Immunologic and Electrophoretic Characteristics of Two Species of Crithidia*†

SYNOPSIS. Crithidia harmosa and Crithidia fasciculata were compared immunologically by the indirect fluorescent antibody (FA) method and by agglutination. The FA technic yielded more specific results with cellular-debris than with whole-cell antigens. The immune sera collected from chickens 9 days after the last inoculations with whole-cell antigens had higher homologous titers than those collected after 4 days. Major antigenic differences between the 2 species were revealed by both methods. The electrophoretic patterns of C. harmosa and C. fasciculata obtained by polyacrylamide gel slab electrophoresis differed in the numbers and relative mobilities of their component bands.     

Simple Nutrition of Crithidia deanei,a Reduviid Trypanosomatid with an Endosymbiont*

Crithidia deanei from the reduviid hemipteron, Zelus leucogrammus, unlike most lower trypanosomatids cultivated in defined medium, required only 2 amino acids, methionine and tyrosine; only 4 vitamins, folic acid, thiamine, biotin, and nicotinamide; and neither hemin nor a purine source. Electron microscopy reveals an endosymbiont, probably bacterial, which presumably provides the other basic trypanosomatid essential nutrients.     

Extra Nutritional Requirements of Artificially Aposymbiotic Crithidia deanei*

SYNOPSIS. Chloramphenicol cured Crithidia deanei of its endosymbiote. The derived aposymbiotic strain had additional growth requirements: purin (as adenine), heme, arginine, histidine, isoleucine, leucine, phenylalanine, threonine, tryptophan, valine, pyridoxine, riboflavin, and pantothenate and liver infusion (replaceable by high nicotinamide).     

Reduced Growth of Blastocrithidia culicis and Crithidia oncopelti Freed of Intracellular Symbiotes by Chloramphenicol*

The intracellular symbiotes of Blastocrithidia culicis and Crithidia oncopelti can be eliminated from cultures of the flagellates by a single chloramphenicol (CAP) treatment. Effective dosages were determined to be 0.01–0.08% (w/v) CAP after a treatment for 2 weeks or more for B. culicis and 0.08% (w/v) after 1 month for C. oncopelti in most cases. Ineffective dosages only lowered the numbers of symbiote-bearing flagellates. Growth of both species of flagellates in the presence of CAP was reduced in proportion to the drug concentration. Repeated subcultures at effective dosages yielded symbiote-free flagellates, which maintained a low level of growth rate. After repeated subcultures at ineffective dosages, the growth rate rose and the symbiote-bearing cells, initially very few, increased in number. The lowest effective dosages proved to be marginal, often producing symbiote-free cultures, but occasionally cultures with a few symbiote-bearing cells. After repeated subcultures at these drug concentrations, symbiote-containing cultures grew faster than the symbiote-free cultures. Hence, the symbiotic bacteria benefit the growth of their hosts, perhaps by supplying essential factors that are inadequate even in a rich blood medium.     

The Ribonucleic Acids of Crithidia fasciculata*

SYNOPSIS. Crithidia fasciculata ribosomes were found to be 80S and to dissociate into 58 and 41S subunits; on 5 to 50% sucrose gradients, rRNA was separated into 25, 18, and 5S components. The molecular sizes of the heavier rRNA species, estimated by polyacrylamide gel electrophoresis were 1.24 and 0.84 M (×10⁶ daltons). The 25S RNA has a tendency to interact with the 18S RNA to give a complex that is difficult to separate by sucrose gradient centrifugation. The 25S RNA is also unstable and dissociates into 0.73 and 0.57 M components. The 18S RNA has molecular size (0.84 M) higher than the 0.7 M reported for most eukaryotes, but similar to that of Euglena and Amoeba. Ribosomal RNA hybridized 0.29% of the nuclear DNA. Mitochondrial RNA, extracted by a rapid procedure was resolved into 16 and 5S components in sucrose gradients.     

Effect of Colchicine on Cell Membrane and on Biopterin Transport in Crithidia fasciculata*

SYNOPSIS. Incorporation of ¹⁴C-labeled biopterin into Crithidia fasciculata was inhibited by 1 mM colchicine or lumicolchicine. These substances do not penetrate the cell membrane, hence they cannot interact with the subpellicular microtubules. In view of this, interference of colchicine with biopterin transport must occur on the outer surface of the cell membrane. Binding of colchicine to Crithidia was not temperature-dependent and did not exhibit saturation kinetics. These facts exclude a binding as in the case of tubulin, or similar proteins which may be present in the membrane. The results suggest an inhibition reflecting steric hindrance of the biopterin carrier system.     

