TURNOVER OF PHOSPHATE COMPOUNDS IN CHLORELLA CELLS UNDER PHOSPHATE EFICIENCY IN DARKNESS

1. With the view to get information as to the mode of phosphatetransfer among the intracellular phosphorous compounds undernonphotosynthesizing conditions, investigation was made on thechanges of P-distribution occurring when the algal cells wereincubated in a P-deficient medium in the dark. 2. During the incubation, appreciable decrease of P-contentwas observed only in the fraction of polyphosphate "B". In parallelwith this decrease, an increase of P occurred only in the RNAfraction,indicating that, under non-photosynthesizing conditions, RNAis synthesized with the expenditure of phosphorus of polyphosphate"B". 3. By comparing the present results with those obtained earlierunder photosynthesizing conditions, the effect of light on themode of intracellular mobilization of phosphorous compoundswas discussed. (Received June 8, 1961; )     

MODES OF FORMATION OF PHOSPHATE COMPOUNDS AND THEIR TURNOVER IN CHLORELLA CELLS DURING THE PROCESS OF LIFE CYCLE AS STUDIED BY THE TECHNIQUE OF SYNCHRONOUS CULTURE

1. Starting with uniformly ³²P-labeled Chlorella cells, a synchronousculture was run in a medium containing non-labeled phosphate.During the synchronous growth and division of the algal cells,the changes in amount of total and labeled P in various phosphatecompounds were followed.
2. Characteristic changes were observedwith (acid-soluble) polyphosphate"A", nucleotidic labile phosphates,(acid-insoluble) polyphosphate"C", DNA-P and protein-P. Thelabeled phosphorus of polyphosphate"C" showed a decrease duringthe earlier phase of experiment,although a considerable uptakeof non-labeled P from the culturemedium into this compoundwas observed throughout the experiment.In parallel with theloss of labeled phosphorus in this compound,the increase oflabeled phosphorus occurred in polyphosphate"A", in the nucleotidiclabile-P compounds, and in DNA, suggestingthat these substancesreceived P from polyphosphate "C". Thelabeled P in polyphosphate"A" and in the nucleotidic labile-Pcompounds increased graduallywith the progress of culture,attained their maximum levelsat the stage of ripening, anddecreased markedly during theprocess of "post-ripening" anddivision of cells, indicatingthat these compounds were in activeturnover and playing someimportant roles in the process ofcell maturation and division.
3. The total amounts of inorganic P, RNA-P and lipid-P increasedcontinuously throughout the experiment and showed no significantchange in the content of labeled P.

(Received June 5, 1961; )     

DISTRIBUTION AND TURNOVER OF OCTOPAMINE IN TISSUES

Abstract— Octopamine is a normally occurring amine in several species of animals. Particularly high concentrations are found in the crustacean central nerve cord. In the rat it is specifically localized to sympathetic nerve endings, has a subcellular distribution similar to that of norepinephrine, and is asymmetrically distributed in the CNS. It has a turnover rate in heart about six times that of norepinephrine. The physiological role of octopamine has not been established but it appears likely that it is a cotransmitter together with norepinephrine in the peripheral sympathetic nervous system.     

SYNTHESIS, MIGRATION AND TURNOVER OF PROTEIN IN RETINAL GANGLION CELLS

Abstract— The synthesis, migration and turnover of proteins in retinal ganglion cells of the adult rabbit was studied after intraocular injections of [³H]leucine. It was shown that the isotope was rapidly incorporated into proteins of the retina and some of the proteins were subsequently transported out into the axons of the retinal ganglion cells down to the terminals. This intra-axonal transport of protein occurred at four different velocities; 150, 40, 6-12 and 2 mm/day respectively. The two most rapidly migrating phases of axonal transport were predominantly associated with light particulate fractions and had a relatively rapid turnover in the nerve terminals in the lateral geniculate body. The third phase of axonal transport which had a rate of 6-12 mm/day was possibly associated with the migration of mitochondria. The most slowly migrating proteins in the axon which moved at an average rate of 2 mm/day carried predominantly soluble proteins down to the nerve terminals. A minor part of this phase was metabolized locally in the axon with a half-life of about 14 days. When this slowly migrating phase had reached the nerve terminals in the lateral geniculate body, it was degraded with a half-life of 9-6 days. The different phases of axonal transport were of different magnitudes. As measured from the maximal amount of radioactivity present in the nerve terminals the relative amounts of radioactivity of the four phases were: 1,1 -8,1 -5 and 8-5.     

