On the relationship between water-soluble protein and calcium in the egg ofOryzias latipes

Summary Quantitative analysis of total and bound calcium in the egg ofOryzias latipes was performed during; the course of early development. The amount of total calcium is nearly constant. 95% of total calcium is bound with the basic substance before fertilization, but 17% becomes free within 10 minutes after fertilization. The rate of bound calcium to total calcium recovers the original value by the blastula stage. The water-soluble protein patterns obtained by paper electrophoresis consist of five anion bands. The protein of band IV increases during the early embryogenesis, while that of band I decreases relatively. There are no remarkable changes in the amount of protein in bands II, III, and V. Among three bands of thin layer chromatogram with Sephadex gel (G-200), the amount of protein of band A (more than 160,000 in molecular weight) reduces gradually after fertilization, while no marked change is observed in bands B (about 100,000 in molecular weight) and C (about 30,000 in molecular weight).The greater part of incorporated⁴⁵Ca is detected in low molecular substance (less than 30,000 in molecular weight), and the rest in bands A, B, and C, respectively.     

Electron paramagnetic resonance study of ferrous cytochrome P-450scc-nitric oxide complexes: effects of cholesterol and its analogues

Electron paramagnetic resonance (EPR) spectra of nitric oxide (NO) complexes of ferrous cytochrome P-450scc were measured at 77 K for the first time without using the rapid-mixing and freeze-quenching technique. Without substrate the EPR spectra were very similar to those of cytochrome P-450cam (from Pseudomonas putida) and cytochrome P-450LM (from rat liver microsomes) with rhombic symmetry; gx = 2.071, gz = 2.001, gy = 1.962, and Az = 2.2 mT for 14NO complexes. Upon addition of substrates [such as cholesterol, 22(S)-hydroxycholesterol, 22(R)-hydroxycholesterol, 25-hydroxycholesterol, and 22-ketocholesterol], the EPR spectra exhibited many variations having rhombic symmetry in the major component and an additional minor component with less rhombic symmetry. Furthermore, addition of 20(S)-hydroxycholesterol caused a striking change in the EPR spectrum. The component with rhombic symmetry disappeared completely, and the component with less rhombic symmetry dominated (gx = 2.027, gz = 2.007, gy = 1.984, and Az = 1.76 mT for 14NO complexes). These observations suggest the existence of the following physiologically important natures: (1) the conformational flexibility of the active site of the enzyme due to the steric interaction between the substrate and the heme-bound ligand molecule and (2) the importance of the hydroxylation of the cholesterol side chain at the 20S position to proceed the side-chain cleavage reaction in cytochrome P-450scc.     

Fragile X expression in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids under low and high thymidylate stress conditions

Expression of the fragile X site fra(X)(q27.3) was studied in thymidine-prototrophic and auxotrophic human-mouse somatic cell hybrids. In these cells, low thymidylate stress, achieved by 5-fluoro-2'-deoxyuridine (FdU) treatment and by limiting the exogenous supply of thymidine (dT), induced fragile X expression. High thymidylate stress, produced by supplying excess amounts of dT, was also effective in inducing fragile X expression, even in a hybrid clone that retained a fragile X chromosome as the only human chromosome; addition of deoxycytidine (dC) completely abolished this effect. In contrast, 5-bromo-2'-deoxyuridine (BrdU) did not induce fragile X expression. Cell-cycle analysis of BrdU-deprived thymidine-auxotrophic hybrid cells indicated that one round of DNA replication under thymidylate stress conditions is sufficient for fragile X expression. Our results suggest that the expression is an intrinsic property of the fragile site itself, which is believed to be composed of replicon clusters with pyrimidine-rich DNA sequence(s).     

Solution Radioactivated by Hadron Radiation Can Increase Sister Chromatid Exchanges

When energetic particles irradiate matter, it becomes activated by nuclear reactions. Radioactivation induced cellular effects are not clearly understood, but it could be a part of bystander effects. This investigation is aimed at understanding the biological effects from radioactivation in solution induced by hadron radiation. Water or phosphate buffered saline was activated by being exposed to hadron radiation including protons, carbon- and iron-ions. 1 mL of radioactivated solution was transferred to flasks with Chinese hamster ovary (CHO) cells cultured in 5 mL of complete media. The induction of sister chromatid exchanges (SCE) was used to observe any increase in DNA damage responses. The energy spectrum and the half-lives of the radioactivation were analyzed by NaI scintillation detector in order to identify generated radionuclides. In the radioactivated solution, 511 keV gamma-rays were observed, and their half-lives were approximately 2 min, 10 min, and 20 min. They respectively correspond to the beta+ decay of ¹⁵O, ¹³N, and ¹¹C. The SCE frequencies in CHO cells increased depending on the amount of radioactivation in the solution. These were suppressed with a 2-hour delayed solution transfer or pretreatment with dimethyl sulfoxide (DMSO). Our results suggest that the SCE induction by radioactivated solution was mediated by free radicals produced by the annihilated gamma-rays. Since the SCE induction and DMSO modulation are also reported in radiation-induced bystander effects, our results imply that radioactivation of the solution may have some contribution to the bystander effects from hadron radiation. Further investigations are required to assess if radioactivation effects would attribute an additional level of cancer risk of the hadron radiation therapy itself.     

