The Role of Germination Inhibitors and Oxygen in the Dormancy of the Light-densitive Seed of Betula spp.

Although unchilled, intact seeds of Betula pubescens and B.verrucosa require light for germination, isolated embryos germinateequally well in both light and darkness. An aqueous extract of these seeds has germination-inhibitoryproperties correlated with the presence of a non-fluorescent,single substance. The light requirement of isolated embryosis restored by the inhibitor. When intact seeds are leachedwith water to remove some inhibitor, it is found that the lightrequirement is reduced, short days and single light periodsthen eliciting greater germination than in unleached seeds. It has been found that scratching, pricking, and cutting theseed coat increases the germination of intact seeds in darkness,and that this is probably due to enhanced oxygen entry. Further,it has been found that germination in short days is increasedin oxygen-enriched atmospheres. It has been found that although the inhibitory effect of theseed coat in intact seeds is partially due to the reductionof the oxygen supply to the embryo, a low oxygen concentrationdoes not prevent germination of isolated embryos. Experimentalresults suggest that the inhibitor in the seed coat increasesthe oxygen requirement of the embryo.     

Natural growth inhibitors associated with the cotyledon effect in light-grown dwarf and tall beans

The contents of non-acidic, acidic and bound growth inhibitorsin hypocotyls and cotyledons were compared between dark- andlight-grown dwarf and tall beans by means of thin-layer chromatographyand bioassay. In the non-acidic fraction, one major inhibitoryactivity appeared on the chromatogram, but its Rf zone was differentbetween hypocotyls and cotyledons. In both the acidic and boundinhibitor fractions, one major inhibitory activity appearedat the Rf zone corresponding to ABA. The ABAIike substance whichwas the major inhibitor in the hypocotyl was more abundant inlight-gorwn than dark-grown ones, especially in the dwarf variety,but light irradiation did not cause its transport from cotyledonsto the hypocotyl. A larger amount of bound ABA-like substance,which was the major inhibitor in the cotyledon, was presentin the dwarf than the tall variety regardless of the light condition.Cotyldon-enhanced photoinhibition of hypocotyl growth couldnot be explained by the levels of the xanthoxinand ABA-likeinhibitors, or the transport of these free inhibitors from cotyledons. (Received September 8, 1977; )     

Effects of Light on the Georeaction and Growth Inhibitor Content of Roots

The positive geotropic response of the apical segments prepared from the primary roots of Zea mays depends upon at least one growth inhibitor, produced by the root cap, moving basipetally into the extending zone of the root in which it accumulates in the lower part. Anjou maize reacts in both darkness and light while Kelvedon maize is, for the first few hours, geotropic only in light. The production (or activity) of the growth-inhibiting substance — tested by using vertical half-decapitated root segments — is quite similar to the georeaction. This finding provides strong evidence that, in the case of Kelvedon maize roots, the inhibitory substance may depend on light. Observations related to the root segment of Anjou and Kelvedon maizes of which the tips are exchanged, are in agreement with the above results.     

STUDIES ON THE MECHANISM OF GROWTH INHIBITING EFFECT OF LIGHT

1. A substance which inhibits indoleacetic acid (IAA)-and naphthaleneaceticacid (NAA)-induced elongation of Avena coleoptile section andIAA-induced Avena coleoptile curvature was found in an ethersoluble neutral fraction of water extract of sunflower leavesand in agar blocks containing the diffusate from young sunflowerleaves.
2. This substance also inhibits the growth of isolatedsunflowerepicotyl.
3. The R_f value (0.9) of the substance ona paper chromatogramdeveloped with ammoniacal iso-propanolindicates that it isidentical with the inhibitor reported byAUDUS et al. (1956),but not with inhibitor-ß.
4. Theinhibitor can be transported from leaf to stem, and thetransportseems to be accelerated by illuminating the leaf.
5. The auxindiffused from sunflower leaf into agar block may beidenticalwith IAA.
6. A substance, which has the same properties as theinhibitorfrom sunflower leaf, was obtained in crystalline formfrom theleaf of Jerusalem artichoke.
7. The mechanism of growthinhibition caused by this crystallinesubstance seems to involveinactivation of a sulfhydryl group.
8. The reason why the stemgrowth of sunflower seedlings is reducedby strong light isdiscussed: the amount of the inhibitor transportedfrom leafto stem is increased under strong light, and in thestem, growthinhibition is caused by a direct effect of thisinhibitor ongrowth and by its inhibiting effect on the transportof IAAfrom leaf to stem.

