Probing the beta-chain hole of fibrinogen with synthetic peptides that differ at their amino termini

In a recent report, we showed that alanine can replace glycine at the amino terminus of synthetic B-knobs that bind to human fibrin(ogen). We now report a survey of 13 synthetic peptides with the general sequence XHRPYam, all tested with regard to their ability to delay fibrinolysis in an in vitro system activated by t-PA, the results being used as measures of binding affinity to the betaC hole. Unexpectedly, some large and bulky amino acids, including methionine and arginine, are effective binders. Amino acids that branch at the beta carbon (valine, isoleucine, and threonine) do not bind effectively. Crystal structures were determined for two of the peptides (GHRPYam and MHRPYam) complexed with fibrin fragment D-dimer; the modeling of various other side chains showed clashing in the cases of beta-carbon substituents. The two crystal structures also showed that the enhanced binding observed with pentapeptides with carboxyl-terminal tyrosine, compared with that of their tetrapeptide equivalents, is attributable to an interaction between the tyrosine side chain and a guanidino group of a nearby arginine (beta406). The equivalent position in gamma-chains of human fibrin(ogen) is occupied by a lysine (gamma338), but in chicken and lamprey fibrin(ogen), it is an arginine, just as occurs in beta chains. Accordingly, the peptides GPRPam and GPRPYam, which are surrogate A-knobs, were tested for their influence on fibrin polymerization with fibrinogen from lamprey and humans. In lampreys, GPRPYam is a significantly better inhibitor, but in humans, it is less effective than GPRPam, indicating that in the lamprey system the same tyrosine-arginine interaction can also occur in the gamma-chain setting.     

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: RESISTANCE OF α2-8-LINKED N-GLYCOLYLNEURAMINIC ACID TO ENZYMATIC CLEAVAGE

The sialic acid (Sia) N-acetylneuraminic acid (Neu5Ac) and its hydroxylated derivative N-glycolylneuraminic acid (Neu5Gc) differ by one oxygen atom. CMP-Neu5Gc is synthesized from CMP-Neu5Ac, with Neu5Gc representing a highly variable fraction of total Sias in various tissues and among different species. The exception may be the brain, where Neu5Ac is abundant and Neu5Gc is reported to be rare. Here, we confirm this unusual pattern and its evolutionary conservation in additional samples from various species, concluding that brain Neu5Gc expression has been maintained at extremely low levels over hundreds of millions of years of vertebrate evolution. Most explanations for this pattern do not require maintaining neural Neu5Gc at such low levels. We hypothesized that resistance of α2-8-linked Neu5Gc to vertebrate sialidases is the detrimental effect requiring the relative absence of Neu5Gc from brain. This linkage is prominent in polysialic acid (polySia), a molecule with critical roles in vertebrate neural development. We show that Neu5Gc is incorporated into neural polySia and does not cause in vitro toxicity. Synthetic polymers of Neu5Ac and Neu5Gc showed that mammalian and bacterial sialidases are much less able to hydrolyze α2-8-linked Neu5Gc at the nonreducing terminus. Notably, this difference was not seen with acid-catalyzed hydrolysis of polySias. Molecular dynamics modeling indicates that differences in the three-dimensional conformation of terminal saccharides may partly explain reduced enzymatic activity. In keeping with this, polymers of N-propionylneuraminic acid are sensitive to sialidases. Resistance of Neu5Gc-containing polySia to sialidases provides a potential explanation for the rarity of Neu5Gc in the vertebrate brain.     

Comparison of Protein Structures in Solution Using Local Conformations Derived from NMR Data: Application to Cytochrome c

Abstract

Structural comparisons of proteins in solution are often required to examine structure-functional relationships, study structural effects of mutations or distinguish between various forms of the same molecule under different conditions. A nuclear magnetic resonance (NMR) based probabilistic strategy is presented and used to study the structural differences between the two redox states of cytochrome C in solution. A probabilistic approach is employed to calculate the main chain conformations of horse ferro- and ferricytochrome C in solution, based on the published sequential d connectivity data. Conformational differences between the two oxidation states of horse cytochrome C in solution are found to be statistically significant. The largest changes in conformation are at residues Lys27, Thr28, Leu32, Gln42, Thr47, Tyr48, Thr49, Glu69, Lys72, Met80, Phe82, Ile85 and Lys86, all of which are close to the heme (within 14 Å of the heme iron in the high resolution Xray structure of tuna cytochrome c). We suggest that these conformational changes may modulate local dipole moments and hence influence the interactions of cytochrome C with its physiological redox partners during the electron transfer process. The oxidation state dependent conformational differences are found to be much greater in solution than in the crystalline state, and the solution and crystal structures differ significantly in regions close to the heme. These results suggest that the highly charged nature of cytochrome C makes this protein particularly sensitive to the ionic strength of its environment and leads to differences between crystal and solution structures in the same oxidation state. In such cases, crystal structures must be used with caution for modeling molecular interactions in vivo. More generally, this analysis indicates that the determination of accurate local conformations based on nmr data can provide useful information about structure-functional aspects of proteins in solution.     

CD28-B7 Interaction Modulates Short- and Long-Lived Plasma Cell Function

The interaction of CD28, which is constitutively expressed on T cells, with B7.1/B7.2 expressed on APCs is critical for T cell activation. CD28 is also expressed on murine and human plasma cells but its function on these cells remains unclear. There are two types of plasma cells: short-lived ones that appear in the secondary lymphoid tissue shortly after Ag exposure, and long-lived plasma cells that mainly reside in the bone marrow. We demonstrate that CD28-deficient murine short- and long-lived plasma cells produce significantly higher levels of Abs than do their wild-type counterparts. This was owing to both increased frequencies of plasma cells as well as increased Ab production per plasma cell. Plasma cells also express the ligand for CD28, B7.1, and B7.2. Surprisingly, deficiency of B7.1 and B7.2 in B cells also led to higher Ab levels, analogous to Cd28(-/-) plasma cells. Collectively, our results suggest that the CD28-B7 interaction operates as a key modulator of plasma cell function.     

PHENMETRAZINE HYDROCHLORIDE—A Clinical Evaluation of a New Anoretic Agent

Phenmetrazine hydrochloride appeared to be a safe anoretic agent and was effective in weight control in 80 per cent of a series of 49 obese patients.Tolerance to the drug did not develop in periods up to 18 weeks of continued treatment.Phenmetrazine was most effective in patients under 45 years of age in this series.Side effects were minimal and easily controlled. No allergic or toxic effects were noted.     

The morphogenetic action of follicle-stimulating hormone on post-nodal fragments of early chick blastoderms

Summary The morphogenetic action of ovine follicle-stimulating hormone has been studied. The hormone is incorporated in known concentrations in agar-albumen-compton base and post-nodal fragments of primitivestreak stage chick blastoderms 0.7 mm behind theHensen's node are cultured on the base for 35–45 hours. The hormone is found to induce the formation of neural tissue, notochord-like tissue and mesodermal aggregations resembling somites.