THE INFLUENCE OF THE SUBSTRATE CONCENTRATION ON THE RATE OF HYDROLYSIS OF PROTEINS BY PEPSIN

1. It is pointed out that the apparent exceptions to the law of mass action found in enzyme reactions may be found in catalytic reactions in strictly homogeneous solutions. 2. These deviations in the rate of reaction from the law of mass action may be explained by the hypothesis that the active mass of the reacting substances is not directly proportional to the total concentration of substance taken. 3. In support of this suggestion it is shown that for any given concentration of pepsin the relative rate of digestion of concentrated and of dilute protein solutions is always the same. If the rate of digestion depended on the saturation of the surface of the enzyme by substrate the relative rate of digestion of concentrated protein solutions should increase more rapidly with the concentration of enzyme than that of dilute solutions. This was found not to be true, even when the enzyme could not be considered saturated in the dilute protein solutions. 4. The rate of digestion and the conductivity of egg albumin solutions of different concentration were found to be approximately proportional at the same pH. This agrees with the hypothesis first expressed by Pauli that the ionized protein is largely or entirely the form which is attacked by the enzyme. 5. The rate of digestion is diminished by a very large increase in the viscosity of the protein solution. This effect is probably a mechanical one due to the retardation of the diffusion of the enzyme.     

THE KINETICS OF STARVATION

1. The relative weight loss of pigeons on starving rations may be represented by a simple modification of the law of autocatalysis. 2. An equation giving the actual weight of the animals during the 53 day period of the experiment is derived by means of the hypothesis that the process of inanition is governed by the simultaneous velocity relations between two independent, homogeneous systems, such that M_T = (A ₀ → 0) + (B ₀ → B_C) in which A is taken proportional to the amount of carbohydrate-fat reserves, and B proportional to the amount of reacting body protoplasm. 3. The course of starvation is governed by the rate of destruction of body protein, but it is modified by the amount and by the rate of destruction of reserve materials. 4. The processes of breaking down tissues are analogous to those by which tissues are synthesized. 5. The close agreement between observed and calculated values suggests that the original assumptions set forth in deriving the foregoing equations were valid. 6. Attention is called to the fact that the time of onset of beriberi in pigeons is coincident with the half period of starvation.     

IMPACT OF IRON LIMITATION ON THE PHOTOSYNTHETIC APPARATUS OF THE DIATOM CHAETOCEROS MUELLERI (BACILLARIOPHYCEAE)

Iron starvation induced marked increases in flavodoxin abundance and decreases in light-saturated and light-limited photosynthesis rates in the diatom Chaetoceros muelleri. Consistent with the substitution of flavodoxin for ferredoxin as an early response to iron starvation, increases of flavodoxin abundance were observed before declines of cell division rate or chl a specific photosynthesis rates. Changes in the abundance of flavodoxin after the addition of iron to iron-starved cells indicated that flavodoxin was not actively degraded under iron-replete conditions. Greater declines in light-saturated oxygen evolution rates than dark oxygen consumption rates indicated that the mitochondrial electron transfer chain was not affected as greatly by iron starvation as the photosynthetic electron transfer chain. The carbon:nitrogen ratio was unaffected by iron starvation, suggesting that photosynthetic electron transfer was a primary target of iron starvation and that reductions in nitrate assimilation were due to energy limitation (the C:N ratio would be expected to rise under nitrogen-limited but energy-replete conditions). Parallel changes were observed in the maximum light-saturated photosynthesis rate and the light-limited initial slope of the photosynthesis-light curve during iron starvation and recovery. The lowest photosynthesis rates were observed in iron-starved cells and the highest values in iron-replete cells. The light saturation parameter, I_k, was not affected by iron starvation, nor was the chl-to-C ratio markedly reduced. These observations were consistent with iron starvation having a similar or greater effect on photochemical charge separation in PSII than on downstream electron transfer steps. Declines of the ratio of variable to maximum fluorescence in iron-starved cells were consistent with PSII being a primary target of iron starvation. The functional cross-section of PSII was affected only marginally (<20%) by iron starvation, with the largest values observed in iron-starved cells. The rate constant for electron transfer calculated from fast repetition rate fluorescence was found to covary with the light-saturated photosynthesis rate; it was lowest in the most severely starved cells.     

