Association of magnetic resonance assessed disc degeneration and late clinical recurrence in dogs treated surgically for thoracolumbar intervertebral disc extrusions.

BACKGROUND: Radiographic signs of intervertebral disc mineralization are thought to indicate sites of future recurrence of disc extrusion (Hansen type I) but the relationship between evidence of disc degeneration on magnetic resonance imaging (MRI) and future disc extrusion with recurrence of clinical signs has not been examined. OBJECTIVES: To examine the relationship between MRI-assessed degeneration of thoracolumbar intervertebral discs and late recurrence of clinical signs in dogs presented with acute thoracolumbar intervertebral disc extrusion and treated by hemilaminectomy alone. ANIMALS: Ninety-two client-owned dogs presented to 2 referral hospitals between 2009 and 2014. METHODS: Retrospective analysis of association between clinical signs consistent with recurrent thoracolumbar intervertebral disc extrusion and MRI evidence of disc degeneration in dogs undergoing hemilaminectomy for acute thoracolumbar intervertebral disc extrusion. Univariable and multivariable Cox regression analyses were used to explore associations between recurrence of clinical signs and several characteristics of T10-L3 discs at initial diagnosis. RESULTS: Ninety-two cases were included, of which 42 (46%) were Dachshunds and median age was 5.3 years. Clinical signs recurred in 33/92 (36%) dogs. Finding a completely degenerate disc in the T10 to L3 region (in addition to the operated site) at the time of surgery was associated with a hazard ratio of 2.92 (95% confidence interval: 1.37-6.20) for recurrence of clinical signs. CONCLUSIONS AND CLINICAL IMPORTANCE: Our results suggest that in cases of thoracolumbar intervertebral disc extrusion in dogs, recurrence of signs is likely if at least 1 completely degenerate disc in addition to the currently symptomatic disc is visible on MRI.

The Structure of the "Vibration Hole" around an Isotopic Substitution-Implications for the Calculation of Nuclear Magnetic Resonance (NMR) Isotopic Shifts.

Calculations of nuclear magnetic resonance (NMR) isotopic shifts often rest on the unverified assumption that the "vibration hole", that is, the change of the vibration motif upon an isotopic substitution, is strongly localized around the substitution site. Using our recently developed difference-dedicated (DD) second-order vibrational perturbation theory (VPT2) method, we test this assumption for a variety of molecules. The vibration hole turns out to be well localized in many cases but not in the interesting case where the H/D substitution site is involved in an intra-molecular hydrogen bond. For a series of salicylaldehyde derivatives recently studied by Hansen and co-workers (Molecules 2019, 24, 4533), the vibrational hole was found to stretch over the whole hydrogen-bond moiety, including the bonds to the neighbouring C atoms, and to be sensitive to substituent effects. We discuss consequences of this finding for the accurate calculation of NMR isotopic shifts and point out directions for the further improvement of our DD-VPT2 method.

Sarcoidosis al descubierto: Lo que deberíamos informar en las imágenes torácicas / Sarcoidosis Uncovered: What we should Report in Thoracic Image

La sarcoidosis es una enfermedad granulomatosa no caseificante, multisistémica, de causa desconocida, que compromete al pulmón y a los ganglios linfáticos mediastinales entre el 90 y el 95% de los casos. También puede afectar otros órganos, como las glándulas salivales, piel, ojos, hígado, bazo, corazón, huesos y sistema nervioso central. La sarcoidosis tiene una baja prevalencia en Latinoamérica y es subdiagnosticada debido a la alta frecuencia de otros trastornos similares, como tuberculosis, lepra y micosis profundas. El diagnóstico presuntivo se establece con hallazgos imagenológicos característicos dentro de un contexto clínico apropiado y se confirma con la evidencia histológica de granulomas no caseificantes de células epiteliales, en ausencia de otras etiologías. Los hallazgos torácicos incluyen la afectación pulmonar, ganglionar y bronquial, los cuales son detectados a través de la radiografía (Rx) y tomografía computada (TC) de tórax, siendo esa última más sensible y específica. En este artículo, resaltamos la importancia de reconocer los patrones de presentación típicos y atípicos de la sarcoidosis en Rx y TC, así como la relevancia de las imágenes torácicas como elemento clave en el algoritmo diagnóstico de esa patología. También describimos la utilidad de la resonancia magnética (RM), como método adicional para el diagnóstico en casos de afectación cardíaca y el papel de la tomografía por emisión de positrones (PET-CT) en el seguimiento terapéutico.

