Localization of the carbon in caffeine biosynthesized from N-methyl carbon of {gamma}-glutamylmethylamide in tea plants

1. Localization of carbon in caffeine molecule biosynthesizedfrom the N-methyl carbon of -glutamylmethylamide in tea plantswas observed. ¹⁴C-Caffeine produced from ¹⁴C--glutamylmethylamidewas isolated and degraded. Approximately 26–55% of the¹⁴C was observed in the three methyl carbons in caffeine, withonly 2–3% at the C-2 carbon, 3–7% at the C-8 carbonposition. The amount of ¹⁴C at the C-4, C-5 and C-6 positionswas calculated from the results obtained. 2. The role of the N-methyl carbon of -glutamylmethylamide inthe formation of RNA in tea plants was examined. Incorporationof the N-methyl-¹⁴C of ¹⁴C--glutamylmethylamide into AMP andGMP in RNA was found. These facts indicate that in tea plants, -glutamylmethylamideis metabolized and most of its N-methyl carbon is utilized asa precursor for caffeine formation and little, if any, as aprecursor for nucleic acid formation. ¹ Present address: Department of Agricultural Chemistry, ShizuokaUniversity, Iwata, Shizuoka 438, Japan. (Received February 2, 1972; )     

The Nitrogen Metabolism of Groundnut Plants: the Role of {gamma}-methyleneglutamine and {gamma}-methyleneglutamic Acid

The changes occurring in the nitrogenous compounds during thegrowth of groundnut seedlings in the dark and light were compared,particular attention being centred on the levels of -methyleneglutamine,the principal amide of these plants, and -methyleneglutamicacid. The distribution of amino acids and amides in the mainorgans of normal young and mature plants was also examined.Suggestions are made concerning the possible pathways of synthesisand the functions of -methyleneglutamic acid and -methyleneglutaminein groundnut plants.     

Expression and location of caffeine synthase in tea plants

Tea caffeine synthase (CS) is a key enzyme in tea plant for synthesis of caffeine. We firstly systematically investigated the gene expression of CS. Northern blot analysis showed that CS gene was not expressed in stems and roots but efficiently expressed in leaves of tea plants. The expression level of CS gene in summer-grown leaves was much higher than in spring leaves. Its expression level in leaves of the shaded or fertilized tea plants was significantly higher. Along the same shoot, CS gene was expressed at much higher level in the young leaves (bud and the first leaf) than in the more mature second and third leaves. RNA in situ hybridization indicated that tea CS gene was mainly expressed in the palisade parenchyma and the epicuticle of leaves but less expressed in the spongy parenchyma and the hypoderm. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 5, pp. 786–789. The text was submitted by the authors in English.     

Crystallin {gamma}B-I4F mutant protein binds to {alpha}-crystallin and affects lens transparency

A new mouse mutant line, Clapper, identified from N-ethyl-N-nitrosurea (ENU)-mutagenized mice, develops a dominant lamellar cataract. The cataract blocks the image of retinal fundus and transmits a fuzzy fluorescein image of retinal vasculature during angiography. The cataractous lens opacity decreases as the mice age. The Clapper mutation has been identified to be a missense mutation of the gammaB-crystallin gene that replaces the 4th isoleucine residue with a phenylalanine (gammaB-I4F). Unlike wild type gammaB, the gammaB-I4F mutant protein binds to alpha-crystallin to form high molecular weight complexes in vivo and in vitro. Circular dichroism measurements indicate that gammaB-I4F protein is less stable than wild type gammaB at high temperature. Darkly stained aggregates, enlarged interfiber spaces, and disorganized and smaller inner mature fibers were found in the regions of the cataract in homozygous Clapper mutant lenses. Thus, the lamellar cataract is likely due to the light-scattering effects of the enlarged interfiber spaces and protein aggregates caused by gammaB-I4F mutant proteins interacting with alpha-crystallin in the lens.     

