Characterization of a substance P-Gly12 amidating enzyme in human cerebrospinal fluid

Enzyme activity capable of converting the glycine-extended substance P precursor, substance P-Gly12, into substance P was purified from human cerebrospinal fluid. The conversion reaction was monitored by radioimmunoassay measurement of substance P formation. The chemical identity of the product was verified by reversed-phase HPLC. The enzyme reaction was stimulated by Cu(II) ion and ascorbic acid and inhibited by the presence of diethyldithiocarbamate. By HPLC molecular sieving, the major enzyme activity appeared as a protein of 26,000 molecular weight.     

Comparison of the effect of excessive light on chlorophyll fluorescence (77K) and photon yield of O2 evolution in leaves of higher plants

High-light treatments (1750–2000 mol photons m^–2 · s^–1) of leaves from a number of higher-plant species invariably resulted in quenching of the maximum 77K chlorophyll fluorescence at both 692 and 734 nm (F _M, 692 and F _M, 734). The response of instantaneous fluorescence at 692 nm (F _O, 692) was complex. In leaves of some species F _O, 692 increased dramatically in others it was quenched, and in others yet it showed no marked, consistent change. Regardless of the response of F _O, 692 an apparently linear relationship was obtained between the ratio of variable to maximum fluorescence (F _V/F _M, 692) and the photon yield of O₂ evolution, indicating that photoinhibition affects these two variables to approximately the same extent. Treatment of leaves in a CO₂–free gas stream containing 2% O₂ and 98% N₂ under weak light (100 mol · m^–2 · s^–1) resulted in a general and fully reversible quenching of 77K fluorescence at 692 and 734 nm. In this case both F _O, 692 and F _M, 692 were invariably quenched, indicating that the quenching was caused by an increased non-radiative energy dissipation in the pigment bed. We propose that high-light treatments can have at least two different, concurrent effects on 77K fluorescence in leaves. One results from damage to the photosystem II (PSII) reaction-center complex and leads to a rise in F _O, 692; the other results from an increased non-radiative energy dissipation and leads to quenching of both F _O, 692 and F _M, 692 This general quenching had a much longer relaxation time than reported for pH-dependent quenching in algae and chloroplasts. Sun leaves, whose F _V/F _M, 692 ratios were little affected by high-light exposure in normal air, suffered pronounced photoinhibition when the exposure was made under conditions that prevent photosynthetic gas exchange (2% O₂, 0% CO₂). However, they were still less susceptible than shade leaves, indicating that the higher capacity for energy dissipation via photosynthesis is not the only cause of their lower susceptibility. The rate constant for recovery from photoinhibition was much higher in mature sun leaves than in mature shade leaves, indicating that differences in the capacity for continuous repair may in part account for the difference in their susceptibility to photoinhibition.Abbreviations and symbols kDa kilodalton - LHC-II light-harvesting chlorophyll-protein complex - PFD photon flux density (photon fluence rate) - PSI, PSII photosystem I, II - F _O, F _M, F _V instantaneous, maximum, variable fluorescence emission - absorptance - _a photon yield of O₂ evolution (absorbed light) C.I.W.-D.P.B. Publication No. 925     

Localization of ornithine decarboxylase in the chick embryo during organogenesis

Summary The localization of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis and thus in cell growth, was determined in the 4.5-day-old chick embryo, using two independent methods of analysis. ODC protein was identified by indirect immunofluorescence with a monospecific ODC antibody, and catalytically active ODC was identified by autoradiography with -(5-³H) difluoromethylornithine. Both methods revealed a basically similar distribution of ODC within the embryo. Among the organs, the brain exhibited the highest ODC levels. ODC levels were also high in spinal cord, mesonephric tubules and heart. Similar levels, but confined to limited areas, were found in liver tissue, head mesenchyme, and the oral and pharyngeal regions. Organs that exhibited high ODC levels are all engaged in rapid growth, as well as in extensive tissue remodeling and differentiation.     

Molecular heterogeneity of angiotensin converting enzyme in human cerebrospinal fluid.

Three different molecular forms of angiotensin converting enzyme (ACE) (approximately Mr 150,000, 80,000 and 40,000, respectively), have been recovered from human cerebrospinal fluid. All three enzymes were inhibited by captopril and enalapril and their activity was potentiated by chloride ions. They were capable of degrading Leu-enkephalin-Arg6 and substance -P, but gave no conversion of neurokinin A. In all these aspects, the CSF enzymes were identical with the human pulmonary enzyme. The Mr 40,000 form of ACE is the smallest active form of the enzyme hitherto reported and is likely to represent a fragment of the C-terminal part of native ACE, where its active center is located.     

