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Novel structures of glycoinositolphosphorylceramide (GIPC) from the infective yeast form of Sporothrix schenckii were determined by methylation analysis, mass spectrometry and NMR spectroscopy. The lipid portion was characterized as a ceramide composed of C-18 phytosphingosine N-acylated by either 2-hydroxylignoceric acid (80%), lignoceric (15%) or 2,3-dihydroxylignoceric acids (5%). The ceramide was linked through a phosphodiester to myo-inositol (Ins) which is substituted on position O-6 by an oligomannose chain. GIPC-derived Ins oligomannosides were liberated by ammonolysis and characterized as: Manpalpha1-->6Ins; Manpalpha1-->3Manpalpha1-->6Ins; Manpalpha1-->6Manpalpha1-->3Manpalpha1-->3Manpalpha1-->6Ins; Manpalpha1-->2Manpalpha1-->6Manpalpha1-->3Manpalpha1-->3Manpalpha1-->6Ins. These structures comprise a novel family of fungal GIPC, as they contain the Manpalpha1-->6Ins substructure, which has not previously been characterized unambigously, and may be acylated with a 2,3 dihydroxylignoceric fatty acid, a feature hitherto undescribed in fungal lipids.

Two-dimensional gel electrophoresis (2-DE) enables separation of complex mixtures of proteins on a single polyacrylamide gel according to isoelectric point, molecular weight, solubility, and relative abundance. For this reason, 2-DE together with mass spectrometry (MS) has become a key technology in proteome analysis. The introduction of immobilised pH gradients (IPGs) for isoelectric focusing of proteins affords improved reproducibility and permits full-scale proteome analyses to be undertaken. Whilst broad-range IPGs are useful for investigating simple proteomes (e.g. Mycoplasma genitalium) it is becoming clear that additional resolving power is needed for separating the more complex proteomes of eukaryotic organisms. The use of narrow-range and very narrow-range IPGs provides the means with which to dissect a complex proteome. We have compared very narrow-range IPGs (3.5-4.5L, 4-5L, 4.5-5.5L, 5-6L, and 5.5-6.7L) with broad- (3-10NL) and narrow-range IPGs (4-7L and 6-9L) for the visualisation of the human heart proteome. The superior ability of very narrow-range IPGs to separate different protein species and isoforms, compared with 3-10NL and 4-7L 2-D gels is demonstrated. The results are supported by MS identifications which further show that reduction of the number of comigrating protein species results in less ambiguous and more reliable database search results.

We have investigated the biological fluids--serum, cerebrospinal fluid, and urine--of three strains of rats; the present data extend our database (also available on-line) and may be of interest for pharmacological and toxicological investigation. Specifically, we have defined reference maps of the major protein components in cerebrospinal fluid and urine. Compartment-specific isoforms were recognized for transferrin and transthyretin. Mass spectrometric data established the cleavage site of the signal peptide and identified the N-terminal blocking group of prostaglandin D synthase from rat cerebrospinal fluid. A previously undescribed member of the family of low molecular mass rat urinary proteins was characterized as containing a sequence similar, but not identical, to the N-terminal region of rat urinary protein-2 (RUP-2), and divergent from RUP-1.

We report the first protein map of human adult lung MRC-5 fibroblasts using isoelectric focusing-immobilized pH gradient-based two-dimensional polyacrylamide gel electrophoresis. MRC-5 is an immortalised cell line used in a wide range of investigations. The two-dimensional gel pattern of proteins generated from any given cell system provides a fingerprint that is unique to those cells. Therefore, the establishment of a protein map for a particular cell system provides a useful reference tool as a "master map" for subsequent studies using those cells. In this map a total of 98 protein spots were identified by comparative searches of the nucleotide and protein database using peptide masses obtained by matrix-assisted laser desorption/ionization time of flight following trypsin digestion. To increase the utility of the reference map, cells were cultured in both Dulbecco's modified Eagle medium (DMEM), the standard medium, and Roswell Park Memorial Institute (RPMI)-1640. Two-dimensional gel protein patterns of MRC-5 cultures were shown to be largely unaffected by the use of RPMI compared to DMEM, respectively. In combination with the reference map, the standardised protocol described provides a tool for comparative studies involving MRC-5 cells in which nonspecific variation is minimized.

Two-dimensional gel electrophoresis (2-DE) remains the method of choice for the Separation of protein mixtures whilst mass spectrometry (MS) is rapidly becoming the premier tool for protein identification. When combined, 2-DE and MS form the current operating paradigm for classical proteomics. One of the key challenges of proteome research is that of detecting and identifying all of the elements (proteins) of a proteome. Silver staining and radiolabelling, e.g. with 35S-methionine ([35S]-met), represent two sensitive methods used to visualise many of the constitutive and synthesised elements of a proteome, respectively. The latter method allows a very low total protein loading on a two-dimensional (2-D) gel and challenges protein identification using current MS-based technology. Therefore, it is necessary to refer to and locate a radiolabelled spot's cognate on a preparatively loaded stained gel, or Western blot, and use that protein spot for identification. Unfortunately, the images of autoradiographs and preparative gels or blots, even of the same sample, often do not correspond making it difficult to accurately locate and select spots of interest by visual comparison. We have established a technique that permits the unambiguous localisation of radiolabelled proteins on the same silver stained 2-D gel. Protein identification of superimposed spots is described by peptide mass fingerprinting and database searching using matrix-assisted laser desorption/ionization-time of flight mass spectrometry and by peptide sequencing using tandem MS by hybrid quadrupole/orthogonal acceleration time of flight MS (Q-TOF).

Skeletal muscle plays a major role in whole body protein metabolism, and changes in the rates of synthesis and degradation of proteins are likely to lead to characteristic changes in the amounts of different proteins in muscle under various physiological and pathological conditions. This paper demonstrates the feasibility of a proteomic approach to analyzing the protein composition of skeletal muscle. We report here the initial establishment of two-dimensional gel electrophoresis (2-D PAGE) reference maps for mixed skeletal muscle taken from the abdominal wall of a normal adult rat. We used immobilized pH gradients of 3-10 (non-linear) and 4-7 (linear), and matrix assisted laser desorption/ionization--time of flight mass spectrometry for protein identification by peptide mass fingerprinting. More than 600 protein spots were detected on each gel, of which 100 were excised and characterized. In-gel digestion followed by peptide mass fingerprinting enabled tentative identification of 74 of these, which included a wide range of myofibrillary and sarcoplasmic proteins. This database should provide the nucleus of a valuable resource for investigation of the biochemical basis of skeletal muscle pathologies in general and such specific disorders as alcoholic myopathy and injury.

