N-Terminal sequencing by mass spectrometry through specific fluorescamine labeling of α-amino groups before tryptic digestion

We present a single-step procedure for the specific mass labeling of unblocked protein N termini. We show that the dye fluorescamine, which is commonly assumed to require mildly alkaline conditions for undergoing a nonspecific reaction with α- and ε-amino groups associated with amino acids, in fact shows a specific reaction only with α-amino groups present at protein N termini when mildly acidic conditions are used. We use this finding to label, identify, and sequence the trypsinolysis-derived N-terminal peptide of lysozyme, using only mass spectrometry, to illustrate how this method could be used with other proteins.     

Serotonin transporter gene,childhood emotional abuse and cognitive vulnerability to depression

Meta‐analyses evaluating the association between the serotonin transporter polymorphism (5‐HTTLPR) with neuroticism and depression diagnosis as phenotypes have been inconclusive. We examined a gene–environment interaction on a cognitive vulnerability marker of depression, cognitive reactivity (CR) to sad mood. A total of 250 university students of European ancestry were genotyped for the 5‐HTTLPR, including SNP rs25531, a polymorphism of the long allele. Association analysis was performed for neuroticism, CR and depression diagnosis (using a self‐report measure). As an environmental pathogen, self‐reported history of childhood emotional abuse was measured because of its strong relationship with depression. Participants with the homozygous low expressing genotype had high CR if they had experienced childhood emotional maltreatment but low CR if they did not have such experience. This interaction was strongest on the Rumination subscale of the CR measure. The interaction was not significant with neuroticism or depression diagnosis as outcome measures. Our results show that 5‐HTTLPR is related to cognitive vulnerability to depression. Our findings provide evidence for a differential susceptibility genotype rather than a vulnerability genotype, possibly because of the relatively low levels of abuse in our sample. The selection of phenotype and environmental contributor is pivotal in investigating gene–environment interactions in psychiatric disorders.     

Fine structural recognition specificities of IgE antibodies distinguishing amoxicilloyl and amoxicillanyl determinants in allergic subjects.

IgE antibodies in the sera of subjects allergic to beta-lactam antibiotics detect a spectrum of specificities ranging from side-chain groups to an entire penicillin or cephalosporin molecule. In addition to such structural heterogeneity of allergenic determinants, IgE antibodies in the sera of different allergic subjects show heterogeneous recognition responses. Detailed immunochemical studies were carried out on the sera of penicillin-allergic subjects that showed selective and unexpected reactions with the frequently prescribed penicillin, amoxicillin. Antibodies from one subject reacted only with the amoxicilloyl determinant while IgE from another subject showed multiple reactivity with penicilloyl and penicillanyl determinants of different penicillins but not with the amoxicilloyl determinant. Quantitative hapten inhibition studies revealed that the combining sites of the former antibodies were complementary to amoxicillin in a form that permits binding to the hydroxyaminobenzyl side-chain and the thiazolidine ring carboxyl. These conditions are satisfied with the drug in the '-oyl' but not in the '-anyl' form which involves linkage through the 2-carboxyl of the thiazolidine ring. With the second serum, adsorption studies showed that the wide-ranging reactivity of IgE was due to a single population of antibodies that detected a common specificity on the different penicillins. Combining site studies revealed clear recognition of the benzyl portion of the side-chain of benzylpenicilloyl, benzylpenicillanyl, ampicilloyl, ampicillanyl and amoxicillanyl determinants when free antibody access to the side-chain was possible but little or no recognition of the ring hydroxyl of amoxicillin. Such uninhibited access may not occur, however, when amoxicillin is conjugated in the '-oyl' form since opening the beta-lactam ring allows increased flexibility and rotation of the molecule and the possibility of close association of the hydroxyaminobenzyl side-chain of amoxicillin with the linked peptide carrier. In such close steric association, H-bonding involving the ring hydroxyl and amino acids of the carrier may prevent antibody access to the side-chain region of the amoxicilloyl determinant.     

