Multiple active site conformers in the carbon monoxide complexes of trematode hemoglobins

Sequence alignment of hemoglobins of the trematodes Paramphistomum epiclitum and Gastrothylax crumenifer with myoglobin suggests the presence of an unusual active site structure in which two tyrosine residues occupy the E7 and B10 helical positions. In the crystal structure of P. epiclitum hemoglobin, such an E7-B10 tyrosine pair at the putative helical positions has been observed, although the E7 Tyr is displaced toward CD region of the polypeptide. Resonance Raman data on both P. epiclitum and G. crumenifer hemoglobins show that interactions of heme-bound ligands with neighboring amino acid residues are unusual. Multiple conformers in the CO complex, termed the C, O, and N conformers, are observed. The conformers are separated by a large difference (approximately 60 cm(-1)) in the frequencies of their Fe-CO stretching modes. In the C conformer the Fe-CO stretching frequency is very high, 539 and 535 cm(-1), for the P. epiclitum and G. crumenifer hemoglobins, respectively. The Fe-CO stretching of the N conformer appears at an unusually low frequency, 479 and 476 cm(-1), respectively, for the two globins. A population of an O conformer is seen in both hemoglobins, at 496 and 492 cm(-1), respectively. The C conformer is stabilized by a strong polar interaction of the CO with the distal B10 tyrosine residue. The O conformer is similar to the ones typically seen in mutant myoglobins in which there are no strong interactions between the CO and residues in the distal pocket. The N conformer possesses an unusual configuration in which a negatively charged group, assigned as the oxygen atom of the B10 Tyr side chain, interacts with the CO. In this conformer, the B10 Tyr assumes an alternative conformation consistent with one of the conformers seen the crystal structure. Implications of the multiple configurations on the ligand kinetics are discussed.     

Mycobacterium tuberculosis complex differentiation using gyrB-restriction fragment length polymorphism analysis

Mycobacterium tuberculosis complex (MTBC) members are causative agents of human and animal tuberculosis. Differentiation of MTBC members is required for appropriate treatment of individual patients and for epidemiological purposes. Strains from six MTBC species -- M. tuberculosis, M. bovis subsp. bovis, M. bovis BCG, M. africanum, M. pinnipedii, and "M. canetti" -- were studied using gyrB-restriction fragment length polymorphism (gyrB-RFLP) analysis. A table was elaborated, based on observed restriction patterns and published gyrB sequences. To evaluate applicability of gyrB-RFLP at Instituto Adolfo Lutz, Sao Paulo, Mycobacterial Reference Laboratory, 311 MTBC clinical isolates, previously identified using traditional methods as M. tuberculosis (306), M. bovis (3), and M. bovis BCG (2), were analyzed by gyrB-RFLP. All isolates were correctly identified by the molecular method, but no distinction between M. bovis and M. bovis BCG was obtained. Differentiation of M. tuberculosis and M. bovis is of utmost importance, because they require different treatment schedules. In conclusion, gyrB-RFLP is accurate and easy-to-perform, with potential to reduce time needed for conventional differentiation methods. However, application for epidemiological studies remains limited, because it cannot differentiate M. tuberculosis from M. africanum subtype II, and "M. canetti", M. africanum subtype I from M. pinnipedii, and. M. bovis from M. bovis BCG.     

NO-Donating Aspirin and Aspirin Partially Inhibit Age-Related Atherosclerosis but Not Radiation-Induced Atherosclerosis in ApoE Null Mice

Background

We previously showed that irradiation to the carotid arteries of ApoE^−/− mice accelerated the development of macrophage-rich, inflammatory atherosclerotic lesions, prone to intra-plaque hemorrhage. In this study we investigated the potential of anti-inflammatory and anti-coagulant intervention strategies to inhibit age-related and radiation-induced atherosclerosis.