Sarcoidosis is a non-caseating granulomatous, multisystemic disease of unknown cause that involves the lung and mediastinal lymph nodes in 90-95% of cases. It can also affect other organs such as the salivary glands, skin, eyes, liver, spleen, heart, bones and the central nervous system. Sarcoidosis has a low prevalence in Latin America and it is underdiagnosed due to the high frequency of other similar disorders such as tuberculosis, leprosy and deep mycosis. The presumptive diagnosis is established based on characteristic imaging findings within an appropriate clinical setting and is confirmed by histological evidence of non-caseating epithelioid cell granulomas, in the absence of other etiologies. Thoracic imaging findings include pulmonary, nodal and bronchial involvement, which are detected on chest radiography (CXR) and computed tomography (CT), this last one having a higher sensitivity and specificity. In this article, we highlight the importance of recognizing the typical and atypical presentation patterns of sarcoidosis on CXR and CT, as well as the relevance of thoracic images as key elements in the diagnostic algorithm of this pathology. We also describe the usefulness of magnetic resonance (MR) imaging as an additional method for diagnosis in cases of cardiac involvement and the role of positron emission tomography (PET-CT) in therapeutic follow-up.

Anti-leprosy drug Clofazimine binds to human Raf1 kinase inhibitory protein and enhances ERK phosphorylation.

Human Raf1 kinase inhibitory protein (hRKIP) is an important modulator of the Ras/Raf1/MEK/ERK signaling pathway. Here, we demonstrated that anti-leprosy drug Clofazimine can bind to hRKIP with a significantly stronger affinity than the endogenous substrate phosphatidylethanolamine (PE) by using Biolayer interference technology. Moreover, we identified that residues P74, S75, K80, P111, P112, V177, and P178 play crucial roles in the binding of hRKIP to Clofazimine by using a combination of Nuclear Magnetic Resonance spectroscopy and molecular docking approach. These residues are located at the conserved ligand-binding pocket of hRKIP. Furthermore, we found that 3.2 µM Clofazimine could significantly increase the ERK phosphorylation level by about 37%. Our results indicate that Clofazimine can enhance Ras/Raf1/MEK/ERK signaling transduction pathway via binding to hRKIP. This work provides valuable hints for exploiting Clofazimine as a potential lead compound to efficiently treat the diseases related to RKIP or the Ras/Raf/MEK/ERK pathway.

Identification and characterization of a new antifungal peptide in fermented milk product containing bioprotective Lactobacillus cultures.

Mold and yeast contamination constitutes a major problem in food commodities, including dairy products, hence new natural preventive measures are in high demand. The aim of the current study is to identify and characterize novel antifungal peptides produced by lactic acid bacteria (LAB) in sour cream. By the use of a newly developed image-based 96-well plate fungal growth inhibition assay targeting Debaryomyces hansenii, combined with a range of analytical tools comprising HPLC-high-resolution mass spectrometry, ultrahigh-performance liquid chromatography-Triple Quadrupole MS and nuclear magnetic resonance spectroscopy, we successfully identified a new antifungal peptide (DMPIQAFLLY; 1211 Da) in sour cream enriched with two bioprotective LAB strains. This peptide represents a fragment of casein, the most abundant protein in milk. Presumably, the proteolytic activity of these bioprotective strains results in the observed 4-fold higher concentration of the peptide during storage. Both bioprotective strains are able to generate this peptide in concentrations up to 0.4 µM, independently of the sour cream starter culture employed. The peptide attenuates the growth rate of D. hansenii at concentrations ≥35 µM, and results in smaller cells and more compact colonies. Hence, the peptide is likely contributing to the overall preserving effect of the investigated bioprotective LAB strains.

Fusarihexins A and B: Novel Cyclic Hexadepsipeptides from the Mangrove Endophytic Fungus Fusarium sp. R5 with Antifungal Activities.

