Nigella sativa itself and as a part of a balm inhibits activity of purified 20S proteasome

Background. Ubiquitin-dependent proteasomal proteolysis is the perspective target for the therapy of the high amount of different diseases. the special attention is paid to the therapy of inflammatory processes of both origins: infection and aseptic. Besides proteolytic processing of the inhibitor of NFkappaB proteasome is important for activation of polymorphonuclear neutrophils (PMN) and neutrophil extrcellular traps (NETs) formation. Basing on the mentioned above information we have evaluated some plant extracts included in the medical compound «Healthy Tonsils» that is used for the treatment of the chronic tonsillitis for their ability to inhibit activity of 20S proteasome. Methods. For in vitro experiments the purified 20S proteasome was used. 20S proteasome was incubated for 30 minutes at 36 C degrees in a mixture with phytobalm components. After the next 30 minutes of incubation with 6 microM solution of corresponded fluorogenic substrates the measurement of fluorescence products was performed (exciting/emission wave length was 360/440) on the spectrofluorimeter Hitachi-4000 with the use of free 7-amido-4-methylcoumarine for a standard curve. Results. In the same concentrations the compounds investigated has demonstrated different ability to inhibit activity of purified 20S proteasome. The most effective compound was the extract of Nigella sativa that inhibits chemotrypsin-like and caspase-like activities of purified 20S proteasome on 69 % and 85 % correspondingly. Conclusions. Thus, our results allow to explaine the anti-inflammatory activity of phytobalm particularly by ability of some of its compounds to inhibit proteasomal proteolysis.

Effect of salinity on modulation by ATP, protein kinases and FXYD2 peptide of gill (Na+, K+)-ATPase activity in the swamp ghost crab Ucides cordatus (Brachyura, Ocypodidae)

ABSTRACTThe gill (Na+, K+)-ATPase is the main enzyme that underpins osmoregulatory ability in crustaceans that occupy biotopes like mangroves, characterized by salinity variation. We evaluated osmotic and ionic regulatory ability in the semi-terrestrial mangrove crab Ucides cordatus after 10-days acclimation to different salinities. We also analyzed modulation by exogenous FXYD2 peptide and by endogenous protein kinases A and C, and Ca2+- calmodulin-dependent kinase of (Na+, K+)-ATPase activity. Hemolymph osmolality was strongly hyper-/hypo-regulated in crabs acclimated at 2 to 35 ‰S. Cl- was well hyper-/hypo- regulated although Na+ much less so, becoming iso-natremic at high salinity. (Na+, K+)- ATPase activity was greatest in isosmotic crabs (26 ‰S), diminishing progressively from 18 and 8 ‰S (≈0.5 fold) to 2 ‰S (0.04-fold), and decreasing notably at 35 ‰S (0.07-fold). At low salinity, the (Na+, K+)-ATPase exhibited a low affinity ATP-binding site that showed Michaelis-Menten behavior. Above 18 ‰S, an additional, high affinity ATP-binding site, corresponding to 10-20% of total (Na+, K+)-ATPase activity appeared. Activity is stimulated by exogenous pig kidney FXYD2 peptide, while endogenous protein kinases A and C and Ca2+/calmodulin-dependent kinase all inhibit activity. This is the first demonstration of inhibitory phosphorylation of a crustacean (Na+, K+)-ATPase by Ca2+/calmodulin-dependent kinase. Curiously, hyper-osmoregulation in U. cordatus shows little dependence on gill (Na+, K+)-ATPase activity, suggesting a role for other ion transporters. These findings reveal that the salinity acclimation response in U. cordatus consists of a suite of osmoregulatory and enzymatic adjustments that maintain its osmotic homeostasis in a challenging, mangrove forest environment.Graphical abstractHighlightsGill (Na+, K+)-ATPase activity is greatest in isosmotic crabs, diminishing in lower and higher salinities.A high affinity ATP-binding site (10-20% of total activity) is exposed above 18 ‰S.Exogenous FXYD2 peptide stimulates activity; endogenous PKA, PKC and CaMK inhibit activity.First demonstration of inhibitory phosphorylation of crustacean (Na+, K+)-ATPase by CaMK.Hyper-osmoregulation shows little dependence on (Na+, K+)-ATPase activity.

