Cell-cycle dependency of radiosensitivity and mutagenesis in fertilized egg cells of rice,Oryza sativa L.

Summary In order to examine changes in survival and mutation rates during a cell cycle in higher plant, fertilized egg cells of rice were irradiated with X-rays at 2 h intervals for the first 36 h after pollination, i.e., at different phases of the first and second cell cycles. The most sensitive phase in lethality was late G₁ to early S, followed by late G₂ to M, which were more sensitive than the other phases. In both M₁ and M₂ generations, sterile plants appeared most frequently when fertilized egg cells were irradiated at G₂ and M phases. Different kinds of mutated characters gave rise to the respective maximum mutation rates at different phases of a cell cycle: namely, albino and viridis were efficiently induced at early G₁, xantha at early S, short-culm mutant at mid G₂, heading-date mutant at M to early G₁. The present study suggests the possibility that the differential mutation spectrums concerning agronomic traits are obtained by selecting the time of irradiation after pollination.     

Systemic effects of chronically administered methyl prednisolonate and methyl 17-deoxyprednisolonate

The systemic activities of methyl prednisolonate and methyl 17-deoxyprednisolonate (1) were studied in rats. Methyl 17-deoxyprednisolonate produced significant changes in the amount of sodium ion (decreased) and potassium ion (increased) in urine; however, methyl prednisolonate had no effect on electrolyte balance. Both methyl prednisolonate and methyl 17-deoxyprednisolonate had no effect on liver glycogen content, plasma corticosterone level and relative adrenal weight. In contrast, the parent compound prednisolone caused a significant decrease in liver glycogen content, plasma corticosterone level and relative adrenal weight.     

Thyroid hormone: Aldosterone antagonism in cultured epithelial cells

Thyroid hormone (T₃) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have exammined the effect of T₃ on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5·10⁻⁸ M T₃ to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T₃ completely inhibits the maximal increase in short-circuit current in response to 1·10⁻⁷ M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T₃ and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5·10⁻⁹ M T₃. T₃ has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T₃ on nuclear binding of [³H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K′_d1 = (0.82 ± 0.36)·10⁻¹⁰ M and K′_d2 = (3.2±0.60)·10⁻⁸ M.The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1·10⁻⁸ M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5·10⁻⁸ M T₃ showed a K′_d1 = (0.15 ± 0.10)·10⁻¹⁰ M and K′_d2 = (3.5 ± 0.10)·10⁻⁸ M. We conclude that T₃ i action of aldosterone on sodium transport at a site after receptor binding in the nucleus.     

Somatostatin: a potential antigrowth factor for the exocrine pancreas

The increases in DNA synthesis and total DNA content after caerulein treatment support the trophic effect of this CCK analog on the pancreas. Over a 15 day caerulein treatment, pancreatic growth plateaued after 5 days and somatostatin is believed to be responsible for this phenomenon. The present study was undertaken to test this possibility. Rats were treated for 2 or 4 days with caerulein (1 μg · kg^?1), somatostatin antiserum plus caerulein or caerulein plus somatostatin (600 μg · kg^?1). Caerulein increased all parameters studied after 2 and 4 days; pancreatic hyperplasia was established after 2 days. The somatostatin antiserum significantly enhanced the effect of caerulein, especially on DNA synthesis and contents after 2 and 4 days. The trophic effect of caerulein was significantly reduced by somatostatin dramatically so with respect to hyperplasia. The effects of the somatostatin antiserum and those of somatostatin on stimulated pancreatic growth support the hypothesis that somatostatin may be considered an endogenous growth inhibitory factor for the pancreas.     

Isolation of methotrexate-resistant cell lines in Petunia hybrida upon stepwise selection procedure

Summary Cell suspensions of Petunia hybrida were subjected to a selection procedure in which the concentration of the selective agent, methotrexate (MTX), was gradually elevated. In mammalian cells, this procedure frequently results in MTX-resistant mutants due to amplification of the gene coding for dihydrofolate reductase (DHFR), the target protein of MTX.Five suspension lines were isolated, with degrees of resistance ranging from 10 to 500 M MTX (in wild type the LD_99.9 is 0.2 M). MTX^R phenotypes were unstable, as manifested by the loss of resistance upon prolonged growth in the absence of drug. All of the mutants also exhibited high values of MTX-binding protein (60- to 400-fold higher than that of the wild type), which declined to intermediate values upon MTX withdrawal. Finally, cellular extracts from all of the mutants also showed high specific staining of DHFR-activity in gels.The results suggest that the resistance of MTX in these plant cell-lines is mediated by the elevation of the amounts of DHFR, probably as a consequence of gene amplification.     

