Freeze-Fracture Study of the Trophozoite and the Cyst of Entamoeba histolytica

ABSTRACT The fine structure of the trophozoite and cyst of Entamoeba histolytica from the stool of a patient was compared using the freeze-fracture method. The intramembranous particles (IMP's) were heterogeneously distributed on the plasma membrane of the trophozoite and their density was 1139 ± 105/μm² on the P face and 27 ± 9/μm² on the E face. Particle-rich depressions and linear particle arrays, reported by other investigators on cultured trophozoites, were also observed on the P face while on the E face such special particle arrangement was not recognized. Particle-free, small protrusions were frequently observed on the P face of the trophozoite membrane. The existence of these protrusions is a new finding. In the cyst, the IMP's were also distributed heterogeneously on both the P and E faces of the plasma membrane. The density of the IMP's, however, was much lower than in the trophozoite: 6 ± 2/μm² on the P face and averaging less than 1/μm² on the E face. In freeze-fracture images, the plasma membrane of the cyst showed a variety of configurations from smooth to uneven or ridged surfaces. These morphological alterations of the plasma membrane may be attributed to the aging of the cyst. The thick wall of the cyst had a filamentous tri- or tetra-lamellar structure. The cytoplasm of the cyst was similar in structure to that of the trophozoite and the diameter of the nuclear pores was equal in both trophozoites and cysts.     

Rhythms in respiratory metabolism of Lemna gibba G3 under continuous illumination

Changes in chemical constituents and respiratory metabolismof a long-day duck-weed, Lemna gibba G₃ exposed to continuousillumination after short-day cultivation were investigated.The dry weight to fresh weight ratio was constant during thefirst 72 hr of continuous illumination. pH of the crude extractwas constant at 6.6, but pH of the culture medium was raisedwith the Lemna growth. Titratable acidity decreased after about44 hr, whereas malic acid content increased in 18 hr. Therewere no significant changes in total reducing sugar and pentose.Total protein content and lipid showed rhythmical changes withcycles of 48 hr. O₂-Uptake gave a damped oscillation with cycles of 24 hr. Itwas low in the first half day and high in the second half. ¹⁴CO₂-Outputfrom glucose-l-¹⁴C showed a similar damped oscillation. ¹⁴CO₂-Outputfrom glucose-2-14^C or glucose-6-₁₄C was almost constant. TheC₆/C₂ ratio, then, showed damped oscillation in the reverseway to O₂- uptake between 0.3–0.5, and the C₈/C₂ ratiowas constant at 0.9. Accordingly, the diurnal rhythm of O₂-uptakewas thought to be brought about by variation in activity ofthe pentose-phosphate pathway. Reproduction of glucose-6-phosphateby the pentose-phosphate pathway was presumably limited in amount. Glyceraldehyde-3-phosphate dehydrogenase activity varied diurnally.The activities of NADP-linked and NAD-linked enzymes increasedand decreased, respectively, in the first half day. Variationsin these enzymatic activities are discussed in correlation withrhythmical changes in O₂-uptake and in the C₆/C₁ ratio. Acidphosphatase activity also followed a diurnal variation. No activitiesof alcohol and formic dehydrogenases were found. The activitiesof NADP glucose-6-phosphate dehydrogenase, 6-phosphogluconatedehydrogenase, pyruvic kinase and NADP isocitric dehydrogenasewere high, but showed no rhythmical variation. ¹Presented at the Annual Meeting of the Botanical Society ofJapan, 1966 (Proceedings, p. 46). Adapted from a thesis submittedby the first author (H. M.) in 1967 to the Biological Institute,Nagoya University in partial fulfillment of the requirementsfor the degree of M. S. (Received May 8, 1969; )     

Induction of Adventitious Buds on Undetached Leaves, Excised Leaves and Leaf Fragments of Heloniopsis orientalis

Adventitious buds were induced on intact, undetached leaves, isolated leaves, and both green and etiolated leaf fragments excised from young plants of Heloniopsis orientalis (Thunb.) C. Tanaka (Liliaceae) in darkness. Morphactin promoted bud initiation on undetached leaves. The regeneration loci on excised leaves were different in darkness and in light, and they were also modified by etiolation and by morphactin or benzyladenine. Experiments with pre-incubation in darkness, with successive treatments by sorbitol and sucrose, and with DCMU-treatment in light, led to the conclusion that bud formation on isolated leaves and leaf fragments is controlled by a photosynthetic system as well as the hormonal level.     

