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.     

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.     

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.     

The promoter of filamentation (POF1) protein from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is an ATPase involved in the protein quality control process

Background

The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.

Results

Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.

Conclusions

Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

     

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.     

Nuovi blastomiceti isolati da mosti del chianti e zone limitrofe

Zusammenfassung Beim Identifizieren von 531 Hefekulturen, die im Herbst 1936 aus Traubenmost des klassischen Chiantigebietes und seiner Umgebung isoliert wurden, fand der Autor folgende neue Arten und Varietäten: Sacch. italicus repräsentiert, soweit heute bekannt, die einzige Spezies der dritten Gruppe nach der Hansen schen Klassifikation der Gattung Saccharomyces. Sacch. ellipsoideus var. major unterscheidet sich von Sacch. ellipsoideus-Typ durch die konstant größeren Zellen und Sporen. Sacch. oviformis var. bisporus zeigt zum Unterschied vom Sacch. oviformis-Typ, außer anderen leichten Abweichungen, Asken mit fast immer zwei, aber nie mehr als zwei Sporen. Zygosaccharomyces florentinus ist ein gutes Beispiel für Übergänge zwischen den Gattungen Torulaspora und Zygosaccharomyces, da sich die seltenen Asken hauptsächlich parthenogenetisch bilden und nur selten das Ergebnis einer Kopulation sind. Zygopichia chiantigiana ermangelt der Fähigkeit zur Alkoholgärung und hat bei der Weinbereitung keine Bedeutung.