Associations among depression, demographic variables, and language impairments in chronic post-stroke aphasia.

INTRODUCTION: Depression may influence treatment participation and outcomes of people with post-stroke aphasia, yet its prevalence and associated characteristics in aphasia are poorly understood. Using retrospective data from an overarching experimental study, we examined depressive symptoms and their relationship to demographic and language characteristics in people with chronic aphasia. As a secondary objective, we compared prevalence of depressive symptoms among the overarching study's included and excluded participants. METHODS: We examined retrospective data from 121 individuals with chronic aphasia including depression scale scores, demographic information (sex, age, time post onset of stroke, education, race/ethnicity, and Veteran status), and scores on assessments of general and modality-specific language impairments. RESULTS: Approximately 50% of participants reported symptoms indicative of depressive disorders: 23% indicative of major depression and 27% indicative of mild depression. Sex (males) and comparatively younger age emerged as statistically significant variables associated with depressive symptoms; naming ability was minimally associated with depressive symptoms. Time post onset of stroke, education level, race/ethnicity, Veteran status, and aphasia severity were not significantly associated with depressive symptoms. Depression-scale scores were significantly higher for individuals excluded from the overarching study compared to those who were included. CONCLUSIONS: The rate of depressive disorders in this sample was higher than rates of depression reported in the general stroke literature. Participant sex, age, and naming ability emerged as factors associated with depressive symptoms, though these links appear complex, especially given variable reports from prior research. Importantly, depressive symptoms do not appear to diminish over time for individuals with chronic aphasia. Given these results and the relatively limited documentation of depression in aphasia literature, depression remains a pressing concern for aphasia research and routine clinical care.

Effects of online augmented kinematic and perceptual feedback on treatment of speech movements in apraxia of speech.

Apraxia of speech (AOS) is a motor speech disorder characterized by disturbed spatial and temporal parameters of movement. Research on motor learning suggests that augmented feedback may provide a beneficial effect for training movement. This study examined the effects of the presence and frequency of online augmented visual kinematic feedback (AVKF) and clinician-provided perceptual feedback on speech accuracy in 2 adults with acquired AOS. Within a single-subject multiple-baseline design, AVKF was provided using electromagnetic midsagittal articulography (EMA) in 2 feedback conditions (50 or 100%). Articulator placement was specified for speech motor targets (SMTs). Treated and baselined SMTs were in the initial or final position of single-syllable words, in varying consonant-vowel or vowel-consonant contexts. SMTs were selected based on each participant's pre-assessed erred productions. Productions were digitally recorded and online perceptual judgments of accuracy (including segment and intersegment distortions) were made. Inter- and intra-judge reliability for perceptual accuracy was high. Results measured by visual inspection and effect size revealed positive acquisition and generalization effects for both participants. Generalization occurred across vowel contexts and to untreated probes. Results of the frequency manipulation were confounded by presentation order. Maintenance of learned and generalized effects were demonstrated for 1 participant. These data provide support for the role of augmented feedback in treating speech movements that result in perceptually accurate speech production. Future investigations will explore the independent contributions of each feedback type (i.e. kinematic and perceptual) in producing efficient and effective training of SMTs in persons with AOS.

The role of the embA and embB gene products in the biosynthesis of the terminal hexaarabinofuranosyl motif of Mycobacterium smegmatis arabinogalactan.

