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A wide range of benefits have been posited from participation in competitive programming contests. However, an analysis of participation in north American regional contests in the International Collegiate Programming Contest (ICPC) shows that participation in these contests is sharply declining, coinciding with the COVID-19 pandemic. Moreover, prior to the pandemic, while the number of teams participating in regional contests was increasing, the number of institutions sending teams to these contests was declining. We find several statistically significant correlations that may underscore structural reasons for this trend. Consistent participation in contests and the number of teams per institution sent to a contest both are correlated with likely participation in future contests. On the other end of the spectrum, institutions sending a team to a contest for the first time in 3 years were much less likely to return in the next year. For this category of teams, if a team is unable to solve any problems in the contest, the institution is significantly less likely to send a team in the next year. Many of these contests have very challenging problem sets, and consequently, have many teams that fail to solve any problems. This result suggests that structuring the problem sets to increase the likelihood that most teams successfully complete problems would broaden participation in these contests., Competing Interests: Competing interestsThe author has been an organizer of large programming contests, but otherwise has no financial or non-financial interests related to this work., (© The Author(s) 2023.)

Background: Treatment of periodontal (gum) disease may lessen the adverse consequences of some chronic systemic conditions., Purpose: To estimate the effects of periodontal therapy on medical costs and hospitalizations among individuals with diagnosed type 2 diabetes (T2D); coronary artery disease (CAD); cerebral vascular disease (CVD); rheumatoid arthritis (RA); and pregnancy in a retrospective observational cohort study., Methods: Insurance claims data from 338,891 individuals with both medical and dental insurance coverage were analyzed in 2011-2013. Inclusion criteria were (1) a diagnosis of at least one of the five specified systemic conditions and (2) evidence of periodontal disease. Subjects were categorized according to whether they had completed treatment for periodontal disease in the baseline year, 2005. Outcomes were (1) total allowed medical costs and (2) number of hospitalizations, per subscriber per year, in 2005-2009. Except in the case of pregnancy, outcomes were aggregated without regard to reported cause. Individuals who were treated and untreated for periodontal disease were compared independently for the two outcomes and five systemic conditions using ANCOVA; age, gender, and T2D status were covariates., Results: Statistically significant reductions in both outcomes (p<0.05) were found for T2D, CVD, CAD, and pregnancy, for which costs were lower by 40.2%, 40.9%, 10.7%, and 73.7%, respectively; results for hospital admissions were comparable. No treatment effect was observed in the RA cohorts., Conclusions: These cost-based results provide new, independent, and potentially valuable evidence that simple, noninvasive periodontal therapy may improve health outcomes in pregnancy and other systemic conditions., (Copyright © 2014 American Journal of Preventive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To assess the role of the glycoprotein PRG4 in joint lubrication and chondroprotection by measuring friction, stiffness, surface topography, and subsurface histology of the hip joints of Prg4(-/-) and wild-type (WT) mice., Methods: Friction and elastic modulus were measured in cartilage from the femoral heads of Prg4(-/-) and WT mice ages 2, 4, 10, and 16 weeks using atomic force microscopy, and the surface microstructure was imaged. Histologic sections of each femoral head were stained and graded., Results: Histologic analysis of the joints of Prg4(-/-) mice showed an enlarged, fragmented surface layer of variable thickness with Safranin O-positive formations sometimes present, a roughened underlying articular cartilage surface, and a progressive loss of pericellular proteoglycans. Friction was significantly higher on cartilage of Prg4(-/-) mice at age 16 weeks, but statistically significant differences in friction were not detected at younger ages. The elastic modulus of the cartilage was similar between cartilage surfaces of Prg4(-/-) and WT mice at young ages, but cartilage of WT mice showed increasing stiffness with age, with significantly higher moduli than cartilage of Prg4(-/-) mice at older ages., Conclusion: Deletion of the gene Prg4 results in significant structural and biomechanical changes in the articular cartilage with age, some of which are consistent with osteoarthritic degeneration. These findings suggest that PRG4 plays a significant role in preserving normal joint structure and function.

