Determination of amide hydrogen exchange by mass spectrometry: a new tool for protein structure elucidation

A new method based on protein fragmentation and directly coupled microbore high-performance liquid chromatography-fast atom bombardment mass spectrometry (HPLC-FABMS) is described for determining the rates at which peptide amide hydrogens in proteins undergo isotopic exchange. Horse heart cytochrome c was incubated in D2O as a function of time and temperature to effect isotopic exchange, transferred into slow exchange conditions (pH 2-3, 0 degrees C), and fragmented with pepsin. The number of peptide amide deuterons present in the proteolytic peptides was deduced from their molecular weights, which were determined following analysis of the digest by HPLC-FABMS. The present results demonstrate that the exchange rates of amide hydrogens in cytochrome c range from very rapid (k > 140 h-1) to very slow (k < 0.002 h-1). The deuterium content of specific segments of the protein was determined as a function of incubation temperature and used to indicate participation of these segments in conformational changes associated with heating of cytochrome c. For the present HPLC-FABMS system, approximately 5 nmol of protein were used for each determination. Results of this investigation indicate that the combination of protein fragmentation and HPLC-FABMS is relatively free of constraints associated with other analytical methods used for this purpose and may be a general method for determining hydrogen exchange rates in specific segments of proteins.     

Comparison of tramadol and tramadol/droperidol mixture for patient-controlled analgesia

PURPOSE: To compare the analgesic efficacy and side effects of tramadol vs tramadol and droperidol for post-operative patient-controlled analgesia (PCA). METHODS: Randomised, double-blind study. Thirty-four patients undergoing elective colorectal or head and neck surgery were allocated to Group 1 (n = 18, PCA bolus 10 mg tramadol) or Group 2 (n = 16, PCA bolus 10 mg tramadol + 0.1 mg droperidol). Anaesthesia was induced with fentanyl and thiopentone and maintained with O2, N2O plus enflurane or isoflurane with iv morphine at doses decided by the attending anaesthetists. Muscle relaxation was achieved with atracurium or vecuronium. Patients were observed four-hourly for pain using an 11-point verbal rating scale (VRS). Nausea and vomiting, and sedation were assessed using four-point scales post-operatively. Vital signs, request for rescue anti-emetic and analgesic, and overall satisfaction were recorded. RESULTS: The mean nausea scores were lower in Group 2 (1.00 +/- 1.33 vs 0.06 +/- 0.25 at 0-8 hr, 1.22 +/- 1.93 vs 0.06 +/- 0.25 at 8-16 hr, P < 0.01; 0.81 +/- 1.68 vs 0 at 32-40 hr, P < 0.05; Group 1 vs Group 2). The vomiting scores were also lower (0.50 +/- 1.04 vs 0 at 0-8 hr, 0.67 +/- 1.50 vs 0, at 8-16 hr, P < 0.05; Group 1 vs Group 2). Seven (39%) patients in Group 1, but none in Group 2 requested rescue anti-emetic (P < 0.01). There were no differences in VRS, sedation score, overall satisfaction or vital signs. CONCLUSION: Tramadol and droperidol combination is superior to tramadol alone for post-operative PCA. It provides a similar quality of analgesia with less nausea and vomiting and without an increase in sedation.     

Genetic studies of spectrin: new life for a ghost protein

Spectrin, together with actin and a number of other accessory proteins, forms a submembrane cytoskeletal network in the human erythrocyte ghost. Through an elegant combination of structural, biochemical, and genetic studies, spectrin was shown to be an important determinant of erythrocyte shape and membrane stability. Genetic studies of a novel nonerythroid spectrin (beta H) in Drosophila and Caenorhabditis elegans now reveal that spectrin can influence the shape and stability of whole organisms. Nonerythroid spectrins are proposed to have roles in cell adhesion, establishment of cell polarity, and attachment of other cytoskeletal structures to the plasma membrane. The phenotypes of the beta H spectrin mutations provide an exciting biological context in which to evaluate these roles and perhaps to uncover new ones.     

