Slow potential changes due to transport number effects in cells with unstirred membrane invaginations or dendrites

Summary Many neurones are extremely invaginated and possess branching processes, axons and dendrites. In general, they are surrounded by a restricted diffusion space. Many of these cells exhibit large, slow potential changes during the passage of current across their membranes. Whenever currents cross membranes separating aqueous solutions, differences in transport numbers of the major permeant ions give rise to local concentration changes of these ions adjacent to the membranes, which will result in various electrical and osmotic effects. These transport number effects are expected to be enhanced by the presence of membrane invaginations. Dendrites are equivalent to reversed invaginations and there should be significant changes in concentrations of permeant ions within them. In general, the effects of such changes on the electrical response of a cell will be greater when the concentration of a major permeant ion is low. The effects have been modelled in terms of two nondimensional parameters: the invagination transport number parameter and the relative area occupied by the invaginations A. If these two parameters are known, the magnitudes and time course of the slow potential changes can immediately be estimated and the time course converted to real time, if the length of the invaginations (l) and ionic diffusion coefficient (D) within them are also known. Both analytical and numerical solutions have been given and predictions compared. It is shown that in the case of large currents and potentials the analytical solution predictions will underestimate the magnitudes and rates of onset of the voltage responses. The relative magnitude of the transport number effect within the invaginations (or dendrites) and other transport number contributions to slow potential changes have also been assessed and order-of-magnitude values of these are estimated for some biological data.     

HLA-JY328: Mapping studies and expression of a polymorphic HLA class I gene

The JY328 clone was identified in a human genomic library using cDNA corresponding to mRNA for HLA-B7 as a probe. The L/328 cell line was established by cotransformation of mouse Ltk^– cells with the herpes thymidine kinase gene and clone JY328. On Northern blots, RNA from,L/328 strongly hybridized to an HLA class I probe, and an antigen was recognized by an anti-HLA class I framework antibody on the cell surface. A DNA probe corresponding to a segment of intron 7 was developed by comparing the nucleotide sequence of clone JY328 with that of other HLA class I-type genes. Using the radiolabeled probe to screen Southern blots of DNA from families with siblings exhibiting intra-HLA recombinations, a restriction fragment length polymorphism was revealed —a 1.4 kb BstE II band not present in all individuals. A corresponding fragment was apparent in the base sequence of clone JY328. The occurrence of this band on Southern blots established that JY328 maps distinct from and centromeric to the HLA-C locus and near to the HLA-B locus. Antibody absorption studies and cytotoxicity tests indicated that the JY328 gene product was not an HLA-B antigen but that it did specifically absorb CW7-specific antibody. In sum, these results suggest a novel, polymorphic HLA class I gene which expresses a product serologically similar to HLA-Cw7 but which does not map within the corresponding locus.     

Multiple conformations of amino acid residues in ribonuclease A

The highly refined 1.26 A structure (R = 0.15) of phosphate-free bovine pancreatic ribonuclease A was modeled with 13 residues having discrete multiple conformations of side chains. These residues are widely distributed over the protein surface, but only one of them, Lys 61, is involved in crystal packing interactions. The discrete conformers have no unusual torsion angles, and their interactions with the solvent and with other atoms of the protein are similar to those residues modeled with a single conformation. For three of the residues--Val 43, Asp 83, and Arg 85--two correlated conformations are found. The observed multiple conformations on the protein surfaces will be of significance in analyzing structure-function relationships and in performing protein engineering.     

Dye permeability at phase transitions in single and binary component phospholipid bilayers

By encapsulating a pH-sensitive dye, phenol red, in multilamellar liposomes of DMPC, DPPC and DMPC/DPPC mixtures, the permeability of these phospholipid bilayers to dye as a function of temperature has been studied. For both DMPC and DPPC liposomes, dye release begins well below the main gel-to-liquid-crystalline phase transition (24°C and 42°C, respectively) at temperatures corresponding to the onset of the pretransition (about 14°C and 36°C, respectively) with DPPC liposomes exhibiting a permeability anomaly at the main phase transition (42°C). The perturbation occurring in the bilayer structure that allows the release of encapsulated phenol red (approx. 5 Å diameter) is not sufficient to permit the release of encapsulated haemoglobin (approx. 20 Å diameter, negatively charged). In liposomes composed of a range of DMPC/DPPC mixtures, dye release commences at the onset of the pretransition range (determined by optical absorbance measurements) and increases with increasing temperature until the first appearance of liquid crystalline phase after which no further dye release occurs. Interestingly, the dye retaining properties of DMPC and DPPC liposomes well below their respective pretransition temperature regions are very different: DMPC liposomes release much encapsulated dye at incubation temperatures of 5°C whilst DPPC liposomes do not.     

