Fine structural alterations in Trypanosoma rhodesiense grown in vitro, treated with WR 163577

Fine-structural alterations in Trypanosoma rhodesiense trypomastigotes exposed to WR 163577, a prophylactic agent against animal African trypanosomiasis, were determined from cells grown in vitro. Exposure of trypomastigotes to a low concentration of drug resulted only in condensation of kinetoplast DNA fibrils. Exposure to higher drug concentrations caused clumping of nuclear chromatin and of cytoplasmic contents. Although alteration of kinetoplast DNA is the first detectable drug-induced change, the function of the kinetoplast in mammalian forms of African trypanosomes is unclear, and the secondary changes in the nucleus and cytoplasm may constitute the functionally significant alterations caused by WR 163577.     

Fine Structural Alterations in Trypanosoma rhodesiense Grown In Vitro,Treated with WR 1635771

Fine-structural alterations in Trypanosoma rhodesiense trypomastigotes exposed to WR 163577, a prophylactic agent against animal African trypanosomiasis, were determined from cells grown in vitro. Exposure of trypomastigotes to a low concentration of drug resulted only in condensation of kinetoplast DNA fibrils. Exposure to higher drug concentrations caused clumping of nuclear chromatin and of cytoplasmic contents. Although alteration of kinetoplast DNA is the first detectable drug-induced change, the function of the kinetoplast in mammalian forms of African trypanosomes is unclear, and the secondary changes in the nucleus and cytoplasm may constitute the functionally significant alterations caused by WR 163577.     

Cytochemical study of free-living flagellates of suborder Bodonina. I. Morphology and nucleic acids]

Nucleic acids (DNA and RNA) have been discovered in the kinetoplast of free-living Bodonina: Bodo caudatus, Pleuromonas jaculans, Rhynchomonas nasuta--by means of cytochemical methods. The kinetoplast has variable contents of nucleic acids whose chemoarchitectonics is due to their non-homogeneous distribution within the kinetoplast. The Feulgen reaction in the kinetoplast is more intensive than in the nucleus. Kinetoplast is closely connected with the cytoplasmic RNA metabolism. Many individuals of R. nasuta were found to have two kinetoplasts, no other signs of cell division being observed. P. jaculans has up to 45% of dyskinetoplastic forms.     

Culture and morphological observations on Trypanosoma melphagium.

The cultural characteristics of Trypanosoma melophagium of sheep were studied. Aspects investigated were size of the inoculum and population growth in Modified Monophasic Medium for Trypanosomes (MMMT), population growth in Medium 199 with 10% inactivated calf serum containing 5, 10, and 15% hemolyzed defibrinated rabbit blood (199-CS-5, 199--CS-10, 199-CS-15) at 27 degrees C, effects on population growth of temperature and hydrogen ion concentration in MMMT, and morphology and morphometrics of the developmental stages found under different experimental conditions. The best growth occurred in medium MMMT at 30 degrees, C, pH 7.25. Temperature seemed to be a critical factor for differentiation of epimastigotes to trypomastigotes. Statistically significant differences were found between the trypomastigotes in MMMT and 199--cs-5 at 37 degrees C on day 4 of incubation for the following measurements: PK (distance from posterior end to kinetoplast), KN (from kinetoplast to middle of nucleus), PN (from posterior end to middle of nucleus), and nuclear and kinetoplastic indices. The trypomastigotes formed in both media were much smaller in size than the blood forms reported by Hoare (1972).     

Mitochondrial development during life cycle differentiation of African trypanosomes: evidence for a kinetoplast-dependent differentiation control point

Life cycle differentiation of African trypanosomes entails developmental regulation of mitochondrial activity. This requires regulation of the nuclear genome and the kinetoplast, the trypanosome's unusual mitochondrial genome. To investigate the potential cross talk between the nuclear and mitochondrial genome during the events of differentiation, we have 1) disrupted expression of a nuclear-encoded component of the cytochrome oxidase (COX) complex; and 2) generated dyskinetoplastid cells, which lack a mitochondrial genome. Using RNA interference (RNAi) and by disrupting the nuclear COX VI gene, we demonstrate independent regulation of COX component mRNAs encoded in the nucleus and kinetoplast. However, two independent approaches (acriflavine treatment and RNA interference ablation of mitochondrial topoisomerase II) failed to establish clonal lines of dyskinetoplastid bloodstream forms. Nevertheless, dyskinetoplastid forms generated in vivo could undergo two life cycle differentiation events: transition from bloodstream slender to stumpy forms and the initiation of transformation to procyclic forms. However, they subsequently arrested at a specific point in this developmental program before cell cycle reentry. These results provide strong evidence for a requirement for kinetoplast DNA in the bloodstream and for a kinetoplast-dependent control point during differentiation to procyclic forms.     

