Partial Characterization of Phytochrome I and II in Etiolated and De-Etiolated Tissues of a Photomorphogenetic Mutant (lh) of Cucumber (Cucumis sativus L.) and Its Isogenic Wild Type

A photomorphogenetic mutant (lh) of cucumber has been suggestedto lack light-stable phytochrome function [Adamse et al. (1987)J. Plant Physiol. 127: 481-491]. The present work reports biochemicaland immunochemical characteristics of phytochrome in this cucumbermutant. Spectrophotometric measurement of phytochrome extractedfrom the etiolated seedlings indicated that the mutant containeda similar amount of phytochrome to that of the wild type. Nosignificant differences in apparent molecular mass and reactivityagainst an anti-pea phytochrome monoclonal antibody were observed.Phytochrome in de-etiolated seedlings was partially purifiedto enable spectrophotometric measurement. The phytochrome contentand difference spectrum for photoconversion was very similarin extracts of the mutant and the wild type. Furthermore, thephytochrome extracted from de-etiolated tissues of the mutantand the wild type appear to contain similar amounts of phytochromeI and II, since in both about one quarter of the phytochromein the fraction could be immunoprecipitated by an antibody whichrecognizes phytochrome I. Two possibilities to explain the Ilphenotype are: (i), the mutation changes the function of phytochromeI and/or II without changing its stability and spectrophotometricalcharacteristics; (ii), the mutation results in modificationof transduction chains between the photo-receptor and physiologicalresponses. (Received February 1, 1989; Accepted April 14, 1989)     

Control of the Entry into S Phase by Phytochrome and Blue Light Receptor in the First Cell Cycle of Fern Spores

Phytochrome- and a blue light receptor-dependent pathway antagonisticallyregulate the first mitosis in spores of the fern Adiantum capillus-venerisL. This study focused on determining which phase(s) of the cellcycle is positively regulated by phytochrome and negativelyregulated by a blue light receptor in germinating spores. Incorporationof the radioactivity of ³H-thymidine into the acid-insolublematerial prepared from the spores indicated that phytochromein the PFR form induced the entry into S phase of the firstcell cycle in the spores 20-28 h after irradiation with redlight. Blue light treatment before or after red light treatmenttotally prevented the P_FR-induced DNA synthesis. Brief irradiationwith red, far-red or blue light showed no effects on mitosisif the irradiation was given 28 h after the red light induction,during S and M phases. These results indicate that phytochromeand a blue light receptor regulate the entry into S phase duringthe first cell cycle of fern spores. ( Accepted July 10, 1997)     

Daily rhythmic change in the transport of histidine by everted sacs of rat small intestine

Fluctuations in the intestinal transport of L- and D-histidine were measured in rats on three feeding schedules under conventional lighting conditions, with a dark night. In rats fed ad libitum, the transport of L-histidine through the everted intestine showed a daily rhythmic change, being high at 4 p.m. and low in the early morning. In rats adapted to daytime feeding, the transport of L-histidine was highest at 6 a.m. and low at night. In starved rats, the rhythmicity was maintained for at least one day of fasting. Transport of D-histidine showed no daily fluctuation.     

Low maternal licking/grooming stimulation increases pain sensitivity in male mouse offspring

Deprivation of maternal care has been associated with higher pain sensitivity in offspring. In the present study, we hypothesized that the maternal licking/grooming behavior was an important factor for the development of the pain regulatory system. To test this hypothesis, we used male F2 offspring of early-weaned (EW) F1 mother mice that exhibit lower frequency of licking/grooming behavior. The formalin test revealed that F2 offspring of EW F1 dams showed significantly higher pain behavior than F2 offspring of normally-weaned (NW) F1 dams. We found that the mRNA levels of transient receptor potential vanilloid 1 (TRPV1), a nociceptor, were higher in the lumbosacral dorsal root ganglion (DRG) of F2 offspring of EW F1 dams than those of F2 offspring of NW F1 dams, suggesting that the higher pain sensitivity may be attributed to low licking/grooming, which may result in developmental changes in nociceptive neurons. In the DRG, mRNA levels of Mas-related G-protein coupled receptor B4 (MrgprB4), a marker of sensory neurons that detect gentle stroking, was also up-regulated in the F2 offspring of EW F1 dams. Considering that gentle touch alleviates pain, Mrgprb4 up-regulation may reflect a compensatory change. The present findings indicate important implications of maternal licking/grooming behavior in the development of the pain regulatory system.     

