Synthesis of ribonucleic acid in the venom gland of Crotalus durissus terrificus (Ophidia, Reptilia) after manual extraction of the venom

1. The incorporation of [5-(3)H]uridine into RNA of the venom gland of Crotalus durissus terrificus was studied after manual extraction (;milking') of the venom. The labelled precursor was injected immediately after milking. 2. The RNA was extracted 1, 2, 4, 6 and 8h after injection of the label and analysed by sucrose-density-gradient centrifugation. 3. The sedimentation analysis showed that 18S rRNA synthesis is higher than 28S rRNA at all time-intervals. The specific radioactivities of both ribosomal components did not reach a plateau even at 8h after injection. 4. An RNA fraction was detected sedimenting between 18S rRNA and 4S tRNA and was called the 10-14S fraction. The specific radioactivity was always higher than that of both classes of rRNA and reached the maximum value at 6h of labelling. 5. The incorporation of the precursor was also studied by radioautography, which helped to elucidate the intracellular origin of the RNA analysed by sucrose-density-gradient centrifugation.     

The linkage between the four-step binding of oxygen and the binding of heterotropic anionic ligands in hemoglobin

The linkage between the four-step binding of oxygen and the binding of heterotropic anionic ligands in hemoglobin was investigated by accurately measuring and analyzing the oxygen equilibrium curves of human adult hemoglobin in the presence and absence of various concentrations of one or two of the following materials: chloride (Cl-), 2,3-diphosphoglycerate (DPG), and inositol hexaphosphate (IHP). Each equilibrium curve was analyzed according to the Adair equation to evaluate the four-step oxygen equilibrium constants (Adair constants) and the median oxygen pressure. The binding constants of the anions for the molecular species of hemoglobin carrying j oxygen molecules, Hb(O2)j(j=0,1,...,4), were evaluated from the dependences of the Adair constants and the median oxygen pressure on the anion concentration by introducing a model which takes the competitive binding of Cl- and DPG or IHP into account. Assumptions made in the model are: (a) the hemoglobin molecule has two oxygen-linked binding sites for Cl- which are equivalent and independent and (b) no Cl- can be bound to hemoglobin to which DPG or IHP is already bound and vice versa. Thus, we could obtain values for the intrinsic binding constants of Cl- and DPG, i.e., the constants in the absence of other competitive anions. For IHP, only the binding constants and apparent binding constants for Hb and Hb(O2)2 were obtained. Values of the Cl- binding constants and apparent binding constants for DPG and IHP, i.e., the binding constants in the presence of Cl- for Hb and Hb(O2)4, were in reasonable agreement with literature values. From the binding constants we calculated anion binding curves for Hb(O2)j(J=0,1,...,4), the number of anions bound to Hb(O2)J, And the relationship between fractional anion saturation of hemoglobin and fractional oxygen saturation. The numbers of released anions are not uniform with respect to oxygenation step. This non-uniformity is the reason for the changes in the shape of the oxygen equilibrium curve with anion concentration changes and for the non-uniform dependences of the Adair constants on anion concentration, and also results in non-linear relations between anion saturation and oxygen saturation. The anion binding constants and various binding properties of the anions derived from those constants are consistent with those observed by other investigators using different techniques, indicating that the present model describes the oxygen-linked competitive anion binding well.     

Differential expression on a daily basis of plastid sigma factor genes from the moss Physcomitrella patens. Regulatory interactions among PpSig5, the circadian clock, and blue light signaling mediated by cryptochromes

The nuclear-encoded plastid sigma factors are supposed to be a regulatory subunit of the multisubunit bacteria-type plastid RNA polymerase. We studied here whether or not three genes, PpSig1, PpSig2, and PpSig5 encoding plastid sigma factors, are controlled by the circadian clock and/or by blue light signaling in the moss Physcomitrella patens. Among the three PpSig genes, only PpSig5 was clearly controlled by the circadian clock. In contrast to the differential regulation on a daily timescale, a pulse of blue light induced the expression of all the three PpSig genes. This induction was significantly reduced in a knockout mutant that lacked the blue light photoreceptor cryptochromes PpCRY1a and PpCRY1b, indicating that PpCRY1a and/or PpCRY1b mediate the blue light signal that induces the expression of the PpSig genes. In a daily cycle of 12-h blue light/12-h dark, the timing of peak expression of PpSig5 and a chloroplast gene psbD, encoding the D2 subunit of photosystem II, advanced in the cryptochrome mutant relative to those in the wild type, suggesting the presence of regulatory interactions among the expression of PpSig5 and psbD, the circadian clock, and the blue light signaling mediated by the cryptochrome(s).     