Two novel cyclic hexadepsipeptides, fusarihexin A (1: ) and fusarihexin B (2: ), and two known compounds, cyclo-(L-Leu-L-Leu-D-Leu-L-Leu-L-Val) (3: ) and cyclo-(L-Leu-L-Leu-D-Leu-L-Leu-L-Ile) (4: ), were isolated from the marine mangrove endophytic fungus Fusarium sp. R5. Their chemical structures were elucidated on the basis of spectroscopic data and Marfey's analysis. In an in vitro bioassay, fusarihexin A (1: ) remarkably inhibited three plant pathogenic fungi: Colletotrichum gloeosporioides (Penz.) Sacc., which causes anthracnose in many fruits and vegetables, Colletotrichum musae (Berk. and M. A. Curtis) Arx, which causes crown rot and anthracnose in bananas, and Fusarium oxysporum Schlecht. f. sp. lycopersici (Sacc.) W. C. Snyder et H. N. Hansen, which causes Fusarium wilt and fruit rot in tomatoes. Fusarihexin B (2: ) strongly inhibited C. gloeosporioides and C. musae. The compounds were more potent than carbendazim, which is widely used as an agricultural and horticultural fungicide worldwide.

Practical considerations for investigation of protein conformational dynamics by 15N R 1ρ relaxation dispersion.

It is becoming increasingly apparent that proteins are not static entities and that their function often critically depends on accurate sampling of multiple conformational states in aqueous solution. Accordingly, the development of methods to study conformational states in proteins beyond their ground-state structure ("excited states") has crucial biophysical importance. Here we investigate experimental schemes for optimally probing chemical exchange processes in proteins on the micro- to millisecond timescale by 15N R 1ρ relaxation dispersion. The schemes use selective Hartmann-Hahn cross-polarization (CP) transfer for excitation, and derive peak integrals from 1D NMR spectra (Korzhnev et al. in J Am Chem Soc 127:713-721, 2005; Hansen et al. in J Am Chem Soc 131:3818-3819, 2009). Simulation and experiment collectively show that in such CP-based schemes care has to be taken to achieve accurate suppression of undesired off-resonance coherences, when using weak spin-lock fields. This then (i) ensures that relaxation dispersion profiles in the absence of chemical exchange are flat, and (ii) facilitates extraction of relaxation dispersion profiles in crowded regions of the spectrum. Further improvement in the quality of the experimental data is achieved by recording the free-induction decays in an interleaved manner and including a heating-compensation element. The reported considerations will particularly benefit the use of CP-based R 1ρ relaxation dispersion to analyze conformational exchange processes in larger proteins, where resonance line overlap becomes the main limiting factor.

Influence of solvent quality on the mechanical strength of ethylcellulose oleogels.

Ethylcellulose (EC) is the only known food-grade polymer able to structure edible oils. The gelation process and gel properties are similar to those of polymer hydrogels, the main difference being the nature of the solvent. The present study examines the influence of solvent quality on the large deformation mechanical behavior of EC oleogels. Two alternative strategies for manipulating the mechanical response of these gels were evaluated; manipulating the bulk solvent polarity and the addition of surface active small molecules. Gel strength was positively correlated to solvent polarity when blending soybean oil with either mineral oil or castor oil. This behavior was attributed to the ability of the polar entities present in the oil phase to interact with the EC gel network. The addition of the small molecules oleic acid and oleyl alcohol resulted in a substantial enhancement in gel strength up to 10wt% addition, followed by a gradual decrease with increasing proportions. Binding interactions between EC and these molecules were successfully modeled using a Langmuir adsorption isotherm below 10wt% addition. Furthermore, the thermal behavior of stearic acid and stearyl alcohol also indicated a direct interaction between these molecules and the EC network. Differences in the mechanical behavior of gels prepared using refined, bleached, and deodorized canola or soybean oils, and those made with cold-pressed flaxseed oil could be attributed to both oil polarity, and the presence of minor components (free fatty acids). Shorter pulsed NMR T2 relaxation times were observed for stronger gels due to the more restricted mobility of the solvent when interacting with the polymer. This work has demonstrated the strong influence of the solvent composition on the mechanical properties of EC oleogels, which will allow for the tailoring of mechanical properties for various applications.

Isolation and characterization of two cellulose morphology mutants of Gluconacetobacter hansenii ATCC23769 producing cellulose with lower crystallinity.