This study specifically investigated a range of vehicle-related factors that are associated with a lower risk of serious or fatal injury to a belted driver in a head-on collision. This analysis investigated a range of structural characteristics, quantities that describes the physical features of a passenger vehicle, e.g., stiffness or frontal geometry. The study used a data-mining approach (classification tree algorithm) to find the most significant relationships between injury outcome and the structural variables. The algorithm was applied to 120,000 real-world, head-on collisions, from the National Highway Traffic Safety Administration's (NHTSA's) State Crash data files, that were linked to structural attributes derived from frontal crash tests performed as part of the USA New Car Assessment Program. As with previous literature, the analysis found that the heavier vehicles were correlated with lower injury risk to their drivers. This analysis also found a new and significant correlation between the vehicle's stiffness and injury risk. When an airbag deployed, the vehicle's stiffness has the most statistically significant correlation with injury risk. These results suggest that in severe collisions, lower intrusion in the occupant cabin associated with higher stiffness is at least as important to occupant protection as vehicle weight for self-protection of the occupant. Consequently, the safety community might better improve self-protection by a renewed focus on increasing vehicle stiffness in order to improve crashworthiness in head-on collisions.

Articular cartilage provides a low-friction, wear-resistant surface for the motion of diarthrodial joints. The objective of this study was to develop a method for in situ friction measurement of murine cartilage using a colloidal probe attached to the cantilever of an atomic force microscope. Sliding friction was measured between a chemically functionalized microsphere and the cartilage of the murine femoral head. Friction was measured at normal loads ranging incrementally from 20 to 100 nN with a sliding speed of 40 microm/s and sliding distance of 64 microm. Under these test conditions, hydrostatic pressurization and biphasic load support in the cartilage were minimized, providing frictional measurements that predominantly reflect boundary lubrication properties. Friction coefficients measured on murine tissue (0.25+/-0.11) were similar to those measured on porcine tissue (0.23+/-0.09) and were in general agreement with measurements of boundary friction on cartilage by other researchers. Using the colloidal probe as an indenter, the elastic mechanical properties and surface roughness were measured in the same configuration. Interfacial shear was found to be the principal mechanism of friction generation, with little to no friction resulting from plowing forces, collision forces, or energy losses due to normal deformation. This measurement technique can be applied to future studies of cartilage friction and mechanical properties on genetically altered mice or other small animals.

Our experience over many years from 2 diabetes clinics with large patient populations indicated that, apparently, excessive doses of intermediate-acting insulin preparations (150-300 U of NPH insulin), alone or in combination with rapid-acting insulin, generally did not result in acceptable control of fasting blood glucose. We hypothesized that insulin resistance at the tissue level and the known variability of insulin absorption were not satisfactory explanations. To deal with the ambiguities of available data on insulin absorption, we elected to measure insulin bioavailability via a different approach. Thirteen publications provided plasma insulin concentrations after the subcutaneous administration of defined doses of insulin. These data were then analyzed by noncompartmental analysis and by standard pharmacokinetic methods. Analyses required only knowledge of the areas under the plasma insulin curve and the metabolic clearance rate of insulin. Both of these are parameters measurable with considerable accuracy. Quantitative pharmacokinetic analysis of published insulin absorption curves for insulin administered subcutaneously revealed mean absorption levels for regular and lispro insulin of 70 to 80%, 30% or less for NPH insulin, and 30 to 40% for lente insulin. In conclusion, poor absorption of intermediate-acting insulin preparations, or combinations of intermediate- and rapid-acting insulin preparations, explains the difficulty in lowering blood glucose in patients with type 2 diabetes mellitus who have had long-standing disease, are insulin resistant, and have a flat insulin response to a glucose load.

