Patients with lymphatic malformations who receive the immunostimulant OK-432 experience excellent long-term outcomes.

AIM: Sclerotherapy is the primary treatment for lymphatic malformations. The aim of this study was to evaluate the long-term outcome in patients with lymphatic malformations treated with the immunostimulant OK-432 as a sclerosant. METHODS: Between 1998 and 2013, we enrolled 131 of 138 eligible patients treated with OK-432 for lymphatic malformations in a retrospective study. The malformations were categorised according to the International Society for the Study of Vascular Anomalies. The outcome was assessed with a clinical examination and a questionnaire. RESULTS: The lymphatic malformations were localised to the head/neck (60%), the trunk (20%) and the extremities (6%) or involved with more than one region (14%). Patients with microcystic (10%), macrocystic (21%) and mixed lymphatic malformations (69%) underwent a median number of three, two and two injection treatments, respectively. The median age at the first injection was 3.4 years. Good or excellent clinical outcomes were seen in 70% of the patients. The number of injections, previous treatment and lesion localisation, but not time to follow-up and cyst size, predicted the clinical outcome. CONCLUSION: OK-432 treatment resulted in a successful outcome in 70% of patients with lymphatic malformations. The long-term outcome was comparable to the short-term outcome.

A novel study of association between Neisseria gonorrhoeae and the human carbohydrate blood groups.

Previous studies of association of ABO blood groups with gonorrhea have shown contradictory results. Despite the interdependencies of ABO, Lewis, and secretor systems, none of the previous studies examined the combined effect of these systems on their proposed association with gonorrhea. This study attempted to redress that and used genotyping in addition to RBC phenotyping to determine correct tissue phenotypes. Samples from 131 gonorrhea-positive individuals and from 175 gonorrhea-negative individuals were typed for ABO and Lewis using routine antisera. Secretor and Lewis genotyping was performed to ensure accurate determination of ABO and Lewis phenotypes. Chi-square and probability values were used to examine whether there is an association of ABO, Lewis, and secretor systems with gonorrhea infection. Neither single nor combined statistical analysis of data sets yielded a significant association of ABO, Lewis, and secretor phenotypes with Neisseria gonorrhoeae. Nevertheless, this study is an example of the approach that should be taken when examining microbial associations with ABO antigens, in turn influenced by coexpression and modification by the interdependent systems of Lewis and secretor, in mucosal tissues.

Proline cis-trans isomers in calbindin D9k observed by X-ray crystallography.

In a structure of recombinant bovine calbindin D9k, determined crystallographically to 1.6 A resolution, a proline in mixed, approximately equally populated, cis and trans conformation is observed. Isomers of this kind have not been reported in structure determinations of calbindin D9k to 2.3 A resolution or in any other crystallographically determined protein structure. The cis-trans isomerization occurs at the peptide bond between Gly42 and Pro43, which is in agreement with results from two-dimensional 1H nuclear magnetic resonance spectroscopy experiments on solutions of calbindin D9k. Alternative backbone stretches have been modeled and refined by stereochemical restrained least-squares refinement for the segment Lys41 to Pro43. The final R-value was 0.188. The structural perturbations accompanying the cis-trans isomerization are found to be very localized. The largest positional differences are observed at residue Gly42, in which the alternative positions of the oxygen atom are 3.6 A apart.

Structure of native and apo carbonic anhydrase II and structure of some of its anion-ligand complexes.

In order to obtain a better structural framework for understanding the catalytic mechanism of carbonic anhydrase, a number of inhibitor complexes of the enzyme were investigated crystallographically. The three-dimensional structure of free human carbonic anhydrase II was refined at pH 7.8 (1.54 A resolution) and at pH 6.0 (1.67 A resolution). The structure around the zinc ion was identical at both pH values. The structure of the zinc-free enzyme was virtually identical with that of the native enzyme, apart from a water molecule that had moved 0.9 A to fill the space that would be occupied by the zinc ion. The complexes with the anionic inhibitors bisulfite and formate were also studied at neutral pH. Bisulfite binds with one of its oxygen atoms, presumably protonized, to the zinc ion and replaces the zinc water. Formate, lacking a hydroxyl group, is bound with its oxygen atoms not far away from the position of the non-protonized oxygen atoms of the bisulfite complex, i.e. at hydrogen bond distance from Thr199 N and at a position between the zinc ion and the hydrophobic part of the active site. The result of these and other studies have implications for our view of the catalytic function of the enzyme, since virtually all inhibitors share some features with substrate, product or expected transition states. A reaction scheme where electrophilic activation of carbon dioxide plays an important role in the hydration reaction is presented. In the reverse direction, the protonized oxygen of the bicarbonate is forced upon the zinc ion, thereby facilitating cleavage of the carbon-oxygen bond. This is achieved by the combined action of the anionic binding site, which binds carboxyl groups, the side-chain of threonine 199, which discriminates between hydrogen bond donors and acceptors, and hydrophobic interaction between substrate and the active site cavity. The required proton transfer between the zinc water and His64 can take place through water molecules 292 and 318.

