First report of Sphingomonas koreensis as a human pathogen in a patient with meningitis.

Sphingomonas koreensis is an aerobic Gram-negative rod originally described in 2001 following isolation from natural mineral water in Korea. Here, we report a case study with Sphingomonas koreensis as the causative agent of meningitis. To our knowledge, this is the first documented case of Sphingomonas koreensis as a human pathogen.

A randomised, double-blind study in adults with major depressive disorder with an inadequate response to a single course of selective serotonin reuptake inhibitor or serotonin-noradrenaline reuptake inhibitor treatment switched to vortioxetine or agomelatine.

OBJECTIVE: This randomised, double-blind, 12-week study compared efficacy and tolerability of flexible-dose treatment with vortioxetine(10-20 mg/day) versus agomelatine (25-50 mg/day) in major depressive disorder patients with inadequate response to selective serotonin reuptake inhibitor (SSRI)/serotonin-noradrenaline reuptake inhibitor (SNRI) monotherapy. METHODS: Patients were switched directly from SSRI/SNRI to vortioxetine or agomelatine. Primary endpoint was change from baseline to week 8 in the Montgomery-Åsberg Depression Rating Scale (MADRS) total score analysed by mixed model for repeated measurements, using a noninferiority test followed by a superiority test. Secondary endpoints included response and remission rates, anxiety symptoms(Hamilton Anxiety Rating Scale), Clinical Global Impression, overall functioning (Sheehan Disability Scale), health-related quality of life(EuroQol 5 Dimensions), productivity (work limitation questionnaire) and family functioning (Depression and Family Functioning Scale). RESULTS: Primary endpoint noninferiority was established and vortioxetine (n = 252) was superior to agomelatine (n = 241) by 2.2 MADRS points (p<0.01). Vortioxetine was also significantly superior in response and remission rates at weeks 8 and 12; MADRS, Hamilton Anxiety Rating Scale, Clinical Global Impression, Sheehan Disability Scale and EuroQol 5 Dimensions scores at week 4 onwards; work limitation questionnaire at week 8 and Depression and Family Functioning Scale at weeks 8 and 12. Fewer patients withdrew because of adverse events with vortioxetine (5.9% vs 9.5%). Adverse events (incidence ≥5%) were nausea, headache, dizziness and somnolence. CONCLUSIONS: Vortioxetine was noninferior and significantly superior to agomelatine in major depressive disorder patients with previous inadequate response to a single course of SSRI/SNRI monotherapy. Vortioxetine was safe and well tolerated.

Lectins as cell recognition molecules.

Lectins on cell surfaces mediate cell-cell interactions by combining with complementary carbohydrates on apposing cells. They play a key role in the control of various normal and pathological processes in living organisms.

Structure of a legume lectin with an ordered N-linked carbohydrate in complex with lactose.

The three-dimensional structure of the lactose complex of the Erythrina corallodendron lectin (EcorL), a dimer of N-glycosylated subunits, was determined crystallographically and refined at 2.0 angstrom resolution to an R value of 0.19. The tertiary structure of the subunit is similar to that of other legume lectins, but interference by the bulky N-linked heptasaccharide, which is exceptionally well ordered in the crystal, forces the EcorL dimer into a drastically different quaternary structure. Only the galactose moiety of the lactose ligand resides within the combining site. The galactose moiety is oriented differently from ligands in the mannose-glucose specific legume lectins and is held by hydrophobic interactions with Ala88, Tyr106, Phe131, and Ala218 and by seven hydrogen bonds, four of which are to the conserved Asp89, Asn133, and NH of Gly107. The specificity of legume lectins toward the different C-4 epimers appears to be associated with extensive variations in the outline of the variable parts of the binding sites.

A 6-Month Open-Label Extension Study of Vortioxetine in Pediatric Patients with Depressive or Anxiety Disorders.

