Massetolide A biosynthesis in Pseudomonas fluorescens.

Massetolide A is a cyclic lipopeptide (CLP) antibiotic produced by various Pseudomonas strains from diverse environments. Cloning, sequencing, site-directed mutagenesis, and complementation showed that massetolide A biosynthesis in P. fluorescens SS101 is governed by three nonribosomal peptide synthetase (NRPS) genes, designated massA, massB, and massC, spanning approximately 30 kb. Prediction of the nature and configuration of the amino acids by in silico analysis of adenylation and condensation domains of the NRPSs was consistent with the chemically determined structure of the peptide moiety of massetolide A. Structural analysis of massetolide A derivatives produced by SS101 indicated that most of the variations in the peptide moiety occur at amino acid positions 4 and 9. Regions flanking the mass genes contained several genes found in other Pseudomonas CLP biosynthesis clusters, which encode LuxR-type transcriptional regulators, ABC transporters, and an RND-like outer membrane protein. In contrast to most Pseudomonas CLP gene clusters known to date, the mass genes are not physically linked but are organized in two separate clusters, with massA disconnected from massB and massC. Quantitative real-time PCR analysis indicated that transcription of massC is strongly reduced when massB is mutated, suggesting that these two genes function in an operon, whereas transcription of massA is independent of massBC and vice versa. Massetolide A is produced in the early exponential growth phase, and biosynthesis appears not to be regulated by N-acylhomoserine lactone-based quorum sensing. Massetolide A production is essential in swarming motility of P. fluorescens SS101 and plays an important role in biofilm formation.

Baerveldt glaucoma implant in paediatric patients.

AIM: To evaluate the Baerveldt glaucoma implant (BGI) in paediatric glaucoma treatment. METHODS: In a retrospective non-comparative case series 55 eyes of 40 consecutive paediatric patients (< or =16 years) with primary or secondary glaucoma underwent Baerveldt (350 mm2) implantation. Surgical outcome was evaluated by Kaplan-Meier table analysis. RESULTS: The overall success rate was 80% at last follow up, with a mean follow up of 32 (range 2-78) months. Cumulative success was 94% at 12 months and 24 months, 85% at 36 months, 78% at 48 months, and 44% at 60 months. 11 eyes (20%) failed postoperatively because of an IOP >21 mm Hg (eight eyes), persistent hypotony (two eyes), and choroidal haemorrhage following cataract surgery (one eye). The most frequent complication needing surgery was tube related (20%). A new observation was mild to moderate dyscoria in 22% of the eyes, all buphthalmic, caused by entrapment of a tuft of peripheral iris in the tube track. CONCLUSIONS: The BGI is effective and safe in the management of primary and secondary glaucoma. When angle surgery has proved to be unsuccessful or inappropriate in paediatric patients, a BGI is a good treatment option. One must be prepared to deal with the tube related problems.

Molecular basis of glycoalkaloid induced membrane disruption.

In this study the interaction between the glycoalkaloids alpha-chaconine, alpha-solanine and alpha-tomatine and sterols in model membranes was analysed systematically using techniques like membrane leakage, binding experiments, detergent extraction, electron microscopy, NMR and molecular modelling. The most important properties for sterols to interact with glycoalkaloids turned out to be a planer ring structure and a 3 beta-OH group, whereas for alpha-chaconine the 5-6 double bond and the 10-methyl group were also of importance. The importance of sugar-sugar interactions was illustrated by the high synergistic effect between alpha-chaconine and alpha-solanine, the leakage enhancing effect of glycolipids, and the almost complete loss of activity after deleting one or more mono-saccharides from the glycoalkaloids. The formed complexes which were resistant against detergent extraction existed of glycoalkaloid/sterol in a 1:1 ratio and formed tubular structures (alpha-chaconine) with an inner monolayer of phospholipids, whereas with alpha-tomatine also spherical structures were formed. Based on the results a molecular model for glycoalkaloid induced membrane disruption is presented.