Molecular heterogeneity of canine cholecystokinin in portal and peripheral plasma

The release of molecular forms of cholecystokinin (CCK) into the portal and peripheral blood in response to an intraduodenal perfusion of sodium oleate (9 mmol X h-1) was studied in six conscious dogs with chronic portal vein catheters. Immunoreactive CCK as concentrated from 20 ml plasma by C18 SEP PAK cartridges and the pattern of molecular forms of CCK were studied by G50 gel filtration. CCK-like immunoreactivity (CCK-LI) was measured in the column eluates with antibody 5135, which measures gastrin and CCK equally and requires the intact carboxyl-terminus for full recognition. Gastrin was measured specifically with antibody 1611. Intraduodenal perfusion with oleate did not alter basal gastrin release. Release of CCK-LI by intraduodenal oleate was calculated by the increments of the integrated CCK-LI peaks over basal. Total CCK-like immunoreactivity (CCK-LI), calculated by integration of all CCK-LI peaks in gel filtration eluates, increased over basal by 12 fmol/ml in the portal and by 6 fmol/ml in the peripheral plasma after intraduodenal perfusion with sodium oleate. The main molecular forms eluted on gel filtration in positions of CCK33,39 and of CCK8. The pattern of CCK in the peripheral plasma was similar to that in the portal plasma except that in the peripheral plasma large molecular forms were more abundant than small forms. This finding was confirmed when CCK39 and CCK8 were infused either into the portal vein or into the peripheral vein and peripheral plasma CCK levels were measured. Elimination of CCK8 after portal vein infusion compared to peripheral vein infusion was about 3 times higher than that of CCK39. The abundance of large molecular forms of CCK in the circulating blood which are similar in potency to small forms, underlines their role in the physiology of CCK.     

Properties of phosphoenolpyruvate carboxylase in desalted extracts from isolated guard-cell protoplasts

Properties of phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) obtained from isolated guard-cell protoplasts of Vicia faba L. were determined following rapidly desalting of the extract on a Sephadex G 25 column. The activity of PEP carboxylase was measured as a function of PEP and malate concentration, pH and K⁺ concentration within 2–3 min after homogenization of the guard-cell protoplasts. The activity of this enzyme was stimulated by PEP concentrations of 0.1 to 0.75 mM and by K⁺ ions (12 mM), but inhibited by PEP concentrations above 1 mM and by malate. Changes in the K_m(PEP) and V_max values with increasing malate concentrations (2.5 and 5 mM) indicate that the malate level, varying in relation to the physiological state of guard cells, plays an important role in regulating the properties of phosphoenolpyruvate carboxylase.Abbreviations CAM Crassulacean acid metabolism - GCP guard-cell protoplast - PEP phosphoenolpyruvate Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

Metabolism of activated oxygen in detached wheat and rye leaves and its relevance to the initiation of senescence

Pollen grains of Plumbago zeylanica L. were serially sectioned and examined using transmission electron microscopy to determine the three-dimensional organization of sperm cells within the microgametophyte and the quantity of membrane-bound organelles occurring within each cell. Sperm cells occur in pairs within each pollen grain, but are dimorphic, differing in size, morphology and organelle content. The larger of the two sperm cells (S_vn) is distinguished by the presence of a long (approx. 30 m) projection, which wraps around and lies within embayments of the vegetative nucleus. This cell contains numerous mitochondria, up to two plastids and, infrequently, microbodies. It is characterized by a larger volume and surface area and contains a larger nucleus than the other sperm cell. The second sperm cell (S_ua) is linked by plasmodesmata with the S_vn, but is unassociated with the vegetative nucleus. It is smaller and lacks a cellular projection. The S_ua contains relatively few mitochondria, but numerous (up to 46) plastids and more microbodies than the other sperm. The degree of dimorphism in their content of heritable cytoplasmic organelles must at fertilization result in nearly unidirectional transmission of sperm plastids into just one of the two female reproductive cells, and preferential transmission of sperm mitochondria into the other.Abbreviations S_ua sperm cell unassociated with the vegetative nucleus - S_vn sperm cell physically associated with the vegetative nucleus 1=Russell and Cass (1981)     

Albumin extinction followed by de novo methylation of its gene in somatic hybrids of a rat hepatoma

We have previously identified an Msp I site at the 5′ end of the rat albumin gene whose undermethylation is necessary but not sufficient for stable albumin expression in rat hepatoma cells [1]. We have also shown that the extinction of albumin expression in somatic hybrids is not the result of methylation at this site, since for two different crosses, rapid extinction was found to occur in the absence of any de novo methylation of the previously active gene[2]. In the present study, we examine albumin expression and albumin gene methylation for independent hybrid clones isolated from crosses between albumin expressing rat hepatoma cells and cells of two different non-expressing lines. The cells from hybrid clones of both crosses are characterized by stable extinction of albumin expression. Moreover, we find that de novo methylation of the “extinguished” albumin gene can occur in somatic hybrids, but only some weeks after the gene has ceased to be expressed.     

Incorporation of [14C]cinnamate into hydrolase-resistant components of the primary cell wall of spinach

[¹⁴C]Cinnamate was taken up very rapidly by cultured spinach cells and completely incorporated into low-MW conjugates within 20 min. The ¹⁴C-labelled products were similar whether the [¹⁴C]cinnamate was supplied continuously over a period of hours via a peristaltic pump or instantaneously. Radioactivity was slowly recruited from the low-MW pool into aromatic components of the cell-wall fraction. Saponification of the radioactive wall fraction yielded, in addition to radioactive ferulate and p-coumarate, large amounts of ethyl acetate-soluble radioactive material with the properties of oxidatively coupled phenols. The coupled material was associated with the most highly ‘Driselase’-resistant fractions of the cell wall. In contrast, ‘Driselase’ released most of the wall's ferulate and p-coumarate on disaccharide fragments. It is suggested that the oxidatively coupled phenols are formed from simpler phenols by peroxidase and that they cross-link the polysaccharides to which they are attached, making these polysaccharides relatively ‘Driselase’-resistant.