In a previous study, a model was developed to investigate the release of luteinizing hormone (LH) from pituitary cells in response to a short pulse of gonadotropin-releasing hormone (GnRH). The model included: binding of GnRH to its receptor (R), dimerization and internalization of the hormone receptor complex, interaction with a G protein, production of inositol 1,4,5-trisphosphate (IP3), release of calcium from the endoplasmic reticulum (ER), entrance of calcium into the cytosol via voltage gated membrane channels, pumping of calcium out of the cytosol via membrane and ER pumps, and release of LH. The extended model, presented in this paper, also includes the following physiologically important phenomena: desensitization of calcium channels; internalization of the dimerized receptors and recycling of some of the internalized receptors; an increase in Gq concentration near the plasma membrane in response to receptor dimerization; and basal rates of synthesis and degradation of the receptors. With suitable choices of the parameters, good agreement with a variety of experimental data of the LH release pattern in response to pulses of various durations, repetition rates, and concentrations of GnRH were obtained. The mathematical model allows us to assess the effects of internalization and desensitization on the shapes and time courses of LH response curves.

We study the improvement in timing accuracy in a neural system having n identical input neurons projecting to one target neuron. The n input neurons receive the same stimulus but fire at stochastic times selected from one of four specified probability densities, f, each with standard deviation 1.0 msec. The target cell fires if and when it receives m inputs within a time window of epsilon msec. Let sigma(n,m,epsilon) denote the standard deviation of the time of firing of the target neuron (i.e. the standard deviation of the target neuron's latency relative to the arrival time of the stimulus). Mathematical analysis shows that sigma(n,m,epsilon) is a very complicated function of n, m, and epsilon. Typically, sigma(n,m,epsilon) is a non-monotone function of m and epsilon and the improvement of timing accuracy is highly dependent of the shape of the probability density for the time of firing of the input neurons. For appropriate choices of m, epsilon, and f, the standard deviation sigma(n,m,epsilon) may be as low as 1/n. Thus, depending on these variables, remarkable improvements in timing accuracy of such a stochastic system may occur.

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.

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.

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 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+).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

L. Donovani promastigotes were grown to late-log and 3-day stationary phase to determine the level of protein tyrosine phosphatase activity in crude extracts and in fractions following gel filtration column chromatography. Over 90% of the activity was soluble in a low salt extraction buffer in both phases of growth. Several peaks of activity were resolved following gel filtration of the crude extracts indicating that multiple tyrosine phosphatases are present in these cells. Tyrosine phosphatase activity was lower in 3-day stationary than in late log-phase cells and a reduction in the major peak of activity, eluting in a gel fraction corresponding to an M(r) of approximately 168 kDa, was observed. In vivo tyrosine phosphorylation was revealed by Western blot analysis. The degree of phosphorylation of at least two proteins differed in cells obtained from late log phase cultures as compared with 3-day stationary phase cultures. These observations indicate that changes in the balance between tyrosine phosphorylation and dephosphorylation occur with increasing culture age.

Leishmania donovani promastigotes in late-stationary phase incorporated label from [2-14C]acetate and [1-14C]laurate into the mannose residues of mannan, thus confirming the presence of a functional glyoxylate bypass in these parasitic protozoa. Isolated, washed calls also incorporated label from [2-14C]acetate and [1-14C]laurate into mannan during a 1-hr incubation in buffer. Glucose had no effect on label incorporation into mannan, but glutamate caused over a four-fold increase in incorporation from [2-14C]acetate and a 2.4-fold increase from [1-14C]laurate. Staurosporine, a protein kinase inhibitor that inhibits glutamate and alanine oxidation, did not inhibit label incorporation from [2-14C]acetate into mannan. Hyperosmolality caused about a 33% inhibition of label incorporation into mannan. These results show the glyoxylate cycle and/or the subsequent biosynthetic pathway from fructose-6-phosphate to mannan are subject to regulation.