Isolation and structure elucidation of an intermediate in the photodegradation of ciprofloxacin

Ciprofloxacin decomposes photochemically in aqueous solutions at acidic pH forming two major degradation products. One of the products, isolated from irradiated solutions by flash chromatography, was 7-[(2-aminoethyl)amino]-1-cyclopropyl-6-fluoro-1, 4-dihydro-4-oxo-3-quinoline carboxylic acid. The compound was an intermediate in the photochemical process, which degraded after longer exposure with a high-pressure mercury lamp to an aromatic amino-compound, 7-amino-1-cyclopropyl-6-fluoro-1, 4-dihydro-4-oxo-3-quinoline carboxylic acid. The structure of the intermediate was elucidated on the basis of information from ultraviolet, mass and nuclear magnetic resonance spectra.     

Fusaricidin A, a new depsipeptide antibiotic produced by Bacillus polymyxa KT-8. Taxonomy, fermentation, isolation, structure elucidation and biological activity

PURPOSE: This study was designed to examine variations in operative mortality among surgical specialists who perform colorectal surgery. METHODS: Mortality rates were compared between six board-certified colorectal surgeons and 33 other institutional surgeons using comparable colorectal procedure codes and a validated database indicating patient severity of illness. Thirty-five ICD-9-CM procedure codes were used to identify 2,805 patients who underwent colorectal surgery as their principal procedure between July 1986 and April 1994. Atlas, a state-legislated outcome database, was used by the hospital's Quality Assurance Department to rank the Admission Severity Group (ASG) of 1,753 patients from January 1989 to April 1994 (higher ASG, 0 to 4, indicates increasing medical instability). RESULTS: Colorectal surgeons had an eight-year mean in-hospital mortality rate of 1.4 percent compared with 7.3 percent by other institutional surgeons (P = 0.0001). There was a significantly lower mortality rate for colorectal surgeons compared with other institutional surgeons in ASG 2 (0.8 and 3.8 percent, respectively; P = 0.026) and ASG 3 (5.7 and 16.4 percent, respectively; P = 0.001). CONCLUSIONS: Board-certified colorectal surgeons had a lower in-hospital mortality rate than other institutional surgeons as patients' severity of illness increased.     

Mass spectrometry of platenolides and their derivatives in connection with structure elucidation

Mass spectra of platenolides--biosynthetic precursors of the 16-membered macrolide antibiotics, platenomycins--and their derivatives are discussed in detail especially in connection with structure elucidation. Mass spectrometry was of great use in establishing the structures for platenolides I (1) and II (2).     

Synthesis and characterization of N-octanoyl-beta-D-glucosylamine, a new surfactant for membrane studies

The new nonionic glycosidic surfactant N-octanoyl-beta-D-glucosylamine (NOGA, molar mass 305.37 g) was synthesized through an easy and efficient two-step procedure. Specifically, beta-D-glucosylamine was obtained by the replacement of the anomeric hydroxyl of D-glucose by an amino group which was then selectively acylated. NOGA was finally purified by silica gel column chromatography and recrystallization. This compound is stable and soluble in water and usual buffers up to 80 mM at 4 degrees C and up to 0.2 M at 37 degrees C. NOGA solutions are also characterized by a low ultraviolet light absorbance above 250 nm (epsilon 280 approximately 1.5 M-1 cm-1). Due to its very high critical micelle concentration (CMC = 80 mM, as determined by spectrofluorimetry), this surfactant may easily be removed from samples by dialysis or, to a lesser extent, by adsorption onto hydrophobic beads. Furthermore, NOGA is colorimetrically titrable by the ninhydrin method and its weak interference in protein determination by the bicinchoninic acid method is easy to overcome. This surfactant exhibits a good solubilizing power toward membrane proteins, with a marked selectivity for spiralin, a bacterial surface antigen. Protein extraction started below the CMC, but was much more effective above this concentration threshold. NADH oxidase activity, ligand binding by the glycine betaine-binding protein, and antigenicity of more than 20 membrane or soluble proteins were not altered by NOGA. Thus, owing to its extraction efficacy and mildness toward protein structure and activity, NOGA should prove useful for membrane studies and offers the additional advantage of being easy to synthesize at low cost.     

