Amino acid transport : the special case of a H/L-glutamate cotransport system in Asparagus sprengeri mesophyll cells /

The addition of L-Glutamate (L-GLU) and L-Hethionine ~ulfoximine (L-HSO) to mechanically isolated. photosynthetically competent, Asparagus sprengeri mesophyll cells ~u~pended in 1mM CaS04 cau~ed an immediate transient alkalinization of the cell su~pension medium in both the light and dark. The alkalinization response was specific and stereospecific as none of the L-isomers of the other 19 protein amino acids tested or D-GLU gave this response. Uptake of 14C-L-GLU was stimulated by the light. The addition of non-radioactive L-GLU. or L-GLU analogs together with 14C-L-GLU showed that only L-GLU and L-HSO stimulated alkalinization whilst inhibiting the uptake of 14C-L-GLU. Both the L-GLU dependent alkalinization and the upt~ke of 14C-L-GLU were stimulated when the external pH was decreased from 6.5 to 5.5. Increasing external K+ concentrations inhibited the uptake of 14C-L-GLU. Fusicoccin (FC) stimulated uptake. The L-GLU dependent alkalinization re~ponse exhibited monophasic saturation kinetics while the uptake of 14C-L-GLU exhibited biphasic saturation kinetics. In addition to a saturable component. the uptake kinetics also showed a linear component of uptake. Addition of L-GLU and L-MSO caused internal acidification of the cell as measured by a change in the distribution of 14C-DMO. There was no change in K+ efflux when L-GLU was added. A H+ to L-GLUinflux stoichiometry of 3:1 wa~ mea~ured at an external I.-GLU concentration of O.5mM and increased with increasing external 13 L-QLU concentration. Metabolism of L-GLU was detected manometrlcally by observing an increase in COa evolution upon the addition of L-QLU and by detection of i*C02 evolution upon the addition of »*C-L-GLU. »*C02 evolution was higher in the dark than in the light. The data are consistent with the operation of a H+/L-QLO cotransport system. The data also show that attempts to quantify the stoichlometry of the process were complicated by the metabolism of L-GLU.

Host-parasite relations in a mycoparasitic system : alterations in membrane permeability and lipid composition in Choanephora cucurbitarum infected by Piptocephalis virginiana /

examined in Choanephora cucurbita rum during the early stages of infection by Piptocephalis virginiana » There was a small but consistent increase in the leakage of electrolytes, amino acids and sugars as a result of infection. These low levels of differential leakage in infected tissues are explained on the basis of the nature of this obligate, biotrophic, mycoparasitic system. Quantitative analysis of the twenty six amino acids and amino compounds detected in the leacheates — showed similar profiles in infected and control host and no new species of amino acids or amino compounds were detected in either infected or control host leacheates. Comparatively high amounts of aspartic acid, glutamic acid and alanine were found in the leacheates of host and infected host . Analyses of the sugars comprising the leacheates of infected and control host showed the presence of eight sugars, among which glucose was found in significant amounts (50-53%) ' The nutritional implication of this preferential leakage is discussed. No significant difference was observed in the leacheates of infected host sugar profiles compared with that of the control host. Profiles of the internal pool sugars of infected and control host did not reflect that obtained from the leacheate data, perhaps owing to leakage of sugars in a selective manner . Membrane lipid analyses yielded higher levels of lipid in infected host compared with the control, both at the 24 h and 36 h analyses. In addition, preliminary investigations of phosphorous-32 incorporation and turnover in phospholipids showed higher levels of 32p incorporation and turnover in infected host compared with the control. No apparent difference was noted in the profiles of the neutral lipid classes and the polar lipid classes of the membrane lipids as determined by one and two dimensional thin-layer chromatography respectively. However, a small but consistently higher degree of unsaturation was detected in the fatty acids of infected tissue compared with the control. Also, '^''-^^''^^'-'-^'^^c acid, a polyunsaturated fatty acid previously reported to show a direct correlation during the early stages of infection and the degree of parasitism of P. virginiana on C. cucurbitarum , was found in higher amounts in infected host membrane lipids compared with that of the control host. The implications of these membrane lipid alterations are discussed with particular reference to the small but consistently higher leakage of electrolytes, amino acids and sugars observed during infection in this study.

