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— We used I-m2 throw traps to examine habitat use by smallsized fishes within a mosaic of wet prairies and sloughs in the headwaters of the St. Johns River, Florida between August 1992 and November 1995. Estimates of total fish density and biomass varied temporally, but did not differ significantly between habitats. Patterns of habitat use, however, differed among the five numerically dominant species. Bluefin killifish, mosquitofish, and golden topminnows were more abundant in sloughs than in wet prairies. In contrast, Everglades pygmy sunfish were more abundant in wet prairies than in sloughs. Finally, the abundance of least killifish did not differ between habitats. Fish densities were positively correlated with plant biomass (i. e., habitat complexity) and negatively correlated with water depth (i. e., hydrology). Species richness and composition were similar among habitats. However, consistent differences in the relative abundance of numerically dominant species between habitats indicated some degree of habitat-specific assemblage structure. Most species were concentrated into deeper sloughs during drying events. This assemblage of small-sized fishes appears to respond relatively rapidly to changes in habitat structure and hydrologic conditions. We therefore recommend that resource managers consider using fishes as indicator taxa to evaluate the efficacy of ongoing restoration and management efforts in wetland systems.

Study objective To compare the effectiveness of a standard jet nebulizer, Respirgard II, and a standard ultrasonic nebulizer, Fisoneb, for the administration of aerosolized pentamidine (AP) as primary and secondary prophylaxis against Pneumocystis carinii pneumonia (PCP) in HIV-infected individuals. Design A retrospective, nonrandomized, parallel group comparative study. Setting Patients were enrolled in a community-based AP program (APP) between May 1989 and April 1992 in Ontario, Canada. They received AP in either (1) a centralized treatment facility (“clinic”) or (2) their attending physician's office or regionalized centers (“nonclinic”). Clinic administration of pentamidine was via Fisoneb; nonclinic via Respirgard II. Patients The study group comprised of 1,762 HIV-infected individuals requiring AP for either primary (CD4 3 ) or secondary PCP prophylaxis. Of these, 1,151 used Fisoneb (clinic) and 611 used Respirgard II (nonclinic). Results In the primary prophylaxis group, 41 of the 892 patients using Fisoneb (4.6%; mean follow-up, 18 months) compared with 16 of 435 patients using Respirgard II (3,7%; mean follow-up, 14.6 months) developed PCP (p=0.44). A total of 28 of 259 (10.8%; mean follow-up, 15.3 months) patients using Fisoneb for secondary prophylaxis compared with 11 of 176 (6.3%; mean follow-up, 14.4 months) patients using Respirgard II for secondary prophylaxis developed PCP (p=0.1). Conclusions Despite the difference in dosage (120 mg/mo vs 300 mg/mo), type of nebulizer (ultrasonic vs jet), and frequency of administration (twice vs once monthly), the results of this study indicate that both regimens of AP provide comparable protection against PCP. This study further supports the effectiveness of AP as a solid second-line prophylaxis for HIV-infected individuals who are intolerant to trimethoprim/sulfamethoxazole or dapsone.

The immune response to a murine myeloid leukemia (cell line C1498) was studied in vitro and in vivo. Natural killer (NK) cells and CD8+ cytotoxic T lymphocytes (CTL) were shown to lyse C1498 in vitro through the binding of leukocyte function antigen-1 (LFA-1) on effectors and intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 on C1498 target cells. However, the ability of nonimmunized mice to resist an in vivo challenge of a low dose (10(4)) of C1498 was NK-cell, but not T-cell dependent. The failure of T cells to participate in the immune surveillance of a low leukemia burden appeared, in part, because of a lack of expansion of leukemia reactive CTL precursors (CTLp). Leukemia reactive CTLp frequency estimations in naive and leukemia bearing mice were not significantly different (range, 1:20,600 to 1:74,000) in contrast to immunized mice (range, 1:1,400 to 1:4,400). Leukemia reactive CTLp could be expanded to a level that could apparently mediate in vivo immune surveillance of 10(5) leukemia cells by injection of irradiated leukemia cells intraperitoneally (IP) or subcutaneously (SC), but not intravenously (IV). However, IV injection of 10(5) live leukemia cells engineered to secrete interleukin-2 (IL-2) resulted in systemic immunity mediated primarily by CD8+ T cells. We conclude that NK cells can mediate immune surveillance of a low leukemia burden. CD8+ CTL-mediated immune surveillance can eliminate a higher leukemia burden than NK cells, but requires T-cell help, which can be delivered by local IL-2. Both NK and CTL-mediated immune surveillance of C1498 murine myeloid leukemia is dependent on recognition through the LFA-1:ICAM adhesion pathway.

