CHANGE OF FATTY ACID COMPOSITION OF CHLORELLA ELLIPSOIDEA DURING ITS CELL CYCLE

The lipids extracted from Chlorella cells at different developmentalstages were separated by chromatography on silicic acid into"nonpolar" (chloroform-eluate) and "polar" (methanol-eluate)lipid fractions. The lipids were also subjected to florisilchromatography to fractionate neutral glycerides and free fattyacids. Gas-liquid chromatographic analysis of these fractions has revealeda marked difference in their fatty acid compositions which werefound to undergo characteristic changes during the course ofalgal cell cycle. It was found that the fatty acids in the "nonpolar"lipid (fat) fraction are synthesized during the growth phasein the light and consumed during the process of cellular division. (Received September 24, 1966; )     

SYNCHRONOUS CULTURE OF CHLORELLA: II. CHANGES IN CONTENT OF VARIOUS VITAMINS DURING THE COURSE OF THE ALGAL LIFE CYCLE

1. Chlorella ellipsoidea was grown synchronously and the changesin content of various vitamins during the algal life cycle werefollowed either by chemical or microbiological assay methods.
2. In terms of µg per gram of cell dry weight, the contentof some vitamins (niacin, biotin, inositol and choline) remainedalmost constant throughout the algal life cycle, while thatof others (vitamin B₆-complex, pantothenic acid, folic acid,thiamine and riboflavin) was found to decrease more or lessmarkedly during the "growing phase" and increase at later phasesof "ripening". The content of p-aminobenzoic acid increasedonly at an early stage of "ripening", and that of ascorbic acidincreased only at the stages in which photosynthesis occurredmost actively.
3. These results were discussed in an attemptto interprete theirrelationship with the previously reportedobservations pertainingto the physiological and biochemicalevents occurring in thelife cycle of the alga.

(Received November 7, 1959; )     

ENZYME PATTERNS DURING A SYNCHRONOUS GROWTH CYCLE OF CHLORELLA PYRENOIDOSA

There have been no studies in which a significant number of isozymes have been investigated during a synchronous growth cycle of any organism. The present study was designed to obtain information on the fluctuations of a broad spectrum of enzymes during a synchronous growth cycle of Chlorella pyrenoidosa. Of the thirty-two enzymic activities investigated, seventeen could be localized on starch gels from a high temperature strain of Chlorella pyrenoidosa. Nine of these activities were found to possess more than one band of activity by starch gel electrophoresis. Seven of these activities were localized on starch gels throughout a synchronous growth cycle of C. pyrenoidosa grown in continuous light. Assay techniques are described. The fluctuations in enzyme activity are discussed with relation to concurrent metabolic and cytological changes during cell maturation in C. pyrenoidosa.     

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; )     

低氧时肺动脉内皮细胞单层通透性变化

目的和方法：研究肺动态内皮细胞（ＰＡＥＣ）在低氧性肺水肿（ＨＰＥ）发生中的作用机制。采用ＯＰＡＥＣ体外培养的方法，观察了ＰＡＥＣ生长状态和特征性蛋白因子ⅧＲ：Ａｇ的变化，并利用ＰＡＥＣ融合单层通透性模型研究了低氧对ＰＡＣＥ融合单层的通透性的影响。结果：ＰＡＥＣ生长数量无明显变化，但细胞的生长质量，ⅧＲ：Ａｇ阳性细胞数明显下降，ＰＡＣＥ通透性明显增加，ＣａＭ阻断剂ＴＦＰ只能部分抑制０这种通透性增加。     

THE PRECISE MEASUREMENT OF HEMOLYSIN

A method is described for the measurement of hemolysin concentration, which makes possible exact comparison of results obtained at different times and with different specimens of erythrocytes and alexin; and gives precise values with an error not greater than 2 per cent.     