Posttranscriptional regulation is important for tumor necrosis factor alpha (TNF-alpha) expression in monocytes and macrophages, and an AU-rich element (ARE) in the 3' untranslated region (UTR) of TNF-alpha mRNA is implicated in control of its translation and mRNA stability. Regulation of mRNA turnover is thought to be mediated by trans-acting proteins, which bind the ARE and stabilize or destabilize the transcript. However, with the exception of the destabilizing factor tristetraprolin, the identity and function of the proteins binding the TNF-alpha mRNA ARE have not been established. To identify other proteins involved in the posttranscriptional control of TNF-alpha, the subcellular location of TNF-alpha mRNA was determined in the macrophage-like cell line RAW 264.7. TNF-alpha mRNA was located in the pellet following centrifugation of cytoplasm at 100,000 x g (P100 fraction). This fraction also contained proteins which formed two distinct ARE-specific complexes with the TNF-alpha mRNA 3' UTR in electrophoretic mobility shift assays (EMSAs). A protein present in these two complexes was purified and identified by peptide mass mapping and tandem mass spectrometry as HuR. In EMSAs both complexes were supershifted by an anti-HuR antibody, while Western blotting also demonstrated the presence of HuR in the P100 extract. A HeLa cell tetracycline-regulated reporter system was used to determine the effect of HuR on mRNA stability. In this system, overexpression of HuR resulted in stabilization of an otherwise unstable reporter-mRNA containing the TNF-alpha ARE. These results demonstrate that the TNF-alpha ARE is a target of the mRNA-stabilizing factor HuR.

Glycoproteins on the cell surface of Trypanosoma cruzi are known to play important roles in the interaction of the parasite with the host cells. We previously determined the structures of the O-glycan chains from the sialoglycoproteins (mucin-like molecules) of the G- and Y-strains and observed significant differences between them. We now report the structures of the sialylated and nonsialylated O-linked oligosaccharides isolated from the cell surface glycoproteins of the myotropic CL-Brener strain grown in the presence of fetal calf serum. The structures of the O-linked oligosaccharide alditols obtained by reductive beta-elimination of the sialoglycoprotein were determined by a combination of methylation analysis, fast atom bombardment-mass spectrometry and nuclear magnetic resonance spectroscopy. The presence of a beta-galactopyranose substituent on the N-acetylglucosamine O-4 position shows that these O-linked oligosaccharides from CL-Brener strain belong to the same family as those isolated from mucins expressed by T. cruzi Y strain, a reticulotropic strain. In addition, novel O-glycans, including alpha2-3 mono-sialylated species are described.

The growing availability of genomic sequence information, together with improvements in analytical methodology, have enabled high throughput, high sensitivity protein identification. Silver staining remains the most sensitive method for visualization of proteins separated by two-dimensional gel electrophoresis (2-D PAGE). Several silver staining protocols have been developed which offer improved compatibility with subsequent mass spectrometric analysis. We describe a modified silver staining method that is available as a commercial kit (Silver Stain PlusOne; Amersham Pharmacia Biotech, Amersham, UK). The 2-D patterns abtained with this modified protocol are comparable to those from other silver staining methods. Omitting the sensitizing reagent allows higher loading without saturation, which facilitates protein identification and quantitation. We show that tryptic digests of proteins visualized by the modified stain afford excellent mass spectra by both matrix-assisted laser desorption/ionization and tandem electrospray ionization. We conclude that the modified silver staining protocol is highly compatible with subsequent mass spectrometric analysis.

The structure of a glycosylphosphatidyl inositol-anchored glucoxylan (GPI-glucoxylan) synthesized by the monogenetic trypanosomatid Leptomonas samueli has been determined. The glucoxylan is anchored to the membrane by phytoceramide and an oligosaccharide core, the structure of which is identical to glycoinositolphospholipids (GIPLs) expressed by this protozoan. The glucoxylan chain is linear, containing -->4Glcalpha1-->, -->4Xylbeta1--> and -->3Xylbeta1--> residues. A well defined sequence heterogeneity was analysed in terms of a series of overlapping trisaccharide substructures. A proportion of the chains are capped with a GlcAalpha1-->3Glcalpha1--> sequence. While an average GlcA-capped chain contained 10 Glc and 16 Xyl residues, uncapped chains have a higher molecular mass with an average of 30 Glc and 50 Xyl per chain. We propose a mode of biosynthesis based on the observed structural heterogeneity.

Background: Neoadjuvant chemotherapy facilitates breast conservation in stage II breast cancer patients, whose primary tumors are assumed to be invasive because they are palpable. However, chemotherapy may not be indicated in the minority of patients whose clinically T2 tumors are completely or predominantly in situ. Almost all previous studies of core needle biopsy in breast cancer have been concerned with nonpalpable, mammographically detected tumors, and none have evaluated its ability to quantitatively determine the amounts of in situ and invasive disease., Methods: From September, 1992 to December, 1997, core needle biopsy was performed on all patients presenting to the Kings County Hospital Breast Clinic with palpable breast masses. Carcinoma was present in both core needle biopsy samples and surgical specimens subsequently obtained from 95 of 99 patients. Each specimen was evaluated for tumor type, histologic grade, and the amounts of in situ and invasive carcinoma it contained, and the results from surgical and core needle biopsy specimens from the same patients were then compared., Results: The surgical specimens of 14 patients had completely or predominantly in situ disease. Completely or predominantly invasive disease was present in 67 specimens, and the remaining 14 had significant amounts of both. The high level of agreement between the amounts of in situ and invasive disease in core needle biopsy and surgical specimens is indicated by Pearson and intraclass correlation coefficients of 0.91 (P < .001 and < .00001, respectively). Tumor type was correctly predicted by core needle biopsy in each case. Variables among these patients, including primary tumor size, interval between biopsy and surgery, or administration of neoadjuvant systemic therapy, did not alter agreement between core needle biopsy and surgical specimens., Conclusions: Core needle biopsy can identify palpable breast tumors that are predominantly or completely in situ, and, thus, avoid unnecessary neoadjuvant chemotherapy. It also can demonstrate that a tumor is predominantly invasive, but cannot rule out small invasive foci. For that purpose, complete surgical excision of the tumor is required.