Synergistic Ca2+ responses by G{alpha}i- and G{alpha}q-coupled G-protein-coupled receptors require a single PLC{beta} isoform that is sensitive to both G{beta}{gamma} and G{alpha}q

Cross-talk between Gα(i)- and Gα(q)-linked G-protein-coupled receptors yields synergistic Ca(2+) responses in a variety of cell types. Prior studies have shown that synergistic Ca(2+) responses from macrophage G-protein-coupled receptors are primarily dependent on phospholipase Cβ3 (PLCβ3), with a possible contribution of PLCβ2, whereas signaling through PLCβ4 interferes with synergy. We here show that synergy can be induced by the combination of Gβγ and Gα(q) activation of a single PLCβ isoform. Synergy was absent in macrophages lacking both PLCβ2 and PLCβ3, but it was fully reconstituted following transduction with PLCβ3 alone. Mechanisms of PLCβ-mediated synergy were further explored in NIH-3T3 cells, which express little if any PLCβ2. RNAi-mediated knockdown of endogenous PLCβs demonstrated that synergy in these cells was dependent on PLCβ3, but PLCβ1 and PLCβ4 did not contribute, and overexpression of either isoform inhibited Ca(2+) synergy. When synergy was blocked by RNAi of endogenous PLCβ3, it could be reconstituted by expression of either human PLCβ3 or mouse PLCβ2. In contrast, it could not be reconstituted by human PLCβ3 with a mutation of the Y box, which disrupted activation by Gβγ, and it was only partially restored by human PLCβ3 with a mutation of the C terminus, which partly disrupted activation by Gα(q). Thus, both Gβγ and Gα(q) contribute to activation of PLCβ3 in cells for Ca(2+) synergy. We conclude that Ca(2+) synergy between Gα(i)-coupled and Gα(q)-coupled receptors requires the direct action of both Gβγ and Gα(q) on PLCβ and is mediated primarily by PLCβ3, although PLCβ2 is also competent.     

Accumulation of {gamma}-Aminobutyric Acid due to Ammonium in Various Cultured Plant Cells

The accumulation of -aminobutyric acid (GABA) resulting fromthe addition of ammonium was investigated with various culturedplant cells including carrot, corn, peanut, rice, rue, soybean,tobacco and wheat. All rice cell lines tested (10 japonica,12 indica types and 3 Oryza species other than 0. saliva) showedmarked accumulation of GABA in the presence of ammonium in themedium. This accumulation was observed in not only seed-derivedrice cells but also anther-derived rice cells. But culturedcells other than rice did not accumulate GABA. Alanine (in cornand wheat) and glutamine in carrot, peanut, rue, soybean andtobacco) were the most abundant free amino acids in those cellsin the presence of ammonium. These results indicate that theaccumulation of GABA due to the addition of ammonium occursspecifically in rice cells and not in the cells of genera otherthan Oryza thus far investigated. (Received January 24, 1987; Accepted September 1, 1987)     

Metabolism of glutamate and {gamma}-aminobutyrate in cultured tobacco cells

In cultured tobacco cells glutamate-U-¹⁴C administrated wasreadily converted to -aminobutyrate (GABA) by decarboxylation,however, GABA-1-¹⁴C remained unchanged. Glutamate decarboxylasewas found in tobacco cells and reached its maximum activityin the rapidly growing stage during culture. Enzyme activityparalleled formation of GABA from glutamate-U-¹⁴C. A high contentof GABA in tobacco cells seems to be due to the rapid decarboxylationof glutamate by glutamate decarboxylase and a slow turn overof GABA. ¹ Present address: The Okayama Tobacco Experiment Station, JapanMonopoly Corp., Tamashima, Kurashiki, Japan. (Received November 20, 1971; )     

Analysis of binding sites in human C-reactive protein for Fc{gamma}RI, Fc{gamma}RIIA, and C1q by site-directed mutagenesis