Mucopolysaccharidosis type I (MPS I) is a progressive, multisystemic, inherited metabolic disease caused by deficiency of alpha-L-iduronidase (IDUA). The most severe form of this disease (Hurler syndrome) results in death by age 10. Current treatments for this disease are ineffective in treating CNS disease due to the inability of lysosomal enzymes to traverse the blood-brain barrier. Our goal is to supplement current therapy, and treat CNS manifestations of the disease, by AAV-mediated gene delivery and expression of IDUA. We have taken a non-invasive and effective approach to the treatment of CNS disease by intranasal administration of an IDUA-encoding AAV9 vector. Adult IDUA-deficient mice were immunotolerized at birth with human iduronidase, to prevent anti-IDUA immune response, and at 3 months of age were infused intranasally with AAV9-IDUA vector. Animals sacrificed 3 months post-infusion exhibited IDUA enzyme activity levels that were 100-fold that of wild type in the olfactory bulb, with wild type levels of enzyme restored in all other parts of the brain. Neurocognitive testing using the Barnes maze demonstrated that treated IDUA-deficient mice were not different from normal control animals, while untreated IDUA-deficient mice exhibited a significant learning deficit. There was strong IDUA immunofluorescence staining observed in tissue sections of the nasal epithelium and olfactory bulb, but no staining was observed in other portions of the brain. This indicates that the widespread distribution of IDUA enzyme most likely was the result of enzyme diffusion from sites of transduction and IDUA expression in the olfactory bulb and the nasal epithelium into deeper areas of the brain. In order to increase access, delivery and vector distribution throughout the brain, IDUA-deficient animals were pretreated with intranasal infusions of an absorption enhancer. At different time points following pretreatment, animals were infused intranasally with AAV9 or AAVrh10 vector encoding IDUA. Animals were sacrificed at 2 months post-infusion, brains microdissected, and assayed for IDUA enzyme activity, clearance of glycosaminoglycans, and immunofluorescence staining for IDUA and GFP. This novel, non-invasive strategy for intranasal AAV9-IDUA administration could potentially be used to treat CNS manifestations of MPS I.

To determine if the density, biomass, species density (number of fish species per square metre), and species composition of juvenile and forage fish (fish ≤ 100 mm SL) differed among macrophyte types, we sampled fishes monthly from beds of Hydrilla verticillata, Panicum hemitomon, and Potamogeton illinoensis in two areas of Lake Okeechobee, Florida, from December 1989 to December 1990. Fish density and biomass differed significantly among the three macrophytes despite our design having relatively low statistical power to detect such differences. Densities and biomass were highest in H. verticillata (196 fish∙m−2, 30 g∙m−2), intermediate in P. hemitomon (60 fish∙m−2, 12 g∙m−2), and lowest in P. illinoensis (31 fish∙m−2, 6 g∙m−2). Trends among macrophytes were consistent throughout the year despite significant variation among months. Fish species density did not vary significantly among macrophyte types. However, the relative abundances of fishes differed significantly among macrophyte types. Patterns in fish abundance and relative species composition led us to infer that beds of different macrophyte types constitute distinct microhabitats for fish and structure the littoral zone of Lake Okeechobee into a mosaic landscape.