Methodology/Principal Findings

ApoE^−/− mice were given 0 or 14 Gy to the neck and the carotid arteries and aortic arches were harvested at 4 or 30 weeks after irradiation. Nitric oxide releasing aspirin (NCX 4016, 60 mg/kg/day) or aspirin (ASA, 30 or 300 mg/kg/day) were given continuously in the chow. High dose ASA effectively blocked platelet aggregation, while the low dose ASA or NCX 4016 had no significant effect on platelet aggregation. High dose ASA, but not NCX 4016, inhibited endothelial cell expression of VCAM-1 and thrombomodulin in the carotid arteries at 4 weeks after irradiation; eNOS and ICAM-1 levels were unchanged. After 30 weeks of follow-up, NCX 4016 significantly reduced the total number of lesions and the number of initial macrophage-rich lesions in the carotid arteries of unirradiated mice, but these effects were not seen in the brachiocephalic artery of the aortic arch (BCA). In contrast, high dose ASA lead to a decrease in the number of initial lesions in the BCA, but not in the carotid artery. Both high dose ASA and NCX 4016 reduced the collagen content of advanced lesions and increased the total plaque burden in the BCA of unirradiated mice. At 30 weeks after irradiation, neither NCX 4016 nor ASA significantly influenced the number or distribution of lesions, but high dose ASA lead to formation of collagen-rich “stable” advanced lesions in carotid arteries. The total plaque area of the irradiated BCA was increased after ASA, but the plaque burden was very low compared with the carotid artery.

Conclusions/Significance

The development and characteristics of radiation-induced atherosclerosis varied between different arteries but could not be circumvented by anti-inflammatory and anti-coagulant therapies. This implicates other underlying mechanistic pathways compared to age-related atherosclerosis.     

GEOGRAPHIC VARIATION IN SKULL MORPHOLOGY OF ADULT STELLER SEA LIONS (EUMETOPIAS JUBATUS)

Data from cranial specimens of adult E. jubatus were analyzed to compare intraspecific morphology of skulls. Males and females were grouped separately to avoid bias from sexual dimorphism. Geographic variation was observed in adult male E. jubutus , indicating the potential presence of three morphologically disparate groups: those from Alaska, those from California, and those from Japan and Russia. Although sample sizes were small, results from cluster and discriminant function analyses indicated that specimens from eastern and western Alaska were morphologically similar, and that the most divergent specimens for the species appeared to be those from Japan. Skulls from Alaska possessed a typically longer, less robust skull, whereas those from Japan appeared smaller, yet most robust. Skulls from California were intermediate.     

Two rapid urine screens for detection of bacteriuria: An evaluation

Five hundred twenty-five random clean catch urine specimens, collected from 339 adult females, 137 adult males, and 49 pediatric patients, were screened for the presence of bacteriuria with the Uriscreen catalase test and with the Chemstrip 2 LN dipstick. Quantitative cultures were performed on all specimens. The sensitivity, specificity, positive predictive value, and negative predictive value for the catalase test, with 10⁵ CFU/ml as the threshold for significant bacteriuria, were 91.3%, 72.3%, 33.7%, and 98.0%, respectively. Values for the dipstick were 83.9%, 77.9%, 43.7%, and 96.0%. when 10⁴ CFU/ml was used as the threshold, the catalase test had a sensitivity of 89.2%, specificity of 70.4%, positive predictive value of 37.3%, and a negative predictive value of 97.0%. Values for the dipstick at that level were 82.3%, 77.5%, 48.6%, and 94.8%. While the catalase test was more sensitive than the dipstick, it was our opinion that high rates of false-negatives associated with these methods negated the convenience of these fast and simple urine screens.The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.     

OMEGA: a three-dimensional databank for protein structures (a complement to PDB)

OMEGA is a compilation of recent structural information on proteinsderived from X-ray crystallography or NMR and published in journalsreferenced by Current Contents. To date, 401 entries have beenincluded (334 X-ray, 28 NMR, 5 NMR + X-ray, S electron microscopy,3 neutron scattering, 2 neutron diffraction, 1 electron microscopy+ X-ray, 12 model, 11 miscellaneous), with 5–10 new proteinsbeing added each week. OMEGA can be accessed on Macintosh andis interrog ated through 32 key words (space group, resolution,secondary structure, number of residues, etc). This pool ofproteins could be used for various purposes, including searchesfor proteins with a particular set of secondary structures.OMEGA will be continuously updated (every 6 months) and maylater include all proteins already reported in the PDB, as wellas structures reported in journals with smaller readerships.     

