Physical mechanism of ATP formation in biomembranes

Membrane-bound and isolated H+ ATPases of various origin are able to synthesize ATP from ADP and Pi after a jump-like pH increase. In the course of this increase the pH of solution (or suspension) must cross a value corresponding to pK of certain acid groups in the catalytic component of ATPase. In the case of isolated soluble enzymes it is possible to obtain up to 10 ATP molecules per one pH jump per one enzyme molecule. A physical mechanism of this phenomenon as well as of oxidative and photosynthetic phosphorylation is suggested.     

2 A prebiotic RNA apparatus functions within the contemporary ribosome

Ribosomes, the universal cellular machines, possess spectacular architecture accompanied by inherent mobility, allowing for their smooth performance as polymerases that translate the genetic code into proteins. The site for peptide bond formation is located within a universal internal semi-symmetrical region, which was identified within all contemporary ribosomes. The high conservation of this region implies its existence irrespective of environmental conditions and indicates that it may represent an ancient RNA molecular apparatus. Hence, we named it the “proto-ribosome”. This prebiotic pocket-like RNA entity is suggested to be capable to accommodate substrates whose stereochemistry enables the creation of chemical bonds. It could have evolved from an earlier catalytic RNA entity that we named the “pre-proto-ribosome”, presumed to be a molecular machine capable of performing various essential tasks in the RNA world, which was snatched by the amino acid invaders for producing proteins.     

Drosophila melanogaster H1 histone is phosphorylated stably.

Phosphorylation of histone H1 is developmentally regulated in Drosophila spp. It cannot be detected in preblastoderm embryos or polytene salivary gland cells, but in cellular blastoderm, postblastoderm embryo, and amitotic adult head nuclei, it occurs with a frequency of roughly 4 x 10(5) molecules per nucleus. We used pulse-labeling to study the relationship between H1 synthesis and modification in cultured cells. These results reveal that the H1-associated phosphate is stable and suggest that Drosophila H1 is synthesized, translocated to the nucleus, associated with chromatin, and then phosphorylated. Partial tryptic digestion of Drosophila H1 revealed that the phosphorylation site is located within the globular, central domain of the protein. Thus, the developmentally regulated phosphorylation of Drosophila H1 presents two contrasts with previously studied H1 phosphorylation. It is not correlated with DNA replication, and it is located in the central domain of the protein.     

Autoradiographic Analysis of [3H]Imipramine Binding in the Human Brain Postmortem: Effects of Age and Alcohol

In vitro quantitative autoradiography of high-affinity [3H]imipramine binding sites was performed on 16 human brains postmortem. The densities of binding sites were highest in the hypothalamus. Next, in descending order, were the basal and lateral nuclei of the amygdala; substantia innominata; insular cortex; the central nucleus of the amygdala; the anterior nucleus of the thalamus; the head of the caudate nucleus; portions of the frontal, parietal, and temporal cortex; claustrum; the granular layer of the dentate gyrus; substantia nigra; the pyramidal layer of CA fields; globus pallidus; red nucleus; and white matter. Imipramine binding was found to increase with age in a region-specific manner. The presence of alcohol had a similar effect, which was most pronounced in the hippocampus. Sex and time from death to autopsy did not affect imipramine binding, in our sample.     

Metastasis-associated alterations in phospholipids and fatty acids of human prostatic adenocarcinoma cell lines.

Metastatic variants of human prostatic adenocarcinoma cell lines (DU-145, LNCaP, and ND-1) were studied by using soft agar colony forming efficiency, nude mice tumorigenicity, in vitro invasion assay, and type IV collagenase assay. The DU-145 and ND-1 cell line showed higher metastatic potential than LNCaP. Lipids from DU-145, ND-1, and LNCaP cells were extracted and analyzed by thin-layer chromatography and gas-liquid chromatography. The major lipids were phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, fatty acids, and cholesterol. The sphingomyelin level was significantly higher in highly metastatic cells (DU-145 and ND-1) compared with the lower metastatic variant (LNCaP). The increase in the synthetic pathway and decrease in degradation pathway of sphingomyelin in microsomal fractions was sufficient to account for the measured increase in sphingomyelin in DU-145 cells compared with LNCaP cells. The major fatty acids of these lipids were palmitic (16:0), stearic (18:0), oelic (18:1), and arachidonic acid (20:4). The arachidonic acid level was significantly decreased in DU-145 and ND-1 compared with LNCaP cells. Electron microscopic studies showed no significant changes in the morphology of DU-145, ND-1, and LNCaP cells. The results of these investigations demonstrate for the first time that sphingomyelin and arachidonic acid contents are different in high and low metastatic variants of human prostatic adenocarcinoma cell lines.     

