Modifications of aldolase during in vivo aging of rabbit red cells.

Crude hemolysates, partially purified aldolase and aldolase purified to homogeneity from reticulocytes and mature erythrocytes, were incubated with a specific antiserum raised against crystalline rabbit muscle aldolase. We show that the same aldolasic activity corresponds to a greater amount of antigen in older than in younger cells, in crude hemolysates as well as in the above mentioned preparations; that is to say, old-cell aldolase contains cross-reacting material (CRM). Properties of purified enzyme from reticulocytes and mature erythrocytes were compared to those of muscle crystalline aldolase: -- the molecular specific activity of purified aldolase from erythrocytes is lower than with crystalline muscle aldolase, i.e. CRM is maintained throughout the purification steps. -- the specific activity of red cell aldolase towards both substrates (FDP and F1P) is lower than that of crystalline muscle aldolase. However, the ratio of activity towards the two substrates FDP/F1P is decreased in erythrocytes and reticulocytes. -- no other difference was found: Michaelis constant towards FDP, thermodenaturation constant and C terminal extremities are identical as are the molecular weights.     

Fine Sampling of Sequence Space for Membrane Protein Structural Biology

We describe an enhancement of traditional genomics-based approaches to improve the success of structure determination of membrane proteins. Following a broad screen of sequence space to identify initial expression-positive targets, we employ a second step to select orthologs with closely related sequences to these hits. We demonstrate that a greater percentage of these latter targets express well and are stable in detergent, increasing the likelihood of identifying candidates that will ultimately yield structural information.     

Biochemical and biological characteristics of cross-reacting material 197 (CRM197), a non-toxic mutant of diphtheria toxin: Use as a conjugation protein in vaccines and other potential clinical applications

The biochemical and biological characteristics of CRM₁₉₇ are reviewed. Polysaccharide protein conjugate vaccines represent an important technological advancement that allowed for protection against dangerous diseases in vulnerable populations such as infants. The first carrier proteins, diphtheria and tetanus toxoids, were chosen in the context of an extensive body of information describing their immunogenicity and safety profiles in clinical use. These carriers perform well, and they require detoxification. A non-toxic mutant of diphtheria toxin, cross-reacting material 197 (CRM₁₉₇), is a useful carrier protein with several manufacturing and other potential advantages over toxoids. For over a decade, several important and widely used routine childhood glycoconjugate vaccines against serious illnesses, including Haemophilus influenzae type b and pneumococcal disease, have employed CRM₁₉₇ as carrier protein. Additional clinical applications of CRM₁₉₇, as in chemotherapy, also exist.     

SUMOylation mediates the disassembly of the Smad4 nuclear export complex via RanGAP1 in KELOIDS

Sentrin/small ubiquitin-like modifier (SUMO) has emerged as a powerful mediator regulating biological processes and participating in pathophysiological processes that cause human diseases, such as cancer, myocardial fibrosis and neurological disorders. Sumoylation has been shown to play a positive regulatory role in keloids. However, the sumoylation mechanism in keloids remains understudied. We proposed that sumoylation regulates keloids via a complex. RanGAP1 acted as a synergistic, functional partner of SUMOs in keloids. Nuclear accumulation of Smad4, a TGF-β/Smad pathway member, was associated with RanGAP1 after SUMO1 inhibition. RanGAP1*SUMO1 mediated the nuclear accumulation of Smad4 due to its impact on nuclear export and reduction in the dissociation of Smad4 and CRM1. We clarified a novel mechanism of positive regulation of sumoylation in keloids and demonstrated the function of sumoylation in Smad4 nuclear export. The NPC-associated RanGAP1*SUMO1 complex functions as a disassembly machine for the export receptor CRM1 and Smad4. Our research provides new perspectives for the mechanisms of keloids and nucleocytoplasmic transport.     

Chemical evidence for covalent linkages of a semisynthetic glycoconjugate vaccine forHaemophilus influenzae type b disease

We have defined the nature of the covalent linkages in aHaemophilus influenzae type b oligosaccharide-CRM₁₉₇ conjugate vaccine, designated HbOC. The conjugate was acid hydrolyzed to release a novel amino-acid derivative,N-(2-hydroxyethyl)lysine (OHEt-Lys), identifiable with an amino-acid analyzer. This amino-acid derivative was formed by reduction of Schiff bases formed betweenH. influenzae type b oligosaccharides (HbO) and the lysyl -amino groups of CRM₁₉₇ (a non-toxic, cross-reactive variant of diphtheria toxin), followed by acid hydrolysis of HbOC. Quantification of OHEt-Lys per CRM₁₉₇ molecule allowed the determination of a covalency ratio, a useful parameter for evaluating the stoichiometry and consistency of HbOC preparations. Covalent association between HbO and CRM₁₉₇ was also demonstrated by the coincidence of immunoreactivity of gelelectrophoresed HbOC on a Western blot probed with anti-CRM₁₉₇ and anti-saccharide antisera.