Muscarinic receptors in the cerebellar vermis modulate the gain of the vestibulospinal reflexes in decerebrate cats.

1. The Purkinje (P)-cells of the cerebellar vermis, which exert a prominent influence on posture as well as on the gain of vestibulospinal (VS) reflexes, are under the control not only of the classic mossy fibers and climbing fibers which liberate excitatory amino acids as neurotransmitter, but also of cholinergic afferents. The role of these afferents was investigated in precollicular decerebrate cats by using the method of local microinjection of cholinergic agents into appropriate areas of the cerebellar cortex. 2. Unilateral injection into the vermal cortex of the culmen of the non-selective cholinergic agonist carbachol (0.25 microliters at 0.5 micrograms/microliters saline) produced a postural asymmetry, characterized by a slight decrease of the extensor tonus in the ipsilateral forelimb and an increased tonus in the contralateral forelimb. Moreover, the gain of the EMG responses of the ipsilateral and the contralateral triceps brachii to animal tilt increased significantly, while no significant changes in the phase angle of the responses were observed. These effects started 5-10 min after the injection and persisted for at least 2 hours before disappearing. Similar but smaller effects were obtained after injection of eserine, an inhibitor of acetylcholinesterase. Thus, the effects could be produced by increasing the naturally present amount of acetylcholine (ACh). 3. The changes in posture and gain of the VS reflexes described above utilized in part at least muscarinic receptors, since effects similar to those induced by carbachol injection were also obtained after unilateral microinjection into the vermal cortex of the culmen of the muscarinic agonist bethanechol (0.25 microliters at 0.1 micrograms/microliters). On the other hand opposite effects, characterized by an increased postural activity in the ipsilateral forelimb associated with a decreased activity in the contralateral forelimb, as well as by a reduced gain of the EMG responses of the triceps brachii of both sides to animal tilt were observed in other experiments after local microinjection of the muscarinic antagonist scopolamine (0.25 microliter at 4-8 micrograms/microliters saline). Evidence for muscarinic supersensitivity was obtained following repetitive injections of scopolamine into the cerebellar vermis. 4. The area which upon injection of the cholinergic agents modified the postural activity as well as the gain of the VS reflexes was located within the third and/or the fourth folium rostral to the fissura prima (culmen), at the laterality of 1.4-1.8 mm with respect to the midline.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of the topological changes induced on cells exposed to adhesive or mechanical stimuli

Fluorescent probes are widely used to study cell structure and function. However, few reports were devoted to a quantitative analysis of the intracellular distribution of fluorescent markers. In the present work, we describe the topographical changes of surface and cytoskeletal markers on individual cells subjected to adhesive or mechanical interaction. Conjugates were prepared with a cytotoxic T-lymphocyte clone and target cells. Specific antigens, membrane phospholipids, surface glycoconjugates, and polymerized actin were labeled with fluorescent antibodies or biochemical probes. The analysis of fluorescence distributions in conjugates demonstrated a selective reorganization of the plasma membrane with a gathering of some molecular species in the intercellular adhesion area. Furthermore, individual phagocytic cells were sucked into glass micropipets, then stained with fluorescent phallacidin to analyze the effect of mechanical efforts on the cytoskeleton organization. The concentration of polymerized actin was found to be similar in mechanicallyinduced protrusions and whole cells. It is concluded that adhesive interactions may result in marked cell polarization and formation of membrane zones with a particular biochemical composition. The submembranar cytoskeleton might play a role in this process.     

The effects of prolonged emersion and submersion by tidal manipulation on marine macrobenthos

Effects of tidal manipulation, resulting in prolonged periods of emersion and submersion or in protracted tidal cycles, on estuarine benthic animals are reviewed.Prolonged submersion periods did not show effects on mortality of most benthic animals tested, with the exception of the crumb-of-bread sponge Halichondrea panicea, which, at low water-flow rates, was covered with a layer of bacteria and subsequently died.Protracted low-water periods of 18 hours during several weeks hardly caused any mortality. However, protracted low-water periods of 30 hours during some weeks or emersion during several days caused a strong increase in mortality, depending on: the duration of emersion, temperature, condition of the animals, species and age. At temperatures below –1 °C and above 24 °C mortality was generally high. Animals with a low glycogen content were more sensitive to emersion than those with a high content. Species with a shell and those that are relatively big were less sensitive than those without a shell or of small size.The reproductive cycle of benthic animals could be delayed or accelerated by both emersion and submersion.     