OBJECTIVES: In this 6-month open-label extension (OLE) of NCT01491035 (a 14-day, open-label, pharmacokinetic/safety lead-in study), the long-term safety and tolerability of vortioxetine (5-20 mg/day) were investigated in children and adolescents with a DSM-IV-TR™ diagnosis of depressive or anxiety disorder in the United States or Germany. The study also was designed to provide data to inform dose selection and titration in future pediatric studies with vortioxetine. METHODS: Safety evaluations included spontaneously reported adverse events (AEs), the Columbia Suicide Severity Rating Scale (C-SSRS), and the Pediatric Adverse Events Rating Scale (PAERS; clinician administered). Clinical effectiveness was determined by Clinical Global Impressions. Comorbid attention-deficit/hyperactivity disorder was permitted, including concomitant use of stimulant medication (US sites only). RESULTS: Of the 47 patients who completed the lead-in period, 41 continued into the OLE. Most patients (n = 39 [95%]) continued their previous dose regimen. Twenty-one patients (51%) withdrew during the OLE; the most common primary reasons were administrative [n = 8], AEs [n = 4], and lack of efficacy [n = 3]. Thirty-five patients (85%) had ≥1 AE, 86% of which were mild or moderate in severity. Five patients (12%) reported a severe AE, none of which was considered related to study medication. The most common AEs (≥10%) were headache (27%), nausea (20%), dysmenorrhea (females; 19%), and vomiting (15%), with no relationship between AE intensity and age or dose. Five patients reported instances of suicidal ideation during the OLE, one of whom also reported this during the lead-in period. Two patients had nonsuicidal self-injurious behavior; one had a nonfatal suicide attempt. Throughout the study, there was a decrease over time in the incidence and intensity of AEs collected using the PAERS. Effectiveness assessment indicated a trend toward improvement based on numeric results. CONCLUSION: This OLE confirms the findings from the lead-in study, which concluded that a dosing strategy of 5-20 mg/day is safe, well tolerated, and suitable for future clinical studies of vortioxetine in pediatric patients.

Pharmacokinetics and Safety of Vortioxetine in Pediatric Patients.

OBJECTIVE: The primary objectives of this study were to evaluate the pharmacokinetics (PK) and tolerability of single and multiple doses of vortioxetine in children and adolescents with a depressive or anxiety disorder and to provide supportive information for appropriate dosing regimens for pediatric clinical trials. METHODS: This prospective, open-label, multinational, multisite, multiple-dose trial enrolled 48 patients (children and adolescents; 1:1 ratio) divided into 8 cohorts (4 adolescent and 4 child), with each cohort including 6 patients. The cohorts in each age group were assigned to receive one of four dosing regimens: vortioxetine 5, 10, 15, or 20 mg q.d. for 14 days. The total treatment period lasted 14-20 days with patients in the higher dose cohorts uptitrated over 2-6 days. Plasma samples for PK analysis were obtained on the first and last days of dosing. RESULTS: Among children and adolescents, respectively, 62% and 92% had depression and 58% and 33% had anxiety disorder. Comorbid attention-deficit/hyperactivity disorder (ADHD) was present in 50% of children and 38% of adolescents. After 14 days q.d. at the target dose, the PK of vortioxetine concentrations was generally proportional to the dose in both age groups. Exposure, as assessed by maximum plasma concentrations and area under the plasma concentration-time curve from time 0 to 24 hours, was 30%-40% lower in adolescents than in children. There was no significant relationship between sex, height, or ADHD diagnosis and PK parameters. Most adverse events were mild in severity and consistent with those seen in adults. CONCLUSION: The results suggest that the dosages of vortioxetine evaluated (5-20 mg q.d.; approved for treatment in adults) and the uptitration schedule used are appropriate for pediatric efficacy and safety trials.

13C-NMR study of the binding of [1-13C]galactose-labelled N-acetyllactosamine and [1-13C]galactose-enriched hen ovalbumin to soybean agglutinin.

The binding of the tide compounds to soybean agglutinin was investigated using 13C-NMR spectroscopy. The equilibrium constant for the binding of N-acetyllactosamine was found to be smaller than that obtained for the binding of ovalbumin (1.1 X 10(3) vs. 7.4 X 10(3) M-1). Only two binding sites per lectin tetramer were determined for the binding of ovalbumin, which is half the number of binding sites reported for the binding of small ligands to the lectin. Steric interference between the bulky ovalbumin molecules is believed to be the reason for the observed decrease in the apparent number of binding sites on the lectin.

Saccharides on teratocarcinoma cell plasma membranes. Their investigation with radioactively labelled lectins.

