Localization of arachidonate 12-lipoxygenase in parenchymal cells of porcine anterior pituitary

12-Lipoxygenases oxygenate arachidonic acid producing its 12S-hydroperoxy derivative and are well known as platelet and leukocyte enzymes. When a peroxidase-linked immunoassay of the enzyme according to the avidin-biotin method was applied to the cytosol fractions from various parts of porcine brain, a considerable amount of the enzyme was found in the anterior pituitary. The enzyme level (about 200 ng/mg cytosol protein) corresponded to about 6% of the enzyme content in porcine peripheral leukocytes. Posterior and intermediate lobes showed about one-tenth of the enzyme level of anterior pituitary. Other parts of porcine brain contained the 12-lipoxygenase in amounts below 7 ng/mg cytosol protein. The cytosol fraction (0.7 mg of protein) of anterior pituitary produced 12S-hydroxy-5,8,10,14-eicosatetraenoic acid from 25 microM arachidonic acid in about 34% conversion at 24 degrees C for 5 min, giving a specific enzyme activity about 3 nmol/min/mg protein. Furthermore, various octadecapolyenoic acids were oxygenated almost as fast as the arachidonate 12-oxygenation. When anterior pituitary was investigated immunohistochemically with anti-12-lipoxygenase antibody, most of the immunostained cells were certain parenchymal cells with granules, which were not blood cells. These biochemical and immunohistochemical results provide a good reason for considering that 12-lipoxygenase does play an important role in pituitary function.     

Purification and characterization of a novel GTP-binding protein with a Mr value of 24,000 from rat liver

About 15% of the total GTP-binding proteins (G proteins) of rat liver homogenate was found in the microsomes-Golgi complex fraction. From this fraction, we purified to near homogeneity and characterized a G protein with a Mr value of 24,000 (24K G). 24K G specifically bound guanosine 5'-(3-Q-thio) triphosphate (GTP gamma S), GTP and GDP with a Kd value for GTP gamma S of about 30 nM. 24K G bound maximally about 0.7 mol of GTP gamma S/mol of protein. 24K G hydrolyzed GTP to liberate Pi with a turnover number of about 0.008 min-1. 24K G was not copurified with the beta gamma subunit of heterotrimeric G proteins. The partial amino acid sequences of 24K G revealed that this protein was a novel small G protein.     

The nucleotide sequence of the mouse immunoglobulin epsilon gene: comparison with the human epsilon gene sequence

We have determined the nucleotide sequence of the immunoglobulin epsilon gene cloned from newborn mouse DNA. The epsilon gene sequence allows prediction of the amino acid sequence of the constant region of the epsilon chain and comparison of it with sequences of the human epsilon and other mouse immunoglobulin genes. The epsilon gene was shown to be under the weakest selection pressure at the protein level among the immunoglobulin genes although the divergence at the synonymous position is similar. Our results suggest that the epsilon gene may be dispensable, which is in accord with the fact that IgE has only obscure roles in the immune defense system but has an undesirable role as a mediator of hypersensitivity. The sequence data suggest that the human and murine epsilon genes were derived from different ancestors duplicated a long time ago. The amino acid sequence of the epsilon chain is more homologous to those of the gamma chains than the other mouse heavy chains. Two membrane exons, separated by an 80-base intron, were identified 1.7 kb 3' to the CH4 domain of the epsilon gene and shown to conserve a hydrophobic portion similar to those of other heavy chain genes. RNA blot hybridization showed that the epsilon membrane exons are transcribed into two species of mRNA in an IgE hybridoma.     

Long terminal repeat-like elements flank a human immunoglobulin epsilon pseudogene that lacks introns

There are at least three immunoglobulin epsilon genes (C epsilon 1, C epsilon 2, and C epsilon 3) in the human genome. The nucleotide sequences of the expressed epsilon gene (C epsilon 1) and one (C epsilon 3) of the two epsilon pseudogenes were compared. The results show that the C epsilon 3 gene lacks the three intervening sequences entirely and has a 31-base A-rich sequence 16 bases 3' to the putative poly(A) addition signal, indicating that the C epsilon 3 gene is a processed gene. The C epsilon 3 gene sequence is homologous to the five separate DNA segments of the C epsilon 1 gene; namely, a segment in the 5'-flanking region (100 bases) and four exons, which are interrupted by a spacer region or intervening sequences. Long terminal repeat (LTR)-like sequences which contain TATAAA and AATAAA sequences as well as terminal inverted repeats are present in both 5'- and 3'-flanking regions. The 5' and 3' LTR-like sequences do not, however, constitute a direct repeat, unlike transposable elements of eukaryotes and retroviruses. The 3' LTR-like sequence is repetitive in the human genome, but is not homologous to the Alu family DNA. Models for the evolutionary origin of the processed gene flanked by the LTR-like sequences are discussed. The C epsilon 3 gene has a new open frame which codes potentially for an unknown protein of 292 amino acid residues.     

