Regulation of diamine oxidase expression by β2-adrenoceptors in normal and hypertrophic rat kidney

The administration of preferential adrenergic receptor antagonists to uninephrectomized rats revealed the $\text{β}{}_2\text{-}\text{adrenergic}$ mediation in diamine oxidase activity increase that occurs in the remaining kidney undergoing compensatory hypertrophy. In fact, $\text{β}{}_1\text{,β}{}_2\text{-}$ or $\text{β}{}_2\text{-}$, but not $\text{α}{}_1\text{-}$, $\text{α}{}_2\text{-}$, or $\text{β}{}_1\text{-}\text{receptor-blocking}$ this enzyme enhancement. Further studies with adrenoceptor agonists, such as epinephrine ($\text{α}{}_1$, $\text{α}{}_2$, $\text{β}{}_1$, $\text{β}{}_2$), isoproterenol ($\text{β}{}_1$, $\text{β}{}_2$) or terbutaline ($\text{β}{}_2$) showed that also in normal rat kidney diamine oxidase activity is under the control of $\text{catecholamine}\text{-β}{}_2\text{-}\text{receptors}$ through a mechanism that involves new synthesis of mRNA and protein. Theophylline, an inhibitor of phosphodiesterase, or forskolin, an activator of adenyl cyclase, increased diamine oxidase activity as does epinephrine or nephrectomy. Thus, catecholamine-triggered $\text{β}{}_2\text{-}\text{receptors}$ coupled to adenyl cyclase are involved in the regulation of diamine oxidase activity in normal and hypertrophic rat kidney.     

In vitro reaction of radioactive 7beta, 8alpha, --dihydroxy--9alpha, 10alpha-epoxy--7,8,9,10--tetrahydrobenzo (A) pyrene and 7beta,8alpha--dihydroxy--9beta, 10beta--epoxy--7,8,9,10--tetrahydrobenzo (A) pyrene with DNA.

The $\text{in vitro}$ reaction of bacteriophage T7-DNA with the radioactive diastereomeric benzo(a)pyrene-diol-epoxides, (±) [${}^3\text{H}{}_9$, ${}^3\text{H}{}_{10}$]-7β,8α-dihydroxy-9α,10β-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene, and (±) [${}^3\text{H}{}_9$, ${}^3\text{H}{}_{10}$]-7β,8α-dihydroxy-9β,19β-epoxy-7,8,9,10-tetrahydrobenzo(1)pyrene, was investigated. Chromatographic analysis of digests of the DNA allowed the distinction of characteristic deoxynucleoside adduct peaks for the two benzo(a)pyrene-diol-epoxides. Our results, together with data from the literature, allow the identification of these adducts as mostly N₂-(10-7β,8α,9α-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine and N₂-(10-7β,8α,9β-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyreney1)deoxyguanosine, respectively. DNA-benzo(a)pyrene adducts with the same chromatographic properties were formed in mouse embryo fibroblasts upon treatment with benzo(a)pyrene.     

The polymorphic phase behaviour and miscibility properties of synthetic phosphatidylethanolamines

(1) The polymorphic phase behaviour of aqueous dispersions of various synthetic phosphatidylethanolamines, both singly and in mixtures, has been investigated by ³¹P-NMR. (2) $\text{14:0}\text{14:0}$ PE remains in the lamellar phase up to 90°C. $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibits a lamellar to hexagonal (H_II) transition between 60°C and 63°C. For $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE, the lamellar to hexagonal (H_II) transition occurs between 7 and 12°C, whereas for $\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}$ PE, the hexagonal (H_II) phase is the preferred structure above ?15°C. (3) Mixtures of $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibit near-ideal miscibility behaviour. For mixtures of $\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}\text{18:1}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{14:0}\text{14:0}$ PE there is evidence of fluid-solid immiscibility at temperatures below the gel-liquid crystalline transition temperature of the $\text{14:0}\text{14:0}$ PE component. Mixtures of $\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}\text{18:2}{}_{\mathrm{<mtext>c</mtext>}}$ PE and $\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}\text{18:1}{}_{\mathrm{<mtext>t</mtext>}}$ PE exhibit complex phase behaviour involving limited fluid-solid immiscibility at low temperatures and formation of a phase allowing isotropic motional averaging at higher temperatures. (4) ³¹P-NMR provides a graphic method for investigating the miscibility properties of mixed PE systems.     

