A Serum-free, Partly Defined Medium, PDM-805, for Axenic Cultivation of Entamoeba histolytica Schaudinn, 1903 and other Entamoeba

We describe the first serum-free, partly defined medium (PDM-805) for cultivating the human enteric pathogen, Entamoeba histolytica , and the reptilian amebae E. barreti, E. invadens , and E. terrapinae. PDM-805 was developed by the stepwise replacement of yeast extract, bovine serum, and a casein peptone digest in TYI-S-33, a medium widely used for the axenic cultivation of these parasites. The defined components include amino acids, carbohydrates, B vitamins, ascorbic acid, tocopherol, thioctic acid, nucleic acid precursors, trace metals, and phosphate buffers. The undefined components include a highly purified bovine serum albumin, a lipoprotein-cholesterol solution from bovine serum, and a dialyzable, autoclavable, water-soluble growth factor(s) having a molecular weight of less than 3,500 prepared from casein peptone. To date, studies on the growth requirements of E. histolytica , strain 200:NIH, show the following are essential for sustained multiplication of this ameba: iron, glucose, biotin, folic acid, niacinamide, pantothenate, pyridoxal, riboflavin, thiamine, cysteine, an ammonium moiety (in addition to that present in cysteine), bovine serum albumin, lipoprotein-cholesterol, and casein peptone dialysate.     

Intracellular potassium compartments in Nitella axillaris

Three intracellular compartments for potassium exchange have been observed in intact cells of the giant-celled alga, Nitella axillaris. These compartments have been compared with the exchange properties of isolated subcellular structures. The smallest and fastest compartment (apparent half-time, 23 seconds) appears to involve passive absorption on the cell wall. The next largest (apparent half-time, 5 hours) may represent exchange with the cytoplasmic layer through the plasma membrane, the chloroplasts being in rapid equilibrium with the surrounding cytoplasm. The largest and slowest compartment (apparent half-time, 40 days) has been identified with the central vacuole. The vacuolar membrane and the plasma membrane have similar properties with respect to K permeability. Thus, the experimental data from the whole cell can be accounted for by a structural model of the compartments. Cyanide in concentrations up to 10(-3)M causes no net loss of K. The fastest compartment in Nitella and in higher plants is compared, and the ecological significance of the slow rate of potassium transport in Nitella is discussed.     

Factors Mediating the Vitamin B12 Requirement of Euglena

SYNOPSIS. Deprived of vitamin B₁₂, Euglena gracilis strain Z ceases to divide which we believe to be a function of the light regime: division inhibition occurs more quickly in continuous light than in alternating (6L : 6D) light and not at all in total darkness. This phenomenon is dependent on the carbon source; cells grown in glutamate-malate medium do not divide regardless of the culture conditions while dl -lactate as carbon source permits growth in darkness in the absence of B₁₂. Conditions which lead to an increased O₂ or decreased CO₂ tension in the medium, such as agitation in darkness or incubation in red or white light, result in inhibition of division. This inhibition can be reversed by re-transferring the cells to still culture in the dark or, in the case of light-induced blockage, by the addition of DCMU.     

The M.ADP.P(i) state is required for helical order in the thick filaments of skeletal muscle

The thick filaments of mammalian and avian skeletal muscle fibers are disordered at low temperature, but become increasingly ordered into an helical structure as the temperature is raised. Wray and colleagues (Schlichting, I., and J. Wray. 1986. J. Muscle Res. Cell Motil. 7:79; Wray, J., R. S. Goody, and K. Holmes. 1986. Adv. Exp. Med. Biol. 226:49-59) interpreted the transition as reflecting a coupling between nucleotide state and global conformation with M.ATP (disordered) being favored at 0 degrees C and M.ADP.P(i) (ordered) at 20 degrees C. However, hitherto this has been limited to a qualitative correlation and the biochemical state of the myosin heads required to obtain the helical array has not been unequivocally identified. In the present study we have critically tested whether the helical arrangement of the myosin heads requires the M.ADP.P(i) state. X-ray diffraction patterns were recorded from skinned rabbit psoas muscle fiber bundles stretched to non-overlap to avoid complications due to interaction with actin. The effect of temperature on the intensities of the myosin-based layer lines and on the phosphate burst of myosin hydrolyzing ATP in solution were examined under closely matched conditions. The results showed that the fraction of myosin mass in the helix closely followed that of the fraction of myosin in the M.ADP.P(i) state. Similar results were found by using a series of nucleoside triphosphates, including CTP and GTP. In addition, fibers treated by N-phenylmaleimide (Barnett, V. A., A. Ehrlich, and M. Schoenberg. 1992. Biophys. J. 61:358-367) so that the myosin was exclusively in the M.ATP state revealed no helical order. Diffraction patterns from muscle fibers in nucleotide-free and in ADP-containing solutions did not show helical structure. All these confirmed that in the presence of nucleotides, the M.NDP.P(i) state is required for helical order. We also found that the spacing of the third meridional reflection of the thick filament is linked to the helical order. The spacing in the ordered M.NDP.P(i) state is 143.4 A, but in the disordered state, it is 144. 2 A. This may be explained by the different interference functions for the myosin heads and the thick filament backbone.     

