Structural parameters of the Pf1 gene 5 protein-DNA complex in solution by neutron scattering

Neutron-scattering experiments have been performed on the intracellular complex formed by the gene 5 protein and single-stranded DNA in cells infected by filamentous bacteriophage Pf1. The contrast matched point of the complex (37% 2H2O) is lower than expected and implies that a substantial fraction of potentially labile hydrogen atoms are unable to exchange with the solvent. The mass/length ratio of the complex (3270 daltons/A) indicates an axial subunit repeat of 5.1 A, a value much larger than the subunit repeat previously determined in fibres. The measured value of the cross-sectional radius of gyration at infinite contrast (Rc = 43.3 A) indicates an outer radius of 60 to 63 A for the complex. The variation in Rc with contrast shows that regions of higher scattering density are located, on average, towards the outside of the complex. The high-angle region of the intensity curve (measured in 2H2O) reveals a clear subsidiary maximum at 0.105 A-1 arising from the 60 A helical pitch of the nucleoprotein complex. The structural parameters of the Pf1 gene 5 protein-DNA complex in solution are compared with those of the fd gene 5 protein-DNA complex.     

Distribution and abundance of young fish in the St. Clair River and associated waters,Ontario

Fish larvae were sampled in 1986 in the St. Clair River, and adjacent waters. Species richness (9 taxa as larvae; 4 others as juveniles) and abundance was lowest in the river, where many larvae (e.g., burbot, rainbow smelt, and yellow perch) were in transit from Lake Huron. The most abundant, and localized, species was gizzard shad, which reached a peak mean density of 4600 larvae 100 m^-3 in an agricultural canal. Adjacent waters contribute greatly to the fish communities of the river and adjoining Lakes Huron and Erie, especially in terms of the number and quantity of forage species.     

A structural basis for the biosynthesis of the major chlorogenic acids found in coffee

Chlorogenic acids (CGAs) are a group of phenolic secondary metabolites produced by certain plant species and an important component of coffee (Coffea spp.). The CGAs have been implicated in biotic and abiotic stress responses, while the related shikimate esters are key intermediates for lignin biosynthesis. Here, two hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyl transferases (HCT/HQT) from coffee were biochemically characterized. We show, to our knowledge for the first time, that in vitro, HCT is capable of synthesizing the 3,5-O-dicaffeoylquinic acid diester, a major constituent of the immature coffee grain. In order to further understand the substrate specificity and catalytic mechanism of the HCT/HQT, we performed structural and mutagenesis studies of HCT. The three-dimensional structure of a native HCT and a proteolytically stable lysine mutant enabled the identification of important residues involved in substrate specificity and catalysis. Site-directed mutagenesis confirmed the role of residues leucine-400 and phenylalanine-402 in substrate specificity and of histidine-153 and the valine-31 to proline-37 loop in catalysis. In addition, the histidine-154-asparagine mutant was observed to produce 4-fold more dichlorogenic acids compared with the native protein. These data provide, to our knowledge, the first structural characterization of a HCT and, in conjunction with the biochemical and mutagenesis studies presented here, delineate the underlying molecular-level determinants for substrate specificity and catalysis. This work has potential applications in fine-tuning the levels of shikimate and quinate esters (CGAs including dichlorogenic acids) in different plant species in order to generate reduced or elevated levels of the desired target compounds.     

Clinical scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells

Dose‐intensive chemotherapy results in an obligatory period of severe neutropenia during which patients are at high risk of infection. While patient support with donor neutrophils is possible, this option is restricted due to donor availability and logistic complications. To overcome these problems, we explored the possibility of large scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells (HPC). CD34⁺ HPC isolated from umbilical cord blood (UCB) and mobilized peripheral blood (mPB) were expanded in serum‐free medium supplemented with stem cell factor, granulocyte colony stimulating factor, and a thrombopoietin peptide mimetic. After 15 days of cultivation a 5,800‐fold expansion in cell number was achieved for UCB, and up to 4,000‐fold for mPB, comprising 40% and 60% mature neutrophils respectively. Ex vivo expanded neutrophils exhibited respiratory burst activity similar to that for donor neutrophils, and were capable of killing Candida albicans in vitro. These yields correspond to a more than 10‐fold improvement over current methods, and are sufficient for the production of multiple neutrophil transfusion doses per HPC donation. To enable clinical scale manufacture, we adapted our protocol for use in a wave‐type bioreactor at a volume of 10 L. This is the first demonstration of a large scale bioprocess suitable for routine manufacture of a mature blood cell product from HPC, and could enable prophylactic neutrophil support for chemotherapy patients. Biotechnol. Bioeng. 2009; 104: 832–840 © 2009 Wiley Periodicals, Inc.     

