The association of H-2Ld with human beta-2 microglobulin induces localized conformational changes in the alpha-1 and- 2 superdomain

We have analyzed changes in the antigenicity of major histocompatibility complex class I molecules resulting from the association of human beta-2 micro-globulin (B2m) with the mouse class I heavy chain. In particular, the H-2L^d molecule exhibited enhanced crossreactivity for the 34-1-2 monoclonal antibody. In order to assess the nature of this structural alteration induced by human B2m, we utilized H-2 class I hybrid molecules in the mapping of the 34-1-2 determinant to the helical region of the alpha-1 domain. H-2L^d class I hybrid molecules were then used to establish the importance of the alpha-2 and- 3 domains in the observed increase of 34-1-2 cross-reactivity following exchange with human B2m. The H-2L^d hybrids suggest that alterations in interdomain contact are responsible for enhanced 34-1-2 cross-reactivity on the H-2L^d molecule. It is likely that this alteration arises through changes in class I conformation at regions of the molecule distant from points of contact between B2m and the class I molecule. This suggests that perturbations induced by association of human B2m with H-2L^d can affect the conformation of the alpha-1 and- 2 superdomain. That class I antigenic determinants are altered by the association of human B2m with mouse class I further suggests that the class I molecule is structurally flexible and may reflect the ability of the class I molecule to bind and present a vast array of disparate peptides to the T-cell receptor.     

Hydrolysis of sunflower oil by means of hydrophobic membrane with lipolytic activity

Polytetrafluoroethylene (PTFE) membranes of different porosity were used for lipase from Rhizopus immobilization. The immobilized enzyme applied to sunflower oil hydrolysis achieved the activity of 1228 U/m² of the membrane area and the half-life time was calculated to be 7 days.     

Photosynthetic properties of leaves of a yellow green mutant of wheat compared to its wild type

We investigated several photosynthetic parameters of a virescent mutant of durum wheat and of its wild-type. Electron transport rate to ferricyanide was the same in the two genotypes when expressed on leaf area basis while O₂ evolution of the leaf tissue in saturating light and CO₂ was slightly higher in the yellow genotype. RuBPCase was also slightly higher. Quantum yield per absorbed light was similar in the two genotypes. P700 and Cyt f were less concentrated in the mutant while PS II was only marginally lower. The light response curve of CO₂ assimilation indicated higher level of photosynthesis of the mutant in high light, which corresponded to a lower non-photochemical quenching compared to the wild-type. It is concluded that the reaction centres, cyt f and chlorophyll are not limiting factors of electron transport in wheat seedlings and that electron transport capacity is in excess with respect to that needed for driving photosynthesis. Since the differences in photosynthesis reflect differences in RuBPCase activity, it is suggested that this enzyme limits photosynthesis in wheat seedlings also at high light intensities.Abbreviations cyt f cytochrome f - chl chlorophyll - PS II photosystem II - Pn_max maximum photosynthesis - RuBCase Ribulose, 1-5,bisphosphate carboxylase     

A mutation in the insulin receptor gene that impairs transport of the receptor to the plasma membrane and causes insulin-resistant diabetes.

Insulin binds to a receptor on the cell surface, thereby triggering a biological response within the target cell. Mutations in the insulin receptor gene can render the cell resistant to the biological action of insulin. We have studied a family in which two sisters have a genetic form of insulin-resistant diabetes mellitus. The technique of homozygosity mapping has been used to demonstrate that the mutation causing diabetes in this consanguineous family is genetically linked to the insulin receptor gene. The two insulin-resistant sisters are homozygous for a mutation encoding substitution of valine for phenylalanine at position 382 in the alpha-subunit of the insulin receptor. Transfection of mutant insulin receptor cDNA into NIH3T3 cells demonstrated that the Val382 mutation impaired post-translational processing and retarded transport of the insulin receptor to the plasma membrane. Thus, the mutation causes insulin resistance by decreasing the number of insulin receptors on the surface of the patients' cells.     