In this study we propose revised structures for the two major compatible solutes of Rhodothermus marinus. We have also examined the accumulation of compatible solutes by the type strains of the slightly halophilic and thermophilic species Rhodothermus marinus and Rhodothermus obamensis at several growth temperatures and salinities. The major solutes of R. marinus were identified as alpha-mannosylglycerate (alpha-MG) and alpha-mannosylglyceramide (alpha-MGA), whereas R. obamensis accumulated only alpha-mannosylglycerate. The total osmolyte content was higher during the early exponential phase and decreased abruptly as growth continued into the stationary phase. At low growth temperatures. R. marinus responded to water stress by accumulation of alpha-mannosylglycerate and its amide, in addition to low levels of trehalose, glutamate, and glucose. At the highest growth temperature, alpha-mannosylglycerate was the major compatible solute and alpha-mannosylglyceramide was not detected. When both compounds were present, an increase in the salinity of the growth medium favored the accumulation of alpha-mannosylglyceramide over alpha-mannosylglycerate. The absence of alpha-mannosylglyceramide in R. obamensis at all growth temperatures and salinities constituted the most pronounced difference in the profiles of compatible solute accumulation by the two strains. Trehalose was also a prominent solute in this organism. Both organisms accumulated higher levels of alpha-mannosylglycerate as the temperature was raised. The importance of the two compounds in the mechanisms of thermoadaptation and osmoadaptation is discussed.

Thin-layer chromatographic analysis of the polar lipids of Meiothermus strains revealed two glycolipid bands with similar chromatographic mobility to the major glycolipid of Thermus strains. In this study the glycolipids from the type strains of Meiothermus ruber, Meiothermus chliarophilus, Meiothermus silvanus and Meiothermus cerbereus were characterized using GC, GC/MS, fast atom bombardment MS and chemical methods. All strains contained dihexosyl-(N-acyl)hexosaminylglucosyl diacylglycerols, related in structure to the major glycolipid of Thermus strains but varying in their fatty acylation pattern. The detection of two glycolipid bands by TLC in Meiothermus spp. was attributable to the invariable presence of 2-hydroxyacyl groups N-linked to the hexosamine of the polar head group which cause the glycolipids to be more strongly retained on silica TLC plates than 3-hydroxy or non-hydroxylated N-acyl glycolipids of similar structure that are also present. M. silvanus contained, in addition to these glyceroglycolipids, several glycolipids which were linked to acylated branched octadecanediols rather than to glycerol. The presence of glycolipids containing 2-hydroxyacyl groups N-linked to hexosamine appears to be a stable phenotypic marker that distinguishes the genus Meiothermus from the genus Thermus.

Chloroform-soluble material was extracted from two strains of L. pneumophila serogroup 1 following growth in continuous culture. The purified material was identified as poly-3-hydroxybutyrate (PHB) by nuclear magnetic resonance spectroscopy and by gas chromatography-mass spectrometry. PHB yields of up to 16% of cell dry weight were extracted from culture samples. The PHB was located in electron-dense intracellular inclusions, which fluoresced bright yellow when stained with the lipophilic dye Nile red. A Nile red spectrofluorometric assay provided a more accurate and reliable determination of the PHB content. PHB accumulation increased threefold during iron-limited culture and was inversely related to the concentration of iron metabolized. Chemostat-grown cells survived in a culturable state for at least 600 days when incubated at 24 degreesC in a low-nutrient tap water environment. Nile red spectrofluorometry and flow cytometry demonstrated that PHB reserves were utilized during starvation. PHB utilization, as revealed by the decline in mean cellular fluorescence and cell complexity, correlated with loss of culturability. Fluorescence microscopy provided visual evidence of PHB utilization, with a marked reduction in the number of Nile red-stained granules during starvation. Heat shock treatment failed to resuscitate nonculturable cells. This study demonstrates that L. pneumophila accumulates significant intracellular reserves of PHB, which promote its long-term survival under conditions of starvation.

A novel phospholipid has been purified from strain Dm 28c of Trypanosoma cruzi, and characterized by fast atom bombardment mass spectrometry and nuclear magnetic resonance spectroscopy as a plasmenylethanolamine with a hexadec-l-enyl group in the sn-1 position and an approximately equimolar mixture of octadecenoate and octadecadienoate esterified to the sn-2 hydroxyl. The purified plasmenylethanolamine reacted positively when probed with sera from patients with chronic Chagas' disease. Since plasmenylethanolamines of similar structure are abundant in mammalian cardiac and neuronal tissues, cross reactions between these epitopes may be a factor in the mechanism of autoimmune pathology in the chronic phase of Chagas' disease.

Glycosphingolipids were extracted from hyphae of Fusarium solani and from an unnamed Fusarium species, and were purified by silica and Iatrobead column chromatography. Their structures were determined by compositional analysis, nuclear magnetic resonance spectroscopy, gas chromatography/mass spectrometry and by fast atom bombardment mass spectrometry of the native and peracetylated materials, which defined their sugar, long-chain base and fatty acid compositions. The locations of the double bonds in the bases were established by 2D NMR spectroscopy and by novel mass spectrometric approaches, including collisional activation of the protonated and lithium-cationized glycosphingolipids, and of the sphingadienene-derived fragment ion at m/z 276. From these results we propose that the structures of the glycosphingolipids from F. solani and Fusarium sp. are N-2'-hydroxyoctadecanoyl-1-O-beta-D-glucopyranosyl-9-methyl-4, 8-sphingadienine and N-2'-hydroxyoctadecenoyl-1-O-beta-D-glucopyranosyl-9-methyl-4, 8-sphingadienine, respectively., (Copyright 1998 Elsevier Science B.V.)

We have characterized glycoinositol phospholipids (GIPLs) from three strains of the trypanosomatid parasites Endotrypanum schaudinni and Endotrypanum monterogeii. Methanolysis of the intact GIPLs liberated methyl esters of tetracosanoic acid, docosanoic acid, octadecanoic acid and hexadecanoic acid and C20 and C21 phytosphingosines. Phosphoinositol oligosaccharides were released from the GIPLs by mild base treatment, and their structures were determined by compositional analysis, fast-atom-bombardment MS and NMR spectroscopy. Similar compounds were detected in all three strains, although their relative proportions varied. The predominant components in E. schaudinni strain LV59 and E. monterogeii LV88 were Galpbeta1-3Galpbeta1-3Manalpha1-3Manalpha1-4G lcNalpha1-6Ins-1-P and Arapbeta1-2Ga lpbeta1-3Galpbeta1-3Manalpha1-3Manalpha1-4Glc Nalpha1-6Ins-1-P, and the major phosphoinositol oligosaccharide in E. schaudinni LV58 was the hybrid-type GIPL Manalpha1-2(EtNP-6)Manalpha1-6(Galpbeta1-3Man alpha1-3)Manalpha1-4GlcN alpha1-6Ins-1-P (where EtNP is ethanolamine phosphate). Several minor oligosaccharides containing additional galactose and/or arabinose residues were also detected.

