Curculin,a sweet-tasting and taste-modifying protein,is a non-functional mannose-binding lectin

A three-dimensional model of curculin, a sweet-tasting and taste-modifying protein from the fruits of Curculigo latifolia, was built from the X-ray coordinates of GNA, a mannose-binding lectin from snowdrop (Galanthus nivalis). The three mannose-binding sites present in GNA were found in curculin but are devoid of mannose-binding activity as shown by docking experiments performed with mannose. Some regions well exposed on the surface of the three-dimensional model of curculin could act as epitopes responsible for the sweet-tasting properties of this protein.     

The Taste-active Regions of Monellin, a Potently Sweet Protein

Monellin, a protein found in the berries of the West Africanplant Dioscoreophyllum cumminsii, is one of the most potentlysweet compounds known. The native three-dimensional structureof monellin is required for sweetness, and this protein hasbeen the subject of intense research in an attempt at understandingthe structural basis for its taste activity. We have used structure-basedsite-directed mutagenesis to delineate the taste-active site(s)of monellin, and we present these results, along with similarwork from M. Kohmura, Y. Ariyoshi and coworkers, in the lightof the three-dimensional structure of this protein. The mutagenesis work suggests that at least four residues, locatedN-terminal to the     

X-Ray Analysis of New Crystal Forms of the Sweet Protein Thaumatin

Abstract

Thaumatin is a plant protein that in the mature form contains 8 disulfide bonds and 207 amino acids. Several forms of this protein occur naturally and each elicits an intense sweetness sensation when tasted in microgram quantities. The two major forms of thaumatin are easily separable by ion exchange chromatography. Crystals of the two proteins (designated here A and B) have been grown by vapor equilibration from solutions containing polyethylene glycol and examined by X-ray diffraction. The thaumatin A crystals are of space group P2₁2₁2₁ with a=44.3Å, b=63.7Åand c=72.7A. The crystals of thaumatin B are of space group C2 with a = 117.7Å, b=44.9Å, and c=38.0Å and β=94.0°. Both crystals diffract to well beyond 2.3Å and appear suitable for high resolution structure analysis. Four heavy atom derivatives of thaumatin B have been generated and diffraction data to 4Å resolution have been collected. This work is designed to provide a basis for studying the 3-dimensional structure of more than 100 genetically generated thaumatin derivatives, several of which show enhanced stability and improved taste characteristics.     

Structural Basis of pH Dependence of Neoculin,a Sweet Taste-Modifying Protein

Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness.     

Fos Protein as a Marker of Neuronal Activity: a Useful Tool in the Study of the Mechanism of Action of Natural Products with Analgesic Activity

Pain treatment is still ineffective in many conditions and remains one of the greatest challenges of modern medicine. Historically, due to the incredible variety of pharmacologically promising natural products (NPs) and the chemical complexity of their compounds, scientists have explored their use as a source of treatment for diseases or symptomatology. Fos protein and its precursor, the gene c-Fos, have been the subject of study in relation to the pathophysiology of pain as a possible tool to aid in its understanding. More recently, it has become a useful tool in the study of NPs with analgesic profile. Thus, this systematic review aimed to investigate the analgesic effect of NPs and derivatives through changes in Fos protein or c-Fos expression in nervous system central. The search terms “analgesics,” “Fos,” and “drug effects” were used in the databases PubMed, MEDLINE, Scopus, and Embase. Forty-six articles were identified. Twenty-five articles investigated Fos expression in the spinal cord, 1 in dorsal root ganglion, 11 in brain areas, and 9 investigated the association between the spinal cord and brain areas. Although Fos protein expression has been used as a tool in the studies of the mechanism of action of pain in relation to NPs with analgesic activity, the associations between brain areas and the spinal cord—and the possible pathways involved—have not yet been fully elucidated and deserve further study.     