Gluconacetobacter hansenii, a Gram-negative bacterium, produces and secrets highly crystalline cellulose into growth medium, and has long been used as a model system for studying cellulose synthesis in higher plants. Cellulose synthesis involves the formation of ß-1,4 glucan chains via the polymerization of glucose units by a multi-enzyme cellulose synthase complex (CSC). These glucan chains assemble into ordered structures including crystalline microfibrils. AcsA is the catalytic subunit of the cellulose synthase enzymes in the CSC, and AcsC is required for the secretion of cellulose. However, little is known about other proteins required for the assembly of crystalline cellulose. To address this question, we visually examined cellulose pellicles formed in growth media of 763 individual colonies of G. hansenii generated via Tn5 transposon insertion mutagenesis, and identified 85 that produced cellulose with altered morphologies. X-ray diffraction analysis of these 85 mutants identified two that produced cellulose with significantly lower crystallinity than wild type. The gene disrupted in one of these two mutants encoded a lysine decarboxylase and that in the other encoded an alanine racemase. Solid-state NMR analysis revealed that cellulose produced by these two mutants contained increased amounts of non-crystalline cellulose and monosaccharides associated with non-cellulosic polysaccharides as compared to the wild type. Monosaccharide analysis detected higher percentages of galactose and mannose in cellulose produced by both mutants. Field emission scanning electron microscopy showed that cellulose produced by the mutants was unevenly distributed, with some regions appearing to contain deposition of non-cellulosic polysaccharides; however, the width of the ribbon was comparable to that of normal cellulose. As both lysine decarboxylase and alanine racemase are required for the integrity of peptidoglycan, we propose a model for the role of peptidoglycan in the assembly of crystalline cellulose.

A simple thermodynamic approach to predict responses from polymer-coated quartz crystal microbalance sensors exposed to organic vapors.

As of lately, the demand for developing artificial sensors with improved capabilities for the detection of explosives, toxics or drugs has increased. Ideally, sensor devices should provide high sensitivity and give a response that is specific to a given target molecule without being influenced by possible interfering molecules in the atmosphere. These properties strongly depend on the structure of the chemical compound used as a sensitive material. It is thus crucial to select the right compound and this step would be facilitated with the aid of predictive tools. The present investigations have been focused on a family of functionalized polysiloxane polymers deposited on a QCM device, producing only weak interactions compatible with reversible sensors. The quartz frequency variation at equilibrium has been linked to the partition coefficient that was evaluated using a thermodynamic description of the adsorption process. We have shown that the relative responses of two polymers can be directly determined from the Gibbs free enthalpy of mixing as determined from NMR measurements performed on neat liquid mixtures. An equivalence of this term-including both enthalpy and entropy contributions-to the energy interaction term calculated using Hansen solubility coefficients, has been demonstrated previously. These results constitute a basis for the development of a numerical program for calculating equilibrium sensor responses. For small molecules, the adsorption kinetics can be easily accounted for by a Fick diffusion coefficient estimated from the Van der Waals volume.

Solid-state tautomeric structure and invariom refinement of a novel and potent HIV integrase inhibitor.

The conformation and tautomeric structure of (Z)-4-[5-(2,6-difluorobenzyl)-1-(2-fluorobenzyl)-2-oxo-1,2-dihydropyridin-3-yl]-4-hydroxy-2-oxo-N-(2-oxopyrrolidin-1-yl)but-3-enamide, C27H22F3N3O5, in the solid state has been resolved by single-crystal X-ray crystallography. The electron distribution in the molecule was evaluated by refinements with invarioms, aspherical scattering factors by the method of Dittrich et al. [Acta Cryst. (2005), A61, 314-320] that are based on the Hansen-Coppens multipole model [Hansen & Coppens (1978). Acta Cryst. A34, 909-921]. The ß-diketo portion of the molecule exists in the enol form. The enol -OH hydrogen forms a strong asymmetric hydrogen bond with the carbonyl O atom on the ß-C atom of the chain. Weak intramolecular hydrogen bonds exist between the weakly acidic α-CH hydrogen of the keto-enol group and the pyridinone carbonyl O atom, and also between the hydrazine N-H group and the carbonyl group in the ß-position from the hydrazine N-H group. The electrostatic properties of the molecule were derived from the molecular charge density. The molecule is in a lengthened conformation and the rings of the two benzyl groups are nearly orthogonal. Results from a high-field (1)H and (13)C NMR correlation spectroscopy study confirm that the same tautomer exists in solution as in the solid state.