The emb genes are conserved among different mycobacteria. In Mycobacterium smegmatis and Mycobacterium tuberculosis, they belong to an operon comprising three genes, embC, embA, and embB. The EmbB protein has been proposed to be the target of ethambutol, a drug which is known to inhibit the synthesis of the arabinan portion of the mycobacterial cell wall arabinogalactan (AG). To further define the role of EmbB protein in arabinan biosynthesis, embA, -B, and -C genes were inactivated individually by homologous recombination in M. smegmatis. All three mutants were viable, and among the three, the slowest growing embB(-) mutant encountered profound morphological changes and exhibited a higher sensitivity to hydrophobic drugs and detergents, presumably due to an increase in cell wall permeability. Furthermore, chemical analyses showed that there was a diminution in the arabinose content of arabinogalactan from the embA(-) and embB(-) mutants. Specifically, in comparison with the wild-type strain, the crucial terminal hexaarabinofuranosyl motif, which is a template for mycolylation, was altered in both embA(-) and embB(-) mutants. Detailed nuclear magnetic resonance studies coupled with enzyme digestion, chromatography, and mass spectrometry analyses revealed that the disaccharide beta-d-Ara(f)-(1-->2)-alpha-d-Ara(f) extension from the 3-position of the 3,5-linked alpha-d-Ara(f) residue is markedly diminished. As a consequence, a linear terminal beta-d-Ara(f)-(1-->2)-alpha-d-Ara(f)-(1-->5)-alpha-d-Ara(f)-(1-->5)-alpha-d-Ara(f) is formed, a motif which is a recognized, nonreducing terminal feature of lipoarabinomannan but not of normal AG. Upon complementation with the embB and embA wild-type genes, the phenotype of the mutants reverted to wild-type, in that normal AG was resynthesized. Our results clearly show that both EmbA and EmbB proteins are involved in the formation of the proper terminal hexaarabinofuranoside motif in AG, thus paving the way for future studies to identify the complete array of arabinosyltransferases involved in the synthesis of mycobacterial cell wall arabinan.

UDP-galactopyranose mutase has a novel structure and mechanism.

Uridine diphosphogalactofuranose (UDP-Galf ) is the precursor of the d-galactofuranose (Galf ) residues found in bacterial and parasitic cell walls, including those of many pathogens, such as Mycobacterium tuberculosis and Trypanosoma cruzi. UDP-Galf is made from UDP-galactopyranose (UDP-Galp) by the enzyme UDP-galactopyranose mutase (mutase). The mutase enzyme is essential for the viability of mycobacteria and is not found in humans, making it a viable therapeutic target. The mechanism by which mutase achieves the unprecedented ring contraction of a nonreducing sugar is unclear. We have solved the crystal structure of Escherichia coli mutase to 2.4 A resolution. The novel structure shows that the flavin nucleotide is located in a cleft lined with conserved residues. Site-directed mutagenesis studies indicate that this cleft contains the active site, with the sugar ring of the substrate UDP-galactose adjacent to the exposed isoalloxazine ring of FAD. Assay results establish that the enzyme is active only when flavin is reduced. We conclude that mutase most likely functions by transient reduction of substrate.

Drug targeting Mycobacterium tuberculosis cell wall synthesis: genetics of dTDP-rhamnose synthetic enzymes and development of a microtiter plate-based screen for inhibitors of conversion of dTDP-glucose to dTDP-rhamnose.

An L-rhamnosyl residue plays an essential structural role in the cell wall of Mycobacterium tuberculosis. Therefore, the four enzymes (RmlA to RmlD) that form dTDP-rhamnose from dTTP and glucose-1-phosphate are important targets for the development of new tuberculosis therapeutics. M. tuberculosis genes encoding RmlA, RmlC, and RmlD have been identified and expressed in Escherichia coli. It is shown here that genes for only one isotype each of RmlA to RmlD are present in the M. tuberculosis genome. The gene for RmlB is Rv3464. Rv3264c was shown to encode ManB, not a second isotype of RmlA. Using recombinant RmlB, -C, and -D enzymes, a microtiter plate assay was developed to screen for inhibitors of the formation of dTDP-rhamnose. The three enzymes were incubated with dTDP-glucose and NADPH to form dTDP-rhamnose and NADP(+) with a concomitant decrease in optical density at 340 nm (OD(340)). Inhibitor candidates were monitored for their ability to lower the rate of OD(340) change. To test the robustness and practicality of the assay, a chemical library of 8,000 compounds was screened. Eleven inhibitors active at 10 microM were identified; four of these showed activities against whole M. tuberculosis cells, with MICs from 128 to 16 microg/ml. A rhodanine structural motif was present in three of the enzyme inhibitors, and two of these showed activity against whole M. tuberculosis cells. The enzyme assay was used to screen 60 Peruvian plant extracts known to inhibit the growth of M. tuberculosis in culture; two extracts were active inhibitors in the enzyme assay at concentrations of less than 2 microg/ml.

