Localization of protein phosphokinase activities in the nucleolus distinct from extra-nucleolar regions in rat ventral prostate nuclei

Rat ventral prostate nucleoli contain protein phosphokinase(s) which have distinctly different characteristics from protein phosphokinase(s) localized in the extra-nucleolar regions of the nucleus. The differences pertain to pH vs activity profiles and activation by divalent cations, utilizing dephospho-phosvitin as substrate. Lysine-rich and arginine-rich F3 histones are also phosphorylated by nucleolar protein phosphokinase(s). Phosphorylation of histones by either nucleolar or extra-nucleolar fractions was not stimulated by cAMP, whereas that of phosvitin was slightly inhibited.     

Wholemount histofluorescence of catecholamine-containing neurones in a hemipteran brain

Whole brains from Rhodnius prolixus treated with glyoxylic acid display catecholamine histofluorescence in eleven bilaterally distributed clusters of neurones, and in three circumscribed neuropile regions. This simple method offers resolution of some features not detected in studies on sectioned insect tissue; cephalic neurosecretory cells in chronically starved animals are histofluorescent and diverse catecholamine-handling fibres converge within the brain of this insect.     

Effect of dietary proteins and carbohydrates on urinary and sympathoadrenal catecholamines

We previously observed that administration of tyrosine to rats or humans elevated urinary dopamine, norepinephrine and epinephrine levels. The present studies examine the effects on these urinary catecholamines of varying the ratio of protein to carbohydrate in the diets.Rats consumed diets containing 0, 18 or 40% protein (76, 58 and 36% carbohydrate respectively) for 8 days. The stress of consuming the protein-free food was associated with a 16% weight reduction, and with significantly lower serum, heart and brain tyrosine levels than those noted in rats eating the 18 or 40% protein diets. Absence of protein from the diet also decreased urinary levels of dopamine and DOPA but increased urinary norepinephrine and epinephrine, probably by increasing sympathoadrenal discharge; it also increased the excretion of DOPA in animals pretreated with carbidopa, a DOPA decarboxylase inhibitor. Carbidopa administration decreased urinary dopamine, norepinephrine and epinephrine as expected; however, among carbidopa-treated rats urinary norepinephrine and epinephrine concentrations were highest for animals consuming the protein-free diet, again suggesting enhanced release of stored catecholamines from sympathoadrenal cells. The changes in urinary catecholamines observed in animals eating the protein-free diet were similar to those seen in rats fasted for 5 days: dopamine levels fell sharply while norepinephrine and epinephrine increased.These data indicate that the effects of varying dietary protein and carbohydrate contents on dopamine secretion from peripheral structures differ from its effects on structures secreting the other two catecholamines. Protein consumption increases dopamine synthesis and release probably by making more of its precursor, tyrosine, available to peripheral dopamine-producing cells; it decreases urinary norepinephrine and epinephrine compared with that seen in protein-deprived animals, probably by diminishing the firing of sympathetic neurons and adrenal chromaffin cells.     

Polyamine-like effects of cobalt(III) hexaammine on various cyclic nucleotide-independent protein phosphokinase reactions

The chemically inert trivalent ion cobalt(III) hexaammine, Co³⁺(NH₃)₆, was found to exert polyamine-like effects in enhancing certain cyclic nucleotide-independent protein kinase reactions catalyzed by nuclear enzyme preparations from rat ventral prostate or liver. At 1 mM, Co³⁺(NH₃)₆ stimulated chromatin- and also non-histone-protein-associated kinase activities with partially dephosphorylated phosvitin as substrate by 38% and 72% respectively, whereas chromatin-associated kinase-catalyzed phosphorylation of lysine-rich histones was not affected under the same conditions. ³²P incorporation (from γ-³²P-ATP) into endogenous protein substrates of chromatin or non-histone protein fractions catalyzed by their erdogencus kinase activity was increased by 47% and 153%, respectively. These effects of Co³⁺(NH₃)₆ were similar to those produced by 1mM spermine. Autoradiographic analysis of endogenous ³²P-labelled nonhistone proteins revealed similar enhancements of the phosphorylation of several of the same proteins, induced by 1mM spermine or 1 mM Co³⁺(NH₃)₆ or 2mM spermidine. The stimulatory actions of polyamines or Co³⁺(NH₃)₆ were not mimicked by raising the ionic strength by addition of comparable concentrations of NaCl. The effects of 1 mM spermine and of 1 mM Co³⁺(NH₃)₆ tested separately were not additive. Phosphorylation of lysine-rich histones by beef heart cyclic AMP-dependent protein kinase was not affected by polyamines or Co³⁺(NH₃)₆ Various findings hint that the enhancement of cyclic nucleotide-independent kinase-catalyzed phosphorylation of certain protein substrates by spermidine, spermine and Co³⁺(NH₃)₆ is primarily due to interaction of these cations with appropriate protein substrates affecting their conformational status. Further, these effects of polyamines may be a reflection of their cationic charge properties rather than being dependent on any particular conformations assumed by the polyamines.     