The effect of prostaglandins E1 and E2 on the human erythrocyte as monitored by spin labels

The effects of the prostaglandins PGE₁ and PGE₂ on the deformability of the human erythrocyte were studied using spin-labeled erythrocytes. Two magnetic resonance parameters were measured: (1) The orientation relaxation time, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$, for the erythrocyte, and (2) the order parameter, S, for a fatty acid spin label bound to the membrane. Prostaglandins PGE₁ and PGE₂ exhibited opposite effects on both $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S. PGE₂ made the cell less deformable (increases of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S) and PGE₁ made the erythrocyte more deformable (decrease of $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ and S).     

Processing of synthetic somatostatin-28 and a related endogenous rat hypothalamic somatostatin-like molecule by hypothalamic enzymes

Synthetic somatostatin-28 (S-28) as well as a related endogenous rat hypothalamic somatostatin-like compound (3K SLI) were incubated with hypothalamic extracts from which endogenous somatostatin-like immunoreactivity (SLI) had been removed by immunoabsorption. The reaction products were analyzed by gel chromatography, HPLC as well as two different radioimmunoassays for tetradecapeptide somatostatin (S-14) in which S-28 crossreacted either 100% (RIA R149) or < 0.001% (RIA S39). The results indicate that incubation of S-28 with SLI free hypothalamic extracts results in a rapid decrease of total immunoreactivity measured with RIA R149 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 14}\text{min}$). By contrast, with RIA S39 a rise from zero to a peak value at 8 min was measured suggesting the formation of S-14. This was confirmed by subsequent analysis by gel chromatography and HPLC. Using endogenous 3K SLI a decrease of total R149-immunoreactivity with a similar time course ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 17}\text{min}$) was observed simultaneously with the emergence of material that corresponded to S-14. This converting activity seems to be specific for SLI-containing tissues since similar rates of conversion were observed with extracts from cerebral cortex and cerebellum but not with lung and liver extracts.It is concluded that (1) S-28 is converted to S-14 by hypothalamic enzymes; (2) the processing of 3K SLI is similar, suggesting the two molecules are closely related, if not identical, and (3) the regulation of S-28 to S-14 conversion could represent an important mechanism for controlling the functional activity of somatostatinergic cells.     

Characterization of the β-adrenergic receptors of hamster white fat cells: Evidence against an important role for the α2-receptor subtype in the adrenergic control of lipolysis

The binding characteristics of the β-adrenergic antagonist, [³H]dihydroalprenolol, to hamster white adipocyte membranes were studied. This binding occurred at two classes of sites, one having high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6±1.3}\text{nM}$) but low capacity ($\text{32±17}\text{fmol/mg}$ membrane protein) and one having low affinity but high binding capacity. While the binding at the high-affinity sites was competitively and stereoselectively displaced by both β-antagonists and β-agonists, competition at the low-affinity sites occurred only with β-antagonists and was non-stereoselective. Thus, the β-agonist (?)-isoproterenol was further used to define nonspecific binding. Under these conditions, saturation studies showed a single class of high-affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1.6±0.5}\text{nM}$) binding sites with a binding capacity of $\text{53 ± 13}\text{fmol/mg}$ membrane protein (corresponding to $\text{4000 ± 980}$ sites per cell), and independent kinetic analysis provided a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ value of 1.9 nM. Competition experiments showed that these binding sites had the characteristics of a $\text{β}{}_1\text{-}\text{receptor}$ subtype, yielding $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ values in good agreement with the $\text{K}{}_{\mathrm{<mtext>act</mtext>}}$ and the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values found for agonist-stimulation and for antagonist-inhibition of adenylate cyclase in membranes and of cyclic AMP accumulation and lipolysis in intact cells. Furthermore, the ability of β-agonists to compete with this binding was severely depressed by p[NH]ppG. These results thus support the contention that the specific [³H]dihydroalprenolol binding sites defined as the binding displaceable by (?)-isoproterenol represent the physiologically relevant β-adrenergic receptors of hamster white adipocytes. Finally, studies of the lipolytic response of these cells to (?)-norepinephrine showed that the inhibitory effect of the $\text{α}{}_2\text{-}\text{component}$ of this catecholamine was apparent only when the effects of endogenous adenosine were suppressed, a result which argues against an important regulatory role for the $\text{α}{}_2\text{-}\text{receptors}$ in the adrenergic control of lipolysis in hamster white adipocytes.     