Galactan biosynthesis in Mycobacterium tuberculosis. Identification of a bifunctional UDP-galactofuranosyltransferase.

The cell wall of Mycobacterium tuberculosis and related genera is unique among prokaryotes, consisting of a covalently bound complex of mycolic acids, D-arabinan and D-galactan, which is linked to peptidoglycan via a special linkage unit consisting of Rhap-(1-->3)-GlcNAc-P. Information concerning the biosynthesis of this entire polymer is now emerging with the promise of new drug targets against tuberculosis. Accordingly, we have developed a galactosyltransferase assay that utilizes the disaccharide neoglycolipid acceptors beta-d-Galf-(1-->5)-beta-D-Galf-O-C(10:1) and beta-D-Galf-(1-->6)-beta-D-Galf-O-C(10:1), with UDP-Gal in conjunction with isolated membranes. Chemical analysis of the subsequent reaction products established that the enzymatically synthesized products contained both beta-D-Galf linkages ((1-->5) and (1-->6)) found within the mycobacterial cell, as well as in an alternating (1-->5) and (1-->6) fashion consistent with the established structure of the cell wall. Furthermore, through a detailed examination of the M. tuberculosis genome, we have shown that the gene product of Rv3808c, now termed glfT, is a novel UDP-galactofuranosyltransferase. This enzyme possesses dual functionality in performing both (1-->5) and (1-->6) galactofuranosyltransferase reactions with the above neoglycolipid acceptors, using membranes isolated from the heterologous host Escherichia coli expressing Rv3808c. Thus, at a biochemical and genetic level, the polymerization of the galactan region of the mycolyl-arabinogalactan complex has been defined, allowing the possibility of further studies toward substrate recognition and catalysis and assay development. Ultimately, this may also lead to a more rational approach to drug design to be explored in the context of mycobacterial infections.

Biosynthesis of the galactan component of the mycobacterial cell wall.

The structural core of the cell walls of Mycobacterium spp. consists of peptidoglycan bound by a linker unit (-alpha-L-Rhap-(1-->3)-D-GlcNAc-P-) to a galactofuran, which in turn is attached to arabinofuran and mycolic acids. The sequence of reactions leading to the biogenesis of this complex starts with the formation of the linker unit on a polyprenyl-P to produce polyprenyl-P-P-GlcNAc-Rha (Mikusová, K., Mikus, M., Besra, G. S., Hancock, I., and Brennan, P. J. (1996) J. Biol. Chem. 271, 7820-7828). We now establish that formation of the galactofuran takes place on this intermediate with UDP-Galf as the Galf donor presented in the form of UDP-Galp and UDP-Galp mutase (the glf gene product) and is catalyzed by galactofuranosyl transferases, one of which, the Mycobacterium tuberculosis H37Rv3808c gene product, has been identified. Evidence is also presented for the growth of the arabinofuran on this polyprenyl-P-P-linker unit-galactan intermediate catalyzed by unidentified arabinosyl transferases, with decaprenyl-P-Araf or 5-P-ribosyl-PP as the Araf donor. The product of these steps, the lipid-linked-LU-galactan-arabinan has been partially characterized in terms of its heterogeneity, size, and composition. Biosynthesis of the major components of mycobacterial cell walls is proving to be extremely complex. However, partial definition of arabinogalactan synthesis, the site of action of several major anti-tuberculosis drugs, facilitates the present day thrust for new drugs to counteract multiple drug-resistant tuberculosis.

The presence of an endogenous endo-D-arabinase in Mycobacterium smegmatis and characterization of its oligoarabinoside product.

Endogenous mycobacterial endo-D-arabinase activity, which degrades cell wall polysaccharide arabinogalactan, was found in Mycobacterium smegmatis. The arabinan product contains 20-30 arabinosyl residues but no galactofuranosyl residues. Recognition of this endogenous activity results in the possibility of developing antituberculosis drugs that do not require bacterial growth for activity.

Construction and use of low-copy number T7 expression vectors for purification of problem proteins: purification of mycobacterium tuberculosis RmlD and pseudomonas aeruginosa LasI and RhlI proteins, and functional analysis of purified RhlI.