Arsenic speciation by ion-exchange chromatography with on- line determination by hydride generation: electronic modification of the D.C. plasma by photon counting

A method using L-cysteine for the determination of arsenous acid (As(III)), arsenic acid (As(V)), monomethylarsonic acid (MMAA), and dimethylarsinic acid (DMAA) by hydride generation was demonstrated. The instrument used was a d.c. plasma atomic emission spectrometer (OCP-AES). Complete recovery was reported for As(III), As(V), and DMAA while 86% recovery was reported for MMAA. Detection limits were determined, as arsenic for the species listed previously, to be 1.2, 0.8, 1.1, and 1.0 ngemL-l, respectively. Precision values, at 50 ngemL-1 arsenic concentration, were f.80/0, 2.50/0, 2.6% and 2.6% relative standard deviation, respectively. The L-cysteine reagent was compared directly with the conventional hydride generation technique which uses a potassium iodide-hydrochloric acid medium. Recoveries using L-cysteine when compared with the conventional method provided the following results: similar recoveries were obtained for As(III), slightly better recoveries were obtained for As(V) and MMAA, and significantly better recoveries for DMAA. In addition, tall and sharp peak shapes were observed for all four species when using L-cysteine. The arsenic speciation method involved separation by ion exchange .. high perfonnance liquid chromatography (HPLC) with on-line hydride generation using the L.. cysteine reagent and measurement byOCP-AES. Total analysis time per sample was 12 min while the time between the start of subsequent runs was approximately 20 min. A binary . gradient elution program, which incorporated the following two eluents: 0.01 and 0.5 mM tri.. sodium citrate both containing 5% methanol (v/v) and both at a pH of approximately 9, was used during the separation by HPLC. Recoveries of the four species which were measured as peak area, and were normalized against As(III), were 880/0, 290/0, and 40% for DMAA, MMAA and As(V), respectively. Resolution factors between adjacent analyte peaks of As(III) and DMAA was 1.1; DMAA and MMAA was 1.3; and MMAA and As(V) was 8.6. During the arsenic speciation study, signals from the d.c. plasma optical system were measured using a new photon-signal integrating device. The_new photon integrator developed and built in this laboratory was based on a previously published design which was further modified to reflect current available hardware. This photon integrator was interfaced to a personal computer through an AID convertor. The .photon integrator has adjustable threshold settings and an adjustable post-gain device.

Developmental variations in the peripheral erythrocytic system of the rainbow trout, Salmo gairdneri

The peripheral circulating erythrocytic system of the rainbow trout, l3 almo gairdner , was examined in vitro in relation differences in the morphology and multiple hemoglobin system organization of adult and juvenile red cells. Cells were separated by velocity sedimentation under unit gravity, a procedure requiring red cell exposure to an incubation medium for periods of at least three hours. Therefore , this must provide an environment in which red cells remain in a condition approximaing normalcy. Previous studies having demonstrated commonly employed media to be ineffective in this regard , a medium was developed through modification of Cortl and saline. One of the principal additions to this me dium , norepinephrine, altered cell regulation of intracellular calcium, magnesium and chloride concentrations. Catecholamine involvement was also suggeste d in the synthes is of hemoglobin . The procedure was found to separtate cells primarily by density and, to a lesser extent, by shape. Characterization of red cells revealed two subpopulations to exist . The first comprised the bulk of the cell population, and were of greater l ength, width, volume and major:minor axis ratio than the smaller population; these were adult cells. The later, juvenile cells were of smaller overall size and were more spherical in shape . Juvenile cells also possessed fewer electrophore tpically distinguishable isomorphs than did adults with only eight of eleven hemoglobin component s typically found With maturation,hemoglobin complement with the development of three more bands. The total complement of the adult cell contained 7 cathodal bands and four anodal hemoglobin isomorphs. Bands acquired with maturation comprised the smallest percentage of the cells hemoglobin. each averaging less than one-percent of the total. Whether these additional bands are derived through degradation and reaggregation of existing components or are the product of pe gQy2 synthesis is not yet known.