Intraocular light scattering in age-related cataracts.

Intraocular light scattering was studied in 34 controls and 65 patients with cortical, nuclear, or posterior subcapsular cataracts by measuring forward scatter and backscatter. Forward scatter was measured by the psychophysical direct compensation method. Backscatter was determined with the Lens Opacity Meter of Interzeag. Contrast sensitivity loss caused by forward scatter was assessed with a glare tester (Vistech MCT 8000). Mean forward scatter was in the upper range for subcapsular cataracts compared to nuclear and cortical cataracts. Experimental results of the glare test (the contrast loss) deviated systematically from expected results based on measured forward scatter. Mean backscatter was largest for nuclear, intermediate for posterior subcapsular, and almost zero for cortical cataracts. Thus, each cataract has a characteristic mean ratio between forward scatter and backscatter. However, this ratio varied considerably among individuals, especially for cortical and posterior subcapsular cataracts. As a rule, forward scatter cannot be derived from backscatter (or the slit-lamp image).

Retinal vasculitis occurring with common variable immunodeficiency syndrome.

PURPOSE: To report severe retinal vasculitis causing decreased vision in three patients with the common variable immunodeficiency syndrome. METHOD: Case report. Three patients with common variable immunodeficiency syndrome developed decreased vision secondary to retinal vasculitis. Fluorescein angiography was performed in all three patients. Peribulbar injections were given in one patient, and two patients were treated with oral steroids and cyclosporin. RESULTS: All three patients were young and had classic common variable immunodeficiency syndrome. Bilateral retinal vasculitis and diffuse retinal edema were present in all three patients, and two patients had retinal neovascularization in the absence of ischemia. No evidence of intraocular infection was present, and none was detected systematically. Visual acuity decreased in five of the six eyes and was responsive to treatment in only one patient (both eyes). CONCLUSION: Retinal vasculitis may be another autoimmune manifestation of common variable immunodeficiency syndrome.

The intraocular straylight function in 129 healthy volunteers; dependence on angle, age and pigmentation.

The direct compensation method allows for an accurate (standard deviation below 0.05 log unit) determination of intraocular light scattering between 3.5 and 25 deg of scattering angle and is suitable for untrained subjects. The method was used to study population behaviour and individual variation in 129 volunteers between 20 and 82 yr of age, visual acuity equal to or better than one and no apparent eye pathology. The results indicate straylight to increase with the 4th power of age, doubling at 70. In addition to the age dependence, there was great variation between individuals. Part of this is due to negative correlation with pigmentation.

Dependence of intraocular straylight on pigmentation and light transmission through the ocular wall.

The straylight function of the human eye depends on eye color, especially at larger angles of scattering. As a potential cause for this dependence, transmission of light through the ocular wall was measured, using a psychophysical method. For a light-blue eye effective transmission of the iris was 1% for red and 0.2% for green light. Also the eyewall around the iris transmits a significant amount of light. For the dark-brown eyes of pigmented individuals transmission is lower by two orders of magnitude. Although important, transmission proved to be only partly responsible for the pigmentation dependence, the other cause probably being reflection from the fundus.

Trypan blue capsule staining to visualize the capsulorhexis in cataract surgery.

A capsulorhexis may be difficult to perform in the absence of a red fundus reflex. Using 0.1 mL of trypan blue 0.1% to stain the anterior capsule in 30 patients with a mature cataract enabled us to visualize the capsulorhexis during phacoemulsification. No adverse reactions were observed up to 12 months after surgery. Trypan blue staining of the anterior capsule appears to be a safe technique to facilitate the performance of a capsulorhexis in the absence of a red fundus reflex.

Surgical revision of filtration blebs: a follow-up study.

