Protein substrate specificity of a calmodulin-dependent protein kinase isolated from bovine heart

The protein substrate specificity of a calmodulin-dependent protein kinase activity from the cytosolic fraction of bovine heart was examined. Prior to the experiments, the kinase activity was purified more than 50-fold with a recovery of greater than 10% of the homogenate activity. Two endogenous protein substrates of molecular weight 57,000 and 73,000 were phosphorylated in these kinase preparations. The kinase preparation was also able to phosphorylate exogenous synapsin, phospholamban, glycogen synthase, MAP-2, myelin basic proteins and κ-casein, but not tubulin, pyruvate kinase, the regulatory subunit of cAMP protein kinase II, myosin light chain or phosphorylase b. High levels of calmodulin were required for activation of the kinase activity toward the 57,000 and 73,000 molecular weight endogenous substrates (K_0.5 = 93 +/- 5 nM), glycogen synthase (K_0.5 = 127 +/- 10 nM), and κ-casein (K_0.5 = 321 +/- 107 nM). The kinase possessed a high affinity for glycogen synthase (half maximal activity at 0.9 +/- 0.4 μM) but a low affinity for κ-casein (21 +/- 2 μM). Sucrose density gradient centrifugation separated the calmodulin-dependent protein kinase activity into two fractions with apparent molecular weights of approximately 900,000 and 100,000. Both fractions phosphorylated the endogenous 57,000 molecular weight substrate and glycogen synthase similarly. These results indicate that cardiac calmodulin-dependent protein kinase previously observed to phosphorylate endogenous protein substrate possesses a wide range of substrate specificity.     

A comparison of protein synthetic patterns in normal and animalized sea urchin embryos

Newly synthesized proteins from normal and animalized sea urchin embryos were compared by the technique of double labeling. Total embryonic protein was solubilized in SDS, urea, and 2-mercaptoethanol. The proteins were examined by coelectrophoresis on an SDS-polyacrylamide gel. The gels were sliced and the radioactivity determined. A standardized ratio of the isotopes served as the basis of comparison. A comparison of newly synthesized proteins from normal embryos 24 and 48 h old showed a shift from larger to smaller molecular weight proteins. Animalized embryos showed a similar shift. When normal and animalized embryos of the same ages were compared, differences were found. The differences were distributed over the entire range of molecular weights. These results show that although differences in protein synthesis between animalized and normal embryos are evident by 24 h, most of the changes in protein synthesis that occur in normal embryos are unaffected by animalization.     

Ionomycin-mediated calcium transport in rigid and fluid liposomes

The antibiotic ionophore ionomycin translocates Ca from an aqueous medium into or across an organic immiscible phase. At pH 8.0, ionomycin translocates less Ca than A23187, the effects of these ionophores being additive to one another. The capacity of ionomycin to translocate Ca across the organic phase is dramatically decreased when the pH of the aqueous media is reduced from 8.0 to 7.5 or lower values. Ionomycin also mediates Ca exchange-diffusion in liposomes, the magnitude of such a process being greater in fluid than in rigid liposomes. At a physiological pH (7.4), ionomycin is unexpectedly as potent as A23187 in mediating Ca transport in fluid liposomes. These findings suggest that the capacity of ionophores to translocate Ca across model membranes depends on both the transverse and lateral mobility of the ionophoretic molecules. The relative importance of the latter phenomenon itself largely depends on the stoichiometry of the Ca-ionophore complex.     

Uptake of the yolk protein, lipovitellin, by developing crustacean oocytes

A variety of cytochemical techniques were used to demonstrate how crustacean lipovitellin accumulates within the egg. It was found that a protein serologically identical to the lipovitellin of yolk spheres was present in the hemolymph of vitellogenic crustaceans, but was absent from the hemolymph of males and immature females.In the three crustacean species studied (Uca pugilator, Cambarus clarkii, and Libinia emarginata), pinocytosis of fluorescein-conjugated lipovitellin and trypan blue occurred only during those periods when oocytes were accumulating yolk.It may be concluded from the present studies that yolk spheres develop in crustacean eggs primarily through micropinocytotic uptake of lipovitellin from the hemolymph, although other oocyte proteins appear to be made in the oocyte.     