In the course of their existence, parasites develop several metabolic pathways that differ significantly from those of their hosts. Despite the fairly close evolutionary kinship between Leishmania donovani and Trypanosoma brucei, the forms that live in the insect vectors have evolved different strategies for the disposition of available food resources. In this brief review, Joseph Blum will focus on the data available from studies on Leishmania spp and will largely ignore the information available from Trypanosoma spp.

Leishmania donovani promastigotes were grown to late log phase, washed and resuspended in iso-osmotic buffer containing L-arginine, and the rate of urea formation was then measured under various conditions. Addition of glucose or mannose activated urea formation, whereas 2-deoxyglucose inhibited and 6-deoxyglucose had no effect. Addition of alanine or of alpha-aminoisobutyrate inhibited urea formation, alanine causing a greater inhibition than alpha-aminoisobutyrate. Addition of leucine, proline, glycine, or lysine had no effect on urea formation. The presence of glutamate also increased the rate of urea formation from arginine, but to a lesser extent than did glucose. The presence of both glucose and alanine caused no net change in urea formation, whereas the inhibitory effect of alanine exceeded the activating effect of glutamate, so that a small inhibition in the rate of urea formation occurred in the presence of both alanine and glutamate. Cells grown to 3-day stationary phase had a markedly reduced rate of arginine catabolism to urea, but the activating effect of glucose and the inhibitory effect of alanine were qualitatively similar to their effects on late log phase cells. Addition of water to cells suspended in buffer also inhibited urea formation, but this appeared to be due primarily to the release of alanine caused by the hypo-osmotic stress. Addition of mannitol to cells suspended in buffer caused a small inhibition of arginine catabolism. Addition of dibutyrylcyclic AMP, 3',5'-cyclic GMP, phorbol myristic acid, or A23187 had no effect on the rate of urea formation from arginine.(ABSTRACT TRUNCATED AT 250 WORDS)

Promastigotes from late-log phase cultures of Leishmania donovani were washed and resuspended in Hanks' Balanced Salt Solution without glucose or phenyl red but with 20 mM (N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid]) (HEPES) (HBSS-, 305 mOsm/kg). They were then added to a solution containing 86Rb such that the final osmolality and ionic composition was as desired. Samples were taken at known times and the amount of intracellular 86Rb was measured. Similarly, experiments were performed in which 86Rb was added to the cultures about 18 hr before collection, and the amount of 86Rb released from the washed cells was measured. Under iso-osmotic conditions only about 1.3% of the intracellular 86Rb was released in 900 sec. This increased about 4-fold if the osmolality was reduced from 305-153 mOsm/kg. This is much slower than the very rapid release of alanine in response to hypo-osmotic stress, indicating that alanine release is not via a non-specific pore. Reducing the temperature from 26 degrees C to 3-4 degrees C completely inhibits 86Rb release under iso-osmotic conditions and largely inhibits it under hypo-osmotic conditions. The rate of 86Rb release was not sensitive to K+ concentration and was not altered if chloride was replaced by sulfamate. Ouabain had no effect on either 86Rb uptake or release, but carbonylcyanide P-trifluoromethoxyphenylhydrazone (FCCP) reduced the rate of 86Rb release and, after about a 300 sec exposure, completely inhibited 86Rb uptake. Amiloride partially inhibited 86Rb release, but had no effect on uptake. A decrease in pH from 7.1-5.9 had little effect on 86Rb release under iso-osmotic conditions and slightly increased the rate of release under hypo-osmotic conditions, but it decreased the rate of uptake under both iso-osmotic and hypo-osmotic conditions. Cells taken from 3-day stationary phase cultures released 86Rb more slowly under iso-osmotic conditions than cells from late log phase cultures, but were more responsive to hypo-osmotic stress than were log phase cells. These data appear to rule out an [Na-K-Cl] transporter or a [K-Cl] cotransporter as the means of K+ release, but are consistent with the possibility that a K+/H+ exchanger is present. The possibility that other carrier systems may be present is also discussed.