THE RATE OF GROWTH OF THE DAIRY COW : IV. GROWTH AND SENESCENCE AS MEASURED BY THE RISE AND FALL OF MILK SECRETION WITH AGE.

Data are presented on the effect of age on milk secretion in the dairy cow. From the age when milk secretion usually begins (2 years) to the age when maximum body weight is reached (about 8 years) increase of milk secretion and increase of body weight with age follow the same exponential course, which is the course of a monomolecular reaction of chemistry. After this age, unlike body weight which remains practically constant, milk secretion declines exponentially, that is, the course of decline follows the course of decline of a monomolecular reaction. The whole course of milk secretion with age was therefore found to follow approximately the course of two simultaneous, consecutive, monomolecular reactions. This is taken to mean that growth and senescence go on simultaneously from the beginning to the end of life, and that each follows an exponential law with age; and therefore perhaps that the course of the two processes are limited by two consecutive chemical reactions.     

THE KINETICS OF SENESCENCE

The course of decline of vitality with age due to the process of senescence, when not complicated by the process of growth, follows a simple exponential law; that is the degree of vitality or of senescence (defining vitality as the reciprocal of senescence) at any moment is, regardless of age, a constant percentage of the degree of vitality or senescence of the preceding moment. This exponential law is the same as the law of monomolecular change in chemistry. During the actively growing period of life the index of vitality rises, due to the process of growth and the course of vitality in the case when the growing period is included in the vitality curve, follows a rising and falling course. This rising and falling course may often be represented by an equation containing two exponential terms which is practically the equation used to represent the course of accumulation and disappearance of a substance as the result of two simultaneous consecutive monomolecular chemical reactions.     