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells

The mucin-type carbohydrate Tn cryptantigen (GalNAc1-O-Ser/Thr,where GalNAc is N-acetyl-D-galactosamine) is expressed in manycarcinomas, in haemopoietic disorders including the Tn syndrome,and on human immunodeficiency virus (HIV) coat glycoproteins,but is not expressed on normal, differentiated cells becauseof the expression of a Tn-processing galactosyltransferase.Using Jurkat T leukaemic cells which express high levels ofTn antigen due to deficient Tn galactosylation, we have establishedthe Tn antigen-mediated gene transfer and demonstrate the considerableefficiency of this approach. We used poly(L-lysine) conjugatesof the monoclonal antibody 1E3 directed against the Tn antigento deliver the luciferase and ß-galactosidase reportergenes to Jurkat cells by receptor-mediated endocytosis. Additionof unconjugated 1E3 reduced transfection efficiency in a concentration-dependentmanner and incubation with free GalNAc abolished DNA transfercompletely, indicating that gene delivery is indeed mediatedby the Tn antigen. Pre-treatment of Jurkat cells with Vibriocholerae sialidase, which uncovers additional Tn antigens, resultedin an improvement of gene transfection. Both human and chickenadenovirus particles attached to the DNA/polylysine complexstrongly augmented transgene expression. When the ß-galactosidase(lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis,up to 60% of the cells were positive in the cytochemical stainusing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside(X-gal) as a chromogenic substrate. The efficiency of the transferrinreceptor-mediated DNA uptake into Jurkat cells was comparativelylow, although these cells were shown to express considerableamounts of transferrin receptor. We show here that a mucin-typecarbohydrate antigen mediates highly efficient DNA uptake byendocytosis into Jurkat T cells. This method represents a 50-foldimprovement of Jurkat cell transfection efficiency over otherphysical gene transfer techniques. Specific gene delivery toprimary cancer cells exhibiting Tn epitopes may especially bedesirable in immunotherapy protocols. adenovirus endocytosis gene transfer T cell Tn antigen     

Contrasting effects of PACAP and carbachol on [Ca]i and inositol phosphates in human neuroblastoma NB-OK-1 cells

The effects of PACAPs on [Ca²⁺]_i were compared to those of carbachol in human neuroblastoma NB-OK-1 cells. PACAP(1–27) and PACAP(1–38) increased [Ca²⁺]_i in a biphasic manner: a transient rise and a secondary plateau. The transient phase reflected the mobilization of [Ca²⁺]_i pool(s) via the inositol phosphate pathway. The modest sustained plateau required extracellular Ca²⁺. Carbachol also increased [Ca²⁺]_i in a biphasic manner, but it mobilized intracellular Ca²⁺ pool(s) with a higher efficacy than PACAPs, then greatly increased Ca²⁺ entry, this being accompanied by a more marked and prolonged elevation of IP₃ and IP₄ than with PACAPs. It is likely that cAMP-mediated phosphorylations due to PACAPs facilitated desensitization at the PACAP receptor-phospholipase C level, so that there was less Ca²⁺ handling through PACAP receptors than with muscarinic M₁ receptors.     

INFLUENCE OF NATURAL ULTRAVIOLET RADIATION ON LOTIC PERIPHYTIC DIATOM COMMUNITY GROWTH,BIOMASS ACCRUAL,AND SPECIES COMPOSITION: SHORT-TERM VERSUS LONG-TERM EFFECTS1, 2

Growth rates, accumulation dynamics, and species succession of periphytic diatom communities were examined in the presence and absence of natural ultraviolet (UV) radiation using a series of outdoor, continuous-flow experimental flumes located on the South Thompson River, British Columbia. In a short-term experiment (2–3 wk), log-phase growth rates of naturally seeded diatom communities comprised of Tabellaria fenestrata (Lyngb.) Kütz., T. flocculosa (Roth) Kütz., Fragilaria crotonesis Kitton, and F. vaucheriae (Ehr.) Peter. exposed to 90% ambient photosynthetically active radiation (PAR) + UV were 30–40% lower than growth rates under 90% PAR alone. UV inhibition of growth rate was independent of the degree of P limitation within the range of relative specific growth rates (μ:μ_max-P) of 0.5–1.0. In a long-term trial, inhibition of attached diatom accumulation under 90% PAR + UV during the first 2–3 wk was corroborated. Reduction of full sunlight to 50% PAR + UV prevented the initial inhibition phase. The initial inihibitory effect of 90% PAR + UV on algal accumulation was reversed after 3–4 wk, and by 5 wk total diatom abundance (chlorophyll a, cell numbers and cell biovolumes) in communities exposed to PAR + UV were 2–4-old greater than in communities protected from UV. Under 90% PAR + UV and 50% PAR + UV, a succession to stalked diatom genera (Cymbella and Gomphoneis) occurred. Species succession under UV radiation doubled the mean cell size of the diatom communities. The shift from inhibition to a long-term increase in the autotrophic community under PAR + UV compared to PAR alone provides further evidence against the use of short-term incubation experiments to define the long-term implications of increases in UVB. These results suggest that the ecological effects of present-day levels of UVB and UVB:UVA ratios on autotrophic communities are not well understood and might be mediated through complex trophic level interactions.     

