Nicotinic receptor-elicited sodium flux in rat pheochromocytoma PC12 cells: Effects of agonists,antagonists, and noncompetitive blockers

Nicotinic agonists stimulate²²Na flux in rat pheochromocytoma PC12 cells. The stimulatory effect of carbamylcholine is maximal at 1 mM, while the stimulatory effect of nicotine and anatoxin maximize at the same level at 100 M and 10 M, respectively. The tertiary amines arecolone and isoarecolone have no effect on flux at 100 M, while the methiodides at 100 M stimulate flux to an extent similar to 1 mM carbamylcholine. Dihydro and alcohol analogues of isoarecolone methiodide have markedly smaller effects on flux. A preincubation for 2 to 20 min with carbamylcholine (2 mM), nicotine (300 M), anatoxin (30 M) or isoarecolone methiodide (100 M) causes marked desensitization to a subsequent carbamylcholine-elicited stimulation of flux. d-Tubocurarine, mecamylamine, hexamethonium, and chlorisondamine inhibit carbamylcholine-elicited flux with IC₅₀ values of 1.0, 0.8, 43, and 0.020 M, respectively. Atropine has no effect at 1 M, but reduces the response to carbamylcholine by 50% at 8.6 M, presumably as a noncompetitive blocker. Other noncompetitive blockers of nicotinic acetylcholine-receptors, such as histrionicotoxins, gephyrotoxin, pumiliotoxin C, phencyclidine, bupivacaine and piperocaine, inhibit carbamylcholine-elicited stimulation of²²Na flux with IC₅₀ values from 0.3 to 1.8 M. In contrast to d-tubocurarine, which inhibits carbamylcholine-elicited desensitization, and mecamylamine, which has no apparent effect on desensitization, chlorisondamine and certain noncompetitive blockers appear to enhance desensitization. The effects of agonists, antagonists and noncompetitive blockers at the neuronal nicotinic acetylcholine receptor-channel of PC12 cells are compared to their effects on binding of [¹²⁵I]-bungarotoxin to agonist-recognition sites and of [³H]perhydrohistrionicotoxin to noncompetitive blocker sites of the nicotinic acetylcholine receptor-channel of electric ray (Torpedo) electroplax membranes. There are marked differences in relative potencies for the two types of nicotinic acetylcholine receptor-channel.     

Determination of the minimal inhibitory concentration of Amphotericin B and Miconazole for 21 strains of Aspergillus

The susceptibility of 21 strains ofAspergillus (11 ofA. fumigatus, 8 ofA. niger, and 2 ofA. flavus) isolated from human pathologic specimens to Amphotericin B and Miconazole has been comparatively studied. Determination of the minimal inhibitory concentration of both drugs in a liquid medium showed a noticeably variability for the different strains. The values obtained for Amphotericin B varied between 0.25g/ml (2 strains) and 1.25g/ml (5 strains) after 48 hours, and between 1.25g/ml (1 strain) and 50g/ml (1 strain) after 10 days. For Miconazole the results varied between 0.1g/ml (1 strain) and 25g/ml (1 strain) after 48 hours of incubation, and between 0.5g/ml (5 strains) and > 100g/ml after 10 days. The variability of these results indicates the usefulness of carrying ourin vitro sensitivity studies whenever it is possible.     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

Coccidia of Brazilian edentates: Eimeria cyclopei n.sp. from the silky anteater,Cyclopes didactylus (Linn.) and Eimeria choloepi n.sp. from the two-toed sloth,Choloepus didactylus (Linn.)