Crystal structure of phospholipase C from Bacillus cereus complexed with a substrate analog.

We report the first crystal structure of a complex between PLC from Bacillus cereus (PLCBc) and a competitive inhibitor that is an analog of the natural phospholipid substrate. The structure has been determined at 1.9 A resolution and refined to a final R-factor of 15.7%. The inhibitor binds with its phosphonyl group to the three Zn ions in the active site of the enzyme and is also involved in a hydrogen bonded network including several water molecules and amino acid side-chains which appear to help orient the substrate for productive binding. The interactions within this complex provide some important information regarding the mechanism of PLC-catalyzed hydrolysis of membrane phospholipids. A water molecule, located approximately apical to the diacylglycerol leaving group, seems to be the most likely candidate for the attacking nucleophile which initiates the reaction.

The symmetry of Escherichia coli cpn60 (GroEL) determined by X-ray crystallography.

The internal symmetries of the Escherichia coli molecular chaperone cpn60 oligomer, also called GroEL, have been examined by X-ray crystallography and self-rotation functions calculated at a resolution of 8.9 A. The oligomer ([cpn60]14) has one 7-fold symmetry axis and seven 2-fold axes that are all perpendicular to the 7-fold. The symmetry can be explained if oligomeric cpn60 is arranged as two heptamers stacked on top of each other, where the heptameric arrangement generates the 7-fold symmetry axis and the head-to-head assembly of two heptamers results in the seven 2-fold axes. This is an agreement with interpretations of electron microscopy data. However, the experimental determination of the symmetries reported here are made with an independent technique and at higher resolution. In addition self-rotation function calculations show that the symmetries observed are valid also for the internal parts of GroEL and not only for surface views. The orientations of the symmetry axes of the two independent cpn60 oligomers in the triclinic unit cell have been determined relative to the crystallographic axes. The planes formed by the 2-fold axes in the two oligomers deviate by about 2 degrees from the plane formed by the crystallographic a and c axes, while the 7-fold axes form angles of about 16 degrees with the b-axis. The two oligomers in the unit cell are arranged with their 7-fold axis parallel, but the second oligomer is rotated 26 degrees around the 7-fold axis relative to the first oligomer. Knowledge of the symmetry and orientation of the oligomers in the unit cell will be of great help in further crystallographic work.

A structural and functional comparison of staphylococcal enterotoxins A and C2 reveals remarkable similarity and dissimilarity.

Staphylococcal enterotoxins and toxic shock syndrome toxin-1 are known as superantigens due to their ability to activate a large number of T-cells by crosslinking the major histocompatibility complex class II molecules with the T-cell receptor. Although superantigens seem to act by a common mechanism, they vary in many of their specific interactions and biological properties. A structural comparison of staphylococcal enterotoxins A and C2, members of the staphylococcal superantigens, has shown large conformational differences at the putative TcR interaction site (loops between alphaN-alpha2, alpha4-beta9 and beta10-alpha5 in staphylococcal enterotoxin A) that could explain the variability in their T-cell receptor specificity. A common Zn2(+)-binding site was identified in both staphylococcal enterotoxin A and C2 that is superimposable but differs somewhat in its coordination geometry between the two molecules.

The crystal structure of staphylococcal enterotoxin H: implications for binding properties to MHC class II and TcR molecules.