Biomechanical stress studies with a new Y-nail

The current success of the treatment of trochanteric fractures of the femur still depends on the type of fracture involved. A number of surgical procedures have proved successful in the treatment of stable fractures (e.g. dynamic hip screw, gamma nail). However, the treatment of unstable fractures remains a problem. With this type of fracture the implant is exposed to very unfavourable biomechanical loading that often leads to failure of the osteosynthesis, and makes removal of the implant necessary. In extensive tests, the biomechanical loading capacity of a new Y-nail has been investigated as a function of the stability and location of the fracture. The results show that this form of intramedullary fixing is suitable for both stable and unstable fractures. Unstable, far distal subtrochanteric fractures of the femur remain borderline indications.     

Properties and structure of a new non-reactive mold flux for high-Al steel

During the conventional continuous casting process of high-aluminum steels(w([Al])＞0.5 wt.％),some components of slag,such as SiO2,B2O3,and TiO2,could be reduced...     

Development of a new cellulose triacetate membrane with a microgradient porous structure for hemodialysis

Dialytic performance and biocompatibility of a newly developed cellulose triacetate (CTA) membrane with a microgradient porous structure, produced without using polyvinylpyrrolidone (PVP) and liquid paraffin, were compared with those of a conventional polysulfone (PS) membrane dialyzer. In vitro and clinical results demonstrated no significant difference in the dialytic performance and biocompatibility of the two dialyzers, but the CTA dialyzer lost less albumin during dialysis than the PS. These results suggest that a CTA membrane dialyzer with a porous microgradient structure attained comparable performance and biocompatibility to PS, and the risk of albumin loss was suppressed by the new CTA membrane.     

Structure elucidation of a photodegradation product of ciprofloxacin

In the photochemical degradation of ciprofloxacin, 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(piperazinyl)-3-quinolone carboxylic acid, two major decomposition products are formed in acidic solution. The main degradation product, after both artificial and daylight exposure, was 7-amino-1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-3-quinolone carboxylic acid. This product was also the dominating compound after more than 5 h irradiation with a high-pressure mercury lamp in aqueous solutions at pH < or = 2 when the solvent additionally contained water-miscible organic solvent. The structure of the isolated compound was elucidated on the basis of the chemical behaviour in thin-layer and high-performance liquid chromatography, and of information from infrared, ultraviolet, mass and nuclear magnetic resonance spectra.     

Repertory grid elucidation of a difficult conjoint therapy

The sacUh, amyB and pap mutations are identical with respect to their pleiotropic phenotype and their genetic location. Strains bearing these mutations overproduce several exocellular enzymes: alpha amylase, lavansucrase and proteases, they are poorly or not at all transformable and most of them are devoid of flagella. These mutations are tightly linked to the sacU- mutations by transformation and therefore lie between the hisA1 and gtaB290 markers. It is possible that the sacUh, amyB and pap mutations on one hand and the sacU- mutations on the other are two different classes of alterations of the same regulatory gene controlling the synthesis of some exocellular enzymes and several other cellular functions. Furthermore an amy- mutation, leading to the lack of alpha-amylase activity, was mapped between the lin2 and aroI906 markers which are not linked to the sacU locus.     

D-glyceric acidemia: biohcemical studies of a new syndrome

Studies of a mentally retarded boy, clinically suffering from nonketotic hyperglycinemia, are reported. Using combined gas chromatography-mass spectrometry, enzyme specificity studies, and spectropolariometry D-glyceric acid in extremely elevated concentrations was demonstrated in both serum and urine (serum: 1.0-1.3 mmol/liter, urine: 33-187 mmol/liter). Hydroxypyruvic acid was not detectable in urine from this boy using a gas chromatographic method with a limit of detection of 0.3 mmol/liter. Enzyme assays of D-glyceric dehydrogenase on blood leukocytes demonstrated significantly lower activity in the patient compared with five normal children.     