De Novo Assessment and Review of Pan-American Pit Viper Anticoagulant and Procoagulant Venom Activities via Kinetomic Analyses

Snakebite with hemotoxic venom continues to be a major source of morbidity and mortality worldwide. Our laboratory has characterized the coagulopathy that occurs in vitro in human plasma via specialized thrombelastographic methods to determine if venoms are predominantly anticoagulant or procoagulant in nature. Further, the exposure of venoms to carbon monoxide (CO) or O-phenylhydroxylamine (PHA) modulate putative heme groups attached to key enzymes has also provided mechanistic insight into the multiple different activities contained in one venom. The present investigation used these techniques to characterize fourteen different venoms obtained from snakes from North, Central, and South America. Further, we review and present previous thrombelastographic-based analyses of eighteen other species from the Americas. Venoms were found to be anticoagulant and procoagulant (thrombin-like activity, thrombin-generating activity). All prospectively assessed venom activities were determined to be heme-modulated except two, wherein both CO and its carrier molecule were found to inhibit activity, while PHA did not affect activity (Bothriechis schlegelii and Crotalus organus abyssus). When divided by continent, North and Central America contained venoms with mostly anticoagulant activities, several thrombin-like activities, with only two thrombin-generating activity containing venoms. In contrast, most venoms with thrombin-generating activity were located in South America, derived from Bothrops species. In conclusion, the kinetomic profiles of venoms obtained from thirty-two Pan-American Pit Viper species are presented. It is anticipated that this approach will be utilized to identify clinically relevant hemotoxic venom enzymatic activity and assess the efficacy of locally delivered CO or systemically administered antivenoms.

The structure, kinetics and interactions of the β-carboxysomal β-carbonic anhydrase, CcaA

CcaA is a β-carbonic anhydrase (CA) that is a component of the carboxysomes of a subset of β-cyanobacteria. This protein, which has a characteristic C-terminal extension of unknown function, is recruited to the carboxysome via interactions with CcmM, which is itself a γ-CA homolog with enzymatic activity in many, but not all cyanobacteria. We have determined the structure of CcaA from Synechocystis sp. PCC 6803 at 1.45 Å. In contrast with the dimer-of-dimers organization of most bacterial β-CAs, or the loose dimer-of-dimers-of-dimers organization found in the plant enzymes, CcaA shows a well-packed trimer-of-dimers organization. The proximal part of the characteristic C-terminal extension is ordered by binding at a site that passes through the two-fold symmetry axis shared with an adjacent dimer; as a result, only one of a pair of converging termini can be ordered at any given time. Docking in Rosetta failed to find well-packed solutions, indicating that formation of the CcaA/CcmM complex probably requires significant backbone movements in at least one of the binding partners. Surface plasmon resonance experiments showed that CcaA forms a complex with CcmM with sub-picomolar affinity, with contributions from residues in CcmM's αA helix and CcaA's C-terminal tail. Catalytic characterization showed CcaA to be among the least active β-CAs characterized to date, with activity comparable with the γ-CA, CcmM, it either complements or replaces. Intriguingly, the C-terminal tail appears to partly inhibit activity, possibly indicating a role in minimizing the activity of unencapsulated enzyme.

Cross-Talk between the Canonical and the Nitrogen-Related Phosphotransferase Systems Modulates Synthesis of the KdpFABC Potassium Transporter in Escherichia coli