Herpesvirus infection modifies adenovirus RNA metabolism in adenovirus type 5-transformed cells.

The effect of herpes simplex virus (HSV) infection of mRNA metabolism was examined in a system where the fate of specific RNA sequence can be assayed. Adenovirus type 5-transformed rat embryo cell line 107 synthesizes adenovirus-specific RNA (ad-RNA), which functions in the cytoplasm as mRNA. We have utilized ad-RNA as a model for mRNA metabolism, and in a preliminiary study we characterized ad-RNA in the nucleus and cytoplasm by hybridization to filter-bound adenovirus DNA. The results indicated the as-RNA accumulates in the nucleus and that cytoplasmic polyadenylic acid [poly(A)]-containing ad-RNA turns over with a half-life of a few hours. Pulse-chase experiments confirmed these observations and a half-life of about h was determined for the poly(A)-containing cytoplasmic ad-RNA. A second class of ad-RNA remains in the nucleus, where it turns over with a longer hlaf-life (about 24 h). The infection of 107 cells by HSV was restricted at 37 degree C, giving a burst size of 5 PFU per cell and allowing continued host DNA synthesis. Protein synthesis was inhibited greater than 50% by 7 h after infection, and total RNA synthesis was 50% inhibited by 4 h after infection. During the first 8 h after infection, HSV has little effect on the rate of synthesis of ad-RNA as determined by hybridization of nuclear RNA samples, but,during the same period, HSV inhibits the accumulation of poly(A)-containing ad-RNA in the cytoplasm. The degree of this inhibition increases steadily throughout this period and reaches 60% by 6.5 to 8 h after infection. Nosignificant effect was seen on the accumulation of total cellular poly(A)-containing RNA. It was concluded from these experiments that HSV infection alters the metabolism of ad-RNA so as to prevent the normal appearance of the poly(A)-containing mRNA in the cytoplasm. The result for ad-RNA may not represent the behavior of total cellular poly(A)-containing RNA under conditions where infection is restricted.     

Evidence that the entire length of a kinetoplast DNA minicircle is transcribed in Trypanosoma cruzi

A 1.3 kb cDNA (cDNA52) was derived from Trypanosoma cruzi trypomastigote mRNA. Using single stranded probes in Northern blots, we identified the putative coding strand of cDNA52. In addition, a minor band was detected in RNA from epimastigotes that was absent in RNA from trypomastigotes. Nucleotide sequence analysis revealed that cDNA52 was highly homologous to T. cruzi kinetoplast DNA minicircle sequences. All four conserved regions of T. cruzi minicircles were identified in cDNA52. Using several criteria, we demonstrated that the hybridization signals were not caused by contaminating minicircle DNA in the RNA preparations. The data provide direct evidence for the unprecedented finding that the entire length of a kDNA minicircle is transcribed in T. cruzi.     

Expression and subcellular localization of kinetoplast-associated proteins in the different developmental stages of Trypanosoma cruzi

Background  

The kinetoplast DNA (kDNA) of trypanosomatids consists of an unusual arrangement of circular molecules catenated into a single network. The diameter of the isolated kDNA network is similar to that of the entire cell. However, within the kinetoplast matrix, the kDNA is highly condensed. Studies in Crithidia fasciculata showed that kinetoplast-associated proteins (KAPs) are capable of condensing the kDNA network. However, little is known about the KAPs of Trypanosoma cruzi, a parasitic protozoon that shows distinct patterns of kDNA condensation during their complex morphogenetic development. In epimastigotes and amastigotes (replicating forms) the kDNA fibers are tightly packed into a disk-shaped kinetoplast, whereas trypomastigotes (non-replicating) present a more relaxed kDNA organization contained within a rounded structure. It is still unclear how the compact kinetoplast disk of epimastigotes is converted into a globular structure in the infective trypomastigotes.     

The parvovirus capsid odyssey: from the cell surface to the nucleus

During cellular entry and infection, the parvovirus capsid follows a complex path from the cell surface to the nucleus, where the DNA is replicated. Various receptors have been characterized that bind to different parvoviruses and mediate their entry into cells. However, the subsequent trafficking pathways within the endosomal system, cytoplasm and into the nucleus are still not well defined. Studies of viruses entering various cell types under different conditions show particles located in many different endosomal compartments, within the cytoplasm and in the nucleus with significant variations in timing and distribution. Here, we define the previously unresolved issues that are now better understood for the infection pathways of these viruses, and outline some of the areas that remain to be clarified in future studies.     