We have studied the interaction of five lectins differing in their sugar specificity, with the surface of clonal cell lines derived from transplantable murine teratocarcinoma. The results show that the differentiation from primitive embryonal carcinoma cells into parietal yolk sac cells is accompanied by changes in cell surface saccharides. These changes consist of a marked decrease in the total number of binding sites for the L-fucose-specific lectin of Lotus tetragonolobus and a large increase in the total number of binding sites for wax bean agglutinin. It is suggested that these differences can be used as markers in the study of this early embryonic differentiation. No agglutination of primitive embryonal carcinoma cells or of parietal yolk sac cells by low concentrations (10mug/ml) of concanavalin A, soybean agglutinin or the fucose binding proteins was observed.

Crystallization and preliminary X-ray diffraction studies of the lectin from Erythrina corallodendron.

The lectin from Erythrina corallodendron, specific for N-acetyllactosamine, crystallizes in the hexagonal space group P6(1) (P6(5)) with unit cell dimensions a = b = 136.3 A, c = 83.2 A and one dimer of Mr 60,000 in the asymmetric unit. The crystals are suitable for high-resolution work.

Crystallization and preliminary X-ray diffraction studies of soybean agglutinin.

Soybean agglutinin crystallizes in the monoclinic space group C2 with unit cell dimensions a = 118.6 A, b = 88.9 A, c = 165.9 A, beta = 103.0 degrees and one tetramer of 120,000 Mr per asymmetric unit. The crystals are suitable for high-resolution work.

Lectins--proteins with a sweet tooth: functions in cell recognition.

Lectins, non-enzymic proteins that bind mono- and oligosaccharides reversibly and with high specificity, occur widely in nature. They come in a variety of sizes and shapes, but can be grouped in families with similar structural features. The combining sites of lectins are also diverse, although they are similar in the same family. The specificities of lectins are determined by the exact shape of the binding sites and the nature of the amino acid residues to which the carbohydrate is linked. Small changes in the structure of the sites, such as the substitution of only one or two amino acids, may result in marked changes in specificity. The carbohydrate is linked to the protein mainly through hydrogen bonds, with added contributions from van der Waals contacts and hydrophobic interactions. Coordination with metal ions may occasionally play a role too. Microbial surface lectins serve as a means of adhesion to host cells of viruses (e.g. influenza virus), bacteria (e.g. E. coli) and protozoa (e.g. amoeba): a prerequisite for the initiation of infection. Blocking the adhesion by carbohydrates that mimic those to which the lectins bind prevents infection by these organisms. The way is thus open for the development of anti-adhesive therapy against microbial diseases. Lectin-carbohydrate mediated interactions between leucocytes and endothelial cells are the first step in the recirculation of lymphocytes and in the migration of neutrophils to sites of inflammation. Such interactions may also feature highly in the formation of metastases. Studies of these processes are expected to lead to the development of carbohydrate-based anti-adhesion drugs for the treatment of inflammatory diseases as well as cancer.

The amino acid sequence of Erythrina corallodendron lectin and its homology with other legume lectins.

The primary sequence of Erythrina corallodendron lectin was deduced from analysis of the peptides derived from the lectin by digestion with trypsin, chymotrypsin, Staphylococcus aureus V8 protease, elastase and lysylendopeptidase-C, and of fragments generated by cleavage of the lectin with dilute formic acid in 6 M guanidine hydrochloride. Purification of the individual peptides was achieved by gel filtration, followed by reverse phase HPLC. The glycosylation site (Asn17-Leu18-Thr19) was deduced from analysis of the glycopeptide isolated from a pronase digest of the lectin before and after deglycosylation of the glycopeptide with endoglycosidase F. Comparison of the sequence of 244 residues thus obtained with those of 9 other legume lectins revealed extensive homologies, including 39 invariant positions and 60 partial identities. These data provide further evidence for the conservation of the lectin gene in leguminous plants.

Identification of glycoproteins that are receptors for peanut agglutinin on immature (cortical) mouse thymocytes.

Binding of peanut agglutinin is being widely used as a marker for immature mouse thymocytes and for the separation of these cells from the mature thymocytes. Two cell surface glycoproteins that bind peanut agglutinin were detected on unfractionated as well as immature thymocytes by lectin overlay and affinity chromatography: one of Mr between 170 000 and 180 000, and the other, a minor component, of Mr 110000, both of which are partially sialylated. No receptors for peanut agglutinin were detected on the mature cells, whereas desialylation experiments revealed the presence of a glycoprotein of Mr 110000. These findings were corroborated by electrophoretic analysis of cell surface glycoproteins of the isolated thymocyte subpopulations labeled in their carbohydrate moieties.

