Phosphorylation of membrane-bound guanylate cyclase of sea urchin spermatozoa

When Arbacia punctulata spermatozoa are incubated in seawater containing ammonium hydroxide (pH 8.8), the sperm plasma membrane-bound guanylate cyclase is dephosphorylated, its electrophoretic mobility increases (from an apparent molecular mass of 160 to 150 kD), and its enzymatic activity decreases 3.5-fold. Transfer of these cells into ammonium-free seawater (pH 7.4) results in the rephosphorylation of the cyclase, its reconversion to 160 kD, and recovery of the enzymatic activity lost upon dephosphorylation. This is the first direct demonstration that the activity of membrane-bound guanylate cyclase can be regulated by phosphorylation. A plasma membrane preparation is described that specifically supports the in vitro phosphorylation of the guanylate cyclase. This preparation will be useful in more detailed studies on the relationship between phosphorylation state and enzymatic activity of membrane-bound guanylate cyclase.     

Acquired drug resistance is accompanied by modification in the karyotype and nuclear matrix of a rat carcinoma cell line

A Walker 256 breast carcinoma cell line (WR) exhibiting a greater than 20-fold resistance to alkylating agents has been selected from a parent cell line (WS). Karyotypic heterogeneity was apparent, with a number of differences evident between WR and WS cells. The modal chromosome number for WS is 62; for WR, 54; double minutes were found only in WR, whereas spontaneous chromosomal aberrations were present in approx. 40% of the WS cells. No similar aberrations were observed in WR. Using SDS-gel electrophoresis and subsequent silver staining, differences in the profile of nuclear matrix proteins in WR and WS were observed. A diffuse band at approx. 70 kD in the WS was absent in WR cells. This protein was phosphorylated, together with a number of the other major matrix polypeptides. Levels of phosphorylated matrix proteins were approximately equivalent in both WR and WS cell lines, but matrix protein phosphorylation levels were approx. 2-fold higher than corresponding values for bulk nuclear proteins. Selective pressure of drug exposure has resulted in enhanced genetic stability in WR cells and observed karyotype differences are accompanied by modifications in the structural proteins of the nuclear matrix. Whether the observed differences are the cause or result of drug resistance remains to be established.     

Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

Oxide-Based Solid-State Batteries: A Perspective on Composite Cathode Architecture

The garnet-type phase Li₇La₃Zr₂O₁₂ (LLZO) attracts significant attention as an oxide solid electrolyte to enable safe and robust solid-state batteries (SSBs) with potentially high energy density. However, while significant progress has been made in demonstrating compatibility with Li metal, integrating LLZO into composite cathodes remains a challenge. The current perspective focuses on the critical issues that need to be addressed to achieve the ultimate goal of an all-solid-state LLZO-based battery that delivers safety, durability, and pack-level performance characteristics that are unobtainable with state-of-the-art Li-ion batteries. This perspective complements existing reviews of solid/solid interfaces with more emphasis on understanding numerous homo- and heteroionic interfaces in a pure oxide-based SSB and the various phenomena that accompany the evolution of the chemical, electrochemical, structural, morphological, and mechanical properties of those interfaces during processing and operation. Finally, the insights gained from a comprehensive literature survey of LLZO–cathode interfaces are used to guide efforts for the development of LLZO-based SSBs.     

Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene

We have analyzed the conserved regions of the gene coding for the circumsporozoite protein (CSP) in 12 species of Plasmodium, the malaria parasite. The closest evolutionary relative of P. falciparum, the agent of malignant human malaria, is P. reichenowi, a chimpanzee parasite. This is consistent with the hypothesis that P. falciparum is an ancient human parasite, associated with humans since the divergence of the hominids from their closest hominoid relatives. Three other human Plasmodium species are each genetically indistinguishable from species parasitic to nonhuman primates; that is, for the DNA sequences included in our analysis, the differences between species are not greater than the differences between strains of the human species. The human P. malariae is indistinguishable from P. brasilianum, and P. vivax is indistinguishable from P. simium; P. brasilianum and P. simium are parasitic to New World monkeys. The human P. vivax-like is indistinguishable from P. simiovale, a parasite of Old World macaques. We conjecture that P. malariae, P. vivax, and P. vivax-like are evolutionarily recent human parasites, the first two at least acquired only within the last several thousand years, and perhaps within the last few hundred years, after the expansion of human populations in South America following the European colonizations. We estimate the rate of evolution of the conserved regions of the CSP gene as 2.46 x 10(-9) per site per year. The divergence between the P. falciparum and P. reichenowi lineages is accordingly dated 8.9 Myr ago. The divergence between the three lineages leading to the human parasites is very ancient, about 100 Myr old between P. malariae and P. vivax (and P. vivax-like) and about 165 Myr old between P. falciparum and the other two.      