Purification of proteins from Escherichia coli under native conditions is often hampered by inclusion-body formation after overexpression from T7 promoter-based expression vectors. This is probably due to the relatively high copy number of the ColE1-based expression vectors. To circumvent these problems, the low-copy-number pViet and pNam expression vectors were constructed. These vectors contain the pSC101 origin of replication and allow the expression of oligohistidine and intein chitin-binding domain fusion proteins, respectively. Since pViet and pNam do not replicate in E. coli B strains, an E. coli K-12 host strain [SA1503(DE3)] was constructed. This strain is defective in the Lon and OmpT proteases and allows IPTG-inducible expression of recombinant proteins from the T7 promoter. The new vectors were successfully tested by purification of three very insoluble proteins (RmlD, LasI and RhlI) under non-denaturing conditions, and all three proteins retained enzymatic activity. The purified hexahistidine (His6)-tagged Pseudomonas aeruginosa RhlI protein was subjected to more detailed analyses, which indicated that (1) only butyryl-acyl carrier protein (ACP) and S-adenosylmethionine (SAM) were required for synthesis of N-butyryl-L-homoserine lactone; (2) when present at physiological concentrations, butyryl-coenzyme A and NADPH were not substrates for RhlI; (3) RhlI was able to synthesize N-hexanoyl-L-homoserine lactone from hexanoyl-ACP and SAM; (4) RhlI was able to direct synthesis of N-butyryl-L-homoserine lactone from crotonyl-ACP in a reaction coupled to purified P. aeruginosa FabI (enoyl-ACP reductase).

Cryptosporidium parvum sporozoite pellicle antigen recognized by a neutralizing monoclonal antibody is a beta-mannosylated glycolipid.

The protozoan parasite Cryptosporidium parvum is an important cause of diarrhea in humans, calves, and other mammals worldwide. No approved vaccines or parasite-specific drugs are currently available for the control of cryptosporidiosis. To effectively immunize against C. parvum, identification and characterization of protective antigens are required. We previously identified CPS-500, a conserved, neutralization-sensitive antigen of C. parvum sporozoites and merozoites defined by monoclonal antibody 18.44. In the present study, the biochemical characteristics and subcellular location of CPS-500 were determined. CPS-500 was chloroform extractable and eluted with acetone and methanol in silicic acid chromatography, consistent with being a polar glycolipid. Following chloroform extraction and silicic acid chromatography, CPS-500 was isolated by high-pressure liquid chromatography for glycosyl analysis, which indicated the presence of mannose and inositol. To identify which component of CPS-500 comprised the neutralization-sensitive epitope recognized by 18.44, the ability of the monoclonal antibody to bind CPS-500 treated with proteases, or with alpha- or beta-glycosidases, was determined. Monoclonal antibody 18.44 did not bind antigen treated with beta-D-mannosidase but did bind antigen treated with alpha-D-mannosidase, other alpha- or beta-glycosidases, or a panel of proteases. These data indicated that the target epitope was dependent on terminal beta-D-mannopyranosyl residues. By immunoelectron microscopy, 18.44 binding was localized to the pellicle and an intracytoplasmic tubulovesicular network in sporozoites. Monoclonal antibody 18.44 also bound to antigen deposited and released onto substrate over the course travelled by gliding sporozoites and merozoites. Surface localization, adhesion and release during locomotion, and neutralization sensitivity suggest that CPS-500 may be involved in motility and invasion processes of the infective zoite stages.

The case of the lawyer's lugubrious language: dysarthria plus primary progressive aphasia or dysarthria plus dementia?

A productive, intelligent, 60-year-old practicing attorney slowly begins to notice that the language that he has commanded throughout his life is beginning to become more difficult to produce, exacting its toll on his mental energy and emotional stability. His search for answers to his diminished "memory for words" leads him through the fetid ranks of traditional medicine and into the search for a differential diagnosis involving clinical neurology, neuropsychology, and speech-language pathology. Consistencies and conflicts in the signs and symptoms between the competing diagnoses raise theoretical and clinical classification issues. A course of treatment for aphasia provides evidence to support the diagnosis of primary progressive aphasia, but the development of concommitant spastic dysarthria and dysphagia challenge current wisdom about the underlying neuropathology of aphasia and support a diagnosis of early dementia. A selective but steady and rapid decline of abilities over the course of 2 years leads to the patient's death and autopsy, from which a neuropathologic analysis was to provide the "final" and "ultimate" diagnosis. But it doesn't!

