Predicting forest floor and woody fuel consumption from prescribed burns in southern and western pine ecosystems of the United States.

We present pre-burn biomass and consumption data from 60 prescribed burns in the southeastern and western United States. The datasets include pre-burn biomass in Mg/ha by fuel category: herbaceous fuels, shrubs, 1-hr, 10-hr, 100-hr, 1000-hr, 10,000-hr, and > 10,000-hr downed wood, litter and duff. Pre-burn depth (cm) and reduction (cm) are provided for litter and duff layers. Day-of-burn fuel moistures and weather are also listed by site.

Thrombolysis of acute ischaemic stroke in a district general hospital: are we doing enough?

Thrombolysis of acute ischaemic stroke is safe, efficacious and licensed for use in the UK. To date, few studies have looked at the rates of thrombolysis within a district general hospital setting. The aim of the study was to identify whether local thrombolysis protocols for stroke are adhered to; the rate of thrombolysis and strategies for implementation focused at improving the provision of thrombolysis of patients with acute ischaemic stroke. The methodology involved in this was a retrospective study within a district general hospital in the South of England. Data on patients admitted between 15 April 2008 and 14 April 2009 including demographics, use of the local thrombolysis protocol and reasons for non-thrombolysis were collected and analysed. Out of a total of 599 patients with a primary presentation of acute stroke, 18 were considered for thrombolysis. Six patients out of these 18 were thrombolysed without complications. Four out of these six patients had an improved National Institutes of Health Stroke Scale (NIHSS) post-thrombolysis and one eventually died due to the extensiveness of the stroke sustained, despite a slightly improved NIHSS. The rate of thrombolysis of acute ischaemic stroke is low in this hospital (1.001%). Various measures will need to be considered for implementation in order to improve the provision of this service.

Scan length optimization for pulmonary embolism at CT angiography: analysis based on the three-dimensional spatial distribution of 370 emboli in 100 patients.

AIM: To determine the size and three-dimensional spatial distribution of pulmonary emboli (PE) at computed tomography angiography (CTA) to optimize the scan length. MATERIALS AND METHODS: Two experienced radiologists jointly reviewed 100 consecutive, positive PE CTA studies performed in the Emergency Department (53 women; age 61±17 years). All studies were conducted on a 16-detector row CT machine. In each case, the number of emboli was counted and the proximal and distal spatial coordinates of each embolus documented. Coordinates of the main pulmonary artery bifurcation (MPAb) and carina were recorded. For normalization, the thoracic cavity height (H)-from inlet to lowest hemidiaphragm-was measured. The minimal scan lengths for (a) capturing all emboli and (b) rendering a positive diagnosis were determined. RESULTS: Three hundred and seventy (370) emboli were detected. The average number of PE per patient was 3.7 (maximum 12, minimum 1). Their average length was 2.7 cm. Nine patients had saddle emboli (9%), and 71% of emboli were at or below the MPAb. An 18 cm (0.90×H) scan length, centred 4 cm (0.18×H) below the carina, captures all PE in this dataset while reducing z-axis coverage by 29% (34% for normalized data). Moreover, a 14.2 cm (0.78×H) scan length appropriately centred captures at least one embolus in all patients while reducing coverage by 44% (43%). Decreasing scan length to the lesser of 14.2 cm and 0.78×H per patient reduces coverage by 47%. CONCLUSION: Scan length at CTA for PE can be reduced by up to 47% while preserving diagnostic accuracy for PE detection.

Diversity in enoyl-acyl carrier protein reductases.

The enoyl-acyl carrier protein reductase (ENR) is the last enzyme in the fatty acid elongation cycle. Unlike most enzymes in this essential pathway, ENR displays an unusual diversity among organisms. The growing interest in ENRs is mainly due to the fact that a variety of both synthetic and natural antibacterial compounds are shown to specifically target their activity. The primary anti-tuberculosis drug, isoniazid, and the broadly used antibacterial compound, triclosan, both target this enzyme. In this review, we discuss the diversity of ENRs, and their inhibitors in the light of current research progress.

Crystallization and rhenium MAD phasing of the acyl-homoserinelactone synthase EsaI.