Promastigotes from late log phase and 3-day stationary phase cultures of Leishmania donovani were collected, washed in buffer, and the cell pellet was treated with boiling KOH. A putative carbohydrate storage material was then precipitated and washed in ethanol/LiBr. This material did not liberate glucose when treated with amyloglucosidase, indicating that it was not glycogen. Acid hydrolysis released a hexose which was identified as mannose by several criteria. Considerably more of this mannan-like carbohydrate is present in cells from 3-day stationary phase than from late log phase cultures, consistent with the ability of 3-day stationary phase cells to survive in non-nutrient buffer and maintain oxygen consumption for longer than log phase cells. The amount of this mannan-like compound decreased by over 50% during a 3-h incubation in buffer of cells from 3-day stationary phase cultures. The presence of glucose during the incubation prevented the utilization of this carbohydrate, consistent with the possibility that it serves as an energy reserve.

We modify our previous mathematical model of axonal transport to analyze data on the fast transport of lipids in rat sciatic nerve given in Toews et al. (J. Neurochem. 40, 555-562 (1983)). The theoretical model accounts well for the shapes of the profiles of phosphatidylcholine, phosphatidylethanolamine, cholesterol and diphosphatidylglycerol. The parameters obtained support the qualitative conclusions of Toews et al. and provide quantitative estimates of the underlying processes, e.g., rates of vesicle and mitochondria translocation, rate constants for association and dissociation between vesicles, kinesin and microtubules, rates of deposition and rates of loss of each class of lipid from the nerve by leakage or via removal by the retrograde transport system. The analysis suggests that two classes of vesicles moving at different speeds may be involved in the transport of phosphatidylcholine and phosphatidylethanolamine.

This study further explores the effects of hypoxia and acute osmotic stress on intermediary metabolism of Leishmania major and Leishmania donovani. Late log phase promastigotes were washed and incubated with glucose as the sole exogenous carbon source, and rates of glucose consumption and product formation were measured as a function of osmotic strength (610, 305, and 167 mOsm kg-1) and pO2 (95, 10, and 0% O2) in the presence of 5% CO2. Very mild hypoxia dramatically altered flux through the pathways of intermediary metabolism and increased the rates of production of the major metabolites, thus confirming the presence of a low-affinity O2 sensor which was active under all osmolalities tested. The data also require that as pO2 is lowered towards anoxia an endogenous carbohydrate source is mobilized. Under aerobic conditions, acute hypo-osmotic stress had little effect on product formation, whereas acute hyperosmotic stress altered metabolism in a manner similar to mild hypoxia, with the exception of decreasing the rates of acetate and succinate production. It was also shown in L. donovani promastigotes that the effects of anoxia and hyperosmolality were not additive. Thus, separate sensors with partially overlapping actions are involved in the metabolic responses to hypoxia and hyperosmolality. There was no apparent species-specificity for the responses to pO2 and osmotic stress. Uncoupling with carbonyl cyanide p-trifluoromethoxyphenylhydrazone caused changes in metabolite flux patterns which differed from the changes caused by either hypoxia or acute osmotic stress, while rotenone and calcium ionophore A23187 had no significant effects. The identity of the sensors responsive to pO2 and osmolality, and the mechanisms by which they regulate flux through the pathways of intermediary metabolism, require further study.

