Two-dimensional crystallization of human vitamin K-dependent gamma-glutamyl carboxylase

Planar-tubular two-dimensional (2D) crystals of human vitamin K-dependent gamma-glutamyl carboxylase grow in the presence of dimyristoyl phosphatidylcholine (DMPC). Surprisingly, these crystals form below the phase transition temperature of DMPC and at the unusually low molar lipid-to-protein (LPR) ratio of 1, while 2D crystals are conventionally grown above the phase transition temperature of the reconstituting lipid and significantly higher LPRs. The crystals are up to 0.75 microm in the shorter dimension of the planar tubes and at least 1 microm in length. Due to the planar-tubular nature of the crystals, two lattices are present. These are rotated by nearly 90 degrees in respect to each other. The ordered arrays exhibit p12(1) plane group symmetry with unit cell dimensions of a=83.7 A, b=76.6 A, gamma=91 degrees. Projection maps calculated from images of negatively stained and electron cryo-microscopy samples reveal the human vitamin K-dependent gamma-glutamyl carboxylase to be a monomer.     

A Marine Mesorhizobium sp. Produces Structurally Novel Long-Chain N-Acyl-l-Homoserine Lactones

Our study focused on a Mesorhizobium sp. that is phylogenetically affiliated by 16S rRNA gene sequence to other marine and saline bacteria of this genus. Liquid chromatography-mass spectrometry investigations of the extract obtained from solid-phase extraction of cultures of this bacterium indicated the presence of several N-acyl homoserine lactones (AHLs), with chain lengths of C₁₀ to C₁₆. Chromatographic separation of the active bacterial extract yielded extraordinarily large amounts of two unprecedented acylated homoserine lactones, 5-cis-3-oxo-C₁₂-homoserine lactone (5-cis-3-oxo-C₁₂-HSL) (compound 1) and 5-cis-C₁₂-HSL (compound 2). Quorum-sensing activity of compounds 1 and 2 was shown in two different biosensor systems [Escherichia coli MT102(pSB403) and Pseudomonas putida F117(pKR-C12)]. Furthermore, it was shown that both compounds can restore protease and pyoverdin production of an AHL-deficient Pseudomonas aeruginosa PAO1 lasI rhlI double mutant, suggesting that these signal molecules maybe used for intergenus signaling. In conclusion, these data indicate that the quorum-sensing activity of compounds 1 and 2 is modulated by the chain length and functional groups of the acyl moiety. Additionally, compound 1 showed antibacterial and cytotoxic activities.     

Leishmania (L.) mexicana Infected Bats in Mexico: Novel Potential Reservoirs

Leishmania (Leishmania) mexicana causes cutaneous leishmaniasis, an endemic zoonosis affecting a growing number of patients in the southeastern states of Mexico. Some foci are found in shade-grown cocoa and coffee plantations, or near perennial forests that provide rich breeding grounds for the sand fly vectors, but also harbor a variety of bat species that live off the abundant fruits provided by these shade-giving trees. The close proximity between sand flies and bats makes their interaction feasible, yet bats infected with Leishmania (L.) mexicana have not been reported. Here we analyzed 420 bats from six states of Mexico that had reported patients with leishmaniasis. Tissues of bats, including skin, heart, liver and/or spleen were screened by PCR for Leishmania (L.) mexicana DNA. We found that 41 bats (9.77%), belonging to 13 species, showed positive PCR results in various tissues. The infected tissues showed no evidence of macroscopic lesions. Of the infected bats, 12 species were frugivorous, insectivorous or nectarivorous, and only one species was sanguivorous (Desmodus rotundus), and most of them belonged to the family Phyllostomidae. The eco-region where most of the infected bats were caught is the Gulf Coastal Plain of Chiapas and Tabasco. Through experimental infections of two Tadarida brasiliensis bats in captivity, we show that this species can harbor viable, infective Leishmania (L.) mexicana parasites that are capable of infecting BALB/c mice. We conclude that various species of bats belonging to the family Phyllostomidae are possible reservoir hosts for Leishmania (L.) mexicana, if it can be shown that such bats are infective for the sand fly vector. Further studies are needed to determine how these bats become infected, how long the parasite remains viable inside these potential hosts and whether they are infective to sand flies to fully evaluate their impact on disease epidemiology.     