Isolation and partial characterization of the basal cell membrane of human placental trophoblast

The function of the syncytiotrophoblast in maternal-fetal exchange is related to the properties of its microvillous (maternal-facing) and basal (fetal-facing) plasma membranes. We have previously reported the properties of the microvillous membrane (Smith, C.H., Nelson, D.M., King, B.F., Donohue, T.M., Ruzycki, S.M. and Kelley, L.K. (1977) Am. J. Obstet. Gynecol. 128, 190–196), and now describe the purification and partial characterization of the basal plasma membrane. Sonication and incubation with EDTA were used to isolate selectively the basal cell membrane. These steps were followed by a more conventional purification by centrifugation. The trophoblast was disrupted and its microvillous membrane and cytoplasmic contents were removed by sonication. The exposed basal cell membrane was selectively released from the underlying basal lamina by sonication in the presence of EDTA and further purified by discontinuous Ficoll gradient centrifugation. The material at the 4–10% Ficoll interface consisted of smooth membrane vesicles with internal microfilaments. It was 45-fold enriched in dihydroalprenolol binding activity and 11-fold enriched in ouabain binding activity. Other enzymatic analyses, including alkaline phosphatase, cytochrome-c oxidase, cytochrome-c reductase and galactosyl transferase indicated low contamination by other organelles. This procedure yields a preparation of relatively high purity which should be suitable for investigation of transport and other functions of the basal surface membrane of trophoblast. In principle, the purification procedures used may be applicable to other transporting epithelia.     

Phytohormones, Rhizobium Mutants, and Nodulation in Legumes : III. Auxin Metabolism in Effective and Ineffective Pea Root Nodules

High specific activity [³H]indole-3-acetic acid (IAA) was applied to the apical bud of intact pea (Pisum sativum L. cv Greenfeast) plants. Radioactivity was detected in all tissues after 24 hours. More radioactivity accumulated in the nodules than in the parent root on a fresh weight basis and more in effective (nitrogen-fixing) nodules than in ineffective nodules (which do not fix nitrogen).

For most samples, thin layer chromatography revealed major peaks of radioactivity at the R_F values of IAA and indole-3-acetylaspartic acid (IAAsp) and further evidence of the identity of these compounds was obtained by chromatography in other systems. Disintegrations per minute due to IAA per unit fresh weight were significantly greater for root than for nodule tissue, but were not significantly different for effective and ineffective nodules. Radioactivity due to IAAsp, expressed both on a percentage basis and per unit fresh weight, was significantly greater for nodule than for root tissue and significantly greater for the effective nodules than for the ineffective nodules. When [³H]IAA was applied to effective nodules, IAAsp was the dominant metabolite in the nodule. The data suggest that metabolism of auxins may be important for the persistence of a functional root nodule.

     

Effect of Elevated Temperature on Extended Enzyme Synthesis Induced by Bacteriophage T4 Amber Mutants Unable to Synthesize Deoxyribonucleic Acid

The extended synthesis of early enzymes by the deoxyribonucleic acid-negative amber mutants of bacteriophage T4 after infection of the nonpermissive host Escherichia coli B was prevented by incubating the infected cells at 44 C. This effect did not occur if the incubation temperature was 43 C or less or if the cells were grown and infected in broth rather than minimal medium (C medium). Once early enzyme synthesis had ceased at 44 C, lowering the incubation temperature to 37 C did not occasion resumption of synthesis. Experiments with chloramphenicol at 44 C indicated that increased degradation of early enzymes is an unlikely explanation for the effect. Examination of pulse-labeled ribonucleic acid and polysomes made at 37 and 44 C in infected cells revealed some differences, but at present there is no obvious way in which these differences may be related to the effect on enzyme formation. There was no discernible difference between the ribosomal ribonucleic acid and ribosomes at the two temperatures, nor was there a difference in the cell-free amino acid-incorporating systems isolated from cells infected at the two temperatures as judged by polyuridylic stimulation of phenylalanine incorporation. Incubation of cells infected with T4amN82 at 44 C with protein synthesis blocked by 5-methyltryptophan for 15 min did not prevent the typical pattern of enzyme synthesis at 44 C when the block was reversed by excess l-tryptophan. The relation of this and other observations relative to the effect at 44 C on the synthesis of early enzymes is discussed.     

Regulation of Newly Evolved Enzymes. I. Selection of a Novel Lactase Regulated by Lactose in ESCHERICHIA COLI

Thirty-four lactose-utilizing strains of E. coli were selected from a lac Z deletion strain. In 31 of these, the synthesis of the newly evolved lactase is regulated by lactose. The lactase activity in all the strains is indistinguishable from the ebg(+) activity identified by Campbell, Lengyel and Langridge (1973).