UV-A induces two calcium waves in Physcomitrella patens

Our understanding of the role of Ca2+ in blue/UV-A photoreceptor signaling in a single cell is limited. Insight into calcium signaling has now been attained in Physcomitrella patens and its cryptochrome and phototropin knock-outs. Physcomitrella patens caulonemal filaments grow in the dark by apical extension and their apical cells are highly polarized. Fura-2-dextran ratio images of the apical cell from wild type (WT), Ppcry1a/1b and PpphotA2/B1/B2 were obtained immediately following UV-A exposure (30 microW cm(-2) at 340 nm for 1,000 ms plus 30 microW cm(-2) at 380 nm for 1,000 ms) [abbreviated as 1,000 ms (340/380 nm)] and demonstrated two intracellular waves: a Ca2+ wave from the growing apical tip through the apical cap, and a wave from the junction of the neighboring cell through the vacuolar, nuclear and plastid regions. In WT, the UV-A-induced tip wave increase had a magnitude of 454.0 +/- 40 nM, traveled at a rate of 3.4 +/- 0.7 microm s(-1) and was complete within 26.6 +/- 2.3 s, while the basal vacuolar wave had a magnitude of 596.8 +/- 110 nM, a rate of 8.4 +/- 0.8 microm s(-1) and duration of 25.3 +/- 4.9 s. Subsequent Ca2+ spikes of similar magnitude followed these waves. The amplitude of the Ca2+ waves in the apical cap and basal vacuolar regions of Ppcry1a/1b were higher than those in the WT, while the duration of those in PpphotA2/B1/B2 was longer. Subsequent Ca2+ spikes occurred in WT and Ppcry1a/1b but not in PpphotA2/B1/B2. When Mn2+ was added to the culture medium, the [Ca2+](cyt) increase was delayed, did not move as a wave and lasted longer. The results indicate that plants respond to blue light and UV-A radiation by generating a wave of changes in the [Ca2+](cyt). The characteristics of these Ca2+ waves were dependent upon cryptochrome and phototropin. Blue/UV-A signaling in P. patens appears to differ from that in Arabidopsis.     

Effects of granulocyte-colony-stimulating factor and interleukin-2 on ascites formation and the survival time of nude mice bearing human ovarian cancer cells

 The aim of this study was to elucidate the effect of intraperitoneal (i.p.) instillations of granulocyte-colony-stimulating factor (G-CSF) and/or interleukin-2 (IL-2) on ascites formation and the survival time of nude mice with malignant ascites, produced by i.p. inoculation of human ovarian cancer cells. When the nude mice were treated with medium alone, ascites was observed in all mice 28 days after tumor inoculation. When the mice were treated with cis-diamminedichloroplatinum(II) (cisplatin) alone, G-CSF alone or IL-2 alone, it took 35 days for the ascites to form in all mice. When cisplatin was combined with G-CSF or IL-2, one of ten mice did not form ascites during the observation period. Surprisingly, when G-CSF and IL-2 were simultaneously administered, ascites formation was not observed in any mice. Although i.p. treatment with cisplatin alone significantly prolonged the survival time, compared to medium alone, the lytic activity of spleen cells against HRA cells was significantly suppressed. When G-CSF or IL-2 was combined with cisplatin, the suppression by cisplatin was eliminated and subsequently resulted in a prolongation of the survival time. When G-CSF was combined with IL-2, both the peritoneal and splenic macrophages/monocytes were stimulated and the splenic lytic activity was about double that following treatment with G-CSF alone on IL-2 alone, suggesting that complete inhibition of ascites formation results not only from a significant increase of the peritoneal macrophages but also from enhancement of the lytic activity. Two mice, died from dissemination of tumor in the abdominal cavity, but eight mice survived without tumor for more than 90 days. As confirmed by monitoring body weight and hematocrit, G-CSF and IL-2 seemed to have no adverse effect. From these results, we conclude that a combination therapy with G-CSF and IL-2 might be of clinical use for inhibiting large amounts of ascites, which may inhibit therapeutic effects for ovarian cancer patients. Received: 20 May 1996 / Accepted: 19 September 1996     

Structure and molecular species composition of three homologous series of alpha-mycolic acids from Mycobacterium spp

Three homologous series of alpha-mycolic acids (dicyclopropanoyl acids, monocyclopropanoyl monoenoic acids and dienoic acids) from 16 rapidly growing and 14 slowly growing mycobacteria were separated by argentation thin-layer chromatography and analysed by gas chromatography/mass spectrometry of their trimethylsilyl ether derivatives. Mycobacterial species were separated into five groups. Strains of group A contained similar amounts of even and odd carbon-numbered dienoic acids, with a methyl branch on the odd acids and a C24-alpha-unit, as typified by Mycobacterium fortuitum and M. chitae. Group B strains possessed similar amounts of even carbon-numbered dicyclopropanoyl alpha-mycolic acids and odd carbon-numbered unsaturated acids with C22- and C24-alpha-units, as found in M. phlei and M. diernhoferi. Group C strains contained mainly even carbon-numbered dicyclopropanoyl acids with C22- and C24-alpha-units, as shown by M. vaccae and M. aurum. Group D strains possessed mainly odd carbon-numbered dienoic acids with a methyl branch and a C24-alpha-unit, as seen in M. triviale and M. nonchromogenicum. Group E strains had mainly even carbon-numbered dicyclopropanoyl acids with C24- or C26-alpha-units, as found in M. avium and M. tuberculosis. Many rapidly growing mycobacteria also produced alpha'-mycolic acids which were shorter in the length of the main carbon chain but whose alpha-units were the same as those in alpha-mycolic acids from the same species. These alpha'-mycolic acids had either one or two double bonds and showed variations in both their unsaturation and overall size, which may be useful in taxonomic studies.     

Phosphorylation of diacylglycerol kinase in vitro by protein kinase C.

We investigated the effects of enzyme phosphorylation in vitro on the properties of diacylglycerol kinase. Diacylglycerol kinase and protein kinase C, both present as Mr-80,000 proteins, were highly purified from pig thymus cytosol. Protein kinase C phosphorylated diacylglycerol kinase (up to 1 mol of 32P/mol of enzyme) much more actively than did cyclic AMP-dependent protein kinase. Phosphorylated and non-phosphorylated diacylglycerol kinase showed a similar pI, approx. 6.8. Diacylglycerol kinase phosphorylated by either protein kinase C or cyclic AMP-dependent protein kinase was almost exclusively associated with phosphatidylserine membranes. In contrast, soluble kinase consisted of the non-phosphorylated form. The catalytic properties of the lipid kinase were not much affected by phosphorylation, although phosphorylation-linked binding with phosphatidylserine vesicles resulted in stabilization of the enzyme activity.