RNA editing in kinetoplastid protozoa.

Mitochondrial transcripts in kinetoplastids undergo remarkable posttranscriptional editing by uridylate insertion and deletion. The often dramatic remodeling of pre-mRNA sequences is directed by small guide RNAs (gRNAs) to produce mature mRNAs. In vitro analyses of editing have been used to determine the mechanism of editing and show that editing occurs by a series of enzyme-catalyzed steps. They also show that chimeric gRNA/mRNA molecules are not editing intermediates as proposed but are aberrant end products of editing. The complexes and molecules that catalyze editing are now being identified and characterized. The origin of editing, its developmental regulation which helps control the switching between terminal respiratory systems during the life cycle of trypanosomes, and other areas for future study are discussed.

Association of guide RNA binding protein gBP21 with active RNA editing complexes in Trypanosoma brucei.

RNA editing in Trypanosoma brucei mitochondria produces mature mRNAs by a series of enzyme-catalyzed reactions that specifically insert or delete uridylates in association with a macromolecular complex. Using a mitochondrial fraction enriched for in vitro RNA editing activity, we produced several monoclonal antibodies that are specific for a 21-kDa guide RNA (gRNA) binding protein initially identified by UV cross-linking. Immunofluorescence studies localize the protein to the mitochondrion, with a preference for the kinetoplast. The antibodies cause a supershift of previously identified gRNA-specific ribonucleoprotein complexes and immunoprecipitate in vitro RNA editing activities that insert and delete uridylates. The immunoprecipitated material also contains gRNA-specific endoribonuclease, terminal uridylyltransferase, and RNA ligase activities as well as gRNA and both edited and unedited mRNA. The immunoprecipitate contains numerous proteins, of which the 21-kDa protein, a 90-kDa protein, and novel 55- and 16-kDa proteins can be UV cross-linked to gRNA. These studies indicate that the 21-kDa protein associates with the ribonucleoprotein complex (or complexes) that catalyze RNA editing.

Complexes from Trypanosoma brucei that exhibit deletion editing and other editing-associated properties.

Transcripts from many mitochondrial genes in kinetoplastids undergo RNA editing, a posttranscriptional process which inserts and deletes uridines. By assaying for deletion editing in vitro, we found that the editing activity from Trypanosoma brucei mitochondrial lysates (S.D. Seiwert and K.D. Stuart), Science 266:114-117,1994) sediments with a peak of approximately 20S. RNA helicase, terminal uridylyl transferase, RNA ligase, and adenylation activities, which may have a role in editing, cosediment in a broad distribution, with most of each activity at 35 to 40S. Most ATPase 6 (A6) guide RNA and unedited A6 mRNA sediments at 20 to 30S, with some sedimenting further into the gradient, while most edited A6 mRNA sediments at >35S. Several mitochondrial proteins which cross-link specifically with guide RNA upon UV treatment also sediment in glycerol gradients. Notably, a 65-kDa protein sediments primarily at approximately 20S, a 90-kDa protein sediments at 35 to 40S, and a 25-kDa protein is present at <10S. Most ribonucleoprotein complexes that form with gRNA in vitro sediment at 10 to 20S, except for one, which sediments at 30 to 45S. These results suggest that RNA editing takes place within a multicomponent complex. The potential functions of and relationships between the 20S and 35 to 40S complexes are discussed.

Amplification of the antitumor activity of phleomycins and bleomycins in rats and mice by caffeine.

While having no antitumor effect per se, caffeine substantially enhanced the antitumor effects of the phleomycins PLM-CHP and PLM-PEP, and the bleomycins BLM-CHP and Blenoxane in rats carrying Walker 256 carcinosarcoma and/or mice carrying Ehrlich ascites tumor, even at doses of phleomycin and bleomycin below the minimum effective level. Positive but less conclusive results were also obtained with PLM-A4A4G and PLM-G.

Kinetoplastid RNA editing: complexes and catalysts.

RNA editing in kinetoplastids involves post-transcriptional insertion and deletion of uridylates (Us) to produce mature mitochondrial mRNAs with sequences specified by trans acting small guide RNAs. In vitro studies indicate the reaction pathway involves endonucleolytic cleavage of the precursor mRNA at the editing site, uridylate addition or removal at the 3' end of the 5' cleavage product, followed by ligation to the 3' cleavage product. This editing is catalyzed by a macromolecular complex that is in the early stages of characterization. Recent studies have resolved the general mechanism of editing, and show that editing occurs in association with a macromolecular complex.

Molecular characterization and overexpression of the hypoxanthine-guanine phosphoribosyltransferase gene from Trypanosoma cruzi.

