A study of functionally active amino acids involved in the interaction of HLA-A2 or HLA-A3 molecules with cytolytic T lymphocytes

A large series of HLA-A2/HLA-A3 recombinant genes were generated by using the in vivo recombination technique. These genes have each been modified in the last two-thirds of the third exon such that one or several HLA-A2-specific substitutions have been made in the HLA-A3 gene and vice versa. The recombinant genes were transfected into the murine cell line P815 and the transfectants were used as targets for a series of 20 human CTL lines or clones specific for HLA-A2 or HLA-A3, or restricted by HLA-A2 and specific for influenza A. Several patterns of anti-HLA-A2, anti-HLA-A3, and HLA-A2-restricted anti-influenza CTL activity were observed and when uncloned cell lines were studied, a progressive selection of some clones with a similar pattern of activity was regularly found. From the comparison of these different patterns the following conclusions can be drawn: 1) In most but not all cases both domains of the class I molecule were essential for CTL recognition, but residue 152 was critically important for the majority of CTL tested; 2) amino acids 114/116 were also critical in most cases, and their position close to amino acid 152 in the tertiary structure of the molecule may have some functional significance; and 3) amino acid 161, although highly conserved, plays an unexpected but very important role in CTL function.     

Heterogeneity of a murine B cell lymphoma. Isolation and characterization of idiotypic variants

mAb directed toward the idiotype of the 38C13 murine B cell lymphoma can be used to treat and cure a high percentage of mice challenged previously with an otherwise lethal dose of tumor cells. Tumors developing in animals despite antibody therapy were examined by immunofluorescence and found to demonstrate either loss of surface Ig, or expression of an altered idiotype that no longer bound the antibody used for treatment. Further immunofluorescence analysis of the variant tumors revealed individual patterns of cross-reactivity with anti-38C13 idiotype mAb other than that used for therapy. The variant tumor cells were fused to myeloma cells and hybrids were isolated which secreted large quantities of the altered idiotype proteins. Polyclonal antibodies and mAb prepared against the mutant proteins demonstrated cross-reactivity with the original 38C13 protein and its other variants. But the variants and wild type cells could be distinguished from each other by their patterns of reactivity with the panels of anti-idiotype antibodies. Differences in apparent m.w. were demonstrated in the L chains of each of the mutant proteins. Southern blot analysis of the H chain locus of these mutants established that they were all clonally related; however, the L chain loci were grossly different. Thus, rare cells with alteration in their Ig L chain genes and expressed proteins can give rise to idiotype variants in this B cell tumor.     

13C NMR assignments of the protonated carbons of [d(TAGCGCTA)]2 by two-dimensional proton-detected heteronuclear correlation

The resonances of the protonated carbons of [d(TAGCGCTA)]2 have been assigned by the two-dimensional proton-detected double-quantum heteronuclear correlation experiment [( 1H-13C]-DQCOSY). 13C-coupled and 13C-decoupled versions of the experiment were used. The assignment method is discussed in detail. The deoxyribose cross peaks segregate into five well-resolved regions, and the base cross peaks have distinct features that are helpful for assignments. The cross peaks from the 1H-13C pairs at the Cyd5, Ado2 and ThdCH3 base positions fall in separate regions of the spectrum from each other; they also are resolved from the closely spaced Ado8, Guo8, Cyd6 and Thd6. Additional parameters for distinction of the base signals are their differing J-coupling values and long-range coupling patterns.     

Phylogeny of metabolic pathways: O-acetylserine sulphydrylase A is homologous to the tryptophan synthase beta subunit

The cysK gene of Escherichia coli K-12 encoding O-acetylserine sulphydrylase A, was cloned and its nucleotide sequence, together with that of the flanking regions, was determined. The deduced amino acid sequence of the carboxy-terminal moiety of O-acetylserine sulphydrylase A shows significant similarity to the amino acid sequence of tryptophan synthase beta chain from several organisms. This sequence similarity is likely to reflect the structural homologies of substrates shared by both enzymes. This may indicate that these proteins, although catalysing different reactions in different metabolic pathways, have evolved from a common ancestral gene.     

The localization of calcium release by inositol trisphosphate in Limulus photoreceptors and its control by negative feedback

Microvillar photoreceptors of invertebrates exhibit a light-induced rise in the intracellular concentration of free calcium (Cai) that results in part from release of calcium from an intracellular compartment. This light-induced release of calcium appears to result from a cascade of reactions that involve rhodopsin, a GTP-binding protein and a phospholipase-C which releases inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) from the plasma membrane; the Ins(1,4,5)P3 acts to release calcium from smooth endoplasmic reticulum. In the ventral photoreceptor of the horseshoe crab Limulus polyphemus not all of the endoplasmic reticulum is subject to calcium release by Ins(1,4,5)P3. Only endoplasmic reticulum in the light-sensitive region of the cell is competent to release calcium in response to Ins(1,4,5)P3. The release of calcium by Ins(1,4,5)P3 in ventral photoreceptors appears to be subject to feedback inhibition through elevated Cai. We suggest that this feedback inhibition contributes to sensory adaptation in the photoreceptor and may account for oscillatory membrane responses sometimes observed with large injections of Ins(1,4,5)P3.     