Blood group MN-dependent difference in degree of galactosylation of O-glycans of glycophorin A is restricted to the GalNAc residues located on amino acid residues 2-4 of the glycophorin polypeptide chain.

Glycophorin A (GPA) of human erythrocytes contains a minor number of unsubstituted GalNAc residues (Tn receptors) which are recognized by Moluccella laevis lectin (MLL). The lectin reacts better with blood group N- than M-type of GPA which suggests a higher number of Tn receptors in GPA-N than in GPA-M. To find out whether this difference is restricted to a defined domain of GPA, the N-terminal tryptic glycopeptides of GPA-M and GPA-N (a.a. residues 1-39) and their fragments obtained by degradation with CNBr (a.a. residues 1-8 and 9-39) were analyzed. The untreated and desialylated glycopeptides were tested as inhibitors of MLL in ELISA, and the content of GalNAc-ol was determined in the products of beta-elimination of the asialoglycopeptides by gas-liquid chromatography/mass spectrometry. The asialoglycopeptides 1-39 and 1-8 derived from GPA-N showed about 2 and 4 times higher content of non-galactosylated GalNAc residues, respectively, and higher reactivity with MLL than their counterparts derived from GPA-M, while asialoglycopeptides 9-39 of GPA-M and GPA-N did not show such differences. These results demonstrate that higher expression of non-galactosylated GalNAc in GPA-N than in GPA-M is confined to GalNAc residues located in the amino-terminal portion of GPA polypeptide chain, between the blood group M- and N-specific amino acid residues 1 and 5.

Mn2+-electron spin resonance spectra of several lectins.

Mn2+-ESR spectra of soybean, wax bean and lima bean agglutinin at Q- and X-band frequencies show nearly axially symmetric zero field splitting (ZFS); the dominant anisotropic term of the spin hamiltonian is the quadratic ZFS interaction. There is a relatively large distribution of ZFS parameters. No effects of specific inhibitor (N-acetylgalactosamine) on the soybean agglutinin spectrum were observed. The stoichiometric complex obtained on addition of Mn2+ to a Mn2+-free sample of this protein has a spectrum similar to that of the native protein. The small changes in the spectrum are interpreted in terms of a wider distribution of the ZFS parameters at the Mn binding site. Addition of Ca2+ to Mn2+-soybean agglutinin sharpens the lines, possibly because Ca2+ increases the rigidity of the complex.

Chiral separation of gamma-butyrolactone derivatives by gas chromatography on 2,3-di-O-methyl-6-O-tert.-butyldimethylsilyl-beta-cyclodextrin.

The chiral GC separation of 2-alkyl-2-keto-gamma-butyrolactone derivatives and their alcohol analogs using 2,3-di-O-methyl-6-O-tert.-butyldimethylsilyl-beta-cyclodextrin (DIMETBCD) as chiral selector was discussed. The results, supported by the ketone preliminary molecular modelling calculations, suggest that the chiral recognition for DIMETBCD depends more on the geometry than on the polarity of the alkyl substituents on the butyrolactones. Hydrogen bonds and alkyl group steric effects should be an important function of the alcohol chiral recognition for DIMETBCD. Comparison of the retention times of the alcohol derivatives, in achiral and chiral stationary phases, suggests a specific structural effect for the cyclodextrin selctor.

Affinity of four immobilized Erythrina lectins toward various N-linked glycopeptides and related oligosaccharides.

The behavior of N-acetyllactosamine-type oligosaccharides and glycopeptides on columns of four different Erythrina agglutinins immobilized on Sepharose was examined. The sugar-binding specificity of the four lectins is very similar and is directed toward unmasked N-acetyllactosamine sequences, the main difference between the four lectins being the relative strength of interaction of the lectins with a given glycan. Substitution of the N-acetyllactosamine sequences by sialic acid residues, either at O-3 or O-6 of galactose completely abolishes the affinity of the lectins for the saccharides. The presence of one or several alpha-Fuc-(1----3)-GlcNAc groups decreases or completely inhibits the interaction between the glycopeptides and the Erythrina lectins. Substitution of the beta-mannose residue by an additional bisecting beta-(1----4)-N-acetylglucosamine residue decreases the affinity of the lectins for these structures as compared to the unsubstituted ones. Surprisingly, the affinity of the lectins for the oligosaccharides tested is higher than for the corresponding glycopeptides. Our findings show that, after careful calibration with well-defined oligosaccharides and glycopeptides, the immobilized Erythrina agglutinin-Sepharose columns provide valuable tools for the fractionation of N-acetyllactosamine-containing oligosaccharides and glycopeptides.