Background and Objectives: Locally advanced breast cancers may form large, infected skin ulcers, which were traditionally treated with radiation therapy. Neoadjuvant chemotherapy is now standard treatment for locally advanced breast cancer., Methods: The response of 33 patients with ulcerated breast cancer to primary chemotherapy was retrospectively analyzed. Antibiotics were not used in primary treatment. Tumor and ulcer responses were evaluated independently., Results: Chemotherapy alone healed 18 of these ulcers. Neither responding nor refractory patients developed sepsis during this treatment., Conclusions: Chemotherapy is safe and effective treatment for patients with infected malignant breast ulcers and does not cause systemic sepsis.

In this study, we purified and characterized tetra- and triglycosyl glycolipids (GL-1 and GL-2, respectively) from two different colonial forms of Thermus scotoductus X-1, from T. filiformis Tok4 A2, and from T. oshimai SPS-11. Acid hydrolysis of the purified glycolipids liberated, in addition to the expected long-chain fatty acids, two components which were identified by gas chromatography-mass spectrometry as 16-methylheptadecane-1,2-diol and 15-methylheptadecane-1,2-diol. Fast atom bombardment mass spectrometry of the intact glycolipids indicated that a major proportion consisted of components with glycan head groups linked to long-chain 1,2-diols rather than to glycerol, although in all cases glycerol-linked compounds containing similar glycan head groups were also present. As in other Thermus strains, the polar head group of GL-1 from T. filiformis Tok4 A2 and from T. scotoductus X-1 colony type t2 was a glucosylgalactosyl-(N-acyl)glucosaminylglucosyl moiety. However, GL-2 from T. scotoductus X-1 colony type t1 and from T. oshimai SPS-11 was a truncated analog which lacked the nonreducing terminal glucose. Long-chain 1,2-diols have been previously reported in the polar lipids of Thermomicrobium roseum and (possibly) Chloroflexus aurantiacus, but to our knowledge, this is the first report of their detection in other bacteria and the first account of the structural determination of long-chain diol-linked glycolipids.

Strains of Deinococcus geothermalis sp. nov. were isolated from the hot spring and runoff at Agnano, Naples, Italy, and from the hot spring at São Pedro do Sul in central Portugal, while strains of Deinococcus murrayi sp. nov. were isolated from the hot springs at São Pedro do Sul, São Gemil, and Alcafache in central Portugal. The strains of D. geothermalis and D. murrayi produce orange-pigmented colonies and have an optimum growth temperature of about 45 to 50 degrees C. The type strains of the two new species are extremely gamma radiation resistant. The fatty acids of these new species are primarily branched-chain fatty acids. The two new species can be distinguished from each other by the lower pH range of D. geothermalis than of D. murrayi, by their fatty acid compositions, and by several biochemical parameters, including the ability of D. geothermalis to grow in minimal medium without yeast extract. 16S rRNA gene sequencing also showed that the isolates constitute two species and that these species are distinct from the other species of the genus Deinococcus. The type strain of D. geothermalis is AG-3a (= DSM 11300), and the type strain of D. murrayi is ALT-1b (= DSM 11303).

Glycoinositolphospholipids (GIPLs) were isolated from promastigotes of the lizard parasites Leishmania adleri by phenol/water extraction. Phosphoinositol oligosaccharides were liberated by mild alkaline hydrolysis, purified by gel filtration and high pH anion exchange chromatography, and characterized by methylation analysis, fast atom bombardment mass spectrometry, and nuclear magnetic resonance spectroscopy. The four major compounds (I-IV) from L. adleri were linked to alkylacyl glycerol, and their glycan moieties had the following structures: Man alpha(1-2)Man alpha(1-6)[Man alpha(1-3)] Man alpha(1-4)GlcN alpha(1-6)Ins-1-PO4 (I), Galp alpha(1-6) Galp alpha(1-3)Galf beta(1-3)Man alpha(1-3)Man alpha(1-4)GlcN alpha(1-6)Ins-1-PO4 (II), Galp alpha(1-3)Galf beta(1-3)Man alpha(1-3) Man alpha(1-4)GlcN alpha(1-6)Ins-1-PO4 (III), Man alpha(1-2)[EtNP(-6)]Man alpha(1-6)[Man alpha(1-3)] Man alpha(1-4)GlcN alpha(1-6)Ins-1-PO4 (IV). These compounds are analogous to the previously characterized GIPLs from New and Old World leishmanial parasites of mammals designated iM4 (identical to compound I), GIPLs 3 and 2 (identical to compounds II and III, respectively), and EPiM4 (identical to compound IV), which is consistent with a close phylogenetic relationship between lizard and mammalian Leishmania. However, in contrast to the mammalian parasites, the abundant surface glycoconjugate known as lipophosphoglycan was either absent or confined to the flagellar pocket region in L. adleri.

A dermatan sulfate-like glycosaminoglycan was isolated from the body of the ascidian Ascidia nigra (J. Biol. Chem. 270: 31027-31036, 1995). 1H NMR and fast atom bombardment mass spectrometry (FAB-MS) spectra of the tetra and disaccharides formed by chondroitin ABC lyase digestion support the proposed repeating disaccharide structure for this glycosaminoglycan, [-->4)-alpha-L-IdoA(2SO4)-(1-->3)-beta-D-GalNAc(6SO4)-(1-->] , which differ from mammalian dermatan sulfate in its sulfation at both 2-position of the alpha-L-iduronic acid and the 6-position of the N-acetyl-beta-D-galactosamine residue.

The molecular weight profiles of low molecular weight heparin samples have been measured by high-performance gel permeation chromatography using as calibrant the heparinase-degraded material (90/686) now established as the 1st International Reference Preparation (IRP) Low Molecular Weight Heparin for Molecular Weight Calibration Use of the calibrant as a broad molecular weight standard is described and a calibration table provided based on data collected over several years in one laboratory. In order to confirm the assignment of degree of polymerisation to resolved oligosaccharide peaks in the calibrant, molecular weights of oligosaccharides fractionated from the 1st IRP were independently determined by fast atom bombardment mass spectrometry (FAB MS). The molecular weight distributions of commercial low molecular weight heparins have been characterized. Measurements of molecular weight parameters of heparin molecular weight standards from several sources provide comparisons between the molecular weight scales of this and other studies.

