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.     

Pattern and Time Course of Cleavages in Early Development of the Ovoviviparous Pond Snail, Sinotaia quadratus historica

The pattern and time course of cleavage during early development of the ovoviviparous pond snail, Sinotaia quadratus historica , in in vitro culture were investigated. The fertilized egg, 25–30 μm in diameter, underwent cleavage by repeated constriction and compaction as in blastocyst formation in mammalian embryos. The cleavage was slightly unequal and dextrally spiral, although a slight time lag in cleavages of blastomeres was observed after the 2nd cleavage. A small polar lobe was formed at the 1st cleavage, but not at the 2nd cleavage. Each cleavage proceeded very slowly under the experimental conditions, the 1st, 2nd, 3rd and 4th cleavages taking 22 hr or more, 9 hr and 10 hr, respectively. The cleavage pattern in in vitro cultures observed by light microscopy was confirmed by scanning electron microscopy.     

Purification and Characterization of Trypsin like Enzyme of Sperm of the Sea Urchin, Hemicentrotus pulcherrimus

Trypsin like enzyme has been isolated from sperm of the sea urchin, Hemicentrotus pulcherrimus , using tryptophane methyl ester-Sepharose 4B and soybean trypsin inhibitor-Sepharose 4B affinity chromatographies.
The isolated enzyme preparation is homogenous in polyacrylamide gel electrohoresis at pH 2.3. The molecular weight of the enzyme estimated by gel filtration is about 33,000, and the enzyme separates into two subunits of 10,900 and 20,500 on sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence of β-mercaptoethanol.
This enzyme is active to N-α-benzoyl arginine ethyl ester (BAEE), N-α-toluenesulfonyl-L-arginine ethyl ester (TAME), and N-α-benzoyl-DL-arginine-p-nitroanilide, but not N-acetyl-L-tyrosine ethyl ester, N-benzoyl-L-tyrosine ethyl ester, Hippuryl-L-arginine, and Hippuryl-L-phenylalanine. The optimal pH of this enzyme is about 8.0. The Michaelis constants for BAEE and TAME are 3.3 × 10⁻⁶M, and 8.2 × 10⁻⁵M, respectively.
Soybean trypsin inhibitor and lima bean trypsin inhibitor completely inhibit the activity of this enzyme, while N-α-tosyl-L-lysine chloromethyl ketone, ovomucoid trypsin inhibitor, and α-1-antitrypsin partially inhibit. L-1-tosylamide-2-phenyl chloromethyl ketone, chyrnostatin, and aporotinine are without effect.
This enzyme is stable at pH 2.0–3.0 and labile at pH 8.0. Ca²⁺ and Mg²⁺ activate this enzyme, but do not stabilize at pH 8.0. Seawater, NaCl, and KCl inhibit this enzyme activity.
Release of this enzyme from the acrosomal vesicle is suggested.     

FLOWER-PROMOTING EFFECT OF GIBBERELLIN IN WINTER WHEAT AND BARLEY

1. Vernalized and unvernalized seeds of barley were sown in greenhouseunder short day or long day condition. At the second leaf stage,a half of the plants in each plot was sprayed with gibberellin.Plants grown from vernalized seeds headed at about the sametime under long day condition, whether they were treated withgibberellin or not. Under short day condition, however, gibberellinpromoted heading. With plants grown from the unvernalized seeds,gibberellin promoted heading under long day condition whereasit delayed heading under short day condition. Thus the substitutionof gibberellin for long day effect and for vernalization effectwas shown.
2. Immature embryos growing on developing ear of wheatand barleywere vernalized with low temperature combined withor withoutgibberellin application. Plants grown from the seedstreatedat their early ripening stage with low temperature combinedwith gibberellin gave earlier heading, associated with the decreasedleaf number, than the plants from seeds treated with low temperaturealone. This effect of gibberellin lasted for a half-year ofseed storage.
3. Gibberellin was applied to germinating seedsunder vernalizationtreatment. Gibberellin promoted floweringin varieties whichpossess high requirement for chilling vernalization,when thechilling was insufficient to satisfy the high requirement.However,the effect of gibberellin was obscure with varietiespossessingrelatively low requirement for vernalization.

(Received September 15, 1964; )