Implementation of hospital-wide computer information system :a case study of this change process

This case study examines the impact of a computer information system as it was being implemented in one Ontario hospital. The attitudes of a cross section of the hospital staff acted as a barometer to measure their perceptions of the implementation process. With The Mississauga Hospital in the early stages of an extensive computer implementation project, the opportunity existed to identify staff attitudes about the computer system, overall knowledge and compare the findings with the literature. The goal of the study was to develop a greater base about the affective domain in the relationship between people and the computer system. Eight exploratory questions shaped the focus of the investigation. Data were collected from three sources: a survey questionnaire, focused interviews, and internal hospital documents. Both quantitative and qualitative data were analyzed. Instrumentation in the study consisted of a survey distributed at two points in time to randomly selected hospital employees who represented all staff levels.Other sources of data included hospital documents, and twenty-five focused interviews with staff who replied to both surveys. Leavitt's socio-technical system, with its four subsystems: task, structure, technology, and people was used to classify staff responses to the research questions. The study findings revealed that the majority of respondents felt positive about using the computer as part of their jobs. No apparent correlations were found between sex, age, or staff group and feelings about using the computer. Differences in attitudes, and attitude changes were found in potential relationship to the element of time. Another difference was found in staff group and perception of being involved in the decision making process. These findings and other evidence about the role of change agents in this change process help to emphasize that planning change is one thing, managing the transition is another.

Investigation of the composition of linear alkylbenzenes with emphasis on the identification and quantitation of some trace compounds using GS/MS system in both electron impact and chemical ionization modes

Linear alkylbenzenes, LAB, formed by the Alel3 or HF catalyzed alkylation of benzene are common raw materials for surfactant manufacture. Normally they are sulphonated using S03 or oleum to give the corresponding linear alkylbenzene sulphonates In >95 % yield. As concern has grown about the environmental impact of surfactants,' questions have been raised about the trace levels of unreacted raw materials, linear alkylbenzenes and minor impurities present in them. With the advent of modem analytical instruments and techniques, namely GCIMS, the opportunity has arisen to identify the exact nature of these impurities and to determine the actual levels of them present in the commercial linear ,alkylbenzenes. The object of the proposed study was to separate, identify and quantify major and minor components (1-10%) in commercial linear alkylbenzenes. The focus of this study was on the structure elucidation and determination of impurities and on the qualitative determination of them in all analyzed linear alkylbenzene samples. A gas chromatography/mass spectrometry, (GCIMS) study was performed o~ five samples from the same manufacturer (different production dates) and then it was followed by the analyses of ten commercial linear alkylbenzenes from four different suppliers. All the major components, namely linear alkylbenzene isomers, followed the same elution pattern with the 2-phenyl isomer eluting last. The individual isomers were identified by interpretation of their electron impact and chemical ionization mass spectra. The percent isomer distribution was found to be different from sample to sample. Average molecular weights were calculated using two methods, GC and GCIMS, and compared with the results reported on the Certificate of Analyses (C.O.A.) provided by the manufacturers of commercial linear alkylbenzenes. The GC results in most cases agreed with the reported values, whereas GC/MS results were significantly lower, between 0.41 and 3.29 amu. The minor components, impurities such as branched alkylbenzenes and dialkyltetralins eluted according to their molecular weights. Their fragmentation patterns were studied using electron impact ionization mode and their molecular weight ions confirmed by a 'soft ionization technique', chemical ionization. The level of impurities present i~ the analyzed commercial linear alkylbenzenes was expressed as the percent of the total sample weight, as well as, in mg/g. The percent of impurities was observed to vary between 4.5 % and 16.8 % with the highest being in sample "I". Quantitation (mg/g) of impurities such as branched alkylbenzenes and dialkyltetralins was done using cis/trans-l,4,6,7-tetramethyltetralin as an internal standard. Samples were analyzed using .GC/MS system operating under full scan and single ion monitoring data acquisition modes. The latter data acquisition mode, which offers higher sensitivity, was used to analyze all samples under investigation for presence of linear dialkyltetralins. Dialkyltetralins were reported quantitatively, whereas branched alkylbenzenes were reported semi-qualitatively. The GC/MS method that was developed during the course of this study allowed identification of some other trace impurities present in commercial LABs. Compounds such as non-linear dialkyltetralins, dialkylindanes, diphenylalkanes and alkylnaphthalenes were identified but their detailed structure elucidation and the quantitation was beyond the scope of this study. However, further investigation of these compounds will be the subject of a future study.

