Pamidronate infusion in patients with systemic sclerosis results in changes in blood mononuclear cell cytokine profiles.

A single infusion of pamidronate was given to patients with systemic sclerosis (scleroderma, SSc) to assess effects on cytokine production by peripheral blood mononuclear cells (PBMC) and lymphocyte subsets. Eighteen patients with SSc received a single intravenous dose of 60 mg of pamidronate and were followed for 6 months. Assessment of cytokine production [interferon (IFN)-gamma, interleukin (IL)-10, transforming growth factor (TGF)-beta1, tumour necrosis factor (TNF)-alpha and IL-4] by PBMC and lymphocyte subsets by flow cytometry was carried out before and after the pamidronate infusion. Unstimulated PBMC produced increased amounts of IFN-gamma and TNF-alpha and reduced levels of TGF-beta1 for up to 24 weeks after the infusion. gammadelta T cells from patients with SSc were activated in vitro and produced increased IFN-gamma. The effects of pamidronate on modulation of cytokine profiles in patients with SSc may merit future study.

Carotenoid accumulation in the psychrotrophic bacterium Arthrobacter agilis in response to thermal and salt stress.

A psychrotrophic strain of Arthrobacter agilis, isolated from Antarctic sea ice, grows from 5 degrees C to 40 degrees C and in culture media containing 0-10% (w/v) NaCl. Maximum growth rate occurred at 30-35 degrees C with a drastic decline as the cultivation temperatures diverged. Adaptation to extremes of low temperature may be partially attributed to the production of the C-50 carotenoid bacterioruberin, and its glycosylated derivatives. Lowering of the cultivation temperature resulted in a concomitant increase in carotenoid production, which may contribute to membrane stabilisation at low temperature. Maximum biomass accumulation occurred at 5-30 degrees C with a tenfold reduction at 40 degrees C. Changes in growth rates were minimal in culture media containing 0-2% (w/v) NaCl at 10 degrees C while a gradual decrease in growth rates occurred at higher salinity. Biomass accumulation at different salinity followed a trend similar to that observed with different cultivation temperatures. Maximum biomass accumulation was observed in culture media containing 0-5% (w/v) NaCl with a tenfold reduction at 10% (w/v) NaCl. Carotenoid production also decreased as salinity increased.

Nuclear magnetic resonance studies of acetic acid inhibition of rec Zymomonas mobilis ZM4(pZB5).

The fermentation characteristics and effects of lignocellulosic toxic compounds on recombinant Zymomonas mobilis ZM4(pZB5), which is capable of converting both glucose and xylose to ethanol, and its parental strain, ZM4, were characterized using 13C and 31P nuclear magnetic resonance (NMR) in vivo. From the 31P NMR data, the levels of nucleoside triphosphates (NTP) of ZM(pZB5) using xylose were lower than those of glucose. This can be related to the intrinsically slower assimilation and/or metabolism of xylose compared to glucose and is evidence of a less energized state of ZM4(pZB5) cells during xylose fermentation. Acetic acid was shown to be strongly inhibitory to ZM4(pZB5) on xylose medium, with xylose utilization being completely inhibited at pH 5.0 or lower in the presence of 10.9 g/L of sodium acetate. From the 31P NMR results, the addition of sodium acetate caused decreased NTP and sugar phosphates, together with acidification of the cytoplasm. Intracellular deenergization and acidification appear to be the major mechanisms by which acetic acid exerts its toxic effects on this recombinant strain.

Effects of singlet oxygen on membrane sterols in the yeast Saccharomyces cerevisiae.