The glycosylation of glycoprotein lectins. Intra- and inter-genus variation in N-linked oligosaccharide expression.

Glycosylated lectins represent a series of glycoproteins with related activities and, in the case of the Leguminosae, related amino acid sequences. Therefore, they offer a model system in which to study the diversity of N-linked oligosaccharide structures of plant glycoproteins. The influence of the polypeptide on the type of oligosaccharide substitution and the problem of inter- and intra-genus variation in glycosylation can also be addressed. Analysis of the glycosylation of 18 lectins has shown that they can be classified into four qualitatively similar groups on the basis of the Bio-Gel P-4 elution profiles of the oligosaccharides released by hydrazinolysis: (a) The Erythrina cristagalli profile, with a major component at 8.8 glucose units (gu) and minor components at 8.0, 7.2, and 5.8 gu. The major component is the heptasaccharide, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-[beta-D-Xyl p-(1----2)]- beta-D-Manp-beta-D-GlcpNAc-(1----4)-[alpha-L-Fucp-(1----3)]- D-GlcNAc. (b) The Phaseolus vulgaris profile, which was characterized by peaks at 12.5, 11.7, 10.8, and 9.9 gu, in addition to the peaks at 8.8, 8.0, 7.2, and 5.8 gu mentioned above. These higher-mol.-wt. components were oligo-D-mannose oligosaccharides containing 9, 8, 7, and 6 D-mannose residues, respectively. (c) The Lonchocarpas capassa profile, which had a major peak at approximately 8 gu. (d) The soybean agglutinin profile, which has a single peak at 12.5 gu. This peak consisted solely of an oligomannose undecasaccharide containing 9 D-mannose residues. This lectin is unique in that it shows no microheterogeneity.

Immunochemical studies on the combining site of the A + N blood type specific Moluccella laevis lectin.

The specificity of the anti A+N lectin of Moluccella laevis (MLL) was examined by hemagglutination experiments with enzyme-modified human erythrocytes and by inhibition of hemagglutination. In addition, binding to various glycoproteins and inhibition by different sugars and glycoproteins were examined by enzyme immunoassay with antibodies to the lectin. Treatment of AMM erythrocytes with proteolytic enzymes increased their agglutinability by MLL 4-16-fold; similar treatment of ONN cells decreased their agglutinability 8-16-fold. This is in line with the known location and enzyme sensitivity of A and N specificity determinants. Treatment of the erythrocytes with sialidase increased their agglutinability and abolished the distinction between N and M cells. Hapten inhibition of hemagglutination of AMM and ONN erythrocytes by the lectin, and its binding to glycoproteins measured by enzyme immunoassay, confirmed the high specificity of MLL for N-acetyl-D-galactosamine (200-500 times more than for D-galactose) and suggested the presence of hydrophobic interactions around HO-2 of the D-galactose unit. The methyl alpha-glycosides of D-galactose and of N-acetyl-D-galactosamine were better inhibitors than the corresponding beta-glycosides; this preference was abolished, and sometimes reversed, when the p-nitrophenyl glycosides of the same monosaccharides were tested, stressing again the importance of hydrophobic interactions in the binding of carbohydrates to MLL. The lectin reacted well with ONN substance and with glycophorin A of the N phenotype (GPAN), but did not react with OMM substance or GPAM. The strongest inhibitor was asialo ovine submaxillary mucin, which contains many unsubstituted alpha-D-GalpNAc-(1-->3)-Ser/Thr residues; calculated per N-acetyl-D-galactosamine residue, it was 1500 stronger than free N-acetyl-D-galactosamine. In accordance with this result, it was found that the lectin strongly agglutinates Tn cells. The specificity of MLL can, thus, be defined as anti-Tn, crossreactive with blood types A and N, and with sialosyl-Tn. The N-specificity can best be explained by assuming that GPAN contains a small number of unsubstituted or partially sialylated alpha-D-GalpNAc-(1-->3)-Ser/Thr residues, which are present in smaller proportions, if at all, in GPAM.