Magnetic and high pressure studies in the YPd5B3C3 system

The macroscopic properties of the superconducting phase in the multiphase compound YPd5B3 C.3 have been investigated. The onset of superconductivity was observed at 22.6 K, zero resistance at 21.2 K, the lower critical field Hel at 5 K was determined to be Hel (5) rv 310 Gauss and the compound was found to be an extreme type-II superconductor with the upper critical field in excess of 55000 Gauss at 15 K. From the upper and lower critical field values obtained, several important parameters of the superconducting state were determined at T = 15 K. The Ginzburg-Landau paramater was determined to be ~ > 9 corresponding to a coherence length ~ rv 80A and magnetic penetration depth of 800A. In addition measurements of the superconducting transition temperature Te(P) under purely hydrostatically applied pressure have been carried out. Te(P) of YPd5B3 C.3 decreases linearly with dTe/dP rv -8.814 X 10-5 J

Response rainbow trout (Salmon gairdneri) blood gas transport system to temperature, oxygen availability and photoperiod

Hematological status in rainbow trout, Salmo gairdneri, was examined in relation to eight combinations of three environmental fa ctors; temperature (5°, 20°C), oxygen availability «35%, >70% saturation) and photoperiod (16L:8D, 8L:16D) and evaluated by 3-factor analysis of variance. Hemog l obin and hematocrit , indicators of oxygenc arrying capacity increased significantly at the higher temperature, following exposure to hypoxia and in relation to reduced light period. Significant variations in mean corpuscular hemoglobin concentration were not detected. The effects of temperature and oxygen availability were more pronounced than that of photoperiod which was generally masked. Although oxygen availability and photoperiod did not interact with temperature, the interaction of the former fac tors was significant. Elec trophoresis revealed twelve hemoglobin isomorphs. Relative concentration changes were found in re lation to the factors c onsidered with temperature>hypoxia>photoperiod. Howeve r , in terms of absolute concentration, effects were hypoxia>temperature>photoperiod. Photoperiod effects were again masked by temperature and (or) hypoxia. Red cell +2 l eve ls of [CI ] and [Mg ], critical elements in the hemoglobin-oxygen affinity regulating system, were also significantly altered. Red cell CI +2 was influenced only by temperature ; Mg by temper ature and oxygen. No photoperiod influence on either ions was observed. Under nominal 'summer' conditions, these changes point to the likelihood of increases in oxygen-c arrying c apac ity coupled with low Hb-02 affinity adjustments which would be expected to increase oxygen delivery rates to their more rapidly metabolising tissues.

Study of the application of the high performance liquid chromatography-particle beam interface-mass spectrometry technique on some pesiticides and the reduction phenomena in the system

This work includes two major parts. The first part of the work concentrated on the studies of the application of the highperfonnance liquid chromatography-particle beam interface-mass spectrometry system of some pesticides. Factors that have effects on the detection sensitivity were studied. The linearity ranges and detection limits of ten pesticides are also given in this work. The second part of the work concentrated on the studies of the reduction phenomena of nitro compounds in the HPLC-PB-MS system. Direct probe mass spectrometry and gas chromatography-mass spectrometry techniques were also used in the work. Factors that have effects on the reduction of the nitro compounds were studied, and the possible explanation is proposed. The final part of this work included the studies of reduction behavior of some other compounds in the HPLC-PB-MS system, included in them are: quinones, sulfoxides, and sulfones.