CHROMATOGRAPHIC ANALYSES OF ACID-SOLUBLE POLYPHOSPHATES IN CHLORELLA CELLS

Using uniformly ³²P-labeled Chlorella cells as material, compositionof acid-soluble inorganic polyphosphates was studied by paperchromatography and ion-exchange chromatography. 2.By the paper chromatographic analysis it was found that theacid-soluble polyphosphates consisted of highly condensed polyphosphates.Ring-forming tri- and tetrametaphosphates, pyrophosphate andtripolyphosphate were not detected in the acid-soluble fractionof the algal cells. 3.By an ion-exchange chromatography with the use of increasingconcentrations of KCl-solution as eluant, it was found thatthe acid-soluble polyphosphate was a mixture of polyphosphateswith a variety of condensation number (n-values). Polyphosphatesof the n-values between 3 and 15 were only 20% of the totalacid-soluble polyphosphate. The majority of the other polyphospateshad greater n-values which was eluted with 0.5–1.0 M KCl. (Received March 2, 1964; )     

QUANTITATIVE SEPARATION OF INORGANIC POLYPHOSPHATES IN CHLORELLA CELLS

Using unicellular green alga, Chlorella ellipsoidea, which hadbeen labeled with ³²P-phosphate, re-examination was performedon our routine method for the quantitative separation of inorganicpolyphosphates (poly-Pi). Confirming the previous results, itwas demonstrated that poly-Pi's in Chlorella cells are clearlyseparated from each other by successive extractions with cold8% TCA (poly-Pi "A"), with cold KOH at pH 9 (poly-Pi "B"), andwith 2 N KOH (poly-Pi's "C" and "D"; the former precipitateson neutralization with PCA, leaving the latter in solution).Use of 2N KOH was found to be most suitable for the purposeof fractionation of poly-Pi's "C" and "D." (Received February 25, 1965; )     

BEHAVIOR OF NUCLEOLI AND CONTRACTING NUCLEOLAR VACUOLES IN TOBACCO CELLS GROWING IN MICROCULTURE

The ability to observe for extended periods of time individual tobacco cells growing in microculture has made it possible to describe the behavior of their nucleoli and contracting nucleolar vacuoles. Nucleoli typically disappeared in prophase and reappeared in telophase. If several nucleoli were present in telophase they generally fused to form only one or two during interphase. In one instance a nucleolus was seen to separate into two nucleoli prior to disappearance in late prophase. In aging and senescent cells the number of nucleoli or bodies similar to normal nucleoli often increased, and occasionally fragmentation of nucleoli was noted prior to death of cells. Budding of solid material from the nucleolus was also observed. The amount of nucleolar material decreased rapidly prior to death of tobacco cells. Nucleolar vacuoles were found to be a general and consistent component of tobacco cells in microculture. Nucleolar vacuoles typically formed and contracted repeatedly in interphase nuclei and apparently released a fluid material into the nucleus. Associated with the contraction of the nucleolar vacuoles was a corresponding decrease in diameter of the nucleolus. Nucleolar vacuoles were observed to occur in about 70% of the actively growing cells examined, whereas they were present in only 33% of the senescent or weakened cells. These data indicate a relationship between nucleolar vacuoles and the morphogenic status of the cells. Since it has been shown by others that the nucleolus is an active site of RNA metabolism, it is suggested that the contracting nucleolar vacuoles may be involved in the controlled release of a soluble product associated with RNA metabolism.     

DEGRADATION AND FORMATION OF SULFOLIPID OCCURRING CONCURRENTLY WITH DE- AND RE-GENERATION OF CHLOROPLASTS IN THE CELLS OF CHLORELLA PROTOTHECOIDES

