The chemical heterogeneity of human hemoglobin F. Direct evidence for the existence of three types of gamma chain, the G gamma I, A gamma I, and A gamma T chains.

Direct evidence is presented for the existence of three types of gamma chain of human hemoglobin F. A modification of a CM-cellulose chromatographic method has allowed the incomplete separation of these gamma chains while high pressure liquid chromatography and fingerprint analyses of tryptic peptides of zones of the isolated gamma chains, and amino acid analyses of isolated peptides were used to identify the chains. These studies have shown that the presence of a glycyl residue in position 136 (G gamma chain) is directly related to that of an isoleucyl residue in position 75 (I gamma chain), thus indicating the existence of an G gamma I chain, and that the presence of an alanyl residue in position 136 (A gamma chain) can be related to that of an isoleucyl residue in position 75, thus suggesting the existence of an A gamma I chain. When the isoleucyl residue at positive 75 is replaced by a threonyl residue, invariably it is related to the alanyl substitution at position 136 (A gamma T chain). These data support indirect evidence from case analyses and family studies which were published before, and indicate that the T gamma chain is an allele of the A gamma which should be renamed the A gamma T chain.     

Application of three-dimensional molecular hydrophobicity potential to the analysis of spatial organization of membrane protein domains. II. Optimization of hydrophobic contacts in transmembrane hairpin structures of Na+, K+-ATPase

A method of packing of transmembrane hairpin helices in proteins is described. The procedure is based on the optimization of hydrophobic contacts calculated using the three-dimensional (3D) molecular hydrophobicity potential technique. To verify the validity of the computational scheme, we calculated relative orientations of membrane-spanning peptides in pairs L2–L3, M2–M3, and M4–M5 from L- and M-subunits of the photoreaction center ofRhodopseudomonas viridis and compared the predicted structures with those derived from atomic coordinates. The results of computer modeling agree with the X-ray data. We applied the approach proposed to study possible interhelical interactions in transmembrane hairpin structures of Na⁺, K⁺-ATPase.     

Effect of lipid composition on the "membrane response" induced by a fusion peptide

To understand the initial stages of membrane destabilization induced by viral proteins, the factors important for binding of fusion peptides to cell membranes must be identified. In this study, effects of lipid composition on the mode of peptides' binding to membranes are explored via molecular dynamics (MD) simulations of the peptide E5, a water-soluble analogue of influenza hemagglutinin fusion peptide, in two full-atom hydrated lipid bilayers composed of dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC, respectively). The results show that, although the peptide has a common folding motif in both systems, it possesses different modes of binding. The peptide inserts obliquely into the DMPC membrane mainly with its N-terminal alpha helix, while in DPPC, the helix lies on the lipid/water interface, almost parallel to the membrane surface. The peptide seriously affects structural and dynamical parameters of surrounding lipids. Thus, it induces local thinning of both bilayers and disordering of acyl chains of lipids in close proximity to the binding site. The "membrane response" significantly depends upon lipid composition: distortions of DMPC bilayer are more pronounced than those in DPPC. Implications of the observed effects to molecular events on initial stages of membrane destabilization induced by fusion peptides are discussed.     

Modeling of peptides and proteins in a membrane environment.II. Structural and energetic aspects of Glycophorin A in a lipid bilayer

The conformational space of a hydrophobic peptide fragment of glycophorin A in a lipid membrane was studied with the Monte Carlo method using the solvation model described in the first communication of this series. The simulation was performed for various starting orientations of the peptide relative to the membrane bilayer: outside, inside, partially immersed, and transbilayer. We showed that the membrane substantially stabilizes the alpha-helical conformation of the central hydrophobic part of the glycophorin A molecule, which for the most part is immersed in the apolar core of the bilayer. For various conformational states, energy values were calculated and the orientations of the peptide relative to the membrane were characterized. Depending on the thickness of the bilayer, either an entirely alpha-helical conformation in transbilayer orientation or a conformation with a kink in the central part of the helix with the N- and C-termini exposed on one side of the membrane corresponds to the minimal-energy structure. The transmembrane orientation of glycophorin A is energetically advantageous when the membrane thickness is close to the length of its hydrophobic helical portion, which is consistent with the effect of "hydrophobic match" observed experimentally. The prospects for further refinement of the model are discussed.     

The immunization of adults against respiratory syncytial virus infection

The results of the comparative study of the immunological effectiveness of experimental samples of respiratory syncytial (RS) viral vaccine, prepared from a live attenuated strain and introduced in a single administration to young adults by the intranasal, intradermal and combined intranasal-intradermal) routes, are presented. The effectiveness of intranasal immunization was inversely related to the level of previously existing humoral (serum, secretory) antibodies. Intradermal immunization enhanced the frequency of the formation of serum antibodies in persons having had such antibodies before the introduction of RS vaccine. The most active formation of serum and secretory antibodies was ensured by the combined (intranasal-intradermal) method of the administration of live RS vaccine which proved to be particularly effective in persons having had antibodies in the blood and secretions prior to immunization.     

Preparation and use of monoclonal antibodies against seal alkaline phosphatase]

Monoclonal antibodies (termed as APP.1 and related to subclass IgG1) against seal alkaline phosphatase, have been obtained. APP.1 did not influence the enzymatic activity of alkaline phosphatase. The dissociation constant for the APP.1 interaction with Greenland seal alkaline phosphatase was equal to 8.5 x 10(-10) M. It was found that APP.1 interact with intestinal isoenzymes of common and fur seal, calf and deer alkaline phosphatases. An APP.1 complex with seal alkaline phosphatase was obtained and successfully applied in immunoenzymatic analysis. The use of this complex made it possible to diminish the limit of detectability of antibodies against peptide fragments of HIV-1 and HIV-2 proteins. Moreover, this complex allowed the identification of cytokeratin-8 and vimentin in human kidney slices and embryonic fibroblast-like cells, respectively.     

Dynamics and stability of polymorphic human telomeric G-quadruplex under tension

As critical DNA structures capping the human chromosome ends, the stability and structural polymorphism of human telomeric G-quadruplex (G4) have drawn increasing attention in recent years. This work characterizes the equilibrium transitions of single-molecule telomeric G4 at physiological K⁺ concentration. We report three folded states of telomeric G4 with markedly different lifetime and mechanical stability. Our results show that the kinetically favored folding pathway is through a short-lived intermediate state to a longer-lived state. By examining the force dependence of transition rates, the force-dependent transition free energy landscape for this pathway is determined. In addition, an ultra-long-lived form of telomeric G4 structure with a much stronger mechanical stability is identified.     

Beta-thalassemia due to a T----A mutation within the ATA box

Sequence analyses of amplified DNA from a Yugoslavian patient with Hb Lepore-beta-thalassemia and from his father with a simple beta-thalassemia trait have revealed a T----A mutation within the ATA box at a position 30 base pairs upstream from the Cap site. The nucleotide substitution was confirmed through dot-blot analysis of amplified DNA with specific 32P-labeled synthetic oligonucleotide probes. The patient had a clinically severe condition; his Hb Lepore-beta-thalassemia was of the beta + type, as about 8-10% of the non-alpha chain was normal beta A. The same T----A mutation at nucleotide -30 was present on both chromosomes of a young Turkish patient who suffered from a thalassemia intermedia with a low level of Hb F (13.1%) and a relatively high beta A chain synthesis. These data are similar to those obtained for other types of beta +-thalassemia caused by comparable substitutions at positions 31, 29, and 28 base pairs upstream from the Cap site of the beta-globin gene.