Nonequivalent effects of PKC activation by PMA on murine CD4 and CD8 cell-surface expression

The membrane glycoproteins CD4 (L3T4) and CD8 (Lyt2) are expressed on distinct populations of mature murine T lymphocytes, and are thought to be receptors for monomorphic determinants expressed on MHC class II and class I molecules, respectively. Although they differ in their ligand specificity, it has been presumed that CD4 and CD8 perform equivalent functions in the T cells that bear them. Since activation of protein kinase C (PKC) is known to cause rapid down-regulation of various receptors, including the T cell receptor complex (TcR complex), we treated cells with phorbol 12-myristate 13-acetate (PMA), a PKC activator, to determine whether cell-surface expression of CD4 and CD8 would be similarly affected by this intracellular mediator. Brief or relatively prolonged treatment with PMA induced mature murine T cells to reduce their surface expression of the TcR complex and of CD4, but not of CD8. Similarly, PMA rapidly induced transfected L cells to down-regulate surface CD4 expression, but had no effect on surface CD8 expression. Most significantly, PMA treatment induced CD4+CD8+ immature thymocytes to rapidly reduce their surface CD4 expression, but, again, it had no immediate effect on the surface expression of CD8. These results indicate that CD4 and TcR complex cell-surface expression are both sensitive to PKC activation by brief treatment with PMA, whereas CD8 expression is not, and suggest that CD4 and CD8 surface expression levels are regulated by distinct intracellular mechanisms.     

Studies on the immediate and delayed leucocytosis elicited by brief (30-min) strenuous exercise

Eight healthy male volunteers exercised for two 30-min sessions starting 3 h apart on an electronically braked cycle ergometer at a work load (mean 155.9 W, SD 33.4 W) which required an oxygen consumption that was 70% of their maximal rate of oxygen uptake. Venous blood samples were taken through an indwelling cannula over a period of 6 h beginning shortly before the first bout of exercise and were analysed for routine haematological parameters and for lactate, noradrenaline, adrenaline and cortisol. Both bouts of exercise induced an immediate leucocytosis due to rises in lymphocytes and neutrophils but only the first exercise bout induced a substantial delayed neutrophilia. In at least five subjects, changes in lymphocyte and platelet numbers were correlated (Spearman's rank procedure, P less than 0.05) with simultaneous changes in the plasma concentrations of lactate, noradrenaline and adrenaline over the 6-h period studied. Increases in the plasma concentration of cortisol due to exercise correlated positively with the percentage changes in neutrophil numbers at 3 h and 6 h. These results are consistent with the suggestion that the immediate and delayed leucocytosis induced by exercise are mediated respectively by catecholamine and by cortisol.     

Interaction of lymphocytes and high endothelial venules in irradiated lymph nodes

After total-body exposure to various doses of ionizing radiation, the ability of lymphocytes to interact specifically with high endothelial venules of rat cervical and mesenteric lymph nodes was analyzed in frozen sections. Following a radiation dose of 1.5 Gy, high endothelial venules remained intact and the binding of unirradiated lymphocytes to the venules was enhanced relative to unirradiated controls. At radiation doses above 5.0 Gy, damage to high endothelial venules was observed histologically as well as assessed functionally. There was a significant decrease in specific lymphocyte-venule binding and a significant increase in nonspecific binding. These findings suggest that radiation-induced damage to high endothelial venules might play a role in radiation-induced immunosuppression by interfering with the normal passage of lymphocytes from the blood into lymph nodes via a specific interaction between lymphocytes and high endothelial venules.     

Apoptosis: final control point in cell biology

The discovery of apoptosis, a widespread and morphologically distinct form of physiological cell death, has had an extraordinary impact on cell biology. The importance of apoptosis stems from its active nature and its potential for controlling biological systems. The growing appreciation of the significance of this process has stimulated intense investigation into the molecular mechanisms involved and into its fundamental implications for developmental biology, immunology and oncology.     

Activin-A binding and biochemical effects in osteoblast-enriched cultures from fetal-rat parietal bone.

Activin, a disulfide-linked polypeptide dimer first isolated from gonadal tissue extracts, has amino acid sequence and structural homology with transforming growth factor beta (TGF beta). Along with other activities, TGF beta regulates replication and differentiation and interacts with a defined set of binding sites on isolated bone cells. To determine if activin shares these properties, recombinant human activin-A (A-chain homodimer) was examined in osteoblast-enriched cultures obtained from fetal-rat parietal bone. After 23 h of treatment, 60 to 6,000 pM activin-A increased the rate of [3H]thymidine incorporation into DNA 1.5- to 4.0-fold, and at 600 to 6,000 pM, it enhanced the rate of [3H]proline incorporation into collagen and noncollagen protein by up to 1.7-fold. Like earlier studies with TGF beta in primary osteoblast-enriched cultures, the stimulatory effects of activin-A on DNA and protein synthesis were opposed by parathyroid hormone, and the influence of activin-A on collagen synthesis was independent of cell replication. Binding studies with 125I-activin-A indicated approximately 8,000 high-affinity (Kd = 0.4 nM) and 300,000 low-affinity (Kd = 40 to 50 nM) binding sites per cell. Polyacrylamide gel analysis revealed 125I-activin-A-binding complexes of Mr greater than 200,000 and 73,000 which did not appear to correspond to primary TGF beta-binding sites. These results indicate that activin-A produces TGF beta-like effects in bone and that some of these effects may be mediated, at least in part, by distinct activin receptors on bone cells.     

