Chemoenzymatic syntheses of water-soluble lipid I fluorescent probes

Peptidoglycan (PG) is unique to bacteria, and thus, the enzymes responsible for its biosynthesis are promising antibacterial drug targets. The membrane-embedded enzymes in PG remain significant challenges in studying their mechanisms due to the fact that preparations of suitable enzymatic substrates require time-consuming biological transformations or chemical synthesis. Lipid I (MurNAc(pentapeptide)-pyrophosphoryl prenol) is an important PG biosynthesis intermediate to study the central enzymes, translocase I (MraY/MurX) and MurG. Lipid I isolated from nature contains the C_(50)- or C_(55)-prenyl unit that shows extremely poor water-solubility that renders studies of translocase I and MurG enzymes difficult. We have studied biological transformation of water soluble lipid I fluorescent probes using bacterial membrane fractions and purified MraY enzymes. In our investigation of the minimum structural requirements of the prenyl phosphates in MraY-catalyzed lipid I synthesis, we found that (2Z,6E)-farnesyl phosphate (C_(15)-phosphate) can be recognized by Escherichia coli MraY to generate the water-soluble lipid I fluorescent probe in high-yields. Under the optimized conditions, the same reaction was performed by using the purified MraY from Hydrogenivirga spp. to afford the lipid I analog with high-yields in a short reaction time

Struktura mezoskalnog strujanja nad sjevernim Jadranom tijekom MAP IOP 15 eksperimenta – slučaj jake bure

We have investigated a strong Bora event over the Northern Adriatic, which occurred on 7 November 1999 during MAP IOP 15. The wind field, potential temperature, vertical component of relative vorticity, and turbulent kinetic energy in the lower troposphere were simulated by a nonhydrostatic mesoscale model (MEMO), which was run at a very fine horizontal resolution with the grid spacing of 1 km. Our model simulation accurately reproduced the timing of the Bora event, and the modeled fields exhibited a good agreement with routinely measured winds along the Croatian coast as well as a large degree of consistence with the MAP measurements offshore. The model results also provided a detailed view of the hydraulic airflow structure and thermodynamic conditions during the Bora event. Our simulation results confirm the existence of topographically induced vorticity filaments originating in the turbulent regions near the coastal mountains, where these turbulent zones are collocated with the hydraulic jump-like flow features. All of the above processes have the highest intensity in the region of Senj.Ispitali smo slučaj jake bure na sjevernom Jadranu, koji se dogodio 7. studenog 1999. tijekom MAP IOP 15 eksperimenta. Polja vjetra, potencijalne temperature, vertikalne komponente relativne vrtložnosti i turbulentne kinetičke energije u donjoj troposferi simulirana su nehidrostatskim mezoskalnim modelom (MEMO) s vrlo finom horizontalnom rezolucijom od 1 km. Simulacija je točno reproducirala nastup bure, a modelirana polja dobro su se podudarala s rutinski mjerenim vjetrom duž hrvatske obale te su pokazala visoku razinu konzistencije s MAP mjerenjima nad morem. Rezultati modela također su omogućili detaljan uvid u strukturu hidrauličkog toka te u termodinamičke uvjete tijekom bure. Rezultati simulacije potvrdili su postojanje topografski induciranih pruga vrtložnosti, koje nastaju u blizini obalnih planina, u turbulentnim područjima čiji položaj se podudara s tokom hidrauličkih svojstava. Svi ti procesi najintenzivniji su u području Senja

Novel FR-900493 Analogues That Inhibit the Outgrowth of Clostridium difficile Spores

The spectrum of antibacterial activity for the nucleoside antibiotic FR-900493 (1) can be extended by chemical modifications. We have generated a small focused library based on the structure of 1 and identified UT-17415 (9), UT-17455 (10), UT-17460 (11), and UT-17465 (12), which exhibit anti-Clostridium difficile growth inhibitory activity. These analogues also inhibit the outgrowth of C. difficile spores at 2× minimum inhibitory concentration. One of these analogues, 11, relative to 1 exhibits over 180-fold and 15-fold greater activity against the enzymes, phospho-MurNAc-pentapeptide translocase (MraY) and polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA), respectively. The phosphotransferase inhibitor 11 displays antimicrobial activity against several tested bacteria including Bacillus subtilis, Clostridium spp., and Mycobacterium smegmatis, but no growth inhibitory activity is observed against the other Gram-positive and Gram-negative bacteria. The selectivity index (Vero cell cytotoxicity/C. difficileantimicrobial activity) of 11 is approximately 17, and 11 does not induce hemolysis even at a 100 μM concentration

Novel FR-900493 Analogues That Inhibit the Outgrowth of Clostridium difficile Spores

The spectrum of antibacterial activity for the nucleoside antibiotic FR-900493 (1) can be extended by chemical modifications. We have generated a small focused library based on the structure of 1 and identified UT-17415 (9), UT-17455 (10), UT-17460 (11), and UT-17465 (12), which exhibit anti-Clostridium difficile growth inhibitory activity. These analogues also inhibit the outgrowth of C. difficile spores at 2× minimum inhibitory concentration. One of these analogues, 11, relative to 1 exhibits over 180-fold and 15-fold greater activity against the enzymes, phospho-MurNAc-pentapeptide translocase (MraY) and polyprenyl phosphate-GlcNAc-1-phosphate transferase (WecA), respectively. The phosphotransferase inhibitor 11 displays antimicrobial activity against several tested bacteria including Bacillus subtilis, Clostridium spp., and Mycobacterium smegmatis, but no growth inhibitory activity is observed against the other Gram-positive and Gram-negative bacteria. The selectivity index (Vero cell cytotoxicity/C. difficileantimicrobial activity) of 11 is approximately 17, and 11 does not induce hemolysis even at a 100 μM concentration

Activation of Heat Shock and Antioxidant Responses by the Natural Product Celastrol: Transcriptional Signatures of a Thiol-targeted Molecule

Stress response pathways allow cells to sense and respond to environmental changes and adverse pathophysiological states. Pharmacological modulation of cellular stress pathways has implications in the treatment of human diseases, including neurodegenerative disorders, cardiovascular disease, and cancer. The quinone methide triterpene celastrol, derived from a traditional Chinese medicinal herb, has numerous pharmacological properties, and it is a potent activator of the mammalian heat shock transcription factor HSF1. However, its mode of action and spectrum of cellular targets are poorly understood. We show here that celastrol activates Hsf1 in Saccharomyces cerevisiae at a similar effective concentration seen in mammalian cells. Transcriptional profiling revealed that celastrol treatment induces a battery of oxidant defense genes in addition to heat shock genes. Celastrol activated the yeast Yap1 oxidant defense transcription factor via the carboxy-terminal redox center that responds to electrophilic compounds. Antioxidant response genes were likewise induced in mammalian cells, demonstrating that the activation of two major cell stress pathways by celastrol is conserved. We report that celastrol's biological effects, including inhibition of glucocorticoid receptor activity, can be blocked by the addition of excess free thiol, suggesting a chemical mechanism for biological activity based on modification of key reactive thiols by this natural product