Utility of field-based artificial streams for assessing effluent effects on riverine ecosystems

Experimentation using field-based artificial streams provides a promising, complimentary approach to biomonitoring assessments because artificial streams provide control over relevant environmental variables and true replication of treatments. We have used large and small artificial stream systems, based in the field, to examine the effect of treated bleached kraft pulp mill effluent (BKME) on the benthos of three large rivers in western Canada. Under natural regimes of temperature, water chemistry, and insolation, these artificial streams provide current velocities and substrata to food chains or food webs that are representative of those in the study river. With these tools we have shown that BKME stimulated mayfly growth in the Thompson River above that which could be accounted for by fertilization of their algal food supply. In contrast, moulting frequency was inhibited at high BKME concentrations. Results from artificial streams also indicate that increased algal biomass and abundances of benthic communities downstream of BKME outfalls were induced by nutrient enrichment from the effluent. BKME treatments did not change diatom species richness in the Fraser River, or diatom species diversity in either the Athabasca or Fraser Rivers. Artificial streams provide a means of understanding the mechanisms of stressor effects over a continuum ranging from single stressor effects on specific taxa to the effects of multiple stressors on communities and ecosystems. Because riverside deployment provides environmental realism within a replicated experimental design, this approach can (i) address questions that cannot be examined using laboratory tests or field observations, (ii) improve our mechanistic understanding of stressor effects on riverine ecosystems, and (iii) can contribute directly to the development, parameterization, and testing of models for predicting ecosystem-level responses.     

Further characterization of nociception-related and arterial pressure-related neuronal responses in the nucleus reticularis gigantocellularis of the rat

The present study was undertaken to further characterize the nucleus reticularis gigantocellularis (NRGC) of the medulla oblongata in the central processing of nociceptive and cardiovascular signals, and its modulation by metenkephalin. In Sprague-Dawley rats anesthetized with pentobarbital sodium, we found that all 125 spontaneously active NRGC neurons that responded to noxious stimuli (tail clamp) also exhibited arterial pressure-relatedness. Forty neurons additionally manifested cardiac periodicity that persisted even during nociceptive responses. While maintaining their cardiovascular responsive characteristics, the nociception-related NRGC neuronal activity was blocked, naloxone-reversibly (0.5 mg/kg, i.v.), by morphine (5 mg/kg, i.v.). Microiontophoretically applied met-enkephalin suppressed the responsiveness of NRGC neurons to individually delivered tail clamp or transient hypertension induced by phenylephrine (5 µg/kg, i.v.). Interestingly, in NRGC neurons that manifested both nociception and arterial pressure relatedness, the preferential reduction in the response to noxious stimuli upon simultaneous elevation in systemic arterial pressure was reversed to one that favored nociception in the presence of met-enkephalin. All actions of met-enkephalin were discernibly blocked by the opioid receptor antagonist, naloxone. Our results suggest that individual NRGC neurons may participate in the processing of both nociceptive and cardiovascular information, or in the coordination of the necessary circulatory supports during nociception. In addition, neuropeptides such as met-enkephalin may exert differential modulation on neuronal responsiveness according to the prevailing physiologic status of the animal. They also showed that NRGC may be a central integrator for pain and cardiovascular-related functions.     

Modulation of the adhesion of hemopoietic progenitor cells to the rgd site of fibronectin by interleukin 3