Ca2+ is Required by Clubroot Resistant Turnip Cells for Transient Increases in PAL Activity that Follow Inoculation with Plasmodiophora brassicae

Phenylalanine ammonia‐lyase (PAL, EC 4.3.1.5) activity in clubroot disease‐resistant turnip calli was transiently increased by 20 h after the inoculation with Plasmodiophora brassicae spores. The magnitude of the increase in PAL activity was four to six times higher than constitutive PAL activity. There was no transient increase in PAL activity in susceptible calli. Preincubation of calli in Ca²⁺‐free medium or the removal of Ca²⁺ from cell surfaces by ethylene glycol bis(2‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid‐chelation, completely inhibited induced PAL activity. The influx of exogenous Ca²⁺ into cells appears necessary for this pathogen induced PAL activity. Verapamil and the calmodulin inhibitor W7 almost completely inhibited induced PAL activity at 1 and 0.1 mm , respectively. Neomycin, ruthenium red and (1‐(6‐[(17β‐3‐Methoxyestra‐1,3,5‐(10)‐trien‐17‐yl)amino]hexyl)‐1H‐pyrrole‐2,5‐dione) did not inhibit induced PAL activity. Thus, verapamil and N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulphonamide hydrochloride‐sensitive Ca²⁺‐mediated signalling process appear necessary for P. brassicae induced PAL activity. As the protein synthesis inhibitor cycloheximide (CHX) blocked the induced increasing PAL activity, de novo synthesis of PAL appears to be required for turnip cell defence reactions against P. brassicae.     

Lectin histochemical study on the infraorbital gland of the Japanese serow (Capricornis crispus)

Histology and lectin histochemistry were performed in the infraorbital gland of the Japanese serow. The gland is composed of glandular tissues and a pouch filled with the secretion. The tissues consist of an inner layer of sebaceous glands and an outer layer of apocrine glands. The male sebaceous layer is made up of the ordinary type, whereas the female's layer consists of the ordinary and modified types. In the apocrine gland stained with Arachis hypogaea (PNA), nine different patterns of glandular tubules were distinguished on the basis of staining of the cytoplasm, the Golgi area of secretory cells and secretion. Secretory modes of apocrine secretion and exocytosis were included in these stainings. Myoepithelial cells stained constantly with Glycine max (SBA) except when only the Golgi area of secretory cells was positive. The modified sebaceous gland was stained with PNA, SBA, Ricinus communis I (RCA), Triticum vulgaris (WGA), Canavalia ensiformis (Con A) and Ulex europaeus I (UEA), while the ordinary type was positive in PNA, RCA, SBA, WGA and Con A. The secretion in the pouch was stained with PNA, RCA, SBA, Dolichos biflorus (DBA), WGA and Con A. These findings suggest that the modified sebaceous gland contains large amounts of glycoconjugates and the apocrine gland shows a cyclic secretory process of apocrine secretion and exocytosis.     

Responses of pyriform cortex neurons to excitatory amino acids: Voltage dependence,conductance changes,and effects of divalent cations

The actions of ionophoretically applied N-methyl aspartate (NMA), quisqualate, and kainate, thought to activate three different types of excitatory amino acid receptors, were studied on pyramidal neurons of the rat pyriform cortex, maintained in an isolated, submerged, and perfused brain slice. Intracellular recordings were made with either K acetate or CsCl electrodes. In most neurons all three agonists elicited monophasic responses which could be evoked at 20-sec intervals. Some neurons showed biphasic responses, most commonly to kainate but, on occasion, also for quisqualate. The slower component appeared to be correlated with excitotoxicity and, consequently, was difficult to study. As a result the kainate responses studied were from neurons selected for having a single component. In neurons selected for having a linear current-voltage relationship or neurons loaded with Cs to suppress K conductance and linearize the current-voltage relationship, the average changes in resistance recorded during ionophoretic responses at resting potential were as follows: NMA, 131.2 +/- 6.7% of control; kainate, 104.7 +/- 5.8% of control; and quisqualate, 92.8 +/- 2.8% of control. The magnitude and direction of the conductance change were very reproducible in any one neuron, but especially for kainate some cells showed clear conductance increases, while others showed clear conductance decreases. Using CsCl electrodes it was possible to reduce K+ conductance and depolarize the neurons over a wider range. By passing depolarizing current it was possible to reverse the responses. The response to all three agonists reversed at the same depolarized potential. This observation indicates that while there are differences in the ionic channels associated with the three agonists at resting potential, the channels have similar properties at more depolarized potentials. Responses to all three agonists were influenced by the concentrations of divalent cations in the perfusion medium. The NMA responses were most sensitive to Mg, increasing in amplitude in the absence of Mg and being depressed by Mg elevation. All responses were sensitive to Ca, with discharges being greatly increased by low Ca and depressed by high Ca. The kainate response was most sensitive to Ca concentration changes. Unlike reports from other preparations the apparent conductance decreases to NMA were not altered by the perfusion of solutions with either no added Mg or no added Ca. The NMA response was very much reduced in either Co (1-2 mM) or Zn (100-200 microM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of a deletion mutant of the prosegment of human prorenin in Chinese hamster ovary cells

Expression plasmids encoding native human preporenin and a mutant deleted in its entire prosegment were transfected into Chinese hamster ovary cells. The cells transfected with the expression plasmid of native preporenin secreted exclusively inactive prorenin, while the cells transfected with the mutant secreted the active enzyme. The secreted amount of renin from the latter cells was much lower than that of prorenin from the former ones, although these two enzymes had little difference in specific activity after trypsin activation. These results suggest that the prosegment plays an important role in the secretory process of renin, although the fully active enzyme can be formed in its absence.