CHANGE IN GLUCOSAMINE CONTENT OF CHLORELLA CELLS DURING THE COURSE OF THEIR LIFE CYCLE

1. By the VAN WISSELINGH color reaction and the chitosan sulfatetest it was revealed that Chlorella cells contain chitosan probablyin their cell walls. 2. By fractionating the cell material into several fractionsfollowed by their hydrolysis, it was revealed that the majorityof glucosamine was present in the residue material remaininginsoluble in ethanol-ether and perchloric acid (PCA) solution.Conceivably, this glucosamine has derived, for the most part,from the chitosan contained in the cell wall material. 3. During the course of life cycle of the algal cells, the increasein content of glucosamine occurred in three steps: first, inproportion to the growth of smaller (young) cells into largercells; second, corresponding to the formation of autosporeswithin ripened cells; and third, in parallel with the growthof newly born daughter cells. 4. Between the first and second phase mentioned above, thereoccurred an abrupt breach in the increase of glucosamine. Thisphenomenon was presumed to be closely related to the profoundchange in the permeability of cell walls occuring at this transitionalstage of cell development. (Received September 5, 1960; )     

SYNCHRONOUS CULTURE OF CHLORELLA: I. KINETIC ANALYSIS OF THE LIFE CYCLE OF CHLORELLA ELLIPSOIDEA AS AFFECTED BY CHANGES OF TEMPERATURE AND LIGHT INTENSITY

1. Using the technique of synchronous culture, investigationsweremade of the effects of temperature and light-intensityon cellularlife cycle of Chlorella ellipsoidea. Some improvementsin theculture technique for obtaining a good synchrony of algalgrowthwere described.
2. By following the changes of averagecell volume and cell numberoccurring during culturing, therates of the following processesof life cycle were determined:(i) "growth" (or the increasein cell mass) occurring from thestage of smaller cells (D_a)to the stage of ripened cell (L₃),(ii) "ripening" (or processofformation of "nuclear substances"as estimated from the averagenumber of daughter cells formedfrom single mother cell), and(iii) " maturing and division" which leads to the full maturationof mother cells (L-cells)and their division into separate daughtercells (D-cells).
3. "Growth"and "ripening" were found to be dependent in light,"maturingand division" light-independent. The time requiredfor "growth"and "ripening" (C) is dependent on temperaturebut independentof light intensity, the onset of "maturing anddivision" occurringat the same time (D) of culturing undervaried light intensities.The average cell volume at this stage(L₃),however, was foundto be markedly modified by light intensity;larger with highertemperatures (see Fig. 4).
4. Changes in incubation temperature(under the condition of saturatinglight intensities) were foundto affect the life cycle in thefollowing way: (i) The timeof onset of "maturing and division"(D), varies markedly withculturing temperature; earlier athigher temperatures, (ii)The average cell volume at this stagealso depends on temperature; smaller at higher temperatures.
5. The average number of daughtercells (n) emerging from singlemother cells, was found to beuninfluenced by culturing temperature;(4.0–4.1 underthe conditions of the present study). Itwas found that thedivision number n is remarkably varied bychanging the lightintensity in the "growth" and "ripening"phases; 2.0 at 1 kilolux,3.7 at 5 kilolux, 4.2 at saturatinglight intensities (10 and25 kilolux). This finding was explainedby assuming a light-dependentformation of "nuclear substances"during the "growth" and "ripening"phases, the quantity of thesubstances in the cell at L₃ stagedeterminig the division number.
6. The experimental data wereanalyzed reaction kinetically, therate constants and othercharacteristics of the reactions constitutingthe processesof life cycle were determined, and values forthe apparent activationenergy for each reaction were computed.The reactions were discussedwith special reference to theirrelationship with photosyntheticprocess was discussed.