Shift-down in growth rate rather than high cell density induces toxic shock syndrome toxin-1 gene expression in Staphylococcus aureus

A luciferase-based reporter system for the expression of the toxic shock syndrome toxin-1 gene (tst) of Staphylococcus aureus FRI 1187 was used in continuous culture to determine whether high cell density on transient shift-down or shift-up of specific growth rate (mu) induced expression of tst. Little expression occurred at steady state at a low dilution rate (D) and in a transient period of increasing mu. However, a rapid and approximately 130-fold increase in expression occurred during a transient shift-down of mu. These findings suggest reduction of mu is a key element in the control of tst expression.     

13[C]-Urea Breath Test as a Novel Point-of-Care Biomarker for Tuberculosis Treatment and Diagnosis

Background

Pathogen-specific metabolic pathways may be detected by breath tests based on introduction of stable isotopically-labeled substrates and detection of labeled products in exhaled breath using portable infrared spectrometers.

Methodology/Principal Findings

We tested whether mycobacterial urease activity could be utilized in such a breath test format as the basis of a novel biomarker and diagnostic for pulmonary TB. Sensitized New-Zealand White Rabbits underwent bronchoscopic infection with either Mycobacterium bovis or Mycobacterium tuberculosis. Rabbits were treated with 25 mg/kg of isoniazid (INH) approximately 2 months after infection when significant cavitary lung pathology was present. [¹³C] urea was instilled directly into the lungs of intubated rabbits at selected time points, exhaled air samples analyzed, and the kinetics of δ¹³CO₂ formation were determined. Samples obtained prior to inoculation served as control samples for background ¹³CO₂ conversion in the rabbit model. ¹³CO₂, from metabolic conversion of [¹³C]-urea by mycobacterial urease activity, was readily detectable in the exhaled breath of infected rabbits within 15 minutes of administration. Analyses showed a rapid increase in the rate of ¹³CO₂ formation both early in disease and prior to treatment with INH. Following INH treatment, all evaluable rabbits showed a decrease in the rate of ¹³CO₂ formation.

Conclusions/Significance

Urea breath testing may provide a useful diagnostic and biomarker assay for tuberculosis and for treatment response. Future work will test specificity for M. tuberculosis using lung-targeted dry powder inhalation formulations, combined with co-administering oral urease inhibitors together with a saturating oral dose of unlabeled urea, which would prevent the δ¹³CO₂ signal from urease-positive gastrointestinal organisms.     

Microarray analysis and functional characterization of the nitrosative stress response in nonmucoid and mucoid Pseudomonas aeruginosa

The type strain of Pseudomonas aeruginosa, PAO1, showed great upregulation of many nitrosative defense genes upon treatment with S-nitrosoglutathione, while the mucoid strain PAO578II showed no further upregulation above its constitutive upregulation of nor and fhp. NO* consumption however, showed that both strains mount functional, protein synthesis-dependent NO*-consumptive responses.     

Method for determining oxygen consumption rates of static cultures from microplate measurements of pericellular dissolved oxygen concentration

We describe a simple protocol for determining the oxygen consumption of cells in static culture. The protocol is based on a noninvasive oxygen-sensing microplate and a simple mathematical model derived from Fick's Law. The applicability of the model is confirmed by showing the correlation of computed oxygen consumption rate (OCR) values to actual cell densities ascertained by direct cell counting and/or MTT for HL60 and U937 cells cultured in suspension. Correlation between computed OCR and these other indications of cell number was quite good, as long as the cultures were not diffusion-limited for oxygen. The impact of the geometric factors of media depth and well size were confirmed to be consistent with the model. Based on this demonstrated correlation, we also developed a simple, completely noninvasive algorithm for ascertaining the per-cell oxygen utilization rate (OUR), which is the ratio of OCR to cell number, and a fundamental cell characteristic. This is accomplished by correlating the known seed densities to extrapolated determinations of OCR at time zero. Such determinations were performed for numerous cell types, in varying well sizes. Resulting OUR values are consistent with literature values acquired by far more painstaking methods, and ranged from <0.01 fmol.min(-1).cell(-1) for bacteria to 0.1-10 fmol.min(-1).cell(-1) for immortalized mammalian and insect cell lines to >10 fmol.min(-1).cell(-1) for primary hepatocytes. This protocol for determining OCR and OUR is extremely simple and broadly applicable and can afford rapid, informative, and noninvasive insight into the state of the culture.     

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.