Antigenic mapping of a human λ light chain: Correlation with three dimensional structure

Although the amino acid sequence and three-dimensional structure of human immunoglobulin light chains have been known for more than 15 years, the location of antigenic markers characteristic of chains has not been determined. Here, we use a set of synthetic overlapping peptides to completely model the sequence of the chain Mcg and test these for the binding or rabbit and goat antisera specific for chain determinants. We assess peptide contributions to -antigenic reactivity and also to identify a portion of C-region where conformational factors contribute to the antigenicity. Specific determinants occur both in the constant and variable (first and third framework) domains of the molecule. The fourth framework of the variable region, a segment specified by the joining gene, is also recognized and cross-reacts antigenically with the homologous region of T cell receptor chains. Major specific determinants are localized in the N- and C-terminal segments, which are linear and devoid of major conformational folding. Other segments that are strongly antigenic, such as the third framework of the V region (residue 78–93) and a segment of the constant region (residues 177–192), show strong conformational dependence in antigenicity.     

Pressure-induced conformational changes in a human Bence-Jones protein (Mcg)

The effect of high static pressures on the internal structure of the immunoglobulin light chain (Bence-Jones) dimer from the patient Mcg was assessed with measurements of intrinsic protein fluorescence polarization and intensity. Depolarization of intrinsic fluorescence was observed at relatively low pressures (less than 2 kbar), with a standard volume change of -93 mL/mol. The significant conformational changes indicated by these observations were not attributable to major protein unfolding, since pressures exceeding 2 kbar were required to alter intrinsic fluorescence emission maxima and yields. Fluorescence intensity and polarization measurements were used to investigate pressure effects on the binding of bis(8-anilino-naphthalene-1-sulfonate) (bis-ANS), rhodamine 123, and bis(N-methylacridinium nitrate) (lucigenin). Below 1.5 kbar the Mcg dimer exhibited a small decrease in affinity for bis-ANS (standard volume change approximately 5.9 mL/mol). At 3 kbar the binding activity increased by greater than 250-fold (volume change -144 mL/mol) and remained 10-fold higher than its starting value after decompression. With rhodamine 123 the binding activity showed an initial linear increase but plateaued at pressures greater than 1.5 kbar (standard volume change -23 mL/mol). These pressure effects were completely reversible. Binding activity with lucigenin increased slightly at low pressures (standard volume change -5.5 mL/mol), but the protein was partially denatured at pressures greater than 2 kbar. Taken in concert with the results of parallel binding studies in crystals of the Mcg dimer, these observations support the concept of a large malleable binding region with broad specificity for aromatic compounds.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phloem transport of 14C-labelled assimilates in Ricinus

Summary Exudate can be obtained from incisions made in the bark of the stem of actively growing Ricinus plants. ¹⁴C-labelled assimilates from a fed leaf are rapidly detected in the exudate. This movement was both acropetal and basipetal from the fed leaf, at rates of over 100 cm h^-1. Estimated rates within intact plants were 80–84 cm h^-1.In contrast with xylem sap obtained from the same plant, the exudate obtained had an alkaline pH (8.2), a high dry matter content (10–12.5%), high sugar content (8–10%) which was predominantly sucrose; high potassium content (60–80 mM) and low calcium content (0.5–1.0 mM).It is concluded, on the basis of the present evidence, that the exudate is a true sample of the sieve tube sap undergoing translocation.     

Evidence for structurally conserved recognition of the major carbohydrate xenoantigen by natural antibodies.

Natural or preformed antibodies that react with oligosaccharides bearing terminal galactose-alpha(1,3)-galactose [Gal alpha(1,3)Gal] stuctures are present in the sera of all humans. Antibodies against Gal alpha(1,3)Gal epitopes initiate hyperacute rejection of xenografts of porcine organs in human recipients. Despite the enormous clinical potential for xenotransplantation, very little is known about the 3D structural basis for natural antibody recognition of the major xenoantigen (i.e. Gal alpha(1,3)Gal). In this review, we discuss general binding patterns that have been repeatedly identified in antibody complexes with small molecules (haptens), carbohydrate and peptide ligands because similar mechanisms will almost certainly mediate recognition of the major xenoantigen by natural antibodies.     