Acyl-homoserine-L-lactones (AHLs) are diffusible chemical signals that are required for virulence of many Gram-negative bacteria. AHLs are produced by AHL synthases from two substrates, S-adenosyl-L-methionine and acyl-acyl carrier protein. The AHL synthase EsaI, which is homologous to the AHL synthases from other pathogenic bacterial species, has been crystallized in the primitive tetragonal space group P4(3), with unit-cell parameters a = b = 66.40, c = 47.33 A. The structure was solved by multiple-wavelength anomalous diffraction with a novel use of the rhenium anomalous signal. The rhenium-containing structure has been refined to a resolution of 2.5 A and the perrhenate ion binding sites and liganding residues have been identified.

The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activity.

Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl-5'-AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site-directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild-type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C-terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild-type BirA. No function has been identified previously for the BirA C-terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C-terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25-fold decrease in the affinity for ATP. Our data indicate that the C-terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain.

Bacterial fatty acid biosynthesis: targets for antibacterial drug discovery.

The increase in drug-resistant pathogenic bacteria has created an urgent demand for new antibiotics. Among the more attractive targets for the development of new antibacterial compounds are the enzymes of fatty acid biosynthesis. Although a number of potent inhibitors of microbial fatty acid biosynthesis have been discovered, few of these are clinically useful drugs. Several of these fatty acid biosynthesis inhibitors have potential as lead compounds in the development of new antibacterials. This review encompasses the known inhibitors and prospective targets for new antibacterials.

Escherichia coli FadR positively regulates transcription of the fabB fatty acid biosynthetic gene.

In Escherichia coli expression of the genes of fatty acid degradation (fad) is negatively regulated at the transcriptional level by FadR protein. In contrast the unsaturated fatty acid biosynthetic gene, fabA, is positively regulated by FadR. We report that fabB, a second unsaturated fatty acid biosynthetic gene, is also positively regulated by FadR. Genomic array studies that compared global transcriptional differences between wild-type and fadR-null mutant strains, as well as in cultures of each strain grown in the presence of exogenous oleic acid, indicated that expression of fabB was regulated in a manner very similar to that of fabA expression. A series of genetic and biochemical tests confirmed these observations. Strains containing both fabB and fadR mutant alleles were constructed and shown to exhibit synthetic lethal phenotypes, similar to those observed in fabA fadR mutants. A fadR strain was hypersensitive to cerulenin, an antibiotic that at low concentrations specifically targets the FabB protein. A transcriptional fusion of chloramphenicol acetyltransferase (CAT) to the fabB promoter produces lower levels of CAT protein in a strain lacking functional FadR. The ability of a putative FadR binding site within the fabB promoter to form a complex with purified FadR protein was determined by a gel mobility shift assay. These experiments demonstrate that expression of fabB is positively regulated by FadR.

The biotinyl domain of Escherichia coli acetyl-CoA carboxylase. Evidence that the "thumb" structure id essential and that the domain functions as a dimer.

Biotin carboxyl carrier protein (BCCP) is the small biotinylated subunit of Escherichia coli acetyl-CoA carboxylase (ACC), the enzyme that catalyzes the first committed step of fatty acid synthesis. Similar proteins are found in other bacteria and in chloroplasts. E. coli BCCP is a member of a large family of protein domains modified by covalent attachment of biotin to a specific lysine residue. However, the BCCP biotinyl domain differs from many of these proteins in that an eight-amino acid residue insertion is present upstream of the biotinylated lysine. X-ray crystallographic and multidimensional NMR studies show that these residues constitute a structure that has the appearance of an extended thumb that protrudes from the otherwise highly symmetrical domain structure. I report that expression of two mutant BCCPs lacking the thumb residues fails to restore growth and fatty acid synthesis to a temperature-sensitive E. coli strain that lacks BCCP when grown at nonpermissive temperature. Alignment of BCCPs from various organisms shows that only two of the eight thumb residues are strictly conserved, and amino acid substitution of either residue results in proteins giving only weak growth of the temperature-sensitive E. coli strain. Therefore, the thumb structure is essential for the function of BCCP in the ACC reaction and provides a useful motif for distinguishing the biotinylated proteins of multisubunit ACCs from those of enzymes catalyzing other biotin-dependent reactions. An unexpected result was that expression of a mutant BCCP in which the biotinylated lysine residue was substituted with cysteine was able to partially restore growth and fatty acid synthesis to the temperature-sensitive E. coli strain. This complementation was shown to be specific to BCCPs having native structure (excepting the biotinylated lysine) and is interpreted in terms of dimerization of the BCCP biotinyl domain during the ACC reaction.