Many Proteobacteria possess the regulatory nitrogen-related phosphotransferase system (PTS^Ntr), which operates in parallel to the transport PTS. PTS^Ntr is composed of the proteins EI^Ntr and NPr and the final phosphate acceptor EIIA^Ntr. Both PTSs can exchange phosphoryl groups among each other. Proteins governing K⁺ uptake represent a major target of PTS^Ntr in <i>Escherichia coli</i>. Nonphosphorylated EIIA^Ntr binds and stimulates the K⁺ sensor KdpD, which activates expression of the <i>kdpFABC</i> operon encoding a K⁺ transporter. Here we show that this regulation also operates in an <i>ilvG</i>^<i>+</i> strain ruling out previous concern about interference with a nonfunctional <i>ilvG</i> allele present in many strains. Furthermore, we analyzed phosphorylation of EIIA^Ntr. In wild-type cells EIIA^Ntr is predominantly phosphorylated, regardless of the growth stage and the utilized carbon source. However, cross-phosphorylation of EIIA^Ntr by the transport PTS becomes apparent in the absence of EI^Ntr: EIIA^Ntr is predominantly nonphosphorylated when cells grow on a PTS sugar and phosphorylated when a non-PTS carbohydrate is utilized. These differences in phosphorylation are transduced into corresponding <i>kdpFABC</i> transcription levels. Thus, the transport PTS may affect phosphorylation of EIIA^Ntr and accordingly modulate processes controlled by EIIA^Ntr. Our data suggest that this cross-talk becomes most relevant under conditions that would inhibit activity of EI^Ntr.

Physalins B and F, seco-steroids isolated from Physalis angulata L., strongly inhibit proliferation, ultrastructure and infectivity of Trypanosoma cruzi

SUMMARYWe previously observed that physalins have immunomodulatory properties, as well as antileishmanial and antiplasmodial activities. Here, we investigated the anti-Trypanosoma cruzi activity of physalins B, D, F and G. We found that physalins B and F were the most potent compounds against trypomastigote and epimastigote forms of T. cruzi. Electron microscopy of trypomastigotes incubated with physalin B showed disruption of kinetoplast, alterations in Golgi apparatus and endoplasmic reticulum, followed by the formation of myelin-like figures, which were stained with MDC to confirm their autophagic vacuole identity. Physalin B-mediated alteration in Golgi apparatus was likely due to T. cruzi protease perturbation; however physalins did not inhibit activity of the trypanosomal protease cruzain. Flow cytometry examination showed that cell death is mainly caused by necrosis. Treatment with physalins reduced the invasion process, as well as intracellular parasite development in macrophage cell culture, with a potency similar to benznidazole. We observed that a combination of physalins and benznidazole has a greater anti-T. cruzi activity than when compounds were used alone. These results indicate that physalins, specifically B and F, are potent and selective trypanocidal agents. They cause structural alterations and induce autophagy, which ultimately lead to parasite cell death by a necrotic process.

PENGGUNAAN DOKSISIKLIN HYCLATE SEBAGAI INHIBITOR MATRIKS METALLOPROTEINASE PADA TERAPI TAMBAHAN PERIODONTITIS

<p>Terbit.com Daily has reported that the prevalence of periodontitis increases related with age person. Found that 35,7% of patients with periodontitis in the group 30-39 years old to 66,5% in the group 50-59 years old, increase</p> <p>89,2% in the group 80-90 years old. Periodontitis that commonly found is a chronic periodontitis which is occurs in individuals older than 45 years, but can also be found in children.</p> <p>The main characteristic of periodontitis is the damage of the periodontal connective tissue, alveolar bone, and the movement of the junctional epithelium to the apical. Bacterial components may directly or indirectly cause tissue damage. Inflammatory mediators product such as proteinases, cytokines, and prostaglandins are part of the host response that can also cause tissue damage. Matrix metalloproteinase (MMP) is a proteinase that can cause periodontal tissue damage by disrupting the extracellular matrix molecules in periodontal tissues. Since is known that MMP has a role in various pathological processes, therapy to inhibit activity MMP in pathological processes has developed. MMP inhibitors will be a useful adjunct therapy for the treatment of periodontal disease. Many products have been introduced as an MMP inhibitor, for example: tetracycline</p> <p>and various derivatives, such as doxycycline and minocycline which are capable of inhibiting the activity of several classes of MMPs. Doxycycline has the ability to inhibit the increase in collagenase activity that is pathological in subgingival and prevent periodontitis to become more severe.</p>