Diurnal variations in learning performance in chickens

Diurnal variations in the learning performance of young chicks were investigated using a visual discrimination task which requires birds to discrminate grains from a background of pebbles. Chicks accustomed to receiving fresh food daily in the morning were found to learn well during the day, in that they pecked almost exclusively at grains; but during the night they pecked indiscriminately at grains and pebbles. This occurred even though food was available ad libitum. Chicks accustomed to receiving fresh food daily in the evening learnt the task during the day, and also late at night. Thus the shape of the performance cycle depends in part on environmental factors. Other factors, such as activity, which may contribute to, or co-vary with, this variation in learning performance were investigated.     

Role of substance P in water homeostasis.

The effect of the naturally occurring peptide substance P on release of antidiuretic hormone (ADH) was studied in anesthetized dogs. Intravenous infusions of substance P in doses of 0.5, 5.0, and 50 ng/kg/min increased plasma concentrations of ADH in a dose-related fashion. At the two low doses, this increase occured in the absence of changes in urine volume, sodium excretion, free water clearance, and urinary cyclic AMP excretion. In addition, when substance P was administered in a concentration of 0.5 ng/kg/min, plasma levels of ADH were increased even though blood pressure did not change, suggesting that substance P may release antidiuretic hormone by a direct mechanism. Intrarenal infusions at a rate of 0.5 and 5 ng/kg/min caused dose-related decreases in free water clearance and significant increases in urinary cyclic AMP excretion. These data suggest that substabce P may play an important role in the regulation of water balance.     

The relation between the increase in reduced nicotinamide nucleotides and the initiation of DNA synthesis in sea urchin eggs

Previous work has suggested that the activation of the sea urchin egg at fertilization is the result of a transient increase in intracellular free calcium and an increase in intracellular pH. We have investigated the absence of nuclear activation in incompletely activated eggs and have found a correlation between nuclear activation and the levels of total reduced nicotinamide nucleotides (NAD[P]H). Eggs activated with ammonia show a similar correlation: besides its action as a weak base in raising intracelluar pH (which we conclude is insufficient to stimulate or maintain nuclear activation as judged by nuclear envelope breakdown or DNA synthesis), ammonia increases NAD(P)H. This increase is associated with the stimulation of 6-³H-thymidine incorporation into egg DNA. Removing ammonia decreases NAD(P)H, and tritiated thymidine incorporation ceases. We conclude that a critical level of NAD(P)H is essential to nuclear activation and that the increase of NAD(P)H at fertilization must be included with the increase in calcium and pH as a causal agent in development.     

Inhibition of the positive inotropic effect of anthopleurin-A (AP-A) by dantrolene

The effect of dantrolene on the positive inotropic effects (PIE) of three cardiotonic agents was assessed on rat and rabbit atria. Dantrolene (10^?5M) had no effect on contractile tension or on the PIE to isoproterenol (10^?10?10^?7M) or ouabain (10^?6?10^?4). The dose-response curve for the PIE of anthopleurin-A (AP-A) was shifted to the right in rat and rabbit atria by dantrolene (10^?4?10^?6M). The maximum effect and the concentration of AP-A required for it remained the same. The results suggest that the PIE of AP-A involves intracellular translocation of calcium, unlike those of isoproterenol and ouabain which require increased transmembrane calcium flux. This conclusion is supported by the observation that the exchange and distribution of the labile calcium involved in excitation-contraction coupling was unaffected by AP-A (10^?8M), by dantrolene (10^?6M) or by the combination. Therefore, AP-A may produce its cardiotonic effect by a action at an intracellular site, a mechanism unlike that of isoproterenol or ouabain.     

The Ca2+-induced membrane transition in mitochondria. II. Nature of the Ca2+ trigger site.