Lipoic acid metabolism in Arabidopsis thaliana: cloning and characterization of a cDNA encoding lipoyltransferase.

Lipoic acid is an essential coenzyme required for activity of several key enzyme complexes, such as the pyruvate dehydrogenase complex, in the central metabolism. In these complexes, lipoic acid must be covalently attached to one of the component proteins for it to have biological activity. We report the cloning and characterization of Arabidopsis thaliana LIP2 cDNA for lipoyltransferase that catalyzes the transfer of the lipoyl group from lipoyl-acyl carrier protein to lipoate-dependent enzymes. This cDNA was shown to code for lipoyltransferase by its ability to complement an Escherichia coli lipB null mutant lacking lipoyltransferase activity. The expressed enzyme in the E. coli mutant efficiently complemented the activity of pyruvate dehydrogenase complex, but less efficiently than that of 2-oxoglutarate dehydrogenase complex. Comparison of the deduced amino acid sequence of LIP2 with those of E. coli and yeast lipoyltransferases showed a marked sequence similarity and the presence of a leader sequence presumably required for import into mitochondria. Southern and northern hybridization analyses suggest that LIP2 is a single-copy gene and is expressed as an mRNA of 860 nt in leaves. Western blot analysis with an antibody against lipoyltransferase demonstrated that a 29 kDa form of lipoyltransferase is located in the mitochondrial compartment of A. thaliana.

Function of Escherichia coli biotin carboxylase requires catalytic activity of both subunits of the homodimer.

Biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin and is one component of the multienzyme complex acetyl-CoA carboxylase that catalyzes the first committed step in fatty acid synthesis. The Escherichia coli biotin carboxylase is readily isolated from the other components of the acetyl-CoA carboxylase complex such that enzymatic activity is retained. The three-dimensional structure of biotin carboxylase, determined by x-ray crystallography, demonstrated that the enzyme is a homodimer consisting of two active sites in which each subunit contains a complete active site. To understand how each subunit contributes to the overall function of biotin carboxylase, we made hybrid molecules in which one subunit had a wild-type active site, and the other subunit contained an active site mutation known to significantly affect the activity of the enzyme. One of the two genes encoded a poly-histidine tag at its N terminus, whereas the other gene had an N-terminal FLAG epitope tag. The two genes were assembled into a mini-operon that was induced to give high level expression of both enzymes. "Hybrid" dimers composed of one subunit with a wild-type active site and a second subunit having a mutant active site were obtained by sequential chromatographic steps on columns of immobilized nickel chelate and anti-FLAG affinity matrices. In vitro kinetic studies of biotin carboxylase dimers in which both subunits were wild type revealed that the presence of the N-terminal tags did not alter the activity of the enzyme. However, kinetic assays of hybrid dimer biotin carboxylase molecules in which one subunit had an active site mutation (R292A, N290A, K238Q, or E288K) and the other subunit had a wild-type active site resulted in 39-, 28-, 94-, and 285-fold decreases in the activity of these enzymes, respectively. The dominant negative effects of these mutant subunits were also detected in vivo by monitoring the rate of fatty acid biosynthesis by [(14)C]acetate labeling of cellular lipids. Expression of the mutant biotin carboxylase genes from an inducible arabinose promoter resulted in a significantly reduced rate of fatty acid synthesis relative to the same strain that expressed the wild type gene. Thus, both the in vitro and in vivo data indicate that both subunits of biotin carboxylase are required for activity and that the two subunits must be in communication during enzyme function.

Inhibition of Escherichia coli acetyl coenzyme A carboxylase by acyl-acyl carrier protein.

Escherichia coli acetyl coenzyme A carboxylase (ACC), the first enzyme of the fatty acid biosynthetic pathway, is inhibited by acylated derivatives of acyl carrier protein (ACP). ACP lacking an acyl moiety does not inhibit ACC. Acylated derivatives of ACP having chain lengths of 6 to 20 carbon atoms were similarly inhibitory at physiologically relevant concentrations. The observed feedback inhibition was specific to the protein moiety, as shown by the inability of the palmitoyl thioester of spinach ACP I to inhibit ACC.

Mutational analysis of protein substrate presentation in the post-translational attachment of biotin to biotin domains.