Biosynthetic origin of mycobacterial cell wall galactofuranosyl residues.

SETTING: Mycobacterial galactofuran is essential to the linking of the peptidoglycan and mycolic acid cell wall layers. Galactofuran biosynthesis should thus be essential for viability. OBJECTIVE: The objective was to determine the pathway of galactofuranosyl biosynthesis and to clone a gene encoding an essential enzyme necessary for its formation. DESIGN: Specific enzymatic conversions involved in formation of galactopyranose and galactofuranose residues in other bacteria were tested for in Mycobacterium smegmatis. M. tuberculosis deoxyribonucleic acid (DNA) was identified by homology. RESULTS: It was shown that the de novo synthesis of the galactose carbon skeleton occurred in M. smegmatis by the transformation of UDP-glucopyranose to UDP-galactopyranose via the enzyme UDP-glucose 4-epimerase (E.C. 5.1.3.2). The N-terminal sequence of this enzyme was obtained after purification. The galactose salvage pathway enzyme, UDP-glucose-galactose-1-phosphate uridylyltransferase (E.C. 2.7.7.12), was also shown to be present. The critical biosynthetic transformation of the galactopyranose to galactofuranose ring form was shown to occur at the sugar nucleotide level via the enzyme UDP-galactopyranose mutase (E.C. 5.4.99.9). The M. tuberculosis DNA encoding this enzyme was sequenced, the gene expressed in Escherichia coli, and the expected enzymatic activity demonstrated. CONCLUSION: Galactofuranose biosynthesis can now be pursued as a potential drug target in M. tuberculosis.

Effects of length and linguistic complexity on temporal acoustic measures in apraxia of speech.

The purpose of this study was to examine the effects of varying length and linguistic utterance types on temporal acoustic characteristics of the imitative speech of apraxic speakers. Vowel duration and two between-word segment durations were examined during the production of three response types: words, word-strings, and sentences. Three length conditions were studied in words, two length conditions for word-strings, and three length conditions for sentences, yielding eight experimental conditions. Apraxic speakers exhibited significantly longer vowel and between-word segment durations than control speakers in all conditions. Apraxic speakers consistently produced longer vowel and between-word segment durations in sentence contexts than in word contexts. Further, intrasubject and intersubject variability for between-word segment durations were substantially greater for the apraxic speakers in sentences compared to word conditions, whereas control speakers exhibited greater homogeneity in sentence production. The differences in duration and variability in sentence production versus word or word-string production imply different mechanisms for executing motor programs for varying linguistic stimuli.

Chemistry of the lyxose-containing mycobacteriophage receptors of Mycobacterium phlei/Mycobacterium smegmatis.

Mycobacterium phlei (strain Timothy) (Mycobacterium smegmatis ATCC 19249) is characterized by the presence of a family of alkali-labile glycolipids, reminiscent of the trehalose-containing lipooligosaccharide class of antigens but lacking the nonreducing trehalose core. Through a combination of methylation analyses, 1H and 13C NMR, two-dimensional 1H/1H and 1H/13C NMR, fast atom bombardment-mass spectrometry, gas chromatography-mass spectrometry, and other analytical techniques, these new structures were shown to possess three distinct features. Firstly, they contained the pentose D-lyxose (Lyx), rarely found in biology, but an epimer of D-arabinose, a key component of the mycobacterial cell wall arabinogalactan and lipoarabinomannnan. Thus, it was apparent that these glycolipids are the same as those described by Bisso et al. and attributed with phage receptor properties [Bisso, G., Castelnuovo, G., Nardelli, M.-G., Orefici, G., Arancia, G., Lanéelle, G., Asselineau, C., & Asselineau, J. (1976) Biochemie 58, 87-97]. Secondly, the complex oligosaccharides within the glycolipids contain the repeating units Lyxn(6-O-CH3-Glc)m and Lyxn(6-O-CH3-Glc)mMan1, where n+m equal to approximately 16 glycosyl residues. Thirdly, the M. phlei glycolipids were found to be heavily O-acylated, such that every D-Lyx residue invariably possesses an acyl function at position -2 and, in some instances, at both positions -2 and -4. The chemical characterization of these glycolipids, not feasible 20 years ago, clearly demonstrates that they are distinct from the type- and species-specific glycopeptidolipids, lipooligosaccharides, phenolic glycolipids, and the genus-specific phosphatidylinositol-based lipoglycans of mycobacteria. This present and previous studies begin to define the precise structural requirements responsible for the attachment of mycobacteriophage to the host cell wall.

