In vivo and in vitro studies on the appearance of LHRH neurons in the hypothalamus of perinatal rats

Summary Ontogenetic development of LHRH-containing neurons was studied by fluorescence and enzyme immunohistochemistry in rats. In in vitro studies, the tissues of the septal-chiasmatic and mediobasal hypothalamic areas of fetal rats on day 16.5 or 18.5 of gestation were trypsinized separately for dissociation of the neural cells, and cultured for several days. Immunopositive reaction against LHRH was first detected in nerve cells derived from both areas of the hypothalamus of the fetuses on days 16.5 and 18.5 of gestation, after 8 and 6 days culture, respectively. The cells were small, and seemed to be bipolar in morphology indicating an axon and arborized dendrites. Immunopositive material occurred in the cell soma as well as in the cellular processes. In in vivo studies, immunopositive material, possibly deposited in nerve fibers, appeared first in OVLT and simultaneously in the external layer of the median eminence of fetuses on day 20.5 of gestation. The immunoreactive fibers increased in number in both parts with development, especially after birth in the median eminence. No immunopositive material was detected within any neural cell bodies nor in the cytoplasm of any ependymal cells.This work was financed by the Ministry of Education, Japan. No. 257008. We would like to thank Dr. Katsuhiko Saito (Department of Surgery, Tokushima University) for his kind advice on the preparation of the antibody used for the immunofluorescence study.     

Origin of hydrogen atoms in the fatty acids synthesized with yeast fatty acid synthetase.

The mechanism of hydrogen incorporation into fatty acids was investigated with an enzyme preparation from baker's yeast. Fatty acids synthesized from malonyl-CoA and acetyl-CoA in the presence of D2O or stereospecifically deuterium-labeled NADPH were isolated and analyzed by mass chromatography to examine the localization of deuterium atoms in the molecule. The following results were obtained: 1. Hydrogen atoms from water were found on the even-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom). The second hydrogen atom was incorporated as the result of hydrogen exchange phenomenon between the methylene group of malonyl CoA and water. 2. HB hydrogen of NADPH was used for beta-ketoacyl reductase. 3. HB hydrogen of NADPH was also used for enoyl reductase. 4. Hydrogen atoms from HB position of NADPH were found on the odd-numbered methylene carbon atoms (2-hydrogen atoms per carbon atom).     

Conversion of DOPA to tetrahydroisoquinolines and stizolobic acid in a callus culture of Stizolobium hassjoo

Isolation and identification of l-3-carboxy-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline and l-1-methyl-3-carboxy-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline from seeds and callus of S. hassjoo are described. Administration of [β-¹⁴C]-labelled DOPA to a callus culture of this legume resulted in the incorporation of radioactivity into l-3-carboxy-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, l-1-methyl-3-carboxy-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline and stizolobic acid, which was confirmed by constant specific radioactivity after co-crystallization with authentic samples of each compound.     

Ultrastructural localization of carbonic anhydrase activity in the rat choroid plexus epithelial cell

Summary Localization of carbonic anhydrase activity was studied electron microscopically on cells of the rat choroid plexus epithelium. For the ultracytochemical detection of these activities, Yokota's technique (1969), which is the modification of Hansson's method (1967) was employed. Numerous electron dense reaction products were observed in the microvilli of the choroidal epithelial cell. The reaction deposits were also remarkably present in the infoldings of the basal plasmalemma but to a lesser extent than in the microvilli. The localization sites were mainly on the plasma membrane, but some reaction products were also observed in the cytoplasm near the plasma membrane. Hardly any reaction product was found in the intracellular organelles except for the mitochondria in which reaction products were occasionally observed on the cristae. These activities were completely inhibited by acetazolamide. As the carbonic anhydrase activity was histochemically seen in the microvilli and the basal infoldings, it is likely that carbonic anhydrase is related to an active transport process in the secretion of cerebrospinal fluid as is Na⁺, K⁺-ATPase (Masuzawa et al. 1980).     

Coupling factor ATPase from Escherichia coli. An uncA mutant (uncA401) with defective alpha subunit.

