Differential susceptibility to recombinant interferon-gamma-induced HLA-DQ antigen modulation among clones from a human metastatic melanoma

Twenty-one clones from an early culture of a histocompatibility leukocyte antigen (HLA) class II negative human metastatic melanoma (Me 9229) were screened for susceptibility to phenotypic modulation induced by recombinant interferon-gamma (rIFN-gamma) by using SPV-L3, a monoclonal antibody to HLA-DQ antigens, in indirect immunofluorescence followed by fluorescence-activated cell sorter analysis. After treatment with 500 U/ml of rIFN-gamma for 3 days one of the clones (9229/18) expressed high levels of DQ antigens, in terms of percentage of positive cells, whereas many other clones were much less susceptible or remained DQ negative. Scatchard analysis of the data of specific binding of 125-I-labeled rIFN-gamma revealed that one clone susceptible (9229/18) and one clone resistant (9229/5) to HLA-DQ modulation expressed similar numbers of interferon-gamma binding sites per cell; dose-response experiments showed that all clones could be induced to express HLA-DR and -DP antigens after exposure to rIFN-gamma. However, the DQ-negative profile of clone 9229/5 was not modified even after incubation with up to 1 X 10(4) U/ml of rIFN-gamma or by extending the culture time in the presence of this lymphokine up to 120 hr. Furthermore, Northern blot analysis indicated a direct correlation between changes in the levels of HLA-DR and -DQ-specific mRNA after rIFN-gamma treatment, and the lack or expression of HLA class II antigens at the cell surface of the two different clones. Karyotype studies did not reveal differences between clones 9229/5 and 9229/18 and Southern blot analysis indicated that both clones had similar EcoRI and HindIII restriction patterns for DR and DQ gene sequences. Finally, strong DQ-specific mRNA signal and antigen expression at the cell surface could be induced even on clone 9229/5 by treating the cells with supernatants from mixed lymphocyte cultures, recently shown to contain a class II-inducing factor different from interferon-gamma. Taken together these results indicate that DQ antigens can be modulated even in clones resistant to rIFN-gamma induction and suggest that the differential susceptibility observed in response to this lymphokine could play a role in the genesis of the phenomenon of intratumor heterogeneity.     

Effect of infusing branched-chain amino acid during incremental exercise with reduced muscle glycogen content

The aim of this study was to investigate whether, when muscle glycogen is reduced, a pre-exercise infusion of branched-chain amino acids (BCAA) modifies exercise performance or the metabolic and respiratory responses to incremental exercise. Six moderately trained volunteers took part in the following protocol on two occasions. On day 1, at 9 a.m. in the postabsorptive state, they performed a graded incremental exercise (increases of 35 W every 4 min) to exhaustion (Ex-1). A meal of 1,000 kcal (4,200 kJ; 60% protein, 40% fat) was consumed at 12 p.m. No food was then allowed until the end of the experiment (20–21 h later). A 90-min period of exercise at alternating high and moderate intensities, designed to deplete muscle glycogen, was performed between 6 p.m. and 7.30 p.m. The morning after (day 2), the subjects randomly received either a mixed solution of BCAA (260 mg × kg^–1 × h^–1 for 70 min), or saline. They then repeated the graded incremental exercise to exhaustion (Ex-2). Metabolic and respiratory measurements suggested a muscle glycogen-depleted state had been achieved. No significant differences were observed in total work performed, maximal oxygen uptake or plasma ammonia, alanine, and blood pyruvate concentrations in the two treatments. After BCAA infusion, higher blood lactate concentrations were observed at maximal power output in comparison with those during saline [BCAA 4.97 (SEM 0.41) mmol × l^–1, Saline 3.88 (SEM 0.47) mmol × l^–1,P < 0.05]. In summary, in conditions of reduced muscle glycogen content, after a short period of fasting, BCAA infusion had no significant effect on the total work that could be performed during a graded incremental exercise.     

Secretion of β-lactamase from K. lactis using pEPS1, a convenient episomal vector designed for the secretion of foreign proteins

Summary A convenient shuttle vector that enables high level secretion of proteins from Kluyveromyces lactis has been developed. The vector, pEPS1, contains a unique cloning site that allows the construction, in a single ligation step, of episomal plasmids capable of directing secretion of foreign gene products from K. lactis. As an example we demonstrate the production of -lactamase and determine optimal conditions for its secretion into the culture media.     

DCCD inhibits proton translocation and electron flow at the second site of the mitochondrial respiratory chain

DCCD inhibits formation of a succinate-driven transmembrane pH gradient in submitochondrial particles, as shown by inhibition of fluorescence quenching of 9-aminoacridine, without concomitant inhibition of succinate oxidation. On the other hand ubiquinol-cytochrome c reductase activity is inhibited by DCCD. Half-inhibition of both fluorescence quenching and ubiquinol-cytochrome c reductase occur at 35 μM DCCD. The results suggest that DCCD inhibits proton pumping activity coupled to electron flow through the bc₁ complex.     

