Entrainment of the circadian locomotor activity rhythm in the Japanese newt by melatonin injections

We examined whether melatonin can act as a synchronizing agent within the circadian system of amphibians by testing the ability of melatonin injections to entrain the circadian locomotor activity rhythm of a newt (Cynops pyrrhogaster). Under constant darkness, all newts (13 cases) showing the free-running rhythms were subcutaneously injected with 10 g melatonin at the same time every other day for at least 30 days. Subsequently, they were injected with vehicle (1% ethanolic saline) instead of melatonin for at least another 30 days. In 10 of the 13 newts, the locomotor activity rhythms could be entrained to a period of 24 h by melatonin injections but not by vehicle injections. During the entrained steady-state, the active phase of an activity-rest cycle preceded the time of melatonin injections as previously reported in other diurnal species. These results suggest that the endogenous circadian rhythm of melatonin concentration may be involved in synchronizing circadian oscillator(s) within the newt's circadian system.     

Production of monoclonal antibodies and development of an ELISA for solamargine

The ratio of hapten and bovine serum albumin in an antigen conjugate was determined by matrix-assisted laser desorption/ionization mass spectrometry. A hybridoma secreting monoclonal antibodies against solamargine was produced by fusing splenocytes immunized with a solamargine-bovine serum albumin conjugate with HAT-sensitive mouse myeloma cell line, P3-X63-Ag8-653. Extensive cross-reaction of anti-solamargine antibodies against solasonine appeared. Aglycone of solamargine, solasodine cross-reacted with anti-solamargine antibodies resulting in a 43.8% cross-reaction. Insignificant cross-reaction appeared with tomatine (2.06%). The full measuring range of the assay extends from 57.5 pmol ml^–1 to 11.5 nmol ml^–1 of solamargine.     

Cellular and subcellular distribution of iron in the lamina propria of rat duodenum

Summary Cellular and subcellular distribution of iron in the lamina propria of rat duodenum was studied after a single i.p. injection of iron dextran, using electron microscopy and peroxidase cytochemistry. X-ray spectrum microanalysis was used for positive identification of iron. Ironcontaining particles (IP) were found in the cytoplasm of three cell types, viz. macrophages, pericytic reticular cells and sheathing fibrocytes. IP-containing organelles in lamina propria cells were more heterogeneous compared to absorptive cells and, in addition, some differences were noted in the subcellular distribution of IP in the 3 cell types. A common denominator in these 3 cell types was the presence of endogenous peroxidase, also shared by Kupffer cells which are known to be involved in iron storage. Peroxidase activity was absent in absorptive epithelial cells. It is hypothesized that the cells of the lamina propria, like Kupffer cells, may be the site of storage of excess iron absorbed, releasing iron upon demand and migrating into the lumen to prevent iron overload. In this fashion they may regulate the exchange of iron with the environment. The presence of peroxidase in these as well as Kupffer cells, and its absence in absorptive cells also raises the possibility that this enzyme may be related to certain aspects of iron storing process.     

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.     

Phase transitions of phospholipid bilayers from an unsaturated fatty acid auxotroph of Escherichia coli

Total phospholipids were extracted from cells of temperature sensitive unsaturated fatty acid auxotrophs of Escherichia coli (K-12 UFA^ts) grown at 28°C (PL28), and at 42°C in the presence of 2% KCl as an osmotic stabilizer (PL42 (KCl)). From the analysis of fatty acids, it was shown that the content of unsaturated fatty acids of PL42 (KCl) is only 9% of the total fatty acids, while that of PL28 is 54%. The thermal phase transitions of the bilayers prepared from the phospholipid fractions were studied by proton magnetic resonance. The line widths of the methylene signals and the sums of the methylene and methyl signal intensities were plotted against reciprocal values of absolute temperature 1/T or temperature itself. From the plots phase transitions were detected at about 19°C for PL28 and at 43°C for PL42 (KCl). In spite of its complex composition of fatty acids a highly cooperative transition was observed in the case of PL42 (KCl). It was also suggested that the phospholipids bilayers in the biomembranes of this strain at the growth temperature (42°C) are in the state where the gel and liquid crystalline phases coexist.     

Modes of DNA replication primed with the oligomer of palindrome

What is the precise molecular mechanism of semi-conservative DNA replication? After the great efforts of the past 20 years, molecular biology has now established the discontinuous syntheses of daughter DNA on both of the parental strands. In order to explain this type of discontinuous replication, we introduce the concept of a palindromic primer.First we focus our attention on various oligomers (RNA or DNA) which appear usually or occasionally in the process of replication. Then we propose the palindromic nature of these oligomers so as to serve as the primer of DNA synthesis. This postulation gives a theoretical reasoning for the discontinuities of both new strands in the fork region of replication.Subsequently we consider Watson's concatemeric intermediate theory, proposed for the explanation of replicative synthesis of phage T7 DNA. By considering the contribution of some sequence-specific endonuclease(s), we suggest the existence of partial palindromic sequences of bases at the connecting region(s) in which the redundant ends of the respective phage DNA molecules are overlapping. Another theory on the replication of linear chromosomal DNA including the concept of the terminal palindromic sequence of bases is also analyzed from the viewpoint of palindromic primer. Further, some recent experimental approaches, especially on the origin(s) of DNA replication, are shown to favour the concept of a palindromic primer.     

Cloning and characterization of the alkaline phosphatase positive regulator gene (phoB) of Escherichia coli

Summary The positive regulator gene (phoB) for alkaline phosphatase of Escherichia coli was cloned into the EcoRI site of pBR322 from the E. coli chromosome by a shotgun method. phoB was then constructed in vitro by replacing the C fragment of gtC by the phoB chromosomal fragment obtained from the hybrid plasmid. When the phoB mutant was lysogenized by phoB, the lysogen became PhoB⁺. The integration site of the phage was identified by P1 phage transduction to be around phoB site on the chromosome. From these results, we conclude that the cloned gene is phoB and not a gene which suppresses phenotypically phoB mutation when it is in a multi-copy state. The restriction map was constructed. Based on this information, several PhoB deletion plasmids and smaller PhoB⁺ plasmids were constructed in vitro. By examining PhoB phenotype when these plasmids were introduced into phoB mutant, we could define the phoB gene locus in 2 kb on the restriction map of the cloned chromosomal fragment. Cells carrying the multi-copy phoB gene produced alkaline phosphatase qualitatively under normal phosphate regulation. The phoB gene product was identified by the maxicell method as a protein with a molecular weight of approximately 31,000 daltons.