CHANGES OF AMINO ACID COMPOSITION OF CHLORELLA CELLS DURING THEIR LIFE CYCLE

The cells of Chlorella ellipsoidea were grown synchronously,and at different stages of their life cycle, the cells wereanalysed for their contents in amino acids existing in freeforms as well as in the fractions of bulk protein and peptides.Throughout the algal life cycle, the content of bulk protein(per unit dry weight of cells) remained relatively constant,being about 20 to 40 times those of peptides and free aminoacids. The amino acid composition of the protein fraction alsoremained fairly constant, the predominant amino acids beingalanine, glutamic acid, glycine and leucine. The contents inthe bulk peptides increased appreciably during the periods ofgrowth and "ripening" (light period), and decreased markedlyduring the periods of "post-ripening" and cellular division(dark period). Similar modes of change in content were alsoobserved in most of the individual amino acids contained inthe peptide fraction. The most abundant component in the peptidefraction was arginine followed by glutamic acid, glycine andcyst(e)ine. Rather irregular was the mode of change of the levelsof individual free amino acids, although, as a whole, theirbehavior was similar to that of bulk peptides, increasing duringthe light period and decreasing during the dark period. Themost predominant free amino acids were glutamic acid and alaninefollowed by proline. Experimental evidence showed that the processes of formationof free amino acids and peptides are for the most part lightdependent, while the synthesis of protein, which is thoughtto be effected using as building blocks mostly free amino acids—formeddirectly or indirectly from early photosynthates or derivedfrom pre-formed peptides—is essentially a light-independentprocess. Peptides, as a whole, seem to have significance asreservoirs of building blocks for the syntheses in the darkof protein and other nitrogenous cellular substances. The synthesisof protein in the dark takes place not only by consuming thefree amino acids and peptides that have been accumulated duringthe light period, but also by assimilating the exogenous nitrogensource (nitrate). The distribution of individual amino acidsin the three main fractions mentioned above as it changed duringthe course of algal cell cycle was followed in detail, and theresults obtained were discussed in relation to various relevantdata reported by other workers. (Received June 29, 1964; )     

Water Stress, Osmolytes and Proteins

Organic osmolytes are small solutes used by cells of numerouswater-stressed organisms and tissues to maintain cell volume.All known osmolytes are amino acids and derivatives, polyolsand sugars, methylamines, and urea; unlike salt ions, most are"compatible," i.e., do not perturb macromolecules. In addition,some stabilize macromolecules and are "counteracting" towardsperturbants, e.g., methylamines can stabilize proteins and ligandbinding against perturbations by urea in elasmobranchs and mammaliankidney, and (our latest findings) high hydrostatic pressurein deep-sea animals. Methylamines appear to coordinate watermolecules tightly, resulting in osmolyte exclusion from hydrationlayers of peptide backbones. This makes unfolded protein conformationsentropically unfavorable (work of Timasheff, Galinski, Bolenand coworkers). These properties have led to proposed uses inbiotechnology, agriculture and medicine, including improvedbiochemical methods, in vitro rescue of misfolded proteins incystic fibrosis and prion diseases (work of Welch and others),and plants engineered for drought and salt tolerance. Theseproperties also explain some but not all of the considerablevariation in osmolyte composition among species with differentmetabolisms and habitats, and among and within mammalian tissuesin development.     

Absorption of Some Amino Acids by Sporophytes Isolated from Polytrichum formosum and Ultrastructural Characteristics of the Haustorium Transfer Cells

The absorption of ¹⁴C-labelled amino acids (glycine, threonineand -aminoisobutyric acid) by the isolated sporophyte of Polytrichumformosum takes place mainly in the haustorium. The isolationof the sporophyte does not alter the absorption capacity ofits haustorium nor its ultrastructure, in particular that ofits peripheral transfer cells. amino acids, transfer cells, sporophyte, Polytrichum formosum, haustorium     

Uptake of Lysine by Wild-Type and S-(2-Aminoethyl)-L-cysteine-Resistant Suspension-Cultured Cells of Triticum aestivum

The kinetics of uptake of L-lysine in wheat (Triticum aestivumcv. Chinese Spring) were analyzed in wild-type cells and inAEC-1 variant cells that are resistant to S-(2-aminoethyl)-L-cysteine(AEC). Uptake of lysine by AEC-1 cells was considerably slowerthan that by the wild-type cells. In the presence of carbonylcyanidem-chlorophenylhydrazone, the rates of uptake by both types ofcell were reduced to a similar linear component. Fitting theuptake data to one linear (diffusional) component and one Michaelis-Menten(active) system showed that, as compared to wild-type cells,AEC-1 cells have a reduced V_max and an increased K_m with respectto the active component, byt they have a similar diffusionalcomponent. Inhibition experiments with various amino acids indicatedthat the active component represents a carrier specific forbasic amino acids, which was competitively inhibited by AEC.The AEC-1 cells also showed reduced uptake of several neutraland acidic amino acids, but the rate of uptake of 3-O-methylglucosewas somewhat higher than that by wild-type cells. (Received May 16, 1989; Accepted September 4, 1989)     

