A simple radioisotope assay for microsomal aryl hydroxylase

A radioassay for liver microsomal aryl hydroxlase activity has been devised which depends on the liberation of tritiated water from generally tritiated benzo[α]pyrene during hydroxylation. The quantity of tritiated water has been shown to be proportional to the amount of 3-hydroxybenzo-[α]pyrene formed. Among the advantages of the radioassay are its speed, simplicity, and the fact that it essentially provides a cumulative measure of the hydroxylation of the benzo[α]pyrene ring. Investigation of a number of variables has made it possible to assay and obtain proportional results with as little as 3 μg of rat liver microsomes. Nucleic acids, but not their component mononucleotides, have been found capable of protecting the enzyme from product inhibition, presumably by interaction with benzo[α]-pyrene oxide, the primary product of benzo[α]pyrene hydroxylation.     

Characterization of the acetylcholine-sensitive muscarinic K+ channel in isolated feline atrial and ventricular myocytes

M₂-cholinergic receptor activation by acetylcholine (ACh) is known to cause a negative inotropic and chronotropic action in atrial tissues. This effect is still controversial in ventricular tissues. The ACh-sensitive muscarinic K⁺ channel (I _K(ACh)) activity was characterized in isolated feline atrial and ventricular myocytes using the patch-clamp technique. Bath application of ACh (1 m) caused shortening of action potential duration without prior stimulation with catecholamines in atrial and ventricular myocytes. Resting membrane potential was slightly hyperpolarized in both tissues. These effects of ACh were greater in atrium than in ventricle. ACh increased whole-cell membrane current in atrial and ventricular myocytes. The current-voltage (I-V) relationship of the ACh-induced current in ventricle exhibited inward-rectification whose slope conductance was smaller than that in atrium. In single channel recording from cell-attached patches, I _K(ACh) activity was observed when ACh was induced in the pipette solution in both tissues. The channel exhibited a slope conductance of 47 ±1 pS (mean ± sd, n=14) in atrium and 47 ±2 pS (n= 10) in ventricle (not different statistically; ns). The open times were distributed according to a single exponential function with mean open lifetime of 2.0±0.3 msec (n= 14) in atrium and 1.9±0.3 msec (n=10) in ventricle (ns); these conductance and kinetic properties were similar between the two tissues. However, the relationship between the concentration of ACh and single channel activity showed a higher sensitivity to ACh in atrium (IC ₅₀ =0.03 m) than in ventricle (IC ₅₀ =0.15 m). In excised inside-out patches, ventricular I _K(ACh) required higher concentrations of GTP to activate the channel compared to atrial channels. These results suggest a reduced I _K(ACh) channel sensitivity to M₂-cholinergic receptor-linked G protein (G_i) in ventricle compared to atrium in feline heart.     

Immunocytochemical localization of trehalase inhibitor in some insect species

Antibody against cockroach trehalase inhibitor was prepared and tested against the plasma of adult locusts and larval silkworms to determine whether these species possess a similar protein. An immunopositive response was elicited in both species. Studies using immunogold labeling show that adult cockroaches have trehalase inhibitor protein in granules of plasmatocytes and in oenocytoid-like structures. Localization of the immunoreactive protein with trehalase inhibitor antibody in locust hemocytes indicated that the protein is also contained in the granules of plasmatocytes. However, in the hemolymph of silkworm larvae, the immunoreactive protein was found only in the spherules of spherulocytes. The results suggest that insect hemolymph commonly contains trehalase inhibitor both in plasma and in certain hemocytes.     

Nuclear receptors for 1,25-dihydroxy-22-oxavitamin D3 (OCT) and 1,25-dihydroxyvitamin D3 in gastric gland neck mucous cells and gastrin enteroendocrine cells

22-Oxacalcitriol the analog with low calcemic effect and the original hormone 1,25(OH)₂ vitamin D₃ were localized by autoradiography in mouse stomach at different time intervals after intravenous injection. Both compunds showed a distinct nuclear concentration and retention in neck mucous cells of gastric and pyloric glands, and in dispersed endocrine cells in the antrum region. When the nuclear binding of radioactively labelled compound was compared between gastric neck cells and duodenal absorptive cells, binding was low but sustained in neck cells. Peak uptake after the injection was between 8 and 12 h in neck cells, but between 15 min and 30 min in duodenal villous epithelium. In the duodenum, weak nuclear labelling appeared at 8 h and was undetectable at 12 h under the conditions of the experiment. Nuclear labelling of neck cells remained detectable at 12 h and even after 24 h, similarly for both OCT and 1,25(OH)₂ vitamin D₃. These results suggest that the stomach is an important target tissue for vitamin D and its analog OCT. Regulation of neck cell functions is suggested, such as proliferation and differentiation of surface epithelium and gastric gland epithelium, and neck cell secretion of acidic mucus. Regulation is also indicated of G-cell gastrin secretion associated with gastrin paracrine effects on parietal cell HCl and intrinsic factor secretion, chief cell pepsinogen secretion, neck cell proliferation, as well as endocrine effects on systemic calcium homeostasis.     