The effect of light levels on daily patterns of chlorophyll fluorescence and organic acid accumulation in the tropical CAM treeClusia hilariana

Sandy plains are characteristic of the coastal region of Brazil. We investigated the diel patterns of changes in organic acid levels, leaf conductance and chlorophylla fluorescence for sun-exposed and shaded plants ofClusia hilariana, one of the dominant woody species in the sandy coastal plains of northern Rio de Janeiro state. Both exposed and shaded plants showed a typical CAM pattern with considerable diel oscillations in organic acid levels. The degradation of both malic and citric acids during the midday stomatal closure period could lead to potential CO₂ fixation rates of 28 mol m^-2 s^-1 in exposed leaves. Moreover, exposed leaves exhibited large increases in total non-photochemical quenching (q_N) accompanied by a substantial decrease in effective quantum yield during the course of the day. However, these potential high rates of CO₂ fixation and the increases inqn of exposed plants were not enough to maintain the primary electron acceptor of photosystem II (q_A) in a low reduction state, similar to that of shaded plants. As a result, there was a moderate increase in the reduction state of q_A throughout the day. Most of the decline in photochemical efficiency of exposed leaves ofC. hilariana was reversible, as evidenced by the high levels of pre-dawn potential quantum yields (F_v/F_m) and their rapid recovery after sunset. However, the depletion of the organic acid pool in the afternoon resulted in an accentuated subsequent drop in F_v/F_m, suggesting that prolonged periods of water stress accompanied by high irradiance levels may expose plants ofC. hilariana in unprotected habitats to the danger of photoinhibition.     

Modeling of adaptations to physical training by using a recursive least squares algorithm

Busso, Thierry, Christian Denis, Régis Bonnefoy,André Geyssant, and Jean-René Lacour. Modeling ofadaptations to physical training by using a recursive least squaresalgorithm. J. Appl. Physiol. 82(5):1685-1693, 1997.The present study assesses the usefulnessof a systems model with time-varying parameters for describing theresponses of physical performance to training. Data for two subjectswho undertook a 14-wk training on a cycle ergometer were used to testthe proposed model, and the results were compared with a model withtime-invariant parameters. Two 4-wk periods of intensive training wereseparated by a 2-wk period of reduced training and followed by a 4-wkperiod of reduced training. The systems input ascribed to the trainingdoses was made up of interval exercises and computed in arbitraryunits. The systems output was evaluated one to five times per week byusing the endurance time at a constant workload. The time-invariantparameters were fitted from actual performances by using the leastsquares method. The time-varying parameters were fitted by using arecursive least squares algorithm. The coefficients of determinationr² were 0.875 and0.879 for the two subjects using the time-varying model, higher thanthe values of 0.682 and 0.666, respectively, obtained with thetime-invariant model. The variations over time in the model parametersresulting from the expected reduction in the residuals appearedgenerally to account for changes in responses to training. Such a modelwould be useful for investigating the underlying mechanisms ofadaptation and fatigue.

     

Studies on kidney sialidase in normal and diabetic rats

Rat kidney cortex sialidase was studied using alpha-sialyl-(2----3)-[3H]lactitol and alpha-sialyl-(2----6)-[3H]lactitol as substrates. The enzyme was found mainly in the lysosomal fraction. Only 23% of the sialidase activity of this fraction could be solubilized by a combination of freezing-thawing, sonication and Triton X-100 treatment. The optimal pH for the lysosomal enzyme activity was 4.2 and the enzyme's Km values for alpha-sialyl-(2----3)-lactitol and alpha-sialyl-(2----6)-lactitol were 0.28 and 0.41 mM, respectively. The specific activity was twice as high with the former substrate than with the latter. Sialidase activities in dialyzed kidney cortex homogenates of streptozotocin-diabetic rats and of age-matched control rats were compared. The specific activity was found to be significantly increased in the diabetic animals when using both substrates 5950 +/- 720 (S.E.) dpm/h per mg protein (n = 7) vs. 3970 +/- 370 in the controls (n = 8) with alpha-sialyl-(2----3)-lactitol (P less than 0.025) and 2870 +/- 300 vs. 1820 +/- 170 with alpha-sialyl-(2----6)-lactitol (P less than 0.02). The activities were also found to be increased when expressed per whole kidney cortex (P less than 0.005 and P less than 0.001, respectively). The elevated sialidase activity in diabetic kidney cortex may be related to the reported decrease in sialic acid content of the glomerular basement membrane, which lowers its negative charges and which may contribute to an increased permeability to proteins.     

Synthesis of subunit III of CF0 by thylakoid-bound polysomes from pea chloroplasts

Summary Wahsed thylakoid membranes from pea chloroplasts incorporate label from (³⁵S)-methionine into protein when supplemented with S-30 soluble factors from E. coli. One of the products associated with the thylakoids is soluble in butanol, precipitated by ether and has an apparent molecular mss of 8200D on urea-lithium dodecyl sulphate (LDS) polyacrylamide gels. In addition, the protein covalently binds dicyclohexylcarbo-diimide (DCCD) which causes it to migrate as two slower forms on gels. Based on these criteria we establish that the proteolipid or subunit III of CF₀ (the intrinsic sector of the ATPase complex) is synthesized by the thylakoid bound polysomes.