1. It has been demonstrated that when the cells of Chlorella protothecoidesare grown mixotrophically under illumination in a medium richin nitrogen source (urea) and poor in glucose, the normal greencells are obtained, while in a medium rich in glucose and poorin the nitrogen source, entirely chlorophyll-less cells withprofoundly degenerated plastids ("glucose-bleached" cells) areproduced, irrespective of whether in the light or in darkness.The "glucose-bleached" cells turn green with regeneration offully organized chloroplasts when incubated in a nitrogen-enrichedmedium in the light ("light-greening"), while in the dark theybecome pale green with formation of only partially organizedchloroplasts ("dark-greening"). When, on the other hand, thegreen cells are transferred into a medium enriched with glucose,they are bleached fairly rapidly with degeneration of chloro-plastsin the light as well as in darkness ("bleaching"). Using ³⁵Sas a tracer, investigations were made on the changes of contentsof the algal cells in sulfolipid and other sulfur compoundsduring the processes of the greening and bleaching.
2. By determiningthe radioactivities of chromatographically separatedsulfur-containingcompounds of the uniformly ³⁵S-labeled green("G") and "glucose-bleached"("W") cells, it was found thatthe concentration of a speciesof sulfolipid (discovered byBENSON et al.) as well as thoseof glutathione, sulfotriosesand most of the other sulfur-containingcompounds were at least5 times higher in the "G" cells thanin the "W" cells, whilesulfoquinovosyl glycerol was presentin approximately equalamounts in the two types of cells.
3. Phospholipidcontents and compositions in the two types of algalcells werefound to be practically identical.
4. The sulfolipid contentof algal cells increased and decreasedalmost in parallel withthe processes of greening and bleaching,respectively.
5. Studyingthe mode of incorporation of radiosulfate into varioussulfurcompounds of algal cells during the processes of "light-anddark-greening" and "bleaching" (lasting about 70 hr), itwasfound that active ³⁵S-incorporation into sulfolipid occurredthroughout the process of "light-greening," while in the "dark-greening"and "bleaching" the active incorporation abruptly ceased afterthe initial 24 hr period of experiments. It was suggested thatthe biosynthesis of the sulfolipid is closely related to theformation of photosynthetic apparatus in chloroplast.
6. Whenthe ³⁵S-labeled green cells were bleached in a medium containingno radiosulfate, the ³⁵S-sulfolipid and most of other ³⁵S-sulfurcompounds decreased markedly but the ³⁵S-sulfoquinovosyl glycerolincreased considerably. It was inferred that the deacylationof the sulfolipid, a surfactant lipid, with formation of watersoluble sulfoquinovosyl glycerol may be a cardinal event ofbleaching process, causing a disintegration of the intact architechtureof photosynthetic apparatus.
7. Based on these observations itwas concluded that the sulfolipidis an integral component ofphotosynthetic structure.

¹This work was partly reported at the Symposium on Biochemistryof Lipids, sponsored by the Agricultural Chemical Society ofJapan, Sapporo, July, 1964.     

DISTRIBUTION AND TURNOVER RATE OF VANILLYL-MANDELIC ACID AND 3-METHOXY- 4- HYDROXYPHENYLGLYCOL IN RAT BRAIN

Abstract— Vanillylmandelic acid (VMA) and 3-methoxy-4- hydroxyphenylglycol (MHPG) were measured in rat brain by a mass fragmentographic procedure. The concentration of VMA and MHPG in whole brain is 11 and 533 pmol/g, respectively. Both compounds were found in all areas of brain studied with VMA, as a percentage of both metabolites, ranging between about 1 and 8%. From the decline of the compounds after pargyline. 75 mg/kg i.p., we calculated that the rate of formation of VMA is 15 and for MHPG 202 pmol/g per h. The fractional rate of elimination of VMA and MHPG is 1.4 and 0.38 h⁻¹, respectively. The rapid rate of loss of VMA suggests that it is transported from brain. However, we were unable to block the elimination of VMA from brain by treatment with probenecid. In contrast, the elimination of MHPG could be blocked by treatment with probenecid. Our study adds support to the notion that MHPG is a major whereas VMA is a minor product of norepinephrine metabolism in brain.     

STUDIES ON THE METABOLISM OF UREA AND OTHER NITROGENOUS COMPOUNDS IN CHLORELLA ELLIPSOIDEA: III ASSIMILATION OF UREA

Assimilation of urea by Chlorella ellipsoidea was investigatedby using ¹⁴C-urea. It was revealed that urea carbon metabolizedby the N-starved cell was almost quantitatively recovered incarbon dioxide under dark-aerobic conditions. The rate of ureaconsumption was considerably influenced by the N-content ofalgal cells, practically no metabolism of urea being observedwith the cells containing more than 7 per cent N on a dry weightbasis. Similar results were also obtained when urea was replacedwith ammonia as nitrogen source. Based on the results obtained,a tentative scheme for the assimilation process of urea in Chlorellawas proposed. (Received February 7, 1960; )     

小鼠肺脏间质树突状细胞形态学分布与观察

目的认识小鼠肺脏间质树突状细胞(dendritic cells,DC)的形态、数量和分布特点。方法运用光镜、电镜观察与免疫组化标记(CD11c、CD205、CD80、CD86及MHC-II类分子I-Ab)方法,观察研究C57BL/6小鼠肺脏间质树突状细胞的形态、数量和分布。结果小鼠肺脏间质树突状细胞具有与免疫器官及外周血中DC相同的超微形态特征;CD11c阳性DC含量约占肺脏总细胞数的8.5‰,广泛分布于肺间质中;表达CD205、CD80、CD86及I-Ab的成熟DC含量稀少,多位于间质血管周围。结论肺脏间质树突状细胞是一种脏器免疫前哨细胞,是肺脏免疫微环境的重要组成部分。     