Polymorphism of Trypanosoma cruzi in the blood of a vertebrate host

Tetramorphism of trypomastigote forms has been discovered in the blood stream of mice infected with Trypanosoma cruzi, that is C slender, C middle, S slender, S middle and forms intermediate between C and S. Regular changes of forms have been observed in the course of the infection. In the middle of the process, at the moment of maximum destruction of parasites affected by immunosystems of the host, C-forms prevailing on the whole substantially give place to S-forms, slender variants being replaced by middle ones within each of them. Polymorphism in the bloodstream of the vertebrate host is the general property of all trypanosomes, with their common biological value: immunological adaptation to parasitism in the blood and preadaptation to the continuation of the life cycle in the invertebrate host.     

Antiparasitic compounds that target DNA

Designed, synthetic heterocyclic diamidines have excellent activity against eukaryotic parasites that cause diseases such as sleeping sickness and leishmania and adversely affect millions of people each year. The most active compounds bind specifically and strongly in the DNA minor groove at AT sequences. The compounds enter parasite cells rapidly and appear first in the kinetoplast that contains the mitochondrial DNA of the parasite. With time the compounds are also generally seen in the cell nucleus but are not significantly observed in the cytoplasm. The kinetoplast decays over time and disappears from the mitochondria of treated cells. At this point the compounds begin to be observed in other regions of the cell, such as the acidocalcisomes. The cells typically die in 24-48h after treatment. Active compounds appear to selectively target extended AT sequences and induce changes in kinetoplast DNA minicircles that cause a synergistic destruction of the catenated kinetoplast DNA network and cell death.     

Anisomorphic cell division by African trypanosomes

In the bloodstream of a mammalian host, African trypanosomes are pleomorphic; the shorter, non-proliferative, stumpy forms arise from longer, proliferative, slender forms with differentiation occurring via a range of morphological intermediates. In order to investigate how the onset of morphological change is co-ordinated with exit from the cell cycle we first characterized slender form cell division. Outgrowth of the new flagellum was found to occur at a linear rate, so by using outgrowth of the new flagellum as a temporal marker of the cell cycle we were able determine the order in which single copy organelles (nucleus, kinetoplast and mitochondrion) were segregated. We also found that flagellar length was an effective marker of the slender to stumpy differentiation and were, therefore, able to study both cell division and differentiation. When these differentiating cells were compared to cells undergoing proliferative cell division, they were found to be anisomorphic – showing discernible differences not only in the length of their new flagella but also in the shape and size of the cells and their nuclei.     

Virus-Specific Ribonucleic Acid in the Nucleus and Cytoplasm of Rat Embryo Cells Transformed by Adenovirus Type 2

Nuclei were isolated from rat embryo cells transformed by adenovirus type 2. Nuclear and cytoplasmic virus-specific ribonucleic acids (RNA) were characterized and quantitated by deoxyribonucleic acid (DNA)-RNA hybrid formation with adenovirus DNA. The results indicate that most, if not all, virus-specific RNA molecules are synthesized in the cell nucleus and subsequently transported into cytoplasm where they degrade with a half-life of 1 to 2 hr. No difference in base sequences between nuclear and cytoplasmic virus-specific RNA species can be detected by hybridization competition experiment with viral DNA.     

三种利什曼原虫的组织化学观察

本文报道了杜氏、砂鼠及墨西哥利什曼原虫体内糖原、DNA,RNA。蛋白质结合的a-氨基、碱性蛋白质、酪氨酸、色氨酸及组氨酸的定位及含量。     

Characterization of RNA molecules restricted to the nucleus in mouse L-cells

RNA molecules which are restricted to the nucleus in mouse L-cells were characterized by the technique of RNA/DNA hybridization. Competition of cytoplasmic RNA with labeled nuclear RNA of various sizes revealed that the RNA restricted to the cell nucleus is heterogeneous in size. Competition for sites on fractions of mouse DNA of various base compositions indicated that this unstable RNA is also heterogeneous in base composition. Fractionation of nuclei into three subfractions failed to separate the uniquely nuclear RNA from the precursors of cytoplasmic RNA. The significance of the selective transport of RNA from the nucleus to the cytoplasm and its importance in the control of gene activity in eucaryotic cells is discussed.