ON THE MECHANISM OF MILK SECRETION : THE INFLUENCE OF INSULIN AND PHLORIDZIN

The four types of experiments on milk secretion herein described really fall into one general class so far as the physiological effects produced are concerned. Starvation lowers the blood sugar and raises the osmotic pressure of the blood. The experiment using parathyroid hormone with or without starvation may have its effects interpreted as simply due to starvation since 1000 units of this hormone produced no visible effects on the blood calcium or milk constituents different from those of starvation. Since insulin produces a marked and rapid drop in blood sugar it too may be looked upon as a rapid starvation effect. It has some other important effects, however. Briggs et al. (21) have shown that potassium and phosphorus of the blood are decreased and Luck, Morrison, and Wilbur (22) indicate a reduction in the amino acids of the blood in insulin treatment. Phloridzin lowers the threshold for sugar retention with the consequence that in time it tends to lower the sugar of the blood to an even greater extent than that noted in starvation. It tends to depress the potassium, to increase the phosphorus content of the blood, and to cause the body to burn protein rather than carbohydrate, thus increasing nitrogen excretion. All of the experiments are characterized by a sharp reduction in the milk yield. Cary and Meigs (23) have studied like reductions in milk yield produced by varying the energy or protein of the diet. They conclude that such decrease in milk production may be interpreted as due to the direct effect of the starvation and the consequent reduction of the energy and protein available to milk secretion. The reduction in milk yield for the experiments herein described can undoubtedly be attributed to the same causes as those cited by Cary and Meigs. The experiment where Cow 47 was given a full ration and at the same time injected with large quantities of insulin is of particular interest in this connection. The ration was adequate and the cow ate well, yet her production declined to a fifth of her normal milk yield. Her chart shows that there was a slight reduction in her blood sugar when insulin was introduced into the blood stream. It seems furthermore likely that this sugar was not as available to milk secretion, since there appears to be more than a corresponding drop in the lactose content of the milk. The work of Luck et al. would seem to indicate that there should be a like drop in the amino acids of the blood. These two conditions would lead, according to the work of Cary and Meigs, to a reduction in the concentration of the nitrogen of the milk. Actually, in the experiment as it was performed, the nitrogen increased to a value about 40 per cent above normal. A somewhat similar conflict is noted in two of the other three insulin experiments where starvation accompanied insulin injection. To this extent it would seem that the factor deserving most emphasis in its immediate effect on milk yield is the energy available, and that the later and more secondary factor is the amino acid concentration of the blood. In the starvation experiments, the butter fat percentage of the milk rises rather uniformly with the duration of starvation. In the insulin experiments, however, the charts appear to show a marked reduction in this butter fat percentage immediately after the introduction of insulin. This is particularly noticed after the second and third injections. Since the dextrose of the blood tends to be reduced and made unavailable to the general physiological processes by the presence of the large excess of insulin, and since this reduction of the butter fat percentage is noted as an accompanying phenomenon, it would appear that the blood dextrose plays a part in the synthesis of milk fat as well as being the source of the milk lactose, possibly as a source of energy in converting body fat to butter fat. In this regard the results for the treatment of Cow 47 with phloridzin are of importance. As noted by others, the introduction of phloridzin causes a marked rise in the fat percentage of the milk. The lactose per cent is also higher than that noted in starvation. Since phloridzin, by lowering the threshold for the blood sugar, causes large quantities of it to be drained from the body through the urine, and therefore reduces the reserve supply, it follows that if the insulin hypotheses are correct we should expect an eventual lowering of the lactose and of the fat below the starvation level. During the last of the experiment this is what was actually observed. The effects of starvation and of insulin furnish concordant proof for the theory that the lactose of milk is derived from the sugar of the blood. The fact that the different constituents of the milk, the fat, the lactose, the nitrogen, and the ash, do not exactly parallel each other in their behavior throughout these experiments indicates that they have in all probability separate origin. This is particularly true of the butter fat percentage, which appears to have a rate of secretion which is more or less independent of the other constituents, and higher in amount. This result would fall in line with the conclusion of the writers in a previous paper in which it was indicated that the fat of the blood was very likely deposited in the udder as fat corresponding to body fat from which source it was metabolized into the fat of milk shortly before it was needed for milk secretion. The wide variation brought about in the constituents of the milk by the treatment all point to the conclusion that in milk secretion a balance is maintained between the osmotic pressure of the milk and of the blood. Thus when the sugar of the milk is reduced either through starvation or by insulin the ash constituents rise to compensate for this reduction and make the osmotic pressure of the milk similar to that of the blood. These results further appear to indicate that the salts and the sugars are more or less independent in their passage and metabolism into milk from the other constituents. These observations are therefore in line with those obtained by Jackson and Rothera (14) and by Davidson (15) in their brilliant experiments where they modified milk secretion by returning milk or milk sugars and salts to the udder. These experiments give direct proof for the conclusion that modifications of the blood of dairy cattle produce direct and predictable modification of the milk secreted.     

THE RATE OF GROWTH OF THE DAIRY COW : V. EXTRAUTERINE GROWTH IN LINEAR DIMENSIONS.

Barring fluctuations due to the cyclic phenomena, the extrauterine course of growth in linear dimensions and in weight of the dairy cow follows an exponential law having the same form as the law representing the course of monomolecular change in chemistry. This suggests the interpretation that the general course of growth is limited by a monomolecular chemical process, and that the cyclic phenomena are due to subsidiary processes in the fundamentally exponential course of growth. The fact that growth follows or tends to follow an exponential course may be stated more simply as follows: if the unit of time is taken sufficiently large so that fluctuations due to the cyclic phenomena are balanced or eliminated, then the amount of growth made during the given unit of time at any age tends to be a constant percentage of the growth made during the preceding unit of time. Thus, the growth in height at withers made during any year is about 34 per cent of the growth made during the preceding year. Similarly the growth in weight made during any year is about 56 per cent of the growth in weight made during the preceding year. This is in accordance with expectations if it is assumed that each animal begins life with a definite endowment of limiting substance necessary for the process of growth, and that this endowment is used up at a constant rate (or percentage) of itself.     

