Mechanism of Ca2+ and monosaccharide binding to a C-type carbohydrate-recognition domain of the macrophage mannose receptor

Site-directed mutagenesis has been used to identify residues that ligate Ca2+ and sugar to the fourth C-type carbohydrate-recognition domain (CRD) of the macrophage mannose receptor. CRD-4 is the only one of the eight CRDs of the mannose receptor to exhibit detectable monosaccharide binding when expressed in isolation, and it is central to ligand binding by the receptor. CRD-4 requires two Ca2+ for sugar binding, like the CRD of rat serum mannose-binding protein (MBP-A). Sequence comparisons between the two CRDs suggest that the binding site for one Ca2+, which ligates directly to the bound sugar in MBP-A, is conserved in CRD-4 but that the auxiliary Ca2+ binding site is not. Mutation of the four residues at positions in CRD-4 equivalent to the auxiliary Ca2+ binding site in MBP-A indicates that only one, Asn728, is involved in ligation of Ca2+. Alanine-scanning mutagenesis was used to identify two other asparagine residues and one glutamic acid residue that are probably involved in ligation of the auxiliary Ca2+ to CRD-4. Sequence comparisons with other C-type CRDs suggest that the proposed binding site for the auxiliary Ca2+ in CRD-4 of the mannose receptor is unique. Evidence that the conserved Ca2+ in CRD-4 bridges between the protein and bound sugar in a manner analogous to MBP-A was obtained by mutation of one of the amino acid side chains at this site. Ring current shifts seen in the 1H NMR spectra of methyl glycosides of mannose, GlcNAc, and fucose in the presence of CRD-4 and site-directed mutagenesis indicate that a stacking interaction with Tyr729 is also involved in binding of sugars to CRD-4. This interaction contributes about 25% of the total free energy of binding to mannose. C-5 and C-6 of mannose interact with Tyr729, whereas C-2 of GlcNAc is closest to this residue, indicating that these two sugars bind to CRD-4 in opposite orientations. Sequence comparisons with other mannose/GlcNAc-specific C-type CRDs suggest that use of a stacking interaction in the binding of these sugars is probably unique to CRD-4 of the mannose receptor.     

Noise power measurements and measurement uncertainties

In RF metrology, noise power measurements consist of intercomparisons between the output noise powers of unknown noise sources and noise standards. With respect to the traceability of the assigned values of noise power, the estimation of uncertainties of measurement plays a decisive role. The evaluation of uncertainties is discussed in relation to the appropriate recommendations of the Comite International des Poids et Mesures (CIPM) and respective guidelines of the Western European Calibration Cooperation (WECC). The authors give specific advice for the treatment of measurement uncertainties in the RF noise power measuring field in order to make the information more readily applicable     

The effects of temperature on the interactions between volatile general anaesthetics and a neuronal nicotinic acetylcholine receptor

1. Completely isolated identified neurones from the right parietal ganglion of the pond snail Lymnaea stagnalis were investigated under two-electrode voltage clamp. Neuronal nicotinic acetylcholine receptor (AChR) currents were studied at low acetylcholine concentrations (< or = 200 nM). 2. Inhibition of the ACh-induced currents by three volatile general anaesthetics (halothane, isoflurane and methoxyflurane) and the specific inhibitor (+)-tubocurarine was studied as a function of temperature (over the range 4-25 degrees C). 3. The inhibition by the volatile anaesthetics increased (inhibition constants decreased) with decreasing temperature while the inhibition by (+)-tubocurarine did not change significantly near room temperature, but decreased at lower temperatures. The (+)-tubocurarine inhibition appeared to be competitive in nature and showed no significant voltage-dependence. 4. The van't Hoff plots (logarithms of the dissociation constants against reciprocal absolute temperature) were linear for the anaesthetics, but markedly non-linear for (+)-tubocurarine. From these plots, values for the changes in the standard Gibbs free energy delta G degrees water-->AChR, enthalpy delta H degree water-->AChR, entropy delta S degree water-->AChR and heat capacity delta Cp degree water-->AChR were determined. Tubocurarine was found to bind very much tighter to the receptor than the volatile anaesthetics due, entirely, to a favourable increase in entropy on binding. 5. A comparison between the temperature-dependence of the anaesthetic inhibition of the ACh receptor and that of general anaesthetic potencies in animals indicates that the temperature-dependence of animal potencies might be simply accounted for in terms of changes in anaesthetic/receptor binding.     