THE GLYCEROL-ISOLATED MITOTIC APPARATUS: A RESPONSE TO PORCINE BRAIN TUBULIN AND INDUCTION OF CHROMOSOME MOTION

The birefringence of the MAs or spindles isolated from sea urchin eggs with the 1 M glycerol-isolation medium was stabilized when more than 0.5 mg/ml tubulin was contained in the medium. The addition of glycerol up to a final concentration of of 4 M strongly stabilized the MAs even in the absence of GTP and tubulin. The birefringence of the spindle and asters was not reduced even for the periods of several hours. The incorporation of heterogeneous tubulin into the isolated anaphase MAs was demonstrated by augmentation of the birefringence at the interzonal region as well as half spindles accompanied by enlargement of spindle and asters. In the anaphase MAs isolated in the absence of brain tubulin, chromosomes moved a short distance toward the poles upon addition of ATP, Mg²⁺ and 0.5 mg/ml tubulin. When the MAs were isolated in the presence of 0.5 mg/ml tubulin, the chromosomes moved in a more regular fashion to half the way to the poles accompanied by an increase in spindle length by 10 to 15%. GTP could not be substituted for ATP for inducing the motion. The chromosome motion of the isolated anaphase spindle was less significant than that of the isolated MA. Increasing tubulin concentration to 3 mg/ml, the chromosomes in the isolated MA separated at random by an unusual growth of the spindle. The stretch of the interzonal region by incorporating heterogeneous tubulin seemed to push the chromosomes apart abnormally. It was suggested that brain tubulin in a range of 0.5 mg/ml supports a tubulin-MA microtubule equilibrium favoring more regular motion of chromosomes in vitro .     

Development of Mitochondrial Activity in Pea Cotyledons Following Imbibition; Influence of the Embryonic Axis

The development of mitochondria in cotyledons during the initialstages following imbibition and the subsequent degenerationwere faster when the embryonic axis was attached. This was moreclearly demonstrated when mitochondrial activity was determinedusing malate or -oxoglutarate as substrate rather than NADHor succinate. Cycloheximide did not inhibit the initial developmentof mitochondria in attached cotyledons, although it inhibitedthe incorporation of ¹⁴C-amino acid into protein. Abscisic acidinhibited almost completely the initial increase in mitochondrialactivities of attached cotyledons. The activities of severalenzymes in mitochondrial fractions were almost the same in attachedand detached cotyledons during 4 d after, imbibition. The degenerationof mitochondria was not accompanied by the loss of enzymes.It was inferred that the initial development and the subsequentdegeneration of mitochondria in cotyledons during 4 d afterimbibition was brought about by the structural improvement anddisorganization of mitochondria, respectively. The initial differencesin the development of mitochondrial activities between attachedand detached cotyledons were suggested to be attributable todifferences in the development of the activities of electrontransfer pathway from endogenous NADH to ubiquinone.     

Regional Difference in Mechanical Properties of the Cell Surface in Dividing Echinoderm Eggs*

Stiffness of the cell was surface was determined in fertilized sea urchin and starfish eggs by measuring the mechanical resistivity of the cell surface against negative pressure applied to a restricted part with a micropipette in contact with the cell surface at its tip (elastimetry). In both sea urchin and starfish eggs, the stiffness of the cell surface changed almost in parallel between the presumptive furrow and polar surfaces before the onset of the first cleavage, and the stiffness of the furrow surface became larger than that of the polar surface when cleavage started, although temporal changes in the stiffness were different between sea urchin and starfish eggs. The stiffness of the cell surface changed almost in parallel between the surfaces at the equator and at the animal pole in starfish eggs before the onset of polar body formation. The stiffness of the cell surface around the forming polar body increased during the formation of the polar body and remained at a high level after the polar body formation. It seems that the stiffness difference responsible for the formation of the contractile ring develops simultaneously with rather than prior to the formation of the cleavage furrow.     

