BIOCHEMICAL CHANGES IN YEAST DURING SPORULATION. I. FATE OF NUCLEIC ACIDS AND RELATED COMPOUNDS

Changes in nuclear figures and in activities of nucleic acid and protein syntheses were observed mainly on Saccharomyces cerevisiae G2-2 during sporogenesis. Patterns of DNA synthesis and of meiosis show that the sporogenic process in yeast was divided into an induction phase (I-phase), a DNA-synthesizing phase (S-phase) and a maturation phase (M-phase). Meiotic figures appeared most frequently at the end of the S-phase at approximately 12 hr in sporulation culture. In M-phase visible spores formed. The amount of protein increased in the initial 7 hr culture of 1-phase, then decreased in the S- and M-phases. But in sporulation culture of the asporogenic diploid strain 3c × a, protein did not decrease. RNA increased within 3 hr of the I-phase then stopped increasing. DNA synthesis occurred critically during S-phase, i.e. between 7 and 12 hr. and was somewhat resumed during the later part of M-phase. Oligodeoxyri-bonucleotide content decreased in the I- and M-phases and increased temporarily. Deoxyribosides decreased linearly during the sporogenic processes. Based on these results and results of experiments estimating the incorporation of ¹⁴C-uracil into nucleic acid and ¹⁴C-amino acid mixture into protein fractions, the roles of nucleic acid synthesis activities in meiosis and in sporulation are discussed.     

The Cell Wall of Rickettsia mooseri I. Morphology and Chemical Composition

Cell walls prepared by mechanically disrupting intact Rickettsia mooseri (R. typhi) were examined in an electron microscope and analyzed chemically. Electron micrographs of metal-shadowed and negatively stained rickettsial cell walls revealed no significant differences, except for smaller size, from bacterial cell walls prepared in a similar manner. The chemical composition was complex, and resembled that of gram-negative bacterial cell walls more closely than that of gram-positive bacterial cell walls. R. mooseri cell walls contained the sugars, glucose, galactose, and glucuronic acid, the amino sugars, glucosamine, and muramic acid, and at least 15 amino acids. Diaminopimelic acid, a compound hitherto found only in bacteria and blue-green algae, was demonstrated in rickettsiae for the first time. Teichoic acids were not detected. The compounds identified accounted for about 70% of the dry weight of the cell walls.     

THE LYSOSOME PERIPHERY: BIOCHEMICAL AND ELECTROKINETIC PROPERTIES OF THE TRITOSOME SURFACE

Normal rat liver lysosomes were isolated by the technique of loading with Triton WR-1339. Purity of the preparation was monitored with marker enzymes; a high enrichment in acid hydrolases was obtained in the tritosome fraction. In 0.0145 M NaCl, 4.5% sorbitol, 0.6 mM NaHCO₃, pH 7.2 at 25°C the tritosomes had an electrophoretic mobility of -1.77 ± 0.02 µm/s/V/cm, a zeta potential of 23.2 mV, a surface charge of 1970 esu/cm², and 33,000 electrons per particle surface assuming a tritosome diameter of 5 x 10^-7 m. Treatment of the tritosomes with 50 µg neuraminidase/mg tritosome protein lowered the electrophoretic mobility of the tritosome to -1.23 ± 0.02 µm/s/V/cm under the same conditions and caused the release of 2.01 µg sialic acid/mg tritosome protein. Treatment of the tritosomes with hyaluronidase did not affect their electrophoretic mobility, while trypsin treatment elevated the net negative electrophoretic mobility of the tritosomes. Tritosome electrophoretic mobilities indicated a homogeneous tritosome population and varied greatly with ionic strength of the suspending media. pH vs. electrophoretic mobility curves indicated the tritosome periphery to contain an acid-dissociable group which likely represents the carboxyl group of N-acetylneuraminic acid; this was not conclusively proven, however, since the tritosomes lysed below a pH of 4 in the present system. Total tritosome carbohydrate (anthrone-positive material as glucose equivalents) was 0.19 mg/mg tritosome protein while total sialic acid was 3.8 µg (11.4 nmol)/mg tritosome protein. A tritosome "membrane" fraction was prepared by osmotic shock, homogenization, and sedimentation. Approximately 25% of the total tritosome protein was present in this fraction. Analysis by gas-liquid chromatography and amino acid analyzer showed the following carbohydrate composition of the tritosome membrane fraction (in microgram per milligram tritosome membrane protein): N-acetylneuraminic acid, 14.8 ± 3; glucosamine, 24 ± 3; galactosamine, 10 ± 2; glucose, 21 ± 2; galactose, 26 ± 2; mannose, 31 ± 5; fucose, 7 ± 1; xylose, 0; and arabinose, 0. The results indicate that the tritosome periphery is characterized by external terminal sialic acid residues and an extensive complement of glycoconjugates. Essentially all the tritosome N-acetylneuraminic acid is located in the membrane and about 53% of it is neuraminidase susceptible.     