Statisches und dynamisches Werkzeugverhalten beim Innenrundschleifen mit axial vorgespannten Werkzeugen

Zusammenfassung Die in diesem Beitrag vorgestellten theoretischen Betrachtungen zum statischen und dynamischen Werkzeugverhalten beim Innenrundschleifen langer Bohrungen nach dem ILB-Verfahren haben wichtige Hinweise für das Proze?verhalten ergeben. Durch geeignete Modellbildung kann hiermit der Einflu? unterschiedlicher Werkzeugspezifikationen sowohl auf die statische Nachgiebigkeit der Werkzeuge und damit letztendlich auf den L?ngsformfehler am Bauteil als auch auf das dynamische Verhalten, insbesondere das Auftreten kritischer Resonanzfrequenzen, vorherbestimmt werden. Die analytischen Berechnungen konnten durch praktische Untersuchungen verfiziert werden. Durchgeführte Schleifversuche haben die Eignung des ILB-Verfahrens als Alternative zur konventionellen Innenrundbearbeitung langer Bohrungen in keramischen Bauteilen best?tigt. Zukünftige Untersuchungen am IFW werden sich auch mit dem Innenrundschleifen langer Bohrungen in Bauteilen aus Hartmetall und geh?rtetem Stahl befassen. Dipl.-Ing.W. Buchholz und Dipl.-Ing.U. Wasmann, wissenschafliche Mitarbeiter des IFW.     

High-performance liquid chromatography-fluorescence assay of pyruvic acid to determine cysteine conjugate beta-lyase activity: application to S-1,2-dichlorovinyl-L-cysteine and S-2-benzothiazolyl-L-cysteine

An HPLC-fluorescence assay has been developed for the determination of the activity of rat renal cytosolic cysteine conjugate beta-lyase. The method is based on isocratic HPLC separation and fluorescence detection of pyruvic acid, derivatized with o-phenylenediamine (OPD), and is shown to be rapid, specific, and very sensitive. The assay has been evaluated with two model substrates for rat renal cytosolic beta-lyase, notably S-1,2-dichorovinyl-L-cysteine (DCVC) and S-2-benzothiazolyl-L-cysteine (BTC). Equimolar formation of pyruvic acid and 2-mercaptobenzothiazole, a chromophoric thiol, indicated that pyruvic acid formation actually reflects the beta-elimination activity of beta-lyase during the beta-elimination of BTC. From this it follows that the pyruvic acid assay can be applied to the measurement of the beta-elimination activity of this enzyme, independent of the presence of chromophoric groups or radiolabels in substrates. Due to the large linear range and the very high sensitivity of the present HPLC-fluorescence assay (detection limit, 7.5 pmol of pyruvic acid), both good and poor substrates of beta-lyase can be measured. Enzyme kinetic data are presented for the model substrates BTC and DCVC and for four structurally related S-2,2-difluoroethyl-L-cysteine conjugates.     

Salmonella arizonae sepsis in a lynx

A 4.5-wk-old lynx (Felis lynx) was presented for necropsy with a history of poor growth, mild diarrhea, anemia, and lethargy. The liver was enlarged and had a 7 mm long fracture that resulted in severe intraabdominal hemorrhage and death. Microscopic lesions were indicative of severe ulcerative cystitis and septicemia. Pure cultures of Salmonella arizonae were isolated from the liver, kidney, and spleen. Based on differences in the chronicity of inflammation in the urinary bladder versus other organs, we speculate that chronic cystitis caused by S arizonae lead to septicemic infection.     

Conjoined gastric and mediastinal benign cystic teratomas. Case report of a rare occurrence and review of literature

In this study, we evaluated the permeation of piperacillin (PIPC), imipenem (IPM), amikacin (AKM), gentamicin (GM), ofloxacin (OFLX), levofloxacin (LVFX), ciprofloxacin (CPFX) and sparfloxacin (SPFX) through Pseudomonas aeruginosa biofilm with a simple new method. Bacteria used were a leucine-requiring mucoid mutant. Bacteria were grown on the membrane of a cell culture insert in chemically defined medium and incubated at 37 degrees C for 5 days. At days 0, 1, 3 and 5, the penetration rates through the biofilms were measured. PIPC and IPM demonstrated relatively high permeation both with penetration rates at day 5 of 50%, whereas AMK and GM, which are aminoglycosides, showed low permeation both with penetration rates after day 1 of less than 25%. Among the 4 fluoroquinolones, LVFX and SPFX demonstrated excellent permeation with penetration rates that reached 100% from day 0 to 5, while OFLX and CPFX showed almost the same permeation as IPM. This method of measuring penetration rates of antimicrobial agents through biofilm is very simple and useful for the evaluation of antibiotics against biofilm-forming bacteria.     