The structures of the glycoinositolphospholipids (GIPLs) from five strains of the protozoan parasite Trypanosoma cruzi have been determined. Two series of structures were identified, all but one containing the same Man4(AEP)GlcN-Ins-PO4 core. Series 1 oligosaccharides are substituted at the third mannose distal to inositol (Man 3) by ethanolamine-phosphate or 2-aminoethylphosphonic acid, as are some glycosyl-phosphatidylinositol-protein anchors of T. cruzi. The core can be further substituted by terminal (1-3)-linked beta-galactofuranose units. In contrast, Series 2 oligosaccharides do not have additional phosphorus-containing groups attached to Man 3, the latter being substituted instead by a single side chain unit of beta-galactofuranose. Series 1 oligosaccharides are present in all strains (G, G-645, Tulahuen CL, and Y) whereas Series 2 structures are present mainly in CL and Y strains. The lipid moiety in the GIPLs from the G, G-645 and Tulahuen strains is predominantly ceramide, as reported for the Y strain, whilst that from the CL strain is a mixture of ceramide and alkylacylglycerol species. The lipid moiety of the GIPLs, and probably also the phosphoinositol-oligosaccharide structures may play an important immunomodulatory role in infection by T. cruzi.

The compositions of the major glycolipids (GL-1) of five strains of Thermus aquaticus, the type strain of T. filiformis, T. oshimai SPS-11, and Thermnus sp. strain CG-2 were examined by gas chromatography, gas chromatography-mass spectroscopy, fast atom bombardment-mass spectroscopy, and chemical methods. The results showed that, with the exception of T. aquaticus 15004, the organisms each have a major glycolipid whose structure was established as diglycosyl-(N-acyl)glycosaminyl-glycosyl diacylglycerol. Glucosamine was present in GL-1 of T. oshimai SPS-11 and Thermus sp. strain CG-2, while galactosamine was present in the GL-1 of T. aquaticus and T. filiformis. The novel major glycolipid of T. aquaticus 15004 was identified as galactofuranosyl-(N-acetyl)galactosaminyl-(N-acyl)galactosaminyl-gluc - osyl diacylglycerol. The hydroxy fatty acids found in the T. aquaticus strains and in the type strain of T. filiformis were exclusively amide linked to the galactosamine of the major glycolipid. Ester-linked hydroxy fatty acids were not detected in the diacylglycerol moiety of GL-1 of these organisms. Hydroxy fatty acids were detected neither in the major glycolipid of T. oshimai SPS-11 and Thermnus sp. strain CG-2, in which glucosamine is present, nor in the major phospholipid of any of the strains examined.

A newly isolated strain of Variovorax paradoxus could grow on homovanillate and several monohydroxylated phenylacetic acids. During growth on homovanillate, the organism formed separate NAD(P)H-dependent hydroxylases with activity towards 4-hydroxyphenylacetic acid and homovanillate. Homovanillate hydroxylase catalysed a typical monooxygenase reaction and had little activity towards 4-hydroxyphenylacetic acid GC-MS and TLC analysis suggested that homovanillate was 1-hydroxylated to yield a dihydroxymonomethoxyphenylacetic acid which served as a substrate for homogentisate 1,2-dioxygenase. Methanol, but not formaldehyde, was released either during ring-cleavage or subsequent metabolism of the ring-cleavage product.

We have investigated the structure of the glycosylphosphatidylinositol (GPI) anchor and the O-linked glycan chains of the 40/45-kDa glycoprotein from the cell surface of the protozoan parasite Trypanosoma cruzi. This glycoconjugate is the major acceptor for sialic acid transferred by trans-sialidase of T. cruzi Y-strain, epimastigote form. The GPI anchor was liberated by treatment with hot alkali, and the phosphoinositol-oligosaccharide moiety was characterized and shown to have the following structure. [formula: see text] Unusually the glucosamine was 6-O-substituted with 2-aminoethylphosphonate, and 2-aminoethylphosphonate was also present on the third mannose residue distal to glucosamine, partially replacing the ethanolamine phosphate. The beta-eliminated reduced oligosaccharide chains showed that two novel classes of O-linked N-acetylglucosamine oligosaccharide were present. The first series had the structures Galp beta 1-3GlcNAc-ol; Galp beta 1-6(Galp beta 1-3)GlcNAc-ol; and Galp beta 1-2Galp beta 1-6(Galp beta 1-3)GlcNAc-ol, whereas the other series had a 1-4 linkage to N-acetylglucosaminitol and had structures Galp beta 1-4GlcNAc-ol, Galp beta 1-6(Galp beta 1-4)GlcNAc-ol, and Galp beta 1-2Galp beta 1-6(Galp beta 1-4)GlcNAc-ol. We have also investigated the kinetics of in vitro sialylation of these O-linked oligosaccharides by the T. cruzi transsialidase and have shown that incorporation of one molecule of sialic acid hinders entry of a second molecule when two potential acceptor sites are present.

The structure of two glycosylinositolphospholipids of the cell surface of the monoxenic protozoan Herpetomonas samuelpessoai have been deduced by methylation analysis, fast-atom bombardment mass spectrometry and two dimensional nuclear magnetic resonance spectroscopy. These glycolipids have features in common with the glycoinositolphospholipids of both Leishmania and Trypanosoma cruzi, resembling the former by the presence of the hybrid type core sequence Man alpha 1-->3(Man alpha 1-->6)Man alpha 1-->4GlcN alpha 1-->6 myo-inositol-1-PO4-lipid, while the 2-aminoethylphosphonate substituent on 0-6 of glucosamine and the presence of ceramide in place of glycerol lipids is more reminiscent of T. cruzi. Possible phylogenetic implications of these observations are discussed.

Lysine vasopressin (LVP) readily reacts with reducing saccharides both in lyophilized preparations and in aqueous solution. Incubation of LVP with, for example, lactose over a pH range of 3.0-8.5 in phosphate buffer or simply in water, gives rise to a number of reaction products, some of which form rapidly (in a matter of hours) even in the frozen state. Reaction mixtures were analysed by reversed-phase HPLC and the structures of the products were deduced from the amino-acid composition of isolated components, by comparison with product profiles obtained with analogues under similar conditions and by FAB mass-spectral analysis of derivatives isolated after reduction with cyanoborohydride. The primary products arise from the formation of Schiff's bases at one or both of the two amino functions. The alpha-amino group of the N-terminal cystine is considerably more reactive than is the epsilon-amino group of lysine and it is the N-terminal adduct which rapidly forms even at -20 degrees C. It is concluded that caution must be shown in using reducing sugars in formulations containing peptides and proteins, particularly the vasopressins and oxytocin.