Galactofuranose biosynthesis in Escherichia coli K-12: identification and cloning of UDP-galactopyranose mutase.

We have cloned two open reading frames (orf6 and orf8) from the Escherichia coli K-12 rfb region. The genes were expressed in E. coli under control of the T7lac promoter, producing large quantities of recombinant protein, most of which accumulated in insoluble inclusion bodies. Sufficient soluble protein was obtained, however, for use in a radiometric assay designed to detect UDP-galactopyranose mutase activity (the conversion of UDP-galactopyranose to UDP-galactofuranose). The assay is based upon high-pressure liquid chromatography separation of sugar phosphates released from both forms of UDP-galactose by phosphodiesterase treatment. The crude orf6 gene product converted UDP-[alpha-D-U-14C]-galactopyranose to a product which upon phosphodiesterase treatment gave a compound with a retention time identical to that of synthetic alpha-galactofuranose-1-phosphate. No mutase activity was detected in extracts from cells lacking the orf6 expression plasmid or from orf8-expressing cells. The orf6 gene product was purified by anion-exchange chromatography and hydrophobic interaction chromatography. Both the crude extract and the purified protein converted 6 to 9% of the UDP-galactopyranose to the furanose form. The enzyme was also shown to catalyze the reverse reaction; in this case an approximately 86% furanose-to-pyranose conversion was observed. These observations strongly suggest that orf6 encodes UDP-galactopyranose mutase (EC 5.4.99.9), and we propose that the gene be designated glf accordingly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified UDP-galactopyranose mutase revealed one major band, and analysis by electrospray mass spectrometry indicated a single major species with a molecular weight of 42,960 +/- 8, in accordance with that calculated for the Glf protein. N-terminal sequencing revealed that the first 15 amino acids of the recombinant protein corresponded to those expected from the published sequence. UV-visible spectra of purified recombinant enzyme indicated that the protein contains a flavin cofactor, which we have identified as flavin adenine dinucleotide.

A unique multifucosylated -3GalNAc beta 1-->4GlcNAc beta 1-->3Gal alpha 1- motif constitutes the repeating unit of the complex O-glycans derived from the cercarial glycocalyx of Schistosoma mansoni.

The entire surface of the cercarial stage of the human blood fluke Schistosoma mansoni is covered by a 1-microns thick, highly immunogenic, fucose-rich glycocalyx (GCX). Using strategies based on enzymatic, chemical, and mass spectrometric analysis, we have defined the structures of the major glycans released by reductive elimination from GCX. They comprise a heterogeneous population of multifocosylated complex oligosaccharides with the following nonreducing terminal sequences: [formula: see text] Our structural data suggest that these tri- to pentafucosylated epitopes are carried on type 1, R-->Gal beta-1-->3GalNAc, and type 2, R-->Gal beta 1-->3(R-->GlcNAc beta-1-->6)GalNAc, core structures via repeat units of (3GalNAc beta 1-->4(Fuc alpha 1-->2Fuc alpha 1-->2Fuc alpha 1-->3)GlcNAc beta-1-->3Gal alpha-->)n, where n is mainly 0 and 1, and all sugars are in the pyranose form. The proposed structure represents the first instance where an alpha-galactosylated beta-GalNAc(1-->4)-beta-GlcNAc sequence occurs as a repeating unit in a glycoprotein. It is also unique in being substituted with oligofucosyl appendages. The unusual oligosaccharide structures described here, particularly the potentially immunodominant oligofucosyl moieties, are most likely responsible for the known potency of GCX in modulating various immune responses including complement activation, B cell mitogenesis, and delayed type hypersensitivity in schistosomiasis.

