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.

Jaagsiekte sheep retrovirus proviral clone JSRV(JS7), derived from the JS7 lung tumor cell line, induces ovine pulmonary carcinoma and is integrated into the surfactant protein A gene.

Ovine pulmonary carcinoma (OPC) is a contagious neoplasm of alveolar epithelial type II (ATII) or Clara cells caused by a type D/B chimeric retrovirus, jaagsiekte sheep retrovirus (JSRV). Here we report the isolation, sequencing, pathogenicity, and integration site of a JSRV provirus isolated from a sheep lung tumor cell line (JS7). The sequence of the virus was 93 to 99% identical to other JSRV isolates and contained all of the expected open reading frames. To produce virions and test its infectivity, the JS7 provirus (JSRV(JS7)) was cloned into a plasmid containing a cytomegalovirus promoter and transfected into 293T cells. After intratracheal inoculation with virions from concentrated supernatant fluid, JSRV-associated OPC lesions were found in one of four lambs, confirming that JSRV(JS7) is pathogenic. In JS7-cell DNA, the viral genome was inserted in the protein-coding region for the surfactant protein A (SP-A) gene, which is highly expressed in ATII cells, in an orientation opposite to the direction of transcription of the SP-A gene. No significant transcription was detected from either the viral or the SP-A gene promoter in the JS7 cell line at passage level 170. The oncogenic significance of the JSRV proviral insertion involving the SP-A locus in the JS7 tumor cell line is unknown.

The functional assessment of deaf and hard of hearing students.

The study reports on a set of questions added to the 1997-98 Annual Survey of Deaf and Hard of Hearing Children and Youth designed to take into consideration the functioning of children in their classroom in nine functional areas. Basing information on 30,198 students, the study describes prevalence rates of reported limitations in these functional areas for deaf and hard of hearing students, compares these to rates resulting from the reporting of categorically defined additional disabilities, and examines interrelationships among the items. Results of school estimates of students' functional hearing abilities are presented. The study's findings suggest a broader range and higher prevalence of functional limitations than would be assumed by analyzing categories of additional disabilities alone. The study's findings support the viability of functional assessment through large surveys. The discussion emphasizes the importance of functional assessment for the provision of appropriate educational services to deaf and hard of hearing children.

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.

Investigating the mechanism and machinery of RNA editing.

RNA editing in trypanosomes produces mature mRNAs by posttranscriptional guide RNA (gRNA)-directed uridylate insertion and deletion. This article describes methods for the study of RNA editing with an emphasis on an in vitro editing system that was used to explore the general mechanism of editing and that can be adapted for more in-depth studies of this intriguing and important process. Methods used to investigate the macromolecular complex that catalyzes RNA editing are also described. This complex is composed of multiple proteins and contains several catalytic activities. It is in the early stages of characterization. The methods described here are intended to assist in its further analysis.

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.

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.

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.

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.

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 gene from Trypanosoma brucei.

The hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme of Trypanosoma brucei and related parasites provides a rational target for the treatment of African sleeping sickness and several other parasitic diseases. To characterize the T. brucei HGPRT enzyme in detail, the T. brucei hgprt was isolated within a 4.2 kb SalI-KpnI genomic insert and sequenced. Nucleotide sequence analysis revealed an open reading frame of 630 bp that encoded a protein of 210 amino acids with a M(r) = 23.4 kd. After gap alignment, the T. brucei HGPRT exhibited 21-23% amino acid sequence identity, mostly in three clustered regions, with the HGPRTs from human, S. mansoni, and P falciparum, indicating that the trypanosome enzyme was the most divergent of the group. Surprisingly, the T. brucei HGPRT was more homologous to the hypoxanthine phosphoribosyltransferase (HPRT) from the prokaryote V. harveyi than to the eukaryotic HGPRTs. Northern blot analysis revealed two trypanosome transcripts of 1.4 and 1.9 kb, each expressed to equivalent degrees in insect vector and mammalian forms of the parasite. The T. brucei hgprt was inserted into an expression plasmid and transformed into S phi 606 E. coli that are deficient in both HPRT and xanthine-guanine phosphoribosyltransferase activities. Soluble, enzymatically active recombinant T. brucei HGPRT was expressed to high levels and purified to homogeneity by GTP-agarose affinity chromatography. The purified recombinant enzyme recognized hypoxanthine, guanine, and allopurinol, but not xanthine or adenine, as substrates and was inhibited by a variety of nucleotide effectors. The availability of a molecular clone encoding the T. brucei hgprt and large quantities of homogeneous recombinant HGPRT enzyme provides an experimentally manipulable molecular and biochemical system for the rational design of novel therapeutic agents for the treatment of African sleeping sickness and other diseases of parasitic origin.

Demographic and audiological profiles of deaf children in Texas with cochlear implants.

This paper reports the findings of a study that examined the demographic, educational, and audiological characteristics of a sample of children in Texas who had received cochlear implants. Children who had received implants and children who had not were compared. Differences between the two groups are discussed in the context of the criteria that have been published in the literature for selecting implant candidates. Results indicate that although the published selection criteria were predictive of the characteristics of the implanted sample, there was a degree of flexibility in applying the criteria to choose implant recipients. Audiological comparisons revealed that cochlear implant recipients experience greater reductions in average hearing thresholds than do users of conventional aids, although these findings should be viewed as preliminary.

Subgroup differences in educational placement for deaf and hard of hearing students.

This study presents crosstabulations of educational placement variables by selected demographic characteristics and reports the different patterns of educational placement for subgroups of the deaf and hard of hearing population. Type of school, amount of academic integration with hearing students, and the number of deaf or hard of hearing students attending the same school building were considered as dependent variables in the analysis. Degree of hearing loss, age, ethnic and racial background, and additional handicap status were considered as independent variables. Data from the 1990-1991 Annual Survey of Hearing Impaired Children and Youth were analyzed. Results indicate that subgroups defined by each of these variables differ markedly with regard to patterns of educational placement. The importance of paying attention to subgroup statistics, as opposed to global population statistics, in the formulation of educational policy is discussed.

Hearing-impaired children under age 6: a demographic analysis.

This paper presents information about hearing-impaired children under the age of 6 who are receiving special educational services in the United States and have been reported to the Annual Survey of Hearing-Impaired Children and Youth conducted by Gallaudet University's Center for Assessment and Demographic Studies. Results indicate an increase in the number of hearing-impaired children under age 6 reported to the survey over the last 10 years, especially children from minority backgrounds. The second section of the analysis focuses on students from schools participating in a model state project that encourages early identification and provision of services to hearing-impaired students and their families. The data pertain only to those children who are receiving services through established programs reporting to the Annual Survey; thus, inferences about the overall prevalence of hearing impairment among this age group should not be drawn.