PHOTOSYNTHETIC NITRITE REDUCTASE II. FURTHER PURIFICATION AND BIOCHEMICAL PROPERTIES OF THE ENZYME

1. Further purification of photosynthetic nitrite reductase (PNiR),which catalyzes the transfer of hydrogen (or electron) fromthe photolytic system to nitrite, is reported in this paper.Chromatography on DEAE- cellulose and Sephadex gel-filtrationwere effective for the purification of PNiR.
2. PNiR could befractionated into two components. It was inferredfrom the dataobtained that one of these components is identicalwith PPNR,and the other one may probably be a hitherto unreportedflavinenzyme containing FMN as prosthetic group.
3. The propertiesof these two components of PNiR were described,and the interrelationshipbetween these catalysts and possibleintermediary carriers ofthis electron transfer system was discussed.

¹Dedicated to Prof. H. TAMIYA on the occasion of his 60th birthday.This investigation was supported by a Grant in Aid for FundamentalScientific Research from the Ministry of Education (No. 407130-1961)and a Grant in Aid for Organized Scientific Research from theMinistry of Education (No. 95037-1960), which are gratefullyacknowledged here. The authors also wish to acknowledge thatthe progress of this study was facilitated by a Grant from theKAISEI-KAI. A preliminary report on this work was read beforethe 25th Annual Meeting of the Botanical Society of Japan (1960,Osaka).     

Fruit Softening I. Changes in Cell Wall Composition and endo-Polygalacturonase in Ripening Pears

Softening of pome fruits during ripening is characterized bythe solubilization of pectin. The activity of endo-polygalacturonase(endo-PG, EC 3.2.1.15 [EC] ) was determined in pears (Pyrus communisL.) ripened at 18 °C, after storage at –1°C. Theenzyme was assayed, using viscometry, in the presence of pectinesterase(EC 3.1.1.11 [EC] ) with citrus pectin as substrate. Endo-PG activitywas not detected in fruit assayed immediately from store at–1 °C but the enzyme was present after 2 d at 18 °Cwhen the fruit had started to soften and degradation of solublepectin was apparent.     

MORPHOLOGICAL AND BIOCHEMICAL CORRELATES OF CEREBRAL MICROSOMES : I. Isolation and Chemical Characterization

Microsomal fractions, both homogeneous in appearance and functionally operative, were isolated from a homogenate of rat cerebral cortex by fractionation in water. The preparations thus obtained contain the membranous elements of the endoplasmic reticulum, synaptic vesicles, and ribosomes. Esterase, ATPase, and glutamine synthetase were found to be present and fully functional in the microsomal fractions isolated in water. The contamination of the water-isolated microsomal fractions by mitochondria and lysosomes was found to be considerably lower than in microsomal pellets isolated in sucrose. The contamination by nerve ending particles, as judged by electron microscopy and by the levels of soluble lactic dehydrogenase entrapped in the cytoplasm of the particles, was also low. Most of the contamination by mitochondria and nerve ending particles could be removed by treatment of the microsomal pellet with 150 mM NaCl. Resistant to elution by this treatment is the lysosomal contamination as well as microsomal esterase and ATPase. Glutamine synthetase, on the other hand, was almost totally solubilized. Microsomal preparations isolated in water are also shown to contain amounts of protein, RNA, phospholipid, and ganglioside comparable to those found in microsomal preparations isolated in sucrose.     

THE BIOCHEMICAL AND PHARMACOLOGICAL PROPERTIES OF 6-AMINODOPAMINE: SIMILARITY WITH 6-HYDROXYDOPAMINE

6-aminodopamine was injected intraperitoneally into male Swiss–Webster mice. At 72 h post injection 6-aminodopamine had caused a reduction in the endogenous content of heart norepinephrine, a decrease in the capacity of heart slices to accumulate [³H]-norepinephrine in vitro, and a virtual disappearance of the adrenergic plexus of the mouse iris as viewed by fluorescence histochemistry. Similar data were obtained with the same dose of 6-hydroxydopamine. These data suggest that 6-aminodopamine causes a destruction of sympathetic nerve terminals. Model experiments showed that 6-aminodopamine, like 6-hydroxydopamine, generated H₂o₂both in vitro and in vivo. 6-Aminodopamine, like 6-hydroxydopamine, also blocked the accumulation of [³H]dopamine into slices of rat brain in vitro.     

