Every kinetoplastid mRNA receives a common, conserved leader sequence via the process of trans-splicing. In Leishmania tarentolae the precursor spliced leader RNA is 96 nucleotides, with a 39-nucleotide exon that is 7meG-capped and methylated on the first 4 nucleotides. trans-Splicing was inferred from the presence of tagged leader in the high molecular weight RNA population and confirmed for accuracy by cDNA cloning. Linker scan substitutions within the exon between positions 10 and 39 did not affect trans-splicing. The trans-splicing efficiency for three of the scan exons was proportional to the tagged:wild type ratio in the spliced leader precursor population. Two scan leader RNAs that were efficiently spliced showed reduced methylation. Longer exons showed reduced splicing, whereas 10-or 20-base pair deletions abolished splicing. These results indicate that size, but not content, of the exon is a constraint on the splicing process. These results, in combination with previous data eliminating a role in transcription initiation, suggest that translation may be the selective pressure on the leader content.The 39-nt 1 spliced leader (SL) in the kinetoplastids is extensively conserved (1-3), such that PCR amplification of SL RNA genes from diverse kinetoplastids can be performed with a single set of oligonucleotide primers (4, 5). These conserved sequences are presumed to be important for one or more basic biological processes, which could include transcription, transsplicing, and translation. In the nematode Ascaris, part of the 22-nt SL sequence functions as a promoter (6); however, a major role for the SL in transcription initiation has been eliminated in Leishmania tarentolae and Trypanosoma brucei, which have upstream promoters (7,8). An Ascaris SL of two nucleotides is accurately trans-spliced in vitro (9), indicating that information for splicing is not contained within the SL. By contrast, the SL sequence is important for both transcription (10) and trans-splicing (11) in some kinetoplastids.Although the U5 small nuclear RNA may be cross-linked to exons near the splice junction (12), the roles of exons in cissplicing are generally constrained by their protein-coding capacities. There are no such constraints apparent in trans-splicing. The kinetoplastid SL functions as a cap 4 donor (13) and as a 5Ј-untranslated region, allowing the possibility for an active role in trans-splicing. Several inter-and intramolecular interactions have been predicted to occur within the SL. Two small RNAs, SL-associated 1 and U5, have been demonstrated through in vivo cross-linking to interact with the SL (14, 15). Intramolecular interactions of the SL include alternation between two structural forms involving SL-SL (Form I) and SLintron (Form II) base pairing in vitro (16) and in vivo (17) and proposed U1 and U5 functions (18).To determine the importance of the conserved kinetoplastid SL in trans-splicing we evaluated several SL mutants for transsplicing in vivo. We show that SLs containing 10-bp replacement mutations between...

In order to determine whether human granulocyte-macrophage colony- stimulating factor (GM-CSF) can enhance phagocytosis, neutrophils were combined with Staphylococcus aureus (S aureus), and both the number of bacteria per neutrophil and the percent of neutrophils phagocytizing were assessed in the absence and presence of GM-CSF. Exposure to GM-CSF did not enable neutrophils to ingest unopsonized bacteria. When bacteria were opsonized with serum, both the number of bacteria per neutrophil and the percent of cells phagocytizing were increased by treatment with GM-CSF. Digestion of extracellular organisms by lysostaphin was used to substantiate phagocytosis. These results indicate that another effect of GM-CSF on the mature neutrophil is the enhancement of phagocytosis.

We conducted a clinical trial of human recombinant granulocyte-macrophage colony-stimulating factor

Microbicidal cationic proteins 1 and 2, peptides derived from rabbit lung macrophages, were tested for bactericidal activity against various bacterial species. Both were highly active against diverse gram-positive and gram-negative organisms under conditions of near-neutral pH (between 7 and 8) and relatively low ionic strength. Susceptible species included Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Listeria monocytogenes, Pseudomonas aeruginosa, Klebsiella pneumoniae, Haemophilus influenzae, Escherichia coli, and Serratia marcescens. Streptococcus agalactiae, type 1A, was less susceptible than the aforementioned organisms or S. agalactiae, type 3. Bordetella bronchiseptica, a common commensal and pathogen of the rabbit respiratory tract, was completely resistant to both peptides.

There is compelling clinical evidence for dysfunction of the mononuclear phagocyte system in patients with AIDS, which is believed due in part to loss of T-cell cooperativity. The direct consequences of human immunodeficiency virus infection on macrophage function are unknown. To address this question we infected normal human macrophages in vitro with a monocytotropic strain of human immunodeficiency virus and performed assays to quantify their extra-and intracellular killing ability. Human immunodeficiency virusinfected macrophages were signflicandy less effective than control cells in mediating antibody-dependent cell-mediated cytotoxicity against leukemic cell targets and intracellular killing of Candidapseudotropicals. The functional defects were profound, related temporarily to active virus production by the macrophages, and could not be overcome by granulocytemacrophage colony-stimulating factor. Treatment of macrophages with 3'-azido-3'-deoxythymidine (AZT) 6 days after infection caused a marked decrease in virus production and prevented development of the intracellular killing functional defect. The results suggest that early antiviral therapy may be useful in preventing or mitigating some virus-induced mononuclear phagocyte dysfunction.The immunopathogenesis of AIDS has been attributed primarily to a selective depletion of T cells with resultant B-lymphocyte and mononuclear phagocyte dysfunction (1)(2)(3)(4)(5). Clinically, the pattern of opportunistic infections seen in patients with AIDS relates to impairment of the T-lymphocyte/monocyte axis as well as diminished antibody responses (3-8). Although mononuclear phagocytes may harbor the human immunodeficiency virus (HIV) in vivo, the incidence and consequences of infection with monocytotropic HIV strains are uncertain (9, 10). Despite compelling clinical evidence of mononuclear phagocyte dysfunction in AIDS, the direct effects of HIV infection on macrophage function are unknown. To address this question we infected normal human monocytes with HIV in vitro and quantified intra-and extracellular killing capacity. Here we show that HIV infection of mononuclear phagocytes causes markedly defective cellular function that may be prevented by treatment with the antiviral agent 3'-azido-3'-deoxythymidine (AZT).MATERIALS AND METHODS Cells and Virus. Peripheral blood was obtained from healthy laboratory volunteers and monocytes were isolated by Ficoll/Hypaque separation and adherence to culture dishes in the presence of5% (vol/vol) human type AB serum.After successive washing procedures cultures contained greater than 97% pure monocytes. Adhered monocytes were cultured in Iscove's modified Dulbecco's medium (Irvine Scientific) supplemented with 15% (vol/vol) heat-inactivated fetal calf serum, 5% human AB serum, 1% glutamine, and antibiotics at 370C in a humidified 5% C02/95% air incubator.The monocytotropic HIV-JRFL virus has been described (11). Two million monocytes were infected with 0.5 ml of a filtered cell-free infecting inocula estimated at 50 ng ...