Chromosome-specific subsets of human alphoid DNA identified by a chromosome 2-derived clone

We have cloned an alphoid DNA fragment, pBS4D, from the DNA of a human-hamster hybrid cell line containing chromosome 2 as its only cytologically detectable human component. Under high stringency conditions, pBS4D hybridized in situ mostly to chromosome 2 and to a lesser extent to chromosomes 18 and 20. Restriction analysis using the DNA from selected somatic hybrid cell lines revealed that the genomic organization of this alphoid DNA differs on each of these three chromosomes.     

DR antigen expression on ovarian carcinoma cells does not correlate with their capacity to elicit an autologous proliferative response

Summary Expression of HLA-DR antigens by purified preparations of human ovarian carcinoma cells freshly isolated from surgical specimens was examined in parallel with the capacity of tumor cells to elicit a blastogenic response from autologous lymphocytes in mixed lymphocyte-tumor culture (MLTC) assay. Of 21 tumor preparations, 11 (52%) reacted with monoclonal antibodies 279 and/or 949 specific for a monomorphic determinant of HLA-DR antigens, with heterogeneous positivity, ranging between 30% and 95%. In this series of patients positive MLTC occurred in 8/21 individual experiments. The HLA-DR expression was proportionally similar in tumors giving positive MLTC (4/8=50%) and negative MLTC (7/13=53%). The lack of correlation between DR expression on tumor cells and stimulatory activity in autologous MLTC and the fact that DR-negative tumors could induce lymphocyte stimulation, support the hypothesis that blastogenesis occurs upon recognition of tumor-associated antigens, different from DR molecules, possibly tumor-specific antigens.     

The peculiar binding properties of 4''-deoxy,4''-iododoxorubicin to isolated DNA and 175 bp nucleosomes.

The thermodynamic parameters governing the interaction of 4'-deoxy,4'-iododoxorubicin (4'-IAM) to double stranded DNA or 175 bp nucleosomes have been evaluated at different ionic strength and temperature conditions by means of fluorescence techniques. The iodo-anthracycline exhibits quite different characteristics from the parent compounds adriamycin (AM) and daunomycin (DM) and other second generation derivatives. In fact, the contribution of electrostatic interactions to the total free energy of binding is rather poor and the changes in enthalpy, usually high and negative, are low and eventually positive. Unlike other members of its family, 4'-IAM exhibits preference for the nucleosomal structure. In addition, its binding to isolated DNA is remarkably cooperative. Circular dichroism studies show changes in the geometry of the intercalation complex when the drug binds to nucleosomes. The possibility for the iodo-sugar moiety to act as an alkylating or free-radical producing species was also considered as an alternative mechanism of action. However, no evidence was obtained to support these hypotheses. Thus the major differences observed in DNA-binding in comparison to parent anthracyclines appear to be mostly related to the lower pKa and higher lipophilicity exhibited by 4'-IAM.     

Effect of protons on the amidase activity of human alpha-thrombin. Analysis in terms of a general linkage scheme

The amidase activity of human alpha-thrombin has been studied in the pH range 5.5 to 10, and at four different chloride concentrations from 5 mM to 1 M. The Michaelis-Menten constant, Km, shows a bell-shaped dependence over the pH range studied, with a minimum around pH 8. The pH dependence of the catalytic constant, kcat, shows multiple inflection points especially at low (less than 0.1 M) chloride concentrations, thereby implicating the existence of multiple catalytic forms of the enzyme. A general linkage scheme is proposed for the analysis of the effect of protons on thrombin amidase activity, and experimental data have globally been analysed over the entire pH range in terms of such a scheme. Four proton-linked ionizable groups seem to be involved in the control of thrombin amidase activity. Two of these groups change their pK value upon substrate binding to the enzyme and account for the pH dependence of Km. All four groups control the catalytic activity of the enzyme which decreases with increasing protonation. Chloride has little effect on Km, while kcat changes significantly at pH less than 8. This effect is due to an increased enzymatic activity of the highly protonated intermediates at high chloride concentrations, as well as to the pK shift of two proton-linked ionizable groups.     