¹ Present address: Botanical Garden, Faculty of Science, Universityof Tokyo, Tokyo (Received February 15, 1961; )     

Properties of the foot inhibitor from hydra

Summary A substance was isolated from crude extracts of hydra that inhibits foot regeneration. This substance, the foot inhibitor, has a molecular weight of 500 daltons. It is a hydrophilic molecule, slightly basic in character and it has no peptide bonds. The pruified substance acts specifically and at concentrations lower than 10^–7 M. At this low concentration only foot and not head regeneration is inhibited. Hydra are sensitive to purified foot inhibitor between the second and eight hour after initiation of foot regeneration by cutting. In normal animals the foot inhibitor is most likely produced by nerve cells. A substance with similar biological and physico-chemical properties is found in other coelenterates.     

Evidence for a proteinase inhibitor binding component associated with murine spermatozoa

The supernatants of frozen-thawed murine epididymal sperm suspensions contain a heat-labile component capable of binding a low molecular weight, acid-stable proteinase inhibitor of seminal vesicle origin. The substance has a molecular weight of approximately 15,000 and can be isolated by affinity chromatography using the inhibitor as the ligand. Although the substance has no inherent enzymatic properties, it will decrease the activity of the seminal inhibitor in the standard N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) assay. Enzyme-linked immunosorbent assays (ELISA) indicate that the substance, when bound to microtiter plates, is capable of binding the seminal vesicle inhibitor. Turkey egg white trypsin inhibitor will decrease the amount of the seminal inhibitor that will bind to the substance, while noninhibitor proteins, e.g., bovine serum albumin or insulin, have no effect. Turkey egg white and lima bean trypsin inhibitor will also decrease the amount of seminal vesicle inhibitor capable of binding to washed sperm. These data indicate the presence of an inhibitor acceptor site associated with murine epididymal spermatozoa.     

An endogenous regulator of diacylglycerol kinase

During the initial steps of the subcellular fractionation of rat brain homogenate, we recovered more than 100% of diacylglycerol kinase activity. The unusually high yields prompted us to examine the possibility that we had removed an endogenous inhibitor from diacylglycerol kinase during those steps. Our study revealed the existence of a potent inhibitor of diacylglycerol kinase in the crude synaptosomal-mitochondrial fraction (P2 pellet). The inhibitory substance was water soluble upon organic solvent extraction. The inhibitory activity of the substance was retained after extensive dialysis, suggesting the macromolecular nature of this compound. This substance may represent an important physiological regulator of diacylglycerol kinase.     

Changes in Endogenous Growth Regulators in the Gladiolus Corm during Dormancy

Changes in endogenous growth regulators in gladiolus corms during dormancy were studied using paper and column chromatography followed by a bioassay with the test for straight growth of Avena coleoptile. Corms were grown in the field or in a glass room of a phytotron at 20°C in the light. Another lot was grown in a dark room at 20°C in the dark. Half of the daughter corms in each lot were cold-treated for about one month and the other half were stored at room temperature after harvest. The earliest sprouting was seen in dark grown corms with cold treatment, and the latest sprouting in light grown corms without cold. This pattern was similar in each cultivar over a period of three years. Corms from both lots contained considerable amounts of inhibiting substance just after harvest. However, dark grown corms treated with cold showed a rapid decrease in inhibitor activity and an increase in promoter activity. On the other hand, in light grown corms without cold treatment there was inhibitor activity found consistently even after two months. —There appear to be two inhibiting zones in the chromato-grams. One of these contains two inhibitory substances, one of which was assumed to be abscisic acid.     

Some Properties of Amylase Inhibitor Produced by Streptomyces sp. No. 280

A strain of Streptomyces produces a new substance capable of inactivating some amylases. This has not been reported by previous workers.

This amylase inhibitor was purified by means of acetone treatment, active carbon adsorption and column chromatography on DEAE-cellulose.

It was dialyzable through a cellophane membrane and soluble in water and methyl alcohol. The inhibitor had a small molecular weight and was a peptide-like substance. The inhibiting substance was resistant to the temperatures, and acted as inhibitor of glucoamylase, bacterial saccharogenic α-amylase, salivary and pancreatic α-amylases.     

Studies on the electroretinogram. II. Model of an a-wave regenerator mechanism

A model is proposed to relate the regeneration of the ERGa-wave after partial light adaptation to the level of the light adaptation. The model assumes that thea-wave amplitude is a function of some reactive substance associated with ana-wave generator. The maximuma-wave amplitude occurs when the eye is fully dark adapted, and thea-wave generator initiator concentration is at a maximum. Thea-wave generator initiator concentration can be decreased by interacting with a product of the rhodopsin-light energy reaction, and increased by removal of this inhibitor. The removal of the inhibitor depends upon the isomerization of the all-trans-retinene to the 11-cis form. An excess of inhibitory material overa-wave generator initiator would cause a delay in the appearance of thea-wave until the excess inhibitory material is removed. This delay is a linear function of the logarithm of the adapting energy. The agreement of this model with the experimental ERG data is very good. Supported in whole by Public Health Service Research Grant No. NB-02522, from the National Institute of Neurological Diseases and Blindness, Bethesda, Maryland.     

