The dhb operon of Bacillus subtilis encodes the biosynthetic template for the catecholic siderophore 2,3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibactin

Bacillus subtilis was reported to produce the catecholic siderophore itoic acid (2,3-dihydroxybenzoate (DHB)-glycine) in response to iron deprivation. However, by inspecting the DNA sequences of the genes dhbE, dhbB, and dhbF as annotated by the B. subtilis genome project to encode the synthetase complex for the siderophore assembly, various sequence errors within the dhbF gene were predicted and confirmed by re-sequencing. According to the corrected sequence, dhbF encodes a dimodular instead of a monomodular nonribosomal peptide synthetase. We have heterologously expressed, purified, and assayed the substrate selectivity of the recombinant proteins DhbB, DhbE, and DhbF. DhbE, a stand-alone adenylation domain of 59.9 kDa, activates, in an ATP-dependent reaction, DHB, which is subsequently transferred to the free thiol group of the cofactor phosphopantetheine of the bifunctional isochorismate lyase/aryl carrier protein DhbB. The third synthetase, DhbF, is a dimodular nonribosomal peptide synthetase of 264 kDa that specifically adenylates threonine and, to a lesser extent, glycine and that covalently loads both amino acids onto their corresponding peptidyl carrier domains. To functionally link the dhb gene cluster to siderophore synthesis, we have disrupted the dhbF gene. Comparative mass spectrometric analysis of culture extracts from both the wild type and the dhbF mutant led to the identification of a mass peak at m/z 881 ([M-H](1-)) that corresponds to a cyclic trimeric ester of DHB-glycine-threonine.     

CIRCADIAN RHYTHM OF PHOTOSYNTHETIC OXYGEN EVOLUTION IN KAPPAPHYCUS ALVAREZII (RHODOPHYTA): DEPENDENCE ON LIGHT QUANTITY AND QUALITY

The rate of oxygen evolution of the tropical red alga Kappaphycus alvarezii (Doty) Doty was measured for 6 days in the laboratory using a computer-aided method for long-term recording. In cool white light, Kappaphycus exhibited a robust circadian rhythm of O₂ evolution in the irradiance range of 100 to 1000 μmol photons·m ^{− 2}·s ^{− 1}. With increasing irradiance, the period of the free-running rhythm, τ, decreased in blue and increased in red light but did not change significantly in green light. The accelerating or slowing action of blue or red light, respectively, points to two photoreceptors used in the light transduction pathway of the circadian oscillator controlling oxygen evolution or the light reactions of photosynthesis in Kappaphycus. No significant changes of τ were observed with increasing irradiance in cool white light, possibly due to the additive opposing responses caused by blue and red light.     

Fluorescence correlation spectroscopy and its potential for intracellular applications

Fluorescence correlation spectroscopy (FCS) is a time-averaging fluctuation analysis of small molecular ensembles, combining maximum sensitivity with high statistical confidence. Among a multitude of physical parameters that are, in principle, accessible by FCS, it most conveniently allows to determine local concentrations, mobility coefficients, and characteristic rate constants of fast-reversible and slow-irreversible reactions of fluorescently labeled biomolecules at very low (nanomolar) concentrations, under equilibrium conditions and without physical separation. Its presently most popular instrumentation by confocal-microscope setups allows for a spatial resolution of fractions of femtoliters for the measurement volumes, containing sparse or even single molecules at any time, and encourages the adaptation of the solution-based technique for cellular applications. The scope of this review is thus, to introduce the FCS technique in particular to the reader with biological background, searching for new methods for a precise quantification of physical parameters governing cellular mechanisms and dynamics, especially if high sensitivity and fast dynamic resolution are required. After a short theoretical introduction, examples are given for the so far most important experimental applications, with respect to their implementation in cellular systems. As an interesting alternative to the confocal instrumentation, two-photon excitation will be introduced, offering a number of important advantages especially in cellular systems with high-noise and low-signal levels.     

The disease business

A report on the 'Host-Pathogen Interactions' minisymposium at the first meeting of the European Life Scientist Organization (ELSO), Geneva, Switzerland, September 2-6, 2000.     