The composition of lipopolysaccharide (LPS) extracted with aqueous phenol from a virulent English plum isolate of Pseudomonas syringae pv. morsprunorum varied according to the partial pressure of oxygen (pO2) in the culture medium at the time of harvest. When pO2 was low, the organism grew slowly and produced smooth LPS bearing rhamnan sidechains. As pO2 was raised, the rate of growth increased and smooth LPS was replaced by a rough species deficient in rhamnose, which co-extracted with a D-glucan. Organization of rhamnose and glucose into separate polymers was shown by the selective susceptibility of the rhamnose-containing polymer to hydrolysis by rhamnanase of the phage A7. By methylation analysis, GC-MS, and 1H- and 13C-NMR spectroscopy, the glucan was shown to consist of alpha (1-->4)-linked residues with alpha (1-->4,6)-branch points and non-reducing terminal residues in the approximate ratio 4:1:1, resembling glycogen in composition. A glucan which co-extracted with LPS using phenol/water from an avirulent plum isolate that was resistant to lysis by phages A1 and A7 was shown by methylation analysis to have a similar structure. Whether the effect on LPS composition was due directly to pO2, or was dependent on the rate of growth, has not been established. It is suggested that, because epiphytic growth would entail exposure to high pO2, English plum isolates growing on the surfaces of host plants might be unable to produce smooth LPS. Since cell surface composition affects virulence in plant-pathogenic pseudomonads, this effect could account for the observed failure of the English plum isolates to enter the host via leaf scars.

In this report we describe studies on the structures of the O-linked carbohydrate units in cell-surface glycoproteins of epimastigote forms of the G-strain of Trypanosoma cruzi. Mild alkaline reductive degradation of the 38/43 kDa glycoproteins resulted in beta-elimination of glycosylated threonine and/or serine residues, and the liberation of N-acetylglucosaminitol, galactobiosyl-, galactotriosyl-, galactotetraosyl- and galactopentaosyl-N-acetylglucosaminitol. The structures of these oligosaccharide alditols were established by n.m.r. spectroscopy and methylation analysis as: Galf beta 1-4(Galp beta 1-6)GlcNAc-ol; Galp beta 1-3Galp beta 1-6(Galf beta 1-4)GlcNAc-ol; [(Galp beta 1-3)(Galp beta 1-2)Galp beta 1-6](Galf beta 1-4)GlcNAc-ol; [(Galp beta 1-3)(Galp beta 1-2)Galp beta 1-6](Galp beta 1-2Galf beta 1-4)GlcNAc-ol.

Mild alkaline hydrolysis of the glycophosphosphingolipids of the protozoan Leptomonas samueli liberated several phosphoinositol-containing oligosaccharides (PI-oligosaccharides), which were purified by high performance anion exchange chromatography. The oligosaccharides in the resulting four fractions were characterized by methylation analysis, fast atom bombardment mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. The oligosaccharides contain the core structure Man alpha (1-4)GlcN alpha (1-6)-myo-inositol-1-OPO3, and are substituted with 2 mol of 2-aminoethylphosphonate per mol of oligosaccharide. The nonreducing ends of the oligosaccharides were terminated by rhamnose branched neutral and acidic xylose-containing penta-, hexa-, hepta- and octasaccharides, of which the three most abundant were shown to have the structures: [formula: see text] More tentative structures are also proposed for three minor oligosaccharides.

Nuclear magnetic resonance (NMR) spectroscopy provides an extremely powerful technique to determine the structure of oligosaccharides, particularly when used in conjunction with other physical techniques such as methylation analysis and fast atom bombardment mass spectrometry (FAB-MS). This brief review describes the application of NMR to the determination of the structure of an oligosaccharide isolated from the glycophosphosphingolipid (GPS) from the monogenetic trypanosomatid Leptomonas samueli. When ambiguities arise in the NMR interpretation, the use of other data will be discussed.

Fast atom bombardment mass spectrometry (FAB MS) enables the rapid, accurate and sensitive determination of the molecular masses of glycosylphosphatidylinositol (GPI)-derived oligosaccharides, from which the composition in terms of monosaccharide residues and non-carbohydrate substituents can be determined. Interpretation of fragment ions in collisional activation mass spectra further enables the determination of residue sequence, the positions of branch points, and the location of non-carbohydrate substituents. We have applied these techniques to the characterization of phosphoinositol oligosaccharides from Leptomonas samueli, Endotrypanum schaudinni and Leishmania adleri. The mass spectral data permit the postulation of candidate structures for the oligosaccharides, which provide a set of constraints that can assist the interpretation of results from other techniques such as NMR.

Glycoinositolphospholipids (GIPLs) were extracted from the trypanosomatid Leishmania adleri by hot phenol extraction and the carbohydrate moieties isolated after base cleavage. Purification of the crude oligosaccharides by high performance anion exchange (HPAE) chromatography yielded four fractions whose structures were determined by a combination of methylation analysis, fast atom bombardment (FAB) mass spectrometry and two-dimensional nuclear magnetic resonance (NMR) spectroscopy.

Recent data suggest that botulinum type-B neurotoxin is a protease which acts on vesicle-associated membrane protein, isoform 2 (VAMP-2). In this report, botulinum type-B neurotoxin is shown to cleave a synthetic fragment (HV62) of VAMP-2, corresponding to the bulk of the hydrophilic domain (amino acids 33-94). The neurotoxin acts at a single site between Gln76 and Phe77. Little or no proteolytic activity by botulinum type-B neurotoxin was observed with peptides containing 7, 10 or 20 amino acids spanning the site of cleavage. The proteolytic action of neurotoxin was strongly inhibited by EDTA and o-phenanthroline whereas captopril and phosphoramidon were ineffective. A series of model peptide substrates were synthesised in order to define the smallest VAMP-2 fragment to be cleaved by botulinum type-B neurotoxin. Data obtained from these substrates suggest that the neurotoxin belongs to a novel class of zinc-endoprotease; more than 12 amino acid residues are required on both the NH2- and COOH-terminal side of the cleavage site for optimal proteolytic activity. The results demonstrate that no other components of cellular vesicles are required for the specific action of the neurotoxin on VAMP-2. The data further show that the highly specific action of the neurotoxin is not dictated solely by the properties of the amino acid residues at the cleavage site but is also dependent on amino acid sequences distal to its site of action.