Experimental allergic encephalomyelitis and cellular immunity in the Lewis rat

The encephalitogenic difference between purified guinea pig and bovine myelin proteins in the Lewis rat is reflected by the two molecules' lack of crossreactivity in the migration inhibition test. Peritoneal exudate cells from rats injected with guinea pig or bovine derived myelin basic protein in Freund's complete adjuvant demonstrate substantial migration inhibition to the sensitizing antigen but little inhibition when cultured in the presence of the other basic protein. The cellular reactivity to guinea pig basic protein is present throughout the induction phase of Experimental Allergic Encephalomyelitis and persists after the recovery of the rats from the paralytic state. Substantial cellular reactivity is also demonstrated to bovine basic protein even though this molecule shows minimal encephalitogenic activity in the Lewis rat. Minimal lymphocyte transformation could be demonstrated to either of the basic proteins, although the immune cells react strongly to the plant mitogen phytohemagglutinin and to a Mycobacterium tuberculosis antigen.     

Stimulatory effect of calcium chelators on Na+-Ca2+ exchange in cardiac sarcolemmal vesicles

The calcium chelators EGTA, EDTA and cyclohexanediamine tetraacetic acid (CDTA) enhance initial rates of Na_i⁺-dependent Ca²⁺ uptake by cardiac sarcolemmal vesicles. The affinity of the exchanger for calcium is increased in the presence of the chelators to an extent dependent on chelator concentration and on the range of free calcium concentrations over which the phenomenon is measured. For free Ca²⁺ in the range of 4 μM or less, the apparent K_m is lowered to approximately 1 μM. The Ca-chelator complex appears to be the species which causes stimulation. The effect is not due to sequestration of contaminating heavy metal ions in the sarcolemmal membrane preparations or the solutions used in experiments. Caution is suggested in the use of EGTA or EDTA as calcium buffers when measuring calcium dependence of phenomena involving calcium binding and transport, because the added chelator may alter the properties of the system.     

The effects of a macrophage-derived cytotoxin on the growth and metabolism of target cells.

Peritoneal macrophages obtained from Sarcoma I (SaI)-immune CS7BL/6 mice release a heat-labile cytotoxin, specific macrophage cytotoxin (SMC), following a two hour interaction with appropriate target cells. Specific macrophage cytotoxin specifically inhibits A/Jax spleen cells from mitogenically responding to concanavalin A, whereas syngeneic CS7BL/6 spleen cells are unaffected. Treatment of target cells with SMC results in early alterations in RNA and DNA metabolism. The uptake and incorporation of ³H-uridine was found to be initially elevated while intracellular levels of ³H-thymidine became markedly reduced. Furthermore, the cytotoxic action of SMC was found to be rapidly accelerated and amplified by low levels of actinomycin-D.     

Antorbital and forehead secretions of black-tailed deer (Odocoileus hemionus columbianus): Their role in age-class recognition

Male black-tailed deer (Odocoileus hemionus columbianus) scent mark with their forehead gland secretion and antorbital sac contents. When forehead and antorbital sections were presented on the same nylon rod on an artificial tree, male deer discriminated between male yearlings' and fawns' secretions. When the secretions were presented separately, male deer discriminated between yearlings' forehead and antorbital secretions and blank controls. Discrimination was indicated by differential rates of sniffing and licking. In addition, the frequency of forehead rubbing of the scent marks appeared to be dependent on the dominance relationships of the male deer. Chemical evidence is presented which suggests that male yearlings' and fawns' antorbital secretions differ quantitatively. Females did not respond preferentially to these secretions and, with the exception of sniffing, responded significantly less than males.     

Migration of thymus cells to the developing gut-associated lymphoid tissues of the young rabbit

Thymus cell migration to the gut-associated lymphoid tissues (GALT) as compared to other lymphoid tissues in young rabbits was determined following in vivo intrathymic inoculation of tritiated thymidine. The GALT received as many or more thymus cells than the spleen or lymph nodes during the first few postnatal days. Migration to the GALT and nonGALT decreased with age, and seeding appeared to be essentially complete by 30–40 days.     

