A three dimensional serial reconstruction of neuronal distributions in the hypostome of a Hydra

The numbers, types, and distributions of neurons in a hypostome of Hydra littoralis were determined from electron micrographs of serial (0.25 μm thick) sections. In 1,080 serial sections examined we found 75 sensory cells and 949 centrally located ganglion cells. More than 96% of the 1,024 neurons identified had a single cilium. Sensory cells were most numerous near the apex of the hypostome. Proceeding away from the apex, they steadily decreased in numbers; at 120 μm they were no longer observed. Ganglion cells were bimodally distributed; some were associated with sensory cells at the apex, but most were found at the sites of tentacle origin. We observed, throughout the hypostome, a total of 64 neuronal clusters (three or more contiguous neurons), with an average of five and a maximum of 11 neurons in a cluster. Clusters were distributed similarly to ganglion cells: an initial concentration of clusters near the apex; the majority at the hypostometentacle junctions. Each neuron identified was traced through succeeding sections in which it was observed. We used a three coordinate system to create a three-dimensional reconstruction of the neuronal locations in the hypostome. Although the functional significance of the neuronal distributions we observed is unknown, we suggest that neurons at the apex of the hypostome transduce sensory information involved in feeding behavior. The neuronal concentrations at sites of tentacle origin may be responsible for initiating Contraction Burst Pulses associated with rhythmic behavioral patterns of Hydra or coordinating tentacle movements involved in prey capture, ingestion or locomotion.     

Kinetic Basis for the Conjugation of Auxin by a GH3 Family Indole-acetic Acid-Amido Synthetase

The GH3 family of acyl-acid-amido synthetases catalyze the ATP-dependent formation of amino acid conjugates to modulate levels of active plant hormones, including auxins and jasmonates. Initial biochemical studies of various GH3s show that these enzymes group into three families based on sequence relationships and acyl-acid substrate preference (I, jasmonate-conjugating; II, auxin- and salicylic acid-conjugating; III, benzoate-conjugating); however, little is known about the kinetic and chemical mechanisms of these enzymes. Here we use GH3-8 from Oryza sativa (rice; OsGH3-8), which functions as an indole-acetic acid (IAA)-amido synthetase, for detailed mechanistic studies. Steady-state kinetic analysis shows that the OsGH3-8 requires either Mg²⁺ or Mn²⁺ for maximal activity and is specific for aspartate but accepts asparagine as a substrate with a 45-fold decrease in catalytic efficiency and accepts other auxin analogs, including phenyl-acetic acid, indole butyric acid, and naphthalene-acetic acid, as acyl-acid substrates with 1.4–9-fold reductions in k_cat/K_m relative to IAA. Initial velocity and product inhibition studies indicate that the enzyme uses a Bi Uni Uni Bi Ping Pong reaction sequence. In the first half-reaction, ATP binds first followed by IAA. Next, formation of an adenylated IAA intermediate results in release of pyrophosphate. The second half-reaction begins with binding of aspartate, which reacts with the adenylated intermediate to release IAA-Asp and AMP. Formation of a catalytically competent adenylated-IAA reaction intermediate was confirmed by mass spectrometry. These mechanistic studies provide insight on the reaction catalyzed by the GH3 family of enzymes to modulate plant hormone action.     

PHYTOTAB — A program package for Braun-Blanquet tables

A computer program package of six programs for vegetation classification is described. The package includes raw field-data input, automatic and user sequencing modes, final table and synoptic table preparation which are camera-ready and also allows the user the option of ordinating by means of DECORANA.     

Simultaneous small-sample multivariate Bernoulli confidence intervals

A technique based on the bootstrap is presented for assessing the simultaneous confidence level of k small-sample confidence intervals for multivariate Bernoulli marginal frequencies. The small-sample intervals used are those of Clopper and Pearson (1934, Biometrika 26, 404-413) and require iterative computation. To estimate the simultaneous confidence level, the multivariate Bernoulli vectors are resampled via the bootstrap and the Clopper-Pearson intervals recomputed on each pseudosample. The bootstrap estimate is then the proportion of times (computed via Monte Carlo) that all the k intervals computed by resampling contain the original sample frequencies. The technique is applied to single-sample HLA data.     

Monitoring plant community change: an application of quadrat classification + discriminant analysis

A procedure for monitoring plant community change was described using data from 189 quadrats (each 0.09 m² in area) from or near 11 Carex exserta meadow sites in the high Sierra Nevada, California, USA. Initially the quadrats were agglomerated into five clusters by the flexible clustering strategy (beta=–0.25) with the standard absolute distance resemblance function. Data for each quadrat were cover percentages for C. exserta, other plants, litter, soil, gravel, and rock. The five clusters appeared to define a cover gradient, from quadrats with mostly gravel and rock to those with mostly C. exserta, and were accordingly designated pioneer, low seral, mid-seral, high seral, and climax.Classification functions (from discriminant analysis) are used with values of the variables to classify individual quadrats on sites used to monitor change. A site is characterized at repeated observations by the proportions of quadrats in each class. Within-class (low seral vs. low seral) rather than between-class (pioneer vs. low seral) tests are made for presence of change. Confidence intervals for differences in proportions of quadrats or individual quadrat probabilities of class membership are computed. If the confidence intervals do not cover zero, values for time one versus time two differ significantly.     