The permeability of isolated mitochondria which have undergone the Ca²⁺-induced transition can be modulated over a wide range simply by adjusting the concentration of free Ca²⁺ in the medium. The effect varies sigmoidally with respect to Ca²⁺ concentration, with an apparent K_m of 16 μm at pH 7.0. It is concluded that the trigger site (by “trigger site” we mean the site of binding of Ca²⁺ which, when Ca²⁺ is bound, will allow the transition in permeability to occur) is possibly also the site for high-affinity Ca²⁺ uptake. Added ADP, NADH and Mg²⁺ inhibit the Ca²⁺-induced permeability of mitochondria which have undergone the Ca²⁺-induced transition. Mg²⁺ and other ions, including H⁺, act like competitive inhibitors of the Ca²⁺ effect. In the presence of Ca²⁺, both neutral and charged molecules of molecular weight <1000 pass readily through the membrane. This response to Ca²⁺ is interpreted as a gating effect at the internal end of hydrophilic channels which span the inner membrane.     

The Ca2+-induced membrane transition in mitochondria. III. Transitional Ca2+ release.

The efflux of Ca²⁺ from mitochondria respiring at steady state, and much of uncoupler-induced Ca²⁺ efflux, is shown to be a consequence of the Ca²⁺-induced membrane transition (the Ca²⁺-induced transition is the Ca²⁺-dependent sudden increase in the nonspecific permeability of the mitochondrial inner membrane which occurs spontaneously when mitochondria are incubated under a variety of conditions (D. R. Hunter, R. A. Haworth, and J. H. Southard, 1976, J. Biol. Chem.251, 5069–5077)). Ca²⁺ release from mitochondria respiring at steady state is shown to be transitional by four criteria: (1) Ca²⁺ release is inhibited by Mg²⁺, ADP, and bovine serum albumin (BSA), all inhibitors of the transition; (2) release is selective for Ca²⁺ over Sr²⁺, a selectivity also found for the transition; (3) the time course of Ca²⁺ release is identical to the time course of the change in the mitochondrial population from the aggregated to the orthodox configuration; and (4) from kinetics, Ca²⁺ release from individual mitochondria is shown to occur suddenly, following a lag period during which no release occurs. Ca²⁺ release induced by uncoupler is shown to be mostly by a transitional mechanism, as judged by four criteria: (1) release of Ca²⁺ is ruthenium red-insensitive and is an order of magnitude faster than Sr²⁺ release which is ruthenium red-sensitive; (2) release of Ca²⁺ is strongly inhibited by keeping the mitochondrial NAD⁺ reduced; (3) the kinetics of Ca²⁺ release indicates a transitional release mechanism; and (4) uncoupler addition triggers the aggregated to orthodox configurational transition which, at higher levels of Ca²⁺ uptake, occurs in the whole mitochondrial population at a rate equal to the rate of Ca²⁺ release. Na²⁺-induced Ca²⁺ release was not accompanied by a configurational change; we therefore conclude that it is not mediated by the Ca²⁺-induced transition.     

Stimulation of prostaglandin E2 synthesis in chondrocytes by a factor derived from activated macrophages

The serum-free spent medium of lipopolysaccharide-activated rabbit peritoneal macrophages contains a proteinaceous factor that stimulates the synthesis of PGE₂ in rabbit articular chondrocytes. Synthesis of this factor by macrophages is inhibited by cycloheximide. Stimulation of PGE₂ in chondrocytes is detected after a four-hour exposure to the macrophage factor and is completely abolished by the addition of either cycloheximide or indomethacin to the chondrocyte cultures. The macrophage derived factor has an apparent molecular weight of 30,000, is heat stable and not inactivated upon reductive alkylation or on treatment with phenylglyoxal. Activity is partially destroyed upon treatment with acid (pH 2.0) and upon trypsin treatment.     

The Ca2+-induced membrane transition in mitochondria. I. The protective mechanisms.