The X-ray structure of the superantigen staphylococcal enterotoxin H (SEH) has been determined at 1.69 A resolution. In this paper we present two structures of zinc-free SEH (apoSEH) and one zinc-loaded form of SEH (ZnSEH). SEH exhibits the conventional superantigen (SAg) fold with two characteristic domains. In ZnSEH one zinc ion per SEH molecule is bound to the C-terminal beta-sheet in the region implicated for major histocompatibility complex class II (MHC class II) binding in SEA, SED and SEE. Surprisingly, the zinc ion has only two ligating amino acid residues His206 and Asp208. The other ligands to the zinc ion are two water molecules. An extensive packing interaction between two symmetry-related molecules in the crystal, 834 A(2)/molecule, forms a cavity that buries the zinc ions of the molecules. This dimer-like interaction is found in two crystal forms. Nevertheless, zinc-dependent dimerisation is not observed in solution, as seen in the case of SED. A unique feature of SEH as compared to other staphylococcal enterotoxins is a large negatively charged surface close to the Zn(2+) site. The interaction of SEH with MHC class II is the strongest known among the staphylococcal enterotoxins. However, SEH seems to lack a SEB-like MHC class II binding site, since the side-chain properties of structurally equivalent amino acid residues in SEH and those in SEB-binding MHC class II differ dramatically. There is also a structural flexibility between the domains of SEH. The domains of two apoSEH structures are related by a 5 degrees rotation leading to at most 3 A difference in C(alpha) positions. Since the T-cell receptor probably interacts with both domains, SEH by this rotation may modulate its binding to different TcR Vbeta-chains.

Crystals of protein S6 from the 30 S ribosomal subunit of Thermus thermophilus.

Crystals of protein S6 from the small ribosomal subunit of an extreme thermophile, Thermus thermophilus, have been obtained by the hanging-drop/vapor diffusion technique using methane pentanediol as a precipitant in the presence of potassium fluoride. The crystals belong to the space group C222 with cell parameters a = 106.7, b = 52.8, c = 41.0 A. They diffract to 2.0 A resolution.

Structural basis for the negative allostery between Ca(2+)- and Mg(2+)-binding in the intracellular Ca(2+)-receptor calbindin D9k.

The three-dimensional structures of the magnesium- and manganese-bound forms of calbindin D9k were determined to 1.6 A and 1.9 A resolution, respectively, using X-ray crystallography. These two structures are nearly identical but deviate significantly from both the calcium bound form and the metal ion-free (apo) form. The largest structural differences are seen in the C-terminal EF-hand, and involve changes in both metal ion coordination and helix packing. The N-terminal calcium binding site is not occupied by any metal ion in the magnesium and manganese structures, and shows little structural deviation from the apo and calcium bound forms. 1H-NMR and UV spectroscopic studies at physiological ion concentrations show that the C-terminal site of the protein is significantly populated by magnesium at resting cell calcium levels, and that there is a negative allosteric interaction between magnesium and calcium binding. Calcium binding was found to occur with positive cooperativity at physiological magnesium concentration.

Crystallization and preliminary X-ray investigation of the Escherichia coli molecular chaperone cpn60 (GroEL).

The Escherichia coli molecular chaperone, cpn60 (GroEL), has been purified from an overproducing E. coli strain and crystallized. Of the two crystal forms that were obtained, one was found to be suitable for crystallographic and structural studies at low resolution. Preliminary X-ray investigation of the crystals show unit cell dimensions: a = 143.3, b = 154.6 and c = 265 A, with alpha = 82 degrees, beta = 95 degrees and gamma = 107 degrees. The space group is P1 and the crystals diffract to a maximum of 7 A when using CuK alpha X-rays from a rotating anode. Both electron microscopy and non-denaturing electrophoretic analysis of redissolved cpn60 crystals show that cpn60 crystallizes in the native oligomeric form. Comparison between the dimensions of oligomeric cpn60 and the crystallographic unit cell volume suggests that the unit cell contains two oligomeric cpn60 molecules. The VM value for two cpn60 molecules per unit cell is 3.5 A3/Da, corresponding to a water content of 65%. Electrophoretic analysis under denaturing conditions shows that the cpn60 in crystals is heterogeneous, and this probably explains the limited resolution of the diffraction data.

Hydrolysis of 3H-bambuterol, a carbamate prodrug of terbutaline, in blood from humans and laboratory animals in vitro.