PURPOSE: To assess the clinical outcome of one technique for surgical revision of filtration blebs in terms of bleb function and intraocular pressure control. METHODS: Retrospective analysis of 36 consecutive cases of leaking, overfiltrating, or oversized blebs treated with bleb excision and conjunctiva and Tenon advancement in a glaucoma referral center between January 1991 and December 1999. Surgical success was defined as a final intraocular pressure between 6 and 22 mm Hg with or without topical antiglaucoma medication, resolution of the bleb leak, hypotony maculopathy and symptoms, and no need for repeat glaucoma surgery. RESULTS: With a minimum of 12 months and an average of 29.5 months of follow-up, the overall success rate was 86.1%, with 51.6% of patients not requiring medication. In the success group, mean (SD) intraocular pressure was 23.7 (5.9) mm Hg before the original trabeculectomy, 4.3 (3.7) mm Hg prior to revision surgery, and 13.5 (SD 3.8) mm Hg at the last follow-up visit after the revision surgery. Mean number of antiglaucoma medications was 2.1 (range, 1-4) before the original trabeculectomy, none before the revision surgery, and 0.8 (range, 0-3) at the last follow-up visit. CONCLUSIONS: The surgical revision technique offers a definitive solution for most of these bleb complications and a satisfactory intraocular pressure control in the majority of patients.

Structural characterisation and enzymic modification of the exopolysaccharide produced by Lactococcus lactis subsp. cremoris B891.

Lactococcus lactis subsp. cremoris B891 grown on whey permeate produced an exopolysaccharide containing D-Gal and D-Glc in a molar ratio of 2:3. The polysaccharide was partially O-acetylated. By means of HF solvolysis, O-deacetylation, enzymic modification, sugar linkage analysis and ID/2D NMR studies the exopolysaccharide was shown to be composed of repeating units with the following structure: [structure: see text].

Measuring non-steady-state metabolic fluxes in starch-converting faecal microbiota in vitro.

This paper explores human gut bacterial metabolism of starch using a combined analytical and computational modelling approach for metabolite and flux analysis. Non-steady-state isotopic labelling experiments were performed with human faecal microbiota in a well-established in vitro model of the human colon. After culture stabilisation, [U-13C] starch was added and samples were taken at regular intervals. Metabolite concentrations and 13C isotopomeric distributions were measured amongst other things for acetate, propionate and butyrate by mass spectrometry and NMR. The vast majority of metabolic flux analysis methods based on isotopomer analysis published to date are not applicable to metabolic non-steady-state experiments. We therefore developed a new ordinary differential equation-based representation of a metabolic model of human faecal microbiota to determine eleven metabolic parameters that characterised the metabolic flux distribution in the isotope labelling experiment. The feasibility of the model parameter quantification was demonstrated on noisy in silico data using a downhill simplex optimisation, matching simulated labelling patterns of isotopically labelled metabolites with measured metabolite and isotope labelling data. Using the experimental data, we determined an increasing net label influx from starch during the experiment from 94±1 µmol/l/min to 133±3 µmol/l/min. Only about 12% of the total carbon flux from starch reached propionate. Propionate production mainly proceeded via succinate with a small contribution via acrylate. The remaining flux from starch yielded acetate (35%) and butyrate (53%). Interpretation of 13C NMR multiplet signals further revealed that butyrate, valerate and caproate were mainly synthesised via cross-feeding, using acetate as a co-substrate. This study demonstrates for the first time that the experimental design and the analysis of the results by computational modelling allows the determination of time-resolved effects of nutrition on the flux distribution within human faecal microbiota in metabolic non-steady-state.

Uveitis-like syndrome and iris transillumination after the use of oral moxifloxacin.

OBJECTIVE: To report a newly recognized adverse effect of oral moxifloxacin. DESIGN: Observational case reports. PARTICIPANTS: Five patients who used oral moxifloxacin therapy. MAIN OUTCOME MEASURES: In five patients, a uveitis-like episode followed oral moxifloxacin therapy, afterwards they experienced photophobia. At slitlamp investigation, the patients showed almost complete iris transillumination, not restricted to one sector, and persistent mydriasis of the pupil, with no reaction to light and no near reflex. Follow-up of 3 years in one of the patients showed no change of symptoms. Only in one patient, with a history of anterior uveitis, an anterior chamber tap was positive for herpes simplex genome. Only after the use of moxifloxacin did she experience continuous photophobia. CONCLUSIONS: Iris transillumination and sphincter paralysis is a newly recognized adverse effect of oral moxifloxacin therapy.

