Phospholipase D (PLD) is present in Leishmania donovani and its activity increases in response to acute osmotic stress.

We report here that the signaling molecule phospholipase D (PLD) is present in the parasitic protozoan Leishmania donovani. In vitro enzymatic activity is dependent on Ca2+ and Mg2+ ions, its basal activity is stimulated by phosphatidyl-inositol-4,5-bisphosphate (PIP2) and its pH optima are pH 8.0 and pH 6.0. PLD activity increases 3-fold about 5 min after an abrupt decrease in osmolality from 317 mOsm (isosmotic) to 155 mOsm and increases 1.5-fold in response to an abrupt increase in osmolality to 617 mOsM. Cells grown for > 24 h under the anisosmotic conditions showed only marginal changes in activity compared to the controls grown under isosmotic conditions, indicating an adaptation to long-term exposure to hypo- or hyper-osmolarity. Immunologically, two isoforms, PLD1 and PLD2, are present. An analysis of in vitro PLD activity in anti-PLD immunocomplexes revealed that either hypotonic (cell swelling) or hypertonic stress (cell shrinking) causes an increase in PLD1 activation but a reduction in PLD2 activity. The interplay between these two isoforms results in a predominance for PLD1 in the observed increase when measuring total PLD activity. Finally, the increase in enzymatic activity in acute hyposmotic shock is accompanied by tyrosyl phosphorylation of the PLD1 isoform, suggesting a role for protein tyrosine kinase in the control of PLD activity in response to osmotic stress.

Compartmental responses to acute osmotic stress in Leishmania major result in rapid loss of Na+ and Cl-.

The elemental composition of the cytoplasm, electron dense vacuoles, and heterochromatin and euchromatin regions of the nucleus of Leishmania major promastigotes was measured by electron probe X-ray microanalysis under iso-osmotic conditions (305 mOsM) and shortly after a sudden increase (to 615 mOsM) or decrease (to 153 mOsM) in the osmolality of the buffer in which they were suspended. In response to acute hypotonicity a complete loss of Na from the electron dense vacuoles and an approximately threefold decrease in the Na concentrations in the cytoplasm and the nuclear regions occurred, together with an approximately threefold decrease in Cl content in each compartment and a smaller (approx. 1.2-fold) decrease in K content. Thus, in addition to the rapid change in shape and release of amino acids known to occur in response to acute hypo-osmotic stress, a major efflux of Na and Cl, and, to a lesser extent, of K, also occurs. In response to acute hypertonicity Na in the acidocalcisomes did not change but Na content of the cytoplasm decreased by 33%. A small increase in the S content of the cytoplasm and the electron dense vacuolar compartments occurred. No changes were detectable in Ca or Zn content in any of the compartments examined in response to hypotonicity or hypertonicity.

Model calculations of time dependent responses to binaural stimuli in the dorsal nucleus of the lateral lemniscus.

In a previous paper (Reed and Blum, 1999), we examined the connectional hypotheses put forward by Markovitz and Pollak (1994) to explain the steady-state behavior of cells in the dorsal nucleus of the lateral lemniscus (DNLL). We found that the steady-state outputs of the four major binaural types of cells found in the DNLL (EI, EI/F, EE/I, and EE/FI) could be accounted for by known connectional patterns using only one or two cells per nucleus and quite simple hypotheses on cell behavior. In this study, we examine the time course of DNLL outputs in response to constant, ongoing, monaural or binaural sounds of various intensities. The model auditory nerve fibers ramp up linearly (usually in 2 ms) to full firing and the anteroventral cochlear nucleus cells have primary-like discharge patterns. Fixed time delays of 1 ms at each synapse are included; other time delays are employed when necessary to understand and reproduce specific features of the experimental data. We find that the connectional patterns utilized in our previous study can account for the rich variety of temporal response patterns found experimentally in the DNLL. Our main findings are: (1) all of the four major binaural types of cells can arise from modifications of the basic connectional pattern that produces EI cells; (2) both excitation and inhibition from the ipsilateral lateral superior olive (LSO) are required to understand DNLL responses; (3) pauser behavior can arise either from time delayed inhibition from a DNLL interneuron or by projection from the LSO; (4) two different mechanisms can account for the ipsilaterally evoked onset response; (5) to explain completely the temporal discharge pattern and binaural interactions of EE/FI cells, a projection from the contralateral DNLL via the commissure of Probst is necessary.