Summary Eimeria cyclopei n.sp. is described from the silky anteater, Cyclopes didactylus, from Pará State, north Brazil. Undifferentiated oocysts, passed in the faeces, complete sporulation in seven days at 26 to 28°C. Oocysts are ellipsoidal to sub-spherical, with a mean size of 28.1 × 23.6 m: the wall is 1.5 to 2.0 m thick, apparently with an outer thin, colourless membrane and two inner, thicker, striated and yellowish layers. There is no micropyle, oocyst residuum or polar body. The mean measurements of sporocysts are 19.0 × 9.0 m, and they are slightly asymmetrical, elongate pear-shape, with a plug-shaped Steida body projecting beyond the end of the sporocyst. Sporozoites are as long as or longer than the sporocysts: The sporocyst residuum is scattered between sporozoites in younger specimens and becomes condensed into rounded mass in older ones. The endogenous stages occur in the epithelial cells of the ileum, on the lumenal side of the host-cell nucleus. Uninucleate meront, microgamont and macrogamont precursors are recognizable morphologically. Mature meronts are 20.0 × 15.7 m some produce 12 to 20 merozoites which are 8.7 × 2.0 m, and others 10 to 26 merozoites which are 11.4 × 2.0 to 15.0 × 3.0 m. Mature microgamonts which are 27.5 × 24.1 m, produce from 150 to 170 microgametes of 7.1 × 1.0 m: microgametes have two flagella of unequal length. Mature macrogamonts are 28.4 × 24.5 m Eimeria choloepi n.sp. is recorded from the two-toed sloth, Choloepus didactylus, from the same area of Brazil. Undifferentiated oocysts, passed in the faeces, complete sporulation in 23 days at 26 to 28°C. Oocysts with a mean size of 23.0 × 20.3 m, have a wall 2.0 to 2.5 m thick which is composed of two thick, yellowish and striated outer layers and a delicate, colourless inner one. There is no micropyle, oocyst residuum or polar granule. Mature sporocysts with a mean size of 11.3 × 7.1 m, are ellipsoidal to egg-shaped and have a poorly developed Steida body. The sporocyst residuum is composed of a small number of large globules: The sporozoites are longer than the sporocyst and strongly recurved. The endogenous stages occur in epithelial cells of the ileum, on the lumenal side of the host-cell nucleus. Dimorphic meronts produce 8 to 18 merozoites which are either 13.0 × 2.0 m or 13.0 × 3.0 m. Microgamonts produce 50 to 80 microgametes of 8.0 × 1.0 m. Mature macrogamonts are 18.3 × 17.9 m. ac]19820212     

The induction and repression of benzene and catechol oxidizing capacity of Pseudomonas putida ML2 studied in perturbed chemostat culture

The oxidation of catechol, an intermediate in benzene catabolism, was studied using transient variations in dissolved oxygen tension (DOT) when a succinate limited steady state culture of Pseudomonas putida ML2 was perturbed with a pulse of another substrate. A model was developed and tested for the effect of fluctuations in oxidizing enzyme activity on DOT. It was found that the rate of induction of catechol oxidizing enzymes was independent of dilution rate up to a relative growth rate /_max of 0.75. Only at higher dilution rates was catabolite repression observed.Abbreviations DOT dissolved oxygen tension - K _L a gas transfer coefficient - specific growth rate - _max maximum specific growth rate - K_s substrate saturation constant     

Methylobacterium rhodesianum MB 126 possesses two acetoacetyl-CoA reductases

Two constitutive acetoacetyl-CoA (AcAc-CoA) reductases were purified from Methylobacterium rhodesianum MB 126, an NADPH-linked d(-)--hydroxybutyryl-CoA forming reductase (enzyme A) and an NADH-and NADPH-linked l(+)--hydroxybutyryl-CoA forming reductase (enzyme B). Enzyme A and B give apparent K _m values of 15 M and 30 M for AcAc-CoA, 18 M for NADPH and 30 M for NADH, respectively. They are inhibited by AcAc-CoA at concentrations higher than 25 M and 50 M, respectively. The contribution of the two reductases to poly--hydroxybutyrate synthesis is discussed.     