Brazzein:一种耐热的甜味蛋白

Brazzein是从非洲西部野生植物Pentadiplandra brazzeana Baillon的果实中提取的一种甜味蛋白。Brazzein由54个氨基酸残基组成,Mr为6500,等电点为5,并且具有很好的热稳定性。本文着重概述Brazzein的结构与功能的关系,及其在基因工程中的研究前景。     

甜蛋白Monellin基因在大肠杆菌中的高效表达

据已报道的单链monellin甜蛋白的氨基酸序列,采用细菌偏爱密码子,人工合成了全长 294bp的 monellin基因。插入到大肠杆菌表达载体Pet_22b中,构建重组分泌型表达载体Petmo。经IPTG诱导Petmo所含有的甜蛋白基因可在大肠杆菌BL21（DE3）中高效表达,表达量占菌体可溶性蛋白的44.8％。且经纯化后测定其甜度是蔗糖的3000倍。得到的甜蛋白热稳性及耐酸性均比天然产物有所提高。     

Non-Acidic Compounds Induce the Intense Sweet Taste of Neoculin,a Taste-Modifying Protein

Neoculin, a sweet protein found in the fruit of Curculigo latifolia, has the ability to change sourness into sweetness. Neoculin turns drinking water sweet, indicating that non-acidic compounds may induce the sweetness. We report that ammonium chloride and certain amino acids elicit the intense sweetness of neoculin. Neoculin can thus sweeten amino acid-enriched foods.     

A Major Jasmonate-Inducible Protein of Sweet Potato, Ipomoelin, is an ABA-Independent Wound-Inducible Protein

Treatment of sweet potato plants cultured in vitro with a vaporof methyl jasmonate (MeJA) induced an accumulation in leavesof a large amount of protein with an apparent molecular massof 18 kDa. This protein, designated ipomoelin, was purified,and the amino acid sequences of proteolytic fragments were determined.Screening a cDNA library of MeJA-treated leaves by oligonucleotideprobes designed from the peptide sequences identified a clonethat could code for a polypeptide with 154 amino acids. Thededuced amino acid sequence of ipomoelin showed an overall aminoacid identity of 25% with the salt-inducible SalT protein ofrice. In addition, the C-terminal 70 amino acid sequence ofipomoelin showed about 50% identity with the C-terminal aminoacid sequences of seed lectins from Moraceae. The gene for ipomoelinwas present in a few copies in the genome of sweet potato. ThemRNA for ipomoelin was detected in leaves and petioles, butnot in stems and tuberous roots, of sweet potato plants grownin the field. Mechanical wounding of leaves induced ipomoelinmRNA both locally and systemically, while treatment of leaveswith ABA, salt, or a high level of sucrose did not induce ipomoelinmRNA. By contrast, ABA-inducible mRNA for sporamin was not inducedby MeJA. These results suggest that ipomoelin is involved indefensive reactions of leaves in response to wounding and thatJA-mediated wound-induction of ipomoelin occurs independentlyof ABA. (Received January 6, 1997; Accepted March 13, 1997)     

Biological Activity and Mode of Action of a Specific Antiphage Substance,AFS

Purified AFS (anti-filamentous phage substance) produced by Streptomyces lavendulae AM–7a showed specific antiphage activity against the male specific, deoxyribonucleic acid-containing filamentous phages of Escherichia coli without any activity against other DNA-phages nor the male-specific ribonucleic acid-containing phages of E. coli. AFS brought about no inactivation of free particles of filamentous phage, fl, nor the receptor of the host cells for the phage, while it showed strong killing effect against the fl-infected host cells at the concentration below 0.01 μg/ml. Antiphage activity of AFS might be due to its highly specific killing effect only on the E. coli cells infected with the filamentous DNA phages, while it exerted no effect on the growth of the unifected E. coli nor other microorganisms. Killing by AFS seemed to require the energy metabolism of the phage-infected host cells. Macro-molecular synthesis and respiration of the infected host cells were inhibited soon after the addition of small amounts of AFS without any cell lysis.     