Glycolytic pathway and hydrogen yield studies of the extreme thermophile Caldicellulosiruptor saccharolyticus.

NMR analysis of (13)C-labelling patterns showed that the Embden-Meyerhof (EM) pathway is the main route for glycolysis in the extreme thermophile Caldicellulosiruptor saccharolyticus. Glucose fermentation via the EM pathway to acetate results in a theoretical yield of 4 mol of hydrogen and 2 mol of acetate per mole of glucose. Previously, approximately 70% of the theoretical maximum hydrogen yield has been reached in batch fermentations. In this study, hydrogen and acetate yields have been determined at different dilution rates during continuous cultivation. The yields were dependent on the growth rate. The highest hydrogen yields of 82 to 90% of theoretical maximum (3.3 to 3.6 mol H(2) per mol glucose) were obtained at low growth rates when a relatively larger part of the consumed glucose is used for maintenance. The hydrogen productivity showed the opposite effect. Both the specific and the volumetric hydrogen production rates were highest at the higher growth rates, reaching values of respectively 30 mmol g(-1) h(-1) and 20 mmol l(-1) h(-1). An industrial process for biohydrogen production will require a bioreactor design, which enables an optimal mix of high productivity and high yield.

Cholangiography during cholecystectomy: a plea for selective use.

Routine peroperative cholangiography was retrospectively studied in 376 patients who underwent elective cholecystectomy. Adequate cholangiograms were obtained in 325 patients using a C-arm image intensifier. Pre- and peroperative indications for common bile duct stones were correlated to the outcome of peroperative cholangiography. The sensitivity and specificity of the cholangiographic technique were 96 and 95 per cent respectively. A false-positive rate of 4.6% and a false-negative rate of 0.6% were found. No serious complications due to the method were observed. Peroperative cholangiography prevented unnecessary exploration of the common bile duct in 70 per cent of the patients with clinical indications for exploration. Abnormal cholangiograms were rare (0.8%) in the absence of clinical signs of stones in the bile ducts. These results demonstrate that selective rather than routine peroperative cholangiography should be performed.

Formation of novel poly(hydroxyalkanoates) from long-chain fatty acids.

Poly(hydroxyalkanoates) (PHAs) were isolated from Pseudomonas aeruginosa 44T1 cultivated on euphorbia oil and castor oil. With the aid of 2-D proton NMR spectra and proton-detected multiple bond coherence NMR spectra the structures of the PHAs were determined. In addition to the usual PHA constituents (C6-C14 3-hydroxy fatty acids), PHAs formed from euphorbia oil contained delta 8,9-epoxy-3-hydroxy-5c-tetradecenoate, and probably delta 6,7-epoxy-3-hydroxydodecanoate and delta 4,5-epoxy-3-hydroxydecanoate. These novel constituents account for approximately 15% of the total amount of monomers and are clearly generated via beta-oxidation of vernolic acid (delta 12,13-epoxy-9c-octadecenoic acid), the main component of euphorbia oil. In PHAs formed from castor oil, 7% of the monomers found were derived from ricinoleic acid (12-hydroxy-9c-octadecenoic acid). The presence of 3,8-dihydroxy-5c-tetradecenoate was clearly demonstrated. Furthermore, NMR analysis strongly suggested the presence of 3,6-dihydroxydodecanoate, 6-hydroxy-3c-dodecenoate, and 4-hydroxydecanoate.

Structural studies on sulfated glycopeptides from the carbohydrate-protein linkage region of chondroitin 4-sulfate proteoglycans of swarm rat chondrosarcoma. Demonstration of the structure Gal(4-O-sulfate)beta 1-3Gal beta 1-4XYL beta 1-O-Ser.