The integrins are a class of adhesion molecules which have been implicated in the homing of hemopoietic stem cells and in their restriction within the bone marrow. Integrins function as mediators of cell-extracellular matrix (ECM) interactions and also of cell-cell interactions. They are unique membrane receptors which are capable of activation, change in affinity, and change in expression. Because of their broad potential for modulation we examined the effect of a cytokine growth factor which is present constitutively in the marrow, interleukin 3 (IL3), on integrin-mediated adherence of hemopoietic progenitor cells to the matrix component fibronectin (FN). The multipotential murine cell line B6Sut and the committed granulocyte progenitor cell line FDCP-1 were used. Both of these cell lines have been shown to bind to FN-coated dishes and to dishes coated with the 120 kDa and 40 kDa chymotryptic fragments of FN. It was found that after a brief withdrawal of IL3 the cells lost 80% adherence to the 120 kDa FN fragment containing the RGD cell binding site. This loss of binding was not related to a loss of viability, appeared unrelated to the growth/survival activity of IL3, and was quickly reversible by readdition of the growth factor. Adhesion of these cells to the RGD site was likely mediated by α5b?1 integrin which was identified in the cell membrane of both cell lines, but present in low copy number in B6Sut cells. Two antibodies against the external and internal domains of α5 and one antibody against b?1 were used to study expression of the integrin. By flow cytometry the expression of α5 was found to decrease in both cell lines by 4 h in the absence of IL3. The relative mean fluorescence intensity for B6Sut cells decreased from 1.0 (control cells always in the presence of IL3) to 0.6 over 4 h, and for FDCP-1 cells the decrement was from 1.0 to 0.8. The loss of RGD-mediated adhesion in the absence of IL3 appeared to proceed through this decrement in expression of the integrin; a loss of affinity of the receptor for its substrate was not detected. The general modulation of integrin activity by growth factors is of great interest because of its potential negative impact on the endothelium in cytokine-treated patients, and also because of its potential positive impact on engraftment during clinical bone marrow transplantation. © 1995 Wiley-Liss, Inc.     

Opioid peptides in the nervous system ofAplysia: A combined biochemical,immunocytochemical, and electrophysiological study

Summary 1. We have used biochemical, immunocytochemical, and electrophysiological techniques to evaluate the role of opioid peptides in the central nervous system of the marine mollusc,Aplysia california.2. Binding studies using³H-d-Ala², met-enkephalinamide (³H-DAMA) showed a single class of high-affinity binding sites with aK _d of 1.3 nM and a binding density of 45 pmol/g.3. HPLC extracts of ganglia revealed multiple peaks with immunoreactivity for either leu (LEU-IR)- or met-enkephalin (MET-IR), but the amounts were not uniformly distributed in all ganglia.4. LEU-IR and MET-IR neurons were demonstrated immunocytochemically in all ganglia, but MET-IR neurons were more frequent and were concentrated in pedal and pleural ganglia. While absorption control studies abolished MET-IR, LEU-IR was only partially abolished in the neuropil.5. In electrophysiological studies, both depolarizing and hyperpolarizing responses were found tod-Ala²-leu-enkephalin (DALEU) andd-Ala²-met enkephalin (DAMET) on some and different neurons.6. HPLC fractions from regions with retention times corresponding to authentic leu- or met-enkephalin showed physiologic responses similar to those of DALEU and DAMET, respectively.7. These studies suggest that a variety of endogeneous opioid peptides play physiologically important roles in the nervous system ofAplysia, including but not necessarily limited to leu- and met-enkephalin.     

The population dynamics of Metapenueus ensis (Crustacea: Decapoda: Penaeidae) in a traditional tidal shrimp pond at the Mai Po Marshes Nature Reserve, Hong Kong

The population dynamics of Metapenaeus ensis in a traditional tidal shrimp pond (locally known as gei wai ) at the Mai Po Marshes Nature Reserve, Hang Kong, were studied over a 29-month period. The population of M. ensis was dominated by either one or two age groups throughout the year. Life span was < 16 months. M. ensis is sexually dimorphic in terms of size, with females growing faster and attaining a larger maximum size than males. The sex ratio did not differ significantly from 1:1. The prawns do not mature and reproduce in the gei wai so that the population is sustained by postlarval immigration which begins in June and ends in December, with peaks in August and November. The gei wai population of M. ensis is characterized by slower growth, lower maximum attainable size, shorter life span, reproductive sterility and an incomplete life cycle, as compared with 'natural' population in the coastal waters of Hong Kong.     