Enzymes of the Ornithine-Arginine Metabolism of Trypanosomatids of the Genus Crithidia*

SYNOPSIS. Twelve strains of Crithidia, which fall into 8 species, were tested for occurrence of enzymes of ornithine-arginine metabolism. The following enzymes were investigated: arginase, ornithine carbamoyltransferase, argininosuccinate lyase, citrulline hydrolase, arginine deiminase and urease. Arginase and argininosuccinate lyase were found in all species. Citrulline hydrolase was also found in all but the 2 strains carrying endosymbiotes C. deanei and C. oncopelti. On the other hand, ornithine carbamoyltransferase was found only in these 2 strains. Arginine deiminase and urease were absent in all strains. The existence of a common enzymatic pattern for species of the genus Crithidia is thus reported.     

Symbiote-Free Hemoflagellates,Blastocrithidia culicis and Crithidia oncopelti: their Liver Factor Requirement and Serologic Identity*

SYNOPSIS. Several aposymbiotic strains of Blastocrithidia culicis and Crithidia oncopelti were cultivated in Trager's chemically defined medium as well as in a blood broth, both supplemented with 0.25% (v/v) liver extract concentrate. For all such strains, the liver extract was found to serve as an essential growth factor in the defined medium and as growth promoting additive in the blood broth. The active molecules were found to be water-soluble, heat stable, dialyzable, and probably nonlipid fractions. Antisera were developed in rabbit against all the available aposymbiotic strains. An almost total cross-reactivity at very high titers was observed in reciprocal agglutination test using strains with and without the bacterial symbiotes. These results indicate that the loss of the symbiotes does not affect the antigenic identity of B. culicis and C. oncopelti.     

水分胁迫下高粱叶片的渗透调节与延伸生长的关系

高粱抗旱品种3197B比不抗旱品种三尺三在水分胁迫条件下ψ_S下降低。在相同ψ_S时,3197B相对含水量高于三尺三。水分胁迫期间,3197B能始终维持比三尺三较高的ψ_P。在中度和严重水分胁迫时,3197B几种渗透物质积累均高于三尺三,其中可溶性糖和K~ 对渗透调节贡献最大。水分胁迫下,3197B正展开叶渗透调节能力较强,ψ_P维持较高,临界膨压低,叶片扩张性能小、故生长速率随ψ_W下降较慢。     

Biochemical Approaches to Problems of Cellular Patterning*†

SYNOPSIS. The expression of intracellular patterning is perhaps nowhere more impressive than in the arrangements of structural elements associated with the cell surface in protozoa. The view is proposed that biochemical studies of protozoan plasma membranes and associated surface structures represent important contributions of potential significance for the understanding of the perpetuation, and expression of positional information at the intracellular level. Some recent work dealing with the isolation, identification, and metabolism of pellicular proteins in Tetrahymena is presented and discussed. Some integral membrane proteins have been identified by iodination and polyacrylamide gel electrophoresis. Labeling studies suggest heterogeneous turnover rates within the group of presently identified membrane proteins. High molecular weight proteins with some similarity to spectrin have been isolated from Tetrahymena epiplasm. It is suggested that the ciliate epiplasm is one example of membrane-associated, actomyosin-like systems found in a variety of cell types. The epiplasm may play a role in the positioning of surface-associated structures and in the control of cell shape.     

The Role of Hormones in Fast Growth Responses of Wheat Plants to Osmotic and Cold Shocks

The effects of nutrient-solution cooling and PEG addition to the nutrient solution on the phytohormone content, the rate of leaf growth, leaf extensibility under the influence of external mechanical action, osmotic potential, and transpiration were studied in seven-day-old wheat plants. Leaf growth rapidly ceased, and the transpiration rate was reduced in both treatments. Growth cessation induced by PEG was transient, and growth resumption was preceded by an increase in the leaf extensibility. The functional role of auxin accumulation in plant shoots in the control of extensibility as well as the relationship between the ABA accumulation and a decrease in the cytokinin content, on the one hand, and reduced transpiration, on the other hand, under stress conditions are discussed.     

The Sites of Action of Allopurinol in Crithidia fasciculata*

Reversal of the growth inhibition of Crithidia fasciculata by allopurinol requires both a purine and a pyrimidine. Hypoxanthine is the most effective purine in the reversal. Cell-free extracts were prepared which were capable of the decarboxylation of orotidine 5′-phosphate. Other enzyme preparations carried out the phosphoribosylation of allopurinol. By the use of [4-¹⁴C] orotidine 5′-phosphate (enzymatically prepared), it was shown that allopurinol ribotide (enzymatically prepared), but not the free base, inhibits orotidine 5′-phosphate decarboxylase.     