Some physiological factors which control the rate of induction of the Ca²⁺-induced membrane transition (the term “Ca²⁺-induced membrane transition” is defined in the introduction) have been systematically investigated. To exclude the complicating factors of electron flow and energization, the transition was studied in mitochondria depleted of endogenous substrate, in the presence of uncoupler. In these mitochondria the transition could be induced by Ca²⁺, whether entry was mediated by ruthenium red-sensitive permeation or by A23187 facilitated diffusion. The rate of the transition was reduced fivefold by any agent which caused complete reduction of endogenous NAD. The rate of the transition was increased threefold by the exchange of endogenous ADP for phosphoenolpyruvate. A further increase was found on the addition of atractyloside, but bongkrekic acid caused inhibition. Addition of uncoupler to energized mitochondria when the endogenous NAD was already fully oxidized caused a stimulation of the transition. From these observations we conclude that mitochondria have a set of protective mechanisms (the term “protective mechanism” refers to the means by which these agents inhibit the Ca²⁺-induced transition; such a mechanism could be through allosteric interactions between the sites of binding of inhibitor and Ca²⁺; it would, however, be premature to conclude this on the basis of this paper) involving endogenous NADH, ADP, and energization which regulate the rate of the Ca²⁺-induced transition. ADP appears to work at two sites: one site which is internal, and another at the ADP/ATP translocase. In addition, we conclude that the transition requires neither electron flow nor energy, but rather the mere accessibility of some internal site to Ca²⁺. Finally, the key roles played by the protective agents in metabolism give the cell great potential flexibility in regulating the Ca²⁺-induced transition. This degree of control suggests that the transition has substantial physiological significance.     

O-Benzylated thioglucoses, intermediates for the synthesis of (1→6)- and (1→4)-linked oligosaccharides: S-benzylation, coupling, and thioglycoside cleavage

The use of the chloroacetyl group as a protecting group has been studied for a 2-methylglyco-[2′,1′:4,5]-2-oxazoline. The reaction of chloroacetyl chloride or chloroacetic anhydride with 2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-β-d-glucopyra-nose provided 2-acetamido-1,3,4-tri-O-acetyl-6-O-(chloroacetyl)-2-deoxy-β-d-glucopyranose which, on treatment with anhydrous ferric chloride in dichloromethane, produced the desired oxazoline. The glycosylating capability of the oxazoline has been investigated with aglycon hydroxides, to give the corresponding 2-acetamido-2-deoxy-β-d-glucopyranosides. The chloroacetyl group can be selectively removed by treatment with thiourea, and migration of O-acetyl groups was not observed under these conditions.     

Neuron differentiation and axon growth in the developing wing of Drosophila melanogaster

Sensory neurons in the wing of Drosophila originate locally from epithelial cells and send their axons toward the base of the wing in two major bundles, the L1 and L3 nerves. We have estimated the birth times of a number of identified wing sensory neurons using an X-irradiation technique and have followed the appearance of their somata and axons by means of an immunohistochemical stain. These cells become immunoreactive and begin axon growth in a sequence which mirrors the sequence of their birth times. The earliest ones are born before pupariation and begin axonogenesis within 1 to 2 hr after the onset of metamorphosis; the last are born and differentiate some 12 to 14 hr later. The L1 and L3 nerves are formed in sections, with specific neurons pioneering defined stretches of the pathways during the period between 0 and 4 hr after pupariation (AP), and finally joining together around 12 hr AP. By 16 hr AP the adult complement of neurons is present and the adult peripheral nerve pattern has been established. Pathway establishment appears to be specified by multiple cues. In places where neurons differentiate in close proximity to one another, random filopodial exploration followed by axon growth to a neighboring neuron soma might be the major factor leading to pathway construction. In other locations, filopodial contact between neighboring somata does not appear to occur, and axon pathways joining neural neighbors by the most direct route are not established. We propose that in these cases additional factors, including veins which are already present at the time of axonogenesis, influence the growth of axons through non-neural tissues.     

Absence of phosphatidylinositol phosphodiesterase in the head of a Drosophila visual mutant, norpA (no receptor potential A)

Phosphatidylinositol phosphodiesterase activity was found to be entirely absent in the head of the Drosophila visual mutant, norpA. It was also demonstrated that the enzyme activity was highly concentrated in the retinular cells of a normal head. Furthermore, the enzyme was revealed to be in a membrane bound form. In view of the present results and our previous work on the reduction of diacylglycerol kinase activity in the norpA mutant, phosphatidylinositol metabolism may play an important role in the generation of photoreceptor potentials.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. II. Inhibition by inhibitors of protein synthesis.