CHANGES IN CONTENTS OF CARBOHYDRATE AND FATTY ACID IN THE CELLS OF CHLORELLA PROTOTHECOIDES DURING THE PROCESSES OF DE- AND RE-GENERATION OF CHLOROPLASTS

1. Previous work has demonstrated that when cells of Chlorellaprotothecoides are grown mixotrophically under illuminationin a medium rich in nitrogen source (urea) and poor in glucose,normal green cells are obtained, while in a medium rich in glucoseand poor in the nitrogen source, strongly bleached cells containingapparently no discernible chloroplast structures — called"glucose-bleached" cells — are produced either in thelight or in darkness. When the green cells are incubated ina glucose-enriched mineral medium without added nitrogen source,they are fairly rapidly bleached with concomitant degenerationof chloroplast structures (" bleaching "). When, on the otherhand, the "glucose-bleached" cells are transferred in a nitrogen-enrichedmedium without added glucose under illumination, they turn greenwith regeneration of chloroplasts (" greening "). In the presentstudy changes in contents of carbohydrate and fatty acid inalgal cells were followed during these processes of "bleaching"and "greening.".
2. During the process of "bleaching", the quantityof glucose existingin the insoluble carbohydrate fraction ofalgal cells increasedrapidly and markedly. A considerable increasewas also observedin the contents of cells in oleic, linoleicand palmitic acids.It was noted, however, that linolenic aciddecreased in quantityduring the most active phase of cell bleaching.
3. During the process of "greening", the glucose in the insolublecarbohydrate fraction rapidly decreased, suggesting that itis utilized, as carbon and energy sources, for the chloroplastregeneration. Linolenic acid was found to be synthesized inparallel with formation of chlorophyll. A peculiar pattern ofchange in contents was observed with oleic and palmitic acids,which was interpreted as being related with the process of cellulardivision occurring incidentally during the process of greening.

(Received September 24, 1966; )     

TRANSPORT, TURNOVER AND DISTRIBUTION OF CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLIN-ESTERASE IN THE VAGUS AND HYPOGLOSSAL NERVES OF THE RABBIT

Abstract— The transport, distribution and turnover of choline O -acetyltransferase (ChAc, EC 2.3.1.6) and acetylcholinesterase (AChE, EC 3.1.1.7) in the vagus and hypoglossal nerves were studied in adult rabbits. The enzymes accumulated proximally and distally to single and double ligatures on both nerves and thus indicated both a proximo-distal and retrograde flow of the enzymes. Double ligature experiments indicated that only 5–20 per cent of the enzymes were mobile in the axon. The rate of accumulation of both enzymes above a single ligature corresponded to the slow rate of axonal flow provided that all the enzymes were mobile, but to an intermediate or fast flow if only a small part of the enzymes was transported. The distribution of ChAc along the hypoglossal neurons was studied and only 2 per cent of ChAc was confined to cell bodies, 42 per cent was localized to the main hypoglossal nerve trunks and 56 per cent to the preterminal axons and axon terminals in the tongue. The ratio of AChE to ChAc was about 3 in the hypoglossal nerve and 32 in the vagus nerve.
Transection of the hypoglossal nerve was followed by a decrease in the activity of ChAc in the hypoglossal nucleus and nerve and in the axons and their terminals in the tongue. The activity of AChE decreased in the hypoglossal nucleus and nerve but not in the tongue. The half-life of ChAc in preterminal axons and terminals of the hypoglossal nerve was estimated to be 16-21 days from the results obtained on transport, axotomy and distribution of the enzyme. Intracisternal injection of colchicine inhibited the cellulifugal transport of both enzymes and led to an increase in enzyme activity in the hypoglossal nucleus.     

CHANGES IN LEVELS OF PYRIDINE NUCLEOTIDES IN CHLORELLA CELLS DURING THE COURSE OF PHOTOSYNTHESIS

1. Using intact cells of Chlorella, the effects of CO₂ on thelevelsof oxidized and reduced forms of DPN and TPN in the lightandin the dark were investigated.
2. It was found that the light-inducedchanges of the DPNH-levelwere not affected by the presenceor absence of CO₂. On theother hand, the light-induced increaseof TPNH was suppressedin the presence of CO₂ and the levelof TPNH which was raisedon illumination in the absence of CO₂was lowered by the provisionof CO₂.
3. On the basis of thesefindings, it was concluded that TPNH,but not DPNH, is participating,in some way, in the mechanismof photosynthesis.
4. Discussionswere made on the difference in the sites of participationofTPNH and of the photogenic reducing agent (R) in the pathofcarbon in photosynthesis.

(Received February 28, 1960; )