High voltage electron stereomicroscopy of the cilium-stereociliary complex of perioral sensory cells in Hydra

The cilium-stereociliary complex in perioral neurons of Hydra was examined by electron microscopy, with emphasis on stereomicrographs of serial, 0.5 micron thick, longitudinal and transverse sections. Longitudinal sections revealed (1) flat-topped cones in which the cilium was bent and the ciliary chamber appeared heart-shaped, and (2) pointed cones in which the cilium was straight and the ciliary chamber appeared triangular. Transverse sections revealed 10-12 stereocilia forming a cone over a central cilium with nine peripheral doublets of microtubules but with often more than two central microtubules. The ciliary membrane was fluted; fine filaments connected the outfoldings of membrane with the center of the microtubule doublets. Thin sections revealed 7 nm microfilaments in the stereocilia cores which branched basally into thick and thin roots; the thick roots surrounded the base of the central cilium. The cilium-stereociliary complex was enveloped by an epitheliomuscular cell sheath with a free margin distally and a septate junction proximally. In flat-topped cones the free margin of the enveloping epitheliomuscular cell was closely applied to the top of the cilium-stereociliary complex, whereas in pointed cones the cilium-stereociliary complex projected above the free margin of the sheath. Thus, the 7 nm actin-like filaments in the stereocilia might function to contract and open the complex in response to favorable stimuli so that the cilium is in contact with the aqueous environment.     

Substrate utilization by Legionella cells after cryopreservation in phosphate buffer

The objective of this study was to evaluate by relatively simple metabolic tests the usefulness of buffers and energy sources commonly used in Legionella growth media. Legionella pneumophila serogroups 1 to 6, Legionella micdadei, and Legionella bozemanii were grown in an enriched charcoal-yeast extract diphasic medium. The cells were washed thrice, suspended in various buffers (pH 6.9) with 1 or 5 mM MgSO4, and used immediately or after controlled-rate cryopreservation. CO2 produced and C incorporated into the cold trichloracetic acid-insoluble fractions from 14C-labeled substrates were determine. Potassium phosphate buffer (0.02 M) was as satisfactory as organic buffers for glutamate metabolism, but the addition of KCl or NaCl reduced activity. Metabolic activity for glutamate was not lost upon cryopreservation, and cryopreserved cells were used to test the utilization of other single or paired substrates. Rates of activity for serine, glutamate, threonine, and pyruvate, in this descending order, were high, and those for alpha-ketoglutarate, succinate, and gamma-aminobutyrate were low. Although glutamine was not used as rapidly as glutamate, when added to glutamate it was preferentially metabolized, possibly because of more rapid transport. When glutamate and serine were combined, glutamate furnished more C for CO2 and less for incorporation, whereas the reverse was true of serine. In conclusion, glutamate as an energy source may in some cases spare other amino acids for synthesis. alpha-Ketoglutarate, a common constituent of Legionella media, may reduce oxygen toxicity but is probably not a chief energy source.     

A pharmacological investigation of the biphasic nature of the contractile response of rabbit and rat vas deferens to field stimulation

It has been demonstrated previously with the vas deferens of the guinea-pig that the first and second phases of the contractile response to motor nerve stimulation are preferentially antagonized by the P₂-purinoceptor antagonist arylazido aminopropionyl ATP (ANAPP₃), and the α₁-adrenoceptor antagonist prazosin, respectively. We have now investigated the effect of the two antagonists on the biphasic contraction in the vas deferens of two other species; rabbit and rat. ANAPP₃, in a concentration which antagonized responses to exogenously applied ATP but not those to exogenous norepinephrine, preferentially reduced the initial phasic response of the rabbit vas deferens to motor nerve stimulation without significantly reducing the secondary, tonic phase of the response. Prazosin had the opposite effect; antagonizing the response to norepinephrine but not to ATP and reducing the tonic response to motor nerve stimulation without significantly reducing the initial phasic response. Results obtained with the rat vas deferens were similar. The present results combined with previous findings suggest that ATP and norepinephrine act as cotransmitters in the vas deferens of several species.     

Morphological and biochemical changes in supersensitive smooth muscle.

The effect of postganglionic denervation on the incidence of nexal contacts in the smooth muscle of the rat vas deferens was investigated. The chronically denervated tissue exhibited twice as many nexuses as control. This increase in the incidence of cell contacts may contribute to the supersensitivity and/or the increase in maximum response of the denervated vas deferens. The effects of denervation, decentralization, and pretreatment with reserpine on the concentration of adenosine triphosphate (ATP) in vasa deferentia of rats and guinea pigs were also investigated. One day after denervation there was a substantial decrease in the endogenous norepinephrine and ATP concentrations. The norepinephrine concentration remained low (less than 10% of control) throughout subsequent days (up to 14 days) whereas the ATP concentration, after the first postoperative day, rose significantly. The rise in ATP concentration was temporally correlated with the development of postjunctional supersensitivity. Decentralization and pretreatment with reserpine both resulted in a significant increase in ATP concentration which preceded by 2 to 3 days a significant increase in sensitivity of the vas deferens. It appears that a change in the tissue concentration of ATP may be one of the initial events that occurs following interruption of the neural contact to the smooth muscle of the vas deferens.