The subunit fine structure of isolated, purified Na,K-adenosine triphosphatase : Freeze-fracture study

The ultrastructural features of a purified fraction of Na⁺,K⁺-adenosine triphosphatase (ATPase) isolated from dog kidney medulla were compared with those of the initial crude microsomal fraction in the purification sequence. Although both fractions consist of vesicular structures, the purified fraction is more homogeneous with respect to overall size and intramembrane protein particle size and distribution. Polyacrylamide gel electrophoresis profiles of both fractions reveal multiple proteins in the microsomal fraction but only two in the final purified fraction. The membranes of the pure fraction comprised one class of particles roughly 95–120 Å in diameter which represent the in vitro configuration of Na⁺,K⁺-ATPase.     

Agarose slab-gel electrophoresis equipment.

Simple slab-gel molds which utilize the electrophoresis apparatus described by F. W. Studier (J. Mol. Biol.79, 237 (1973)) have been designed for pouring and running agarose slab-gels. Analytical gels in which many samples are run simultaneously facilitate the assay of many enzymes which lead to physical changes in DNA, whereas the preparative gels allow the separation of large quantities (1–20 mg) of DNA fragments.     

Structural studies on H+,K+-ATPase: determination of the NH2-terminal amino acid sequence and immunological cross-reactivity with Na+,K+-ATPase

The NH2-terminal amino acid sequence of the 100 kilodalton subunit of porcine gastric H+,K+-ATPase has been determined to be YKAENYELYQVELGPGP. Although the NH2-terminal region of this protein is not similar to the same region of the lamb kidney Na+,K+-ATPase catalytic subunit, other regions of these ATPase proteins appear to be homologous. Both monoclonal and polyclonal antibodies raised to lamb kidney Na+,K+-ATPase and its alpha, but not beta, subunit cross-react with the 100 kilodalton protein of H+,K+-ATPase.     

Induction of VCAM-1 and ICAM-1 on human neural cells and mechanisms of mononuclear leukocyte adherence.

We propose that leukocyte-derived cytokines induce the expression of adhesion molecules on the surface of neural cells that facilitates the subsequent attachment of leukocytes. Leukocyte adherence may contribute to some of the neural cell injury seen with various inflammatory diseases of the nervous system. With an in vitro model system, we have shown that mononuclear leukocytes bind to human neuroblastoma and cortical neuron cells only after the neural cells are stimulated with TNF-alpha. TNF-alpha stimulates expression of vascular cell adhesion molecule-1 (VCAM-1) in both of these neural cell lines. VCAM-1 mRNA is increased and VCAM-1 protein can be identified on the neural cell membranes with a new VCAM-1-specific mAb, CL40/2 F8. TNF-alpha also induces ICAM-1 in both of these neural cell lines. Leukocyte beta 1 (CD29) and beta 2 (CD18) integrins and their respective ligands, ICAM-1 and VCAM-1, on neural cells appear to be the dominant ligands mediating MNL:neural cell adhesive interactions. mAb to CD18 block 32 to 57% of the MNL binding to neural cells; similar inhibition is seen with mAb to ICAM-1. mAb to CD29 block 16 to 17% of the MNL binding to the neural cells suggesting that leukocyte beta 1 integrins and neural VCAM-1 may be a second route for MNL:neural cell interactions. Addition of both anti-CD18 and anti-CD29 mAb have an additive blocking effect; both ligand pairs may participate in MNL adhesion to neural cells, reminiscent of the multiplicity of ligands used by MNL when binding to endothelium.     

Response to Preuss and Zuccarello (2020): biological definitions that can be unambiguously applied for red algal parasites

In response to a comment in this issue on our proposal of new terminology to distinguish red algal parasites, we clarify a few key issues. The terms adelphoparasite and alloparasite were previously used to identify parasites that infected close or distant relatives. However, most red algal parasites have only been studied morphologically, and molecular tools have shown that these binary terms do a poor job at representing the range of parasite–host relationships. We recognize the need to clarify inferred misconceptions that appear to be drawing from historical terminology to contaminate our new definitions. We did not intend to replace the term adelphoparasite with neoplastic parasites and the term alloparasites with archaeplastic parasites. Rather, we seek to establish new terms for discussing red algal parasites, based on the retention of a native plastid, a binary biological trait that is relatively easy to identify using modern methods and has biological implications for the interactions between a parasite and its host. The new terminology can better account for the spectrum of relationships and developmental patterns found among the many independently evolved red algal parasites, and it is intended to inspire new research, particularly the role of plastids in the survival and evolution of red algal parasites.     

Expression of Telomerase and Telomere Length Are Unaffected by either Age or Limb Regeneration in Danio rerio

Background

The zebrafish is an increasingly popular model for studying many aspects of biology. Recently, ztert, the zebrafish homolog of the mammalian telomerase gene has been cloned and sequenced. In contrast to humans, it has been shown that the zebrafish maintains telomerase activity for much of its adult life and has remarkable regenerative capacity. To date, there has been no longitudinal study to assess whether this retention of telomerase activity equates to the retention of chromosome telomere length through adulthood.

Methodology/Principal Findings

We have systematically analyzed individual organs of zebrafish with regard to both telomere length and telomerase activity at various time points in its adult life. Heart, gills, kidney, spleen, liver, and intestine were evaluated at 3 months, 6 months, 9 months, and 2 years of age by Southern blot analysis. We found that telomeres do not appreciably shorten throughout the lifespan of the zebrafish in any organ. In addition, there was little difference in telomere lengths between organs. Even when cells were under the highest pressure to divide after fin-clipping experiments, telomere length was unaffected. All aged (2 year old) tissues examined also expressed active amounts of telomerase activity as assessed by TRAP assay.

Conclusions/Significance

In contrast to several other species including humans, the retention of lifelong telomerase and telomeres, as we have reported here, would be necessary in the zebrafish to maintain its tremendous regenerative capacity. The ongoing study of the zebrafish''s ability to maintain telomerase activity may be helpful in unraveling the complexity involved in the maintenance (or lack thereof) of telomeres in other species such the mouse or human.     

Inhibition or down-regulation of protein kinase C attenuates late phase p70s6k activation induced by epidermal growth factor but not by platelet-derived growth factor or insulin.

The late phase of the time-dependent epidermal growth factor (EGF)-induced biphasic activation of the p70s6k is selectively attenuated by the specific PKC inhibitor, CGP 41,251, a staurosporine derivative. At a 40-fold lower concentration than CGP 41,251, staurosporine inhibits both phases of S6 kinase activation to the same extent, whereas the inactive staurosporine derivative CGP 42,700 shows no effect on either phase. Platelet-derived growth factor (PDGF) and insulin also induce biphasic S6 kinase activation, but in neither case is either phase of activation affected by the presence of CGP 41,251. This finding was unexpected in the case of PDGF, which is a potent activator of PKC and whose receptor directly interacts with phospholipase C gamma 1. However, similar results were obtained following down-regulation of PKC by prolonged 12-O-tetradecanoylphorbol-13-acetate treatment. Therefore, even though EGF and PDGF induce PKC activation, PDGF, unlike EGF, does not appear to use this signaling pathway for late phase p70s6k activation.