Human C-reactive protein (CRP) is a classical, acute phase serum protein synthesized by the liver in response to infection, inflammation, or trauma. CRP binds to microbial antigens and damaged cells, opsonizes particles for phagocytosis and regulates the inflammatory response by the induction of cytokine synthesis. These activities of CRP depend on its ability to activate complement and to bind to Fcgamma receptors (FcgammaR). The goal of this study was to elucidate amino acid residues important for the interaction of CRP with human FcgammaRI (CD64) and FcgammaRIIa (CD32). Several mutations of the CRP structure were studied based on the published crystal structure of CRP. Mutant and wild-type recombinant CRP molecules were expressed in the baculovirus system and their interactions with FcgammaR and C1q were determined. A previous study by our laboratory identified an amino acid position, Leu(176), critical for CRP binding to FcgammaRI and work by others (Agrawal, A., Shrive, A. K., Greenhough, T. J., and Volanakis, J. E. (2001) J. Immunol. 166, 3998-4004) determined several residues important for C1q binding. The amino acid residues important to CRP binding to FcgammaRIIa were previously unknown. This study newly identifies residues Thr(173) and Asn(186) as important for the binding of CRP to FcgammaRIIa and FcgammaRI. Lys(114), like Leu(176), was implicated in binding to FcgammaRI, but not FcgammaRIIa. Single mutations at amino acid positions Lys(114), Asp(169), Thr(173), Tyr(175), and Leu(176) affected C1q binding to CRP. These results further identify amino acids involved in the binding sites on CRP for FcgammaRI, FcgammaRIIa, and C1q and indicate that these sites are overlapping.     

Metabolic conversion of 24-methyl-Delta25-cholesterol to 24-methylcholesterol in higher plants

Feeding of chemically synthesized [27-13C]codisterol ([27-13C]2), [27-13C]24-epicodisterol ([27-13C]3), [23,24-2H2]codisterol ([23,24-2H2]2), and [26,27-2H6]24-methyldesmosterol ([26,27-2H6]8) to Oryza sativa cell cultures, followed by MS and NMR analysis of the biosynthesized dihydrobrassicasterol (9)/campesterol (10), revealed that both (24R)- and (24S)-epimers of 24-methyl-Delta25-cholesterol (2/3) were converted to 9 and 10 via the common intermediate 24-methyldesmosterol (8).     

Observation volumes and {gamma}-factors in two-photon fluorescence fluctuation spectroscopy

Fluorescence fluctuation spectroscopy has become an important measurement tool for investigating molecular dynamics, molecular interactions, and chemical kinetics in biological systems. Although the basic theory of fluctuation spectroscopy is well established, it is not widely recognized that saturation of the fluorescence excitation can dramatically alter the size and profile of the fluorescence observation volume from which fluorescence fluctuations are measured, even at relatively modest excitation levels. A precise model for these changes is needed for accurate analysis and interpretation of fluctuation spectroscopy data. We here introduce a combined analytical and computational approach to characterize the observation volume under saturating conditions and demonstrate how the variation in the volume is important in two-photon fluorescence correlation spectroscopy. We introduce a simple approach for analysis of fluorescence correlation spectroscopy data that can fully account for the effects of saturation, and demonstrate its success for characterizing the observed changes in both the amplitude and relaxation timescale of measured correlation curves. We also discuss how a quantitative model for the observed phenomena may be of broader importance in fluorescence fluctuation spectroscopy.     

Ligand- and subunit-specific conformational changes in the ligand-binding domain and the TM2-TM3 linker of {alpha}1 {beta}2 {gamma}2 GABAA receptors