An absorbable catheter system for use in microvascular and peripheral vascular surgery: an experimental study in the canine.

An absorbable catheter for use in regional anticoagulation in microvascular and peripheral vascular surgery was studied in 20 sites in 10 adult beagle dogs to answer three questions: (1) Could the polyglycolic acid and trimethylene carbonate catheter withstand intraarterial pressures of infusion and completely absorb over a predictable time interval? (2) Could the catheter be filled with heparin and maintain patency for reuse after a 24-hour interval? (3) Could the catheter be placed in a side branch of a major artery and, after catheter dissolution, maintain long-term patency of the primary feeding artery? The catheters were completely absorbed from 24 to 34 weeks following implantation. The catheters were able to withstand intraarterial pressures, and no evidence of significant thrombosis of the primary feeding artery was seen in any animal studied. No complications of catheter leak, hematoma formation within the catheter placement sites, or sepsis were noted in any of the 20 catheter sites studied.     

Identification and characterization of a potent, selective, and orally active antagonist of the CC chemokine receptor-1

The CC chemokine receptor-1 (CCR1) is a prime therapeutic target for treating autoimmune diseases. Through high capacity screening followed by chemical optimization, we identified a novel non-peptide CCR1 antagonist, R-N-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl ]-2-oxoethoxy]phenyl]urea hydrochloric acid salt (BX 471). Competition binding studies revealed that BX 471 was able to displace the CCR1 ligands macrophage inflammatory protein-1alpha (MIP-1alpha), RANTES, and monocyte chemotactic protein-3 (MCP-3) with high affinity (K(i) ranged from 1 nm to 5.5 nm). BX 471 was a potent functional antagonist based on its ability to inhibit a number of CCR1-mediated effects including Ca(2+) mobilization, increase in extracellular acidification rate, CD11b expression, and leukocyte migration. BX 471 demonstrated a greater than 10,000-fold selectivity for CCR1 compared with 28 G-protein-coupled receptors. Pharmacokinetic studies demonstrated that BX 471 was orally active with a bioavailability of 60% in dogs. Furthermore, BX 471 effectively reduces disease in a rat experimental allergic encephalomyelitis model of multiple sclerosis. This study is the first to demonstrate that a non-peptide chemokine receptor antagonist is efficacious in an animal model of an autoimmune disease. In summary, we have identified a potent, selective, and orally available CCR1 antagonist that may be useful in the treatment of chronic inflammatory diseases.     

Substituted 1,6-naphthyridines as human cytomegalovirus inhibitors: conformational requirements

Substituted 1,6-naphthyridine derivatives, a new class of human cytomegalovirus inhibitors, were prepared to demonstrate the role of intramolecular hydrogen bonds to maintain the compounds in their active conformation.     

Rainbow trout p53: cDNA cloning and biochemical characterization.

We have cloned and sequenced the p53-encoding cDNA of rainbow trout (Salmo gairdneri). The encoded product contains the characteristics found in all p53 proteins: (i) the five highly conserved domains, (ii) an acidic N terminus, (iii) a hydrophilic C terminus, and (iv) a penultimate serine residue. Furthermore, we demonstrate that the rainbow trout p53 is able to specifically interact with the SV40 large T antigen.     

The preparation of bile acid amides and oxazolines, II, the synthesis of the amides and oxazolines of ursodeoxycholic acid, deoxycholic acid, hyodeoxycholic acid and cholic acid

Bile acid amides and oxazolines were synthesized by a sequence of steps involving the reaction of the free bile acid with formic acid to yield the formyloxy derivative, preparation of the formyloxy acid chloride, condensation of the acid chloride with 2-amino-2-methyl-1-propanol to give the amide and, finally, cyclization of the amide with thionyl chloride to give the oxazoline. The oxazolines were characterized by physical constants, thin layer and gas-liquid chromatography and identified by elemental analysis and gas-liquid chromatography-mass spectrometry. Some of the bile acid oxazoline derivatives alter the activity of bacterial 7-dehydroxylases $\text{in vitro}$, and inhibit the growth of certain anaerobic bacteria in pure culture.