Problems in estimating growth rates of marine phytoplankton from short-term14C assays

Growth rate estimates () of phytoplankton populations that were sampled from nitrogen-limited continuous cultures and then incubated for short durations in batch culture with added¹⁴C-HCO₃ ^– were significantly different than steady-state growth rates () for 3 of 5 marine phytoplankton species. Two diatoms,Thalassiosira weissflogii andChaetoceros simplex, displayed virtually identical growth rates (=) over a wide range of, whereas for a third diatom,Phaeodactylum tricornutum, was overestimated by an average of 40% compared to. In contrast, was underestimated by the¹⁴C technique for the two remaining species: up to 40% at a steady-state of 1.0 day^–1 for the chlorophyteDunaliella tertiolecta and up to 100% at of 1.4 day^–1 for the haptophytePavlova lutheri. For the latter two species the divergence between and appeared to increase with increasing steady-state. A simple model of labeled and total carbon flow between the aqueous phase and cellular biomass was constructed to demonstrate that respiration was negligible when=, but was significant when>. In the cases in which<, a rapid physiological alteration presumably took place once the steady state was disturbed and cells were placed in the incubation chambers, which perhaps was related to the nutritional state of the cultures at the time of sampling. Questions thus are raised regarding our ability to measure accurately primary productivity from shipboard experiments with confined samples of phytoplankton from nutrient-impoverished waters that probably are less hardy than the laboratory cultures used in these studies.     

Enhancement of somatic embryogenesis in orchardgrass leaf cultures by epinephrine

Epinephrine at 10–100 M stimulated somatic embryogenesis from orchardgrass (Dactylis glomerata L.) leaves cultured on SH medium with 30 M of indole-3-acetic acid (IAA). Ethylene emanation was increased at epinephrine concentrations greater than 10 M. Decarboxylation by the leaves of [1-¹⁴C]IAA included in the medium was decreased almost 3-fold by 10 M epinephrine. Epinephrine at 10 M enhanced the number of regenerated plants on SH medium with 30 M dicamba (SH-30). Ethylene emanation was increased by epinephrine concentrations of 500 M and greater included in SH-30 but somatic embryogenesis was decreased. Addition of 8 M CoCl₂, 6H₂O (an ethylene biosynthesis inhibitor) to medium with 500 M epinephrine decreased ethylene emanation to the control level but did not alleviate the decreased embryogenic response.     

Plant regeneration via somatic embryogenesis in Styrian pumpkin: cytological and biochemical investigations

Somatic embryo formation was induced from cotyledon explants of Styrian pumpkin (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) by using a solid MS medium supplemented with 16.11M NAA and 4.44M BA or 26.85M NAA and 13.32M BA. The callus proliferation was more efficient on medium supplemented with 26.85M NAA and 13.32M BA. In contrast, the embryogenic response was higher on medium with lower concentrations of growth regulators (16.11M NAA and 4.44M BA). The time needed for embryo induction did not depend on medium composition. Embryos in globular stage were transferred to three different maturation media, containing 2.89M GA₃ in combination with 0.54M NAA, 11.42M IAA and growth regulator-free medium. The germination rate was the highest when embryos were cultured on medium with 11.42M IAA. Plantlets grown on this medium achieved maturity suitable for transplantation into soil within 9 to 10weeks. The regenerated plants were successfully transferred into field and developed fertile flowers and set fruits. Biochemical analysis showed significant lower total glutathione levels among in vitro grown plantlets compared to seedlings grown in soil. When the plantlets were transferred into soil, they reached a normal size within a month and the glutathione concentration was comparable to seed-derived plants at the same developmental stage. Transmission electron microscopy was used to investigate possible differences in the ultrastructure of cells from callus cultures, and leaf cells of regenerated and seed-derived plants. Differences in the ultrastructure were found within chloroplasts which contained only single thylakoids, large starch grains and small plastoglobuli in callus cells in comparison to leaf cells, which possessed a well developed thylakoid system, small starch grains and large plastoglobuli.     