Crude Exopolysaccharides (EPS) from Xanthomonas campestris pv. manihotis, Xanthomonas campestris pv. Campestris and Commercial Xanthan Gum as Inducers of Protection in Coffee Plants against Hemileia vastatrix

Foliar-applied exopolysaccharides, obtained from bacterial cells of either Xanthomonas campestris pv. manihotis (EPS-Xcm) or Xanthomonas campestris pv. campestris (EPS-Xcc), isolate NRRL B-1459, were tested for their ability to induce local and systemic protection against coffee leaf rust caused by Hemileia vastatrix. Both preparations of EPS were effective in inducing local and systemic protection when applied 72 h before challenge with the pathogen. Protection was also observed when plants were treated with different concentrations of a commercially available preparation of xanthan gum (CXG).
Systemic protection was induced by EPS-Xcm, EPS-Xcc and CXG even after the removal of the treated leaves immediately before the challenge. Local protection lasted at least 5 weeks, when EPS-Xcm was applied at the concentration of 100 μg equivalents of glucose/ml. Fluorescent microscopic studies of pathogen development in protected and control leaves indicated that neither the germination, appressoria formation nor the number of infection sites were affected by treatment with EPS-Xcm.     

Pyruvate kinase isozymes: Ancient diversity retained in modern plant cells

Pyruvate kinase is a key regulatory enzyme of glycolysis. Phylogenetic analyses of the sequences coding for pyruvate kinase from plants and other organisms revealed unexpected diversity of this glycolytic enzyme in the progenitor of the present-day eukaryotes. Plants contain an ancient lineage of cytosolic pyruvate kinase, which may have diverged from the animal pyruvate kinase genes prior to the plant-animal divergence. The plant cytosolic pyruvate kinase genes are no more closely related to the animal and fungal pyruvate kinase genes than to the prokaryotic pyruvate kinase genes. The results suggest that the plant pyruvate kinase genes and the animal-fungal pyruvate kinase genes have descended from divergent isozymes which existed in the progenitor of the present-day eukaryotes and prokaryotes.     

BG-1 ovarian cell line: An alternative model for examining estrogen-dependent growth in vitro

Summary Examination of estrogen-responsive processes in cell culture is used to investigate hormonal influence on cancer cell growth and gene expression. Most experimental studies have used breast cancer cell lines, in particular MCF7 cells, to investigate estrogen responsiveness. In this study we examined an ovarian cancer cell line, BG-1, which is highly estrogen-responsive in vitro. This observation, plus the fact that the cells are of ovarian rather than mammary gland origin, makes it an attractive alternative model. 17β-Estradiol, epidermal growth factor, and insulin-like growth factor induced proliferation of BG-1 and MCF7 cells. Viability was dependent on these growth factors in BG-1 cells, but not in MCF7 cells. Therefore, we examined the differences between these two cell lines with respect to estrogen and growth factor receptors. BG-1 cells have twice as many estrogen receptors as MCF7 cells, and BG-1 cells have higher insulin-like growth factor-1 and epidermal growth factor receptor levels than MCF7 cells. This may also explain why BG-1 cells proliferate 56% more robustly in serum and show more serum dependence in culture. In both BG-1 and MCF7 cells, epidermal growth factor receptor number is low (<20 000/cell), while insulin-like growth factor-1 receptor level was highest in estrogen receptor positive cell lines. For example, insulin-like growth factor-1 receptor was higher in BG-1 and MCF7 cells than in estrogen receptor negative cells (HeLa>MDA-MB-435>HBL100). In conclusion, BG-1 cells are an excellent model for understanding hormone responsiveness in ovarian tissue and an alternative for examining estrogen receptor-mediated and insulin-like growth factor-1/epidermal growth factor/estrogen cross-talk processes because of their sensitivity to these factors.