CELL PROLIFERATION IN COMPLEX TISSUES: THE CONTROL OF THE MITOTIC CYCLE OF CELL POPULATIONS IN THE CULTURED ROOT MERISTEM OF SUNFLOWER (HELIANTHUS)

Experiments were performed with cultured primary root tips of sunflower (Helianthus annuus var. Russian Mammoth) to determine: (1) if progression in the mitotic cycle of meristematic cells was nutritionally controllable by carbohydrate starvation and replenishment; (2) where in the mitotic cycle control was effected; and (3) whether nutritional deprivation could be used to detect phenotypically different subpopulations in a complex tissue. Meristematic cells were rendered stationary by carbohydrate starvation, as indicated by the absence of cell division; this condition was reversed by carbohydrate provision. After 72 or 96 hr of starvation most cells stopped in G1 (80–90%) and G2 (10–20%), and a very few (“leaky” cells) continued to enter S. “Leaky” cells represent a small population with an S period of approximately 4.1 hr that either lack a principal control point in G1 or have an unusual metabolism whereby the control point requirements are met and have a carbohydrate dependence for mitosis. Though phenotypically different, no specific functions can be attributed to “leaky” cells at this time.     

EFFECT OF PHOSPHATE CONCENTRATION ON THE FINE STRUCTURE OF THE CYANOBACTERIUM,MICROCYSTIS AERUGINOSA KÜTZ. EMEND. ELENKIN

SUMMARY

Microcystis aeruginosa toxic strain UV-006 stored a fixed amount of polyphosphate in spherical granules located in the centroplasm. Twenty four hours of phosphate starvation induced use of stored polyphosphate, manifested by reduction in granule numbers. Reintroduction of 2, 4 or 8 mg l^?1 K₂HPO₄ resulted in redeposition of polyphosphate in a critical number of centroplasmic polyphosphate granules. Growth rate was unaffected by phosphate concentrations, although the final cell yield was slightly lower at 8 mg l ^?1

Continued starvation decreased photosynthetic rate and growth ceased. Cells appeared senescent. Cyanophycin and polyglucoside reserves apparently increased in these cells, whilst thylakoids were reduced in number and reorientated away from. the cell wall and polyhedral bodies were lost. After the initial decrease, centroplasmic polyphosphate bodies increased to about half of the maximum numbers stored in cells grown in the presence of phosphate, suggesting that translocation of phosphorus from other areas in the phosphate-starved cell occurred.

Two further polyphosphate deposition areas were observed. DNA fibrils may have represented nucleation sites for developing polyphosphate granules. Intrathylakoidal deposits were rare.     

THE USE OF GLUCOSE INORGANIC SALTS MEDIA IN THE CLASSIFICATION OF THE COLI-AEROGENES BACTERIA. I. THE METHYL RED AND VOGES-PROSKAUER REACTIONS

SUMMARY: A chemically defined glucose ammonium phosphate medium gave methy red and Voges-Proskauer reactions which were superior to those in glucose peptone phosphate. Optimum condition for both tests require a starting pH of 6·8 and incubation for 3 days at 30°. The medium gave a negative correlation between the two reactions with 34 out of 35 strains that were positive to both tests in the peptone medium. The addition of glutamic acid helped to eliminate doubtful positive M. R. reactions but seemed to suppress acetylmethylcarbinol production by weakly V.-P. positive strains.     