Biotinylation in vivo is an extremely selective post-translational event where the enzyme biotin protein ligase (BPL) catalyzes the covalent attachment of biotin to one specific and conserved lysine residue of biotin-dependent enzymes. The biotin-accepting lysine, present in a conserved Met-Lys-Met motif, resides in a structured domain that functions as the BPL substrate. We have employed phage display coupled with a genetic selection to identify determinants of the biotin domain (yPC-104) of yeast pyruvate carboxylase 1 (residues 1075-1178) required for interaction with BPL. Mutants isolated using this strategy were analyzed by in vivo biotinylation assays performed at both 30 degrees C and 37 degrees C. The temperature-sensitive substrates were reasoned to have structural mutations, leading to compromised conformations at the higher temperature. This interpretation was supplemented by molecular modeling of yPC-104, since these mutants mapped to residues involved in defining the structure of the biotin domain. In contrast, substitution of the Met residue N-terminal to the target lysine with either Val or Thr produced mutations that were temperature-insensitive in the in vivo assay. Furthermore, these two mutant proteins and wild-type yPC-104 showed identical susceptibility to trypsin, consistent with these substitutions having no structural effect. Kinetic analysis of enzymatic biotinylation using purified Met --> Thr/Val mutant proteins with both yeast and Escherichia coli BPLs revealed that these substitutions had a strong effect upon K(m) values but not k(cat). The Met --> Thr mutant was a poor substrate for both BPLs, whereas the Met --> Val substitution was a poor substrate for bacterial BPL but had only a 2-fold lower affinity for yeast BPL than the wild-type peptide. Our data suggest that substitution of Thr or Val for the Met N-terminal of the biotinyl-Lys results in mutants specifically compromised in their interaction with BPL.

Postcatheterization pseudoaneurysms: new developments in the diagnosis and treatment with ultrasound.

Postcatheterization pseudoaneurysms are an increasingly common complication of endovascular procedures. Ultrasound (US) is essential in diagnosis and in playing the key role in the noninvasive treatment of such pseudoaneurysms. The past decade has seen a shift from surgical management to US-guided therapy initially using manual compression. Recently, the minimally invasive technique of percutaneous thrombin injection has been described as an alternative to US-guided compression. This review article discusses the cause and natural history of pseudoaneurysms and describes the technique of US-guided thrombin injection.

Pre-operative localization of parathyroid adenomas: a comparison of power and colour Doppler ultrasonography with nuclear medicine scintigraphy.

AIM: To compare power and colour Doppler ultrasonography (US) with nuclear medicine scintigraphy (NM) in the preoperative localization of parathyroid adenomas in patients with primary hyperparathyroidism (PHPT). MATERIALS AND METHODS: Thirty-one patients with biochemical evidence of PHPT underwent pre-operative US and NM for parathyroid adenoma localization. Both studies were interpreted independently without prior knowledge of the other study's findings. All patients had surgical removal of the parathyroid adenoma utilizing standard neck exploration or minimally invasive unilateral surgical techniques with rapid serum assay of circulating parathyroid hormone levels. RESULTS: All patients had single parathyroid adenomas at surgery. Prospective sensitivities for US, NM and both studies combined were 65%, 68%, and 74%, respectively, with a positive predictive value of 100% each. The adenoma was localized by only one imaging modality in 16% of cases. CONCLUSIONS: US and NM provide complementary roles in the pre-operative localization of parathyroid adenomas in patients with PHPT.

Escherichia coli LipA is a lipoyl synthase: in vitro biosynthesis of lipoylated pyruvate dehydrogenase complex from octanoyl-acyl carrier protein.