Tritiated bambuterol, a bis-dimethylcarbamate prodrug of terbutaline, was incubated in vitro with blood from both sexes of the following species: man, guinea pig, rat, mouse, dog and rabbit. The rates of hydrolysis of bambuterol to its monocarbamate derivative and further to terbutaline were measured. Large species variations were observed, e.g. blood from two of the human subjects was 15-fold more active than blood from the male rats. The rate of terbutaline formation as a function of initial bambuterol concentration was investigated in human plasma, and was found to describe a bell-shaped curve. Several pieces of evidence indicated that butyrylcholinesterase (EC 3.1.1.8) is the blood enzyme predominantly responsible for hydrolysis of bambuterol, although minor contributions from other esterases cannot be excluded. An exception may be blood from the rabbit, where the kinetics of the hydrolysis was different than in blood from the other species. The kinetics of bambuterol hydrolysis is discussed on basis of the established mechanism of carbamate interactions with cholinesterases, and the high affinity of bambuterol for butyrylcholinesterase.

Prevalence of cdtABC genes encoding cytolethal distending toxin among Haemophilus ducreyi and Actinobacillus actinomycetemcomitans strains.

The aim of this study was to investigate the presence of the three cdtABC genes responsible for production of cytolethal distending toxin (CDT) in Haemophilus ducreyi and Actinobacillus actinomycetemcomitans strains. Of 100 H. ducreyi strains from the culture collection of the University of Göteborg (CCUG), 27 strains with low or intermediate cytotoxic titre (< 1 in 10(4)) and 23 of the remaining isolates with a high cytotoxic titre (> or = 1 in 10(4)) were selected. Twenty-nine strains of H. ducreyi were isolated recently from patients with chancroid and 50 A. actinomycetemcomitans strains from patients with periodontitis. The cytotoxic activity on HEp-2 cells and the presence of cdtABC genes were studied by cytotoxicity assay of bacterial sonicates and PCR with primers specific for individual cdtA, B, and C genes of H. ducreyi in bacterial DNA preparations, respectively. All strains that manifested a cytotoxic titre in sonicate > or = 1 in 100 possessed all the three cdt genes. Eighteen of the 50 strains selected from the culture collection were negative and 32 positive for cdt genes. As all strains with a high cytotoxic titre gave positive PCR results, it can be assumed that the remaining 50 strains, which have high cytotoxic titre, would have been positive as well. Thus, it can be estimated that 82% of the culture collection strains had cdtABC genes. Similarly, 24 (83%) of 29 recent H. ducreyi isolates expressed the CDT activity and displayed all cdtABC genes. Forty-three (86%) of 50 strains of the closely related A. actinomycetemcomitans, expressing a cytotoxic activity > or = 1 in 100, also possessed all three genes. Furthermore, the nucleotide sequence of the cdtABC genes was highly conserved among H. ducreyi strains from different geographic areas. These results indicate that the majority of pathogenic H. ducreyi and A. actinomycetemcomitans strains express a CDT activity encoded by all three cdtABC.

Immobilisation and evaluation of a vancomycin chiral stationary phase for capillary electrochromatography.

The macrocyclic antibiotic, vancomycin, is covalently bonded to LiChrospher diol silica packed columns and evaluated in capillary electrochromatography (CEC) both in the reversed-phase and polar organic mode. Initially, capillaries were packed with 5 microm LiChrospher 100 A diol silica and evaluated in CEC with a reversed-phase biphenyl-pyrene achiral test resulting in resolution and efficiency values of ca. 2.5 and 100000 plates meter(-1), respectively. Repeatability for this test (resolution and efficiency) was also examined and found to be acceptable for both run-to-run (n=5, 0.74% and 1.5%) and column-to-column (n=5, 3.4% and 9.0%), respectively. Similar results were obtained when the 10 microm LiChrospher 1000 A diol silica was examined with the exception of efficiency, where a reduced plate height value of four times lower was obtained compared to the 100 A material. A simple three step in-situ vancomycin immobilisation procedure was subsequently carried out on these packed diol columns. Selectivity was obtained for thalidomide enantiomers on this vancomycin chiral stationary phase in reversed-phase CEC with resolution and efficiency values of ca. 2.5 and 80000 plates meter(-1), with acceptable repeatability (n=8) 0.9% and 3.0%, respectively. Selectivity was also obtained for thalidomide enantiomers on this phase in the polar organic mode with resolution and efficiency values of ca. 2.5 and 120000 plates meter(-1), with acceptable repeatability (n=7) 0.9% and 2.0%, respectively. It was possible to deduce from a chemometric design carried out for evaluating the mobile phase component effects that organic modifier ratio, MeOH/MeCN, played a significant role in controlling both resolution and efficiency. It was also possible to separate a number of basic analytes including four beta-adrenergic blocking agents in the polar organic mode albeit with lower resolution and efficiency values, ca. 1.5 and 45000 plates meter(-1), respectively.