饥饿对太平洋鲑生长、机体组成及血浆相关生化指标变化研究

实验研究了太平洋鲑鱼(Oncorhynchus spp.)经8-32d饥饿后对其生长、体组成与血浆生化指标变化的影响。90尾初始重约为217g的太平洋鲑鱼放养于0.25m3的水族箱中0-32d,水温为(17.0±2.9)℃。实验分5组,分别为对照组(饥饿0d)、实验1组(饥饿8d)、实验2组(饥饿16d)、实验3组(饥饿24d)、实验4组(饥饿32d)。每组3个平行,每箱6尾鱼。结果表明:饥饿期间,太平洋鲑鱼存活率均为100%,相对体重损失率与饥饿时间直线回归方程为y=0.0086x(R2=0.9177),呈显著的正相关(P0.05);肠系膜脂肪是太平洋鲑鱼最主要的能量来源,与饥饿时间呈显著的负相关(P0.05),其直线回归方程y=-0.0719x±4.11(R2=0.9732);饥饿初期太平洋鲑鱼主要消耗肝脏糖原和脂肪、部分肠系膜脂肪和少许肌肉中脂肪维持生命活动,能耗较低;饥饿后期主要以消耗肠系膜脂肪、部分肌肉脂肪和少量蛋白质维持生命活动,能耗较高;血浆中脂肪分解酶和白蛋白无显著变化(P0.05),胆固醇、甘油三酯和高低密度脂蛋白有显著波动(P0.05),表明脂肪代谢活跃,免疫功能未受明显影响。研究表明太平洋鲑鱼能够有效地利用体内储存的脂肪,对饥饿的耐受能力较强。       

EFFECT OF NITROGEN STARVATION ON THE BIOCHEMISTRY OF PHORMIDIUM LAMINOSUM (CYANOPHYCEAE)1

Cells of the non-N₂-fixing cyanobacterium Phormidium laminosum (Agardh) Gomont (strain OH-1-pCl₁) showed doubling times of 24 h in media containing nitrate and 120 h in media without a nitrogen source. Nitrogen starvation resulted in a drastic decrease in the cellular content of chlorophyll, phycobiliproteins (phycocyanin and allophycocyanin), and other soluble proteins, although the total protein of cells was unchanged. N-starved cells showed an exocellular layer of mucilage that rapidly increased with starvation time. The appearance of N deficiency symptoms was strongly dependent on culture conditions, and it was faster under the optimal conditions used for cell growth. The relative content of C and N of nitrate-grown cells remained more or less constant during all growth phases (C/N ratio of ca. 5) but diminished at different rates in N-starved cells. Cells subjected to N starvation for 48 h had a C/N ratio of more than 10. N starvation also resulted in the selective degradation of soluble poly-peptides of masses lower than 20 kDa (which include those constituting phycobiliproteins), whereas the relative content of soluble polypeptides of greater size increased.     

低龄草鱼鳞片环纹的增长及幼轮和年轮特征的初步研究

幼草鱼鳞片环纹,以孵出后第二个月生长最快,往后生长减慢,冬季停止生长。幼鱼体长一般在47—70毫米形成幼轮。鳞片的“切割相”是区别年轮和幼轮的标志。幼鱼在饥饿时,环片不仅不增长,反而出现环片被吸收的现象。鳞片上各种副轮标记的形成与摄食条件的变化有关。计算了体长与鳞径、环纹数以及鳞径与环纹数的迴归方程,它们之间都呈直线正相关,相关显著性均在99%以上。       

RESPONSE OF THE PHOTOSYNTHETIC APPARATUS OF PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE) TO NITRATE,PHOSPHATE, OR IRON STARVATION1