Lifetime of microsomal cytochrome P-450 and steroidogenic enzymes in rat testis as influenced by human chorionic gonadotrophin

The lifetime of different microsomal steroidogenic enzymes and the cytochrome components of the NADPH-cytochrome P-450 pathway have been determined in rat testis by measuring their decrease logarithmically after hypophysectomy. Although both cytochrome P-450 and 17α-hydroxylase show biphasic decay curves, the first decay curve contains 89–94% of the cytochrome P-450 and 17α-hydroxylase levels. Steroidogenic enzymes which are located mainly in the leydig cells, decay much faster than microsomal protein, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 12}$ days, which represents mainly decay of tubular protein. The similarity between the major half-life of cytochrome P-450, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 3.3}$ days, 17α-hydroxylase, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 2.3}$ days and the C₁₇–C₂₀ lyase, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 3.4}$ days and the uniformity of their response to human chorionic gonadotrophin (HCG) provides additional evidence that these two steroidogenic enzymes require cytochrome P-450. Both the 17α-hydroxylase and the C₁₇–C₂₀ lyase were shown to have a constant activity per nmole of cytochrome P-450 during a sixfold change in the level of cytochrome P-450 brought about by HCG treatment of rats with intact pituitaries. The decay of 17β-hydroxysteroid dehydrogenase, $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4.5}$ days, was slower than P-450 dependent enzymes. Rats with intact pituitaries are not under maximal stimulation by endogenous LH because addition of HCG increases the levels of microsomal and mitochondrial cytochrome P-450 220 and 1620%, respectively. The rates of synthesis during the increase from one cytochrome P-450 level to another was calculated at $\text{0.118}\text{2}$ testes/day for microsomal cytochrome P-450 and 0.10 nmoles/2 testes/day for mitochondrial cytochrome P-450. Treatment of hypophysectomized rats with HCG results in large increases of cytochrome P-450, 17α-hydroxylase, C₁₇–C₂₀ lyase and 5α-reductase, but not cytochrome b₅, microsomal protein, 7α-hydroxylase, or the 17β-hydroxysteroid dehydrogenase. While it is clear that the two cytochrome P-450 dependent hydroxylases involved in steroidogenesis and the 5α-reductase are under the control of gonadotrophin, it is not clear how 17β-hydroxysteroid dehydrogenase levels are maintained or in what manner the 5α-reductase level is controlled in mature animals.     

Cytoplasmic DNA: Differentiation by reassociation kinetics

Rat liver nuclear and cytoplasmic DNA samples were denatured and the kinetics of their reassociation was measured. About 85% of the soluble cytoplasmic (mitochondrial) DNA reannealed rapidly with a $\text{C}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.03}$ while 65% of the particulate (microsomal) DNA reassociated with a $\text{C}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.14}$ Both nucleic acids were clearly differentiated from nuclear DNA in their reassociation kinetics. The results indicate that both mitochondrial and microsomal DNA consist mainly of single components or closely related families with repetitive sequences.     