A mathematical model quantifying GnRH-induced LH secretion from gonadotropes.

A mathematical model is developed to investigate the rate of release of luteinizing hormone (LH) from pituitary gonadotropes in response to short pulses of gonadotropin-releasing hormone (GnRH). The model includes binding of the hormone to its receptor, dimerization, interaction with a G protein, production of inositol 1,4, 5-trisphosphate, release of Ca(2+) from the endoplasmic reticulum, entrance of Ca(2+) into the cytosol via voltage-gated membrane channels, pumping of Ca(2+) out of the cytosol via membrane and endoplasmic reticulum pumps, and release of LH. Cytosolic Ca(2+) dynamics are simplified (i.e., oscillations are not included in the model), and it is assumed that there is only one pool of releasable LH. Despite these and other simplifications, the model explains the qualitative features of LH release in response to GnRH pulses of various durations and different concentrations in the presence and absence of external Ca(2+).

Model calculations of steady state responses to binaural stimuli in the dorsal nucleus of the lateral lemniscus.

Several studies have been performed in which both the time-dependent and steady state output of cells in the dorsal nucleus of the lateral lemniscus (DNLL) have been measured in response to binaural sound stimulation. In this paper, a mathematical and computational model for the steady state output of DNLL cells is formulated. The model includes ascending connections from both lateral and medial superior olives (LSO and MSO) as well connections from interneurons in the DNLL and connections from the contralateral DNLL through the commissure of Probst. Our intent is to understand how the steady state behavior arises from the cell properties in and connectional patterns from lower brainstem nuclei. In particular, we examine the connectional hypotheses put forward by Markovitz and Pollak (1994) to explain the observed behavior of EI, EI/F, EE/I and EE/FI cells. Using these connections (with minor modifications) and cells with simple input-output relations, we are able to account for the steady state behavior of these cell types. We are able to explain interesting features of the data not commented on before, for example, the initial dip in spike output for EE cells at low ipsilateral sound levels. The presence of an inhibitory interneuron in the DNLL is essential for facilitation. In addition, we examine the effects of the MSO and the commissure of Probst on DNLL output. Furthermore, we propose a simple mechanism by which the cells of the DNLL and LSO could create a topographic place map in the inferior colliculus.

Effects of wide band inhibitors in the dorsal cochlear nucleus. II. Model calculations of the responses to complex sounds.

In a previous modeling study of signal processing in the dorsal cochlear nucleus [Reed and Blum, J. Acoust. Soc. Am 96, 1442-1453 (1997)] it was shown that inclusion of a wideband inhibitor (WBI) greatly improved the fit between model response maps and the experimental response maps of type IV units to pure tones. In this study we examine the effect of the WBI on the responses to complex sound stimuli such as broadband noise (BBN), notch noise, noise bands, and band/notch combinations. A new and more realistic model for auditory nerve (AN) response in the presence of different levels of noise is used. It is shown that one can explain and understand the qualitative features of virtually all the published data on type II and type IV unit responses to BBN, notch noise and noise bands. The monotone decreasing response of the maximum firing rate of type II units to noise bands of increasing width that is observed experimentally occurs in the model due to the increasing inhibition of type II cells by the WBI. Similarly, the various nonmonotone patterns of maximum firing rate of type IV units to noise bands of increasing width is shown to arise from the complex and highly nonlinear effects of inhibition from the type II to type IV and the WBI to type IV cells and the nonlinear direct excitation from the AN to the type IV cells. A number of experiments using double notches, double noise bands, or notch-noise band pairs are suggested which, by comparison with model results, would allow one to infer probable connectional patterns between type II and type IV units and between the WBI units and the type IV units.