The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme in Trypanosoma cruzi is a rational target for the treatment of Chagas disease. To evaluate the T. cruzi HGPRT in detail, the HGPRT gene (hgprt) was cloned from a genomic library of T. cruzi DNA and sequenced. Translation of the nucleotide sequence of the hgprt revealed an open reading frame of 663 bp that encoded a 25.5-kDa polypeptide of 221 amino acids. The T. cruzi HGPRT exhibited only 24%, 25%, and 21% amino acid sequence identity to its human, Plasmodium falciparum, and Schistosoma mansoni counterparts, respectively, but was 50% identical to the T. brucei HGPRT protein. Northern analysis of T. cruzi RNA revealed a 1.8-kb hgprt transcript, while Southern blots of genomic DNA suggested that hgprt was a single copy gene within the T. cruzi genome. The T. cruzi hgprt was inserted into the pBAce expression plasmid and transformed into Escherichia coli that are deficient in hypoxanthine and guanine phosphoribosylating activities. High levels of soluble, enzymatically active T. cruzi HGPRT were obtained, and this expression complemented the bacterial phosphoribosyltransferase deficiencies. The recombinant HGPRT was purified to apparent homogeneity by GTP-agarose affinity chromatography and recognized hypoxanthine, guanine, and allopurinol, but not adenine or xanthine, as substrates. The availability of the hgprt clone and large amounts of pure HGPRT protein provide a foundation for a structure-based drug design strategy for the treatment of Chagas disease.

Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase from Leishmania donovani.

The gene encoding the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme from Leishmania donovani has been cloned and sequenced. The hgprt open reading frame encoded a polypeptide of 211 amino acids that exhibited 3 regions of significant homology with other eukaryotic HGPRTs and a C-terminal tripeptide compatible with a glycosomal targeting signal. Northern blot analysis of L. donovani RNA revealed two hgprt transcripts, a 1.9-kb mRNA and a 1.7-kb transcript. The expression of the 1.7-kb hgprt mRNA and the activity of HGPRT enzyme were both augmented approx. 5-fold in parasites incubated in the absence of purines. Southern blots of genomic DNA indicated only a single hgprt locus within the L. donovani genome. Overexpression of L. donovani hgprt in E. coli complemented genetic deficiencies in hypoxanthine and guanine phosphoribosylating activities and yielded abundant quantities of enzymatically active HGPRT. The recombinant HGPRT was purified to homogeneity and recognized hypoxanthine, guanine and allopurinol, but not adenine or xanthine, as substrates. The hgprt clone and pure HGPRT protein provide essential reagents for validating HGPRT as a therapeutic target for the treatment of leishmaniasis and other diseases of parasitic origin.

Colostral transmission of maedi visna virus: sites of viral entry in lambs born from experimentally infected ewes.

Maedi visna virus (MVV) vertical transmission in sheep via infected colostrums is a very important route of infection in lambs. To verify colostral transmission and to study early viral entry in lambs, colostrum samples, and small intestine and mesenteric lymph nodes of lambs born from experimentally infected ewes were examined by histopathology, immunohistochemistry (IHC) and in situ hybridisation (ISH) studies. In particular, newborn lambs were naturally fed maternal colostrum and humanely killed at 10, 24, 48, 72, 96 h and 7 and 10 days after birth; two caesarian-derived lambs served as uninfected controls. No lesions suggestive of MVV infection were found, but marked immunoreactions for MVV capsid antigen (CA, p28) were detected in lambs fed maternal colostrum and in macrophages cultured from colostrum. IHC results in lambs suggest an initial viral absorption by intestinal epithelial cells at the tip of the villi, passage to mononuclear cells in the lamina propria and involvement of ileum Peyers' patches and mesenteric lymph nodes, with different staining patterns depending on infection times. ISH on intestinal sections of the 72 h lamb revealed the presence of proviral DNA in epithelial cells at the tip of the villi, suggesting a role for these cells in early MVV replication. The results contribute to knowledge about the pathogenesis of ovine lentivirus infection suggesting that the small intestine and mesenteric nodes are the sites of entry and propagation of MVV in lambs fed colostrums from infected ewes.

Amplification of the antitumor activity of phleomycins in rats and mice by heterocyclic analogues of purines.

Significant and often substantial enhancement of the antitumor properties of several individual phleomycins , by co- administration via intraperitoneal injection of a number of purine analogues, is demonstrated in rats and mice having three diverse tumors. It is evident that the dose levels of both the phleomycin and the amplifier are very significant and that optimal levels vary widely with the actual agents used. Constant serum levels of amplifier can be maintained for several days by administration via silastic-pellet implantation rather than injection, and this route of administration is an effective alternative for amplifiers of low solubility.

Metabolie and Cardiorespiratory Responses of Young Women to Skipping and Jogging.

In brief: To compare jogging and skipping with respect to metabolic demand, musculoskeletal stress, and perceived exertion, nine women (aged 18 to 29) were studied while jogging and skipping at treadmill speeds of 4.0, 4.8, and 5.4 mph. Data for V O2, heart rate, and perceived exertion were collected, and the subjects provided subjective ratings of musculoskeletal stress. The results showed that skipping imposed significantly greater metabolic demands and caused higher heart rates than jogging at each speed. In additon, skipping was rated as more stressful to the legs and ankles and less enjoyable. Thus skipping appears to be a stronger cardiorespiratory training stimulus than jogging for a given pace, but a less enjoyable activity.