Abstract. To identify and localize the protein products of genes encoding distinct L-type calcium channels in central neurons, anti-peptide antibodies specific for the class C and class D otl subunits were produced. Anti-CNC1 directed against class C immunoprecipitated 75 % of the L-type channels solubilized from rat cerebral cortex and hippocampus. Anti-CND1 directed against class D immunoprecipitated only 20% of the L-type calcium channels. Immunoblotting revealed two size forms of the class C L-type al subunit, L¢~ and Lc2, and two size forms of the class D L-type t~l subunit, Lo~ and Lm. The larger isoforms had apparent molecular masses of •200-210 kD while the smaller isoforms were 180-190 kD, as estimated from electrophoresis in gels polymerized from 5 % acrylamide.Imrnunocytochemical studies using CNC1 and CND1 antibodies revealed that the o~1 subunits of both L-type calcium channel subtypes are localized mainly in neuronal cell bodies and proximal dendrites. Relatively dense labeling was observed at the base of major dendrites in many neurons. Staining in more distal dendritic regions was faint or undetectable with CND1, while a more significant level of staining of distal dendrites was observed with CNC1, particularly in the dentate gyrus and the CA2 and CA3 areas of the hippocampus. Class C calcium channels were concentrated in clusters, while class D calcium channels were generally distributed in the cell surface membrane of cell bodies and proximal dendrites. Our results demonstrate multiple size forms and differential localization of two subtypes of L-type calcium channels in the cell bodies and proximal dendrites of central neurons. The differential localization and multiple size forms may allow these two channel subtypes to participate in distinct aspects of electrical signal integration and intracellular calcium signaling in neuronal cell bodies. The preferential localization of these calcium channels in cell bodies and proximal dendrites implies their involvement in regulation of calciumdependent functions occurring in those cellular compartments such as protein phosphorylation, enzyme activity, and gene expression.

Previous molecular cloning experiments showed that multiple isoforms of the al subunit of voltagegated Ca channels are expressed in the mammalian brain (designated rbA, rbB, rbC, and rbD). We report here the isolation and characterization of cDNAs encoding the rat brain class A (rbA) Ca channel. The rbA-126 cDNA encodes a 2212-amino acid protein that shares 33% sequence identity with the al subunits of cardiac and skeletal muscle dihydropyridine-sensitive Ca channels. When compared with other Ca channels, the rbA channel is notably different in both the carboxyl terminus and in a large (474-amino acid) hydrophilic segment between domains II and HI. Northern blot analysis shows that rbA transcripts are expressed in all regions of the rat central nervous system, but most prominently in the cerebellum. A more widespread distribution of rbA Ca channels is indicated by PCR analysis, which demonstrates the presence of class A transcripts in the rat heart and pituitary but not in the spleen, kidney, or liver. The rbA cDNA appears to encode a Ca channel al subunit that is distinct from the dihydropyridine-sensitive Ca channel sequences and that is expressed in a variety of excitable cells.Voltage-gated Ca channels form a diverse set of molecules that mediate Ca entry into a wide variety ofcell types. In neurons, Ca entry both contributes to the electrical properties of cells and affects the level of intracellular Ca, which acts as a second messenger (for reviews see refs. 1 and 2). On the basis of electrophysiological and pharmacological criteria, four major types ofCa channels have been described in neurons (designated T, L, N, and P; reviewed in ref.3). However, Ca channels from different neuronal cell types often exhibit pharmacological and electrophysiological profiles that do not correlate exactly with any one of the four types, which suggests that additional Ca channel diversity exists (3,4,39).Detailed biochemical studies of the dihydropyridine (DHP)-sensitive Ca channel from skeletal muscle T-tubule membranes (L-type Ca channel) have shown that it is composed of five distinct subunits (al, a2, ,, y, and 8; reviewed in refs. 5 and 6). Molecular cloning has revealed that the al subunit is a 212-kDa protein (7) that is structurally homologous to the a subunit of voltage-gated Na channels (8,9). Both the Ca channel al subunit and the Na channel a subunit contain four repeated domains, each containing six to eight putative transmembrane segments. More recently, fulllength cDNAs for the al subunits of cardiac and smooth muscle L-type Ca channels have also been reported (10-12). Expression of these cloned al-subunit cDNAs in a number of test systems shows that this subunit encodes a voltage-gated, 11,13).Utilizing low-stringency hybridization with a skeletal muscle probe, Snutch et al. (14) isolated a family of cDNAs from rat brain that are related to the al subunits of L-type Ca channels. With these probes we have begun to address a number of basic questions concerning the structure, expression, and regulation...

Five wetland designs, based on conventional surface flow (SF) and subsurface flow (SSF) approaches, were assessed for nitrogen and phosphorus removal from greenhouse wastewater. Results indicated none of the individual designs assessed was capable of providing the highest treatment effect for all nutrients of concern; however, the SF wetland emerged as the most appropriate design for the treatment of greenhouse wastewater. The highest mean phosphorus reduction of 65% was observed in the unplanted SF wetlands. Peak nitrate reductions of 54% were observed in the 15-cm deep SF wetlands and ammonia removal of 74% was achieved in the unplanted SF wetlands. Nitrate concentration in the greenhouse effluent can be reduced to acceptable levels for the protection of freshwater aquatic life (i.e., less then 40 ppm) using a loading rate of 1.65 g NO3-N/m2/day and a design water depth of 30 cm or greater. Based on available literature and the results of this research project, a multistage design, consisting of an unplanted pre-treatment basin followed by a 25 to 35 cm deep surface flow marsh with open water components, is recommended.

Some of the largest oil and gas projects in Canada are currently being proposed in British Columbia. Establishing a fulsome and scientifically and socially defensible scope for environmental assessments in the oil and gas sector is a serious challenge for government and proponents. The approach taken by the federal National Energy Board to scope effects assessments on pipelines is quite different than the approach taken by the British Columbia Environmental Assessment Office on other types of oil and gas projects. The NEB has published guidelines for scoping and conducting environmental and socio-economic assessments within its Filing Manual (National Energy Board [NEB] 2014). This manual sets out the expectations for scoping, baseline information, and effects assessments to be submitted as part of approval applications. Proponents are expected to provide all information necessary to meet the guidelines. In British Columbia, the environmental assessment process is dictated by the British Columbia Environmental Assessment Act and includes a negotiated terms of reference for the assessment, called the Application Information Requirements (AIR). The approach to selection of valued components is guided by provincial guidelines (EAO, 2013). The first draft of the AIR is prepared by the proponent and is then amended to address matters raised by federal and provincial agencies, local governments, and representatives of potentially affected First Nations. Through two to three revisions, the scope of assessment is jointly established and then formally issued by the government. While there are valid reasons for the differing federal and provincial approaches to scoping environmental assessments, each of these processes create risks for proponents in terms of project timelines and costs for preparing the environmental assessment. More specifically, the use of generic and negotiated guidelines can result in a number of issues including: • A scope of assessment that is broader than necessary to understand the potential for significant adverse effects • Inclusion of issues that are “near and dear” to a specific regulator or community but has no direct relationship to the effects of the project itself • Selection of valued components that do not allow for defensible quantification of effects or use of directly relevant significance thresholds • Selection of valued components that are only of indirect concern as opposed to focusing the assessment on the true concern. • Double counting of environmental effects • Risks in assessing cumulative effects This paper discusses where and how these risks occur, and provides examples from recent and current environmental assessments for pipelines and facilities in British Columbia. Opportunities to manage the scope of assessment while providing a fulsome, efficient, effective and scientifically/socially defensible assessment are discussed.

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