Sphere-Rod Morphogenesis in Arthrobacter crystallopoietes I. Cell Wall Composition and Polysaccharides of the Peptidoglycan

Cell walls of Arthrobacter crystallopoietes were prepared from cells grown as spheres and from peptone- and succinate-induced rod stage cells. Undegraded polysaccharide backbones of the peptidoglycans were isolated from myxobacter AL-1 protease digests by ECTEOLA cellulose and Sephadex G-50 chromatography. The polysaccharide backbones of the sphere cell wall peptidoglycan are heterogeneous in their size, and average less than 40 hexosamines per chain. Those of the rod cell walls are homogeneous in size and average 114 to 135 hexosamines per chain.     

Combined Effects of Partial Defoliation and Nutrient Availability on Cloned Betula pendula Saplings: II. CHANGES IN NET PHOTOSYNTHESIS AND RELATED BIOCHEMICAL PROPERTIES

The combined effects of partial defoliation and nutrient availabilityon net photosynthesis and related biochemical variables werestudied in cloned Betula pendula Roth saplings. The saplingswere randomly assigned to different nutrient levels (5, 1·5and 0·5 mol N m^–3) in aerated nutrient cultureand to the following defoliation treatments: (1) control (nodamage), (2) damage of the developing main stem leaves (halfof the leaf lamina removed), and (3)removal of the developingmain stem leaves (entire leaf lamina removed). The leaf immediatelybelow the damaged area in the treated plants, and the correspondingleaf in the control plants, were selected for study. Net photosynthesismeasurements and biochemical determinations were made 2, 8 and14 d after assigning the treatments. At intermediate and lownutrient levels the final net photosynthetic capacity was significantlyhigher in the saplings with the topmost leaves removed thanin the undamaged control saplings, indicating that the expressionof compensatory photosynthesis after partial defoliation isnot inhibited by nutrient deficiency. The photosynthetic enhancementwas closely associated with the increased initial activity ofribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). However,the increased activity of Rubisco was not exclusively the resultof a higher amount of Rubisco. The expression of compensatoryphotosynthesis after partial defoliation in our study cannotunequivocally be attributed to an increased flow of nitrogento the remaining leaves. Key words: Partial defoliation, nutrient availability, net photosynthesis, nitrogen, Rubisco     

MORPHOMETRY OF ORCHID SEEDS. I. PAPHIOPEDILUM AND NATIVE CALIFORNIA AND RELATED SPECIES OF CYPRIPEDIUM

Examination of differences in morphology, size and color between seeds of the genera Paphiopedilum and Cypripedium (family: Orchidaceae; subfamily: Cypripedoideae) and among several Cypripedium species confirm present taxonomic relationships. Additionally, the shape, size and relatively large air volume in the testae (79–96% of available space) provide an explanation for their long flotation periods in the atmosphere, which is an adaptation for dispersal by wind.     

失水-吸水过程中集球藻的生理生化特性研究

荒漠结皮藻类广泛分布于干旱、半干旱地区,经常面临生存水源的短缺。以一种从荒漠结皮中分离的典型绿藻-集球藻(Palmellococcus sp.)为材料,研究其在失水-吸水过程中的某些生理生化特性。即在温室模拟条件下设置相对湿度分别为0%(极度失水)、43%(适度失水)和98%(水合,对照),测定不同失水处理对集球藻光合活性、膜脂过氧化产物丙二醛含量、细胞内可溶性物质含量和抗氧化酶活性的影响,并测定蒸馏水、BBM培养基、0.2 g/L蔗糖溶液、0.2 g/L蓝藻多糖、0.2 g/L脯氨酸和2.5 mmol/L氯霉素及50μmol/L敌草隆进行吸水处理时对失水藻体光合活性的恢复效果。结果表明,与对照处理相比,集球藻在失水过程中光合活性迅速降低,SOD和CAT活性大量升高,细胞内可溶性糖含量和可溶性蛋白含量明显增加,并且膜脂丙二醛含量出现大量积累。不同的失水-吸水处理发现,外源蔗糖和胞外多糖吸水对集球藻的光合活性有较好的恢复作用,添加脯氨酸和氯霉素吸水时光合活性也获得了一定程度的恢复,采用敌草隆吸水时则出现光合活性的明显抑制。研究结果对于更好地理解荒漠结皮绿藻对干旱的生理适应机制提供了有意义的参考。     

Cell Wall Monosaccharide Composition and Muskmelon Resistance to Myrothecium roridum1