Silica Triggers Inflammation and Ectopic Lymphoid Neogenesis in the Lungs in Parallel with Accelerated Onset of Systemic Autoimmunity and Glomerulonephritis in the Lupus-Prone NZBWF1 Mouse

Genetic predisposition and environmental factors influence the development of human autoimmune disease. Occupational exposure to crystalline silica (cSiO₂) has been etiologically linked to increased incidence of autoimmunity, including systemic lupus erythematosus (SLE), but the underlying mechanisms are poorly understood. The purpose of this study was to test the hypothesis that early repeated short-term cSiO₂ exposure will modulate both latency and severity of autoimmunity in the lupus-prone female NZBWF1 mouse. Weekly intranasal exposure to cSiO₂ (0.25 and 1.0 mg) for 4 wk beginning at 9 wk of age both reduced latency and increased intensity of glomerulonephritis. cSiO₂ elicited robust inflammatory responses in the lungs as evidenced by extensive perivascular and peribronchial lymphoplasmacytic infiltration consisting of IgG-producing plasma cells, and CD45R+ and CD3+ lymphocytes that were highly suggestive of ectopic lymphoid tissue (ELT). In addition, there were elevated concentrations of immunoglobulins and the cytokines MCP-1, TNF-α and IL-6 in bronchoalveolar lavage fluid. cSiO₂-associated kidney and lung effects paralleled dose-dependent elevations of autoantibodies and proinflammatory cytokines in plasma. Taken together, cSiO₂-induced pulmonary inflammation and ectopic lymphoid neogenesis in the NZBWF1 mouse corresponded closely to systemic inflammatory and autoimmune responses as well as the early initiation of pathological outcomes in the kidney. These findings suggest that following airway exposure to crystalline silica, in mice genetically prone to SLE, the lung serves as a platform for triggering systemic autoimmunity and glomerulonephritis.     

Energy Intake and Physical Activity in Pima Indians: Comparison with Energy Expenditure Measured by Doubly-Labeled Water

To test the validity of survey techniques for measuring diet and activity patterns of Pima Indians, sequential 24-hour recalls, a food frequency ques tionnaire (FFQ), and an activity questionnaire were compared to free-living energy expenditure. Total energy expenditure (TEE) measured by doubly labeled water was 13.27 ± 2.95 MJ/d for the 12 males (mean±SD: 35 ± 14 yr; 97 ± 35 kg; 32 + 9% body fat) and 11.67 ± 1.85 MJ/d for the 9 females (31 ± 13 yr; 106 ± 32 kg; 49 ± 6% body fat). Energy intake assessed by 24-hour recall was 13.59 ± 7.81 MJ/d for men and 9.29 ± 2.77 MJ/d for women, compared to 12.84 ± 2.85 and 9.40 ± 2.61 MJ/d for men and women, respectively, by FFQ. Both dietary methods indicated significant underreporting by women when compared to TEE. Energy intake assessed by FFQ was significantly correlated with TEE (r=0.48, p=0.03). This was true with 24-hour recall energy intake only when data from two extremely large alcohol consumers were eliminated (r=0.64, p=0.03, N=19). Although a low level of activity was apparent, the activity questionnaire produced significant correlations with measurements of energy expenditure and therefore represents an important tool for examining the relationship between physical activity and diseases.     

