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.     

Carbodiimide inactivation of Na,K-ATPase, via intramolecular cross-link formation, is due to inhibition of phosphorylation

We have recently shown that inactivation of renal Na,K-ATPase by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide occurs via an intramolecular cross-link formed between an activated carboxyl group and an endogenous nucleophile (Pedemonte, C.H., and Kaplan, J.H. (1986) J. Biol. Chem. 261, 3632-3639). The modified enzyme shows the same level of Rb+ binding as untreated enzyme: 3.16 and 2.93 ATP-sensitive mumol of Rb+ binding/mumol of phosphoenzyme, respectively. Thus, the Rb+ binding site and the transition accomplished by low affinity nucleotide binding which accelerates de-occlusion are not greatly affected by the carbodiimide inactivation. 1 mM K+ reduces the ADP binding to the high affinity nucleotide binding site to the same extent in normal and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-treated enzyme and Na+ counteracts this effect. Thus, the competition between Na+ and K+ ions for binding to the free enzyme are also largely unaltered by the modification. Phosphorylation from ATP (microM) in the presence of Na+ and Mg2+ ions and from inorganic phosphate in the presence of Mg2+ ions (in the absence or presence of ouabain) is greatly inhibited (85%) following carbodiimide treatment. The extent of inhibition of phosphorylation quantitatively correlates with the residual Na,K-ATPase activity (15%). Consequently, the rate of inactivation by carbodiimide is reduced when a greater proportion of the enzyme is in the phosphorylated form. Fluoroscein isothiocyanate, which inhibits the Na,K-ATPase by covalently modifying a lysine residue close to the high affinity binding site for ATP in the alpha-subunit does not bind to the carbodiimide-inactivated enzyme. Since high affinity nucleotide binding is only partially inhibited by the modification produced by the carbodiimide this suggests that the lysine residue to which fluoroscein isothiocyanate binds is not specifically required for competent nucleotide binding.     

Single unit activity in the guinea-pig cochlear nucleus during sleep and wakefulness.

The effects of waking and sleep on the response properties of auditory units in the ventral cochlear nucleus (CN) were explored by using extracellular recordings in chronic guinea-pigs. Significant increases and decreases in firing rate were detected in two neuronal groups, a) the "sound-responding" and b) the "spontaneous" (units that do not show responses to any acoustic stimuli controlled by the experimenter). The "spontaneous" may be considered as belonging to the auditory system because the corresponding units showed a suppression of their discharge when the receptor was destroyed. The auditory CN units were characterized by their PSTH in response to tones at their characteristic frequency and also by the changes in firing rate and probability of discharge evaluated during periods of waking, slow wave and paradoxical sleep. The CNS performs functions dependent on sensory inputs during wakefulness and sleep phases. By studying the auditory input at the level of the ventral CN with constant sound stimuli, it was shown that, in addition to the firing rate shifts, some units presented changes in the temporal probability of discharge, implying central actions on the corresponding neurons. The mean latency of the responses, however, did not show significant changes throughout the sleep-waking cycle. The auditory efferent pathways are postulated to modulate the auditory input at CN level during different animal states. The probability of firing and the changes in the temporal pattern, as shown by the PSTH, are thus dependent on both the auditory input and the functional brain state related to the sleep-waking cycle.     

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)     

Multidrug-resistance (MDR) phenotype of human osteosarcoma cells evaluated by quantitative morphological and electron microscopy analyses

Summary— Multidrug-resistant (MDR) variants of a human osteosarcoma cell line (U-2 OS) have been recently obtained by continuous exposure to doxorubicin (DX). The growth and phenotypic characteristics of these cell lines have been demonstrated to be related to the level of expression of P-glycoprotein. In this work, the morphological changes associated with MDR have been evaluated by quantitative image analysis and transmission electron microscopy. Resistant cells present morphological changes with respect to sensitive cells at both cytoplasmic and nuclear level. Some of these changes appear to be related to the degree of resistance but not to the direct presence of DX, since deprived cells maintain some modified characters, while others are partly lost. These findings suggest that DX exposure affects cell metabolism causing progressive changes of the cell morphotype.     

Regulation of Na,K-ATPase Transport Activity by Protein Kinase C

Considerable evidence indicates that the renal Na⁺,K⁺-ATPase is regulated through phosphorylation/dephosphorylation reactions by kinases and phosphatases stimulated by hormones and second messengers. Recently, it has been reported that amino acids close to the NH₂-terminal end of the Na⁺,K⁺-ATPase α-subunit are phosphorylated by protein kinase C (PKC) without apparent effect of this phosphorylation on Na⁺,K⁺-ATPase activity. To determine whether the α-subunit NH₂-terminus is involved in the regulation of Na⁺,K⁺-ATPase activity by PKC, we have expressed the wild-type rodent Na⁺,K⁺-ATPase α-subunit and a mutant of this protein that lacks the first thirty-one amino acids at the NH₂-terminal end in opossum kidney (OK) cells. Transfected cells expressed the ouabain-resistant phenotype characteristic of rodent kidney cells. The presence of the α-subunit NH₂-terminal segment was not necessary to express the maximal Na⁺,K⁺-ATPase activity in cell membranes, and the sensitivity to ouabain and level of ouabain-sensitive Rb⁺-transport in intact cells were the same in cells transfected with the wild-type rodent α1 and the NH₂-deletion mutant cDNAs. Activation of PKC by phorbol 12-myristate 13-acetate increased the Na⁺,K⁺-ATPase mediated Rb⁺-uptake and reduced the intracellular Na⁺ concentration of cells transfected with wild-type α1 cDNA. In contrast, these effects were not observed in cells expressing the NH₂-deletion mutant of the α-subunit. Treatment with phorbol ester appears to affect specifically the Na⁺,K⁺-ATPase activity and no evidence was observed that other proteins involved in Na⁺-transport were affected. These results indicate that amino acid(s) located at the α-subunit NH₂-terminus participate in the regulation of the Na⁺,K⁺-ATPase activity by PKC. Received: 10 July 1996/Revised: 19 September 1996