Fourteen Legionella-like strains isolated from aquatic sources have been characterized serologically, biochemically, and in terms of DNA relatedness. The strains grew on buffered charcoal-yeast extract agar but not on blood agar and displayed phenotypic characteristics typical of the family Legionellaceae, including a requirement for cysteine, cellular fatty acid compositions in which branched-chain acids predominate, and the possession of isoprenoid quinones of the ubiquinone series with more than 10 isoprene units in their side chains. All were nonfermentative, lacked urease, were incapable of nitrate reduction, and reacted positively with a DNA probe specific for the Legionellaceae. DNA hybridization studies in which the hydroxyapatite method was used demonstrated that the strains represented five new species of the genus Legionella. Nine of the strains were more than 90% interrelated, and the name Legionella londiniensis sp. nov. is proposed for this group. Two strains formed a second hybridization group, for which the name Legionella nautarum sp. nov. is proposed, while the three remaining species, Legionella geestiana sp. nov., Legionella quateirensis sp. nov., and Legionella worsleiensis sp. nov., are each represented by a single strain. The levels of relatedness of the new species to each other are 23% or less, and the levels of relatedness to other members of the genus ranged from 0 to 36%. L. geestiana, L. nautarum, and L. londiniensis are serologically unrelated to all other known Legionella species. L. worsleiensis cannot be separated from Legionella pneumophila serogroup 4 by serological methods and is also serologically indistinguishable from L. quateirensis; distinctions may be made on the basis of fatty acid composition and biochemical reactions.

Mass spectrometry (MS) is a sensitive and powerful analytical technique, in which ionized sample molecules are separated according to their mass to charge ratios (m/z) by the application of electric and/or magnetic fields. If the ionization regime deposits sufficient excess energy, a proportion of the sample molecules will dissociate, the pattern of product ions formed being dependent on the structure of the intact compound (Fig. 1). A mass spectrum thus consists of the masses (strictly mass to charge ratios, m/z) of these ions plotted against abundance. Interpretation of the spectrum thus affords information about both the mol wt and the structure of the sample. By the standards of most other physical methods, mass spectrometry is fairly sensitive, requiring somewhere between low picomoles and nanomoles of material, depending on the ionization method employed, but against this must be set its destructive nature. The present introduction aims to provide a brief overview of the technique, to define some of the key terms, and to offer a short tour of some of the different instruments that are more or less legitimately called mass spectrometers. Readers wishing a more detailed account should consult refs. 1-9. Arecent volume of Methods in Enzymology (5) devoted entirely to mass spectrometry is particularly recommended, since both instrumentation and applications are comprehensively covered.

The contribution of mass spectrometry to the solution of problems in protein biochemistry was limited until the development of methods of ionization that do not require derivatization or prior vaporization of the sample. Fast atom bombardment (FAB), introduced by Barber et al. in 1981 (1), is one of the most important of these methods, and has been widely applied in the peptide and protein field. In the FAB experiment (Fig. 1), the sample is dissolved in a liquid of low vapor pressure, often glycerol or thioglycerol ("the matrix"), and is bombarded by a beam of energetic particles, such as xenon atoms that sputter sample molecules from the surface layers of the matrix into the mass spectrometer vacuum. Proton or other cation attachment produces abundant (positive) ions characteristic of the sample's molecular mass. A proportion of these molecular ions dissociate, producing structurally informative fragments that are generally less intense than the molecular ions, since the ionization process imparts relatively little excess energy. Negatively charged ions are also generated, and spectra may be recorded in either mode by appropriate selection of the polarity of the ion extraction voltages. At low-mass FAB, spectra are generally dominated by signals attributable to ionization of the matrix. The background of "chemical noise" extending to high mass, which gives FAB spectra their characteristic peak-at-every-mass appearance, is probably attributable to direct hits on sample and matrix molecules by the bombarding species. Figure 2 shows a typical FAB spectrum of the cyclic heptapeptide microcystin-LR, obtained from the cyanobacterium Microcystis aeruginosa.

Aqueous phenol extraction of the lower trypanosomatid Leptomonas samueli released into the aqueous layer a chloroform/methanol/water-soluble glycophosphosphingolipid fraction. Alkaline degradation and purification by gel filtration chromatography resulted in a tetrasaccharide (phosphatidylinositol (PI)-oligosaccharide A), and a pentasaccharide (PI-oligosaccharide B), each containing 2 mol of 2-aminoethylphosphonate and 1 mol of phosphate. Nuclear magnetic resonance spectroscopy and fast atom bombardment-mass spectrometry suggested that the structure of PI-oligosaccharide A is [formula: see text] and that of PI-oligosaccharide B is as shown. [formula: see text] Both compounds contain an inositol unit linked to ceramide via a phosphodiester bridge. The major aliphatic components of the ceramide portion are stearic acid, lignoceric acid, and C20-phytosphingosine. These novel glycolipids fall within the glycosylated phosphatidylinositol (GPI) family, since they contain the core structure Man alpha (1-->4)GlcNH2 alpha (1-->6)myo-inositol-1-PO4, which is also found in the glycoinositolphospholipids and lipophosphoglycan of Leishmania spp., the L. major promastigote surface protease, the glycosylphosphatidylinositol anchor of Trypanosoma brucei variant surface glycoprotein, and the lipopeptidophosphoglycan of Trypanosoma cruzi. The glycophosphosphingolipids of Leptomonas have features in common with the glycolipids of both Leishmania and T. cruzi, resembling the former by the alpha (1-->3) linkage of mannose to the GPI core, while the 2-aminoethylphosphonate substituent on O-6 of glucosamine and the presence of ceramide in place of glycerol lipids is more reminiscent of T. cruzi. Thus these data lend some support to the hypothesis that both T. cruzi and Leishmania evolved from a Leptomonas-like ancestor.

Two strains of Legionella pneumophila serogroup 1 monoclonal subgroup Pontiac were grown for the first time in continuous culture using a chemically defined medium. The influence of temperature on physiology and morphology was investigated by fixing the growth rate (equal to the dilution rate, D) at 0.08 h-1 and controlling the pH and dissolved oxygen concentration of the culture. Serine provided the principal source of carbon and energy but growth was limited by tyrosine. The bacterium behaved as a microaerophile in this medium, with maximal growth occurring at 0.31 (mg O2)I-1 (equivalent to a dissolved oxygen tension of 4% (v/v) air saturation at 30 degrees C). The cultures consisted of flagellated, short rods at 24 degrees C, but exhibited an increased level of pleomorphism and the loss of flagella as the temperature was increased to 37 degrees C. The presence of intracellular granules was noted, and their abundance was temperature-dependent. Polyhydroxybutyrate was present in L. pneumophila, and the proportion of the cell dry weight that it accounted for varied with temperature, being maximal at 24 degrees C. The ratio of saturated to unsaturated fatty acids in the cells decreased as the temperature was reduced towards 24 degrees C, so as to maintain membrane fluidity at low growth temperature.