‘Osmotic pump‘ feeding by coreids

Species of Coreidae (Heteroptera) cause ‘water soaked’ lesions in their food plants. Such insects typically feed from parenchyma in and surrounding vascular tissues and also cause acropetal wilting and necrosis of small diameter shoots. Feeding byMictis profana (Fabr.) in South Australia on the shoots ofAcacia iteaphylla F. Muell. ex Benth. was found to cause a local, concurrent increase in both water content and free amino acid concentration, consistent with phloem unloading. Coreids, unlike other groups of phytophagous Heteroptera, secrete a salivary sucrase (α-D-glucohydrolase, EC 3.2.1.48) as probably the sole salivary carbohydrase, and tissues attacked byM. profana showed more sucrose hydrolysing activity than unattacked. The salivary enzyme is postulated to cause unloading of solutes into the apoplast due to the osmotic effects of conversion of endogenous sucrose to glucose and fructose, allowing the insect to suck the leaked contents of many cells from a single locus. The term ‘osmotic pump feeding’ is proposed for such a process. In demonstrations of its feasibility, infiltration of shoots with mixtures of glucose and fructose stoichiometrically equivalent to isosmotic sucrose increased the amounts of tissue sap and amino acid that could be sucked from the tissues; similarly, invertase and 1 M sugars forced into the extracellular space of stem sections increased the amino acids offloaded into the bathing solutions.     

Role of Arabidopsis UV RESISTANCE LOCUS 8 in Plant Growth Reduction under Osmotic Stress and Low Levels of UV-B

In high-light environments, plants are exposed to different types of stresses, such as an excess of UV-B, but also drought stress which triggers a common morphogenic adaptive response resulting in a general reduction of plant growth. Here, we report that the Arabidopsis thaliana UVRESISTANCE LOCUS 8 （UVR8） gene, a known regulator of the UV-B morphogenic response, was able to complement a Saccharomyces cerevisiae osmo-sensitive mutant and its expression was induced after osmotic or salt stress in Arabidopsis plants. Under low levels of UV-B, plants overexpressing UVR8 are dwarfed with a reduced root development and accumulate more flavonoids compared to control plants. The growth defects are mainly due to the inhibition of cell expansion. The growth inhibition triggered by UVR8 overexpression in plants under low levels of UV-B was exacerbated by mannitol-induced osmotic stress, but it was not significantly affected by ionic stress. In contrast, uvr8-6 mutant plants do not differ from wild-type plants under standard conditions, but they show an increased shoot growth under high-salt stress. Our data suggest that UVR8-mediated accumulation of flavonoid and possibly changes in auxin homeostasis are the underlying mechanism of the observed growth phenotypes and that UVR8 might have an important role for integrating plant growth and stress signals.     

Dense Crithidia Growth and Heme Sparing: Relation to Fe,Cu, Mo Chelation*

SYNOPSIS. Heme, intrinsically required by Trypanosomatidae, is unstable, especially in conventional alkaline (pH 7.2–8.0) media. Low solubility of heme in a pH 6.5 basal medium (developed to assay biopterin with Crithidia fasciculata) posed a problem: in media acidified during growth because of glycolysis, heme precipitated, perhaps contributed to acid-limited growth and interfered with densitometric estimation of growth. The remedy was to: replace glucose with less rapidly metabolized mannitol; distribute media in thin layers to promote oxidation of acetate, fumarate, and malate (presumably leaving an alkaline residue); and buffer heavily with histidine + Good zwitterionic buffers, and superimpcse physiological buffering by arginine + asparagine whose catabolism appeared to yield an excess of NH⁺₄ over acid. Thereupon, Fe and Cu deficiencies sharply limited growth in the medium whose main chelators were: (a) 2,3–dihydroxybenzoic + 5-sulfosalicylic acids (which preferentially bind transitional elements at their higher valences; (b) malic and gluconic acids; and (c) histidine. With unconventionally heightened concentrations of Fe, Cu, and Mo (the latter serving as Cu buffer as well as nutrient per se), the hemin concentration could be lowered, widening the margin of safety for heme solubility. Growth then reached 1.4 × 10⁸ cell/ml. This medium may serve to screen for ligands promoting uptake or release of Fe and Cu. The increased growth is a step towards improving the assay medium for biopterin and practical use of Crithidia to assay several B vitamins and essential amino acids for metazoa.     