What is Unique About Superoxide Toxicity as Compared to Other Biological Reductants? — A Hypothesis

Usually the toxicity of superoxide is attributed lo its ability to reduce metal ions and subsequently reoxidation of the metal by hydrogen peroxide yields deleterious oxidizing species. As many other nontoxic biological reductants reduce metal compounds, we suggest that part of the mechanism of superoxide toxicity results from its ability to oxidize metal ions bound to biological targets, which subsequently degrade the target via an intramolecular electron Transfer reaction.     

Localization of the pupil trigger in insect superposition eyes

Summary In the superposition eyes of the sphingid moth Deilephila and the neuropteran Ascalaphus, adjustment to different intensities is subserved by longitudinal migrations of screening pigment in specialized pigment cells. Using ophthalmoscopic techniques we have localized the light-sensitive trigger that controls pigment position.In both species, local illumination of a small spot anywhere within the eye glow of a dark-adapted eye evokes local light adaptation in the ommatidia whose facets receive the light. Details of the response pattern demonstrate that a distal light-sensitive trigger is located axially in the ommatidium, just beneath the crystalline cone, and extends with less sensitivity deep into the clear zone. The distal trigger in Deilephila was shown to be predominantly UV sensitive, and a UV-absorbing structure, presumably the distal trigger, was observed near the proximal tip of the crystalline cone.In Ascalaphus we also found another trigger located more proximally, which causes local pigment reaction in the ommatidia whose rhabdoms are illuminated (the centre of the eye glow). The light-sensitive trigger for this response appears to be the rhabdom itself.     

Essential fatty acid deficiency lowers the activity of the acetylated low density lipoprotein receptor of rat peritoneal macrophages.

We compared phospholipid fatty acid composition, cholesterol ester accumulation, and receptor-mediated binding, internalization, and degradation of acetylated low-density lipoprotein (acetyl-LDL) in cultured peritoneal macrophages from rats fed an essential fatty acid deficient or control diet. The deficient diet increased the 5,8,11-eicosatrienoic acid and decreased the omega 6 fatty acid content of macrophage phospholipid relative to control. The deficient diet did not affect macrophage uptake of [1-14C]oleate; however, it lowered the accumulation of intracellular labelled cholesteroyl oleate to 66% of the control. This effect was attributed to a diminution of the specific binding of acetyl-LDL, and not to acetyl-LDL internalization nor to degradation. The results demonstrate the sensitivity of the acetyl-LDL receptor to changes in its membrane environment, brought about through dietary means.     

Inhibition of human immunodeficiency virus replication in acutely infected CD4+ cells by CD8+ cells involves a noncytotoxic mechanism.

The mechanism by which CD8+ T cells from human immunodeficiency virus (HIV)-infected individuals suppress HIV replication in acutely infected CD4+ T cells was investigated. Cytotoxicity was not involved, as the antiviral activity of the CD8+ cells did not correlate with the ability to lyse HIV-infected or uninfected CD4+ T cells. In addition, the frequency of HIV-infected CD4+ cells increased during coculture with CD8+ T cells even in the absence of detectable levels of virus replication. Moreover, separation of the CD4+ and CD8+ cells by a 0.4-micron-pore-size filter delayed HIV replication, indicating a role, at least in part, for a soluble factor. However, cell contact was required for optimal antiviral activity. These results extend further the observation on the mechanism of antiviral HIV activity by CD8+ cells from infected individuals. They support the conclusion that CD8+ cells can play a major role in preventing development of disease in HIV-infected individuals.     

Several CD4 domains can play a role in human immunodeficiency virus infection in cells.

The human immunodefiency virus (HIV) uses the human CD4 glycoprotein as a receptor for infection of susceptible cells. Cells expressing a series of mutated forms of the CD4 gene have shown a variability in their ability to support replication of three HIV type 1 (HIV-1) and three HIV-2 strains. Moreover, when different stages of virus production were examined by a variety of assays, a consistent delay was observed in all cell lines containing CD4 mutants compared with those with intact full-length CD4. Cells expressing the CD4.415 mutant (modified at the serine 415 corresponding to a phosphorylation site of the cytoplasmic domain) showed only a minimal effect on virus replication. Cells expressing CD4.403 and CD4.401 mutants (lacking the whole cytoplasmic domain) manifested a moderate delay in production of virus progeny. The most substantial effect on HIV replication was observed in cells expressing a chimeric hybrid containing sequences corresponding to the first 177 residues of the N-terminal CD4 fused to CD8 sequences encoding the hinge, transmembrane, and cytoplasmic domains of the human CD8. Furthermore, in a cell-to-cell contact assay, fusion was absent when the CD4 proximal membrane domain was replaced by the CD8 counterpart. In addition, a strong correlation between the down-modulation of the surface CD4 and HIV expression was observed. These observations suggest that in addition to the known binding region, other domains of CD4 could play an important role in regulating HIV entry of cells.