Cilia internal mechanism and metachronal coordination as the result of hydrodynamical coupling.

We present a simple but realistic model for the internal bend-generating mechanism of cilia, using parameters obtained from the analysis of data of the beat of a single cilium, and incorporate it into a recently developed dynamical model. Comparing the results to experimental data for two-dimensional beats, we demonstrate that the model captures the essential features of the motion, including many properties that are not built in explicitly. The beat pattern and frequency change in response to increased viscosity and the presence of neighboring cilia in a realistic fashion. Using the model, we are able to investigate multicilia configurations such as rows of cilia and two-dimensional arrays of cilia. When two adjacent model cilia start beating at different phase, they synchronize within two cycles, as observed in experiments in which two flagella beating out of phase are brought close together. Examination of various multicilia configurations shows that metachronal patterns (i. e., beats with a constant phase difference between neighboring cilia) evolve autonomously. This provides modeling evidence in support of the conjecture that metachronism may occur as a self-organized phenomenon due to hydrodynamical interactions between the cilia.

Model calculations of the effects of wide-band inhibitors in the dorsal cochlear nucleus.

In two previous papers [Reed and Blum, J. Acoust. Soc. Am. 97, 425-438 (1995), Blum et al., J. Acoust. Soc. Am. 98, 181-191 (1995)] a computational model for signal processing in the dorsal cochlear nucleus (DCN) was developed. In those modelling studies, stellate cells inhibited only type II cells. In this study, the effect of including wide-band inhibitory (WBI) connections from stellate cells to type IV cells, as proposed by Nelken and Young [J. Neurophysiol. 71, 2446-2462 (1994)], is examined. Inclusion of the WBI connections improves the fit to the experimental pure tone response maps for both the "standard" and "non-standard" cells examined by Spirou and Young [J. Neurophysiol. 66, 1750-1768 (1991)]. Thus, these modelling studies support the hypothesis of Nelken and Young [J. Neurophysiol. 71, 2446-2462 (1994)]. The degree of improvement is greatest for cells with prominent upper inhibitory sidebands. The qualitative features of the pure tone response map and the theoretical model allow one to deduce the probable frequency bias of the type II to type IV and stellate to type IV connections.

Oxidation of alanine, acetate, glutamate, and succinate by digitonin-permeabilized Leishmania major promastigotes.

Leishmania major promastigotes were treated with digitonin and the rates at which [1-14C]acetate, [1,4-14C]succinate, [1-14C]glutamate, and [U-14C]alanine are oxidized were measured in the presence of suitable cofactors. Acetate was oxidized at the lowest rate of the four substrates examined, even in the presence of added NAD, CoA, ADP and acetyl-CoA synthase. Its rate of oxidation was negligible if the permeabilized cells were washed before the cofactors were added, indicating the requirement for an as yet unknown factor. Succinate was oxidized at a rate much higher than the very slow rate at which it is oxidized by intact cells. Its rate of oxidation was strongly inhibited by antimycin A, but that of glutamate was scarcely affected. Fumarate inhibited the rate of oxidation of acetate, glutamate, and succinate, but increased that of alanine. Ca++ inhibited the rates of oxidation of alanine and succinate, but not of acetate or glutamate. Increasing the osmolality by addition of mannitol partially inhibited the rate of oxidation of alanine but had little effect on that of glutamate. These results show that appreciable transaminase activity remains in the permeabilized cells and support earlier data indicating the presence of a branched NAD-to-cytochrome oxidase system. These results also provide preliminary information on the sensitivity of the two branches to Ca++, hyperosmolality, and Krebs cycle intermediates.

Uptake and release of 2-aminoisobutyrate by Leishmania donovani: culture-age dependent effects of osmolality and of protein kinase inhibitors.