Antimicrobial activities of neo- and 1-epineo-isoshinanolones from Plumbago zeylanica roots.

CONTEXT: The roots of Plumbago zeylanica Linn. (Plumbaginaceae) are reputed to have a wide spectrum of therapeutic properties in the Ayurvedic system of medicine. They are useful in curing many ailments such as skin diseases, diarrhea, plague and leprosy. OBJECTIVE: The study was aimed at isolating, separating and evaluating the antimicrobial properties of compounds such as neoisoshinanolone and 1-epineo-isoshinanolone from the roots of P. zeylanica. MATERIALS AND METHODS: The crude petroleum ether extract of roots of P. zeylanica was subjected to repeated chromatographic techniques to separate compounds 2 and 3 along with plumbagin. Structure elucidation was carried out using nuclear magnetic resonance (NMR), infra red (IR) and mass spectroscopy. The serial dilution method was used to test antimicrobial activities and their minimum inhibitory concentration (MIC) expressed in microg/mL. RESULTS: 1-Epineo-isoshinanolone is more active with a MIC of 12.5-25 microg/mL whereas neoisoshinanolone has recorded a MIC of 50-100 microg/mL. The activities are compared with plumbagin (0.78-3.13 microg/mL) and standards streptomycin for bacteria and nystatin for fungi. DISCUSSION: Earlier researchers have established the presence of plumbagin in the roots of P. zeylanica and its antimicrobial activities. The structure elucidation of two more biologically active biogenetic precursors along with their activities in the root extracts has been established for the first time in the present study. CONCLUSION: The root extract of P. zeylanica possesses good antimicrobial activity, which suggests its therapeutic use in the Ayurvedic system of medicine to cure skin diseases.

Synthesis, characterization and antimicrobial activity of water soluble dendritic macromolecules.

Several families of water soluble dendrimers were synthesized based on poly(propyleneoxide) amines (Jeffamines) (P(1)). P(1)-core and branched units were constructed from both methylacrylate and ethylenediamine (P(2)-P(9), and generations 0-3 with -NH(2), -COOH functionalities). They were characterized by elemental analysis (EA), gel permeation chromatography (GPC), FT-IR, (1)H, and (13)C NMR. The antimicrobial activities of only water soluble compounds (P(1), P(3), P(4), P(6), P(7) and P(9)) were evaluated using disk diffusion method in water as well as the minimal inhibitory concentration (MIC) dilution method against 9 bacteria. The obtained results from disk diffusion method are assessed in side-by-side comparison with those of Penicillin-g, Ampicillin, Cefotaxime, Vancomycin, Oflaxacin, and Tetracycline, well-known antibacterial agents. The results from dilution procedure are compared with Gentamycin as antibacterial and Nystatin as antifungal. The antifungal activities are reported on five yeast cultures namely, Candida albicans, Kluyveromyces fragilis, Rhodotorula rubra, Debaryomyces hansenii, and Hanseniaspora guilliermondii, and the results are referenced with Nystatin, Ketaconazole, and Clotrimazole, commercial antifungal agents. In most cases, the compounds show broad-spectrum (gram-positive and gram-negative bacteria) activities that are comparatively higher or equipotent to the antibiotic and antifungal agents in the comparison tests.

Synthesis, Raman, FT-IR, NMR spectroscopic data and antimicrobial activity of mixed aza-oxo-thia macrocyclic compounds.