The effects of nitrate, phosphate, and iron starvation and resupply on photosynthetic pigments, selected photosynthetic proteins, and photosystem II (PSII) photochemistry were examined in the diatom Phaeodactylum tricornutum Bohlin (CCMP 1327). Although cell chlorophyll a (chl a) content decreased in nutrient-starved cells, the ratios of light-harvesting accessory pigments (chl c and fucoxanthin) to chl a were unaffected by nutrient starvation. The chl a-specific light absorpition coefficient (a*) and the functional absorption cross-section of PSII (σ) increased during nutrient starvation, consistent with reduction of intracellular self-shading (i.e. a reduction of the “package effect”) as cells became chlorotic. The light-harvesting complex proteins remained a constant proportion of total cell protein during nutrient starvation, indicating that chlorosis mirrored a general reduction in cell protein content. The ratio of the xanthophylls cycle pigments diatoxanthin and diadinoxanthin to chl a increased during nutrient starvation. These pigments are thought to play a photo-protective role by increasing dissipation of excitation energy in the pigment bed upstream from the reaction centers. Despite the increase in diatoxanthin and diadinoxanthin, the efficiency of PSII photochemistry, as measured by the ration of variable to maximum fluorescence (F_v/F_m) of dark-adapted cells, declined markedly under nitrate and iron starvation and moderately under phosphate starvation. Parallel to changes in F_v/F_m were decreases in abundance of the reaction center protein D1 consistent with damage of PSII reaction centers in nutrient-starved cells. The relative abundance of the carboxylating enzyme, ribulose bisphosphate carboxylase/oxygenase (RUBISCO), decreased in response to nitrate and iron starvation but not phosphate starvation. Most marked was the decline in the abundance of the small subunit of RUBISCO in nitrate-starved cells. The changes in pigment content and fluorescence characteristics were typically reversed within 24 h of resupply of the limiting nutrient.     

COMPLEX TRADE-OFFS AND THE EVOLUTION OF STARVATION RESISTANCE IN DROSOPHILA MELANOGASTER

The measurement of trade-offs may be complicated when selection exploits multiple avenues of adaptation or multiple life-cycle stages. We surveyed 10 populations of Drosophila melanogaster selected for increased resistance to starvation for 60 generations, their paired controls, and their mutual ancestors (a total of 30 outbred populations) for evidence of physiological and life-history trade-offs that span life-cycle stages. The directly selected lines showed an impressive response to starvation selection, with mature adult females resisting starvation death 4–6 times longer than unselected controls or ancestors—up to a maximum of almost 20 days. Starvation-selected flies are already 80% more resistant to starvation death than their controls immediately upon eclosion, suggesting that a significant portion of their selection response was owing to preadult growth and acquisition of metabolites relevant to the stress. These same lines exhibited significantly longer development and lower viability in the larval and pupal stages. Weight and lipid measurements on one of the starvation-selected treatments (SB_1–5), its control populations (CB_1–5), and their ancestor populations (B_1–5) revealed three important findings. First, starvation resistance and lipid content were linearly correlated; second, larval lipid acquisition played a major role in the evolution of adult starvation resistance; finally, increased larval growth rate and lipid acquisition had a fitness cost exacted in reduced viability and slower development. This study implicates multiple life-cycle stages in the response to selection for the stress resistance of only one stage. Our starvation-selected populations illustrate a case that may be common in nature. Patterns of genetic correlation may prove misleading unless multiple pleiotropic interconnections are resolved.     

饥饿对大鼠胰岛素分泌的影响及其机制的分析

本工作探讨了大鼠饥饿时胰岛素分泌减少的机制。大鼠连续饥饿1至4d,其血浆胰岛素水平的变化不与血糖平行,二者虽均于饥饿第1天即显著下降,但胰岛素在饥饿期间一直维持于低水平。而血糖则于饥饿第3天开始回升,第4天接近正常水平。在离体实验中发现,禁食3d大鼠的离体灌流胰腺对精氨酸的胰岛素分泌反应明显地低于正常摄食大鼠。看来,饥饿时胰岛素分泌的减少似不是由于营养物质对胰岛β细胞刺激作用的减弱,而可能是由于局部抑制性影响加强所致。 我们进一步观察了胰内生长抑素的作用,发现饥饿3d的大鼠,在胰岛素分泌减少的同时,胰腺生长抑素的含量增高,如预先给动物注射半胱胺以耗竭内源性生长抑素,则使饥饿大鼠胰岛素分泌的抑制现象明显减轻。在离体实验中也发现,预先注射半胱胺的饥饿大鼠,其离体灌流的胰腺对精氨酸的反应恢复正常。这些结果提示,胰内生长抑素对β细胞局部抑制作用的增强,可能是饥饿引起胰岛素分泌减少的机制之一。     