Immunohistochemical localization of porcine diazepam-binding inhibitor (DBI) to rat endocrine pancreas

Summary The occurrence of diazepam-binding inhibitor (DBI), isolated and characterized from porcine upper intestine, was examined in the pancreas of Sprague-Dawley albino rats using indirect immunofluorescence. The polypeptide was found in the endocrine Langerhans islets and, utilizing double-labelling controls, it was shown to be present within the peripherally located glucagon-containing cells. Regulation of islet hormone production may therefore be under DBI control.     

The pollination ecology of two nectarless Cimicifuga sp. (Ranunculaceae) in North America

The pollination ecology of Cimicifuga rubifolia Kearney and C. elata Nutt., endemic nectarless species in Tennessee and the Pacific Northwest, respectively, was studied. Both species are pollinated by bumblebee workers, which run over the flowers pressing their bodies against them. Large syrphids act as copollinators. Extremely high pollen/ovule ratios are explained as optimal sex allocation in response to pollen-tolling by the pollen-collecting pollinators. Facultative geitonogamy in the self-compatible C. elata seems to be evolved in response to very low pollinator density in the habitat. Because of the pollinator behavior, geitonogamy virtyally always precedes xenogamy. C. rubifolia encounters higher pollinator visit frequency than C. elata , and self-incompatibility prevents selfing. Since it is nectarless, C. rubifolia is inferior in competion for bumblebees with most other entomogamous plants. It appears to have a commensalistic relationship with its constant associates, Impatiens pallida (Balsaminaceae) and/or Polymnia canadensis (Asteraceae), which attract large numbers of bumblebees. C. rubifolia is pollinated by bumblebees encountering them when scouting around the nectar-flower stand. This conclusion is based on: 1) Negative correlation for distance between nectar-flowers and Cimicifuga and the fruit set of the latter, 2) non-skewed flowering period, 3) extremely extended flowering on both the ramet and population levels, only limited by flowering period of nectar-flower associates and by pollinator abundance, and 4) fruit set rates independent of visual display size, and also of flower density in the habitat (i.e., the pollination rate was directly proportional to the density).     

Polyamine depletion increases cellular ribonucleotide levels

Summary Depletion of the putrescine and spermidine content of Ehrlich ascites tumor cells by -difluoromethylornithine (DFMO) treatment results in at least a 1500-fold increase in the decarboxylated S-adenosylmethionine (deSAM) content. The accumulation of this adenine nucleoside occurs because of the absence of putrescine and spermidine to act as aminopropyl group acceptors in the spermidine and spermine synthase reactions and because of an increase in S-adenosylmethionine decarboxylase activity. The fact that the synthesis of deSAM continues in DFMO-treated cells makes the pathway an adenine trap. This prompted a study of the adenine nucleotide pools. High-performance liquid chromatographic analysis showed that the total adenine nucleotide pool increased, rather than decreased, as a result of DFMO treatment; the major contributors to the increase being ATP and ADP, which increased 2.6 and 1.9 times, respectively. The cellular content of other ribonucleotides increased as well, particularly that of UTP and CTP. When putrescine was added together with DFMO, the increases in cellular ribonucleotide contents were prevented, showing that they were indeed caused by polyamine depletion.     

Neuropeptide Y,enkephalin and noradrenaline coexist in sympathetic neurons innervating the bovine spleen

Summary The subcellular distribution of noradrenaline (NA), neuropeptide Y (NPY), Met and Leu-enkephalin (ENK), substance P (SP), somatostatin (SOM), and vasoactive intestinal polypeptide (VIP) was investigated in homogenates of bovine splenic nerve. The distribution of noradrenergic peptide-containing nerves in the bovine celiac ganglion, splenic nerve and terminal areas in spleen was studied by indirect immunofluorescence histochemistry using antisera to tyrosine hydroxylase (TH), dopamine--hydroxylase (DBH), NPY, enkephalin peptides, SP, SOM, VIP and peptide HI (PHI).After density gradient centrifugation, high levels of NPY and ENK-like immunoreactivity (LI) were found in high-density gradient fractions, coinciding with the main NA peak. SP, SOM and VIP were found in fractions with a lower density, VIP being also enriched in a heavy fraction; the latter three peptides were present in low concentrations.Immunohistochemistry revealed that staining for NPYLI and ENK-LI partly overlapped that for TH and DBH in celiac ganglia, splenic nerve axons and terminal areas of spleen. Almost all principal ganglion cells were TH- and DBH-immunoreactive. Many were also NPY-immunoreactive, whereas a smaller number were ENK-positive. In the celiac ganglion patches of dense SP-positive networks and some VIP/PHI- and ENK-immunoreactive fibers were seen around cell bodies.The results indicate that NPY and ENK are stored with NA in large dense-cored vesicles in unmyelinated axons of bovine splenic nerve. SP, SOM and VIP appear in different organelles in axon populations separate from sympathetic noradrenergic nerves.