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.     

Light and electron microscopic observation on the appearance of immunoreactive LHRH in perinatal rat hypothalamus

Summary The appearance and localization of LHRH were studied in the developing hypothalamus of perinatal rats using the unlabelled antibody method. By light microscopy, immunoreactive LHRH was first detected as brown dots on day 18.5 of gestation in the OVLT and on day 19.5 in the median eminence, respectively. When the median eminence was examined by the preembedding immunohistochemistry technique for electron microscopy, the occurrence of immunoreactive LHRH fibers could be demonstrated on day 18.5. These fibers were thin and very occasionally encountered near the surface of the lateral regions of the median eminence. The axoplasm contained a few immunopositive secretory granules and also extragranular immunoreactive products. With development, a gradual increase was noted both in number and size of nerve fibers with a concomitant accumulation of secretory granules within the axoplasm.A possible physiological significance of LHRH is discussed in relation to the onset of hypothalamo-hypophysial system in fetal life.     

A new technique of depositing phospholipid bilayers on quartz surfaces: its use in membrane spin-label studies

We have developed a new improved technique termed the parallel-beam spattering (PBS) method for depositing phospholipid bilayers on quartz surfaces. This technique involves atomizing the phospholipid mixture with a stream of nitrogen gas and passing this atomized mixture through two orifices separated by a distance to achieve a parallel beam of atomized particles before deposition on the quartz plate. A static electric field can easily be applied to the quartz surface. Also a goniometer of new design has been constructed to allow precise positioning of the deposited phospholipid bilayers with reference to the magnetic field. We have utilized the PBS method to deposit phosphatidylcholine/nitroxyl labeled cholestane mixtures on quartz plates and have found that hydrated bilayers of these mixtures yield ESR spectra with essentially the same characteristics as those obtained using more conventional techniques. The distinct advantage of the new technique for depositing bilayers is that there is no spectral anomaly present which usually is present when the more conventional method of depositing bilayers is used. The spectral anomaly is apparently caused by a portion of the bilayers aligned in directions not directly parallel to the quartz surface. For precision work the spectral anomaly is unacceptable. It is not observed with the new PBS method which has yielded highly reproducible results.     

Production of itaconic acid by Aspergillus terreus immobilized in polyacrylamide gels

Summary Living Aspergillus terreus cells were entrapped in polyacrylamide gels and employed in both replacement batch and continuous column reactors to produce itaconic acid from glucose.With the replacement batch reactor, maximum itaconic acid productivity was observed under the following conditions: pH 2.50, temperature at 35°C, addition of NH₄H₂PO₄ and MgSO₄·7H₂O. Using the continuous reactor, the maximum itaconic acid yield was 60 mg/h/40 g of gel. The biocatalyst activity or half-life was about 10 days.     

Preparation and properties of a lysozyme derivative in which two domains are cross-linked intramolecularly between Trp62 and Asp101.

A lysozyme derivative in which two domains were cross-linked intramolecularly was newly prepared by means of a two-step reaction. First, the beta-carboxyl group of Asp101 in lysozyme was selectively modified with 2-(2-pyridyldithio)ethylamine in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride. After reduction of the pyridyldithio moiety of Asp101 modified lysozyme at pH 4.5 with dithiothreitol, the derivative was allowed to cross-link intramolecularly by reaction with 1,3-dichloroacetone at pH 7. Intramolecularly cross-linked lysozyme thus formed was purified by gel chromatography followed by ion-exchange chromatography. Based on the results of 1H-NMR and peptide analyses, it was concluded that Asp101 was cross-linked to Trp62 with a -CH2COCH2SCH2CH2NH-bridge in this derivative. The derivative showed minor but distinct activity against Micrococcus lysodeikticus and glycol chitin. Its melting temperature for thermal denaturation was higher by 7.3 degrees than that of native lysozyme at pH 3.     