Ouabain binding sites and the (Na+,K+)-ATPase of brain microsomal membranes

Beef brain microsomes bound approximately 180–220 pmoles of [³H]ouabain per mg of protein in the presence of either MgCl₂ and inorganic phosphate or ATP, MgCl₂ and NaCl. The ouabain-binding capacity and the ouabain-membrane complex were more stable than the (Na⁺,K⁺)-ATPase activity to treatment with agents known to affect the membrane integrity, such as, NaClO₄, sodium dodecyl sulfate, $\text{p-}\text{chloromercuribenzoate}$, urea. ultrasonication, heating, pH and phospholinase C.The presence of binding sites that were normally inaccessible to ouabain in brain microsomes was demonstrated. These sites appeared after disruption of microsomes with 2 M NaClO₄ as evidenced by increased binding of [³H]ouabain. These sites may be buried during the subcellular fractionation procedure and could be accessible in the intact cell.     

Keratan sulfate-like glycosaminoglycan in the cerebral cortex of the brain and its variation with age

A keratan sulfate-like substance was found in the cerebral cortex proteoglycans of rats. This substance tripled during the rapid growth phase from 1 to 3 monts of age and then decreased steadily to a negligible quantity in the senescent rat, aged 25 months. In contrast, the uronic acid-containing glycosaminoglycans remained constant during the life span studied.The marked decrease observed in the proportion of keratan sulfate-like moiety to the other glycosaminoglycans in the cerebral cortex of the senescent rat (aged 25 months) compared with the young adult rat (aged 3 months) may explain the reduced extracellular volume observed by others in the senescent brain.     

Breakdown of brain tubulin by cerebral cathepsin D

The breakdown of cytoplasmic tubulin from brain (purified by ammonium sulfate fractionation and DEAE cellulose chromatography) by cathepsin D from brain (purified by ammonium sulfate fractionation and pepstatin Sepharose chromatography) was studied; changes in the intensity of tubulin gel bands were determined. The pH optimum of hemoglobin breakdown by cathepsin D was 3.2; the pH optimum for tubulin breakdown was 5.8; at pH 5.8 there was no significant hemoglobin breakdown by the enzyme. Tubulin breakdown had an apparent K_m of 1.8 × 10⁻⁵ M and a V_max of 0.56 μg tubulin (μg enzyme per min). The rate of breakdown was heterogeneous and studied on length of incubation; the major portion of tubulin was rapidly broken down and a smaller portion was more stable. The rate under our experimental conditions was 18%/h in the 1–4 h period and 2%/h after 4 h. This was not due to enzyme instability: after 4 h of inhibition freshly added tubulin was rapidly broken down, whereas freshly added enzyme did not increase the rate of breakdown. Thus breakdown heterogeneity was due to substrate (tubulin) heterogeneity. Pepstatin inhibited cathepsin D breakdown of tubulin at acid pH; at pH 7.6 it had no effect. Leupeptin was not inhibitory. We calculated that the cathepsin D content in brain, if fully active, could break down cytoplasmic tubulin with a half-life of 24 h, but it is likely that under in vivo conditions enzyme activity is greatly modified.     

Effects of angiotensin II and dibutyryl cyclic adenosine monophosphate on phosphatidylinositol metabolism, 45Ca2+ fluxes, and aldosterone synthesis in bovine adrenal glomerulosa cells

Angiotensin II (AII) and N6,O2'-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP) both stimulated aldosterone synthesis in bovine adrenal glomerulosa cells. AII altered 45Ca2+ fluxes and increased 32PO4 incorporation into phosphatidylinositol in these cells, whereas dibutyryl cyclic AMP did not affect either process. Neither AII nor dibutyryl cyclic AMP increased the mass of phosphatidylinositol. Both agents are known to stimulate pregnenolone synthesis. Thus, although dibutyryl cyclic AMP and AII may increase aldosterone synthesis at a common site (pregnenolone synthesis), they do so by different mechanisms. AII stimulation of phosphatidylinositol labeling by 32PO4 (the "PI effect") was blocked when cells were incubated in a medium containing both EGTA and the calcium antagonist, 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxy-benzoate hydrochloride (TMB-8), suggesting a calcium requirement for the PI effect.     