The pancreatic islet system of the rock bass /|nby Edward Francis Squires. -- 260 St. Catharines, Ont. : [s. n.],

The endocrine pancreas of the rock bass (Ambloplites rupestris) was examined by light and electron microscopy. Two cell types with staining properties similar to mammalian A and B cells, and a third, non-staining cell type were found in the spherical pancreatic islets that were surrounded by a connective tissue capsule and embedded in two small masses of exocrine tissue. From an analysis of the ultrastructure of the A and B cells, a secretory cycle for each of these cell types was proposed. The secretory cycle of the A cell consisted of three well defined stages: (1) A cell production stage: during which A granule formation occurred in the sacs of the Golgi apparatus and the cell was characterized by the presence of numerous secretory granules, some elements of lamellar endoplasmic reticulum, and a homogeneously granular nucleus. The cytoplasm contained few distended cisternae, variable numbers of free ribosomes, microtubules and small vesicles. (2) A cell release stage: during which the release of A granules occurred and the cell usually contained several large distended cisternae and variable numbers of secretory granules. Granule release mechanisms included exocytosis, by which individual granules were released into the extracellular space after their membranes fused with the plasmalemma, and emiocytosis, by which one or more granules were released into a large cisterna whose membrane fused with the plasmalemma and formed a pore through which the cisternal contents passed out of the cell. (3) A cell reorganization stage: during which the changeover from the release stage to the production stage occurred and the reorganization of organelles and membrane structures took place. The cell contained few secretory granules and numerous small endoplasmic reticular cisternae. The cytoplasm exhibited less electron density than either of the other two stages. The A granule after formation underwent a series of morphological changes which were described in four numerically identified phases. The secretory cycle of the B cell consisred of two stages: (1) B cell production stage: during which the B granule formation occurred in the sacs of the Go1gi apparatus. The cell was characterized by an irregular outline, the presence of numerous secretory granules, and an irregularly shaped nucleus which contained variable amounts of clumped chromatin. The cytoplasm contained moderate amounts of lamellar endoplasmic reticulum studded with ribosomes, several small vesicles, and an active Go1gi apparatus. (2) B cell release stage: during which the release of B granules occurred. The cell contained a rounded nucleus with dispersed chromatin, several distended endoplasmic reticular cisternae and a variable number of secretory granules. Granule release occu~ by emiocytosis and exocytosis similar to that found for the A cell.

Understanding the portfolio in the secondary school classroom

Four secondary school teachers were involved in this case study. Individual interviews, group reflective sessions, and participant portfolios were transcribed verbatim and analyzed. The use of the portfolio in the secondary school classroom was then discussed in relation to emergent themes. These themes included teacher attitude, portfolio structure, portfolio purpose, challenges, effect, and professional development. Teachers were able to individualize the portfolio structure to meet both program and students' needs. The portfolio structure enabled both teachers and students to assume control over the learning process. The portfolio informed teachers about their teaching. This, in tum, challenged them to reflect on their teaching practices and enabled them to redesign curriculum implementation. A collaborative professional development structure fostered a learning environment that enabled teachers to experience success, despite the challenges that they inevitably encountered. These findings were related to contemporary literature. Finally, implications for theory and practice related to portfolio use in the secondary school classroom and professional development for secondary school teachers were considered.

The role of the dopaminergic system in the production of ultrasonic calls in adult rats /

Ultrasonic vocalizations (USV) are emitted by rats in a number of social situations such as aggressive encounters, during sexual behavior, and during play in young rats, situations which are predominantly associated with strong emotional responses. These USV typically involve two distinct types of calls: 22 kHz calls, which are emitted in aversive situations and 50 kHz calls, which are emitted in non-aversive, appetitive situation. The 50 kHz calls are the focus of the present study and to date both the glutamatergic and the dopaminergic systems have been independently implicated in the production of these 50 kHz calls. The present study was conducted to examine a possible relationship between glutamate (GLU) and dopamine (DA) in mediating 50 kHz calls. It was hypothesized that the dopaminergic system plays a mediating role in 50 kHz calls induced by injections ofGLU into the anterior hypothalamic/preoptic area (AHPOA) in adult rats. A total of 68 adult male rats were used in this study. Rats' USV were recorded and analyzed in five experiments that were designed to test the hypothesis: in experiment 1, rats were treated with systemic amphetamine (AMPH) alone; in experiment 2, intra- AHPOA GLU was pretreated with systemic AMPH; in experiment 3, intra-AHPOA GLU was pretreated with intra-AHPOA AMPH; in experiment 4, rats were treated with high and low doses of intra-AHPOA AMPH only; in experiment 5, rats were treated with systemic haloperidol (HAL) as a pretreatment for intra-AHPOA GLU. Analysis of the results indicated that AMPH has a facilitatory effect on 50 kHz USV and that a relationship between DA and GLU in inducing 50 kHz calls does exist. The effect, however, was only observed when DA receptors were antagonized with HAL and was not seen with systemic AMPH pretreatments of intra-AHPOA GLU. The DAGLU relationship at the AHPOA was unclear.