Early degradation of photosynthetic membranes in carob and sunflower cotyledons

The assimilatory activity of cotyledons can play an essential role in the survival of seedlings with a slow and delayed development of primary leaves. Changes in the photosynthetic activity of the cotyledon, from the onset of greening through senescence, were studied in two such plants, carob and sunflower, in order to determine its efficiency and duration, also in connection with the achievement of assimilatory autonomy by the plantlet. Chlorophyll analyses showed that the cotyledon's chloroplasts reached maximal greening in plantlets with a pair of expanded leaves. In contrast, the cotyledon's photosynthetic activity, measured as the rate of oxygen release, started to decrease early, before expansion of primary leaves. The decrease was due to the inactivation of a number of photosystem II (PSII) units, as revealed by immunodetection of breackdown products of the reaction centre's D1 and D2 thylakoid proteins. No signals of PSII alteration were noticed in the primary leaf chloroplasts that differentiated under the same environmental conditions. The damage to the cotyledon PSII, occurring in a non-photoinhibitory situation, might be due to a slower rate of turnover of D1 polypeptide than in the leaf thylakoids. The differential turnover of this protein in cotyledons and in leaves might represent an organ-specific regulation of the photosynthetic activity. The peculiarity of the cotyledon thylakoids make these organs useful objects for studying the metabolic cycle of both D1 and D2 proteins in vivo, under non-photoinhibiting conditions.     

Role for the Vacuolar H+-ATPase in Regulating the Cytoplasmic pH: An in Vivo Study Carried out in chl1, an Arabidopsis thaliana Mutant Impaired in NO-3 Transport

The effects of the growth in a medium containing NH₄NO₃ as nitrogensource were studied on cell sap pH, cytoplasmic pH and malatecontent in chl1, an Arabidopsis thaliana mutant impaired inchlorate and nitrate transport. In all the conditions testedthe pH of the cytoplasm in chl1 was more alkaline, and thatof the vacuole was more acidic as compared with those measuredin wt. Treatment with bafilomycin A1, a specific inhibitor ofthe vacuolar H⁺-ATPase, induced a small alkalinization of thevacuole, and a significant acidification of the cytoplasm, theseeffects being greater in chl1 than in wt. The greater responseof the mutant to bafilomycin Al suggests that, in the absenceof the inhibitor, the activity of the tonoplast H⁺-ATPase inchl1 is higher than in wt, this diversity being a possible reasonfor the differences in intracellular pH detected between thetwo strains. A possible role for the vacuolar H⁺-ATPase in regulatingthe cytoplasmic pH is discussed. (Received August 2, 1995; Accepted February 1, 1996)     

Theory of multivalent binding in one and two-dimensional lattices

Ligand binding to a linear lattice composed of N sites, under general conditions of cooperativity and number of sites covered upon binding, m, is approached in terms of the theory of contracted partition functions. The partition function of the system obeys a recursion relation leading to a generating function that provides an exact analytical solution for any case of interest. Site-specific properties of the lattice are derived from simple transformations of the analytical expressions. The McGhee-von Hippel model is obtained as a special case in the limit N --> infinity. The derivation is straightforward and involves no combinatorial arguments. Partition functions and site-specific properties are also derived for the case of non-cooperative binding to a two-dimensional torus of length N, containing s sites in its section for a total of sN sites. The torus provides a relevant model for ligand binding to double-stranded DNA (s = 2) or protein helices (s = 3,4). It is proved that non-cooperative binding to the two-dimensional torus can mimic cooperative binding to a one-dimensional linear lattice when m = s. The dimensional embedding of the lattice and the geometry of interaction of its sites play a crucial role in defining the binding properties of the system accessible to experimental measurements. Hence, caution must be exercised in the interpretation of Scatchard plots in terms of the one-dimensional McGhee-von Hippel model, especially when m < or = 4 and the geometry of the system is clearly two-dimensional.     

Map analysis of ligand binding to a linear lattice

A one-dimensional mapping of the binding properties of a linear lattice offers an exact analytical solution for the site-specific properties of the lattice once the length N and the parameter for nearest neighbor interactions are specified. The solution is derived independent of the definition of the partition function or the transfer matrix, nor does it involve combinatorial arguments. This result provides a simple and effective way of analyzing experimental data for protein-ligand interactions and broadens our understanding of site-specific properties in biological macromolecules.