Immunohistochemical studies on the serotonergic innervation of the pia mater

Summary The direct innervation of the pial blood vessels by serotonin neurons has been demonstrated with a modified peroxidase-antiperoxidase technique in the mammalian central nervous system. The pia mater covering the ventrolateral surface of the medulla oblongata is innervated by numerous varicose serotonin fibers originating from the serotonin neurons of the lower brainstem. Scattered serotonin fibers were observed in the pia mater in every part of the brain and spinal cord.     

Effect of calcium and magnesium ions on the interaction of corticosterone with the cytosol receptor(s) in the rat brain

The effects of calcium and magnesium ions on the corticosterone binding to rat brain cytosol receptor protein(s) were investigated. The increasing amounts of CaCl2 or MgCl2 up to 5.0 mM were added, the specific [3H] corticosterone binding increased 1.3-fold and 1.5 respectively. The addition of MnCl2 and KCl did not affect this binding. The binding of corticosterone with rat brain cytosol receptor(s) were decreased by increasing amounts of EDTA and complete inhibition was observed at concentration equal to and greater than 2.5 mM. Inhibition of this binding by EDTA was less than by EGTA. Either theophylline or dibutyryl cyclic AMP had no effect on this binding.     

Effect of aliphatic alcohols on the conformation of poly (l-ornithine hydrobromide) as studied by square-pulse and reversing-pulse electric birefringence

The electric birefringence and circular dichroism spectra of poly(l-ornithine hydrobromide) have been measured in ethanol/water, 2-propanol/water and tertiary butyl alcohol/water mixtures of various compositions. This charged polypeptide underwent a transition from the coil conformation to the helical conformation at high alcohol content in every case tested. Anomalous birefringence signals, indicative of a field-induced helix-to-coil transition. were observed at high electric fields only in the case of ethanol/water mixtures. The reversing-pulse electric birefringence of this polypeptide has been studied in ethanol/water mixtures and in neutral aqueous solution. Upon rapid reversal of the pulse field, no transient could be observed. This confirms that the electric-field orientation of poly(l-ornithine hydrobromide) results predominantly from the contribution of the counterion-induced dipole moment, regardless of its molecular conformations. It is very probable that the backbone permanent dipole moment of the helical conformation is largely suppressed by the counterion-induced dipole moment in the ionized form.     

Interfacial preference of anesthetic action upon the phase transition of phospholipid bilayers and partition equilibrium of inhalation anesthetics between membrane and deuterium oxide

The half-height linewidth (v 1/2) of the 1H-NMR spectra of dipalmitoylphosphatidylcholine vesicles changes abruptly at the phase transition temperature. In the absence of inhalation anesthetics, proton signals from the choline head group (hydrophilic interface) and acyl-chain tails (lipid core) change at the same temperature of 39.6 degrees C. The present study compared the effect of four inhalation anesthetics, i.e., methoxyflurane, chloroform, halothane and enflurane, upon the ligand-induced phase transition of phosphatidylcholine vesicle membranes at 37 degrees C. The anesthetics showed differential action upon the phase transition of the phospholipid vesicle membranes between the lipid core and the hydrophilic interface. The concentrations of anesthetics which induced the phase transition of the lipid core were about 2-fold greater than those required for the phase transition of the interfacial choline head groups. From the area under the proton signals of inhalation anesthetics in the NMR spectra, the maximum solubilities of methoxyflurane, chloroform and halothane in 2H2O at 37 degrees C were determined to be 0.671 . 10(-4), 2.637 . 10(-4) and 1.398 . 10(-4) (expressed as mole fractions), or 3.35, 13.17 and 6.98 mmol/1000 g 2H2O, respectively. The solubilities of the anesthetic vapor in 2H2O expressed as mole fractions according to Henry's law ere 9.586 . 10(-4), 6.432 . 10(-4) and 2.311 10(-4)/atm (1.013 . 10(5) Pa) partial pressure, respectively. The presence of phospholipid vesicles in 2H2O increased the solubility of the inhalation anesthetics. From difference between solubility in 2H2O and a dipalmitoylphosphatidylcholine vesicle suspension, the partition coefficients of methoxyflurane, chloroform and halothane between the phospholipid vesicle membranes and 2H2O were estimated. These values, calculated from the mole fractions, were 3364, 1660 and 3850, respectively at 37 degrees C.