Contrasting levels of DNA polymorphism at the autosomal and X-linked visual color pigment loci in humans and squirrel monkeys

The X-linked color pigment (opsin) locus is known to be highly polymorphic in the squirrel monkey and other New World monkeys. To see whether this is also the case for the autosomal (blue) opsin locus, we obtained 32 squirrel monkey and 30 human blue opsin gene sequences. No amino acid polymorphism was found in either the squirrel monkey sample or the human sample, contrary to the situation at the X-linked opsin locus. This sharp contrast in the level of polymorphism might be due to differences in gene expression between the autosomal and the X-linked loci. At the X-linked locus, heterozygote advantage can occur because, owing to X-inactivation, the two alleles in a heterozygote are expressed in different cone cells, producing two types of cone cell, whereas at the autosomal locus, heterozygote advantage cannot occur because the two alleles in a heterozygote are expressed in the same cone cells, producing only one type of cone cell (i.e., phenotypically a homozygote). From the sequence data, the levels of nucleotide diversity (pi, i.e., the number of nucleotide differences per site) are estimated: for the human sample, pi = 0.00% per nondegenerate site, 0.00% per twofold degenerate site, and 0.04% per fourfold degenerate site in the coding regions and 0.01% per site in intron 4; for the squirrel monkey sample, pi = 0.00% per nondegenerate site, 0.00% per twofold degenerate site, and 0.15% per fourfold degenerate site in the coding regions and 0.17% per site in intron 4. The blue opsin genes from the common and pygmy chimpanzees, the gorilla, the capuchin, and the howler monkey were also sequenced. Features critical to the function of the opsin are well conserved in all known mammalian sequences. However, the interhelical loops are, on average, actually more conservative than the transmembrane helical regions. In addition, these sequence data and those from some other genes indicate that the common and pygmy chimpanzees are not closely related, their divergence data being from one third to one half the date of the human-chimpanzee divergence.      

Axenic Cultivation of Trichomonas tenax, the Oral Flagellate of Man I. Establishment of Cultures

SYNOPSIS. A technique for the axenic cultivation of Trichomonas tenax , the oral flagellate of man, is presented. The medium employed consists of a nutrient broth supplemented with horse serum and a cell-free extract of chick embryo. Three strains established in this medium have been maintained for 16, 23 and 48 months respectively.
All the cultures were initiated with trichomonads grown in association with Trypanosoma cruzi. Attempts to establish axenic cultures with trichomonads obtained from xenic cultures containing a bacterial flora of unknown composition met with failure. This suggests that the successful cutcome of the process of axenization is to a certain extent dependent upon the type of organism(s) with which T. tenax is associated in culture. Furthermore, these findings may serve to explain earlier failures since not only were all of these attempts made with media lacking tissue extract supplements but all were made using bacteria-trichomonad cultures as a source of trichomonads.     

Attachment of Entamoeba histolytica to Glass in a Defined Maintenance Medium: Specific Requirement for Cysteine and Ascorbic Acid*

SYNOPSIS. Cysteine and ascorbic acid were previously shown to be required by Entamoeba histolytica trophozoites for attachment to glass, elongation, and ameboid movement as well as for short-term (12–24 h) survival in a balanced salt solution containing bovine serum albumin and a vitamin solution (Maintenance Medium 1). If the only function of cysteine and ascorbate was to decrease the redox potential, other reducing agents should be effective. However, the requirement for cysteine in the presence of ascorbic acid was highly specific. Equally effective were D- and L-cysteine; however, of many other compounds tested, only thioglycolic acid, ascorbic acid, or L-cystine (in decreasing order) were somewhat active. Under N₂ atmosphere, cysteine and ascorbic acid were still required, although their concentrations could be halved. The ability to attach in the maintenance medium was irreversibly lost after only 5 min of cysteine-ascorbic acid deprivation; however, there was no decrease in viability when the amebae were transferred to growth medium within 30 min. Cysteine thiol groups in the medium were oxidized rapidly regardless of the concentration of ascorbic acid or the presence of amebae; however, ascorbic acid prolonged attachment of amebae.