Phycological studies in the North Channel,Lake Huron

The phytoplankton of North Channel in Lake Huron and its productivity was studied at 8 stations distributed across the channel during May to October, 1974. The phytoplankton analysis was conducted using the Utermohl technique. The mean percent biomass at each station indicated Diatomeae (59–77%) and phytoflagellates such as Chrysophyceae (4–21%) and Cryptophyceae (7–19%) as the dominant contributors. Seasonal variations of biomass ranged from 0.2 to 0.35 g·m^–3 with a single peak during stratified conditions. Diatomeae dominated throughout the period of investigation followed by Chrysophyceae and Cryptophyceae. Biomass composition by size revealed the dominance of ultraplankton (5–20 m) which contributed 29–68% to the total biomass. Species such as Fragilaria crotonensis, Tabellaria fenestrata, Synedra acus var. radians, Cyclotella comta and C. bodanica made substantial contributions during the unstratified and stratified conditions.Ultraplankton contributed overwhelmingly to the primary productivity as measured by carbon-14 uptake. The contaminant bioassays with single metals, metals in combination and a mixture of metals demonstrated that the ultraplankton's carbon assimilation was inhibited significantly, revealing their sensitivity to contaminants. Phytoplankton ecology of the Channel appears to be affected by tributary inflows, industrial/municipal inputs, and short flushing rates. However, statistical treatment of the ultraplankton biomass showed correlations with temperature and nutrients. Based on phycological and limnological characteristics, the Channel appears to be oligotrophic. The chlorophyll/biomass ratios and Activity Coefficient (P/B) align it with the most oligotrophic Lake Superior in its metabolic efficiency.     

Genetic transformation system in the archaebacterium Methanobacterium thermoautotrophicum Marburg.

A wild-type strain of Methanobacterium thermoautotrophicum Marburg was transformed by DNA from strains resistant to 5-fluorouracil. Recipient cells were grown without selection on gellan gum (GELRITE) plates with DNA. Drug-resistant cells were recovered by replica plating the resulting colonies onto drug plates. Transformation required high-molecular-weight DNA with appropriate markers and was not observed on agar or in liquid media under a variety of conditions.     

Structural studies of alpha-bungarotoxin. 3. Corrections in the primary sequence and X-ray structure and characterization of an isotoxic alpha-bungarotoxin

The most plausible set of chemical shift assignments for alpha-bungarotoxin as deduced from the combined use of two-dimensional J-correlated and two-dimensional nuclear Overhauser effect 1H nuclear magnetic resonance (NMR) spectroscopy was in conflict with the accepted amino acid sequence between residues 8 and 12 and residues 66 and 70 [Basus, V. J., Billeter, M., Love, R. A., Stroud, R. M., & Kuntz, I. D. (1988) Biochemistry (first paper of three in this issue]). Furthermore, NMR spectra of alpha-bungarotoxin, purified by conventional methods, evidenced a second species at the level of approximately 10% total protein. The minor component was separated from alpha-bungarotoxin by Mono-S (cationic) chromatography. Sequencing of Mono-S-purified alpha-bungarotoxin and one of its tryptic peptides showed that the correct sequence for alpha-bungarotoxin is Ser-Pro-Ile at positions 9-11 and Pro-His-Pro at positions 67-69. The electron density map of alpha-bungarotoxin [Love, R. A., & Stroud, R. M. (1986) Protein Eng. 1, 37] was refined with the new sequence data. Improvements in the structure were found primarily for residues 9-11. Sequence analysis of two overlapping tryptic peptides proved that the minor species differed from alpha-bungarotoxin by replacement of a valine for an alanine at position 31. This new toxin, alpha-bungarotoxin(Val-31), binds to the acetylcholine receptor with an affinity that is comparable to that of alpha-bungarotoxin.     

Alternative model for the internal structure of laminin

A monoclonal antibody to laminin, LMN-1, was generated by immunizing rats with laminin from the EHS tumor and fusing the rat spleen cells with mouse NS-1 myeloma cells. Laminin fragments were generated by proteolytic digestion with thrombin, thermolysin, and chymotrypsin. Monoclonal antibody binding fragments were identified by immunoblotting. Fragments which bound monoclonal antibody LMN-1 included a 440-kilodalton (kDa) chymotrypsin fragment and thermolysin fragments of 440 and 110 kDa. These fragments could also be generated from within a 600-kDa thrombin fragment. Digestion of the 440-kDa chymotrypsin fragment with thermolysin generated the 110-kDa antibody binding fragment and a 330-kDa nonbinding fragment. Immunoblotting was performed on extracts of PYS-2 cells and EHS cells using polyclonal and monoclonal antibodies to laminin. Polyclonal antibodies stained the intact 850-kDa complex and the 200- and 400-kDa subunits, while monoclonal LMN-1 stained only the 400-kDa subunit and the complete molecule. Rotary shadowing of monoclonal LMN-1 bound to laminin molecules indicated that the binding site was within the long arm of laminin. Changes in the model of the internal organization of the laminin molecule are proposed, based on the binding of LMN-1 to the 400-kDa subunit and specific proteolytic fragments. The locations of the major thrombin and chymotrypsin fragments in the model are rotated 180 degrees relative to the previously described model [Ott, U., Odermatt, E., Engel, J., Furthmayr, H., & Timpl, R. (1982) Eur. J. Biochem. 123, 63-72] to include part of the 400-kDa subunit of laminin.