In vitro growth of Myrothecium roridum, a pathogen of muskmelon (Cucumis melo L.), on media supplemented with eight cell wall-related monosaccharides revealed that germination and germ tube elongation were enhanced in the presence of arabinose, galactose and glucose. Colony expansion of established mycelia of M. roridum was also enhanced by arabinose and glucose but inhibited by galactose, Non-cellulosic neutral sugar analysis of fruit cell walls from muskmelon cultivars resistant or susceptible to M. roridum revealed that susceptible cultivars had consistently higher arabinosyl, galactosyl and glucosyl residue content than resistant cultivars, while a net loss of galaciosyl and arabinosyl residues occurred in cell walls of fruits between 20- and 27-days post-anthesis. M. roridum germinated more rapidly on isolated fruit cell walls from susceptible than resistant cultivars, but no correlation was found between cultivar resistance to M. roridum and inhibitin of fungal colony expansion on cell walls. Although factors affecting spore germination and mycelial growth of M. roridum, in vitro and in vivo, may differ, any factor that increases cell wall polysaccharide hydrolysis may contribute to ability of M. roridum to become established in immature fruit of muskmelon.     

The Monosaccharide Composition of Cell Wall Material in Cassava Tuber (Manihot utilissima)

The monosaccharide composition of cell wall material (CWM) in the cassava tuber and the contents of the other constituents were determined for more advanced industrial utilization. Starch, 80% ethanol–soluble sugar, uronic acid, lignin, ash, and CWM contents in the cassava tuber 86.1, 2.4, 3.4, 0.5, 0.9, and 4.5%, respectively. Rhamnose, fucose, arabinose, xylose, mannose, galactose, and glucose contents in CWM were 1.9, 1.2, 2.6, 4.2, 2.0, 12.8, and 52.7%, respectively. Then, the degradation pattern of CWM by enzymatic and sequential acid hydrolysis was studied. Aspergillus niger cellulase preparation was the most effective, and 57.1 % of CWM was degraded by the enzyme preparation. On the other hand, about 50% of the hemicellulose part was extracted from CWM by hot water only.     

ION SERIES AND THE PHYSICAL PROPERTIES OF PROTEINS. I

1. This paper contains experiments on the influence of acids and alkalies on the osmotic pressure of solutions of crystalline egg albumin and of gelatin, and on the viscosity of solutions of gelatin. 2. It was found in all cases that there is no difference in the effects of HCl, HBr, HNO₃, acetic, mono-, di-, and trichloracetic, succinic, tartaric, citric, and phosphoric acids upon these physical properties when the solutions of the protein with these different acids have the same pH and the same concentration of originally isoelectric protein. 3. It was possible to show that in all the protein-acid salts named the anion in combination with the protein is monovalent. 4. The strong dibasic acid H₂SO₄ forms protein-acid salts with a divalent anion SO₄ and the solutions of protein sulfate have an osmotic pressure and a viscosity of only half or less than that of a protein chloride solution of the same pH and the same concentration of originally isoelectric protein. Oxalic acid behaves essentially like a weak dibasic acid though it seems that a small part of the acid combines with the protein in the form of divalent anions. 5. It was found that the osmotic pressure and viscosity of solutions of Li, Na, K, and NH₄ salts of a protein are the same at the same pH and the same concentration of originally isoelectric protein. 6. Ca(OH)₂ and Ba(OH)₂ form salts with proteins in which the cation is divalent and the osmotic pressure and viscosity of solutions of these two metal proteinates are only one-half or less than half of that of Na proteinate of the same pH and the same concentration of originally isoelectric gelatin. 7. These results exclude the possibility of expressing the effect of different acids and alkalies on the osmotic pressure of solutions of gelatin and egg albumin and on the viscosity of solutions of gelatin in the form of ion series. The different results of former workers were probably chiefly due to the fact that the effects of acids and alkalies on these proteins were compared for the same quantity of acid and alkali instead of for the same pH.     

COMPARATIVE STUDIES ON VARIOUS CALLUSES AND CROWN GALL OF JERUSALEM ARTICHOKE II. SOME BIOCHEMICAL PROPERTIES

A callus isolated from Jerusalem artichoke tuber was found tohave much more cell wall material and tyrosinase activity thanthe original tuber, and its dry weight was dependent on thesucrose concentration in the culture medium. All calluses anda crown gall tested contained no detectable inulin. Even thoughdifferent cultures were widely different in their dry weight,this was closely related to their total hexose content, andthe latter in turn was proportional to their free hexose content. (Received January 18, 1967; )