The Escherichia coli lipA gene product has been genetically linked to carbon-sulfur bond formation in lipoic acid biosynthesis [Vanden Boom, T. J., Reed, K. E., and Cronan, J. E., Jr. (1991) J. Bacteriol. 173, 6411-6420], although in vitro lipoate biosynthesis with LipA has never been observed. In this study, the lipA gene and a hexahistidine tagged lipA construct (LipA-His) were overexpressed in E. coli as soluble proteins. The proteins were purified as a mixture of monomeric and dimeric species that contain approximately four iron atoms per LipA polypeptide and a similar amount of acid-labile sulfide. Electron paramagnetic resonance and electronic absorbance spectroscopy indicate that the proteins contain a mixture of [3Fe-4S] and [4Fe-4S] cluster states. Reduction with sodium dithionite results in small quantities of an S = 1/2 [4Fe-4S](1+) cluster with the majority of the protein containing a species consistent with an S = 0 [4Fe-4S](2+) cluster. LipA was assayed for lipoate or lipoyl-ACP formation using E. coli lipoate-protein ligase A (LplA) or lipoyl-[acyl-carrier-protein]-protein-N-lipoyltransferase (LipB), respectively, to lipoylate apo-pyruvate dehydrogenase complex (apo-PDC) [Jordan, S. W., and Cronan, J. E. (1997) Methods Enzymol. 279, 176-183]. When sodium dithionite-reduced LipA was incubated with octanoyl-ACP, LipB, apo-PDC, and S-adenosyl methionine (AdoMet), lipoylated PDC was formed. As shown by this assay, octanoic acid is not a substrate for LipA. Confirmation that LipA catalyzes formation of lipoyl groups from octanoyl-ACP was obtained by MALDI mass spectrometry of a recombinant PDC lipoyl-binding domain that had been lipoylated in a LipA reaction. These results provide information about the mechanism of LipA catalysis and place LipA within the family of iron-sulfur proteins that utilize AdoMet for radical-based chemistry.

Conversion of Escherichia coli pyruvate oxidase to an 'alpha-ketobutyrate oxidase'.

Escherichia coli pyruvate oxidase (PoxB), a lipid-activated homotetrameric enzyme, is active on both pyruvate and 2-oxobutanoate ('alpha-ketobutyrate'), although pyruvate is the favoured substrate. By localized random mutagenesis of residues chosen on the basis of a modelled active site, we obtained several PoxB enzymes that had a markedly decreased activity with the natural substrate, pyruvate, but retained full activity with 2-oxobutanoate. In each of these mutant proteins Val-380 had been replaced with a smaller residue, namely alanine, glycine or serine. One of these, PoxB V380A/L253F, was shown to lack detectable pyruvate oxidase activity in vivo; this protein was purified, studied and found to have a 6-fold increase in K(m) for pyruvate and a 10-fold lower V(max) with this substrate. In contrast, the mutant had essentially normal kinetic constants with 2-oxobutanoate. The altered substrate specificity was reflected in a decreased rate of pyruvate binding to the latent conformer of the mutant protein owing to the V380A mutation. The L253F mutation alone had no effect on PoxB activity, although it increased the activity of proteins carrying substitutions at residue 380, as it did that of the wild-type protein. The properties of the V380A/L253F protein provide new insights into the mode of substrate binding and the unusual activation properties of this enzyme.

Percutaneous injection of thrombin for the treatment of pseudoaneurysms after catheterization: an alternative to sonographically guided compression.

OBJECTIVE: The purpose of our study was to determine the efficacy of percutaneous thrombin treatment for iatrogenic pseudoaneurysms of the femoral artery in comparison with sonographically guided compression repair. SUBJECTS AND METHODS: Twenty-three pseudoaneurysms occurring after catheterization were treated percutaneously with an initial injection of 1.0 mL of thrombin solution via a 22-gauge spinal needle under continuous sonographic guidance. Four patients required the additional injection of 1.0-4.0 mL of thrombin for complete thrombosis. Repeated sonography was performed 24 hr after injection. Additionally, we compared our results with those of a control group by reviewing the imaging findings and medical records of 16 patients who underwent sonographically guided compression of iatrogenic pseudoaneurysms between January 1998 and July 1998. RESULTS: Twenty-two of 23 pseudoaneurysms occurring after catheterization were successfully treated with percutaneous thrombin injection. One recurrence was identified 24 hr after injection in a patient who experienced a significant complication. Procedure time was limited to 15 min with an overall success rate of 96%. Retrospectively, 18 iatrogenic pseudoaneurysms were identified in 16 patients. Six (60%) of 10 pseudoaneurysms were successfully compressed under sonographic guidance, with an average time to thrombosis of 32 min. Compression was unsuccessful for four pseudoaneurysms with an average compression time of 45 min. Compression could not be performed in seven patients (39%). The overall success rate of sonographically guided repair was 60%. CONCLUSION: Preliminary evidence suggests that sonographically guided percutaneous thrombin injection is a safe and effective method of treatment for iatrogenic pseudoaneurysms and offers significant advantages over conventional sonographically guided compression.