Selective inactivation of heterokaryons of Candida albicans by anaerobiosis

Heterokaryons (hets), but not monokaryons of Candida albicans die when grown anaerobically on minimal medium. Their rates of inactivation increase with decreases in growth temperatures from 37°C to 25°C. At 10°C, however, anaerobiosis is not lethal and suppresses the inactivation which normally occurs among hets cultured aerobically at that temperature. Killing of hets by anaerobiosis can be altered significantly by certain exogenously provided amino acids or intermediates of oxidative respiration. Aspartic acid alone promotes inactivation whereas alanine, glutamic acid or lysine individually have no effects. However, glutamate and lysine combined afford slight protection against inactivation while aspartate and glutamate combined, with or without lysine, are highly protective: the activity of the aspartate-glutamate combination is completely negated by the addition of alanine. Other common amino acids have no effects on het responses to anaerobiosis other than the ability, when combined, to relieve the antagonism of alanine for the aspartate-glutamate combination. Anaerobic survivals are also enhanced by oxalacetic acid or -ketoglutaric acid, and even more so by a combination of these two intermediates. The resistances to inactivation elicited by the oxalacetate -ketoglutarate or aspartate-glutamate combinations are not additive. These relationships are interpreted to signify that inactivation of hets by anaerobic growth is largely, if not exclusively, due to depletion of their oxalacetic acid and -ketoglutaric acid contents for amino acid biosyntheses, and the unique inability of het cells to replenish those keto acids upon subsequent return to aerobic conditions. The observations are consistent with previous indications that mitochondria formed by hets are functionally abnormal.     

Variation in adhesion and cell surface hydrophobicity in Candida albicans white and opaque phenotypes

A previous study had established that a select group of pathogenic isolates of Candida albicans was capable of switching heritably, reversibly and at a high frequency (10^–2 to 10^–3) between two phenotypes (white or opaque) readily distinguishable by the size, shape, and color of colonies formed on agar at 25°C. This paper describes experiments designed to determine the ability of these two phenotypes to attach to buccal epithelial cells (BECs) and plastic, and to compare the cell surface hydrophobicities of white and opaque phenotypes from three clinical isolates. White cells were found to be significantly more adhesive to BECs, and a strong correlation was also found between phenotype adhesiveness and the percentage of BECs to which C. albicans had attached. The percentage of BECs with one or more attached C. albicans was approximately 90% for the white phenotype and approximately 50% for the opaque phenotype. Opaque cells, in contrast, were twice as hydrophobic as white cells, and the percentage of opaque cells bound to BECs by coadhesion was also double that of white cells. The differences in adhesion to plastic between the two phenotypes were not statistically significant and there was no distinct trend to suggest which phenotype might be more adhesive to plastic. These results indicate that several factors are involved in the adhesion of C. albicans to plastic, and confirm the hypothesis that cell surface hydrophobicity is of minor importance in direct adhesion to epithelial cells but that it may contribute to indirect attachment to epithelial cells by promoting yeast coadhesion. Moreover, the data presented in this paper also revealed that under identical growth conditions, adhesion of C. albicans was significantly altered depending on the phenotypic state of the organism tested. Therefore, because C. albicans can switch at a high frequency to various phenotypes in vitro, it may be that in future adhesion studies involving Candida the phenotypic state of the organism at the time of testing will have to be determined. Otherwise, the results, even within the same laboratory, may be difficult to interpret.     

Extracellular Amino Acid Concentrations in the Dorsal Spinal Cord of Freely Moving Rats Following Veratridine and Nociceptive Stimulation

In vivo microdialysis was used to sample extracellular concentrations of amino acids in the dorsal lumbar spinal cord of freely moving rats. Changes in the extracellular concentrations of amino acids were measured in response to infusion of veratridine (180 microM), a sodium channel activator, as well as during acute noxious stimulation by an injection of 5% formalin into the metatarsal region of the hindleg. Veratridine produced a tetrodotoxin (TTX)-sensitive increase in the extracellular concentration of Glu. Concentrations of Asp, taurine, Ala, Asn, and Gly were not significantly elevated following veratridine stimulation. Intradermal injection of formalin produced a TTX-sensitive increase in Asp concentration and a non-TTX-sensitive increase in Glu concentration. These data support the hypothesis that Glu and Asp are dorsal horn neurotransmitters involved in nociception.     

Alternative Developmental Pathways Determined by Environmental Conditions in the Cellular Slime Mold Dictyostelium discoideum

The cellular slime mold Dictyostelium discoideum grows in the soil as a population of independent, uninucleate amoebae. Upon entrance to the stationary phase, the amoebae collect in multicellular aggregates to form organized fruiting bodies composed of spores and stalk cells. Depending upon environmental conditions, the developing aggregate either constructs the fruiting body at the site of aggregation or transforms into a structure that can migrate to a more favorable location. Environmental conditions that favor migration are (i) the accumulation of metabolite(s) produced by the aggregate and (ii) a low ionic strength in the substratum. Conditions that prevent migration or that stop a migrating slug are (i) the presence of buffer and (ii) illumination by overhead light.     

