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Aquatic acidification by deposition of airborne pollutants emerged as an environmental issue in southeastern Canada during the 1970s. Drawing information from the extensive research and monitoring programs, a sequence of issue assessments demonstrated the necessity of reducing the anthropogenic emissions of acidifying pollutants, particularly sulphur dioxide (SO2). The 1991 Canada-U.S. Air Quality Agreement (AQA) was negotiated to reduce North American SO2 emissions by approximately 40% relative to 1980 levels by 2010, and at present, both countries have reduced emissions beyond their AQA commitment. In response to reduced SO2 emissions, atmospheric deposition of sulphate (SO4(2-)) and SO4(2-) concentrations in many lakes have declined, particularly in south-central Ontario and southern Québec. Sulphate deposition still exceeds aquatic critical loads throughout southeastern Canada however. Increasing pH or alkalinity (commonly deemed 'recovery') has been observed in only some lakes. Several biogeochemical factors have intervened to modify the lake chemistry response to reduced SO4(2-) input, notably release of stored SO4(2-) from wetlands following periods of drought and reduction in the export of base cations from terrestrial soils. Three examples from Ontario are presented to illustrate these responses. Significant increases in pH and alkalinity have been observed in many lakes in the Sudbury area of Ontario due to the large reductions in local SO2 emissions; 'early-stage' biological recovery is evident in these lakes. An integrated assessment model predicts that AQA emission reductions will not be sufficient to promote widespread chemical or biological recovery of Canadian lakes. Monitoring and modeling are mutually supporting assessment activities and both must continue.

By using high-performance size exclusion chromatography (HPSEC) and reversed-phase high-performance liquid chromatography with on-line three-dimensional excitation emission matrix fluorescence detection, we measured fluorescence properties of natural organic matter (NOM) as a function of molecular size (MS) and polarity. The work was carried out with Suwannee River Fulvic Acid, Aldrich Humic Acid, and a naturally occurring river NOM sample. Significant differences in fluorescence maximum pattern were found as NOM was separated chromatographically based on MS and polarity. There existed a strong relationship between MS, fluorescence pattern, and polarity. Humic-, fulvic-, and protein-like fluorescence fractions had distinct hydrophilic/hydrophobic nature. The results suggest that HPSEC may be better for characterizing major fulvic-like fluorescence and smaller MS fractions but not those having humic- and protein-like fluorescence and larger MS, which may be strongly adsorbed onto the HPSEC column because of their hydrophobic nature. This study has significant implications for further understanding the nature of NOM and its complexation with trace metals.

The accumulation of iron (Fe) in several lakes in Ontario, Canada was determined by two independent approaches. First, Fe accumulation was calculated in cores collected from several sites in each lake by integrating Fe concentration profiles with sediment accumulation rates determined from Pb210 dating. These site-specific accumulation rates were corrected for sediment focussing so that whole-lake Fe accumulation values could be derived. Using this approach, recent whole-lake Fe accumulation in eight lakes ranged between approximately 750 and 4000 mg/m2 per year. Second, whole-lake Fe accumulation was estimated from lake mass budgets, which were measured over a maximum of 14 years. Accumulation measured using the mass balances ranged from 10 to 1330 mg/m2 per year. Comparison of the two approaches indicated that retentions calculated from the sediment cores were much greater than those estimated from the mass balances. The most likely explanation for this difference is that, in the two decades since the cores were collected, there has been a substantial decline in Fe retention (in mass units but not percent) in the study lakes, principally as a result of reduced inputs of Fe from the catchments.

We have developed a rapid assay for single nucleotide polymorphism (SNP) detection that utilizes electronic circuitry on silicon microchips. The method was validated by the accurate discrimination of blinded DNA samples for the complex quadra-allelic SNP of mannose binding protein. The microchip directed the transport, concentration, and attachment of amplified patient DNA to selected electrodes (test sites) creating an array of DNA samples. Through control of the electric field, the microchip enabled accurate genetic identification of these samples using fluorescently labeled DNA reporter probes. The accuracy of this approach was established by internal controls of dual labeled reporters and by using mismatched sequences in addition to the wild-type and variant reporter sequences to validate the SNP-genotype. The ability to customize this assay for multiple genes has advantages over other existing approaches.

