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.     

Superhelical DNA studied by solution scattering and computer models

We present here recent results on the structure of superhelical DNA and its changes with salt concentration between 0.01 and 1.5 M NaCl. Scattering curves of two different superhelical DNAs were determined by static light scattering. The measured radii of gyration do not change significantly with salt concentration. Small-angle neutron scattering, together with calculations from a Monte Carlo model, allows to determine the superhelix diameter. Measured and simulated scattering curves agreed almost quantitatively. Experimentally we find that the diameter decreases from 16.0±0.9 nm at 10 mM to 9.0±0.7 nm at 100 mM NaCl. The superhelix diameter from the simulated conformations decreased from 18.0±1.5 nm at 10 mM to 9.4±1.5 nm at 100 mM NaCl. At higher salt concentrations up to 1.5 M NaCl, the diameter stays constant at 9 nm.This revised version was published online in October 2005 with corrections to the Cover Date.     

The tumescent technique: the effect of high tissue pressure and dilute epinephrine on absorption of lidocaine

Injection of lidocaine into the subcutaneous tissues by the tumescent technique results in a delayed absorption of the local anesthetic and has allowed clinicians to exceed the maximum recommended dose of lidocaine without reported complications. However, little knowledge exists about the mechanisms that permit such high doses of lidocaine to be used safely with this technique. The presence of low concentration epinephrine and the increased tissue pressure resulting from the tumescent injection have both been implicated as important factors, but neither has been studied in patients whose results were not altered by the variability of the suction procedure. The purpose of this work was to determine the effect of tissue pressure during tumescent injection and presence of low concentration epinephrine on the absorption of lidocaine from subcutaneous tissues in human volunteers. Twenty healthy female human volunteers were randomized into four study groups. After body fat measurements, all subjects received an injection of 7 mg/kg of lidocaine into the subcutaneous tissues of both lateral thighs. The injected solution consisted of 0.1% lidocaine and 12.5 meq/liter sodium bicarbonate in normal saline with or without 1:1,000,000 epinephrine. Tissue pressure was recorded during injection using a specially designed double-barreled needle. The time required for injection was also recorded. Subjects in group 1 received lidocaine with epinephrine injected by a high-pressure technique. Group 2 subjects received lidocaine with epinephrine injected by a low-pressure technique. Group 3 subjects received lidocaine without epinephrine injected under high pressure. Group 4 subjects received lidocaine without epinephrine injected under low pressure. Following injection, sequential blood samples were drawn over a 14-hour period, and plasma lidocaine concentrations were determined by gas chromatography. No suction lipectomy was performed. Maximum tissue pressure during injection was 339 +/- 63 mmHg and 27 +/- 9 mmHg using high- and low-pressure techniques, respectively. Addition of 1:1,000,000 epinephrine, regardless of the pressure of injected fluid, significantly delayed the time to peak plasma concentration by over 7 hours. There was no significant difference in the peak plasma concentration of lidocaine among the four groups. Peak plasma concentrations greater than 1 mcg/ml were seen in 11 subjects. Epinephrine (1:1,000,000) significantly delays the absorption of lidocaine administered by the tumescent technique. High pressure generated in the subcutaneous tissues during injection of the solution does not affect lidocaine absorption. The delay in absorption may allow time for some lidocaine to be removed from the tissues by suction lipectomy. In addition, the slow rise to peak lidocaine concentration in the epinephrine groups may allow the development of systemic tolerance to high lidocaine plasma levels.     

Flap perfusion mapping: TRAM flap after abdominal suction-assisted lipectomy

This technique or its modification (using other dyes) may play a beneficial role in other clinical scenarios where the reconstructive plastic surgeon preoperatively needs to know the integrity of vessels that are too small to image using standard angiographic techniques. In addition, flap perfusion mapping can demonstrate the pattern of skin that is physiologically perfused by the intact vessels. Knowledge of the perfusion characteristics of the tissues to be transferred before surgery may, at the least, alter the design of the tissues to be transferred and, in the extreme case, could affect the nature of the operative choice altogether.     

AUX1 regulates root gravitropism in Arabidopsis by facilitating auxin uptake within root apical tissues

Plants employ a specialized transport system composed of separate influx and efflux carriers to mobilize the plant hormone auxin between its site(s) of synthesis and action. Mutations within the permease-like AUX1 protein significantly reduce the rate of carrier-mediated auxin uptake within Arabidopsis roots, conferring an agravitropic phenotype. We are able to bypass the defect within auxin uptake and restore the gravitropic root phenotype of aux1 by growing mutant seedlings in the presence of the membrane-permeable synthetic auxin, 1-naphthaleneacetic acid. We illustrate that AUX1 expression overlaps that previously described for the auxin efflux carrier, AtPIN2, using transgenic lines expressing an AUX1 promoter::uidA (GUS) gene. Finally, we demonstrate that AUX1 regulates gravitropic curvature by acting in unison with the auxin efflux carrier to co-ordinate the localized redistribution of auxin within the Arabidopsis root apex. Our results provide the first example of a developmental role for the auxin influx carrier within higher plants and supply new insight into the molecular basis of gravitropic signalling.     

Coding theory based models for protein translation initiation in prokaryotic organisms

Our research explores the feasibility of using communication theory, error control (EC) coding theory specifically, for quantitatively modeling the protein translation initiation mechanism. The messenger RNA (mRNA) of Escherichia coli K-12 is modeled as a noisy (errored), encoded signal and the ribosome as a minimum Hamming distance decoder, where the 16S ribosomal RNA (rRNA) serves as a template for generating a set of valid codewords (the codebook). We tested the E. coli based coding models on 5' untranslated leader sequences of prokaryotic organisms of varying taxonomical relation to E. coli including: Salmonella typhimurium LT2, Bacillus subtilis, and Staphylococcus aureus Mu50. The model identified regions on the 5' untranslated leader where the minimum Hamming distance values of translated mRNA sub-sequences and non-translated genomic sequences differ the most. These regions correspond to the Shine-Dalgarno domain and the non-random domain. Applying the EC coding-based models to B. subtilis, and S. aureus Mu50 yielded results similar to those for E. coli K-12. Contrary to our expectations, the behavior of S. typhimurium LT2, the more taxonomically related to E. coli, resembled that of the non-translated sequence group.