Fluorescence-activated cell sorting of human T and B lymphocytes: I. Direct evidence that lymphocytes with a high density of membrane-bound immunoglobulin are precursors of plasmacytes

Human peripheral lymphocytes bearing either a high or a low amount of membrane-bound immunoglobulin were studied. Cells were “tagged” with fluorescein-labeled antiimmunoglobulin reagents and separated by means of a new electronic instrument, a fluorescence-activated cell sorter (FACS), into populations with either > 10⁵ or < 5 × 10³ immunoglobulin molecules per cell. Fractions of high purities were obtained. (>80% and >99.9%, respectively). In vitro, different functional properties were observed: lymphocytes with high densities of membrane-Ig gave a late proliferative response after stimulation with Pokeweed mitogen (PWM). A considerable proportion of stimulated cells developed into mature plasmacytes as detected by cytoplasmic staining. Those lymphocytes with a low density or complete absence of membrane-Ig could be stimulated by both Phytohemagglutinin (PHA) and Pokeweed mitogen, but no differentiation into plasmacytes occurred. The functions are similar to those of bone marrow-derived (B) and thymus-derived (T) lymphocytes in mice. Thus, the designation as B lymphocytes for human lymphocytes with a large quantity of membrane-bound immunoglobulin seems justified.     

Differential effects of phenylmethanesulfonyl fluoride (PMSF) on carbachol and potassium stimulated phosphoinositide turnover and contraction in longitudinal smooth muscle of guinea pig ileum

Phenylmethanesulfonyl fluoride (PMSF) (2 mM), a putative inhibitor of phosphatidylinositol-specific phospholipase C, almost completely inhibited carbachol-stimulated inositol incorporation into phosphatidylinositol (PI) of longitudinal smooth muscle of guinea pig ileum, while it had no effect on potassium-stimulated inositol incorporation. This suggests that the two stimuli may affect phosphoinositide turnover by different mechanisms, distinguishable by PMSF. In contrast to its specific inhibition of carbachol-stimulated phosphoinositide turnover, PMSF produced a transient inhibition of contraction by both carbachol and potassium. The non-selective effect of PMSF on contraction suggests that it is not the result of its inhibitory effect on phosphoinositide breakdown. PMSF (2 mM) inhibited carbachol-stimulated inositol phosphate accumulation in the presence of Li+ by only 15%-19%, indicating that PMSF inhibition of phosphoinositide turnover was not due to its inhibition of phosphoinositide phosphodiesterase, but to one or more steps following phosphoinositide breakdown.     

Macrophage migration-inhibition in desensitized guinea pigs

The migration of peritoneal exudate cells obtained from guinea pigs with delayed skin reactivity to egg albumin (EA) and diphtheria toxoid (DT) was inhibited in the presence of antigen. A dose of 2 mg of EA given intravenously 8 days after sensitization specifically abolished the migration inhibition tested 5 weeks later. When the challenge was given into a foot pad 6 weeks after sensitization the migration inhibition was partially suppressed 3 to 28 days later.Repeated skin testing did not affect the migration results of the challenged or unchallenged guinea pigs.The demonstration in vitro of desensitization argues that the mechanism is either a reduced number or a reduced responsiveness of the specific effector cells of delayed hypersensitivity, or an inhibitory effect of cells stimulated by the specific antigen. If a humoral inhibitory factor is involved, it is either tightly bound by the cells or produced during the migration assay.     

Activation of protein kinase in the guinea pig fundic gastric mucosa by histamine.

The effect of histamine, 1,4-methylhistamine and ethanol on cyclic AMP levels and protein kinase activation was measured in tissue strips from the fundic region of guinea pig gastric mucosa. Histamine induced a significant elevation of tissue cyclic AMP levels and also $\text{in situ}$ activation of the protein kinase. 1,4-methylhistamine, an inactive analog of histamine, and ethanol had no effect on these two parameters. Results suggest that protein kinase activation is involved in the cyclic AMP-mediated action of histamine on the gastric fundic mucosa.     