A four-year-old boy was admitted to intensive care via casualty having suffered an apnoeic episode due to compression of his chest by a heavy cattle bar. A petechial rash was noted on the upper chest and arms. A chest radiograph showed pulmonary oedema which resolved over a few hours following positive pressure ventilation and positive end expiratory pressure. We believe this to be an unusual presentation of obstructive pulmonary oedemia (Negative Pressure Pulmonary Oedema).

Although the polymerase chain reaction (PCR) (1,2) is invaluable for the cloning and manipulation of existing DNA sequences, PCR also makes it possible to create new DNA fragments consisting of a nucleic acid sequence that is specified entirely by the investigator. In this chapter we describe a simple two-step PCR method for the rapid construction of synthetic genes (3). This method is based on early observations by Mullis et al. (4) in which multiple overlapping oligonucleotides could be used to generate synthetic DNA through several sequential rounds of Klenow-based PCR amplification. The method described in this chapter utilizes the thermostabile Taq polymerase and allows for the generation of synthetic genes in as little as 1 d. This method has proven useful in studies in which synthetic genes were constructed for the HIV-2 Rev protein (3,5) and the Wilms' tumor locus zinc finger protein (6). Furthermore, this method has been successfully employed in extensive mutagenesis of the HIV-1 rev response element (7).

As a consequence of decreases in the emission rate of sulphur in eastern North America in the late 1970s and early 1980s, sulphate deposition in central Ontario declined by about 40%, but has remained constant for about six years. Plastic Lake, a small, dilute lake on the Precambrian shield that the authors have studied since 1979, acidified between the start of the study and about 1986, but since then has not changed. The authors also monitored the chemistry of streamwater draining the Plastic Lake catchment. Water quality of runoff from an upland site improved rapidly (pH and alkalinity increased, SO4(2-) and Al decreased), but two factors offset these improvements. A small wetland area downstream reversed most of these changes, resulting in a constant output of strong acid from the catchment. In addition, in extremely dry years (1983, 1987, 1989) there were very high concentrations of SO4(2-) in the streamwater, suggesting substantial re-oxidation of reduced S in the catchment.

Examination of the life cycle of the human immunodeficiency virus (HIV) has shown that multiple levels of regulation exist, including some which require the virus-encoded Rev protein. In the absence of Rev, mRNAs encoding the structural proteins remain untranslated, a phenomenon which appears, in part, to be caused by nuclear entrapment of these RNA species. To examine the basis for repression of structural gene mRNA expression, a heterologous assay system was utilized to determine whether regions present within gag and pol contain elements capable of suppressing gene expression when present in cis. Both genes were found to contain cis-acting repressor sequences (CRS) that block gene expression when present within the 3' untranslated portion of a heterologous gene transcript. The element within pol was found to have the strongest repressive effect. While Rev alone was unable to reverse the repression observed with the pol sequence, addition of the env Rev-responsive element (RRE) in cis and Rev in trans did cause reversal of inhibition. Deletion mutagenesis defined a 260-bp element within the 3' portion of pol that contains a potent CRS which functions when present in the sense orientation. The corresponding region in HIV-2 pol was found to contain a functionally similar CRS element. To examine the mechanism of repression, the effects of the CRS elements on both the abundance and subcellular distribution of the mRNAs were examined. Neither was dramatically altered when examined in the context of a heterologous reporter (chloramphenicol acetyltransferase) mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

A replication-competent Akv murine leukemia virus-based vector encoding the human immunodeficiency virus tat cDNA under control of the simian virus 40 early promoter sequences was constructed. The simian virus 40 tat sequences were placed within the U3 region of the 3' long terminal repeat. The resulting virus, derived by transfection, replicated efficiently in mouse NIH 3T3 cells and maintained the tat cDNA insert. It has been suggested that Tat function requires the presence of a human-specific cofactor, which is absent in murine cells. However, infection of murine cells with the Akv virus encoding tat resulted in significant transactivation of a human immunodeficiency virus long terminal repeat-driven reporter gene, indicating that human cofactors are not always required for Tat function. The vector system described may be useful for introduction of foreign genes in vivo and in whole animals when virus spread is required for efficient infection and levels of gene expression.