Stimulation of liver glycogen particle synthase D phosphatase activity by caffeine, AMP, and glucose 6-phosphate

Caffeine stimulates synthase and phosphorylase phosphatase activities by independent mechanisms. Both effects of caffeine are concentration-dependent with different apparent A_0.5 for each reaction. Stimulation of the synthase phosphatase reaction was independent of the initial phosphorylase a concentration, was immediate, and did not follow in sequence the depletion of phosphorylase a. Glucose 6-phosphate also was stimulatory to the synthase phosphatase reaction (A_0.5 = 0.14 mM) with little effect on phosphorylase phosphatase. In combination glucose 6-phosphate and caffeine effects were additive suggesting the existence of separate binding sites. The synthase phosphatase reaction also was stimulated by AMP (binding affinity 2.3 mm) but with no effect on phosphorylase phosphatase activity. Together caffeine and AMP effects were not additive suggesting that they share a common binding site or closely interrelated sites. The location of the AMP and caffeine site(s) has not yet been determined.     

Desensitization of the D-2 dopamine receptor and the β2-adrenoceptor in the intermediate lobe of the rat pituitary gland

A D-2 dopamine receptor and a β₂-adrenoceptor occur in the intermediate lobe of the rat pituitary gland (IL). Exposure of intact IL tissue to a D-2 agonist diminished the ability of dopaminergic agonists [but not 5′-guanylyl imidodiphosphate (Gpp(NH)p)] to inhibit adenylate cyclase activity. Conversely, exposure of intact IL tissue to a β-adrenergic agonist diminished the ability of a β-adrenergic agonist (but not forskolin) to stimulate adenylate cyclase activity. Treatment of ovariectomized rats with 17β-estradiol desensitizes the β₂-adrenoceptor but not the D-2 receptor. Desensitization of the IL catecholamine receptors is discussed within the framework of a previously published “working model” of these receptors.     

Effect of sanguinarine,a benzophenanthridine alkaloid,on frog skin potential difference and short circuit current

Sanguinarine, a benzophenanthridine alkaloid, causes an initial stimulation of frog skin short circuit current $\text{I}{}_{\mathrm{<mtext>sc</mtext>}}$ when present in the mucosal bathing medium at 10^?4 M. The stimulation is accompanied by an increase in spontaneous potential difference (PD) and increase in D.C. resistance. No effects are seen with sanguinarine in the serosal bathing medium. The initial stimulation is followed by a decrease in $\text{I}{}_{\mathrm{<mtext>se</mtext>}}$ and PD, but a continued increase in resistance. In skins whose initial spontaneous PD is high, no initial stimulation in ${}_{\mathrm{<mtext>se</mtext>}}$ and PD is seen; however, clamping these skins to a lower potential does not alter their initial inhibitory response to sanguinarine. Likewise, clamping the lower potential skins to higher potential does not alter their initial stimulatory response. Sanguinarine seems to be acting on the permeability barriers at the outer surface of the frog skin.     

Computer simulation of the P31 NMR equations governing the creatine kinase reaction

A method for solving the Bloch equations that govern magnetization transfer NMR experiments is presented. It requires the numerical evaluation of a matrix exponential and lends itself to computer simulation. It is a simple, versatile method for evaluating P31-NMR magnetization transfer experiments designed to measure biochemical exchange rates. We apply the method to the saturation and inversion transfer experiments and find that the "initial slope" method of determining flux is subject to at least two interpretations. This verifies the accepted concept that biochemical models representing compartmentation and competing reaction hypotheses cannot be reliably distinguished by simply selecting values for NMR and biochemical parameters that give a "best fit" to experimental data. However, our results do indicate that a controlled manipulation of biochemical exchange rate may distinguish between these two models.     