Water Stress and Protein Synthesis: V. Protein Synthesis, Protein Stability, and Membrane Permeability in a Drought-sensitive and a Drought-tolerant Moss

The effects have been studied of water stress and desiccation on protein synthesis in the drought-tolerant moss Tortula ruralis and the drought-sensitive moss Hygrohypnum luridum. At any particular level of steady state water stress, the inhibition of protein synthesis was greater in H. luridum than in T. ruralis. Water stress-induced changes in the pattern of protein synthesis, as determined by the double label ratio technique, were minor in T. ruralis, but major in H. luridum. Proteins of both mosses were found to be stable during desiccation and subsequent rehydration. Changes in membrane permeability, as indicated by the leakage of amino acid, were observed during rehydration of desiccated moss and were dependent on the rate of desiccation. The leakage was small and reversible in T. ruralis but large and irreversible in H. luridum. Although H. luridum failed to recover from complete desiccation (80% loss in fresh weight), it was able to recover fully from steady state stress under conditions where a maximum loss of 55% in fresh weight was recorded.     

Zif,the Zoocin A Immunity Factor,Is a FemABX-Like Immunity Protein with a Novel Mode of Action

Producer cell immunity to the streptococcolytic enzyme zoocin A, which is a d-alanyl-l-alanine endopeptidase, is due to Zif, the zoocin A immunity factor. Zif has high degrees of similarity to MurM and MurN (members of the FemABX family of proteins), which are responsible for the addition of amino acids to cross bridges during peptidoglycan synthesis in streptococci. In this study, purified peptidoglycans from strains with and without zif were compared to determine how Zif modifies the peptidoglycan layer to cause resistance to zoocin A. The peptidoglycan from each strain was hydrolyzed using the streptococcolytic phage lysin B30, and the resulting muropeptides were separated by reverse-phase high-pressure liquid chromatography, labeled with 4-sulfophenyl isothiocyanate, and analyzed by tandem mass spectrometry in the negative-ion mode. It was determined that Zif alters the peptidoglycan by increasing the proportion of cross bridges containing three l-alanines instead of two. This modification decreased binding of the recombinant target recognition domain of zoocin A to peptidoglycan. Zif-modified peptidoglycan also was less susceptible to hydrolysis by the recombinant catalytic domain of zoocin A. Thus, Zif is a novel FemABX-like immunity factor because it provides resistance to a bacteriolytic endopeptidase by lengthening the peptidoglycan cross bridge rather than by causing an amino acid substitution.During streptococcal peptidoglycan synthesis, monomer subunits are generated inside the cell, with nonribosomal peptidyl transferases responsible for the addition of amino acids onto the epsilon amino group of lysine in the subunits. These nonribosomal peptidyl transferases are part of the FemABX family of proteins, some of which have been implicated in penicillin resistance (5, 26). In Streptococcus pneumoniae peptidoglycan synthesis, MurM attaches either an l-alanine or an l-serine to the epsilon amino group of lysine, and MurN then adds an l-alanine (11, 26).Zoocin A is a d-alanyl-l-alanine endopeptidase produced by Streptococcus equi subsp. zooepidemicus 4881 that hydrolyzes peptidoglycan cross bridges of susceptible streptococci (12). Zoocin A has two functional domains (18). The N-terminal catalytic domain (CAT) has high degrees of similarity to several other bacteriolytic endopeptidases, including the staphylolytic enzyme lysostaphin. The C-terminal target recognition domain (TRD), which facilitates binding of the enzyme to peptidoglycan (1), has very little similarity to any characterized conserved domain.Producer cell immunity to zoocin A is due to zif (zoocin A immunity factor), which is adjacent to zooA on the chromosome and is transcribed divergently (4). Zif has high degrees of similarity to MurM and MurN and also to the lysostaphin resistance protein and other FemABX-like immunity proteins (23). Previously characterized FemABX-like immunity proteins provide resistance to peptidoglycan cross-bridge hydrolases by inserting an amino acid different from those specified by the normal FemABX-like proteins (6, 9, 15, 25), whereas Zif does not (4). It has been shown previously that Zif-specified resistance to zoocin A is an intrinsic characteristic of the peptidoglycan layer (12). Therefore, Zif must modify the peptidoglycan layer in a novel way that provides resistance to zoocin A. In the present study, Zif was shown to insert an additional l-alanine into the peptidoglycan cross bridges, which inhibited both binding of the zoocin A TRD and the ability of the zoocin A CAT to hydrolyze the cross bridge.     