Developmental changes in the calcium currents in embryonic chick ventricular myocytes

Summary Using the patch-clamp technique, we recorded whole-cell calcium current from isolated cardiac myocytes dissociated from the apical ventricles of 7-day and 14-day chick embryos. In 70% of 14-day cells after 24 hr in culture, two component currents could be separated from totalI _Ca activated from a holding potential (V _h) of –80 mV. L-type current (I _L) was activated by depolarizing steps fromV _h –30 or –40 mV. The difference current (I _T) was obtained by subtractingI _L, fromI _Ca.I _T could also be distinguished pharmacologically fromI _L in these cells.I _T was selectively blocked by 40–160 m Ni²⁺, whereasI _L was suppressed by 1 m D600 or 2 m nifedipine. The Ni²⁺-resistant and D600-resistant currents had activation thresholds and peak voltages that were near those ofI _T andI _L defined by voltage threshold, and resembled those in adult mammalian heart. In 7-day cells,I _T andI _L could be distinguished by voltage threshold in 45% (S cells), while an additional 45% of 7-day cells were nonseparable (NS) by activation voltage threshold. Nonetheless, in mostNS cells,I _Ca was partly blocked by Ni²⁺ and by D600 given separately, and the effects were additive when these agents were given together. Differences among the cells in the ability to separateI _T andI _L by voltage threshold resulted largely from differences in the position of the steady-state inactivation and activation curves along the voltage axis. In all cells at both ages in which the steady-state inactivation relation was determined with a double-pulse protocol, the half-inactivation potential (V _1/2) of the Ni²⁺-resistant currentI _L averaged –18 mV. In contrast,V _1/2 of the Ni²⁺-sensitiveI _T was –60 mV in 14-day cells, –52 mV in 7-dayS cells, and –43 mV in 7-day NS cells. The half-activation potential was near –2 mV forI _L at both ages, but that ofI _T was –38 mV in 14-day and –29 mV in 7-day cells. Maximal current density was highly variable from cell to cell, but showed no systematic differences between 7-day and 14-day cells. These results indicate that the main developmental change that occurs in the components ofI _Ca is a negative shift with, embryonic age in the activation and inactivation relationships ofI _T along the voltage axis.     

Comparison of cytochrome P-450 species which catalyze the hydroxylations of the aromatic ring of estradiol and estradiol 17-sulfate

For identification of microsomal cytochrome P-450 (P-450) enzymes which catalyze 2- or 4-hydroxylations of estrogens in the rat liver, estradiol (E₂) and estradiol 17-sulfate (E₂-17-S) were selected as the substrates and incubated with various kinds of purified P-450 enzymes: PB-1, PB-2, PB-4 and PB-5 obtained from phenobarbital-treated male rats (Sprague-Dawley); MC-1 and MC-5 from 3-methylcholanthrene-treated male rats; and UT-1, UT-2, UT-4 and UT-5 from untreated animals. The reactions were carried out under the P-450-reconstructed system, and the resulting products were determined by HPLC using electrochemical detection. All the enzymes tested were shown to have varying degrees of catalytic activities for 2-hydroxylation of the two substrates; UT-1 and UT-2 had the highest activity. Of the induced P-450 enzymes, PB-2 and MC-1 showed fairly high catalytic activity for 4-hydroxylation of E₂. The P-450 enzymes obtained from the untreated male rats, especially UT-4, showed the highest catalytic activity for 4-hydroxylation of the two substrates. From these results and also from kinetic experiments, the P-450 enzymes which catalyze 2- and 4-hydroxylations of estrogen were considered to be different species. A part of E₂ was converted to such metabolites as estrone and those having a hydroxyl group at positions 6β, 15 or 16, each production of which was estimated to be catalyzed by single or multiple P-450s.     

Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca²⁺-dependent nuclease and a 23 kDa Zn²⁺ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.     

Biosystematic studies onDisporum (Liliaceae-Polygonatae)

Both the floral biology and morphometrics of two Japanese species of AsianDisporum (sectionEudisporum) are presented. These two species,D. sessile andD. smilacinum, represent extremes in both floral morphology and divergence in pollination within the section. The inverted flowers ofD. sessile have an elongate floral tube formed by the imbrication of the oblanceolate tepals. The tepal bases are modified into well developed, saccate nectaries. The stamens have rigid, vertical filaments which tightly encircle the ovary-style axis, and extrorse anthers located within a floral cavity which can accommodate a large pollinator (cross-pollination). The stigma is exserted and the depth of its cleft formation constant.D. smilacinum, in contrast, has an open, nodding campanulate flower with lanceolate tepals which have only shallow nectaries at their bases. The stamens have widely divergent filaments with versatile anthers that have laterally introrse dehiscence (wind and/or self-pollination). The depth of the stigma cleft is variable. For both species, the pattern of differential UV absorption and reflectance is similar. It is suggested on morphological grounds and by pollinator observation, thatD. sessile with a high energy flower requiring specialized visitors represents a more advanced condition than that observed inD. smilacinum, which is more generalized and primitive. Seasonal herbivore pressure on the tepal nectaries ofD. sessile is discussed in relation to its pollination.