The metabolism of leucine by Leishmania donovani was investigated. Washed promastigotes were incubated with [1-14C]- or [U-14C]leucine or [1-14C]alpha-ketoisocaproate (KIC) and 14CO2 release was measured. The amount of KIC-derived acetyl-CoA oxidized in the citric acid cycle was computed. Promastigotes from mid-stationary phase cultures oxidized each of these labeled substrates less rapidly than cells from late log phase cultures, and significantly less acetyl-CoA derived from KIC oxidation was oxidized in the citric acid cycle. Glucose was a stronger inhibitor than was acetate of CO2 formation in the citric acid cycle in log phase promastigotes, but the reverse was observed in cells from mid-stationary phase. Alanine also inhibited leucine catabolism, but glutamate had little effect. Acute hypo-osmotic stress did not affect leucine catabolism, but hyper-osmotic stress caused appreciable inhibition of leucine oxidation. Cells grown under hypo- or hyper-osmotic conditions showed no changes in the effects of hypo- or hyper-osmotic stress on leucine catabolism, i.e. L. donovani is not an osmoconformer with respect to leucine metabolism. Leucine utilization in L. donovani was insensitive to a number of drugs that affect leucine metabolism in mammalian cells, indicating that the leucine pathway in L. donovani is not regulated in the same manner as in mammalian cells.

A number of investigators have published measurements of the outputs of single neurons from the lateral superior olive (LSO) of the cat for a variety of auditory signals. The response curves show a very wide range of shapes and thresholds. In this paper, the single neuron response curves predicted by a previously published model for the encoding of azimuthal location by the LSO to the experimental curves are compared. The predicted curves are in good qualitative agreement with the experiments and, in addition, the model provides an explanation of the seemingly paradoxical drop in output as interaural intensity difference (IID) is held fixed and absolute intensities are raised. The particular shape of the response curve depends on the location of the LSO neuron examined. In the model, two possible developmental programs to form the adult pattern of connections from the anteroventral cochlear nucleus and the medial nucleus of the trapezoid body onto the LSO are also examined. In the first, connections are made by a forward stochastic process in which there is a limit on the numbers of synapses formed on each LSO cell. In the second, there is no such limit, but in later developmental stages pruning of synapses occurs which reduces their number to the limit. Both schemes give similar adult connectional patterns. The LSO response curves generated by the model are similar to those observed in LSO neurons of the developing gerbil by Sanes and Rubel [J. Neurosci. 8, 682-700 (1988)]. Thus the model mechanism not only encodes azimuthal location by activity across the population of neurons, but is also consistent with single unit neurophysiological measurements of LSO output in both developing and adult animals.

Leishmania major promastigotes were grown to late log phase, washed and resuspended in Hanks' balanced salt solution, and incubated with glucose at various pO2s in the presence of 5% CO2. Samples were taken at times from 0-40 min and assayed for fructose 2,6-bisphosphate (Fru(2,6)P2), glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), phospho(enol)pyruvate (PEP), and ATP. At 95% O2 ATP remained constant throughout the incubation. It did not decrease significantly at 10% O2, but decreased by about 20% and 30% at 6% and 0% O2, respectively. At 95% O2, Fru(2,6)P2 increased about 15-fold within 5 min after the addition of glucose and remained at this high level. At 10%, 6%, and 0% O2 Fru(2,6)P2 rose about 5-fold within 5 min and then declined slightly during the remainder of the incubation. G6P increased from about 0.5 to 12 nmol (mg protein)-1 at 5 min in cells incubated under 95% O2 and then declined to about 5 nmol (mg protein)-1. It increased to about 8 nmol (mg protein)-1 at 5 min and then declined slightly in cells incubated under 10% O2. F6P levels were approximately one-eighth of G6P levels under all conditions, suggesting that phosphohexoseisomerase was not subject to regulation. PEP levels were initially high, but at 95% O2 there was a 50% drop in PEP at 5 min, while at 10%, 6%, and 0% O2 there was less of a decline. The observation that the rise in Fru(2,6)P2 levels at 10%, 6%, or 0% O2 is the same at 5 min and less than the rise at 95% O2 supports the presence of a low affinity oxygen sensor. The different time course of changes in G6P, F6P, and PEP levels suggests that in addition to an activation of pyruvate kinase by Fru(2,6)P2, other regulatory events are also operative at low pO2.

Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer. The rate of oxidation of 14C-labeled substrates was then measured as a function of osmolality. An acute decrease in osmolality (achieved by adding H2O to the cell suspension) caused an increase in the rates of 14CO2 production from [6-14C]glucose and, to a lesser extent, from [1,(3)-14C]glycerol. An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of 14CO2 production from [1-14C]alanine,[1-14C]glutamate, and [1,(3)-14C]glycerol. The rates of 14CO2 formation from [1-14C]laurate,[1-14C]acetate, and [2-14C]glucose (all of which form [1-14C]acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality. These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress. Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.

Earlier studies showed that Leishmania major promastigotes are sensitive to osmotic conditions. A reduction in osmolality caused the cells to shorten and to rapidly release most of their large internal pool of alanine. In this study some effects of hyper-osmotic stress were examined. An increase in osmolality of the culture medium from 308 to 625 mOsm/kg caused only a small decrease in growth rate. When cells grown in the usual culture medium (308 mOsm/kg) were washed, resuspended in iso-osmotic buffer, and subjected to acute hyper-osmotic stress by addition of mannitol, the alanine content increased even in the absence of exogenous substrate. Promastigotes, depleted of alanine by a 5-min exposure to hypo-osmotic conditions, also synthesized alanine when resuspended in iso-osmotic buffer. Washed cells resuspended in iso-osmotic buffer consume their internal pool of alanine under aerobic conditions. Rates of consumption decreased on addition of mannitol, becoming zero at about 440 mOsm/kg. At higher osmolalities, alanine synthesis occurred. To estimate whether proteolysis could account for alanine synthesis in the absence of exogenous substrate, cells that had been grown with [1-14C]leucine were washed and resuspended under hypo-, iso-, and hyper-osmotic conditions and the amounts of 14CO2 and 14C-labelled peptides released in 1 h were measured. Little proteolysis occurred under these conditions, but the possibility that proteolysis was the source of the alanine increase, observed in response to hyper-osmotic stress, cannot be ruled out.

Leishmania major promastigotes were grown to late-log phase and washed and resuspended in an isosmotic buffer. When osmolality was suddenly decreased by 50%, the cells rapidly became shorter and increased in width. Cell volume, calculated assuming a prolate-ellipsoidal shape, increased 1.4 times after 1 min. Over the next several minutes, the average length and width returned to control values while the volume returned to baseline, indicating the ability to regulate volume. Concomitantly with the swelling, large amounts of alanine and other ninhydrin-positive substances were released. All of the alanine pool was released within 1 min after reduction of the osmolality by 66%. Cells pre-loaded with [14C]-aminoisobutyric acid also released it very rapidly upon hypo-osmotic stress. Release of ninhydrin-positive substances resulted from decreased osmolality rather than changes in ionic composition. The same results were obtained if osmolality was decreased by reducing only the NaCl content of the buffer instead of diluting it with water, and mannitol could substitute for the NaCl. Promastigotes were able to grow well over several days in media as low as 154 mOsm/kg. The nature of the signalling mechanisms(s) that initiates the rapid shape change and efflux of ninhydrin-positive substances in response to hypo-osmotic stress is at present unknown.

The effect of culture age on the rate of oxidation of short-, medium, and long-chain fatty acids by Leishmania major promastigotes was investigated. Promastigotes from 5-day stationary phase cultures oxidized several saturated fatty acids about 3-to-4-fold faster than cells from late log phase cultures, but [10-14C]oleate was oxidized 9-fold faster. The increase in rate of oxidation was partially reversed within 5 h and almost completely reversed within 30 h after resuspending cells from a 5-day stationary culture in fresh medium. Addition of acetate, leucine, or alanine caused moderate inhibitions of [1-14C]palmitate oxidation, while glycerol had little effect. Glucose, however, was a powerful inhibitor of the oxidation of [1-14C]palmitate and of [1-14C]octanoate. Mannose and fructose were also strong inhibitors of palmitate oxidation, but neither galactose, 2-deoxyglucose or 6-deoxyglucose caused appreciable inhibition. The extent of inhibition by acetate increased with increasing culture age, whereas inhibition by glucose decreased. In addition to demonstrating a reversible rise in beta-oxidation capacity with culture age, these data also demonstrate a hitherto unrecognized strong and culture age-dependent inhibition of fatty acid oxidation by glucose.