Physico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies.

Although irreversible damage to the central nervous system has been considered inevitable after 6 min of circulatory deprivation, there has been evidence for almost 20 years that this may be the consequence of postischemic events. Recent investigations have implicated calcium-mediated phenomena as responsible for damage to neuronal cells in the reperfusion period. We studied the effect of verapamil on the neurologic sequelae of spinal cord ischemia using somatosensory evoked potential monitoring in a canine preparation of spinal cord ischemia. Ten mongrel dogs weighing between 20 and 30 kg each were divided into two groups. The experimental group was pretreated with 0.4 mg/kg verapamil and the control group received no treatment. The thoracic aorta was then occluded. Flow was restored 17 min after there was complete loss of somatosensory evoked potentials. Experimental dogs received additional doses of verapamil upon reperfusion and at 1, 2, 3, 4, 5, 6, and 10 hr after reperfusion. Four of five verapamil-treated dogs were able to walk postoperatively, whereas all of the control dogs suffered dense paraplegias (p = .02). We conclude that verapamil can ameliorate the sequelae of spinal cord ischemia and that this preparation is suitable for the study of the mechanisms of ischemic neuronal damage in an area outside the brain.

The metabolism of ursodeoxycholic acid by Pseudomonas sp NCIB 10590 has been studied in phosphate-buffered mineral salts. The organism completely metabolized ursodeoxycholic acid in 24 hr, and time-course experiments revealed that maximum product formation occurred at 14 hr. The major products detected and identified at 14 hr were 7 beta-hydroxychol-4-en-3-one-24-oic acid, 7 beta-hydroxy-3-oxo-pregna-1,4-diene-20-carboxylic acid, and 7 beta-hydroxyandrosta-1,4-diene-3,17-dione. Several minor intermediates were isolated and evidence is given for the following structures: 7 beta-hydroxy-5 beta-cholan-3-oxo-24-oic acid, 7 beta-hydroxyandrost-4-en-3,17-dione, 7 beta,17 beta-dihydroxyandrosta-1,4-diene-3-one, 3-hydroxy-1,3,5(10)-9,10-seco-androstatriene-3,17-dione-7 beta-ol, and 22 alpha-hydroxymethylpregna-1,4-diene-3-one-7 beta-ol.

Population samples from 4 study areas showing a 3-fold variation in large bowel cancer incidence were investigated for fecal bile acid loss and concentration, fecal neutral steroid loss and concentration, and urinary loss of volatile phenols. Each sample consisted of 30 randomly selected men, aged 50-59 years. Daily loss of bile acids was found to be identical in the 4 areas, but due to differences in fecal bulk, the fecal bile acid concentration varied, showing a positive correlation with large bowel cancer incidence. No significant difference between the 4 populations emerged with regard to fecal neutral steroids or urinary volatile phenols.

An Arthrobacter sp. has been shown to dehalogenate 4-chlorobenzoate yielding 4-hydroxybenzoate. Experiments with 18O indicate that, in the presence of cell-free extracts, the hydroxyl group which is substituted onto the aromatic nucleus during dehalogenation is derived from water and not from molecular oxygen. Dehalogenation therefore is not catalysed by a mixed-function oxidase; instead a novel aromatic hydroxylase is implicated in the reaction.

The sidechain of lipopolysaccharide from Erwinia amylovora T was composed of D-fucose, D-galactose and D-glucose in equimolar proportions. Using NMR spectroscopy, methylation analysis, mass spectrometry, Smith degradation and optical rotation data, the repeat unit was shown to have the following most probable structure: (formula; see text)

Organic solvents are used frequently in industry and workers are often exposed to various combinations of these chemicals. Several are CNS depressants, and the purpose of this experiment was to assess the behavioral effects of 4-hour inhalation exposures to two solvents, toluene and methyl ethyl ketone (MEK) alone and combined. Ethanol at 0.08% blood levels was used as a positive control. A total of 144 paid volunteers were randomly assigned to one of eight treatment combinations in a series of four two-group between subjects studies. Testing was carried out in an exposure chamber, and participants were tested before, during, and after the treatment or control condition on three performance tasks. The tasks measured alertness and psychomotor function and produced a total of 28 measures on each individual over the approximate 8 h of testing. Results indicated that toluene at 100 ppm produced a small but significant impairment on one measure of a visual-vigilance task by lowering the percentage of correct hits. MEK at 200 ppm produced no interpretable significant effects on any of these measures. Additivity was not evident when individuals were exposed to MEK (100 ppm) and toluene (50 ppm) in combination, as no significant performance differences were noted. Ethanol, at 0.08%, affected both the visual-vigilance and a choice-reaction time task at statistically significant levels on two measures, confirming the sensitivity of these two tasks to CNS depressants.

A strain of Legionella isolated from the environment which could not initially be identified was shown by restriction fragment length polymorphisms to be a Legionella spiritensis. This was confirmed by DNA homology studies, cell wall fatty acid composition and isoprenoid quinone analysis. This strain, which is only the second reported representative of the species, was shown to be serologically distinct from the type strain of L. spiritensis and all other serogroups of Legionella.

The spontaneous appearance of unusual colony forms was observed during prolonged growth of Bacteroides gingivalis W50 in a chemostat. Two variants were selected for further study which could be distinguished from the parent strain by the rate and intensity of pigmentation of their colonies. For example, after anaerobic incubation for 14 days, variant W50/BR1 produced brown colonies whereas those of the parent strain were black; in contrast, variant W50/BE1 did not show signs of pigmentation until incubation had continued for 21 days. In subsequent studies in the chemostat, variant W50/BE1 bred true even after prolonged growth whereas other colony forms appeared after incubation of variant W50/BR1 for 14 days. The relatedness of W50/BR1 and W50/BE1 to the parent strain was confirmed by comparisons of the whole-cell fatty-acid profiles, the patterns of pre-formed enzymes and by the metabolic end products after growth. However, the variants did differ from the parent strain in their virulence in a mouse pathogenicity model. The parent strain killed all mice given infective doses greater than 5 x 10(8) cfu whereas W50/BR1 was much less virulent (2 out of 10 mice killed and higher infective doses needed for higher mortality rates) and W50/BE1 was avirulent at all infective doses tested.