Mixed aza-oxo-thia macrocyclic ligands 1,3,5,11,13,15-hexaaza-6,10,16,20-tetraoxo-8,18-dithia-2,3,4:12,13,14-dipyridine cyclocosane (L(1)); 1,3,5,12,14,16-hexeaza-6,11,17,22-tetraoxo-8,9,19,20-tetrathia-2,3,4:13,14,15-dipyridine cyclodocosane (L(2)); 1,3,5,13,15,17-hexaaza-6,12,18,24-tetraoxo-9,21-dithia-2,3,4:14,15,16-dipyridine cyclotetracosane (L(3)) and 1,3,5,14,16,18-hexaaza-6,13,19,26-tetraoxo-9,10,22,23-tetrathia-2,3,4:15,16,17-dipyridine cyclohexacosane (L(4)) were synthesised. The structural features of the ligands have been studied by elemental analyses, Raman, IR, (1)H and (13)C NMR spectroscopy. The antimicrobial activities of the ligands were evaluated using disk diffusion method in dimethyl sulfoxide (DMSO) as well as the minimal inhibitory concentration (MIC) dilution method, against nine bacteria. The obtained results from disk diffusion method were assessed in side-by-side comparison with those of penicillin G, ampicillin, cefotaxime, vancomycin, ofloxacin, and tetracycline well known antibacterial agents. The results from dilution procedure were compared with gentamycin as antibacterial and nystatin as antifungal. The antifungal activities are reported on five yeast cultures namely Candida albicans, Kluyveromyces fragilis, Rhodotorula rubra, Debaryomyces hansenii, and Hanseniaspora guilliermondii, and the results are referenced with nystatin, Ketoconazole, and clotrimazole, commercial antifungal agents. In most cases, the compounds show broad-spectrum (Gram(+) and Gram(-) bacteria) activities that were more active or equipotent to the antibiotic and antifungal agents in the comparison tests.

Efficient chemical synthesis of a dodecasaccharidyl lipomannan component of mycobacterial lipoarabinomannan.

Lipomannan (LM) is one of the domains of lipoarabinomannan (LAM) glycolipids, the latter being one of several cell surface organic molecules that fortify mycobacterial species against external attack. Some members of mycobacterial families are pathogenic, most notably Mycobacterium tuberculosis and Mycobacterium leprae, while others are nonpathogenic, and used in the clinic, such as Mycobacterium smegmatis. Additional biological significance arises from the fact that LM has been implicated in several health disorders outside of those associated with mycobacterial pathogens, notably for treatment of bladder cancer. LM is comprised of a heavily lipidated phosphoinositide dimannoside headgroup, from which a mannan array, of varied complexity, extends. The latter consists of a 1,6-alpha-linked backbone flanked at position O2, not necessarily regularly, with alpha-linked mannosides. This paper gives an example of lipomannan synthesis in which all of the sugar components, whether functioning as donors or acceptors, are obtained from n-pentenyl orthoesters, themselves in turn prepared in three easy steps from D-mannose. Assembly of the mannan array is facilitated by the exquisite regioselectivity occasioned by the use of ytterbium triflate/N-iodosuccinimide as the trigger for reaction of n-pentenyl orthoesters.

Characterization of two heparan sulphate-binding sites in the mycobacterial adhesin Hlp.

BACKGROUND: The histone-like Hlp protein is emerging as a key component in mycobacterial pathogenesis, being involved in the initial events of host colonization by interacting with laminin and glycosaminoglycans (GAGs). In the present study, nuclear magnetic resonance (NMR) was used to map the binding site(s) of Hlp to heparan sulfate and identify the nature of the amino acid residues directly involved in this interaction. RESULTS: The capacity of a panel of 30 mer synthetic peptides covering the full length of Hlp to bind to heparin/heparan sulfate was analyzed by solid phase assays, NMR, and affinity chromatography. An additional active region between the residues Gly46 and Ala60 was defined at the N-terminal domain of Hlp, expanding the previously defined heparin-binding site between Thr31 and Phe50. Additionally, the C-terminus, rich in Lys residues, was confirmed as another heparan sulfate binding region. The amino acids in Hlp identified as mediators in the interaction with heparan sulfate were Arg, Val, Ile, Lys, Phe, and Thr. CONCLUSION: Our data indicate that Hlp interacts with heparan sulfate through two distinct regions of the protein. Both heparan sulfate-binding regions here defined are preserved in all mycobacterial Hlp homologues that have been sequenced, suggesting important but possibly divergent roles for this surface-exposed protein in both pathogenic and saprophic species.

Investigating the function of the putative mycolic acid methyltransferase UmaA: divergence between the Mycobacterium smegmatis and Mycobacterium tuberculosis proteins.