Biogenic magnetite as a basis for magnetic field detection in animals

Bacteria, sharks, honey bees, and homing pigeons as well as other organisms seem to detect the direction of the earth's magnetic field. Indirect but reproducible evidence suggests that the bees and birds can also respond to very minute changes in its intensity. The mechanisms behind this sensitivity are not known. Naturally magnetic, biologically precipitated magnetite (Fe₃O₄) has been found in chitons, magnetotactic bacteria, honey bees, homing pigeons, and dolphins. Its mineralization in localized areas may be associated with the ability of these animals to respond to the direction and intensity of the earth's magnetic field. The presence of large numbers (～10⁸) of superparamagnetic magnetite crystals in honey bees and similar numbers of single-domain magnetite grains in pigeons suggests that there may be at least two basic types of ferrimagnetic magnetoreceptive organelles. Theoretical calculations show that ferrimagnetic organs using either type of grain when integrated by the nervous system are capable of accounting for even the most extreme magnetic field sensitivities reported. Indirect evidence suggests that organic magnetite may be a common biological component, and may account for the results of numerous high field and electromagnetic experiments on animals.     

Natural cell-mediated cytotoxicity in mice treated with total lymphoid irradiation (TLI)

Fractionated total lymphoid irradiation (TLI) of adult (BALB/c × C57BL/6)F₁ mice resulted in transiently augmented natural killer (NK) and natural cytotoxic (NC) cell activities. Thus, 1 day after completion of TLI, NK and NC activities in the spleens of treated mice were lower than controls but values increased and reached a maximum level of 23- to 190-fold above control at 6 days after irradiation, returning to normal levels 9 days later. Cytotoxicity was enhanced after removal of the plastic adherent population. No cytotoxicity was observed against P 815 target cells, which are sensitive to activated macrophages but not to NK. The significance of this modulation of natural cell-mediated cytotoxicity following TLI is discussed.     

Female secondary sexual coloration and sex recognition in the keeled earless lizard, Holbrookia propinqua

Relationships between male social behaviour and female reproductive coloration in the iguanid lizard Holbrookia propinqua were examined by introduction of tethered non-resident lizards into the territories of adult males. Introduced lizards were plainly coloured females, females having bright reproductive coloration, adult males, plain females painted to resemble brightly coloured females, bright females painted to mimic plain females, and males painted with the bright yellow and orange secondary sexual coloration of females. Resident males courted all unpainted females, despite being aggressively rejected by the brightly coloured ones. They also courted all but two females in each of the painted groups. All unaltered non-resident males were challenged, attacked or subjected to other aggressive behaviour by residents, but all non-resident males painted to resemble bright females were courted. This differential treatment is highly significant. It clearly demonstrates that the bright female pigmentation functions in sex recognition, identifying a female to males. Other stimuli, especially pheromonal and behavioural cues, may contribute to sex recognition. Because sex recognition alone seems insufficient to account for the evolution of bright female coloration in H. propinqua and several other iguanids, several proposed additional functions are discussed.     

Mechanism of action of agents which uncouple oxidative phosphorylation: Direct correlation between protoncarrying and respiratory-releasing properties using rat liver mitochondria

The proton-carrying properties of uncoupling agents were investigated by measuring passive mitochondrial swelling under conditions where electrogenic proton transport was rate limiting. The ability of uncoupling agents to transport protons into mitochondria, measured in this way, was compared with respiratory stimulation. The results show that with the single exception of arsenate, all agents tested which uncouple oxidative phosphorylation demonstrate a very close correlation between release of respiration and proton transport. These findings are in support of Mitchell's original proposal that uncoupling agents act by promoting electrogenic hydrogen ion transport across the mitochondrial inner membrane.     

Specific protein-binding reactions monitored with ligand-ATP conjugates and firefly luciferase

A new method was developed to monitor specific protein binding reactions with an ATP-labeled ligand and firefly luciferase. The ligand, 2,4-dinitrobenzene, was covalently coupled to four ATP derivatives and three of these conjugates were measured quantitatively at nanomolar levels with firefly luciferase. Incubation of the conjugates with antibody to the 2,4-dinitrophenyl residue diminished the peak light intensities produced in the bioluminescent assay, whereas incubation with immunoglobulin from a nonimmunized rabbit did not affect light production. Therefore, the antibody-bound ligand-ATP conjugates were inactive in the bioluminescent assay and levels of unbound conjugate could be measured in the presence of the bound form. The firefly luciferase was used to monitor competitive binding reactions between the antibody, the conjugates, and N(2,4-dinitrophenyl)-β-alanine.