Photodynamic treatment of the yeast Saccharomyces cerevisiae with the singlet oxygen sensitizer toluidine blue and visible light leads to rapid oxidation of ergosterol and accumulation of oxidized ergosterol derivatives in the plasma membrane. The predominant oxidation product accumulated was identified as 5alpha, 6alpha-epoxy-(22E)-ergosta-8,22-dien-3beta,7a lpha-diol (8-DED). 9(11)-dehydroergosterol (DHE) was identified as a minor oxidation product. In heat inactivated cells ergosterol is photooxidized to ergosterol epidioxide (EEP) and DHE. Disrupted cell preparations of S. cerevisiae convert EEP to 8-DED, and this activity is abolished in a boiled control indicating the presence of a membrane associated enzyme with an EEP isomerase activity. Yeast selectively mobilizes ergosterol from the intracellular sterol ester pool to replenish the level of free ergosterol in the plasma membrane during singlet oxygen oxidation. The following reaction pathway is proposed: singlet oxygen-mediated oxidation of ergosterol leads to mainly the formation of EEP, which is enzymatically rearranged to 8-DED. Ergosterol 7-hydroperoxide, a known minor product of the reaction of singlet oxygen with ergosterol, is formed at a much lower rate and decomposes to give DHE. Changes of physical properties of the plasma membrane are induced by depletion of ergosterol and accumulation of polar derivatives. Subsequent permeation of photosensitizer through the plasma membrane into the cell leads to events including impairment of mitochondrial function and cell inactivation.

Kinetic and nuclear magnetic resonance studies of xylose metabolism by recombinant Zymomonas mobilis ZM4(pZB5).

The specific rates of growth, substrate utilization, and ethanol production as well as yields of biomass and ethanol production on xylose for the recombinant Zymomonas mobilis ZM4(pZB5) were shown to be much less than those on glucose or glucose-xylose mixtures. Typical fermentations with ZM4(pZB5) growing on glucose-xylose mixtures followed two-phase growth kinetics with the initial uptakes of glucose and xylose being followed by slower growth and metabolic uncoupling on xylose after glucose depletion. The reductions in rates and yields from xylose metabolism were considered in the present investigation and may be due to a number of factors, including the following: (i) the increased metabolic burden from maintenance of plasmid-related functions, (ii) the production of by-products identified as xylitol, acetate, lactate, acetoin, and dihydroxyacetone by (13)C-nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography, (iii) growth inhibition due to xylitol by the putative inhibitory compound xylitol phosphate, and (iv) the less energized state of ZM4(pZB5). In vivo (31)P-NMR studies have established that the levels of NTP and UDP sugars on xylose were less than those on glucose, and this energy limitation is likely to restrict the growth of the recombinant strain on xylose media.

Beta-human chorionic gonadotropin in semen: a marker for early detection of prostate cancer?

BACKGROUND AND PURPOSE: The beta subunit of human chorionic gonadotropin (beta-hCG) has been detected in prostate cancer by immunologic and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques. Recently, prostate cells have been detected in human ejaculate. This study aimed to determine if beta-hCG could be detected by RT-PCR from prostatic mRNA isolated from semen, thus providing a noninvasive procedure for prostate cancer detection. RESULTS: Expression of beta-hCG in prostate cancer was confirmed by immunohistochemistry methods. The protein was associated with low-grade disease: Gleason Score 4 through 7 (N = 26; 69%) but not high-grade (Gleason 8 or 9) or metastatic (lymph node) disease (N = 12; 8%). Normal prostate tissue was negative for beta-hCG (N = 14). The beta-hCG RT-PCR was performed on RNA extracted from seven human prostate cancer cell lines, which showed variable expression of beta-hCG mRNA. Semen was collected from patients suspected of having carcinoma of the prostate (N = 94) and from volunteers who were under the age of 30 years and had no family history of prostate cancer (N = 9). mRNA for beta-hCG was detected in the ejaculates of 12% of the patients with confirmed prostate cancer (N = 42) but not in any patients found to be negative for cancer (N = 52). Expression of beta-hCG mRNA was found in 22% of the control samples. CONCLUSIONS: The beta-hCG protein is expressed in low-grade prostate cancer and can be detected by RT-PCR in both prostate cancer cell lines and human ejaculate. However, the low percentage of detection in ejaculate suggests that beta-hCG in semen does not provide a useful marker for early prostate cancer detection.

The selective enzymatic synthesis of lipophilic esters of swainsonine.

The potent and specific inhibitor of Golgi alpha-mannosidase II, swainsonine (SW) has been isolated in high yield from Swainsona procumbens and derivatised by regiospecific enzymatic reactions. In this study the regioselectivity of three commercially available enzymes, subtilisin Carlsberg, porcine pancreatic lipase (PPL) and Candida cylindracea lipase was determined for the acylation of swainsonine in predominantly anhydrous organic medium. The use of subtilisin in pyridine facilitated the single step synthesis of 2-O-butyryl-SW in a 23% yield, whilst catalysis by PPL in tetrahydrofuran gave 2-O-butyryl-SW (6%) and 1,2-di-O-butyryl-SW (31%).