A new interpretation of the structure of the mycolyl-arabinogalactan complex of Mycobacterium tuberculosis as revealed through characterization of oligoglycosylalditol fragments by fast-atom bombardment mass spectrometry and 1H nuclear magnetic resonance spectroscopy.

Previous structural analysis of small oligosaccharide fragments had allowed the recognition of several small structural motifs within arabinogalactan, the dominant cell was structural polysaccharide of Mycobacterium tuberculosis. To determine how these motifs are connected to one another to form the complete polymer, oligosaccharide fragments containing up to 26 glycosyl residues were released by gentle acid hydrolysis of the per-O-methylated arabinogalactan, converted to fully per-O-alkylated oligoglycosylalditols, and purified by high-performance liquid chromatography, and the molecular weights and alkylation patterns of the resultant oligoglycosyl fragments were determined by fast atom bombardment mass spectrometry. The results, combined with previous studies, allowed further understanding of the intricate structural features of the nonreducing ends of arabinogalactan. Thus, the extended nonreducing ends of the arabinan were shown to consist of a tricosaarabinoside (23-mer). We reason that three such arabinan motifs are attached to the homogalactan component or arabinogalactan, which was previously shown to consist of alternating 5- and 6-linked beta-D-galactofuranosyl residues. Using the same approach as applied to the arabinan branches, an extended stretch of the galactan was isolated that consisted of at least 23 alternating beta-1,6 and beta-1,5 D-Galf residues, devoid of any branching, demonstrating that the points of attachment of the arabinan chains to galactan are close to the reducing end of galactan, which itself is linked to peptidoglycan via the linker disaccharide phosphate L-Rhap-(1-->3)-alpha-D-GlcNAc-P. By nuclear magnetic resonance analysis, the L-Rhap was shown to be in the alpha configuration. The long-chain alpha-alkyl-beta-hydroxy mycolic acids, known to occupy the 5-positions of both the terminal beta-D-Araf and internal 2-alpha-D-Araf residues of the terminal branched pentaarabinosyl motif, are now shown to be nonacylated at the beta-hydroxy function. Lack of acylation points to intramolecular hydrogen bonding between the beta-hydroxyl and carbonyl functions of the mycolic acid, providing a highly ordered arrangement of mycolic acids in accord with evolving models of the orientation of the cell wall polymers in mycobacterial cell walls. A revised model is proposed for the composition and orientation of the mycolyl-arabinogalactan in the cell walls of M. tuberculosis, which should increase our understanding of cell wall hydrophobicity, impermeability, and role in disease pathogenesis.

Recognition of multiple effects of ethambutol on metabolism of mycobacterial cell envelope.

Ethambutol is known to rapidly inhibit biosynthesis of the arabinan component of the mycobacterial cell wall core polymer, arabinogalactan (K. Takayama and J. O. Kilburn, Antimicrob. Agents Chemother. 33:1493-1499, 1989). This effect was confirmed, and it was also shown that ethambutol inhibits biosynthesis of the arabinan of lipoarabinomannan, a lipopolysaccharide noncovalently associated with the cell wall core. In contrast to cell wall core arabinan, which is completely inhibited by ethambutol, synthesis of the arabinan of lipoarabinomannan was only partially affected, demonstrating a differential effect on arabinan synthesis in the two locales. Further studies of the effect of ethambutol on cell wall biosynthesis revealed that the synthesis of galactan in the cell wall core is strongly inhibited by the drug. In addition, ethambutol treatment resulted in the cleavage of arabinosyl residues present in the mycobacterial cell wall; more than 50% of the arabinan in the cell wall core was removed from the wall 1 h after addition of the drug to growing mycobacterial cultures. In contrast, galactan was not released from the cell wall during ethambutol treatment. The natural function of the arabinosyl-releasing enzyme remains unknown, but its action in combination with inhibition of synthesis during ethambutol treatment results in severe disruption of the mycobacterial cell wall. Accordingly, ethambutol-induced damage to the cell wall provides a ready molecular explanation for the known synergetic effects of ethambutol with other chemotherapeutic agents. Nevertheless, the initial direct effect of ethambutol remains to be elucidated.