Hormone-sensitive cAMP phosphodiesterase in liver and fat cells

Summary The enzymatic characteristics and the mode of hormone-dependent stimulation of cAMP phosphodiesterase are reviewed. The hormone-sensitive phosphodiesterase is a low Km enzyme, which has been found in liver and fat cells. The fat cell enzyme is mostly associated with the endoplasmic reticulum. The liver cell enzyme is also associated with certain subcellular structures.The hormone-sensitive phosphodiesterase appears to have catalytic and regulatory domains and is thought to be attached to subcellular structures at the regulatory portion of the enzyme. The catalytic domain of the fat cell enzyme can be obtained in a soluble form from the microsomal preparation by mild proteolysis or by dithiothreitol treatment at 0–4 °C. The catalytic domain of the liver enzyme can be solubilized by either hypotonic treatment or mild trypsin digestion. The catalytic domains solubilized from the basal and hormonally activated forms of the enzyme are apparently identical.The membrane-bound basal enzyme from adipocytes is activated in a concentrated salt solution without being solubilized. On the other hand, the plus-insulin activity is deactivated in a low salt solution or by a short dithiothreitol treatment at 37°, apparently without suffering any changes in the catalytic domain. In contrast, p-chloromercuriphenyl sulfonate seems to inactivate the enzyme by interacting with SH-groups in the catalytic domain. Although the liver enzyme is not similarly affected by salt concentrations, its catalytic activity is blocked by p-chloromercuribenzoate.The adipocyte enzyme can be solubilized with a mixture of Lubrol WX and Zwittergent 3–14. The apparent Stokes radius of the basal enzyme is approximately 87 A, while that of the hormone-stimulated enzyme is approximately 94 A.Apparently, the same species of phosphodiesterase is activated by both insulin and epinephrine in fat cells and by insulin and glucagon in liver, possibly being mediated by reactions involving phosphorylation. However, it is yet to be ascertained how phosphorylation is involved and how the apparent Stokes radius of the adipocyte enzyme is increased as a result of stimulation.     

Comparison of antiviral and antitumor activity of activated macrophages.

The antiviral and antitumor activity in vitro of normal, stimulated, vaccinia virus “immune”, and activated peritoneal macrophages were compared. Activated (pyran or corynebacteria induced) PEC exhibited both antitumor and antiviral activity. Stimulated (thioglyocollate) and vaccinia virus “immune” PEC inhibited virus growth but did not possess antitumor activity. Normal (unstimulated) PEC were relatively ineffective in either activity. The antiviral activity was nonspecific, being expressed against herpes simplex and EMC viruses in addition to vaccinia. Although a possible role for interferon was suggested by the lack of activity of mouse PEC on vaccinia virus growth in heterologous FLK cells, definitive proof was not obtained. The activity was most pronounced against multiple cycles of viral infection initiated at a low multiplicity of infection. Single cycle virus growth was not affected, suggesting that the major inhibition was on subsequent cycles of virus growth.     

Transmembrane ferricyanide reduction in tobacco callus cells

Transmembrane ferricyanide reduction in whole cells of normal and of transformed tobacco (Nicotiana tabacum) callus tissue was compared. It was found that low concentrations of indoleacetic acid (IAA, 0.1 μM), gibberellic acid (GA, 0.3 μM), and benzyl adenine (BA, 0.03 μM) stimulate external ferricyanide reduction in normal tobacco callus cells, but inhibit this reaction up to 67% in transformed cells when hormones are applied to cells 10 min prior to assay. Higher concentrations of these growth regulators (1 μM or greater) inhibit transmembrane ferricyanide reduction in both types of cells, with the exception of IAA, giving an initial stimulation of the rate (12%), followed by 24% inhibition after 2 min. The observed external ferricyanide reduction by whole tobacco callus cells may be explained on the basis of a transplasmalemma redox system, which may be associated with the iron metabolism of these cells.     

Calmodulin antagonists inhibit electron transport in photosystem II of spinach chloroplasts

Chlorpromazine, phenothiazine and trifluoperazine, known as calmodulin antagonists, inhibit electron transport in Photosystem II of spinach chloroplasts in concentrations from 20–500 μM. The inhibition site is located on the diphenyl carbazide to indophenol pathway in Tris-treated chloroplasts, indicating that water oxidation is not affected by these drugs. Ca²⁺ ions, bound to chloroplast membranes before the addition of calmodulin antagonists, can protect against inhibition up to 25% of the electron transport rate. In presence of A23187, the Ca²⁺-specific ionophore, Ca²⁺ ions provide less protection against inhibition by the 3 calmodulin antagonists used. A possible role of a calmodulin-like protein in spinach chloroplasts is postulated.     

Ca2+ and calmodulin antagonists inhibit the proton gradients associated with non-cyclic and cyclic photophosphorylation in spinach chloroplasts

The synthesis of benzylpenicillin (BP) after mixing phenyl-acetyl-glycine(PAG), 6-aminopenicillanic acid (6-APA) and free or immobilized penicillin amidase (E.C.3.5.1.11.) was studied as a function of pH and ionic strength. Before the final equilibrium was reached a kinetically controlled synthesis of BP was observed. Then a transient maximum concentration in BP much larger than the final equilibrium content was synthesized in the acyl-transfer process. The factors influencing this maximum have been analyzed. Increasing ionic strength markedly decreased the maximum in BP and the rate of deacylation of phenyl-acetyl-penicillin amidase by 6-APA. The change was largest when the enzyme was immobilized in a positively charged support, where at low ionic strength the concentration of 6-APA around the enzyme is larger than the bulk concentration due to the partitioning of charged solutes.     