Oligomeric forms of the membrane-bound acetylcholine receptor disclosed upon extraction of the M(r) 43,000 nonreceptor peptide

Oligomeric forms of the acetylcholine receptor are directly visualized by electron microscopy in receptor-rich membranes from torpedo marmorata. The receptor structures are quantitatively correlated with the molecular species so far identified only after detergent solubilization, and further related to the polypeptide composition of the membranes and changes thereof. The structural identification is made possibly by the increased fragility of the membranes after extraction of nonreceptor peptides and their subsequent disruption upon drying onto hydrophilic carbon supports. Receptor particles in native membranes depleted of nonreceptor peptides appear as single units of 7-8 nm, and double and multiple aggregates thereof. Particle doublets having a main-axis diameter of 19 +/- 3 nm predominate in these membranes. Linear aggregates of particles similar to those observed in rotary replicas of quick-frozen fresh electrolytes (Heuser, J.E. and S. R. Salpeter. 1979, J. Cell Biol. 82: 150-173) are also present in the alkaline-extracted membranes. Chemical modifications of the thiol groups shift the distribution of structural species. Dithiothreitol reduction, which renders almost exclusively the 9S, monomeric receptor form, results in the observation of the 7-8 nm particle in isolated form. The proportion of doublets increases in membranes alkylated with N-ethylmaleimide. Treatment with 5,5’-dithiobis-(nitrobenzoic acid) increases the proportion of higher oligomeric species, and particle aggregates (n=oligo) predominate. The nonreceptor v-peptide (doublet of M(r) 43,000) appears to play a role in the receptor monomer-polymer equilibria. Receptor protein and v-peptide co-aggregate upon reduction and reoxidation of native membranes. In membranes protected ab initio with N- ethylmaleimide, only the receptor appears to self-aggregate. The v-peptide cannot be extracted from these alkylated membranes, though it is easily released from normal, subsequently alkylated or reduced membranes. A stabilization of the dimeric species by the nonreceptor v-peptide is suggested by these experiments. Monospecific antibodies against the v-peptide are used in conjunction with rhodamine- labeled anti-bodies in an indirect immunoflourescence assay to map the vectorial exposure of the v-peptide. When intact membranes, v-peptide depleted and “holey” native membranes (treated with 0.3 percent saponin) are compared, maximal labeling is obtained with the latter type of membranes, suggesting a predominantly cytoplasmic exposure of the antigenic determinants of the v-peptide in the membrane. The influence of the v-peptide in the thiol-dependent interconversions of the receptor protein and the putative topography of the peptide are analyzed in the light of the present results.     

The cytolytic isolation of the cortex of the sea urchin egg

When the vitelline layer of sea urchin eggs (Lytechinus pictus) is disrupted by trypsin or dithiothreitol and the eggs are placed in an isosmotic medium devoid of Ca²⁺, cytolysis of the eggs occurs. During lysis the entire egg cortex peels off in one piece. Lysis is temperature and pH dependent and is inhibited by cytochalasin B. Cortices from unfertilized eggs contain seven major macromolecular components. A 42K-dalton component is believed to be actin, representing between 12 and 27% of the total protein. Cortices from fertilized eggs may contain between 50 and 65% actin. The actin appears to increase the strength of its attachment to the cortex after fertilization. This method of isolating the entire cortex may be useful for studying structural and enzymatic changes which may occur in the cortex during the cell cycle.     

The accessory function of murine intercellular adhesion molecule-1 in T lymphocyte activation. Contributions of adhesion and co-activation.

These studies demonstrate that the murine intercellular adhesion molecule-1 (ICAM-1) performs at least two roles in enhancing T cell activation. These two roles are evident in both of our experimental systems: with ICAM-1 expressed on the surface of transfected fibroblast cells, and with purified ICAM-1 immobilized on plastic. First, as has been documented by many investigators, ICAM-1 mediates adhesion between ICAM-1- and lymphocyte function-associated Ag-1 (LFA-1)-bearing cells. This adhesive interaction occurs even in the absence of T cell stimulation, although it is increased by addition of phorbol ester and calcium ionophore. Although ICAM-1 expression does markedly increase intercellular adhesion, the increase is significantly less than the improvement ICAM-1 expression makes in the Ag-presenting ability of MHC class II-transfected fibroblast cells. We have investigated whether this difference is due to LFA-1-mediated signaling, and we present data that demonstrates that although ICAM-1 does not deliver costimulatory signals required for T cell activation, the interaction of LFA-1 with ICAM-1 does synergize with TCR-transduced signals. This synergy is observed for ICAM-1 on live and on chemically fixed accessory cells, and for purified ICAM-1 molecules, but in all cases occurs only when the ICAM-1 and the TCR ligands are on the same surface. Finally, when the ICAM-1 is present on the surface of accessory cells, it enhances T cell activation by changing the Ag dose-dependence of the T cell, but when ICAM-1 and CD3 mAb are co-immobilized, ICAM-1 increases the peak response of the T cell without affecting the dose dependence of the response.     

In vitro phosphorylation of sea urchin sperm adenylate cyclase by cyclic adenosine monophosphate-dependent protein kinase

Addition of [gamma -32P]ATP to a 2% Brij-78 40,000g supernatant of sea urchin sperm results in the cAMP-dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC). An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a Km for ATP of 5.2 microM and is maximally stimulated by 4-8 microM cAMP. The protein kinase inhibitors H8 (9 microM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine cAMP-dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the Km or Vmax of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2(+) -activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by cAMP-dependent protein kinase.