Intracellular Distribution of Free Amino Acids between the Vacuolar and Extravacuolar Compartments in Internodal Cells of Chara australis

Distribution of free amino acids between the vacuolar and extra-vacuolar(cytoplasmic) compartments in internodal cells of Chara australiswas studied. Under the control conditions (14-h light : 10-hdark), most (90%) of the cellular free amino acids were foundin the extra-vacuolar compartment. The reverse was true forammonia. The major amino acids were isoasparagine, alanine,glutamic acid, aspartic acid, serine and glycine. The contentsof hydrophobic and basic amino acids were minor and relativelygreater proportions were found in the vacuole except when theircontents were extremely low. When cells were kept for 3 days under continuous light or incontinuous darkness, the total free amino acid content increasedto about 120% (light) and about 150% (dark) that of the control.These increases mainly took place in the vacuole, but the aminoacid species responsible for the increments differed with thelight conditions. In contrast, the cytoplasmic content was relativelyconstant (50–60 mM) even under continuous light or darkness.The results suggest that the vacuole acts in the homeostasisof the cytoplasmic amino acid content. As anion, amino acidsin the cytoplasm compensated for about 10–20% of the reported"anion deficiency" in the cytoplasm. (Received June 7, 1984; Accepted September 11, 1984)     

Regulation of Aspartokinase Isoenzyme Levels in Cultured Cells of Vinca rosea

The levels of aspartokinase isoenzymes were followed as a functionof days after transfer of V. rosea cells to fresh medium. Whencells were subcultured in a 7-day cycle, both isoenzymes showedpeaks at early, but not identical, stages of cell proliferation.Levels of the intracellular amino acids lysine, threonine, isoleucineand methionine decreased as the cellular level of protein increased.As soon as the increase in protein ceased, the amino acid levelbegan to increase. The stationary cells accumulated large amountsof free amino acids. When late stationary cells were used asthe inoculum, growth was slow and, as expected, it took longerbefore the depletion of endogenous free amino acids and thedevelopment of aspartokinase isoenzymes was significantly retarded.These results are further evidence that the syntheses of aspartokinaseisoenzymes are under repression-derepression control in higherplants as they are in bacterial systems. (Received April 22, 1981; Accepted August 31, 1981)     

Chemical Properties and the Physiological and Pharmacological Actions of Urophysial Peptides

Recent advances in urophysial research are reported and currentresearch in this laboratory on the urophysial peptides (urotensinI, urotensin II) of Catostomxts commersoni is summarized. UrotensinI has been purified by gel filtration, electrofocussing, ultrafiltrationand ion exchange chromatography. Three similar utotensin I peptideswere found. The main peptide was shown to be a straight chainpeptide with about 36 amino acid residues. Pressor-diureticeffects in fish have been observed. The peptide produces a long-lastinglowering of mammalian blood pressure by peripheral vasodilatation.Urotensin II has been isolated and purified. It appears to contain23 amino acids including two disulphidebridges. It is a potentpressor-diuretic-smooth muscle contracting agent in the fish,without any known pharmacological actions in mammals. UrotensinIII: the existence of this activity as a separate chemical entityhas not been established, but urophysectomy was shown to lowerblood Na⁺ without an increase in renal excretion. UrotensinIV has been shown to be similar to, or identical with, argininevasotocin. Urotensin IV could not be found in the urophysisof Catostomus (gel filtration, bioassays, immunohistochemistry).Similarities and differences between the hypothalamic and caudalneurosecretory systems and their active principles are analysed.     