Differential effects of cholesterol and oxidised-cholesterol in egg lecithin bilayers.

Low frequency impedance measurements of pure egg lecithin (phosphatidylcholine) bilayers have revealed the presence of four layers which can be attributed to the acyl chain, carbonyl, glycerol bridge and phosphatidylcholine regions of the lecithin molecule. Measurements on bilayers formed in the presence of unoxidised-cholesterol revealed that cholesterol molecules were located in the hydrocarbon region of the bilayer with its hydroxyl groups aligned with the carbonyl region of the lecithin molecules. Measurements of oxidised-cholesterol lecithin bilayers revealed that these molecules protruded less into the hydrocarbon region and their polar hydroxyl group aligned with the glycerol bridge region of the lecithin molecule.

Characterisation of the anti-bladder-cancer monoclonal antibody BLCA-8: identification of its antigen as a neutral glycolipid.

A monoclonal antibody, BLCA-8, was raised against the human bladder cancer cell line, UCRU-BL-17CL. By flow cytometry and immunoperoxidase staining, this antibody was found to possess high specificity for bladder tumours, some reactivity with fetal tissues, and no reactivity with normal bladder, or any normal or malignant tissue. This high specificity and the stability of the antigen to the urinary environment suggest that BLCA-8 may have potential for use as an anti-bladder-cancer therapeutic agent. By thin-layer chromatography and autoradiography, BLCA-8 was found to bind four components within the neutral lipid fraction of a bladder cancer cell line, UCRU-BL-17/23 alpha. These components had RF values of 0.22, 0.16/0.15 (doublet), 0.12 and 0.08, and migrated below globoside, indicating the presence of more than four sugars. By enzyme-linked immunosorbent assay and thin-layer chromatography it was found that the binding of BLCA-8 to the lipid extract was increased by both mild alkaline hydrolysis and enzymatic treatments, indicating that adjacent phospholipids and glycolipids interfere with the accessibility of the antibody-binding site. Full biochemical characterisation of the BLCA-8 antigen is currently underway.

Floridoside, L-Isofloridoside, and D-Isofloridoside in the Red Alga Porphyra columbina (Seasonal and Osmotic Effects).

The quantitative heteroside distribution in Porphyra columbina Montagne and Bangia atropurpurea (Roth) C. Agardh (Bangiales, Rhodophyta) has been measured using 13C-nuclear magnetic resonance spectroscopy and gas-liquid chromatography. In P. columbina, floridoside and both D- and L-isofloridoside were recorded, with concentrations of L-isofloridoside exceeding those of floridoside. All three compounds were also measured in B. atropurpurea. Marked changes in the relative amounts of the heterosides were recorded throughout the season. The role of L-isofloridoside in the osmotic acclimation of P. columbina has been demonstrated.

Determination of hopanoid levels in bacteria using high-performance liquid chromatography.

A reverse-phase HPLC method to detect and quantify levels of hopanoids in bacteria has been developed. Chromophores have been introduced by derivatization and the levels of the C35 hopanoids and their conjugates can be measured in bacterial lipid extracts down to picomole levels. Some structural variations of the complex lipids were detected after derivatization and were easily purified using the same HPLC system. Zymomonas mobilis and Rhodospirillum rubrum extracts were examined using this system and different structural examples of the lipids were detected. The relative levels of the different triterpenes were very dependent on the growth conditions.

Distribution and chemical composition of the toxic skin secretions from trunkfish (family Ostraciidae).

The components of the mucus skin secretions from eight species of trunkfish found in the coastal waters of Australia were analyzed by combined chemical ionization-gas chromatography mass spectrometry. The species investigated were Anoplacapros lenticularis, Aracana aurita, Aracana ornata, Lactoria fornasini, Ostracion cubicus, Rhinesomas reipublicae, Strophiurichthys inermis and Strophiurichthys robustus. The beta-substituted choline chloride esters (mainly acetoxy, but with some species having butyryloxy, valeryloxy and one species with caproyloxy) of palmitic acid were the predominant components in almost all cases. High concentrations of monounsaturated palmitic acid were present in S. inermis and S. robustus. Trace quantities of C14, C17 and C18 choline chloride esters were also detected as were compounds where the choline moiety was modified by addition of one extra carbon.