Isolation and structure elucidation of 7,8-dideoxy-6-oxo-griseorhodin C produced by Actinoplanes ianthinogenes

A novel product, isolated from a culture broth of Actinoplanes ianthinogenes fermented for producing purpuromycin, was purified and its structure established on the basis of physico-chemical data and chemical reactions. The new product resulted to be structurally related to griseorhodins, a group of hydroquinonic antibiotics obtained from Streptomyces californicus. This compound showed a weak activity against Gram-positive and resulted inactive against Gram-negative bacteria and Candida albicans.     

Antineoplastic agents. 398. Isolation and structure elucidation of cephalostatins 18 and 19

Continued investigation of murine leukemia (P-388) active fractions from the African marine worm Cephalodiscus gilchristi has resulted in the discovery of cephalostatins 18 (1b) and 19 (1c). The structures were determined by interpretation of their highfield (500 MHz) 1H, 13C, and 2D NMR and HRMS. Both of these new methoxy steroidal alkaloids exhibited strong activity against the murine P-388 lymphocytic leukemia cell line (ED50 ca. 10(-3) microg/mL), a mini panel of human cancer cell lines (GI50 <10(-3) microg/mL), and the U.S. National Cancer Institute's 60 human cancer cell line panel (mean panel GI50 ca. 10(-9) M).     

新型LiB化合物晶体结构和形貌研究

3.3 电子密度函数分析 电子密度函数的计算是确定晶胞内电子云分布情况最好的方法,它可以使键性和结构的测定更为精确。计算电子密度函数的公式为     

Laboratory studies with a new broad-spectrum penicillin, pirbenicillin

Pirbenicillin {6-[d-2-phenyl-2(N-4-pyridylformimidoylaminoacetamido) -acetamido]-penicillanic acid} showed broad-spectrum antibacterial activity in vitro and also in the treatment of experimental infections after parenteral administration to mice. Against Pseudomonas aeruginosa, a three- to fourfold potency advantage over carbenicillin was seen both in vitro and in vivo. The in vitro antibacterial spectrum of pirbenicillin includes Escherichia coli, Serratia, Citrobacter, and Enterobacter isolates, against which it exhibited minimal inhibitory concentration values comparable to those of carbenicillin. However, mice infected with E. coli and Serratia were protected at doses of pirbenicillin that were two to four times lower than those required of carbenicillin. Pirbenicillin was more active than carbenicillin against gram-positive bacteria, especially Streptococcus faecalis. It was less active than carbenicillin against Proteus spp. and was inactive against ampicillin-resistant E. coli strains. Pirbenicillin was bactericidal at concentrations generally equal to or only two-fold higher than the minimal inhibitory concentration. With appropriately buffered media, pirbenicillin demonstrated eight- and fourfold better minimal bactericidal concentration values towards Pseudomonas isolates than those of carbenicillin and ticarcillin, respectively.     

Response time studies of a new, portable mass spectrometer

Needleless connectors have been widely introduced into clinical practice to allow the connection of syringes and luers to peripheral and central vascular catheters. The potential for microbial contamination of catheters via these devices is currently unclear. A recently introduced connector, the 'Connecta Clave', was assessed by various in-vitro methods. The 'Connecta Clave' is specifically devised to separate external components from the fluid pathway. The compression seals of 50 devices were contaminated with 1 x 10(4) cfu Staphylococcus epidermidis, disinfected with isopropanol, and fluid passed through. Only one device allowed organisms to pass through, despite this challenge, representing a contamination rate of 2%. In comparison, when 50 connectors were challenged with 20 cfu of S. epidermidis, no organisms passed through the device during use. In the clinical situation, after manipulation, < 16 cfu of skin organisms were found associated with the compression seal of the devices. It is, therefore, likely that the contamination rates in clinical practice will be extremely low. Three methods of disinfecting the compression seals and associated rims were also evaluated. A combination of alcohol chlorhexidine spray, followed by a 70% isopropanol swab, resulted in the most efficacious disinfection. The isopropanol swabs produced an adequate disinfection rate. The overall results suggest that by use of specially designed connectors, not only are needlestick injuries reduced, but the likelihood of microbial contamination of catheters via the internal route may also be diminished.