Nonsulfated, monosulfated, and disulfated glycopeptides containing the entire carbohydrate sequence of the glycosaminoglycan-specific linkage region were isolated after exhaustive enzymatic digestions of Swarm rat chondrosarcoma proteoglycans with chondroitinase ABC, papain, and Pronase. Their structures were examined by 500 MHz 1H NMR spectroscopy. The nonsulfated compound has the following structure with trace amounts of a few additional amino acids: delta 4,5-GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser. The monosulfated compound has an ester sulfate on C-4 of the GalNAc residue and the disulfated compound has an additional hitherto unrecognized ester sulfate on C-4 of the second galactose residue which is remote from the innermost xylose. This new structure was confirmed by two-dimensional homonuclear Hartmann-Hahn spectroscopy. The molar ratio of the isolated nonsulfated, monosulfated, and disulfated compounds was 53:37:10 based on the serine contents. Biological significance of the newly found sulfated linkage structure is discussed.

Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. I. Six compounds containing 0 or 1 sulfate and/or phosphate residues.

Shark cartilage proteoglycans bear predominantly chondroitin 6-sulfate. After exhaustive protease digestion, reductive beta-elimination, and subsequent chondroitinase ABC digestion, 13 hexasaccharide alditols, which are nonsulfated, sulfated, and/or phosphorylated, were obtained from the carbohydrate-protein linkage region. Six compounds, containing 0 or 1 sulfate and/or phosphate residue, represent approximately 40% of the isolated linkage hexasaccharide alditols. They were analyzed by chondroitinase ACII or alkaline phosphatase digestion in conjunction with high performance liquid chromatography, and by 500 MHz one- and two-dimensional 1H NMR spectroscopy. All six compounds have the conventional structure in common. Delta 4,5-GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl-ol One compound has no sulfate nor phosphate. Two of the monosulfated compounds have a O-sulfate on C-6 or on C-4 of the GalNAc residue. The third monosulfated compound has a novel O-sulfate on C-6 of the Gal residue attached to xylitol. The two phosphorylated compounds have O-phosphate on C-2 of Xyl-ol, and one of them has in addition sulfate on C-6 of GalNAc.

Structural studies on sulfated oligosaccharides derived from the carbohydrate-protein linkage region of chondroitin 6-sulfate proteoglycans of shark cartilage. II. Seven compounds containing 2 or 3 sulfate residues.

Shark cartilage proteoglycans bear predominantly chondroitin 6-sulfate. After exhaustive protease digestion, reductive beta-elimination and subsequent chondroitinase ABC digestion, 13 hexasaccharide alditols were obtained from the carbohydrate-protein linkage region and six of them contain 0 or 1 sulfate and/or 1 phosphate residue (Sugahara, K., Ohi, Y., Harada, T., de Waard, P., and Vliegenthart, J. F. G. (1992) J. Biol. Chem. 267, 6027-6035). The other seven compounds, which represent approximately 60% of the isolated linkage hexasaccharides, were analyzed by chondroitinase ACII digestion in conjunction with high performance liquid chromatography and by 500-MHz one- and two dimensional 1H NMR spectroscopy. All seven compounds have the following conventional structure in common. [formula: see text] Two disulfated compounds have an O-sulfate on C-6 of the Gal-2 residue attached to xylitol in combination with an O-sulfate on C-4 or on C-6 of the GalNAc residue. The third disulfated compound has O-sulfate on C-6 of Gal-2, and also on C-6 of Gal-3. Two of the trisulfated compounds also have O-sulfate on C-6 of both Gal-2 and Gal-3 with in addition sulfate on C-6 or C-4 of GalNAc. The other two trisulfated compounds have O-sulfate on C-6 of Gal-2 and on C-4 of Gal-3 in conjunction with sulfate on C-6 or C-4 of GalNAc.