饥饿状态下草鱼生长激素的分泌

以两种规模草鱼为对象,研究了饥饿对其生长激素的影响。检测由背大动脉导管抽取的连续血样的结果表明;饥饿状态下草鱼（体重为0．5－1．0kg）生长激素分泌仍是间歇性的,但饥饿明显提高其总体生长激素平均值、基础生长激素平均值和其最大峰值。对于草鱼鱼种（体重为25．30g）,饥饿也明显提高其血液中生长激素水平,但草鱼种的肥满度系数和血糖浓度却。在体外灌流实验中,饥饿的草鱼种脑垂体碎片生长激素基础分泌值明显高于正常投喂的对照组。这些结果表明：饥饿状态下草鱼生长激素分泌增强。     

STUDIES ON LUMINOUS BACTERIA : II. THE INFLUENCE OF TEMPERATURE ON THE INTENSITY OF THE LIGHT OF LUMINOUS BACTERIA.

1. A method has been described whereby the intensity of the light of luminous bacteria may be measured in a quantitative manner. 2. It is pointed out that the temperature coefficients for light intensity do not follow the van''t Hoff rule, but are higher and vary with each 10° temperature interval. 3. From a comparison with other data it is found that the process is not a simple one, but that the observed curve is the resultant of several reactions which proceed simultaneously. 4. The discrepancies in the temperature coefficients in the neighborhood of the "optimum temperature" may be due to a process of coagulation of the colloidal particles of the enzyme. This coagulation will tend to cause a deviation of the curve away from that normal for chemical reactions.     

INFECTIVE ORGANISMS IN THE CYTOPLASM OF AMOEBA PROTEUS

Evidence from electron and phase microscopy is given which shows that infective organisms are present in the cytoplasm of Amoeba proteus. Vesicles containing living organisms have been observed after repeated washing and starvation of the amebae for a period of 2 weeks. Exposure to γ-radiation in conjunction with starvation, repeated washing, isolation of single amebae, refeeding with contaminant-free Tetrahymena, and clone selection has produced clones with reduced cytoplasmic infection. These findings are discussed in regard to the autoradiographic studies of other investigators on Amoeba proteus. The controversies over whether DNA and RNA are synthesized in the cytoplasm may be resolved by the finding of cytoplasmic infection.     

METABOLIC RESPONSES OF THE DIATOM ACHNANTHES BREVIPES (BACILLARIOPHYCEAE) TO NUTRIENT LIMITATION

The diatom Achnanthes brevipes C.A. Ag. was cultured in the presence of limiting concentrations of nitrogen (N) or inorganic phosphate (P_i). Growth, in terms of final yield, was more affected by N limitation than P_i limitation; N limitation had a greater effect also on protein and chlorophyll content. Carbohydrate concentrations increased under both nutrient starvation treatments, but N or P_i limitation had different effects. Total (intracellular plus extracellular) sugar content increased when cells were exposed to both types of nutrient limitation, but the extracellular polysaccharide fraction increased only in the presence of P_i starvation. Analyses were performed to identify the metabolic changes occurring in cells exposed to low phosphate because this was the main condition that affected carbohydrate extrusion. Activities of several enzymes involved in carbohydrate metabolism showed that under P_i limitation there was no activation of alternative reactions that were found to result in P_i liberation, instead of its consumption, in some higher plants and in the green alga Selenastrum minutum Naeg. Collins. Results showed that activities of pyruvate kinase, phosphorylating NAD-dependent 3-phosphate-glyceraldehyde dehydrogenase, and 3-phospho-glycerate kinase were inhibited under P_i-limited conditions compared with control cells, indicating limited glucose catabolism. Activity of uridine diphosphate glucose pyrophosphorylase, a key enzyme for the biosynthesis of the storage compound crysolaminarin, was also partly inhibited in P_i-stressed cells. Our findings suggest that carbohydrate catabolism in A. brevipes is limited under P_i deficiency, whereas extracellular extrusion of carbohydrate is favored.