The Smc5/Smc6/MAGE Complex Confers Resistance to Caffeine and Genotoxic Stress in Drosophila melanogaster

The SMC5/6 protein complex consists of the Smc5, Smc6 and Non-Smc-Element (Nse) proteins and is important for genome stability in many species. To identify novel components in the DNA repair pathway, we carried out a genetic screen to identify mutations that confer reduced resistance to the genotoxic effects of caffeine, which inhibits the ATM and ATR DNA damage response proteins. This approach identified inactivating mutations in CG5524 and MAGE, homologs of genes encoding Smc6 and Nse3 in yeasts. The fact that Smc5 mutants are also caffeine-sensitive and that Mage physically interacts with Drosophila homologs of Nse proteins suggests that the structure of the Smc5/6 complex is conserved in Drosophila. Although Smc5/6 proteins are required for viability in S. cerevisiae, they are not essential under normal circumstances in Drosophila. However, flies carrying mutations in Smc5, Smc6 and MAGE are hypersensitive to genotoxic agents such as ionizing radiation, camptothecin, hydroxyurea and MMS, consistent with the Smc5/6 complex serving a conserved role in genome stability. We also show that mutant flies are not compromised for pre-mitotic cell cycle checkpoint responses. Rather, caffeine-induced apoptosis in these mutants is exacerbated by inhibition of ATM or ATR checkpoint kinases but suppressed by Rad51 depletion, suggesting a functional interaction involving homologous DNA repair pathways that deserves further scrutiny. Our insights into the SMC5/6 complex provide new challenges for understanding the role of this enigmatic chromatin factor in multi-cellular organisms.     

Refinement of Imaging Predictors of Recurrent Events following Transient Ischemic Attack and Minor Stroke

Background

TIA and minor stroke have a high risk of recurrent stroke. Abnormalities on CT/CTA and MRI predict recurrent events in TIA and minor stroke. However there are many other imaging abnormalities that could potentially predict outcome that have not been assessed in this population. Also the definition of recurrent events used includes deterioration due to stroke progression or recurrent stroke and whether imaging is either of these is not known.

Aims

To improve upon the clinical, CT/CTA and MRI parameters that predict recurrent events after TIA and minor stroke by assessing further imaging parameters. Secondary aim was to explore predictors of stroke progression versus recurrent stroke.

Methods

510 consecutive TIA and minor stroke patients had CT/CTA and most had MRI. Primary outcome was recurrent events (stroke progression or recurrent stroke) within 90 days. Further imaging parameters were assessed for prediction of recurrent events (combined outcome of stroke progression and recurrent stroke). We also explored predictors of symptom progression versus recurrence individually.

Results

36 recurrent events (36/510, 7.1% (95% CI: 5.0–9.6)) including 19 progression and 17 recurrent strokes. On CT/CTA: white matter disease, prior stroke, aortic arch focal plaque≥4 mm, or intraluminal thrombus did not predict recurrent events (progression or recurrent stroke). On MRI: white matter disease, prior stroke, and microbleeds did not predict recurrent events. Parameters predicting the individual outcome of symptom progression included: ongoing symptoms at initial assessment, symptom fluctuation, intracranial occlusion, intracranial occlusion or stenosis, and the CT/CTA metric. No parameter was strongly predictive of a distinct recurrent stroke.

Conclusions

There was no imaging parameter that could improve upon our original CT/CTA or MRI metrics to predict the combined outcome of stroke progression or a recurrent stroke after TIA and minor stroke. We are better at using imaging to predict stroke progression rather than recurrent stroke.     

Ectopic expression of the Drosophila Cdk1 inhibitory kinases,Wee1 and Myt1, interferes with the second mitotic wave and disrupts pattern formation during eye development

Wee1 kinases catalyze inhibitory phosphorylation of the mitotic regulator Cdk1, preventing mitosis during S phase and delaying it in response to DNA damage or developmental signals during G2. Unlike yeast, metazoans have two distinct Wee1-like kinases, a nuclear protein (Wee1) and a cytoplasmic protein (Myt1). We have isolated the genes encoding Drosophila Wee1 and Myt1 and are using genetic approaches to dissect their functions during normal development. Overexpression of Dwee1 or Dmyt1 during eye development generates a rough adult eye phenotype. The phenotype can be modified by altering the gene dosage of known regulators of the G2/M transition, suggesting that we could use these transgenic strains in modifier screens to identify potential regulators of Wee1 and Myt1. To confirm this idea, we tested a collection of deletions for loci that can modify the eye overexpression phenotypes and identified several loci as dominant modifiers. Mutations affecting the Delta/Notch signaling pathway strongly enhance a GMR-Dmyt1 eye phenotype but do not affect a GMR-Dwee1 eye phenotype, suggesting that Myt1 is potentially a downstream target for Notch activity during eye development. We also observed interactions with p53, which suggest that Wee1 and Myt1 activity can block apoptosis.     

SIR2-induced inviability is suppressed by histone H4 overexpression

We have identified histone H4 as a high-expression suppressor of Sir2-induced inviability in yeast cells. Overexpression of histone H3 does not suppress Sir2-induced lethality, nor does overexpression of histone H4 alleles associated with silencing defects. These results suggest a direct and specific interaction between Sir2 and H4 in the silencing mechanism.