Derepression of Phosphomannose Isomerase by Regulator Gene Mutations Involved in Capsular Polysaccharide Synthesis in Escherichia coli K-12

A regulator gene mutation (capR) that causes increased synthesis of capsular polysaccharide and derepressed synthesis of several enzymes involved in polysaccharide synthesis also derepresses phosphomannose isomerase (PMI) synthesis. In contrast, a second mutation (capS, which maps separately from capR) that causes increased production of the same polysaccharide does not lead to increased synthesis of PMI (nor of several of the other enzymes involved in polysaccharide synthesis). Introduction of the capR9 allele by transduction or mutation of capR(+) to capR can change the phenotype of a mannose-negative nonmucoid strain to a mannose-positive mucoid phenotype. Thus, genotype capR(+)man-2 is mannose-negative and nonmucoid, but genotype capR9 man-2 is mannose positive and mucoid. Other interactions between these alleles in the synthesis of capsular polysaccharide are recorded.     

The Creatine Phosphoryltransfer Reaction in Iodoacetate-Poisoned Muscle

The iodoacetate-nitrogen-poisoned muscle offers the possibility of studying the stoichiometry of the single muscle twitch since metabolic resynthesis by glycolysis and oxidative phosphorylation are blocked, and there remains as an energy source only the creatine phosphoryltransfer system, creatine phosphate reacting with adenosinediphosphate to give the triphosphate and creatine. It is shown, preparatory to a determination of the amount of phosphocreatine split in a single twitch, that iodoacetate does not inhibit creatine phosphoryltransferase at concentrations which block glycolysis. An analysis is developed which assumes that the transferase maintains the creatine phosphoryl transfer reaction in equilibrium following contraction, and further that the creatine phosporyltransfer reaction and the myokinase reaction are isolated in muscle. On the basis of this analysis and the data obtained, an estimate of the equilibrium constant of the creatine phosphoryl reaction in muscle is obtained which agrees with values determined in vitro. Using the estimated equilibrium constant, and the concentrations of creatine, creatine phosphate, and adenosinetriphosphate found, a value for the concentration of free adenosinediphosphate is obtained which is considerably less than that found by direct chemical analysis.     

Localization of melanin synthesis within the pigment cell: Determination by a combination of electron microscopic autoradiography and topographic planimetry

Summary Localization of melanin synthesis within the pigment cells of the Cloudman S-91 mouse melanoma was determined by means of a combination of high resolution autoradiography and topographic planimetry. Initial melanin biosynthesis occurred predominantly in the endoplasmic reticulum and associated ribonucleoprotein particles of the melanocytes. By measuring a number of cell organelles and employing the index of relative specific localization it could be shown that the nucleus and mitochondria are of little or no importance in the process of melanogenesis.This investigation has been supported by the following research grants: CA 06548 CB, NIH, PHS and an Institutional Grant from the American Cancer Society (to H. M. H.); CA-05887, NIH, PHS (to A. S. Z.); M-00388 and NB-00782, NIH, PHS (to J. F. H.). One of us (H. M. H.) holds a Research Cancer Development Award (5-K 3-GM-2634) of the National Institute of General Medical Sciences, Public Health Service.We are grateful to Mr. Ronald Abler for his help with the topographic measurements; to Dr. Erhard Haus for help and advice; to Mr. J. Thornby and Mr. A. P. Basu for assistance with the statistical aspects of this study; and to Mrs. Lenore Mottaz, Miss Bernice Uittenbogaard, and Mrs. Judith Strong for careful technical assistance.     

Homogenisation and oxygen transfer rates in large agitated and sparged animal cell bioreactors: Some implications for growth and production

Because of concern for cell damage, very low agitation energy inputs have been used in industrial animal cell bioreactors, typical values being two orders of magnitude less than those found in bacterial fermentations. Aeration rates are also very small. As a result, such bioreactors might be both poorly mixed and also unable to provide the higher oxygen up-take rates demanded by more intensive operation. This paper reports experimental studies both of K _L a and of mixing (via pH measurements) in bioreactors up to 8 m³ at Wellcome and of scaled down models of such reactors at Birmingham. Alongside these physical measurements, sensitivity of certain cell lines to continuously controlled dO₂ has been studied and the oxygen up-take rates measured in representative growth conditions. An analysis of characteristic times and mixing theory, together with other recent work showing that more vigorous agitation and aeration can be used especially in the presence of Pluronic F-68, indicates ways of improving their performance. pH gradients offer a special challenge.