Fine structure of abscission zones

Summary Electron micrographs of the zone of separation in flower pedicels of Lycopersicon esculentum and Nicotiana tabacum are presented with particular reference to the indentation of epidermal tissue in the abscission zone, subcellular organelles, and the cell wall. The indentation or groove which delineates the abscission zone extends some distance into the pedicel with branchings off the main groove. These branches are approximately 20 m in width while the main groove averages approximately 200 m in width. Invaginations of the plasmalemma are observed with considerable frequency. within these invaginations one observes a material of about the same density as the cell wall except that it is more fibrillar. Plasmodesmata are also observed, with considerable branching into middle lamellae of cells comprising the abscission zones. Microbodies with crystalloid cores appear with considerable frequency in cells of the abscission zone. The crystalloids appear to be cubical in shape and are composed of parallel sheets of osmiophilic material. The sheets average about 6 m in thickness and are spaced at 4 m intervals. The microbodies with crystalloid cores are observed to be characteristically of two size groupings. In tobacco the microbodies average 900 m and 1,500 m in profile. In tomato they average 300 m and 500 m. Chloroplasts contain a granular component which is membrane-enclosed. The component is large in comparison with the plastid in which it occurs, averaging 1.2–1.4 in diameter in chloroplasts ranging from 1.6 to 2.2 in diameter. The inner membrane of the chloroplast is highly invaginated, and DNA- and phytoferritin-like materials are observed within the plastids. Microtubules with an average diameter of 20 m are observed adjacent and parallel to the plasmalemma, primarily in the corners of the cells. Micrographs of other normally occurring cytoplasmic inclusions are also presented.     

Image processing technique for measuring specific growth rates of Gibberella fujikuroi

Summary Two methods were compared for estimating of Gibberella fujikuroi grown with different proportions of glucose and starch. They were, =Ln2 (V_r/Le) on Petri dish (V_r= rate of tip extension and L_e= mean hyphal length) and, ₂=d (LnX)/dt in stirred fermenter. Values of ₁ and ₂ were in close agreement with each other.     

Control of yeast fed-batch process through regulation of extracellular ethanol concentration

At high growth rates, the biomass yield of bakers yeast (Saccharomyces cerevisiae) decreases due to the production of ethanol. For this reason, it is standard industrial practice to use a fed-batch process whereby the specific growth rate, , is fixed at a level below the point of ethanol production, i.e., _crit. Optimally, growth should be maintained at _crit, but in practice, this is difficult because _crit is dependent upon strain and culture conditions. In this work, growth was maintained at a point just above _crit by regulating ethanol concentration in the bioreactor. The models used for control design are shown, as are the experimental results obtained when this strategy was implemented. This technique should be applicable to all microorganisms that exhibit an overflow type metabolism.     

Changes of photosynthetic parameters in cucumber leaves under Cu, Cd, and Pb stress

The effects of Cu, Cd, and Pb toxicity on photosynthesis in cucumber leaves (Cucumis sativus L.) were studied by the measurements of gas exchange characteristics, chlorophyll (Chl) fluorescence parameters, and Chl content. Concentrations of metals in sequence of 20 M Cu, 20 and 50 M Cd, and 1 000 M Pb decreased the plant dry mass to 50–60 % after 10 d of treatment whereas 50 M of Cu decreased it to 30 %. The content of Cd in leaves of plants treated with 50 M Cd was three times higher than the contents of Cu and Pb after plant treatment with 50 M Cu or 1 000 M Pb. Hence Cd was transported to leaves much better than Cu and Pb. Nevertheless, the net photosynthetic rate and stomatal conductance in leaves treated with 50 M Cu or Cd were similarly reduced. Thus, Cu was more toxic than Cd and Pb for photosynthesis in cucumber leaves. None of the investigated metals decreased internal CO₂ concentrations. Also the effect of metals on potential efficiency of photosystem 2, PS2 (F_v/F_m) was negligible. The metal dependent reduction of PS2 quantum efficiency (_PS2) after plant adaptation in actinic irradiation was more noticeable. This could imply that reduced demand for ATP and NADPH in a dark phase of photosynthesis caused a down-regulation of PS2 photochemistry. Furthermore, in leaves of metal-treated plants the decrease in water percentage as well as lower contents of Chl and Fe were observed. Thus photosynthesis is not the main limiting factor for cucumber growth under Cu, Cd, or Pb stress.This revised version was published online in March 2005 with corrections to the page numbers.     