The rate of uptake of 2-aminoisobutyrate (AIB), a non-metabolizable analogue of alanine, by Leishmania donovani was investigated as a function of culture age and osmolality in the presence of protein kinase inhibitors and of glucose, glutamate or proline. Hyperosmolality inhibited AIB uptake by cells of both growth stages, but to a greater extent for log than for stationary cells. Staurosporine, a protein kinase C inhibitor, had no effect on AIB uptake by cells of either culture age, but it reduced the rate of AIB release in response to hypo-osmolality, and more so in stationary cells than in log cells. Genistein, a protein-tyrosine kinase inhibitor, caused a small increase in AIB uptake by log cells under both iso- and hyperosmotic conditions, but had no effect on AIB uptake by stationary cells. Genistein also caused a small increase in the rate of release of AIB in response to a decrease in osmolality. Brief preincubation with glucose, glutamate or proline of cells that had been depleted of their osmolytes by prior exposure to hypo-osmolality caused an increase in AIB release by log cells, but a decrease by stationary cells. Similarly, whereas glucose and glutamate increased the rate of AIB uptake by log cells, and proline inhibited it, they had no effect on AIB uptake by stationary cells. Thus AIB uptake and release are sensitive to changes in osmolality, to protein kinase inhibitors, and to certain nutrients in a manner that changes markedly with culture age.

Effects of osmotic stress on metabolism, shape, and amino acid content of Leishmania.

An acute decrease in osmolality causes a rapid change in the shape of the parasitic protozoan Leishmania donovani as determined by light microscopy and by flow cytometry. Incubation of the cells is an isotonic buffer supplemented with glucose. 2-deoxyglucose (2-DG), alanine, or proline also causes a shape change, presumably due to the swelling caused by the water that accompanies these substrates as they are actively transported into the cells. Hypo-osmolality also causes a rapid release of alanine and several other amino acids via a swelling activated amino acid channel. A sudden increase in osmolality causes a change in shape, an inhibition in the rates of oxidation of alanine, proline, leucine, and glucose, and in the rates of uptake of 2-aminoisobutyrate (AIB) and 2-DG. The protein kinase inhibitors staurosporine and genistein inhibited the rates of oxidation of alanine, glucose, and proline in a culture-age dependent manner and also altered the rate of release of AIB in response to hypo-osmotic stress. The possible roles of protein kinases in the culture-age dependent changes in the uptake, release and metabolism of several amino acids and of glucose are discussed.

A computational model for signal processing by the dorsal cochlear nucleus. II. Responses to broadband and notch noise.

In a previous paper a computational model was developed which was shown to account for most of the essential features of the variety of experimentally observed response maps of type-IV cells in the dorsal cochlear nucleus to pure tones. In the present study, the responses of the same model DCN to broadband noise and notch noise are investigated. It is shown that the previous model qualitatively accounts for the observed responses to these more complex sounds. Predictions of the model for inverted notches and for the behavior of type-IV cell output as notch center is varied for different amplitudes are presented. It is shown that the model is suitable for feature detection of auditory signals and an expansion is given as to how this ability arises from the properties of the stellate, type-II, and type-IV cells and the variations in the connectional patterns that were previously shown to account for the response patterns of type-IV cells to pure tones.

The NADP-linked aldehyde reductase of Leishmania donovani.

An enzyme that oxidizes ethanol to acetaldehyde in the presence of NADP (but not NAD) and reduces acetaldehyde to ethanol in the presence of NADPH (but not NADH) is present in Leishmania donovani promastigotes. The activity is present only in the supernatant fraction obtained from sonication of the cells and high speed centrifugation. The Km and Vm values were evaluated for propanol and propionaldehyde as well as for ethanol and acetaldehyde in cells obtained from late log and 3-day stationary phase cultures. There was no significant change in Km or Vm values for any of these four substrates with culture age. Since the Km values for ethanol and propanol are much higher than for the corresponding aldehydes and higher than any physiological range of alcohol concentration likely to be encountered, this enzyme is considered to function as an aldehyde reductase.

A computational model for signal processing by the dorsal cochlear nucleus. I. Responses to pure tones.