Allylmercapturic acid as urinary biomarker of human exposure to allyl chloride

OBJECTIVE: To evaluate the use of urinary mercapturic acids as a biomarker of human exposure to allyl chloride (3-chloropropene) (AC). During three regular shut down periods in a production factory for AC, both types of variables were measured in 136 workers involved in maintenance operations. METHODS: Potential airborne exposure to AC was measured by personal air monitoring in the breathing zone. In total 205 workshifts were evaluated. During 99 workshifts no respiratory protection equipment was used. Mercapturic acid metabolites were measured in urinary extracts by gas chromatography-mass spectrometry (GC-MS). RESULTS: During 86 work shifts when no respiratory protection was used the air concentrations of AC were below the Dutch eight hour time weighted average (8 h-TWA) occupational exposure limit (OEL) of AC (3 mg/m3), whereas in 13 workshifts the potential exposure, as measured by personal air monitoring, exceeded the OEL (3.3 to 17 mg/m3). With the aid of GC-MS, 3-hydroxypropylmercapturic acid (HPMA) was identified as a minor and allylmercapturic acid (ALMA) as a major metabolite of AC in urine samples from the maintenance workers exposed to AC. The concentrations of ALMA excreted were in a range from < 25 micrograms/l (detection limit) to 3550 micrograms/l. The increases in urinary ALMA concentrations during the workshifts correlated well with the 8h-TWA air concentrations of AC (r = 0.816, P = 0.0001, n = 39). Based on this correlation, for AC a biological exposure index (BEI) of 352 micrograms ALMA/g creatinine during an eight hour workshift is proposed. In some urine samples unexpectedly high concentrations of ALMA were found. Some of these could definitely be attributed to dermal exposure to AC. In other cases garlic consumption was identified as a confounding factor. CONCLUSION: The mercapturic acid ALMA was identified in urine of workers occupationally exposed to airborne AC and the increase in ALMA concentrations in urine during a workshift correlated well with the 8 h-TWA exposure to AC. Garlic consumption, but not smoking, is a potential confounding factor for this biomarker of human exposure to AC.     

Anaerobic bacteria as a gene delivery system that is controlled by the tumor microenvironment

A fundamental obstacle in gene therapy for cancer treatment is the specific delivery of an anticancer gene product to a solid tumor. Although several strategies exist to control gene expression once a vector is directly introduced into a tumor, as yet no systemic delivery system exists that specifically targets solid tumors. Nonpathogenic, obligate anaerobic bacteria of the genus Clostridium have been used experimentally as anticancer agents because of their selective growth in the hypoxic regions of solid tumors after systemic application. In this report we further describe a novel approach to cancer gene therapy in which genetically engineered clostridia are used as tumor-specific vectors for the delivery of antitumor genes. We have introduced into a strain of C. beijerinckii the gene for an E. coli nitroreductase known to activate the nontoxic prodrug CB 1954 to a toxic anticancer drug. Nitroreductase produced by these clostridia enhanced the killing of tumor cells in vitro by CB 1954, by a factor of 22. To demonstrate the specificity of this approach for tumor targeting, we intravenously injected the inactive spore form of C. beijerinckii, which upon transition to a reproductive state will express the E. coli nitroreductase gene. Nitroreductase activity was detectable in 10 of 10 tumors during the first 5 days after intravenous injection of inactive clostridial spores, indicating a rapid transition from spore to reproductive state. Tumors harboring clostridial spores which did not possess the E. coli nitroreductase gene were devoid of nitroreductase activity. Most importantly, E. coli nitroreductase protein was not found in a large survey of normal mouse tissues following intravenous injection of nitroreductase containing clostridia, strongly suggesting that obligate anaerobic bacteria such as clostridia can be utilized as highly specific gene delivery vectors for cancer therapy.