Morphological and Physiological Responses of Echinoderm Larvae to Nutritive Signals

SYNOPSIS. Echinoid larvae have been found previously to developshorter arms as phytoplankton concentrations increase. In thepresent study, the skeletal dimensions of larvae of the seaurchins Lytechinus pictus and Strongylocentrotus purpuratuswere measured after exposure to dissolved organic compoundsin seawater. The presence of glucose or individual amino acids(at 1 or 2 µM), a mixture of 16 different amino acids(100 nM each), or algal exudate resulted in larvae with shorterarms (by 12 to 88%), relative to larvae in seawater with noadditions. Larvae exposed to mixtures of amino acids also hadchanges in the constituents of their internal free amino acidpools (as determined by HPLC). For another echinoid, Dendrasterexcentricus, amino acid transport (T, from 500 nM) by individuallarvae (n = 47) scaled to arm length (L) as follows: T = 2.06L^0.81(0.81 ± 0.26,95% confidence intervals). Mass (M) andmetabolic rate (MR) did not scale in the same manner (as transport)to arm length for larvae of the echinoid Centrostephanus coronatus(MR = 148L^1.51; M = 1.7L^2.01). These characteristics may scaleto arm length independently from transport rate under certainconditions. Larvae of echinoderms respond morphologically to"signals" in their environment that may indicate the availabilityof dissolved and paniculate nutrients. This in turn will haveconsequences for their ability to take up and metabolize thesenutrients. The response may be mediated by surface receptorsfor dissolved organic compounds, or by changes in the sizesof intracellular substrate pools.     

烟夜蛾组织蛋白酶B酶原基因的克隆、序列分析和原核表达

利用反转录多聚酶链式反应（RT-PCR）技术从烟夜蛾Helicoverpa assulta (Guenée)雌虫卵巢中扩增得到了组织蛋白酶B酶原基因（cathepsin B,CB）cDNA片段,将其克隆至pMD19-T载体。测序结果表明, 该片段长度为1 017 bp,含有组织蛋白酶B酶原基因完整开放阅读框架（ORF）（GenBank登录号：EF154237）。序列分析结果显示：烟夜蛾组织蛋白酶B（HassCB）编码338个氨基酸残基,预测N-末端含有长度为21个氨基酸残基的信号肽序列;去除信号肽序列后,预测成熟蛋白分子量为35.5 kDa,等电点为5.96。氨基酸序列比对结果表明,HassCB与其他昆虫的组织蛋白酶B酶原氨基酸序列有较高的一致性。将去除信号肽序列的HassCB（HassCBa）重组到表达载体pGEX-4T-1中,并转入原核细胞中表达,SDS-PAGE和Western印迹分析表明：该基因能在大肠杆菌BL21中表达,电泳检测到一条大约61 kDa的目的条带,与预测的融合蛋白分子量相符。用该基因制备多克隆抗体并测得该抗体对重组表达的HassCBa的效价为1∶51 200。通过免疫印迹检测证实,此抗体既能识别重组表达的HassCBa,又能识别烟夜蛾卵巢匀浆液中的HassCB。     

Changes in the Levels of Free Amino Acids in Various Regions of Cuscuta during Growth

The angiospermous plant parasite Cuscuta derives reduced carbonand nitrogen compounds primarily from its host. Free amino acidsalong Cuscuta vines in three zones, viz., 0 to 5 cm, 5 to 15cm, and 15 to 30 cm, which in a broad sense represent the regionof cell division, cell elongation and differentiation and vasculartissue differentiation respectively, were quantitatively estimated.The free amino acid content was the highest in the 0 to 5 cmregion and progressively decreased along the posterior regionsof the vine. The haustorial region showed the lowest contentof free amino acids. In general, the free amino acid contentin samples collected at 7 p.m. was found to be higher than thatin the samples collected at 7 a.m. Three basic amino acids,histidine, the uncommon amino acid -hydroxyarginine, and arginineconstituted more than 50% of the total free amino acids in allthe zones studied except the haustorial region. Aspartic acidand glutamic acid constituted the major portion in the acidicand neutral fraction of amino acids. Glutamine, asparagine,threonine, and serine were eluted together and occurred in substantialamounts. -Hydroxyarginine constituted the largest fraction inthe cut end exudate of Cuscuta and presumably appeared to bethe major form of transport amino acid. -Hydroxyarginine wasalso a major constituent of the basic amino acids in Cuscutavines parasitizing host plants from widely separated families,suggesting that this amino acid is a biosynthetic product ofthe parasite rather than that of the hosts. Also, U-¹⁴C argininewas converted to -hydroxyarginine by cut Cuscuta vines, suggestingthat -hydroxyarginine is synthesized de novo from arginine byCuscuta. (Received March 30, 1987; Accepted June 7, 1988)     

Origin of Amino Acids in Datura stramonium Seeds

¹⁴C isotope studies show that the seeds of Datura stramoniumL. can produce a number of amino acids (particularly alanine,glutamate, phenylalanine, and tyrosine) from a supply of sucroseand nitrate. These amino acids can be incorporated into theseed protein. The bulk of the amino acids incorporated into the seed proteinmust, however, be supplied by adult leaves in the proximityof the fruit, either as the amino acids themselves, or theirimmediate precursors. The major free-amino-acid products of Datura leaves are theamides asparagine and glutamine.     