Structure Activity Relationship of Dendrimer Microbicides with Dual Action Antiviral Activity

Background

Topical microbicides, used by women to prevent the transmission of HIV and other sexually transmitted infections are urgently required. Dendrimers are highly branched nanoparticles being developed as microbicides. However, the anti-HIV and HSV structure-activity relationship of dendrimers comprising benzyhydryl amide cores and lysine branches, and a comprehensive analysis of their broad-spectrum anti-HIV activity and mechanism of action have not been published.

Methods and Findings

Dendrimers with optimized activity against HIV-1 and HSV-2 were identified with respect to the number of lysine branches (generations) and surface groups. Antiviral activity was determined in cell culture assays. Time-of-addition assays were performed to determine dendrimer mechanism of action. In vivo toxicity and HSV-2 inhibitory activity were evaluated in the mouse HSV-2 susceptibility model. Surface groups imparting the most potent inhibitory activity against HIV-1 and HSV-2 were naphthalene disulfonic acid (DNAA) and 3,5-disulfobenzoic acid exhibiting the greatest anionic charge and hydrophobicity of the seven surface groups tested. Their anti-HIV-1 activity did not appreciably increase beyond a second-generation dendrimer while dendrimers larger than two generations were required for potent anti-HSV-2 activity. Second (SPL7115) and fourth generation (SPL7013) DNAA dendrimers demonstrated broad-spectrum anti-HIV activity. However, SPL7013 was more active against HSV and blocking HIV-1 envelope mediated cell-to-cell fusion. SPL7013 and SPL7115 inhibited viral entry with similar potency against CXCR4-(X4) and CCR5-using (R5) HIV-1 strains. SPL7013 was not toxic and provided at least 12 h protection against HSV-2 in the mouse vagina.

Conclusions

Dendrimers can be engineered with optimized potency against HIV and HSV representing a unique platform for the controlled synthesis of chemically defined multivalent agents as viral entry inhibitors. SPL7013 is formulated as VivaGel® and is currently in clinical development to provide protection against HIV and HSV. SPL7013 could also be combined with other microbicides.     

Transport of a Sweet Potato Storage Protein, Sporamin, to the Vacuole in Yeast Cells

The propeptide of a precursor to sporamin, a storage proteinof sweet potato, is required for targeting of sporamin to thevacuole in transformed tobacco cells (Matsuoka and Nakamura1991). A fusion gene consisting of an inducible GAL 10 promoterand sporamin cDNA was introduced into Saccharomyces cerevisiaeby use either of a multiple-copy plasmid (YEpSAD16) or of asingle-copy plasmid (YCpSAD16) to control the level of expressionof the precursor. Although we could not detect any sporamin-relatedpolypeptides in cells that harbored YCpSAD16, extracts fromcells that harbored YEpSAD16 contained multiple forms of sporaminrelatedpolypeptides: preprosporamin, prosporamin and several polypeptidesthat were smaller than prosporamin. However, YCpSAD16 directedthe accumulation of prosporamin in pep4 mutant yeast cells thatlack vacuolar proteases, andpep4 mutant cells that harboredYEpSAD16 did not contain any sporamin-related polypeptides smallerthan prosporamin. The vacuole fractions isolated from the wild-typeand pep4 mutant cells contained sporamin-related polypeptidessmaller than prosporamin and prosporamin, respectively. Theseand other results suggest that, at a low level of expressionof the precursor, prosporamin is transported to the vacuoleand degraded by vacuolar proteases. A mutant precursor to sporamin,in which the propeptide and the N-terminal region of maturesporamin were replaced by an unrelated sequence of four aminoacid residues, directed the secretion of sporamin to the culturemedium in transformed tobacco cells. However, this mutationdid not affect the transport of sporamin to the vacuole in yeastcells and none of the sporamin-related polypeptides were secretedto the extracellular space. (Received July 16, 1991; Accepted March 25, 1992)     

Action Research as a Professional Development Activity

Reflective teachers are always searching for ways to improve their teaching. When this reflection becomes intentional and systematic, they are engaging in teacher research. This type of research, sometimes called action research, can help bridge the gap between theory and practice by addressing topics that are relevant to practicing teachers. This article synthesizes literature within music education and general education to address (a) the conceptual underpinnings of action research, (b) characteristics of action research and teacher research, (c) action research critiques, and (d) implications for teacher research as a form of professional development.