The role of the GABAergic system in the production of ultrasonic vocalization in rats /

Ultrasonic vocalization plays an important role in intraspecies communication for rats. It has been well demonstrated that rats will emit 22kHz vocalization in stressfiil or threatening situations. Although the neural mechanism underlying vocahzation is not well understood, it is known that chohnergic input to the basal forebrain induces such alarm calls. A number of experiments have found that intracerebral injection of carbachol, a predominantly muscarinic agonist, into die anterior hypothalamic/preoptic area (AH/POA) rehably induces vocalization similar to naturally emitted ultrasonic calls. It has also been shown that carbachol has extensive inhibitory effects on neuronal firing in the same area. This result impUes that the inhibitory effects of carbachol in the AH/POA could trigger vocahzation, and that the GABAergic system could be involved. The purpose of this study is to investigate the effects ofGABA agonists and antagonists on flie production of carbachol induced 22kHz vocalization. The following hypotheses were examined: 1) apphcation ofGABA (a naturally occurring inhibitory neurotransmitter) will have a synergistic effect with carbachol, increasing vocalization; and 2) tiie apphcation ofGABA antagonists (picrotoxin or bicuculline) will reduce caibachol-induced vocalization. A total of sixty rats were implanted with stainless steel guide cannulae in the AH/POA area. After recovery, animals were locally pretreated with 1) GABA (l-40ng), 2) picrotoxin (1 .5^g) or bicuculhne (0.03ng), or 3) sahne; before injection with carbachol (1 .5^g). The resulting vocalization was measured and quantitated. The results indicate that pretreatment with GABA or GABA antagonists had no significant effect on vocalization. Local pretreatment with GABA did not potentiate the vocal response as measured by its duration, latraicy, and total number of calls. Similarly, pretreatment with picrotoxin or bicuculline had no effects on the same measures of vocalization. The results suggest tfiat chohnoceptive neurons involved in the production of alarm calls are not under direct GABAergic control.

Hydrogen sulphide as inhibitor and substrates for the cytochrome c-cytochrome c oxidase system /

It has previously been recognized that the major biochemical toxicity induced by sulphide is due to an inhibition of cytochrome ~ oxidase. Inhibition of this enzyme occurs at 30°C and pH 7.4 with a Ki of approximately 0.2 ~M, and a kon of 104 M-1 s-l, under catalytic conditions. However, the equimo1ar mixture of sulphide and the enzyme shows identical catalytic behaviour to that of the native enzyme. This cannot readily be attributed to rapid dissociation of sulphide, as both spectroscopic and plot analysis indicate the koff value is low. The addition of stoichiometric sulphide to the resting oxidized enzyme gives rise to the appearance of a low-spin ferric-type spectrum not identical with that seen on the addition of excess sulphide to the enzyme aerobically. Sulphide added to the enzyme anaerobically gives rise to another low-spin, probably largely ferric, form which upon admission of oxygen is then converted into a 607 nm species closely resembling Compound C. The 607 nm form is probably the precursor of oxyferricytochrome aa3. The addition of successive a1iquots of Na2S solution to the enzyme induces initial uptake of approximately 3 moles of oxygen per mole of the enzyme. Thus, it is concluded that: 1. the initial product of sulphide-cytochrome c oxidase interaction is not an inhibited form of the enzyme, but the low-spin (oxyferri) ~3+~+ species; 2. a subsequent step in which sulphide reduces cytochrome ~ occurs; 3. the final inhibitory step, in which a further molecule of sulphide binds to the cytochrome ~ iron centre in the cytochrome ~2+~+ species, gives the cytochrome a2+~+-H2S form which is a half-reduced fully inhibited species;4. a 607 run form of the enzyme is produced which may be converted into a catalytically active low-spin (oxyferri) state; and therefore 5. liganded sulphide may be able to reduce the cytochrome 33 -Cu centre without securing the prior reduction of the cytochrome a_ haem group or the Cud centre associated with it.