Evidence for chromosomal replicons as units of sister chromatid exchanges

Chromosomal replicons have been described as the cytological counterpart of DNA replicon clusters and have previously been studied in vitro using premature chromosome condensation-sister chromatid differentiation (PCC-SCD) techniques. Chromosomal replicons are visualized as small SCD segments in S-phase cells, and measurement of these segments can provide estimates of relative chromosomal replicon size corresponding to DNA replicon clusters functioning coordinately in S-phase. Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the PCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cyclophosphamide (CP) for two cell cycles. We have been able to visualize chromosomal replicons, as well as SCEs which have been induced in vivo by CP treatment, simultaneously in the same cells. Chromosomal replicons visualized as small SCD segments were measured in PCC cells classified at early or late S-phase based on SCD segment size prevalence. Early S-phase (E/S) PCC cells contained 90% of the SCD segments measured clustered in a segment size range of 0.1 to 0.8 m with a peak value around 0.3 to 0.6 m regardless of CP treatment. As the cells progressed through S-phase, late S-phase (L/S) PCC cells were characterized by the appearance of larger SCD segments and even whole SCD chromosomes in addition to small SCD segments. A concentration of units around 0.4 to 1.0 m was found for L/S SCD segment size distributions regardless of CP treatment with an apparent bimodal profile. Our in vivo data support the existence of a subunit organization of chromosomal replication with a basic functional unit being 0.3 to 0.6 m in size. In addition, we have found that this chromosomal unit of replication or chromosomal replicon does not seem to be functionally perturbed by the mutagen CP. We also found that small SCD segments of 0.4 to 0.7 m in length were involved in the formation of an SCE, suggesting that both spontaneous and CP-induced SCEs occur between chromosomal replicons. These findings provide direct cytogenetic evidence to support a replicon cluster/chromosomal replicon model for SCE formation.     

The polyene antibiotic amphotericin B acts as a Ca2+ ionophore in sterol-containing liposomes

Amphotericin B (AmB) was shown to induce a Ca2+ influx across ergosterol- and cholesterol-containing large unilamellar liposomes, by following spectrophotometrically the formation of the Arsenazo III-Ca2+ complex. At equivalent antibiotic concentrations the Ca2+ influx was much more extensive through ergosterol-containing membranes (almost 100% with 1 microM AmB, 160 microM lipid) than through cholesterol-containing membranes (below 0.5 microM the influx of Ca2+ was negligible). In the presence of ergosterol-containing membranes the initial rate of Ca2+ influx had the same linear dependence on the ratio antibiotic/lipid whatever the lipid concentration, which was not the case in cholesterol-containing membranes. These results suggest that the channels responsible for the AmB-induced Ca2+ permeability across cholesterol- and ergosterol-containing liposomes have different structures.     

Size variation in Brachionus plicatilis resting eggs

The effect of temperature and salinity on resting egg size of two Brachionus plicatilis (Rotifers) clones was investigated. Clones were selected according to their different behaviour in laying resting eggs: one clone ejects them, whereas they remain inside the females body in the other clone. The difference in resting eggs size between the two clones is noticeable, although the difference is not as great as that between female body size. An important temperature-salinity interaction on resting egg size has been observed. The general inverse relationship between size and temperature is only true at lower temperatures. At high temperatures size varies around the mean although could be greater than at intermediate temperatures. This is more evident at the intermediate salinity tested which is considered to be the closest to the optimum in our experiments. This pattern of variation suggests that mean size is bigger than expected, in relation to temperature and salinity, when these factors have values close to the extremes of their range, normally found in nature, and to which adaptative mechanisms can evolve. Size is bigger at the salinity — temperature low - low and high - high combinations which are the most commonly found in the temperate environments.     

Substitution of a proline for alanine 183 in the hinge region of phosphoglycerate kinase: Effects on catalysis, activation by sulfate, and thermal stability

A hinge-bending domain movement has been postulated as an important part of the catalytic mechanism of phosphoglycerate kinase (PGK) (Bankset al., 1979). In order to test the role of the flexibility of a putative interdomain hinge in the substrate- and sulfate-induced conformational transitions, alanine-183 was replaced by proline using site-directed mutagenesis. The maximal velocity of the Ala 183Pro mutant, measured at saturating concentrations of ATP and phosphoglycerate (5 mM and 10 mM, respectively) and in the absence of sulfate ions, is increased approximately 21% in comparison to the wild type PGK. TheK _m values for both substrates are essentially unchanged. The effect of sulfate on the specific activity of the Ala 183Pro mutant and the wild type PGK was measured in the presence of 1 mM ATP and 2 mM 3-phosphoglycerate (3-PG). A maximum activation of 70% was observed at 20 mM sulfate for the mutant enzyme, as compared to 130% activation at 30 mM sulfate for the wild type PGK. These results demonstrate that the increased rigidity of the putative hinge, introduced by the AlaPro mutation, does not impair catalytic efficiency of phosphoglycerate kinase, while it appears to decrease the sulfate-dependent activation. The differential scanning calorimetry (DSC) studies demonstrate an increased susceptibility of the Ala 183 Pro mutant to thermal denaturation. In contrast to one asymmetric transition observed in the DSC scan for the wild type PGK, withT _m near 54°C, two transitions are evident for the mutant enzyme withT _m values of about 45 and 54°C. Using a thermodynamic model for two interacting domains, a decrease in the free energy of domain-domain interactions of about 2 kcal was estimated from the DSC data.