Mycolic acids are major and specific lipid components of the cell envelope of mycobacteria that include the causative agents of tuberculosis and leprosy, Mycobacterium tuberculosis and Mycobacterium leprae, respectively. Subtle structural variations that are known to be crucial for both their virulence and the permeability of their cell envelope occur in mycolic acids. Among these are the introduction of cyclopropyl groups and methyl branches by mycolic acid S-adenosylmethionine-dependent methyltransferases (MA-MTs). While the functions of seven of the M. tuberculosis MA-MTs have been either established or strongly presumed nothing is known of the roles of the remaining umaA gene product and those of M. smegmatis MA-MTs. Mutants of the M. tuberculosis umaA gene and its putative M. smegmatis orthologue, MSMEG0913, were created. The lipid extracts of the resulting mutants were analyzed in detail using a combination of analytical techniques such as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and proton nuclear magnetic resonance spectroscopy, and chemical degradation methods. The M. smegmatis mutants no longer synthesized subtypes of mycolates containing a methyl branch adjacent to either trans cyclopropyl group or trans double bond at the "proximal" position of both alpha- and epoxy-mycolates. Complementation with MSMEG0913, but not with umaA, fully restored the wild-type phenotype in M. smegmatis. Consistently, no modification was observed in the structures of mycolic acids produced by the M. tuberculosis umaA mutant. These data proved that despite their synteny and high similarity umaA and MSMEG0913 are not functionally orthologous.

Analysis of the structure of mycolic acids of Mycobacterium simiae reveals a particular composition of alpha-mycolates in strain 'habana' TMC 5135, considered as immunogenic in tuberculosis and leprosy.

Structural analysis of mycolic acids from Mycobacterium simiae (including some 'habana' strains) was carried out using (1)H-NMR and MS. Results indicated that this species presents a general pattern of alpha-, alpha'- and keto-mycolates. alpha-Mycolates were composed of a complex mixture of 82 to 89 carbon atoms (C82-C89), with the predominant molecular species containing two di-substituted cyclopropane rings. Among keto-mycolates (C84-C89), those containing one trans di-substituted cyclopropane ring were the most abundant. The alpha'-mycolates were monounsaturated (C64, C66). According to MS and (1)H-NMR data, the strains studied differed in fine structural details of alpha-mycolates and keto-mycolates. Notably, strain 'habana' TMC 5135 (belonging to the 'habana' group, and considered as highly immunogenic in tuberculosis and leprosy) presented a particular composition of alpha-mycolates, with a major component (C87) containing one cis plus one trans di-substituted cyclopropane ring, unlike the type strain of M. simiae and other strains of the 'habana' group (IPK-220 and IPK-337R), in which the major component (C84) contained two cis di-substituted cyclopropane rings. In spite of this finding, the 'habana' strains were closely related to each other and mainly differed from the type strain of M. simiae in some details of the fine structure of keto-mycolates. The present work indicated that within an identical general pattern of mycolic acids, there is a complex composition in M. simiae and structural variation among different strains, as reported for pathogenic species of the genus. Noteworthy was the particular composition of alpha-mycolates in strain 'habana' TMC 5135.

Glycosylation of Pseudomonas aeruginosa strain Pa5196 type IV pilins with mycobacterium-like alpha-1,5-linked d-Araf oligosaccharides.

Pseudomonas aeruginosa is a gram-negative bacterium that uses polar type IV pili for adherence to various materials and for rapid colonization of surfaces via twitching motility. Within the P. aeruginosa species, five distinct alleles encoding variants of the structural subunit PilA varying in amino acid sequence, length, and presence of posttranslational modifications have been identified. In this work, a combination of mass spectrometry and nuclear magnetic resonance spectroscopy was used to identify a novel glycan modification on the pilins of the group IV strain Pa5196. Group IV pilins continued to be modified in a lipopolysaccharide (wbpM) mutant of Pa5196, showing that, unlike group I strains, the pilins of group IV are not modified with the O-antigen unit of the background strain. Instead, the pilin glycan was determined to be an unusual homo-oligomer of alpha-1,5-linked d-arabinofuranose (d-Araf). This sugar is uncommon in prokaryotes, occurring mainly in the cell wall arabinogalactan and lipoarabinomannan (LAM) polymers of mycobacteria, including Mycobacterium tuberculosis and Mycobacterium leprae. Antibodies raised against M. tuberculosis LAM specifically identified the glycosylated pilins from Pa5196, confirming that the glycan is antigenically, as well as chemically, identical to those of Mycobacterium. P. aeruginosa Pa5196, a rapidly growing strain of low virulence that expresses large amounts of glycosylated type IV pilins on its surface, represents a genetically tractable model system for elucidation of alternate pathways for biosynthesis of d-Araf and its polymerization into mycobacterium-like alpha-1,5-linked oligosaccharides.