(Received November 7, 1959; )     

ROLE OF DICTYOSOMES IN WALL FORMATION DURING CELL DIVISION OF CHLORELLA VULGARIS

The behavior of dictyosomes in wall formation during cell division of Chlorella vulgaris follows a definite pattern. During formation of the partition membrane they migrate into the equatorial plane and pair. There is a close spatial relationship between the dictyosomes and the partition membrane which, itself, may be derived from the fusion of dictyosomal vesicles. Dictyosomes also may participate significantly in the deposition of new wall material.     

CHANGES IN CELL PROLIFERATION KINETICS OCCURRING DURING THE LIFE HISTORY OF MONOLAYER CULTURES OF A MOUSE TUMOUR CELL LINE

Following inoculation of monolayer cultures of EMT6 mouse tumour cells at 10⁵ cells, a short lag is followed by 3 days of exponential growth with a population doubling time of 12 hr. A plateau cell number is reached between days 4 and 5 and is maintained for at least 8 days. During exponential growth, the pulse ³H-TdR labelling index is 55–60%, all cells are in cycle, and the median cycle time is 11–12 hr. For the first 3 days of plateau phase, the labelling index is about 25 % and there is considerable cell loss. The cell cycle is 32–40 hr, and S-phase is very long. Later in plateau phase, the labelling index falls to <2 % and there is little cell loss. The changes in kinetics occurring in EMT6 cultures are discussed with reference to reported changes occurring in other cell lines.     

离体细胞共培养中科间细胞共质体的形成

离体培养下选出的绿色胡萝卜（Ｄａｕｃｕｓ ｃａｒｏｔａ）细胞系和白色普通烟草（Ｎｉｃｏｔｉａｎａｔａｂａｃｕｍ ）细胞系,各自具有独特的细胞结构标志,在愈伤组织、光镜和电镜３ 个水平上均可区分。对两个细胞系进行分散、混合、Ｋ＋ 液低渗处理后在固体培养基上共培养,１０—１５ ｄ 后可观察到两种细胞的镶嵌生长。光镜和电镜下均观察到烟草细胞和胡萝卜细胞之间隔离层的存在与消失。在隔离层消失的区域可见到异种细胞间次生胞间连丝的形成,从而将独立的两个共质体连成一个统一的共质体。对科间细胞共质联系的建立过程进行了讨论,认为细胞接触后首先非特异粘连——以隔离层形成并适度加厚为标志,然后特异的细胞识别在隔离层中启动,从而导致隔离层或消失而重新建立共质联系或加厚、木质化、木栓化     

ROLE OF SULFUR IN THE CELL DIVISION OF CHLORELLA, WITH SPECIAL REFERENCE TO THE SULFUR COMPOUNDS APPEARING DURING THE PROCESS OF CELL DIVISION II.

1. Chlorella cells, which had been grown synchronously undersulfur-deficient conditions and thus rendered unable to performcell division, were made capable of nuclear and cellular divisionby being supplied with ³⁵S-labeled sulfate and nitrate underphotosynthesizing conditions, and the fate of sulfur duringthese recovery processes was followed. 2. When the S-starved cells were provided with sulfate aloneunder photosynthesizing conditions, cells grew appreciably inmass performing nuclear division but remaining incapable ofcellular division. During these processes most of the ³⁵S wasfound to be incorporated into the protein fraction of algalcells. 3. When the cells which had been stalemated at the above-mentionedstage were supplied with nitrate, they grew further in massand eventually performed cellular division. During this periodthe ³⁵S was found to be distributed not only in the proteinfraction, but also in an appreciable amount in the cold andhot acid-soluble fractions. 4. By paper-electrophoretic experiments it was found that thenature of the sulfur substances appearing in the hot acid-solublefraction changed strikingly during the process of cellular division.Zone electrophoresis and an anion-exchange chromatography ofthese substance isolated from the cells at the completion ofcellular division, disclosed that they were most probably deoxypentosepolynucleotides containing sulfur in some form yet unidentified. 5. It was demonstrated that there exist some antagonistic relationsbetween the protein synthesis and the formation of these sulfur-containingdeoxypentose polynucleotides, and that the former predominatesunder photosynthesizing conditions while the latter outweighsunder nonphotosynthesizing conditions. (Received August 9, 1960; )     

ROLE OF SULFUR IN THE CELL DIVISION OF CHLORELLA, WITH SPECIAL REFERENCE TO THE SULFUR COMPOUNDS APPEARING DURING THE PROCESS OF CELL DIVISION. I.