The effect of arachidonic acid and prostanoids on collagen dissolution in human uterine cervix in vitro

Explants of human non-pregnant cervix produce collagenolytic enxymes which degrade collagen over a 10 day period in culture. This is significantly enhanced by the presence of very low concentrations of arachidonic acid (10⁻¹⁶−10⁻¹¹M). Prostaglandin E₂, F_2α and 6-keto-F_1α were synthesised in declining amounts over the 10 day period and synthesis was not increased by adding arachidonic acid (10−11M). Meclofenamic acid (10⁻⁶M) and indomethacin (10⁻⁵M), but not tranylcypromine (10⁻⁵) suppressed prostaglandin synthesis yet all reduced collagen dissolution. Mepacrine (phospholipase A₂ inhibitor) also suppressed collagen dissolution. Remodelling of the structure of the cervix matrix may, in part, depend upon arachidonic acid or one of its cyclo-oxygenase or lipoxygenase derived products.     

Role of Ca in stimulus-secretion coupling in the gastric oxyntic cell: Effect of A23187

The effects of Ca⁺⁺ ionophore A23187 on H⁺ secretion and histamine release were studied in the isolated gastric mucosa of the toad . A23187 added from the mucosal side stimulated H⁺ secretion. At high concentrations, A23187 also caused histamine release. This histamine was not sufficient to explain the effects of A23187 on H⁺ secretion. Metiamide, only partially inhibited the effect of ionophore. There was summation and/or potentiation of effects between A23187 and histamine. The results are consistent with the hypothesis that Ca⁺⁺ acts as a second messenger in stimulus-secretion coupling in the oxyntic cell. It is possible that Ca⁺⁺ and cAMP may interact as parallel second messengers in the control of gastric H⁺ secretion.     

Ionic shift in cerebral ischemia

Electrolyte and water contents were measured in gerbil brain after unilateral cerebral ischemia. Increase of Na⁺ and water, and decrease of K⁺ occurred after an ischemic period of 30 minutes. However, these abnormalities disappeared within 3 hours. When the ischemic period was extended to 3 hours, the abnormalities observed after ischemia for 30 minutes were again encountered, but more significant alterations occurred immediately after re-establishment of blood flow. In addition to more pronounced increase of Na⁺ and decrease of K⁺, Ca²⁺ became significantly elevated after recirculation for 15 minutes and progressively increased during recirculation for 3 hours. The steady rise of Ca²⁺ appears to be related to the irreversibility of cerebral ischemia.     

Ecdysteroids in developing ovaries and eggs of the tobacco hornworm

Ecdysteroids of ovaries and newly-laid eggs (0- to 1-hour-old) of the tobacco hornworm are present mainly as conjugates (greater than 95%). Newly-laid eggs contain ecdysteroid conjugates equivalent to 21 micrograms of 26-hydroxyecdysone and 0.73 micrograms of ecdysone per gram of eggs. These levels are similar in ovaries of 93-hour-old adult females. In 1- to 18-hour-old eggs more than 63% of the ecdysteroids exist in the free form and the proportion is similar in 48- to 64-hour-old eggs. The ratio of 26-hydroxyecdysone to ecdysone in the conjugated form remains constant during oocyte maturation and embryogenesis. Though 26-hydroxyecdysone is without molting hormone activity in the house fly assay, the exceptionally high concentration of 26-hydroxyecdysone conjugate(s) in ovaries and newly-laid eggs, together with the fact that it is being released during embryogenesis, indicate some physiological role for 26-hydroxyecdysone.     

Passive avoidance performance correlates with catecholamine turnover in discrete limbic brain regions

Experimentally naive male rats were sequentially tested for an exploratory (open-field) and a one-trial learning passive avoidance behavior. Subsequently, α-MPT-induced disappearance of noradrenaline (NA) and dopamine (DA) was determined in microdissected brain regions. The animals were classified as good or poor avoiders on the basis of their performance in passive avoidance retention test. Trained controls were subjected to the same training except of electric foot-shock during the learning trial. The rate constant of NA disappearance was higher in the hippocampal dentate gyrus of the good vs. poor avoiders. In the good avoiders, the rate constant of DA disappearance was significantly higher in the central nucleus of the amygdala. The different turnover of catecholamines in the dorsal hippocampus and the amygdala in relation to passive avoidance performance suggests that individual differences in memory and/or learning may correlate with the catecholamine turnover of certain limbic structures.