Proteolytic modification of mouse liver catalase

When catalase was immunoprecipitated from different subfractions of mouse liver homogenates, the enzyme which was obtained from extracts of the large granular fraction exhibited a lower molecular weight than that from either the cytosol or purified peroxisomal fractions, as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis. This modification of the enzyme could be prevented by the addition of proteolytic inhibitors to extraction buffers; and consequently, unmodified catalase was able to be purified in the presence of 5 mM iodoacetamide. Electrophoretic comparison of the catalases against standards of known molecular sizes indicated that the unmodified enzyme had a subunit mass approximately 2,000 daltons larger than the modified enzyme. The significance of these proteolytic modifications has been discussed in relation to the involvements of catalase and peroxisome turnover.     

Analysis of the patterns of inheritance of splenomegaly and serum IgM levels in the Watut of Papua New Guinea

Hyperreactive malarious splenomegaly (HMS) reflects abnormal immune responses to malarial infection. The central question is whether HMS results from unusual patterns of malarial infection or from immune incompetence in the host. Family distributions of two features of the syndrome, splenomegaly and excessively high IgM levels, have been examined in a Papau New Guinea population in which HMS is exceptionally common. Segregation analysis of spleen grade shows that a major sex-linked gene controls hyperresponsiveness to malaria. This finding is supported by additional segregation analysis, which shows that an autosomal locus cannot account for a significant proportion of variation in spleen grade, and by path analysis, which rejects a model that assumes that parents contribute equally to the child's genotype. The sex-linked gene contributing to HMS was not mediated through sex linkage of a major gene for IgM concentrations, as shown by segregation analysis. It has yet to be determined whether this pattern of inheritance also applies to HMS occurring sporadically in other less severely affected populations. The applicability of these findings to the general variability in "normal" IgM responses to malaria also remains to be established.     

Cerebral Metabolic Effects of Monoamine Oxidase Inhibition in Normal and 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Acutely Treated Monkeys

The neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces dopaminergic cell death in the substantia nigra pars compacta (SNpc) and clinical parkinsonism in humans and experimental animals. Pretreatment with monoamine oxidase inhibitors prevents this cell death and associated parkinsonism by blocking the oxidation of MPTP to a toxic intermediate. The 2-deoxyglucose method was used to study the acute effects of MPTP in the monkey brain and the effects of monoamine oxidase inhibition on local cerebral glucose utilization in both normal and MPTP-treated monkeys. MPTP administration alone caused a major increase in glucose utilization in the SNpc and smaller increases in some subnuclei within the ventral tegmental area in which eventual dopaminergic cell loss also occurs. Pretreatment with pargyline abolished these metabolic increases, a finding suggesting both that the oxidized product of MPTP generates the metabolic increases and that the increased glucose consumption may contribute to cell toxicity. On the other hand, in most cortical, thalamic, striatal, brainstem, and cerebellar areas MPTP alone caused reductions in glucose utilization, and pargyline failed to prevent these effects. Pargyline alone depressed metabolism in the locus coeruleus and a few other monoaminergic structures.     

Cell Damage Associated with Changing the Medium of Mesencephalic Cultures in Serum-Free Medium Is Mediated via N-Methyl-D-Aspartate Receptors

Dopaminergic neurons from embryonic rat mesencephalon were grown in simple serum-free media. The cells develop over a period of several weeks in vitro, particularly between day 14 and day 23. Removing the culture medium and replacing it with fresh medium during this interval caused severe damage to the cultures; this damage is mediated by excitatory amino acids acting through glutamate receptors. Damage could be completely prevented by antagonists of the N-methyl-D-aspartate subtype of glutamate receptor. As expected, medium that contains glutamate (i.e., Ham's F-12 medium) caused damage; however, medium that contains no glutamate or aspartate (i.e., Dulbecco's modified Eagle medium) also caused severe damage, and most of the damage was dependent on the presence of glutamine in the medium. The presence of the antibiotics penicillin and streptomycin greatly enhanced damage caused by medium change.     

Right-side dominance for song control in the zebra finch.

Adult male zebra finches underwent unilateral denervation of the syrinx or unilateral lesion of the forebrain nucleus HVC known to be important for song control. Disruptive effects on song were greater after right-side than after left-side operations. After denervation of the right half of the syrinx, the fundamental frequencies of all syllables within a song converged on a value near 500 Hz, and nearly all syllables were altered in type. In contrast, the syllables produced after denervation of the left side of the syrinx largely maintained their preoperative frequencies, and fewer syllables changed in type. Unlike nerve sections, HVC lesions did not result in strikingly lateralized effects on syllable phonology; however, HVC lesions did affect the temporal patterning of a bird's song, whereas nerve sections did not, and changes in temporal patterning were more marked after right than after left HVC lesions. Right-side dominance for zebra finch song control is the reverse of that described in other songbird species with lateral asymmetry for vocal communication. We suggest that the need for a dominant side is more important than the side of dominance.