Reduction in antiviral activity of human interferon-gamma in acidic media with reference to structural change

The fluorescence intensity of a unique tryptophan 36 in human interferon-gamma was drastically decreased below pH 4 with a concomitant decrease of antiviral activity. The region of residues 32-42 of human interferon-gamma was found by calculation to have a low hydrophobicity together with a high helical hydrophobic moment, and the net electric charge of this region having an amphiphilic helical structure changed significantly near pH 4. These results suggest that the region of residues 32-42 plays an important role in exhibiting antiviral activity.     

The structure ofHLA-B35 suggests that it is derived fromHLA-Bw58 by two genetic mechanisms

The primary structure ofHLA-B51 andHLA-Bw52 suggested thatHLA-B51 was derived fromHLA-Bw52 by the combination of a genetic exchange withHLA-B8 and a point mutation. To investigate the evolution of theHLA-B5 cross reactive group, theHLA-B35 gene was cloned and the primary structure was determined.HLA-B35 is identical toHLA-Bw58 except in the α₁ domain. The α₁ domain ofHLA-B35 except Bw4/Bw6-associated amino acids is identical to that ofHLA-B51 ^*, which was suspected to be an intermediate gene betweenHLA-B51 andHLA-Bw52. These data suggest thatHLA-B35 has evolved fromHLA-Bw58 in two steps; an in vivo replacement of the α₁ domain withHLA-B51 and genetic exchange with one of theHLA-Bw6 genes. These three genes andHLA-Bw58 are postulated to share a common ancestor.     

A newly established human acute lymphoblastic leukemia cell line with characteristics of the earliest B-cell maturation

A new human acute lymphoblastic leukemia (ALL) cell line, designated HBL-3, was established from the bone marrow of a patient with non-T-ALL. The HBL-3 cell line expressed B4 (CD 19), BA-1 (CD 24) and HLA-DR antigens, but not surface immunoglobulin (SIg) or cytoplasmic immunoglobulin (CIg). The cell line lacked the common acute lymphoblastic leukemia antigen (CALLA) and antigenic markers characteristic of T-cell and myeloid cell lineages. The HBL-3 cells had structural rearrangements of both the homologous chromosome 9s, including a translocation with chromosome 1 which has been reported in a patient with common ALL. The cell line had rearranged immunoglobulin heavy chain genes but retained germ-line κ light chain genes and germ-line T-cell receptorβ- and γ-chain genes. The HBL-3 cell line was strongly positive for terminal deoxynucleotidyl transferase (TdT). These findings indicate that the HBL-3 cell line is derived from the earliest B-cell committed to B-cell lineage.     

Involvement of the histidine residues in the pH-induced conformational change of histone H5

Comparative studies on the conformational stability of histones H1 and H5 have been carried out by monitoring the pH-induced conformational transitions of the proteins by CD and 1H NMR spectroscopies. The transition point of H1 agrees with the pKa of the carboxyl groups of the acidic residues. In contrast, the transition of H5 is associated with the ionization of the histidine residues which exist exclusively in H5, as well as the deionization of the acidic residues. These observations, combined with the result of the deuterium exchange rates of the histidine C-2 protons, led us to conclude that His-25 and His-62, which are buried in the globular domain, play an important role in the conformational stability of histone H5.     

Distinction and similarity in the structure of histones H1 and H5 as indicated by 13C nuclear-magnetic-resonance spectroscopy

The 13C nuclear magnetic resonance studies have been carried out on histones H1 and H5, by focusing our interest on possible formation of specific salt bridges between acidic and basic amino acid residues in the proteins and also on the structural difference between the two proteins. The 13C chemical shift and pKa values of the carboxyl group of glutamic acid residues in the histones coincided with those of free glutamic acid. Based on this result and another experiment using completely modified lysine residues in the histones, no evidence for a specific interaction between acidic and basic residues has been found. It has also been shown that the pH-effects of aliphatic and aromatic resonances are quite different between H1 and H5, suggesting that the globular domain of H5 is more stable than that of H1. The correlation time (1.5 ns) for the alpha-carbons of H5 estimated from 13C nuclear Overhauser enhancement was twice as long as that of H1 (0.9 ns), indicating that the backbone in the N-terminal and C-terminal domains of H5 is less mobile than that of H1.     