Effect of {alpha}-Naphthalene Acetic Acid on Variations in Free Amino Acid Contents in Developing Fruits of Capsicum annuum L.

Capsicum annuum seeds were pretreated with three concentrationsof -naphthaleneacetic acid (NAA). The fruits of the controland treated plants showed differences in the quantities of freeamino acids during three developmental stages. From a relativelyhigh amount of total free amino acids in the early phase offruit development, there was lowering in the mid-phase and furtherlowering in the terminal phase. There were eight prominent ninhydrin-reactingspots which were identified as tryptophan, glycine, glutamicacid, valine-methionine, -aminobutyric acid, leucine(s), alanine,and aspartic acid and eleven trace spots attributable to tyrosine,serine, lysine, asparagine, cystine, thrconine histidine, phenylalanine,arginine, glutamine, and ornithine. Hormone treatment tendedto decrease the total amino acids in the early- and mid-phasesof fruit development compared with the control fruits. Asparticacid was present in greater quantities in the treatments thanin the controls. In the terminal phase, the treated fruit didnot show any significant change in the total free amino acidcontents. The significance of the NAA-included variation infree amino acids is discussed. Capsicum annuum, amino acids, -naphthaleneacetic acid     

Metabolism of Arginine and Ornithine in Suspension-Cultured Cells and Aseptic Roots of Intact Plants of Atropa belladonna

Suspension-cultured cells and aseptically cultured roots ofintact plants of Atropa belladonna L. removed tropane alkaloidprecursors arginine (Arg) and ornithine (Orn) at nearly an equalrate from the feeding medium. A great part of Arg- and Orn-derived¹⁴C-label was found in ethanol-insoluble compounds, mostly inproteins already after 2 h feeding. Ethanol-soluble label inthe roots was found mainly in amino acids (e.g. glutamine, Gln)after 2 h feeding, and after 20 h also in some intermediatesof the urea cycle (e.g. argininosuccinate). In suspension cultures, subculturing of the initiation callusdecreased both the uptake of the basic amino acids tested andtheir binding on to the apoplastic space. After 20 h feedingwith Arg more label was found in organic acids in stationaryphase suspension cultures with repressed alkaloid synthesisthan in roots producing alkaloids. The growth phase and passagenumber also affected into which amino acids the label was incorporated.When the initiation callus was young (the 3rd passage), theintermediates of the urea cycle were actively labelled, butwhen the initiation callus was older (the 8th passage) and thesuspension formed roots, especially Gln was labelled. Only tracesof -N-methylornithine were detected in feeding experiments withOrn and Arg. Considerable arginase activity with a high pH optimumwas observed in cell suspensions and roots of A. belladonna. Key words: Atropa, arginine, ornithine, roots, suspension culture     

The Amino Acid Sequence of Ascorbate Peroxidase from Tea Has a High Degree of Homology to That of Cytochrome c Peroxidase from Yeast

The chloroplast isozyme of ascorbate peroxidase from tea leaveswas digested with lysyl endopeptidase, and the amino acid sequencesof the peptide fragments were determined. These sequences accountedfor 64% of the amino acids in the entire protein. The sequenceof one of the peptides can be aligned with the region whichincludes the proximal histidine that serves as the fifth ligandof the heme iron in guaiacol peroxidases and cytochrome c peroxidase.The sequences of the peptides from ascorbate peroxidase exhibita higher degree of homology to the sequence of cytochrome cperoxidase from yeast than to those of guaiacol peroxidasesfrom plants. In addition, three of the peptides from ascorbateperoxidase show a high degree of homology to triose-phosphateisomerase from maize. From the available amino acid sequencesand the enzymatic and molecular properties of ascorbate andcytochrome c peroxidases, we propose that these hydrogen peroxide-scavengingperoxidases that use either cytochrome c or ascorbate as theelectron donor originated from the same ancestral protein. (Received July 5, 1991; Accepted December 6, 1991)     

东亚飞蝗中肠几丁质酶基因的克隆、序列分析及组织定位

通过RACE方法,克隆了东亚飞蝗Locusta migratoria manilensis (Meyen)几丁质酶基因 (LmChi)cDNA全序列 (GenBank 登录号：EF092841)。获得的cDNA全长1 604 bp,其中可读框1 452 bp, 编码483个氨基酸。推测其氨基酸序列与18家族昆虫几丁质酶有较高的相似性。与其他几丁质酶一样,东亚飞蝗几丁质酶序列也包含一个信号肽、一个几丁质酶活性位点、一个碳端丝氨酸富集区和一个几丁质结合域。半定量RT-PCR研究表明,LmChi基因只在东亚飞蝗不同发育阶段的中肠组织中表达,而在东亚飞蝗体壁、前肠和后肠均没有发现LmChi基因的转录。     

