Effects of Carboxylemethylation and Polyamine Synthesis Inhibitors on Methylation of Trypanosoma brucei Cellular Proteins and Lipids

ABSTRACT. The fate of methionine in eukaryotic cells is divided between protein synthesis and the branched pathway encompassing polyamine synthesis, methylation of proteins and lipids, and transsulphuration reactions. Aside from protein synthesis, the first step to all other uses of methionine is conversion to S-adenosylmethionine. Blockade of polyamine synthesis in African trypanosomes by the ornithine decarboxylase inhibitor DL-α-difluoromethylomithine (Ornidyl, DFMO) the AdoMet decarboxylase inhibitor 5′-{[(Z)-4-amino-2-butenyl]-methylamino}-5′-deoxyadenosine or the protein methylase inhibitor sinefungin induces dramatic increases in intracellular AdoMet. In a previous study, distribution and pool sizes of [¹⁵S] or [U-¹⁴C]methionine were followed in bloodform trypanosomes as incorporation into the total TCA precipitable fraction. In the present study, the effects of pretreatment with DFMO (1 mM), MDL 73811 (1 μM) and sinefugin (2 nM) on [³⁵S] and [U-¹⁴C]methionine incorporation were studied in blood forms. DFMO or MDL 73811 pretreatment increased protein methylation 1.5-fold through incorporation of [U¹⁴C]methionine, while sinefungin caused a 40% reduction of incorporation. The increases in incorporation of [U-¹⁴C]methionine due to DFMO and MDL 73811 were reduced 40% to 70% by including cold AdoMet (1 mM) in the incubation medium, an indication of AdoMet transport by bloodform trypanosomes and the utilization of [U-¹⁴C]methionine as AdoMet. Exogenous AdoMet had no effect on [³⁵S]methionine incorporation. The agents studied are curative for African trypnosomiasis infections, either clinically (DFMO) or in model infections (MDL 73811, sinefungin) and thus highlight interference with AdoMet metabolism and methylation reactions as biochemical consequences of these agents.     

Development of a Rapid Detection Procedure for Ctyptosporidium, Using In Vitro Cell Culture Combined with PCR

A detection, viability, and infectivity assay was developed for Cryptosporidiurn parvum. Oocysts or excysted sporozoites were inoculated onto monolayers of CaCo-2 cells grown on chamber slides. C. parvum infection was monitored by three methods: a) application of a fluorescein-labeled anti-sporozoite antibody; b) PCR of a heat-shock protein gene fragment; and c) detection of mRNA from the heat-shock protein gene by RT-PCR.     

The Remote Borderland: Transylvania in the Hungarian Imagination.

The Remote Borderland: Transylvania in the Hungarian Imagination. László Kürti. New York: State University of New York Press, 2001. 259 pp.     

The Evolution of Avian Senescence Patterns: Implications for Understanding Primary Aging Processes

SYNOPSIS. The long life spans of birds relative to those ofmammals are intriguing to biogerontologists, particularly inlight of birds' high body temperatures, high blood glucose levels,and high metabolic rates—all of which should theoreticallyincrease their biochemical liability for rapid aging. The comparativelongevity of birds and other flying homeotherms is consistentwith evolutionary senescence theory, which posits that specieswith low mortality rates from predation or accident will bereleased from selection for rapid maturity and early reproduction,and will exhibit retarded aging. Comparative analyses of avianlife history parameters to date, although not as extensive asthose for mammals, broadly support an association between lowmortality rates, slow reproduction, and long lifespan. The diversityof bird life histories suggests the importance of developinga diversity of avian models for studies of aging mechanisms,both proximate and ultimate, and for using wild as well as domesticrepresentatives. Birds studied in the laboratory thus far showmany of the same manifestations of aging as mammals, includinghumans, and many ornithologists are beginning to document actuarialevidence consistent with aging in their study populations. Weencourage greater communication and collaboration among comparativegerontologists and ornithologists, in the hope that the studyof aging in birds will lead to an integrated understanding ofphysiological aging processes well grounded in an evolutionaryparadigm.     

Inhibition of Cell Wall-Associated Peroxidase Isozymes with British Anti-Lewisite

Conditions were established for the use of British Anti-Lewisite(BAL) to inhibit preferentially the extracellular isoperoxidasesin pea stems. After a 2 h incubation period with 9.09 x 10⁴M BAL, approximately half of the activity of cell wall-associatedperoxidase was lost, while little inhibition of cytoplasmicenzyme was observed. Four isozymes from cell wall material wereidentified on starch gel e0lectrophoresis and stained with eugenol.Activity was tested with eugenol, ferulic and caffeic acids,and several redox dyes to probe the possible involvement ofthese isozymes in lignin biosynthesis. All isozymes had similarK_m values with eugenol and caffeic acid as compared to benzidine,a standard redox dye.     

The Response of Contractile and Non-Contractile Vacuoles of Paramecium calkinsi to Widely Varying Salinities

ABSTRACT. Paramecium calkinsi from tidal marshes survive a wide salinity range. Fluid output of contractile vacuoles of these cells decreased as salinity of the medium to which they were acclimated increased, and both pulse rate and vacuole volume were used to regulate output. When cells were first exposed to more dilute medium, contractile vacuoles greatly increased volume so that fluid output increased even though pulse rate decreased. In cells shifted to a more concentrated medium, contractile vacuole output decreased by decreasing pulse rate. The contractile vacuole is surrounded by a set of collecting structures which change form as the salinity changes. Distensible ampullae are found in media of low salinity and collecting canals are found in media of high salinity. When cells are shifted from high salinity to low, the number of ampullae increases and the number of canals decreases. When cells are shifted from low salinity to high, the number of ampullae decreases and the number of canals decreases. Other non-contracting vacuoles also appear in response to a hypoosmotic shock. These include vacuoles within the cell as well as "blisters" on the surface. The number and frequency of blisters increases with the size of the hypoosmotic shock. They detach from cells without resulting in any visible loss of cytoplasm. Non-contractile vacuoles may play a role in sequestering and removing excess water that the contractile vacuoles cannot handle.