The effect of inhibitors of protein synthesis on the killing of tumor cells by in vitro activated macrophages was determined. Cytotoxicity was inhibited by concentrations of puromycin, pactamycin, and actinomycin D that almost completely inhibited protein synthesis by guinea pig macrophages, but not by concentrations of drug that inhibited protein synthesis by only ± 50%. Cytotoxicity was inhibited when the effector macrophages were pretreated with the metabolic inhibitors, but not when the drugs were added 30 to 60 min after the initiation of the reaction. Pretreatment with puromycin or pactamycin also markedly inhibited the binding of tumor cells by mediator activated macrophages. These results are consistent with the hypothesis that one possible mechanism by which inhibitors of protein synthesis inhibit macrophage mediated cytotoxicity is by inhibiting close contact between effector and target cells. The finding that pretreatment of activated macrophages with trypsin also inhibits tumor cell killing suggests that protein synthesis may be necessary to maintain an adequate number of “recognition structures” on the macrophage membrane, structures that mediate the initial contact between the activated macrophage and the target tumor.     

A lipid peroxide inhibits the enzyme in blood vessel microsomes that generates from prostaglandin endoperoxides the substance (prostaglandin X) which prevents platelet aggregation

Microsomal fractions from arterial walls of pigs and rabbits and fundus of rat stomach generate from prostaglandin endoperoxides (PGG₂ or H₂) an unstable substance, prostaglandin X (PGX) which is a potent inhibitor of platelet aggregation induced by several different substances.     

Cyclic AMP dependent regulation of mitosis in human lymphoid cells

Intracellular levels of cyclic AMP (cAMP), cAMP-dependent phosphodiesterase activity, and adenylate cyclase activity are examined in an established line of human lymphoid cells synchronized by either excess thymidine or by colcemid treatment. cAMP levels and adenylate cyclase activities during the two G periods are high when compared with the values in M. cAMP-dependent phosphodiesterase activity, which is low during early G 2, is shown to increase during G 2 and reach a maximum activity during M. Agents such as dibutyryl cAMP, 1-methyl-3-isobutyl xanthine, noradrenaline, and isopropyl noradrenaline, which increase the levels of intracellular cAMP were examined to determine their effects on mitosis and on DNA synthesis. In thymidine-synchronized cells the onset of mitosis is prevented by increasing or maintaining high levels of cAMP during G 2. The specificity of inhibition of DNA synthesis or mitosis by dibutyryl cAMP is a function of the time, during the cell cycle, when the analogue is added. The elevation of cAMP by methyl xanthine results in a more general inhibition of nucleic acid synthesis and mitosis. Although both catecholamine hormones inhibit mitosis, isopropylnoradrenaline also inhibits DNA synthesis while noradrenaline treatment does not result in such inhibition.     

An unexpected response of Torulopsis glabrata fusion products to x-irradiation

Intra-species fusion products of Saccharomyces cerevisiae, Saccharomyces unisporus and Torulopsis glabrata have been isolated following polyethylene glycol-induced fusion of protoplasts and selection for prototrophic colonies. Staining with lomofungin showed that all fusion products were uninucleate. Measurement of DNA content mostly gave values between haploid and diploid levels indicating that the majority of fusion products were aneuploid. Nevertheless fusion products of S. cerevisiae and S. unisporus were, as expected, more resistant to X-irradiation than their haploid parents. By contrast, the X-ray doze—response curve of all T. glabrata fusion products was indistinguishable from their progenitors despite the fact that mitotic segregants could be recovered amongst the survivors to X-rays. A possible explanation for the behaviour towards X-rays of T. glabrata fusion products is that this species lacks a DNA repair pathway involving recombination between homologous chromosomes. We conclude from this study that the shape of the X-ray dose—response curve should not be taken to indicate the ploidy of new yeast isolates without supporting data.     

Proteins foretelling head or abdomen development in the embryo of Smittia spec. (Chironomidae,Diptera)