Structure determination of N-linked oligosaccharides engineered at the CH1 domain of humanized LL2

Two humanized antibody mutants, hLL2HCN1 and hLL2HCN5, engineeredwith CH₁ domain-appended carbohydrates (CHOs) were generatedto facilitate site-specific conjugation of radionudides andanti-cancer drugs to antibodies. Such site-specific conjugationmay minimize the incidence of immunoreactlvity perturbationas is often observed with random conjugation. Since the compositionsand structures of CHOs are important in determining the chemistry,efficiency, and extent of conjugation, the sequences of theCH₁-appended CHOs were determined by exoglycosidase digestionsand fluorophore-assisted CHO electrophoresis (FACE). The CHOspecies attached at HCN1 and HCN5 sites in hLL2HCN1 and IJLL2HCN5,respectively, were distinct from each other, heterogeneous,and extensively processed. All of these CHOs were corefucosylatedcomplex-type oligosaccharides and contained Gal (galactose)and GlcNAc (N-acetylglucosamine) residues in the outer branches.Some of the outer branches were composed of Gal     

Angiotensin II Receptor Type I-Regulated Anion Secretion in Cystic Fibrosis Pancreatic Duct Cells

The β-adrenergic (cAMP-dependent) regulation of Cl⁻ conductance is defective in cystic fibrosis (CF). The present study explored alternative regulation of anion secretion in CF pancreatic ductal cells (CFPAC-1) by angiotensin II (AII) using the short-circuit current (I _SC ) technique. An increase in I _SC could be induced in CFPAC-1 cells by basolateral or apical application of AII in a concentration-dependent manner (EC₅₀ at 3 μm and 100 nm, respectively). Angiotensin receptor subtypes were identified using specific antagonists, losartan and PD123177, for AT₁ and AT₂ receptors, respectively. It was found that losartan (1 μm) could completely inhibit the AII-induced I _SC , whereas, PD123177 exerted insignificant effect on the I _SC , indicating predominant involvement of AT₁ receptors. The presence of AT₁ receptors in CFPAC-1 cells was also demonstrated by immunohistochemical studies using specific antibodies against AT₁ receptors. Confocal microscopic study demonstrated a rise in intracellular Ca²⁺ upon stimulation by AII indicating a role of intracellular Ca²⁺ in mediating the AII response. Depletion of intracellular but not extracellular pool of Ca²⁺ diminished the AII-induced I _SC . Treatment of the monolayers with a Cl⁻ channel blocker, DIDS, markedly reduced the I _SC , indicating that a large portion of the AII-activated I _SC was Cl⁻-dependent. AII-induced I _SC was also observed in monolayers whose basolateral membranes had been permeabilized by nystatin, suggesting that the I _SC was mediated by apical Cl⁻ channels. Our study indicates an AT₁-mediated Ca²⁺-dependent regulatory mechanism for anion secretion in CF pancreatic duct cells which may be important for the physiology and pathophysiology of the pancreas. Received: 17 June 1996/Revised: 14 November 1996     

Effectiveness of acoustic “prey”: Environmental enrichment for a captive African leopard (Panthera pardus)

In the course of developing active naturalistic exercise opportunities for zoo felines at moderate cost, a computer-controlled acoustic prey device was established. Changes in the behavior of a 16-year-old melanistic leopard (Sabrina) were studied as she learned to actively pursue bird sounds and obtained food treats as a function of the activity. By the twenty-ninth day she began to capture all 24 bird parts supplied on the feeder belt and continues to actively use the opportunity on a daily basis. General activity and apparent well-being have been enhanced, while stereotypic behaviors have decreased. © 1995 Wiley-Liss, Inc.     

Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase.

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5'-[beta gamma-imido]triphosphate or 20 mM-guanosine 5'-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP-binding site of the EGF-receptor kinase with apparent high affinity and that this analogue is an effective photoaffinity label for the kinase. Furthermore, these studies demonstrate that the EGF receptor, identified by using monoclonal antibodies, contains an ATP-binding site, providing further confirmation that the EGF receptor and EGF-dependent protein kinase are domains of the Mr 170000 protein.