Substance P contracts the human iris sphincter: possible modulation by endogenous enkephalinase.

Substance P-immunoreactive neurons have been found in the irides of many species including humans. In several species, substance P has been shown to induce contraction of the sphincter muscle but this action of substance P has not been previously demonstrated in the human eye. Using an eye cup model in which the sensitivity of the iris muscle to substance P is increased compared to the isolated sphincter muscle, we have observed that nanomolar amounts of substance P induced contraction of the sphincter in the human iris. This contractile response was enhanced in eyes pretreated with thiorphan, an enkephalinase inhibitor, suggesting that endogenous enkephalinase (E.C. 3.4.24.11) may modulate the substance P contraction in the human iris. Further support for this hypothesis was the finding of enkephalinase-like immunoreactivity and enzyme activity in the human iris sphincter muscle.     

Inhibition of Trypanosoma cruzi alpha-hydroxyacid dehydrogenase-isozyme II by N-isopropyl oxamate and its effect on intact epimastigotes

The effect of N-isopropyl oxamate on the activity of alpha-hydroxyacid dehydrogenase-isozyme II (HADH-isozyme II) from Trypanosoma cruzi was investigated. The kinetic studies showed that this substance was a competitive inhibitor of this isozyme. The attachment of the nonpolar isopropylic branched chain to the nitrogen of oxamate increased 12-fold the affinity of N-isopropyl oxamate for the active site of T. cruzi HADH-isozyme II. N-isopropyl oxamate was a selective inhibitor of HADH-isozyme II, since other T. cruzi dehydrogenases were not inhibited by this substance. Since HADH-isozyme II participates in the energy metabolism of T. cruzi, a trypanocidal effect can be expected with inhibitors of this isozyme. However, although it was not possible to detect any trypanocidal activity with N-isopropyl oxamate when the ethyl ester was tested as a possible trypanocidal prodrug, the expected trypanocidal effect was obtained, comparable to that obtained with nifurtimox and benznidazole.     

Antiviral substance from silkworm faeces: characterization of its antiviral activity

The antiviral activity of a substance (L4-1) purified from silkworm faeces was examined in an HVJ (Sendai virus)-LLC-MK2 cell system. Its antiviral effect depended on the period of light irradiation and was inhibited by sodium sulfite and anaerobic conditions. These results indicate that the antiviral activity of L4-1 is associated with active oxygen species produced from the substance. SDS-polyacrylamide gel electrophoretic analysis showed that viral proteins were damaged by this substance under light irradiation. The results suggest that the antiviral activity is due to damage to viral protein(s) caused by active oxygen species produced from L4-1.     

Light-induced ATP release from the lens

The recent discovery of the photoreceptor melanopsin in lens epithelial cells has opened the possibility of modulating this protein by light stimulation. Experiments carried out on New Zealand white rabbits have demonstrated that the release of ATP from the lens to the aqueous humor can be reduced either when a yellow filter or a melanopsin antagonist is used. Compared to control (1.10?±?0.15 μM ATP), the application of a yellow filter (λ465–480) reduced ATP in the aqueous humor 70%, while the melanopsin antagonist AA92593 reduced the presence of ATP 63% (n?=?5), an effect which was also obtained with the PLC inhibitor U73122. These results indicate that when melanopsin is blocked either by the lack of light, a filter, or an antagonist, the extracellular presence of ATP is significantly reduced. This discovery may be relevant, on the one hand, because many ocular physiological processes are controlled by ATP and, on the other hand, because it is possible to stimulate ATP release with just light and without using any added substance.     

A selective inhibitor of cAMP-dependent protein kinase isolated from an alkalophilic strain of Bacillus species

Many alkalophilic bacteria were found to produce inhibitors of protein kinases. We isolated a novel inhibitor of protein kinase from an alkalophilic strain of Bacillus species. This substance was A heat-stable peptide with a molecular weight of 13,000 daltons. It was found to be a selective inhibitor of cyclic AMP-dependent protein kinase (A kinase). The inhibition of a kinase by this substance was non-competitive with histone or ATP. It behaved distinctly; other known inhibitors such as H-7, H-8, Staurosporine, K-252 and Erbstatine inhibit protein kinase less selectively and their functions are competitive with either substrate or ATP. This inhibitor was found to bind to the regulatory subunits of A kinase and markedly inhibited the separation of the catalytic subunits from A kinase induced by the binding of cAMP despite of no effect on the binding of cAMP. Thus, the activation step of A kinase was influenced by this inhibitor. This molecule had no effect on the inhibition by cAMP of CHO cell proliferation. This may have been due to the inability of this molecule to reach the target in the cell. Modification of the molecule itself or the administration method is needed for cellular or animal application.     