Inactive coupling factor ATPase (F1) was prepared from an uncoupled mutant (uncA401) of Escherichia coli. Reconstitution of ATPase activity was observed when alpha subunit from wild-type F1 was added to the dissociated inactive F1 and the mixture was dialyzed against buffer containing ATP and Mg2+. ATPase was also reconstituted when the mixture of alpha subunit (wild type) and crude extract from the mutant was dialyzed against the same buffer. These results indicate that the mutant is defective in alpha subunit, suggesting that the uncA401 locus carries the structural gene for alpha subunit, and that this polypeptide plays an essential role in ATPase activity in F1 molecule.     

Membrane asymmetry and enhanced ultrastructural detail of sarcoplasmic reticulum revealed with use of tannic acid

Fixation of purified sarcoplasmic reticulum (SR) membrane vesicles, using glutaraldehyde supplemented with 1% tannic acid, reveals newly visualized ultrastructure in thin sections. The trilaminar appearance of the membrane is highly asymmetric; the outer electron-opaque layer is appreciably wider (70 A) than the inner layer (20 A). The asymmetry is not referable to lack of penetration of the tannic acid since: (a) SR vesicles made permeable with 1 mM EDTA, pH 8.5, show similar asymmetry; (b) treatment of SR with trypsin results in progressive loss in protein content and decrease in the thickness of the outer layer, until in the limit the trilayer has a symmetric appearance; (c) within the same muscle section, the SR membrane appears highly asymmetric whereas the sarcolemma has a more symmetric appearance; (d) reconstituted SR vesicles have a symmetric appearance with equally broad inner and outer layers (approximately 70 A); the symmetric structure is confirmed by freeze-fracture and negative staining electron microscopy. Heavy and light SR vesicles obtained by isopycnic density sedimentation of purified SR have the same asymmetric appearance of the membrane and seem to differ mainly in that the heavy vesicles contain internal contents consisting largely of Ca++-binding protein. The asymmetry of the SR membrane is referable mainly to the unidirectional alignment of the Ca++ pump protein, the major component (90% of the protein) of the membrane. The asymmetry of the SR membrane can be visualized now for the first time in situ in thin sections of muscle.     

Membrane lipid biosynthesis in Acholeplasma laidlawii B: Investigations into the in vivo regulation of the quantity and hydrocarbon chain lengths of de novo biosynthesized fatty acids in response to exogenously supplied fatty acids

The regulation of the nature and quantity of the fatty acids produced in vivo by Acholeplasma laidlawii B in the presence of various exogenous fatty acids has been investigated. In the presence of exogenous medium- or long-chain fatty acids, the organism appears to reduce the amounts of de novo biosynthesized fatty acids in its cellular lipid pool by two distinct mechanisms: an excretion of biosynthesized fatty acids to the growth medium as free fatty acids, and a reduction in total de novo biosynthetic output. These two mechanisms do not suffice to maintain constant total membrane lipid levels, but they do appear to significantly moderate the effect of exogenous fatty acids on the level of membrane lipid. In the presence of short-chain fatty acids, total membrane lipid levels are not elevated. Exogenous fatty acids can cause shifts in the average chain length of de novo biosynthesized fatty acids; the magnitudes and directions of these shifts can be correlated with the specificity of the exogenous species for esterification to the 1- or the 2-position of the glycerol moiety of membrane glycerolipids. As the various endogenously synthesized fatty acids differ in their positional specificity for glycerolipid esterification, we propose that the competition of an exogenous species with significant specificity for a particular position with the endogenously derived fatty acids specific for that position can selectively depress the synthesis of such endogenously derived species, thereby altering the overall product spectrum of de novo fatty acid biosynthesis in vivo.     

Effect of furosemide on urinary excretion of prostaglandin E in normal volunteers and pateints with essential hypertension

Urinary excretion of prostaglandin E was measured radioimmunologically in 19 healthy persons ( 15 men and 4 women ) and in 16 patients ( 10 men and 6 women ) with essential hypertension before and after the administration of furosemide. The excretion rates were increased from 26.3±3.0 to 64.5±11.3 ng/hr in the former and from 11.9±2.7 to 26.9±85 ng/hr in the latter. There was a significant difference between them, healthy subjects showing a greater increase than patients with essential hypertension.There was an obvious sexual difference in urinary excretion of prostaglandin. In men, greater increase in the excretion rates was found than in the women. Greater increases were also obtained in healthy men than in hypertensive men and in healthy women than in hypertensive women. The present results suggest that furosemide enhances urinary excretion of prostaglandin E by mechanisms which entails either an increase in prostaglandin synthesis or a decrease in renal metabolism.