Purification and characterisation of an inducible β-galactosidase from Corynebacterium murisepticum

The -galactosidase (EC 3.2.1.32) of Corynebacterium murisepticum (inducible by lactose and galactose) was purified by successive column chromatography on Sephadex G-200, DEAE-Sephadex A-50 and DEAE-cellulose (DE52). The enzyme was found to be a dimer of identical subunits of molecular mass 100,000 daltons. The K _m values of the enzyme for the substrates lactose and o-nitrophenyl--d-galactopyranoside (ONPG) are 16.7 mM and 4.4 mM, respectively, indicating, its low affinity for the substrates. The Ouchterlony immunodiffusion method exhibited immunological homogeneity of the enzyme preparation. The catalytic site of the enzyme does not take part in antigen-antibody reaction.     

Size fractionation of thermal aggregates of immunoglobulin G

Purified pooled human immunoglobulin G (IgG) in solution, when extensively heated at high temperatures or for long periods, irreversibly aggregates and insoluble precipitates result. However, when IgG solutions are heated in the temperature range 55-65 degrees C for more limited time periods, soluble turbid polydispersed aggregate mixtures are obtained. Gel filtration of such aggregate mixtures on calibrated Bio-Rad A-150m columns demonstrates a continuous size distribution from dimers to aggregates as large as 4 X 10(7) Da (200-mers) with no particular size predominant. Chromatographically reproducible cuts of narrow size heterogeneity can be obtained by short-time fraction collection. Elution-time reproducibility is excellent both for mixture and for individual cuts. Stability studies indicate that reproducible and stable aggregates may be made from purified IgG and that fractionated aggregates should be stored quick-frozen until needed. Sized IgG aggregates have proved useful in reactivity studies with rheumatoid factor, animal anti-IgG antibodies, and complement.     

The effect of feedback-assisted reduction in heart rate reactivity on videogame performance

In 67 male volunteers, we examined the reduction of cardiovascular responsivity to a psychomotor challenge (videogame) achieved by use of heart rate (HR) feedback and effects of these procedures on concomitant behavioral performance. Each subject participated in a pretraining assessment of his cardiovascular responses to the videogame, a training condition, and a posttraining assessment identical to the initial evaluation. During training, subjects were assigned to one of four conditions: (a) a habituation control group receiving no instructions to alter HR (HC); (b) an instructions-only control group receiving instructions to maintain a low or unchanged HR during videogame presentations (IC); (c) a feedback group receiving instructions to reduce HR using ongoing HR feedback (FB–); or (d) a feedback group receiving instructions to lower HR and given HR feedback plus a score contingency in which total game score was jointly determined by subjects' game performance and success at HR control (FB+). Subjects receiving feedback (FB+, FB–) exhibited greater reductions in HR response to the videogame in the posttraining assessment than control (HC, IC) subjects; FB+ subjects showed greater HR reductions than subjects in any other group. FB+ and FB– subjects showed a lower SBP at posttraining relative to the two control groups, but no reduction in task-induced blood pressure reactivity. There were no group differences in videogame performance, either before or following training.The authors wish to thank Fred Claus, who served as a research assistant for this study.     

Particle sources and trace element reactivity in the Humber plume

Samples of suspended particulate matter (SPM) collected from the Humber Estuary had higher concentrations of particulate metals than SPM from Holderness coastal waters (U.K.). Characterised SPM from both sources was used in laboratory experiments involving the uptake of radiotracer¹⁰⁹Cd,¹³⁷Cs,⁵⁴Mn and⁶⁵Zn. Kinetic experiments, over five days, showed that the rate and extent of uptake was highly dependent on particle type, with¹⁰⁹Cd,⁵⁴Mn and⁶⁵Zn being more reactive with Humber Estuary particles than those from Holderness and¹³⁷Cs having the opposite trend. Adsorption experiments were also carried out on suspensions in which SPM from the Humber Estuary and Holderness coastal water were mixed in various proportions. These experiments revealed that K_d for⁶⁵Zn increased linearly with the proportion of Humber SPM, K_d for¹³⁷Cs decreased linearly with increase in Humber SPM and K_d for⁵⁴Mn and¹⁰⁹Cd displayed non-linear behaviour. The results of the study were used to develop an algorithm for predicting the partition coefficients in the Humber Plume based on the extent of particle mixing from the two source regions. The use of^206/207Pb ratios in determining the extent of particle mixing is discussed, along with the application of the algorithm to the modelling of particulate trace metal behaviour in the Humber-Wash coastal zone.