Effect of Light on Cell Division in Developing Callus Cultures

Explants removed from the Jerusalem artichoke tuber and exposedto white light in the presence of 2, 4-D, when cultured in liquidmedia, exhibit much smaller dividing populations than similartissue not exposed to light. White light, which is effectiveonly in the presence of 2, 4-D and during the period beforethe onset of DNA replication, is required only in small amountsto promote a maximum effect, although inhibition of cell divisionwas never complete Light does not interfere with the timingof the cell cycle but exerts an influence on the size of thedividing population. The results presented are consistent witha hypothesis which postulates that a substance or substancesessential for cell division is reduced in amount by exposureto light. The extent of the first synchronous division is probablytherefore determined by the supply of this substance.     

Purification of a Na+/K+ ATPase inhibitor from borderline hypertensives' plasma

Increasing experimental evidences suggest an involvement of an endogenous Na+/K+ ATPase inhibitor in regulating water and electrolytes balance as well as in the pathogenesis of hypertension. However, conflicting results on the nature and the chemical structure of this substance still make it difficult to understand exactly its physiological mechanism of action. In the present study an attempt was made to purify a Na+/K+ ATPase inhibitor from hypertensives' plasma by solid phase extraction followed by 2 HPLC steps using reverse and normal phase columns. The fractions, from both columns, were able to inhibit Na+/K+ ATPase, 3H-ouabain binding to enzyme, ouabain sensitive 86Rb uptake and pNPPase activity in a manner not affected by boiling. Ultrafiltration experiments demonstrate that inhibitory activity is largely due to a low-molecular weight substance. These findings seem to confirm the presence in hypertensives plasma of a Na+/K+ ATPase inhibitor with some similarities with ouabain.     

Studies of the Growth in Culture of Excised Wheat Roots

Excised wheat roots in culture release a substance inhibitory to their growth and which can be absorbed on activated charcoal. This growth inhibitor can be isolated in pure form by diethylether extraction followed by DEAE cellulose and thin-layer chromatography. At very low concentration it inhibits lateral root development. The significance of this inhibitor to the problem of the continuous growth of cultured wheat roots is discussed.     

Effect of mitogen-activated protein kinases on chemokine synthesis induced by substance P in mouse pancreatic acinar cells

Substance P, acting via its neurokinin 1 receptor (NK1 R), plays an important role in mediating a variety of inflammatory processes. Its interaction with chemokines is known to play a crucial role in the pathogenesis of acute pancreatitis. In pancreatic acinar cells, substance P stimulates the release of NFκB-driven chemokines. However, the signal transduction pathways by which substance P-NK1 R interaction induces chemokine production are still unclear. To that end, we went on to examine the participation of mitogen-activated protein kinases (MAPKs) in substance P-induced synthesis of pro-inflammatory chemokines, monocyte chemoanractant protein-1 (MCP-I), macrophage inflammatory protein-lα (MIP-lα) and macrophage inflammatory protein-2 (MIP-2), in pancreatic acini. In this study, we observed a time-dependent activation of ERK1/2, c-Jun N-terminal kinase (JNK), NFκB and activator protein-1 (AP-1) when pancreatic acini were stimulated with substance P. Moreover, substance P-induced ERK 1/2, JNK, NFκB and AP-1 activation as well as chemokine synthesis were blocked by pre-treatment with either extracellular signal-regulated protein kinase kinase 1 (MEK1) inhibitor or JNK inhibitor. In addition, substance P-induced activation of ERK 112, JNK, NFκB and AP-1-driven chemokine production were attenuated by CP96345, a selective NK1 R antagonist, in pancreatic acinar cells. Taken together, these results suggest that substance P-NK1 R induced chemokine production depends on the activation of MAPKs-mediated NFκB and AP-1 signalling pathways in mouse pancreatic acini.     

Root Cap Inhibitor Formation in Isolated Root Caps of Zea mays

Root caps were isolated and cultured aseptically on variousdefined media. Under appropriate culture conditions plus suitableillumination a substance able to produce a positive geotropicresponse (i.e. a downward bending of roots) was formed in isolatedroot caps. The presence of this substance, or root cap inhibitor,was assessed by substituting cultured caps in place of capsof dark-grown roots. Normally these roots if kept in continuousdarkness will not respond to gravity (i.e. no bending). Optimalroot cap inhibitor production occurs on a relatively simplemedium, lacking sucrose, but supplemented with 10^–9 MIAA. Protein synthesis is necessary for inhibitor productionand/or expression, whereas DNA synthesis is not.