Cys-loop receptor ligand binding sites are located at subunit interfaces where they are lined by loops A-C from one subunit and loops D-F from the adjacent subunit. Agonist binding induces large conformational changes in loops C and F. However, it is controversial as to whether these conformational changes are essential for gating. Here we used voltage clamp fluorometry to investigate the roles of loops C and F in gating the α1 β2 γ2 GABA(A) receptor. Voltage clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Previous attempts to define the roles of loops C and F using this technique have focused on homomeric Cys-loop receptors. However, the problem with studying homomeric receptors is that it is difficult to eliminate the possibility of bound ligands interacting directly with attached fluorophores at the same site. Here we show that ligands binding to the β2-α1 interface GABA binding site produce conformational changes at the adjacent subunit interface. This is most likely due to agonist-induced loop C closure directly altering loop F conformation at the adjacent α1-β2 subunit interface. However, as antagonists and agonists produce identical α1 subunit loop F conformational changes, these conformational changes appear unimportant for gating. Finally, we demonstrate that TM2-TM3 loops from adjacent β2 subunits in α1 β2 receptors can dimerize via K24'C disulfides in the closed state. This result implies unexpected conformational mobility in this crucial part of the gating machinery. Together, this information provides new insights into the activation mechanisms of Cys-loop receptors.     

The Enzymic Decarboxylation of {gamma}-methyleneglutamic Acid by Plant Extracts

-Methyleneglutamic acid, an acidic amino-acid isolated fromgroundnut plants, was decarboxylated by enzymes present in extractsof Capsicum fruits, barley roots, and tulip leaves, and alsoby intact cells of Clostridium welchii S.R. I2. The amino-acidwas attacked in a similar manner to, but in all cases at a slowerrate than, l-glutamic acid. The nature of the enzyme responsiblefor the decarboxylation of -methyleneglutamic acid was furtherinvestigated using preparations from barley roots (which donot contain the amino-acid) and from tulip leaves (in whichthe amino-acid is normally present, together with larger amountsof its amide form, -methyleneglutamine). The effects of pH,inhibitors, and partial heat denaturation upon the enzyme systemspresent in the barley and tulip extracts indicated that a singleenzyme was responsible for the decarboxylation of both l-glutamicacid and -methyleneglutamic acid. Although the Cl. welchii rapidlydeamidated and then decarboxylated l-glutamine, -methyleneglutaminewas not attacked by the organism.     

G-protein {beta}{gamma} Subunit Genes Expressed in Olfactory Receptor Neurons

The expression of genes encoding G-protein ß subunitswas investigated in isolated olfactory receptor neurons fromchannel catfish. DNA sequencing of PCR products showed thatthe ß1, ß2, 2 and 3 genes were expressedin the neurons. Western blotting showed that at least threeof these subunit proteins were expressed. This first analysisof the expression of ß genes in olfactory receptorneurons suggests that these subunits may be involved in a varietyof transduction events in these cells. Chem. Senses 22: 587–592,1997.     

Accumulation of {gamma}-aminobutyric acid due to adding ammonium or glutamine to cultured rice cells

-Aminobutyric acid (GABA) was accumulated in rice cell cultureswhen ammonium was added to the medium. After the addition ofammonium, a temporary increase in the glutamine (Gln) pool wasobserved before the accumulation of GABA. GABA also was markedlyaccumulated when Gln was added to the medium in place of ammonium.When glutamic acid (Glu) was added without ammonium, no accumulationof GABA occurred. When L-methionine-DL-sulfoximine (MSO), an inhibitor of glutaminesynthetase, was added to the nitrate medium, the ammonium poolincreased with no accumulation of Gln and GABA. Even when ammoniumwas supplied to the medium, no GABA accumulated in the presenceof MSO. When azaserine (AZ), which inhibits the transamidationof Gln, was added with Gln, no GABA was accumulated, althoughthe Gln pool in the cell cultures increased significantly. The accumulation of GABA in cultured rice cells produced byammonium as the nitrogen source probably is related directlyto the Gln pool size, which is increased when ammonium is suppliedto the medium. (Received August 6, 1979; )     

PDCD10/CCM3 acts downstream of {gamma}-protocadherins to regulate neuronal survival

γ-Protocadherins (PCDH-γ) regulate neuronal survival in the vertebrate central nervous system. The molecular mechanisms of how PCDH-γ mediates this function are still not understood. In this study, we show that through their common cytoplasmic domain, different PCDH-γ isoforms interact with an intracellular adaptor protein named PDCD10 (programmed cell death 10). PDCD10 is also known as CCM3, a causative genetic defect for cerebral cavernous malformations in humans. Using RNAi-mediated knockdown, we demonstrate that PDCD10 is required for the occurrence of apoptosis upon PCDH-γ depletion in developing chicken spinal neurons. Moreover, overexpression of PDCD10 is sufficient to induce neuronal apoptosis. Taken together, our data reveal a novel function for PDCD10/CCM3, acting as a critical regulator of neuronal survival during development.     