Saturation kinetics of coenzyme Q in NADH and succinate oxidation in beef heart mitochondria.

The saturation kinetics of NADH and succinate oxidation for Coenzyme Q (CoQ) has been re-investigated in pentane-extracted lyophilized beef heart mitochondria reconstituted with exogenous CoQ10. The apparent 'Km' for CoQ10 was one order of magnitude lower in succinate cytochrome c reductase than in NADH cytochrome c reductase. The Km value in NADH oxidation approaches the natural CoQ content of beef heart mitochondria, whereas that in succinate oxidation is close to the content of respiratory chain enzymes.     

Expression of anchorin CII mRNA by cultured chondrocytes

The establishment of a cell culture system promoting chondrocyte differentiation has been utilized to better characterize phenotypic stages of chondrogenesis at the cellular level. Although the expression of the type II collagen gene has been studied during “in vitro” chondrocyte differentiation, little is known about the expression of the gene coding for its receptor: anchorin CII. The modulation of the anchorin mRNA steady state level in chick embryo chondrocytes at different developmental stages is described here.The anchorin mRNA level was low in dedifferentiated chondrocytes, progressively increased after the cell transfer into suspension (a condition promoting differentiation), reached its maximal value after 4 weeks and decreased after 5 weeks.Therefore anchorin CII mRNA reaches its maximum level in hypertrophic stage II chondrocytes.     

Impact of antigen presentation on TCR modulation and cytokine release: implications for detection and sorting of antigen-specific CD8+ T cells using HLA-A2 wild-type or HLA-A2 mutant tetrameric complexes

Soluble MHC class I molecules loaded with antigenic peptides are available either to detect and to enumerate or, alternatively, to sort and expand MHC class I-restricted and peptide-reactive T cells. A defined number of MHC class I/peptide complexes can now be implemented to measure T cell responses induced upon Ag-specific stimulation, including CD3/CD8/zeta-chain down-regulation, pattern, and quantity of cytokine secretion. As a paradigm, we analyzed the reactivity of a Melan-A/MART-1-specific and HLA-A2-restricted CD8(+) T cell clone to either soluble or solid-phase presented peptides, including the naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV or the peptide analog ELAGIGILTV presented either by the HLA-A2 wild-type (wt) or mutant (alanineright arrowvaline aa 245) MHC class I molecule, which reduces engagement of the CD8 molecule with the HLA-A2 heavy chain. Soluble MHC class I complexes were used as either monomeric or tetrameric complexes. Soluble monomeric MHC class I complexes, loaded with the Melan-A/MART-1 peptide, resulted in CD3/CD8 and TCR zeta-chain down-regulation, but did not induce measurable cytokine release. In general, differences pertaining to CD3/CD8/zeta-chain regulation and cytokine release, including IL-2, IFN-gamma, and GM-CSF, were associated with 1) the format of Ag presentation (monomeric vs tetrameric MHC class I complexes), 2) wt vs mutant HLA-A2 molecules, and 3) the target Ag (wt vs analog peptide). These differences are to be considered if T cells are exposed to recombinant MHC class I Ags loaded with peptides implemented for detection, activation, or sorting of Ag-specific T cells.     

Experimental and simulative dissociation of dimeric Cu,Zn superoxide dismutase doubly mutated at the intersubunit surface

The equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase mutant bearing two negative charges in the amino acid clusters at the association interface has been studied, experimentally and computationally, and compared to those of the native enzyme. Pressure-dependent dissociation is observed for the mutant, as observed by the fluorescence shift of the unique tryptophan residue located at the intersubunit surface. The spectral shift occurs slowly, reaching a plateau after 15-20 min, and is fully reversible. Measurement of the degree of dissociation allows us to calculate the standard volume variation upon association and the dissociation constant at atmospheric pressure. On the other hand the native protein is undissociable at any pressure. In the simulative approach, the dissociation free energy has been calculated through the blue moon calculation method for the case of a multidimensional reaction coordinate, corrected for the rotational contribution within the semiclassical approximation for a free rigid-body rotor. The scheme permits to define a definite path for the rupture of the dimer and to calculate the effective force involved in the process. The calculated free energy difference is close to the experimental one, and the value obtained for the mutant is well below that obtained for the native protein, indicating that the theoretical reaction scheme is able to reproduce the experimental trend. Moreover, we find that, when the separation distance increases, the protein structure of the monomer is stable in line with the fast recovery of the original fluorescence properties after decompression, which excludes the presence of partly unfolded intermediates during the dimer-monomer transition.