The Rev proteins of the human immunodeficiency virus (HIV) are necessary for expression of viral structural gene products. Site-directed mutations were made within the HIV-2 rev gene to identify functional domains. We observed that similar to HIV-1 Rev, the HIV-2 Rev protein was phosphorylated, albeit to a much lesser extent than was HIV-1 Rev. We also found that like HIV-1 Rev, HIV-2 Rev localized to the nucleus, with a marked accumulation in the nucleolus. Mutations within a stretch of basic residues prevented both nuclear and nucleolar localization. Furthermore, mutant Rev proteins able to localize in the nucleus but unable to localize in the nucleolus were nonfunctional.

The interaction of the human immunodeficiency virus type 1 (HIV-1) Rev protein with a structured region in env mRNA (the Rev-responsive element [RRE]) mediates the export of structural mRNAs from the nucleus to the cytoplasm. We demonstrated that unlike HIV-1 Rev, which functions with both the HIV-1 and HIV-2 RREs, HIV-2 Rev functions only with the HIV-2 RRE. Rev-RRE binding studies suggested that the lack of nonreciprocal complementation stems from the inability of HIV-2 Rev to interact with HIV-1 RRE RNA. Maintenance of RNA secondary structure, rather than the primary nucleotide sequence, appeared to be the major determinant for interaction of both HIV-1 and HIV-2 Rev with the HIV-2 RRE. Moreover, the binding domain of the HIV-2 RRE recognized by HIV-1 Rev was dissimilar to the binding domain of the HIV-1 RRE, in terms of both secondary structure and primary nucleotide sequence. Our results support the hypothesis that function of HIV Rev proteins and possibly the functionally similar Rex proteins encoded by the human T-cell leukemia viruses (HTLVs) HTLV-I and HTLV-II is controlled by the presence of RNA secondary structure generated within the RRE RNA.

Interaction of the human immunodeficiency virus type 1 (HIV-1) Rev protein with a structured region within env mRNA (termed RRE) mediates the export of virus structural mRNAs from the nucleus to the cytoplasm. We show that the region encompassing the basic stretch of amino acids is essential for the ability of Rev to bind to RRE RNA and function in vivo. By use of a functional truncated Rev protein in conjunction with authentic Rev, effects on gel mobilities of the Rev-RRE RNA complex attributable to multimerization of Rev protein were observed. Rev proteins, unable to multimerize, failed to bind RRE RNA. Identification of Rev mutants capable of forming multimers, but unable to bind RRE RNA, suggests that the multimerization and RNA-binding domains can be distinguished and that multimerization is likely a prerequisite for formation of the RRE RNA-binding site. A mutant Rev protein, shown previously to function as a trans-dominant inhibitor of Rev function, bound to RRE RNA as a multimer to a similar extent as wild-type Rev. This observation is consistent with the hypothesis that regulation of HIV gene expression by Rev involves the interaction with cellular factors and that the trans-dominant Rev is probably defective in this function.

The human immunodeficiency virus (HIV) tat protein (Tat) is a positive regulator of virus gene expression and replication. Biotinylated Tat was used as a probe to screen a lambda gt11 fusion protein library, and a complementary DNA encoding a protein that interacts with Tat was cloned. Expression of this protein, designated TBP-1 (for Tat binding protein-1), was observed in a variety of cell lines, with expression being highest in human cells. TBP-1 was localized predominantly in the nucleus, which is consistent with the nuclear localization of Tat. In cotransfection experiments, expression of TBP-1 was able to specifically suppress Tat-mediated transactivation. The strategy described may be useful for direct identification and cloning of genes encoding proteins that associate with other proteins to modulate their activity in a positive or negative fashion.