Much information is available on the anatomical organization and neurophysiological properties of the major cell types in the dorsal cochlear nucleus (DCN). The complicated response properties of individual cells and units in the DCN indicate that substantial information processing already occurs at the level of the DCN. A large number of connectional hypotheses have been put forward to explain various aspects of the response characteristics of DCN cells, but many of the consequences of these hypotheses have not been investigated quantitatively. In this paper, we investigate these hypotheses by constructing and testing mathematical and computational models and compare our results to those of previous modeling studies. The simplest versions of our models include auditory nerve (AN) fibers, type II cells (inhibitory interneurons) and type IV cells (fusiform and giant cells). The model response maps, i.e., the pattern of output of model type IV cells, generated by the simplest model have some but not all the features of the experimental response maps of type IV neurons. In particular, the excitatory region which occurs at best frequency is not isolated and the excitatory region at low frequencies and high amplitude is narrower than observed. Since experimental evidence exists that some of the connections between these cell types are divergent or convergent across adjacent isofrequency sheets, the effect of such convergence and divergence was then investigated. Response maps so obtained reproduce many of the qualitative features of the experimental maps. Enlargement of the model by including inhibitory interneurons (e.g., stellate cells) that receive convergent input from AN fibers and which inhibit type II cells results in the generation of response maps that, with some variations in connectional patterns and physiological properties of neurons, match most of the essential features seen in the large variety of experimental maps obtained from the cat DCN.

Aerobic and anaerobic glucose metabolism of Phytomonas sp. isolated from Euphorbia characias.

Metabolic studies on Phytomonas sp. isolated from the lactiferous tubes of the latex-bearing spurge Euphorbia characias indicate that glucose is the preferred energy and carbon substrate during logarithmic growth. In stationary phase cells glucose consumption was dramatically reduced. Glucose consumption and end-product formation were measured on logarithmically growing cells, both under aerobic (air and 95% O2/5% CO2) and anaerobic (95% N2/5% CO2 and 100% N2) conditions. The rate of glucose consumption slightly increased under anaerobic conditions indicating that Phytomonas lacks a 'reverse Pasteur' effect contrary to the situation encountered in Leishmania major. Major end-products of glucose catabolism under aerobic conditions, detected by enzymatic and NMR measurements, were acetate, ethanol and carbon dioxide and under anaerobic conditions ethanol, glycerol and carbon dioxide. Smaller amounts of pyruvate, succinate, L-malate, L-lactate, phosphoenolpyruvate, alanine and aspartate were also detected.

Secretion of sucrase by Leishmania donovani.

Leishmania donovani promastigotes were collected, washed, resuspended in buffer, and assayed for sucrase activity. No activity was observed in the intact washed cells, but activity was measurable when the cells were permeabilized with Triton X-100. Intracellular sucrase activity was highest in promastigotes grown at pH 7.4, somewhat lower in promastigotes grown at pH 5.5, and significantly lower in "amastigotes" grown at pH 5.5. No trehalase, lactase, or maltase activities were observed. Assay of the medium in which the cells had grown showed that most the sucrase activity was extracellular, i.e. was secreted into the medium during growth.

Energy metabolism in Leishmania.

Alanine plays a key role in the response of promastigotes to osmotic stress and to hypoxia. It is rapidly released in response to hypo-osmolality, is consumed from its large intracellular pool under iso-osmotic conditions even in the presence of glucose, and is synthesized under hyperosmotic conditions even in the absence of glucose. Its rate of oxidation, in the presence or absence of any of ten other amino acids tested, is strongly inhibited by hyperosmolality. Glucose oxidation is also inhibited by hyperosmolality, but to a lesser extent than that of alanine, and is inhibited by alanine, glutamate, and aspartate. Hyperosmolality also inhibits the incorporation of label from [2-14C]acetate into the putative storage carbohydrate, mannan, which occurs via the glyoxylate bypass and the as yet unexplored "mannoneogenic" pathway. The rates of glycolysis and of oxidation of several amino acids decrease with increasing culture age, but the capacity to oxidize fatty acids increases, and in cells from 3-day stationary phase cultures hyperosmolality enhances rather than inhibits alanine oxidation.