NMR studies of [1-2H]glucose metabolism in Zymomonas mobilis.

In complementary experiments the metabolism of [1-2H]glucose in H2O and of unlabelled glucose in 2H2O by Zymomonas mobilis was examined. The utilization of [1-2H]glucose by Z. mobilis was monitored by high-resolution 2H NMR. The deuterium-labelling pattern and stereochemistry of the ethanols produced from the metabolism of [1-2H]glucose and unlabelled glucose in 2H2O were determined by a combination of 13C and 1H NMR and selective enzyme action. The labelling patterns were explained in terms of enzyme mechanisms and stereospecificity, and metabolite enolization.

The structure of a novel polysaccharide isolated from Zymomonas mobilis determined by nuclear magnetic resonance spectroscopy.

A novel polysaccharide has been observed in vivo by 13C nuclear magnetic resonance (NMR) spectroscopy of the gram-negative bacterium, Zymomonas mobilis. The polysaccharide, which was not removed from the Z. mobilis cell by washing with 0.86% saline, was extracted by mild acid treatment. The structure was elucidated by a combination of NMR techniques, including proton and proton-carbon two-dimensional methods. The structure was determined to be -alpha-fructofuranosyl-(2-1)-beta-fructofuranosyl-(2-6)-. The polysaccharide is unlike levan, a beta-(2-6)-fructose polymer which is produced by many bacteria, including Z.mobilis, when grown on sucrose. The polysaccharide isolated here is produced by cells cultured on glucose, fructose or sucrose.

31P nuclear magnetic resonance studies of the fermentation of glucose to ethanol by Zymomonas mobilis.

High resolution 31P nuclear magnetic resonance spectroscopy has been employed to study the fermentation of glucose to ethanol by Zymomonas mobilis, strain ZM4, a bacterium which uses the Entner-Doudoroff pathway. The levels of nucleoside triphosphates, sugar phosphates, UDP-sugars and Pi in intact fermenting cells have been studied with a time resolution of 1 min. It is suggested that a pH gradient is established across the cell membrane during fermentation and that the intracellular pH does not rise above approximately 6.4. 31P resonances from most phosphorus-containing intermediates in the Entner-Doudoroff pathway, as well as adenosine and uridine nucleotides and a number of other intracellular metabolites, have been assigned in perchloric extracts of fermenting cells by means of a number of techniques, including two-dimensional homonuclear J-resolved and two-dimensional homonuclear shift-correlated spectroscopy. Quantification of these metabolites in spectra of extracts of fermenting cells indicates that the rate-limiting steps in the Entner-Doudoroff pathway in Z. mobilis are the conversions of glucose 6-phosphate to 6-phosphogluconate and of 3-phosphoglycerate to 2-phosphoglycerate.

Uridine and inosine: pressor nucleosides from man, rat and dog.

Short-acting pressor compounds isolated from rat kidney, brain, heart and spleen have been identified as inosine and uridine by gas chromatography, mass spectrometry, high pressure liquid chromatography and analysis of ultraviolet spectra. Inosine was further identified by nuclear magnetic resonance. These compounds have also been found in kidneys from hypertensive man, rejected renal transplants and dog and beef kidney. Tissues were extracted by acid ethanol extraction followed by gel filtration and high voltage paper electrophoresis. Compounds found to be pressor in the anaesthetised rat resisted proteolytic enzymes, boiling for 10 min, extremes of pH and incubation with plasma from the source species for up to 20 min. The pressor effects of bolus injections of active gel filtration fractions, uridine and inosine were short-lived with a maximum effect at 5-6 s. Intravenous (I.V.) infusions of extracts gave a sustained pressor response without a concurrent change in heart rate. The effect on blood pressure was not accompanied by increased heart rate and persisted when the pressor effects of angiotensin II, noradrenaline and 5-hydroxy-tryptamine were blocked pharmacologically.