Ab initio phase determination for X-ray diffraction data from crystals of a native protein.

An efficient algorithm for the determination of an everywhere positive electron-density distribution that agrees with observed structure amplitudes has been used to determine the phases of X-ray diffraction data from recombinant bovine chymosin, a protein with 323 amino-acid residues in the molecular chain whose structure was recently determined using molecular replacement methods. A systematic procedure for testing the signs of centric reflections, using the total entropy of the map as a figure of merit, was used to produce a low-resolution map. The phases of acentric and additional centric reflections were then chosen by adding them to the map with various possible phases and computing the total entropy of the resulting map. Of 159 centric reflections whose phases were chosen by this procedure, 141 had the same phase as in the refined structure. The median absolute phase difference for 1811 acentric reflections was 32 degrees. A map produced from these 1970 reflections, out of 12,346 reflections in the data set, showed a remarkable agreement with the refined structure. This molecule is many times larger than any whose structures have previously been determined without the use of isomorphous replacement, molecular replacement or anomalous dispersion, and the map demonstrates the potential of maximum-entropy methods in macromolecular structure determination.

Direct separation of captopril diastereoisomers including their rotational isomers by RP-LC using a teicoplanin column.

A direct reversed-phase liquid chromatography (LC) method has been developed for the separation and analysis of captopril and its 2R,2S diastereoisomer using a teicoplanin stationary phase. The proline containing diastereoisomers, which are known to form conformers in aqueous solution, were also separated from their rotational isomers. The influence of temperature, different organic modifiers and buffer type, concentration and pH were optimised to obtain a working resolution between the two diastereoisomers and their respective rotational isomers. The diastereoisomeric purity of several commercial captopril batches was subsequently evaluated using a 0.05% triethylammonium acetate (TEAA) buffer (pH 3.8) run at 1.0 ml/min with mobile phase reservoir and column temperature controlled at 0 degrees C. Throughout the study online UV diode array and mass spectrometry detection was carried out simultaneously to confirm that peaks eluting from the teicoplanin column were in fact captopril and not its readily converted disulphide dimer. Additionally, as a result of the greater detection sensitivity of mass spectrometry, it also facilitated a more accurate optimisation study where trace amounts of the rotational isomers were found to be present in the baseline at temperatures higher than optimum.

Identification of a new by-product detected in metoprolol tartrate.

A new impurity has been found in some batches of metoprolol tartrate. As the amount exceeded 0.1% it was of interest to deduce the structure. Techniques involved in solving the problem were LC, LC-MS and GC-MS. The LC systems showed that the impurity and metoprolol behaved differently to modifications of the mobile phase, indicating that there were differences in the functional groups. LC-MS was used to determine the molecular weight, which was 74 mass units higher than metoprolol. A hydrogen-deuterium shift technique using micro column LC-MS gave the information that three hydrogen atoms were bound to heteroatoms, i.e. one more than in metoprolol. This led to the conclusion that the impurity had three extra carbon and two extra oxygen atoms. It was supposedly a by-product in the synthesis. Knowledge of the synthesis steps for beta-receptor blocking drugs suggested three possible structures. Two were independently synthesized and one of these was found to be identical to the impurity.

Bambuterol, a bronchodilator prodrug with sustained action, enhances delivery of active drug to the lung.

As exemplified with bambuterol, a bronchodilator prodrug with sustained action, increased therapeutic selectivity can be obtained as a result of altered tissue distribution and pharmacokinetics rather than of increased beta 2-adrenoceptor selectivity.