The Role of Glutamate Decarboxylase and {gamma}-Aminobutyric Acid in Germinating Barley

The variations in glutamate decarboxylase activity and in glutamicacid and -ABA concentration have been measured in barley embryosduring the uptake of water and in the roots and shoots for upto 6 days of growth. Glutamate decarboxylase activity was relativelysteady in the embryos during soaking but rose rapidly once growthbegan. This development paralleled an increase in the concentrationof glutamic acid in both roots and shoots at a time when theconcentration of -ABA was falling. During soaking in aeratedwater, the -ABA content of the embryos rose for 36 h, at whichpoint it accounted for 35 per cent of the soluble amino acids.-ABA was found to be a major free amino acid in roots but notin shoots. Experiments in vivo involving ¹⁴C-labelled glutamicacid and -ABA indicated that carbon from -ABA passed very rapidlyinto the citric-acid cycle intermediates and also that, throughoutthe period studied, -ABA was formed from glutamic acid despitethe alterations in relative concentrations of these amino acidsin the growing tissue.     

{gamma}-Protocadherins, presenilin-mediated release of C-terminal fragment promotes locus expression

gamma-Protocadherins (gamma-pcdhs) are type I membrane-spanning glycoproteins, widely expressed in the mammal and required for survival. These cell adhesion molecules are expressed from a complex locus comprising 22 functional variable exons arranged in tandem, each encoding extracellular, transmembrane and intracellular sequence, and three exons for an invariant C-terminal domain (gamma-ICD). However, the signaling mechanisms that lie downstream of gamma-pcdhs have not been elucidated. Here we report that gamma-pcdhs are subject to presenilin-dependent intramembrane cleavage (PS-IP), accompanied by shedding of the extracellular domain. The cleaved intracellular domain (gamma-ICD) translocates to the cell nucleus and was detected in subsets of cortical neurons. Notably, gene-targeted mice lacking functional gamma-ICD sequence showed severely reduced gamma-pcdh mRNA levels and neonatal lethality. Most importantly, inhibition of gamma-secretase decreased gamma-pcdh locus expression. Luciferase reporter assays demonstrated that gamma-pcdh promoter activity is increased by gamma-ICD. These results reveal an intracellular signaling mechanism for gamma-pcdhs and identify a novel vital target for the gamma-secretase complex.     

G protein {beta}{gamma} gating confers volatile anesthetic inhibition to Kir3 channels

G protein-activated inwardly rectifying potassium (GIRK or Kir3) channels are directly gated by the βγ subunits of G proteins and contribute to inhibitory neurotransmitter signaling pathways. Paradoxically, volatile anesthetics such as halothane inhibit these channels. We find that neuronal Kir3 currents are highly sensitive to inhibition by halothane. Given that Kir3 currents result from increased Gβγ available to the channels, we asked whether reducing available Gβγ to the channel would adversely affect halothane inhibition. Remarkably, scavenging Gβγ using the C-terminal domain of β-adrenergic receptor kinase (cβARK) resulted in channel activation by halothane. Consistent with this effect, channel mutants that impair Gβγ activation were also activated by halothane. A single residue, phenylalanine 192, occupies the putative Gβγ gate of neuronal Kir3.2 channels. Mutation of Phe-192 at the gate to other residues rendered the channel non-responsive, either activated or inhibited by halothane. These data indicated that halothane predominantly interferes with Gβγ-mediated Kir3 currents, such as those functioning during inhibitory synaptic activity. Our report identifies the molecular correlate for anesthetic inhibition of Kir3 channels and highlights the significance of these effects in modulating neurotransmitter-mediated inhibitory signaling.