In previous studies characterizing intron-dependent expression of the human purine nucleoside phosphorylase-encoding gene (PNP), we identified a putative enhancer sequence in the first intron which was capable of mediating increased cat reporter gene expression in transfected murine NIH 3T3 cells in a position- and orientation-independent manner. In order to further characterize this enhancer activity, the nucleotide sequence was determined for the region of intron 1 to which this activity was originally ascribed. The sequence was analyzed for the presence of binding sites for known transcription factors, but none were identified. A 444-bp downstream portion of the intron-1 sequence enhanced cat expression either in conjunction with a human PNP promoter sequence or with a 105-bp heterologous herpes simplex virus thymidine kinase (TK) promoter. Nested deletions of the downstream intron-1 sequence fused to a TK::cat fusion gene localized the enhancer activity to a 170-bp sequence in intron 1. A 154-bp HgiAI fragment (bp 424 to 577 of intron 1) excised from this region contained enhancer activity which varied directly with the number of fragments inserted upstream from the TK::cat fusion gene. However, inversion of the HgiAI fragment in a PNP abbreviated gene, or relocation of the HgiAI fragment from intron 1 to a position upstream from the PNP promoter, reduced or eliminated PNP expression. The effect of the intron-1 enhancer element on PNP expression is thus maximized in a position- and orientation-dependent manner.

SUMMARYA simple test for diagnosing Nosema apis infections in honey bees, which does not require a microscope, is described. Bee abdomens are crushed in water in plastic 1.5 ml microcentrifuge tubes, nigrosin stain is added and the tube left to stand vertically overnight. Centrifugation of the tubes eliminates the need for an overnight wait. The nigrosin stains any bee tissues and most pollen a dark blue/black and the N. apis spores settle to form a pale grey, unstained pellet at the bottom of the tube. Eight million or more spores per bee can be detected (P < 0.05) and the size of the pellet gives an indication of the magnitude of the spore load. Pooled samples of bees crushed in a plastic bag may be similarly assessed, although some accuracy is lost.

Abbreviated purine nucleoside phosphorylase (PNP) genes were engineered to determine the effect of introns on human PNP gene expression. PNP minigenes containing the first intron (complete or shortened from 2.9 kb down to 855 bp), the first two introns or all five PNP introns resulted in substantial human PNP isozyme expression after transient transfection of murine NIH 3T3 cells. Low level human PNP activity was observed after transfection with a PNP minigene containing the last three introns. An intronless PNP minigene construct containing the PNP cDNA fused to genomic flanking sequences resulted in undetectable human PNP activity. Heterogeneous, stable NIH 3T3 transfectants of intron-containing PNP minigenes (verified by Southern analysis), expressed high levels of PNP activity and contained appropriately processed 1.7 kb message visualized by northern analysis. Stable transfectants of the intronless PNP minigene (40-45 copies per haploid genome) contained no detectable human PNP isozyme or mRNA. Insertion of the 855 bp shortened intron 1 sequence in either orientation upstream or downstream of a chimeric PNP promoter-bacterial chloramphenicol acetyltransferase (CAT) gene resulted in a several-fold increase in CAT expression in comparison with the parental PNP-CAT construct. We conclude that human PNP gene expression at the mRNA and protein level is dependent on the presence of intronic sequences and that the level of PNP expression varies directly with the number of introns included. The disproportionately greatest effect of intron 1 can be explained by the presence of an enhancer-like element retained in the shortened 855 bp intron 1 sequence.

Purine nucleoside phosphorylase (PNP) is a ubiquitously expressed enzyme which contributes to the catabolism and recycling of nucleotides. To characterize the promoter region of the human PNP gene, the nucleotide sequence from a BamHI site located in the 5' untranslated region extending 2237 bp upstream to an XbaI site was determined. The transcriptional start site as determined by primer extension was 119 bp upstream of the coding sequence and consisted of a 5'-CA-3' dimer with A at +1. A TATA box was identified -24 to -29 bp upstream of the transcriptional start site. A CCAAT pentamer sequence in the inverted orientation was present at -51 to -55 bp and two GC rich regions were identified at -68 to -81 bp and -168 to -187 bp. Progressive 5' deletions of the 5' flanking region were fused to the chloramphenicol acetyltransferase (CAT) reporter gene and transient expression measured after transfection of murine NIH/3T3 fibroblasts. A 91 bp promoter (the shortest tested) provided CAT activity at 60% the level of a 216 bp promoter, possibly due to removal of the GC rich region between -168 and -187 bp. Longer promoters resulted in CAT expression at similar or lower levels than the 216 bp promoter indicating that this region contained all of the 5' flanking sequences affecting transcription from the PNP promoter.