Faculty Bibliography

The purpose of this study was to determine the incidence of deep venous thrombosis in medical intensive care unit patients receiving deep venous thrombosis prophylaxis. This was a prospective cohort study of 141 consecutive adult patients anticipated to remain in the medical intensive care unit for >48 hours. Deep venous thrombosis prophylaxis was provided using subcutaneous unfractionated heparin or a sequential compression device according to risk-stratified protocol. Compression ultrasound was performed. Fourteen patients (9.9%) developed deep venous thrombosis on follow-up studies. Incidence of deep venous thrombosis was 7.9% per person year (95% confidence interval, 4.8-12.8). Two of 14 developed pulmonary embolism. Eight patients required full anticoagulation with intravenous heparin or coumadin. In-hospital mortality was similar in both groups. Patients with deep venous thrombosis had a statistically higher risk of pulmonary embolism: 14.2% (95% confidence interval, 2.0-43.0) versus 0.0% (95% confidence interval, 0-3; P = .009). Incidence of deep venous thrombosis is high in medical intensive care unit patients receiving standard prophylaxis. Adherence to strict deep venous thrombosis prophylaxis protocol and exploration of other prophylaxis regimens should be pursued

Trypanosoma brucei evades its host's immune response by utilizing the system of antigenic variation, whereby the organism sequentially expresses antigenically distinct variant surface glycoproteins (VSGs). Actively expressed VSG genes are found in VSG expression sites (ESs), and transcription of these ESs is directed by a small promoter composed of two essential cis-acting elements, the VSG ES promoter upstream element (VUE) and VSG ES promoter downstream element (VDE). Using electrophoretic mobility shift assays, we have identified double-stranded DNA binding activity in bloodstream-form trypanosome nuclear extracts. This activity, the VEP complex, is specific for the VSG ES promoter, and requires the intact sequences of the VUE and VDE in the appropriate spacing. These requirements of VEP Complex formation parallel the requirements for promoter function, suggesting that the VEP complex may be composed of functionally significant trans-acting factors. Furthermore, the requirement of both elements suggests that the binding of factors to the promoter may be cooperative. However, subtly different binding characteristics were observed when we used nuclear extracts derived from procyclic trypanosomes

The African trypanosome Trypanosoma brucei is a protozoan parasite that causes the disease African sleeping sickness. The parasite avoids the host's immune response by the process of antigenic variation, or by sequentially expressing antigenically different cell-surface coat proteins. These proteins, called variant surface glycoproteins (VSGs), are expressed from a specific locus, the VSG gene expression site (ES). In an attempt to understand expression of VSG genes, we expanded on earlier investigations of the promoter that controls the large VSG gene expression site transcription unit. We studied VSG ES promoter function both in transient transfection assays, and after stable integration at a chromosomal locus. Analysis of closely spaced deletion mutants showed that the minimum VSG ES promoter fragment that gives full activity is extremely small, and mapped precisely to a fragment that contains no more than -67 bp 5' to the putative transcription initiation site. The promoter lacked an upstream control element, or UCE, an element found at the PARP promoter, and at most eukaryotic Pol I promoters. Furthermore, linker scanning mutagenesis demonstrated that the VSG ES promoter contains at least two essential regulatory elements, including sequences within the region -67/-60 and the region -35/-20, both numbered relative to the initiation site. An altered promoter with mutated nucleotides surrounding the transcription initiation site still directed wild-type levels of expression. In this study, the results were similar for both insect and bloodstream form trypanosomes, suggesting that the same basic machinery for expression from the VSG ES promoter is found in both stages of the parasite

We show that the ribosomal RNA (rRNA) promoter can efficiently direct expression of protein-coding genes in the parasitic protozoan Trypanosoma brucei. The rRNA promoter was characterized by: (i) point mutations at the rRNA transcription initiation site which completely abolished its promoter function in transient CAT transformation assays; (ii) the alpha-amanitin resistance of transcription of rRNA promoter-neomycin phosphotransferase (neo) genes in stably transformed trypanosomes; and (iii) the nucleolar location of neo RNA, synthesized under the control of the rRNA promoter. The rRNA promoter-derived CAT mRNA required a 3' splice acceptor site and the neo mRNA was trans-spliced and polyadenylated. In situ hybridization revealed neo RNA at the nucleolus in stably transformed trypanosomes in which rRNA promoter-neo constructs were integrated either at a rRNA locus or at a locus for the procyclic acidic repetitive protein (PARP) coding genes. We postulate that trans-splicing, by uncoupling the requirement for transcription of protein-coding genes by RNA polymerase II, allows RNA polymerase I mediated protein-coding gene transcription, presumably because a 5' cap can be transferred to the pre-mRNA by trans-splicing