Binding of [3H]muscimol to calf cerebrocortical synaptic membranes and the effects of sulphur-containing convulsant and non-convulsant compounds

Endogenous and xenobiotic sulphur-containing convulsant and non-convulsant compounds containing structural moieties of, or bearing a structural resemblance to, GABA and homocysteine were tested in binding studies for their potency in displacing the GABA-mimetic [³H]muscimol from specific, high-affinity sites (K _d=3.6 nM;B _max=3.94 pmol/mg protein) on freeze-thawed, Triton-treated calf-brain synaptic membranes. The xenobiotic convulsants, 4-mercaptobutyric acid (MBA), 3-mercaptopropionic acid (3-MPA) and 2-mercaptopropionic acid (2-MPA) were found to be two-site competitive inhibitors exhibiting apparent inhibition affinity constants (K _i ^app ) of 5000 M, 3750 M, and 4800 M, respectively; while homocysteic acid (K _i ^app =4800 M) was shown to be a one-site partial competitive inhibitor. Intermediary metabolites of methionine: S-adenosyl-l-homocysteine,l-cysteine, the convulsantl-homocysteine, and its non-convulsant disulphide oxidation product, homocystine, were found to be one-site partial competitive inhibitors exhibitingK _i ^app values of 5750 M, 8350 M, 5000 M, and 510 M, respectively. The endogenous anticonvulsant neuroeffector, taurine, and the tripeptide, reduced glutathione (GSH) were shown to be, respectively, one-site (K _i=20 M) and two-site (K _i ^app =4300 M) competitive inhibitors of [³H]muscimol binding. These findings are discussed with regard to a previously proposed mechanism for the convulsant action of homocysteine.     

Somatic embryogenesis in callus cultures of Rosa hybrida L. cv. Landora

Callus was initiated from immature leaf and stem segments of rose (Rosa hybrida cv. Landora) and subcultured every four weeks on a basal medium of half-strength Murashige & Skoog (1962) salts plus 30 g l^-1 sucrose (1/2 MS) and supplemented with 2.2 M BA, 5.4 M NAA and 2.2–9.0 M 2,4-D. Embryogenic callus and subsequently somatic embryos were obtained from 8-week-old callus culture on 1/2 MS+2.2 M BA+0.05 M NAA+0.3 M GA₃+200–800 mg l^-1 L-proline. Long-term cultures were established and maintained for up to 16 months by repeated subculture of embryogenic callus on L-proline deficient medium. About 12% of cotyledonary stage embryos taken from cultures cold-stored at 8±1°C for 4 days germinated on 1/2 MS+2.2 M BA+0.3 M GA₃+24.7 M adenine sulphate.Abbreviations BA benzyladenine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid     

β-Glucuronidase activity in human T and B lymphocytes and the Tμ and Tγ subpopulations

Summary The -glucuronidase staining characteristics of isolated T cell populations and the T and T enriched fractions derived of them were studied. T lymphocytes were obtained from purified T lymphocytes by ox-IgG rosette sedimentation. The rosette-forming cells in the pellet were referred to as T lymphocytes, whereas the lymphocytes in the interface were referred to as T depleted or T lymphocytes. B cells were studied on rosetted mononuclear cells with either mouse erythrocytes or with Staphylococcus Aureus (Cowan I) bacteria, after a preceeding polyvalent anti-human Ig treatment of the cells. While B cells showed mostly no reactivity, T and T cells were respectively characterised by a dot-like and granular pattern of reactivity. These findings are in agreement with those observed by others after -naphthyl-acetate esterase or acid phosphatase staining. Within the T lymphocyte fraction, the T non-, non lymphocytes seemed to have a granular pattern of reactivity. The same staining pattern was found in non-B, non-T lymphocytes.     

