP21A2). The CYP21A2 gene and its inactive pseudogene (CYP21A1P) are located within the HLA class III region of the major histocompatibility complex (MHC) locus on chromosome 6p21.3. In this study, we describe chimeric CYP21A1P/CYP21A2 genes detected in our patients with 21-hydroxylase deficiency (21OHD). Chimeric CYP21A1P/CYP21A2 genes were present in 171 out of 508 mutated CYP21A2 alleles (33.8%). We detected four types of chimeric CYP21A1P/CYP21A2 genes: three of them have been described previously as CH-1, CH-3, CH-4, and one type is novel. The novel chimeric gene, termed CH-7, was detected in 21.4% of the mutant alleles. Possible causes of CYP21A1P/CYP21A2 formation are associated with 1) high recombination rate in the MHC locus, 2) high recombination rate between highly homologous genes and pseudogenes in the CYP21 gene area, and 3) the existence of chi-like sequences and repetitive minisatellite consensus sequences in CYP21A2 and CYP21A1P which play a role in promoting genetic recombination.     

Post-translational modifications regulate signalling by Ror1

Aim: In this study, we analysed the post‐translational modification of receptor tyrosine kinase‐like orphan receptor (Ror1). Ror1 is highly upregulated in B cells of patients with chronic lymphocytic leukaemia (CLL). Molecularly, Ror1 acts as the Wnt receptor in the non‐canonical Wnt pathway. Methods: The level of Ror1 glycosylation in HEK293 cells and in primary human CLL cells was analysed by treatment of inhibitors interfering with different steps of glycosylation process and by direct treatment of cell lysates with N‐glycosidase. Ror1 ubiquitination was determined by ubiquitination assay. Functional consequences of post‐translational modifications were analysed by immunohistochemistry and by analysis of cell surface proteins. Differences in Ror1 glycosylation were confirmed by analysis of 14 samples of B cells from CLL patients. Results: We demonstrate that Ror1 is extensively modified by N‐linked glycosylation. Glycosylation produces several variants of Ror1 with electrophoretic migration of approx. 100, 115 and 130 kDa. Inhibition of glycosylation interferes with cell surface localization of the 130‐kDa variant of Ror1 and prevents Ror1‐induced formation of filopodia. Moreover, we show that 130‐kDa Ror1 is mono‐ubiquitinated. Furthermore, individual CLL patients show striking differences in the electrophoretic migration of Ror1, which correspond to the level of glycosylation. Conclusion: Our data show that Ror1 undergoes complex post‐translational modifications by glycosylation and mono‐ubiquitination. These modifications regulate Ror1 localization and signalling, and are highly variable among individual CLL patients. These may suggest that Ror1 signals only in a subset of CLL patients despite Ror1 levels are ubiquitously high in all CLL patients.     

Amyotrophic lateral sclerosis immunoglobulins G enhance the mobility of Lysotracker-labelled vesicles in cultured rat astrocytes

Aim: We examined the effect of purified immunoglobulins G (IgG) from patients with amyotrophic lateral sclerosis (ALS) on the mobility and exocytotic release from Lysotracker‐stained vesicles in cultured rat astrocytes. Methods: Time‐lapse confocal images were acquired, and vesicle mobility was analysed before and after the application of ALS IgG. The vesicle counts were obtained to assess cargo exocytosis from stained organelles. Results: At rest, when mobility was monitored for 2 min in bath with Ca²⁺, two vesicle populations were discovered: (1) non‐mobile vesicles (6.1%) with total track length (TL) < 1 μm, averaging at 0.33 ± 0.01 μm (n = 1305) and (2) mobile vesicles (93.9%) with TL > 1 μm, averaging at 3.03 ± 0.01 μm (n = 20 200). ALS IgG (0.1 mg mL^?1) from 12 of 13 patients increased the TL of mobile vesicles by approx. 24% and maximal displacement (MD) by approx. 26% within 4 min, while the IgG from control group did not alter the vesicle mobility. The mobility enhancement by ALS IgG was reduced in extracellular solution devoid of Ca²⁺, indicating that ALS IgG vesicle mobility enhancement involves changes in Ca²⁺ homeostasis. To examine whether enhanced mobility relates to elevated Ca²⁺ activity, cells were stimulated by 1 mm ATP, a cytosolic Ca²⁺ increasing agent, in the presence (2 mm ) and in the absence of extracellular Ca²⁺. ATP stimulation triggered an increase in TL by approx. 7% and 12% and a decrease in MD by approx. 11% and 1%, within 4 min respectively. Interestingly, none of the stimuli triggered the release of vesicle cargo. Conclusion: Amyotrophic lateral sclerosis‐IgG‐enhanced vesicle mobility in astrocytes engages changes in calcium homeostasis.