In vitro Transcription of Kinetoplast and Nuclear DNA in Kinetoplastida*†

SYNOPSIS. DNA-dependent RNA polymerases have been solubilized from homogenates of Crithidia fasciculata using gentle extraction procedures. RNA polymerase I and II are separated on DEAE cellulose at 0.07M (NH₄)₂SO₄ and 0.13M (NH₄)₂SO₄ respectively. RNA polymerase II is inhibited 80% by α-amanitin (25 μg/ml). Both RNA polymerases require DNA as a template, ribonucleoside triphosphates and Mn²⁺. The synthesis of RNA as a product is inhibited by DNase. RNase, pronase and actinomycin D. Purified kinetoplast and nuclear DNA can serve as templates for the RNA polymerases. Denatured DNA templates are preferred. The synthesis of RNA continues for at least an hour and is inhibited by trypanocidal drugs including suramin. antrycide, acriflavine, ethidium bromide and berenil. Complementary RNA synthesized in vitro from C. fasciculata kinetoplast DNA hybridizes with C. fasciculata kinetoplast DNA but not with C. fasciculata nuclear DNA or Blastocrithidia culicis kinetoplast DNA, Escherichia coli, T4 or calf thymus DNAs. The complementary RNA synthesized in vitro from C.fasciculata kinetoplast DNA sediments at 4–5S.     

The Effects of Insect Juvenile Hormone on the Growth of Some Protozoans in vitro. I. Crithidia sp.

SYNOPSIS. Experiments were designed to investigate the effects of insect juvenile hormone (JH) on the over-all growth and macromolecular synthesis of Crithidia sp. in vitro. Cells grown in the presence of 10⁻⁵M-10⁻³M JH showed a concentration-dependent inhibition of growth, which appeared to result from both a prolongation of generation time and a delay in the onset of logarithmic growth. Juvenile hormone (10⁻³M) inhibited the incorporation of [³H]thymidine, [³H]uridine and [³H] leucine into logarithmically growing cells by 50, 70 and 40% respectively. The incorporation of [³H]uridine into acid insoluble material could be stopped within 1 hr of application of the hormone (10⁻³M). The inhibitory effect was reversible in terms of cell numbers in subcultures of washed cells but an examination of the reversibility of RNA synthesis inhibition suggested that the resumption of RNA synthesis at an optimal level would require a lag period of at least 1–3 hr. It is suggested that JH may act by interfering with RNA synthesis either directly or indirectly by primarily acting at the level of the plasma membrane.     

Contractility and its Control in Peritrich Ciliates*†

A review of the recent literature on the structure and physico-chemical properties of the myoneme and its specialization, the spasmoneme, of peritrich ciliates was made. Myonemes are composed of tightly packed bundles of 3–5 nm microfibrils which parallel, more or less, the long axis of the bundle and are of indefinite length. The presence of contractility in these ciliates is correlated with the presence of myonemes. Associated with the microfibrils is a system of membrane-bound tubules and saccules continuous with the endoplasmic reticulum (ER). This system is known to accumulate calcium. Myonemes differ from muscles in their structure, solubility properties, birefringence pattern, and in the time during the contraction-relaxation cycle at which they require energy. They may be related more closely to the cytochalasin B-sensitive microfibrils of higher organisms than to muscles. Contraction of extracted stalks can be induced solely by raising the calcium ion concentration above a certain threshold. Thus the calcium-accumulating myoneme-associated ER would appear to play an important role in the control of myoneme contractility. A specialization at the interface between the myoneme and the ER membranes is described as it appears in Vorticella and Opercularia. This structure, called a linkage complex, is found both in the body myonemes and the spasmoneme and links the membranes of the ER to the microfibrils. It also has microfilaments that pass from the ER-myoneme interface to the surface membranes. The uniqueness of this structure and its location suggests that it may play a role in controlling the movements of calcium between the ER and myoneme and also in transmission of messages from the pellicular membranes, possibly the alveolar sacs, to the ER.     

The Induction of a Phospholipid Requirement and Morphological Abnormalities in Tetrahymena pyriformis by Growth at Supraoptimal Temperatures*†

SYNOPSIS. Supplementation of a chemically-defined medium that supported excellent axenic growth of Tetrahymena pyriformis, mating type II, variety 1, at 35 C, with nucleic acid derivatives allowed full growth at 37, and further addition of phospholipids permitted equivalent growth at 39 and 1/3 of that at 40. No other nutrients tested were active at 40. Population growth could be sustained continuously by serial sub-culture every 48 hr at 40 only if the medium contained synthetic phospholipids or phospholipids isolated from natural sources. The requirement was specific for phosphatidyl choline and phosphatidyl ethanolamine. Phospholipids which contained only saturated fatty acids, and ones which contained unsaturated acids were active. Phospholipids and neutral lipids isolated from 35-grown T. pyriformis were also effective. Phospholipid precursors, sterols, neutral lipids and fatty acids were not. A 48–72 hr exposure to 40 in either the unsupplemented medium or the nucleic acid derivatives-phospholipid-supplemented medium caused extreme variation in size and shape, fractured and displaced kineties, and abnormalities of karyokinesis. The frequency and degree of teratology was greater in the unsupplemented medium, but not markedly so. A possible metabolic basis for the temperature-induced phospholipid requirement and the morphological abnormalities is discussed.