A general equation for the evaluation of the error that affects the value of the maximum specific growth rate

A general equation is proposed to evaluate the absolute error that affects the maximum specific growth rate calculated from batch or continuous experiments. This error depends on the relative errors of the cell concentration measurements and on the duration of the test.Nomenclature X ₁ Cell concentration at the beginning of the exponential growth phase or of the washing-out period - X ₁ Lowest value of X ₁ due to experimental errors - X ₁ Highest value of X ₁ due to experimental errors - X ₂ Cell concentration at the end of the exponential growth phse or of the washing-out period - X ₂ Lowest value of X ₂ due to experimental errors - X ₂ Highest value of X ₂ due to experiment errors - Relative error that affects X ₁ - Relative error that affects X ₂ - t Exponential growth stage or washing-out duration - Asolute error that affects - Maximum specific growth rate - ₁ Lowest value of due to experimental errors - ₂ Highest value of due to experimental errors The author is with the Instituto Mauá de Technologia, Estrada das Lágrimas 2035, 09580-900, São Caetano do Sul, SP, Brazil     

Saccharomyces cerevisiae 2-mum DNA. An analysis of the monomer and its multimers by electron microscopy.

Summary The non-tandem inverted duplication in the 2-m DNA of Saccharomyces cerevisiae has a length of 0.19 m and is located asymmetrically along the molecule. The majority of the dumb-bell structures that are formed upon denaturation and selfannealing of the 2-m monomer consists of the renatured inverted duplication sequences as double stranded stem and two single stranded loops of 0.67 m±0.06 m (S-loop) and 0.86 m±0.05 m (L-loop) length. Two additional size classes which comprised 5–10% of the measured molecules had contour lengths of around 1.7 m and 2.1 m. The smaller dumb-bells contained two S-loops and the larger dumb-bells contained two L-loops as was shown by heteroduplex mapping with an HindIII fragment from the L-loop. Two models which assume illegitimate or site specific recombination, are presented to explain the generation of double S-loop and double L-loop molecules. At least part of the 4-m and 6- circular molecules present in the yeast supercoiled DNA fraction are shown to be dimers and trimers of 2-m monomers, but often with inverted loop segments most probably due to intramolecular recombination between sequences of the inverted duplication.2-m DNA is used to indicate the supercoiled DNA fraction although in our measurements the average monomeric length is 1.9 mPart of this work has been presented at the Conference: The Genetics and Biogenesis of Chloroplasts and Mitochondria, Munich, August, 1976     

Genes of the constant regions of functional immunoglobulin heavy chain of Japanese flounder,<Emphasis Type="Italic"> Paralichthys olivaceus</Emphasis>

IgM and IgD genes of the Japanese flounder were cloned and characterized from a genomic bacterial artificial chromosome (BAC) library. The gene contained four constant region exons (C1–C4), and two transmembrane exons (TM1 and TM2), which conforms to the organizational pattern of all other vertebrate -chain genes examined so far. In the same BAC clone, the gene, which is homologous to the IgD gene in mammals and teleost fish, was found immediately (0.9 kb) downstream of the IgM gene. This gene encoded seven exons (C1–C7) and two TM exons (TM1 and TM2) and had no duplication of C1-C2, as found in Atlantic cod, or C2-C3-C4, as found in Atlantic salmon and channel catfish. Phylogenetic and sequence analyses strongly suggest that teleost is more closely related to non-IgM isotypes than IgM isotypes. The heavy chain (IgH) locus of Japanese flounder, which encodes m, s and m, was found to be fully functional.     

Bacterial symbionts on green hydra and their effect on phosphate uptake

Gram-negative rod-shaped bacteria (1.5–2m long and 0.5m wide) have been found associated with green hydra. They are always present on the hydra surface delineating the ectodermal cells, on animals in culture, and also on those sampled from a natural habitat. The bacteria could be removed by a 30-min treatment with antibiotics (50/ml polymyxin B and 50/ml streptomycin). Antibiotic-treated hydra took up 55% less phosphate from the medium than control hydra. The nutritional relationship between the bacteria and green hydra and possible routes of infection of the hydra by these prokaryotic symbionts are discussed. Their importance in interpreting results of certain types of physiological experiments using aquatic organisms is emphasized.