Magnetic field affects the fluorescence yield in reaction center preparations from Rhodopseudomonas sphaeroides R-26

Purified photochemical reaction centers from Rhodopseudomonas sphaeroides R-26 were reduced with Na₂S₂O₄ so as to block their photochemical electron-transfer reactions. The magnetic field induced an increase in the emission yield. Our results support the hypothesis that under these conditions, charge recombination in the singlet radical pair composed of the oxidized primary donor and reduced primary acceptor predominantly generates the excited singlet state of the reaction center bacteriochlorophyll.The maximum relative fluorescence change and the value of the magnetic field at which half-saturation of the effect is achieved ($\text{B}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) at room temperature are 5.5% and 75 G, respectively. For the whole cells of Rps. sphaeroides R-26 these parameters are 1.2% and 120 G.The relative fluorescence change at 600 G, $\text{ΔF}\text{F(600)}$, and $\text{B}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ are studied as functions of temperature. The temperature dependencies of $\text{ΔF}\text{F(600)}$ for reaction centers and whole cells of Rps. sphaeroides R-26 are qualitatively the same, with the maximum effect (8% for reaction centers) occurring at 230 K. However, the $\text{B}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ curves for the two preparations are different.     

Synthesis and characterization of a DNA probe complementary to rat liver 28S ribosomal RNA.

Conditions for the production of a complementary DNA sequence for use in studies of ribosomal RNA are described. $\text{E}$. $\text{coli}$ DNA polymerase I is used to transcribe highly purified 28S ribosomal RNA from rat liver. The reaction is sensitive to the tertiary structure of the rRNA template-primer. The complementary DNA hybridizes to its rRNA template with a $\text{R}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.02. The hybrid formed between 28S ribosomal RNA and complementary DNA has a T_m of 73°C. The probe reacts with total rat nuclear RNA with a $\text{R}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.0.     

Characterization and application of a radioimmunoassay for beta-endorphin using an antiserum with negligible cross-reactivity against beta-lipotropin

High avidity antisera against β-endorphin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}\text{)}$ were obtained in two of five rabbits immunized with unconjugated synthetic human $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. One of these antisera (K-7762) cross-reacted 1.5% on a molar basis with β-lipotropin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}\text{)}$ and did not recognize leucine-enkephalin in a concentration as high as 0.2 mmol/l. The cross-reaction with methionine-enkephalin $\text{(β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}\text{61–65)}$ was 9%, while that with α-endorphin ($\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}$ 61–76) was 69%. This implied that the specific recognition site was in the amino-terminal region of $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. Although this sequence is present in $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{LPH}$ it was poorly recognized by the antiserum, suggesting that the free amino-terminal is essential. This interpretation was supported by the finding that $\text{α-N-}\text{acetyl}\text{-β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was equally poorly recognized by the antiserum. The sensitivity of the radioimmunoassay was 1.9 pmol/l. $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was not detectable (< 3 pmol/l) in 26 of 27 extracted plasma samples in healthy blood donors, in one it was 5 pmol/l. In five of six patients with an enlarged sella turcica, but without clinical and laboratory evidence of pituitary dysfunction, $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was detectable ($\text{5 ± 3}\text{pmol/l}$; mean ± S.D.) after metyrapone. $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ was elevated in Addison's disease (23, 54 and 76 pmol/l), Nelson's syndrome (37, 39 and 109 pmol/l), ectopic ACTH production (27, 59 and 76 pmol/l), but only detectable in one of three samples from patients with Cushing's disease (7 pmol/l). Gel chromatography of extracts of porcine pituitary revealed only one immunoreactive peak co-eluting with synthetic human $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$. The specificity of the antiserum K-7762 was such that the $\text{β}{}_{\mathrm{<mtext>h</mtext>}}\text{-}\text{EP}$ concentration in plasma extracts could be reliably estimated by radioimmunoassay without prior chromatography.     