1. The role of sulfur in the cell division of Chlorella was studiedby following the fate of the sulfur supplied to the sulfur-deficientcells using ³⁵S as a tracer.
2. The sulfur-deficient cells whichwere unable to perform celldivision were made capable of divisionby the provision of ³⁶S-labeledsulfate under non-photosynthesizingconditions. Soon after theprovision of sulfate the labeledsulfur went rapidly into thecold perchloric acid (PCA)-solublefraction of algal cells,almost entirely in the form of sulfateand/or some other inorganicsulfur substance (s). With the lapseof time, more or less remarkablechanges occurred in the patternof ³⁵S-distribution in differentfractions of cell material.It was noticed that, at the onsetof cell division, a sulfur-containingpeptide-nucleotide compound(s)(SPN), which has been reportedearlier, appeared in a largequantity in the cold PCA-solublefraction, and that its quantitydecreased gradually during thesubsequent process of cell division,suggesting that the compoundwas transformed into some othersubstance (s), presumably withits nucleotide moiety going intonucleic acids and the peptidemoiety going into some essentialproteins.
3. Another noteworthyphenomenon observed during the process ofcell division wasthe incorporation of ³⁶S in a group of hotPCA-soluble substances.These sulfur substances were revealedto be sulfur-containingnucleotidic compounds, which might possiblybe some essentialcomponents of, or substances in close relationto, deoxypentosenucleic acid (DNA).

(Received March 1, 1960; )     

AGE-DEPENDENT CHANGE IN THE TRANSDIFFERENTIATION ABILITY OF CHICK NEURAL RETINA IN CELL CULTURE*

We examined how the transdifferentiation ability of neural retinal cells into lens and/or pigment cells in call culture is changed with the development of the donor. Cells dissociated from neural retinas of chick embryos ranging from 3-day-old to the stage immediately before hatching and of 3-day-old chicks were cultured for about 60 days. The results clearly indicated that the transdifferentiation ability decreased with age. The latest developmental stage at which the differentiation of lens cells took place was in 18-day-old embryos. A gradual decrease in this ability was shown by the comparison of crystallin content in cultures prepared from embryos at different stages. The differentiation of pigment cells was recognized in cultures of neural retinas earlier than in 15-day-old embryos. Such loss of the ability of neural retinal cells to transdifferentiate into pigment cells earlier than into lens cells can be partially attributed to inhibitory factors accumulated in medium conditioned with many neuronal cells present in cultures.     

A STATISTICAL METHOD TO COMPARE THE DEGREE OF MUSCLE CELL MULTIPLICATION IN DIFFERENT CULTURE DISHES

A method was developed for comparing two groups of numbers of cultured muscle cells which were counted under a microscope. Practically important problems for this purpose were: how many fields per dish should be observed, and how many dishes should be prepared under the same conditions, when given test criteria were set.
In the present experiment, 4 dishes were prepared under the same conditions. From each of the dishes, 20 fields were selected, and the numbers of muscle cells *** were counted and separately recorded. Since the purpose was to compare two groups of dishes, the design was a simple case of a nested one. From the experiment, the type of distribution seemed approximately a long-normal distribution with constancy of variance (homoscedasticity). Since the distribution of the cells in dishes belonging to the same group could be considered to be the same, the numbers from each dish could be pooled within a group. Therefore, if the test criteria for Student's t test and the sensitivity to descriminate the ratio of the number of groups are given, the number of fields to be observed per dish times that of dishes can be uniquely determined. This method can be applied for the same purpose to other kinds of cells with log-normal distribution.