Proliferation of Pneumocystis Trophozoites in Human Lymph Nodes and in Nude Mice Lungs

Pneumocystis infection in athymic nude mice lungs showed a particularly high trophozoite to cyst ratio. A similar observation was obtained from a study of a patient with lymph node infection with Pneumocystis . Eosinophilic foamy masses in these sites were observed by light microscopy. With the electron microscope, the masses were seen to be composed of large aggregates of trophozoites. Cystic forms (precysl, cyst and empty cyst) were extremely scarce in comparison with the huge numbers of trophozoites. These cystic forms were mostly undergoing degeneration. These observations indicate that the mode of proliferation in both situations was predominantly asexual, that is, proliferation by trophozoites, suggesting that certain conditions may enhance asexual reproduction or depress the fonnation of cysts.     

Processing and Secretion of Lysosomal Acid α-Glucosidase In Tetrahymena Wild Type and Secretion-Deficient Mutant Cells

ABSTRACT. The proteolytic processing and secretion of a lysosomal enzyme, acid α-glucosidase, was studied by pulse-chase labeling with [³⁵S]methionine in Tetrahymena thermophila CU-399 cells treated with ammonium chloride. This cell secreted a large amount of acid α-glucosidase into the cultured medium during starvation. the secretion was found to be repressed by addition of ammonium chloride (NH₄Cl). Acid α-glucosidase was produced as a precursor form (108 kDa) and then processed to a mature polypeptide (105 kDa) within 60 min. This mature enzyme was secreted into the media within 2-3 h after chase, whereas the precursor form was not secreted by either control cells or NH₄Cl-treated cells. NH₄Cl did not affect the processing of the precursor acid α-glucosidase. Processing profile of this enzyme was apparently indistinguishable from that of the mutant MS-1 defective in lysosomal enzyme secretion. Furthermore, the purified extracellular (CU-399) and intracellular (MS-1) acid a-glucosidases were the same in molecular mass (105 kDa) and enzymatic properties. They contained no mannose 6-phosphate residues in N-linked oligosaccharides. These results suggested that unlike mammalian cells, Tetrahymena acid α-glucosidase may be transferred to lysosomes by a mannose 6-phosphate receptor-independent mechanism, and also that low pH was not essential for the proteolytic processing of precursor polypeptide.     

RESPIRATORY METABOLISM IN THE PHLOEM, XYLEM AND CAMBIUM OF CARROT ROOT

Comparisons of respiratory metabolism among the phloem, cambiumand xylem of mature carrot root were carried out and the followingresults were obtained. 1) The activity of O₂ uptake on a dry weight basis in the cambiumwas higher than that in the xylem. The phloem showed the lowestactivity. 2) RQ was close to unity in all the three tissues.3) The inhibition rate of O₂ uptake by malonate was considerablyhigh in the three tissues. The highest inhibition was observedin the cambium. 4) In the phloem and xylem the malonic inhibitionrate of ¹⁴CO₂ release from G-U-¹⁴C was not so high as comparedwith that of O₂ uptake. In the cambium, however, those of O₂uptake and ¹⁴CO₂ output were comparable. 5) Malonate treatmentdid not cause any significant change in RQ. 6) The C₆/C₁ ratiowas highest in the cambium and lowest in the phloem. From these it is concluded that the participation of the TCAcycle in respiration is great in all the three tissues of carrotroot, and among the three the cambium has the highest activityof the cycle. As for the PP pathway, the xylem and phloem havea higher activity than the cambium. (Received April 11, 1967; )     

Cell Adhesion and Fusion Induced by Insemination in Zona-Free Hamster Eggs: Electrophysiological Monitoring of the Fusion Process

Cell adhesion and fusion were found to occur in zona-free hamster eggs placed in contact and then inseminated. A cytoplasmic fusion occurred in 7% of paired eggs mainly between 10 and 50 min after insemination. All other eggs that failed to fuse adhered tightly to one another within about 1 hr. Electrophysiological monitoring of the fusion process revealed that an electrical coupling (EC) suddenly appears between apposed eggs and completed within 2–15 min as the first step of cell fusion. Periodic hyperpolarizing responses (HRs), which have been found previously in fertilized hamster eggs to reflect a periodic increase in cytoplasmic Ca²⁺ ions, become gradually synchronized and completely coincide in paired eggs 10–30 min after the establishment of EC. Thus the paired eggs become an electrically single cell as the second step. Then the boundary between the eggs breaks down, resulting in formation of a single, spherical egg in several minutes. On the other hand, most of adhered eggs showed neither EC nor synchronization of HRs. Some adhered eggs showed only a low efficiency EC.