Inhibition of Endothelial Cell Differentiation on a Glycosylated Reconstituted Basement Membrane Complex

The vascular basement membrane is involved in the regulation of endothelial cell differentiation. The accumulation of advanced glycosylation endproducts (AGEs) has been demonstrated on these basement membranes in patients with diabetes. We examined the effect of AGEs on endothelial cell behavior on reconstituted basement membrane, Matrigel. Human umbilical vein-derived endothelial cells (HUVECs) stopped proliferating and differentiated into capillary-like tube-shaped structures on Matrigel. Laminin antibody partially blocked this process. HUVECs cultured on glycosylated Matrigel, however, proliferated and formed a monolayer without tube formation. The inclusion of aminoguanidine, an inhibitor of AGE formation, during the glycosylation of Matrigel restored HUVEC differentiation. Although the laminin adsorbed onto the plastic culture wells promoted HUVEC attachment and spreading, glycosylated laminin reduced HUVEC attachment by 50% and abolished cellular spreading. These effects were restored by aminoguanidine. HUVEC attachment to glycosylated laminin was further reduced by AGE-modified albumin, poly I, acetylated low-density lipoprotein, or maleylated albumin, ligands for a scavenger receptor. Coating the culture dishes with the laminin peptides RGD, YIGSR, and SIKVAV supported the attachment of HUVECs that was unaffected by glycosylation. Results suggest that AGE accumulation on the basement membranes inhibits endothelial cell differentiation by impairing the normal interactions of endothelial cell receptors with their specific matrix ligands. This process may be involved in diabetic angiopathy.     

Refined peptide HLA-B*3501 binding motif reveals differences in 9-mer to 11-mer peptide binding

 HLA-B^*3501 is associated with subacute thyroiditis and fast progression of AIDS. An important prerequisite to investigate the T-cell recognition of HLA-B^*3501-restricted antigens is the characterization of peptide-HLA-B^*3501 interactions. In this study, peptide-HLA-B^*3501 interactions were determined in quantitative peptide binding assays. The results were statistically analyzed to evaluate the influence of both anchor and nonanchor positions and the predictability of peptide binding. The binding data demonstrated that all anchor residues at position 2 and the C-terminus found in 9-mers functioned equally as anchors in 10-mers and 11-mers. These minimum requirements of peptide binding were refined by assessing positive and negative effects of nonanchor residues. Aliphatic hydrophobic residues at positions 3, 5, and 8 of 10-mers and position 3 of 11-mers significantly enhanced HLA-B^*3501 binding. Similar effects rendered aromatic, bulky residues, acidic or polar residues of 11-mers at position 1 as well as at positions 4, 8, and 10, respectively. Negative effects were observed for residues carrying positively charged side-chains at position 7 of 11-mers. The refined HLA-B^*3501 peptide binding motifs enhanced the identification of potential T-cell epitopes. The disparity between positive effects at the middle and C-terminal part (positions 5 – 8 and 10) of 11-mers and shorter peptides supports the extrusion of 11-mer residues at positions 5, 6, and 7, away from the HLA-B^*3501 binding cleft. Received: 29 May 1996 / Revised: 5 August 1996     

Binding of nonamer peptides to three HLA-B51 molecules which differ by a single amino acid substitution in the A-pocket

The interaction between 9-mer peptides and HLA-B51 molecules was investigated by quantitative peptide binding assay using RMA-S cell expressing human β2-microglobulin and HLA-B51 molecules. Of 147 chemically synthesized 9-mer peptides possessing two anchor residues corresponding to the motif of HLA-B^*5101 binding self-peptides, 27 paptides bound to HLA-B^*5101 molecules. Pro and Ala at position 2 as well as Ile at position 9 were confirmed to be main anchor residues, while Gly at position 2 as well as Val, Leu, and Met at position 9 were weak anchor residues for HLA-B^*5101. The A-pocket is suspected to have a critical role in peptide binding to MHC class I molecules because this pocket corresponds to the N-terminus of peptides and has a strong hydrogen bond formed by conserved Tyr residues. Further analysis of peptide binding to HLA-B^*5102 and B^*5103 molecules showed that a single amino acid substitution of Tyor for His at residue 171(B^*5102) and that of Gly for Trp at residue 167 (B^*5103) has a minimum effect in HLA-B51-peptide binding. Since previous studies showed that some HLA-B51 alloreactive CTL clones failed to kill the cells expressing HLA-B^*5102 or HLA-B^*5103, these results imply that the structural change of the A-pocket among HLA-B51 subtypes causes a critical conformational change of the epitope for TCR recognition rather than influences the interaction between peptides and MHC class I molecules.