Solitary chemoreceptor cells of Ciliata mustela (Gadidae, Teleostei) are tuned to mucoid stimuli

Summed potentials were recorded from the dorsal recurrent facialnerve innervating the solitary chemoreceptor cells on the anteriordorsal fin (ADF), from the ventral recurrent facial nerve innervatingboth taste buds and solitary chemoreceptor cells on the pectoral(PEC) and pelvic (PEL) fins, and from the anterior dorsal finmuscles in the rockling, Ciliata mustela. There is little overlapbetween the sumulus spectra of solitary chemoreceptor cellsand taste buds. The ADF solitary cells are particularly sensitiveto body mucus (skin water) of non-congeners like Gadus, Solea,Cottus, Mugil, Zoarces, Gaidropsarus, and Encheliopus, but insensitiveto amino acids and a variety of body fluids of fish, invertebrates,and extracts of potential stimuli like algae and sand. Pectoraland pelvic fins are particularly sensitive to amino acids, bodyfluids of fish and invertebrates, but less sensitive to skinmucus of fish, probably due to the abundance of taste buds.Active sampling by undulation of the anterior dorsal fin isessential for proper functioning; it induces disadaptation ofthe receptor elements. Solitary chemoreceptor cells provide,apparently, cues to discriminate between conspecifics and non-conspecifics.It is unlikely that they are involved in pheromone detection.     

Comparison of Free and Bound Amino Acids in White and Green Tissues of Variegated Tobacco Plants

Qualitative and quantitative comparisons of free and bound aminoacids and soluble proteins in white and green tissues of variegatedtobacco leaves were made. White tissue contained more free andless bound amino acids than green tissue, although the sum ofthe total amino acids did not differ significantly between thetwo tissues. The major free amino acids in white tissue wereglutamine and asparagine, whereas those in green tissue wereglutamic acid, aspartic acid and -aminobutyric acid. The contentsof fraction 1 protein and 70 S ribosomes in white tissue werenegligible in comparison with those found in green tissue, butthe amounts of other soluble protein components and the 80 Sribosomes were at the same level in both tissues. (Received October 21, 1981; Accepted January 28, 1982)     

Fungicidal Activity of Oxadiazolyl Sulfides

Glycyl-l-leucine is one of the best substrates for peptide hydrolases in the intestinal mucosa. Its absorption and hydrolysis were investigated in epithelial cells isolated from the rat intestine in the presence of bestatin, a specific inhibitor of certain peptide hydrolases, Bestatin competitively inhibited dipeptide hydrolase activities in isolated cells with a K_i value of 10^?8 m, but noncompetitively inhibited, and less significantly, the dipeptide absorption by isolated cells. At 10^?4 m bestatin inhibited half the dipeptide absorption, but only minimally inhibited the absorption of constituent amino acids. In the presence of bestatin at substantial concentrations the isolated cells took up a significant amount of the intact dipeptide, which otherwise appeared entirely in the form of free amino acids. These results are interpreted to substantiate a notion that a dual mechanism is operative for the absorption of readily-hydrolysable peptides: the peptide hydrolysis followed by uptake of thereby released amino acids, and the peptide transport followed by cytosolic hydrolysis.     

Amino-Acid Composition of Wood Proteins

The ammo-acid compositions of proteins present in the sapwoodand heartwood from three species of Eucalyptus, in normal, reaction,and opposite wood from Pinus radiata seedlings, and in reactionand opposite wood from Eucalyptus goniocalyx, are listed. Thequestion of the location of protein within wood cell walls isdiscussed. The amounts of tyrosine found in the woods from P.radiata seedlings are incompatible both with the belief thatit determines the intensity of staining of reaction wood cellwalls with Millon's reagent, and that it is the source of thep-hydroxybenzaldehyde obtained when milled wood lignin is oxidizedwith nitrobenzene. Loss of amino acids in the presence of milledwood lignin, -cellulose, hemicellulose, glucose, mannose, galactose,xylose, arabinose, glucuronic, and galacturonic acids is demonstrated.The presence of numerous peaks which are not indentifiable withknown amino acids in the chromatograms of wood hydrolysatesis believed to be due largely to the products of interactionof amino acids and sugars. Some retention of nitrogen by theresidues of wood hydrolysates occurs. The significance of theresults obtained with the wood samples listed above is discussedagainst this background.