Cockroaches on a treadmill: Aerobic running

Five species of cockroach were tested on a miniature treadmill at three velocities as O₂ consumption () was measured: Gromphadorhina chopardi, Blaberus discoidalis, Eublaberus posticus, Byrsotria fumagata and Periplaneta americana. All cockroaches showed a classical aerobic response to running: increased rapidly from a resting rate to a steady-state on-response varied from under 30 s to 3 min. Recovery after exercise was rapid as well; $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ off-response varied from under 30 s to 6 min. These times are faster or similar to mammalian values. varied directly with velocity as in running mammals, birds and reptiles. during steady-state running was only 4–12 times higher than at rest. Running is energetically much less costly per unit time than flying, but the cost of transport per unit distance is much more expensive for pedestrians. The minimal cost of transport (M_run), the lowest necessary to transport a given mass a specific distance, is high in cockroaches due to their small size. The new data suggest that insects may be less economical than comparable sized vertebrates.     

Optimizing enzyme assays with one or two coupling enzymes

The cost of assays using one or two coupling enzymes is optimized by using equations to calculate the minimum amount(s) of enzyme(s) which should be used to obtain a given time (t₉₉) in which 99% of the rate V₀ of the first reaction is obtained. Using two coupling enzymes and given a value of t₉₉, the induction period L = L₁ + L₂ fulfills the requirement $\text{t}{}_{99}\text{2}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{4.6}\text{≥ L ≥}\text{t}{}_{99}\text{4.6}$, allowing one to choose a cost lower than that derived from the until-now generally applied assumption of t₉₉ = 4.6L. Being $\text{α =}\text{L}{}_1\text{L}{}_2$, in optimized assays the values α, t₉₉, and L are related by $\text{T}{}_{99}\text{=4.6}\text{(1+α)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{1+α}\text{L}$, thus allowing (graphical) calculation of the amounts of coupling enzymes which will minimize the cost for every chosen t₉₉ or L. Maximum practical rates, allowed in some supposed interesting cases, have been calculated.     

Acetylcholine receptors at the neuromuscular junction: Developmental change in receptor turnover

The turnover of acetylcholine receptors labeled with ¹²⁵I-labeled α-bungarotoxin was measured in the developing posterior latissimus dorsi muscle of the chick. The degradation rates for acetylcholine receptors at the neuromuscular junction and in extrajunctional regions of the muscle cell were determined. One week after hatching, the rates of junctional and extrajunctional receptor degradation are identical ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 30}\text{hr}$). Three weeks weeks after hatching, however, the rate of junctional receptor degradation is considerably slower ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{≥ 5}\text{days}$) and different than the rate of extrajunctional receptor degradation ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 30}\text{hr}$). Thus, receptors which are localized at the neuromuscular junction early in embryonic life only become stable several weeks after hatching.     

Properties of bilayer membranes in the presence of dipicrylamine. A comparative study by optical absorption and electrical relaxation measurements

In order to test the question if a pool of lipophilic ions may exist in black lipid membranes which cannot be detected by electrical relaxation measurements we have performed simultaneously measurements of the optical absorption of a lipophilic ion. The absorbance of membrane-bound dipicrylamine at 410 nm was measured with a sensitive spectrophotometer which can detect absorbance changes $\text{? 4 · 10}{}^{\mathrm{?5}}$. A minimal concentration of about 6 · 10¹¹ dipicrylamine ions per cm² of the membrane could be detected with this instrument. The dipicrylamine concentration in the membrane obtained with the optical method $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ is compared with the concentrations $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$ obtained from simultaneous electrical relaxation measurements. $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ and $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$ agreed at low dipicrylamine concentrations (10^?8–10^?7 M in the aqueous phase) and showed saturation at higher concentrations (up to 5 · 10^?6 M). In the saturation range $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>opt</mtext>}}$ was maximally four times higher than $\text{N}{}_{\mathrm{<mtext>t</mtext>}}{}^{\mathrm{<mtext>el</mtext>}}$. The significance of this difference is discussed together with general aspects of the saturation phenomenon.