Light Regulation of Hypocotyl Elongation and Greening in Transgenic Tobacco Seedlings That Over-Express Rice Phytochrome A

Previous analysis of a transgenic tobacco line (BN1) that over-expressedrice phytochrome A (PhyA) indicated that the introduced PhyAwas spectrally and biologically active [Kay et al. (1989) PlantCell 1: 775, Nagatani et al. (1991) Proc. Natl. Acad. Sci. USA88: 5207]. In the present study, we have further investigatedresponses of the BN1 plants to light. Fluence rate dependenceanalysis of the inhibition of hypocotyl elongation indicatedthat the response is biphasic. The amplitude of the low fluencerate component increased by 2 to 3 fold in the BN1 plants comparedto the wild type. In contrast, the presence of rice PhyA didnot alter the level of chlorophyll in the BN1 seedlings grownunder the same light conditions. Ultrastructure studies showedthat chloroplasts in the BN1 plants were not significantly differentfrom those in the wild type plants, except that chloroplastsin the guard cells of the BN1 plants appeared to be more developedthan those of the wild type plants. The fluence response analysisof the potentiation of chlorophyll accumulation indicated nosignificant difference between the BN1 and the wild type plants.Thus, the introduced rice PhyA greatly influenced hypocotylelongation but did not significantly affect the greening process. ⁴Present address: NSFC Center for Biological Timing, Universityof Virginia Charlottesville, VA 22901, U.S.A. ⁵Present address: Advanced Research Laboratory, Hitachi Ltd.Hatoyama, Saitama, 350-03 Japan     

Characterization of ATPases Associated with Various Cellular Membranes Isolated from Etiolated Hypocotyls of Vigna radiata (L.) Wilczek

Cellular membrane fractions, including endoplasmic reticum (ER),Golgi-enriched membrane, plasma membrane and tonoplasts, wereisolated from Vigna radiata seedlings. Each of these membranefractions was associated with specific ATPases which were highlydependent on Mg²⁺. ATPases of ER, Golgi-enriched membrane andplasma membrane were sensitive to vanadate but the tonoplastATPase was not. ATPases were mostly dependent on Cl¹, but aslight stimulation by K⁺ was observed in the case of ATPasesof Golgi-enriched membrane and plasma membrane. KNO₃ inhibitedtonoplast ATPase but stimulated the other ATPases. ER ATPasecan be distinguished from other ATPases by the following characteristics:specific inhibition by KNO₂ and Triton X-100, stimulation bylow concentrations of diethylstilbestrol and 4,4'-diisothiocyanostilbene-2,2'-disulfonicacid, and high sensitivity to heat. The ATPases showed typicalMichaelis-Menten kinetics and had K_m values of 0.5 to 0.6 ITIMMg²⁺-ATP for ER, Golgienriched-membrane and tonoplast ATPases,and 2.27 msi Mg²⁺-ATP for plasma membrane ATPase. ATPases ofGolgi-enriched membranes and plasma membranes had similar properties,but they were still distinguishable by the differences in theirK_m values and their responses to Triton X-100. Based on theseresults, it is postulated that each cellular membrane is associatedwith a specific ATPase in cells of V. radiata. ¹Contribution No. 3171 from the Institute of Low TemperatureScience. (Received April 22, 1988; Accepted September 28, 1988)     

LrrkA,a kinase with leucine‐rich repeats,links folate sensing with Kil2 activity and intracellular killing

Phagocytic cells ingest bacteria by phagocytosis and kill them efficiently inside phagolysosomes. The molecular mechanisms involved in intracellular killing and their regulation are complex and still incompletely understood. Dictyostelium discoideum has been used as a model to discover and to study new gene products involved in intracellular killing of ingested bacteria. In this study, we performed random mutagenesis of Dictyostelium cells and isolated a mutant defective for growth on bacteria. This mutant is characterized by the genetic inactivation of the lrrkA gene, which encodes a protein with a kinase domain and leucine‐rich repeats. LrrkA knockout (KO) cells kill ingested Klebsiella pneumoniae bacteria inefficiently. This defect is not additive to the killing defect observed in kil2 KO cells, suggesting that the function of Kil2 is partially controlled by LrrkA. Indeed, lrrkA KO cells exhibit a phenotype similar to that of kil2 KO cells: Intraphagosomal proteolysis is inefficient, and both intraphagosomal killing and proteolysis are restored upon exogenous supplementation with magnesium ions. Bacterially secreted folate stimulates intracellular killing in Dictyostelium cells, but this stimulation is lost in cells with genetic inactivation of kil2, lrrkA, or far1. Together, these results indicate that the stimulation of intracellular killing by folate involves Far1 (the cell surface receptor for folate), LrrkA, and Kil2. This study is the first identification of a signalling pathway regulating intraphagosomal bacterial killing in Dictyostelium cells.