Site-directed mutagenesis of Met243, a residue of myeloperoxidase involved in binding of the prosthetic group

 The optical absorbance spectrum of reduced myeloperoxidase is red-shifted with respect to that of other haemoproteins, and has the Soret band at 472 nm and the α band at 636 nm. The origin of the red shift is poorly understood, but the interaction of the protein matrix with the chromophore is thought to play an important role. Met243 is one of the three residues in close proximity to the prosthetic group of the enzyme, and we have examined the effect of a Met243Gln mutation on the spectroscopic properties and catalytic activity of the enzyme. The mutation has a large effect on the position of the Soret band in the optical absorbance spectrum of the reduced mutated enzyme, which shifts from 472 nm to 445 nm. The alkaline pyridine haemochrome spectrum is greatly affected and similar to that of protohaem. The mutation also drastically affects the resonance Raman (RR) spectrum, which is indicative of an iron porphyrin-like chromophore. The mutant enzyme is unable to peroxidise chloride to hypochlorous acid. We conclude that there are two factors involved which account for the red-shifted Soret band. One of them is the interaction of Met243 with the prosthetic group via a special sulfonium linkage. The other factor which contributes is the presence of ester linkages between hydroxylated methyl groups on the haem and glutamate and aspartate residues, respectively. The results, combined with those of previous studies, now give us a comprehensive picture of the various factors which contribute to the unusual optical properties of myeloperoxidase. Received: 17 July 1996 / Accepted: 28 November 1996     

Is aspirin useful in patients on lithium? A pharmacoepidemiological study related to bipolar disorder

ObjectivesAdministration to rats of mood stabilizers approved for bipolar disorder (BD) downregulates markers of the brain arachidonic acid (AA, 20:4n-6) metabolic cascade, including phospholipase A₂ (PLA₂) and cyclooxygenase (COX) expression. We hypothesized that other agents that target the brain AA cascade, nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids, also would ameliorate BD symptoms.MethodsMedication histories on subjects who had been prescribed lithium were collected from the Netherlands PHARMO Record Linkage System. Data were stratified according to drug classes that inhibit PLA₂ and/or COX enzymes, and duration of use. Incidence density (ID) of medication events (dose increase or substance change) was used as a proxy for clinical worsening. ID ratios in patients with the inhibitors plus lithium were compared to ratios in patients using lithium alone.ResultsLow-dose acetylsalicylic acid (aspirin) significantly reduced the ID ratio of medication events, independent of use duration. The ID ratios of NSAIDs and glucocorticoids did not differ significantly from 1.0 if prescribed for ≥180 or ≥90 days, but exceeded 1.0 with shorter use. Selective COX-2 inhibitors had no significant effect and multiagent administration increased the ID ratio above 1.0.ConclusionsLow-dose aspirin produced a statistically significant duration-independent reduction in the relative risk of clinical deterioration in subjects on lithium, whereas other NSAIDs and glucocorticoids did not. These tentative findings could be tested on larger databases containing detailed information about diagnosis and disease course, as well as by controlled clinical trials.     

Phytotoxin production by Pseudomonas syringae pv. syringae: Syringopeptin production by syr mutants defective in biosynthesis or secretion of syringomycin

Abstract Syringomycin and syringopeptin are lipodepsipeptide phytotoxins produced by Pseudomonas syringae pv. syringae . Four syr genes were identified previously and hypothesized to be involved in the regulation ( syrA ), biosynthesis ( syrB and syrC ), or export ( syrD ) of syringomycin. This study determines the influence of syr mutations on the composition of phytotoxic metabolites produced by P. syringae pv. syringae strain B301D-R. Levels of syringomycin and syringopeptin produced in liquid cultures were estimated by reverse phase HPLC analyses and differential antimicrobial assays. Significant quantities of syringopeptin were produced by both syrB and syrC mutants despite their inability to produce syringomycin. Only trace quantities of both lipodepsipeptides were produced by syrA and syrD mutants of P. syringae pv. syringae . These results indicate that syringomycin and syringopeptin are synthesized by separate pathways, but may share common mechanisms for secretion and regulation.