Properties of heart fibroblasts of adult rats in culture

Summary In the heart of the adult rat, fibroblasts are mainly responsible for the synthesis and deposition of the collagenous matrix. Because these cells in vitro may serve as an important model system for studies of collagen metabolism in heart tissue, we have cultured and characterized rat-heart fibroblasts from young adult and old animals. Conditions included use of media of different compositions with and without addition of ascorbate. Cell used were either cultured directly from fresh tissues or thawed previously frozen cells. Cultured cells were studied with respect to growth properties, morphology and ultrastructure and patterns of collagen. Heart fibroblasts generally resembled fibroblasts cultured from other tissues, but were more like skeletal muscle fibroblasts in that they deposited, in addition to type I collagen, type IV collagen and laminin. The fibroblasts showed a typical appearance in phase-contrast microscopy and electron microscopy. In the case of cells grown with added ascorbate, aligned collagen fibrils in the extracellular matrix showed a periodicity typical of type I collagen. The deposition of type I collagen occurred only in medium supplemented with ascorbate, and in that circumstance increased as a function of time past confluence; this was independent of the age of the animal from which the cells were obtained or of other changes of medium composition studied. Immunofluorescence studies with specific antibodies revealed that the cells deposited types I and IV collagens, laminin and fibronectin. In contrast to the case of type I collagen, the deposition of type IV collagen occurred in cells grown either with or without ascorbate. Direct observation of type IV collagen is consistent with the previous finding of type IV mRNA in cardiac fibroblasts in situ and in freshly isolated populations of these cells.     

Effect of Chronic Desipramine Treatment on Dihydroalprenolol, Imipramine, and Desipramine Binding Sites: A Quantitative Autoradiographic Study in the Rat Brain

Desmethylimipramine (DMI) administered once daily for 10 days caused a significant decrease in beta-adrenergic receptor binding, as measured by quantitative autoradiography in discrete brain regions. The decrease was observed 72 h after the last injection throughout the cortex and in hippocampus but not in other regions, much richer in beta-receptors, such as the caudate, olfactory tubercle, superior colliculus, dorsomedial thalamus, substantia nigra, or pineal. The same paradigm did not affect imipramine (IMI) binding in the cortex or in regions with high concentrations of IMI binding sites. DMI binding was not decreased, either. Significant increases in DMI binding were observed in frontal cortex and in the ventral aspect of the bed nucleus of the stria terminalis. We conclude that a reduction in tricyclic binding is not a general phenomenon following chronic treatment with tricyclic antidepressants, and changes in binding, when they do occur, are not correlated with areas of high binding site density.     

Salt-dependent conformational transitions in the self-complementary deoxydodecanucleotide d(CGCAATTCGCG): Evidence for hairpin formation

Differential scanning calorimetry (DSC), temperature-dependent uv-absorption spectroscopy, and temperature-dependent CD were used to monitor and characterize the salt-dependent, thermally induced structural transitions in the deoxydodecanucleotide d(CGCGAATTCGCG). At the high oligomer concentrations required for DSC, the calorimetric scans revealed a single, monophasic transition curve at all salt concentrations. Based on previous nmr melting studies under similar conditions, we conclude that these monophasic transitions correspond to the cooperative duplex-to-single-strand conversion of the dodecamer. By contrast, at the lower oligomer concentrations used for the spectroscopic studies, the shapes of the uv and CD melting curves were found to depend on the concentration of the added salt. At high salt (≥0.1M Na⁺), a single, monophasic transition curve was observed. At lower salt (?0.01M Na⁺), the CD and uv melting curves exhibit biphasic behavior. Based on the concentration dependence, the enthalpy, and the cooperativity of each transition in the biphasic curve, we conclude that at low salt and low oligomer concentrations, the dodecamer melts in a sequential manner involving initial disruption of a duplex structure and subsequent disruption of a hairpin structure.     

The chimpanzee M blood-group antigen is a variant of the human M-N glycoproteins

Chimpanzee erythrocytes express strong M but weak, occasional N blood-group activity, as detected by anti-M and anti-N reagents. We have found that the M activity is carried by a major membrane glycoprotein that is similar but not identical to the human MM glycoprotein (glycophorin A). We have isolated and characterized this glycoprotein from erythrocyte membranes of four individual chimpanzees. The purified glycoproteins strongly inhibited agglutination of M cells by rabbit anti-human M sera and only weakly inhibited the agglutination of N cells by rabbit anti-human N sera. They also displayed medium-to-strong inhibitory activity against chimpanzee iso- and crossimmune antisera tested with chimpanzee erythrocytes of various V-A-B-D and W^c specificities, which are known as chimpanzee extensions of the human type M-N system and the Miltenberger counterpart, respectively. Each glycoprotein was cleaved with CNBr into three fragments, whose size, solubility, and composition were analogous to those obtained by similar treatment of the human M-N antigens. The amino-terminal fragment was found to be a glycooctapeptide whose amino acid composition and partial sequence indicated that it is an intermediate form of the human M and N glycooctapeptides. Its carbohydrate content comprised two threonine-linked saccharide units that, although similar in composition to the human threonine-linked units, were fewer in number than the three units found in the corresponding human glycooctapeptides. Structural similarities to the human antigens strongly suggest that the amino terminus bears the major antigenic determinants of the molecule, and the occurrence in this region of numerous, albeit rare, variants among humans and in chimpanzees indicates that the corresponding coding sequence of the structural gene is particularly susceptible to mutational events. We conclude that the chimpanzee M gene product is a variant of the human type and that the chimpanzee gene is an allele of the human polymorphic M-N locus.This research was supported by National Institutes of Health Grants GM 16389 and HL 19011 and March of Dimes Grant 1-661.