Cold lability of pyruvate, orthophosphate dikinase in the maize leaf

Cold lability of pyruvate, orthophosphate dikinase was investigated using a homogeneous, purified enzyme preparation from maize (Zea mays L. var. Golden Cross Bantam T51) leaves. Its stability was markedly reduced below about 10 C and the rate of cold inactivation followed first order kinetics at a concentration lower than about 0.1 milligram of enzyme per milliliter. Cold inactivation was little affected by pH in the range which gives good stability for the enzyme at warm temperatures and the enzyme activity was protected strongly by inclusion of substrates (pyruvate and phosphoenolpyruvate) and polyols such as sucrose, sorbitol, and glycerol. Loss of catalytic activity was accompanied by an apparent dissociation of a tetrameric form of the enzyme (9S form) into a new, more slowly sedimenting (5.1S) component. Inclusion of pyruvate at 4 mM in the cold-treated enzyme had no effect on the sedimentation value. A sharp change in activation energy of the dikinase-catalyzed reaction was observed near 12 C and its break point appears to be close to the generally accepted critical low temperature limit for the growth of maize plants.     

Role of beta-1 and beta-2 adrenoceptors in isoproterenol-induced renin release in conscious dogs.

The present experiments were designed to classify the ß-adrenoceptors pertaining to the renin release induced by isoproterenol in conscious dog. Atenolol (ß-1 adrenoceptor antagonist), in oral dose of 6 mg/kg, produced a significant inhibition of renin release caused by isoproterenol. This dose of atenolol suppressed effectively the tachycardia of isoproterenol. On the other hand, the renin release produced by isoproterenol was not modified significantly by a ß-2 adrenoceptor antagonist, IPS-339, at a oral dose of 3 mg/kg which fully antagonized hypotensive response to isoproterenol. These results strongly suggest that the renin release induced by isoproterenol is largely due to stimulation of ß-1 type adrenoceptors.     

Properties of antigen-specific suppressive T cell factor in the regulation of antibody response of the mouse. II.In vitro activity and evidence for the I region gene product.

An antigen-specific suppressive T cell factor, which was extracted from carrier-primed T cells, was further characterized in an in vitro secondary antibody response. The factor was capable of suppressing secondary IgG antibody response of primed spleen cells when it was added to the culture together with relevant antigen. The suppressive T cell factor was not released from primed T cells by a short-term culture with antigen, but was kept bound to the membrane of the residual cultured cells, only the physical disruption of which can release the T cell factor. The target of the suppressive T cell factor was determined as being the helper T cell, since the factor did not exert any effect in the absence of the helper T cell with identical specificity to that of the factor. The suppressive activity was completely absorbed with alloantisera specific for products of the I region of H-2 complex, although various anti-immunoglobulin antisera failed to do so. Close analysis of the specificity of alloantisera capable of absorbing the suppressor molecule indicated that the suppressive T cell factor may, in fact, be an I region gene product probably coded for by genes in I-A and/or I-B (including I-E) subregions.     

Syntheses and conformational studies of poly- ,N-alkyl L-asparagines

Several β,N-alkyl L -asparagines were prepared from the phthalyl and benzyloxycarbonyl derivatives. High-molecular-weight poly-β,N-benzyl L -asparagine and poly-β,N-(1)-phenethyl L -asparagine were prepared from the corresponding N-carboxyanhy-drides. From the results obtained by a study of the infrared absorption spectra and the optical rotatory dispersion, poly-β-N-benzyl L -asparagine was found to be a random coil structure in dichloroacetic acid and the optical rotatory dispersion curves gradually changed into the left-handed α-helix structure when chloroform was added to the solution. The coil-to-helix transition was observed in the vicinity of 20% dichloroacetic acid in a dichloroacetic acid-chloroform mixture. Poly-β,N-(d), (l), and (d + l, 1:1)

1 (d + l, 1:1): mixed polymer containing the same weighed poly-β,N-(d) and (l)-(1)-phenethyl L -asparagines.

-(1)-phenethyl L -asparagines showed a nearly constant specific rotation in the dichloroacetic acidchloroform solvent system. Poly-β,N-(dl)-(1)-phenethyl L -asparagine caused a gradual folding of the helix at dichloroacetic acid content of less than 20%.