The development of the segment pattern in Smittia embryos can be manipulated experimentally. Centrifugation during intravitelline cleavage leads to a mirror image duplication of most of the head in the absence of abdominal segments (“double cephalons”). Conversely, mirror image duplications of abdominal segments in the absence of head and thorax (“double abdomens”) can be generated by UV-irradiation of the anterior pole before blastoderm formation. By subsequent exposure to blue light, UV-irradiated embryos can be reprogrammed for normal development (photoreversal). We have characterized an “anterior indicator” protein (designated AI₁; M_r ? 35,000; IEP ? 4.9). Its synthesis was restricted to anterior fragments of embryos during a late blastoderm stage (Bl_VI). This protein was synthesized, however, in both anterior and posterior fragments of prospective double cephalons. Conversely, this protein was synthesized neither in anterior nor in posterior fragments of UV-induced double abdomens. Upon photoreversal, the protein was synthesized again in anterior fragments. Thus, synthesis of this protein in a given fragment always indicated development of head and thorax there. Likewise, we have characterized a “posterior indicator protein” (designated PI₁, M_r ? 50,000, IEP ? 5.5). Its synthesis during early blastoderm stages (Bl_I and Bl_II) was restricted to posterior fragments but not to pole cells in normal embryos. In UV-induced double abdomens, PI_I was synthesized in both anterior and posterior fragments at stage Bl_II. Photoreversal again led to restriction of PI_I synthesis to posterior fragments. Thus, the synthesis of PI_I in a given fragment at stage Bl_II always foreshadowed the formation of an abdomen several hours before this can be discerned morphologically. The synthesis of two other proteins (designated a₁ and p₁) was also restricted, during certain blastoderm stages, to anterior or posterior fragments, respectively. However, UV-irradiation or centrifugation had little or no effect on the synthesis of these proteins. Conversely, programming embryos for double abdomen development by UV-irradiation caused a set of reproducible, and mostly photoreversible, changes in the pattern of proteins synthesized in anterior embryonic fragments. However, the synthesis of most of the affected proteins was not region-specific in normal embryos.     

Regulation of the in vitro anamnestic antibody response by cyclic AMP. II. Antigen-dependent enhancement by exogenous prostaglandins of the E series.

The spleen cells from CFW/D mice injected with dimethylbenzanthracene-induced leukemia virus exhibited a progressive decline in the in vitro response to heterologous erythrocyte antigens in parallel with tumor growth. Cell transfer experiments revealed that this immunodepressed state may involve a B-cell defect rather than extrinsic factors in the cellular environment since: (i) nonresponsiveness could be transferred to irradiated non-tumor-bearing mice with spleen cells, and (ii) T cells from tumorbearing mice cooperated with normal bone marrow cells, but bone marrow from tumorbearing mice did not cooperate with normal T cells. In addition, T cells from the thymic tumor could cooperate with normal bone marrow cells upon transfer to irradiated recipients. TL 485-2 cells, a T-cell line derived from the tumor, could be specifically activated with SRBC thereby indicating that the virus transformed T cells were immunocompetent. Suppressor cells, which appeared in the spleen concomitant with immunodepression and tumor development, may directly raise B-cell thresholds for T-dependent triggering signals since the antibody response of spleen cells from tumor-bearing mice could be restored by adding agents such as LPS, 2 mercaptoethanol, or T cells exogenously preactivated in normal animals. The suppressor cell could be enriched by adherence to plastic and was removed by treatment with carbonyl iron. In addition, it was unlikely that the suppressor cell was a virus-infected cell since transformed, virus-infected cells from the tumor or TL 485-2 cells were not suppressive when added to spleen cells in vitro but rather resulted in a marked, polyclonal enhancement of the PFC response. The interaction of TL 485-2 cells and normal spleen cells resulted in the release of a stimulatory factor which increased DNA synthesis in resting cells as well as increasing PFC. The role of these enhancing factors and suppressor cells in controlling tumor growth remains to be elucidated.     

Difference in androgen-dependent change of non-histone proteins between dorsolateral and ventral prostates of rats

A novel species of non-histone protein having a molecular weight of approximately 20,000 (20K), abundantly localized in the dorsolateral prostate, was found to be decreased in the content by castration and to be restored by replacement of androgen, in addition to non-histone proteins of molecular weights ≥ 34,000. The content of $\text{20K}\text{DNA}$ was more rapidly decreased by castration, but more slowly restored by replacement of androgen, with the dorsolateral prostate than the ventral prostate. Of other nuclear proteins, non-histone proteins of molecular weights ≥ 90,000 in the dorsolateral prostate were more susceptible to the decrease by castration, whereas those of all kinds of histones were hardly dependent on the androgen level.