A structural model is proposed for the processing of interaural intensity differences by the lateral superior olive. One fundamental assumption is that the incoming excitatory projections from the ipsilateral anteroventral cochlear nucleus innervate columns of LSO neurons serially according to threshold. A second fundamental assumption is that the inhibitory innervation from the ipsilateral medial nucleus of the trapezoid body is also serially arranged according to threshold but in the opposite direction along the LSO column. Using neurophysiological and neuroanatomical data for neuronal response curves, connectional patterns, and cell and synapse numbers, the model was formulated quantitatively and implemented for machine computation. Azimuthal location is encoded by the position along the LSO column where LSO cell firing first goes to zero. Accuracy of coding was tested for three different connectional schemes, for variations in neuronal parameters, and for cell and synapse death. Encoding is shown to be independent of absolute sound level and to vary linearly with interaural intensity difference.

Leishmania major promastigotes in late-log phase are generally long and slender, and remain so during a 1 h incubation in buffer without exogenous substrate. When glucose, 2-deoxyglucose, fructose, mannose, or proline are added, the cells become shorter and more rounded. The shape change in response to glucose is complete within 20 min and is reversible upon incubating the cells without substrate. Galactose, 3-O-methylglucose, 6-deoxyglucose, sucrose, maltose, ribose, glycerol, alanine, glutamate or aspartate do not cause the shape change. Decreasing the osmolarity of the medium causes a rounding of the cells similar to that observed in the presence of glucose, and increasing the osmolarity inhibits the shape change in response to glucose. Inhibitors of glucose transport and 2nd messenger analogs do not affect the shape change.

Leishmania major promastigotes contain electron-dense vacuoles. The elemental composition of these vacuoles and of the cytoplasm was measured by electron probe X-ray microanalysis, using rapid cryopreservation techniques to prevent alterations in composition due to diffusion. The electron-dense vacuoles are rich in P, presumably present as polyphosphate (poly P). Mg is present at about 9 times its cytoplasmic level. There is sufficient Mg to largely neutralize most of the negative charge of the Poly P. The electron-dense vacuoles also contain appreciable amounts of Ca and Zn, which are not detectable in the cytoplasm, as well as Na, K, and Cl, the latter two at concentrations below that of the cytoplasm. These results suggest that the vacuolar membranes have at least one cation transport system. Incubation of the promastigotes for 1 h in the absence of phosphate in the presence or absence of glucose did not cause significant changes in the vacuolar contents of P, Mg, or Zn, but changes in K and Cl content were observed in both the electron-dense vacuoles and in the cytoplasm.

Leishmania major promastigotes grown in late log phase were incubated with glucose as sole exogenous carbon source in the presence of 5% CO2 and the amounts of glucose consumed and of the major products formed--succinate, pyruvate, alanine, acetate, glycerol, and D-lactate--were measured as a function of pO2. Glucose consumption increased as pO2 was lowered to 6% (a positive Pasteur effect) and then declined to the same level at 95% N2 as at 95% O2. The production of D-lactate and of glycerol increased as pO2 dropped from 95%, reaching a maximum at about 2% O2. Succinate production, however, increased dramatically when pO2 was reduced to 6% and remained at that level with further reduction of pO2. The amount of succinate produced relative to the amount of glucose carbon consumed suggests utilization of an endogenous carbon source. Acetate production did not change between 95% O2 and 6% O2 and then declined with decreasing pO2. These observations suggest the presence of two sensors, one with a high and one with a low affinity for oxygen. When glycerol or alanine were the only exogenous sources of carbon, the primary products released were acetate and succinate. Acetate production from alanine declined slightly as pO2 was reduced to 2%, and then dropped markedly when pO2 was reduced to 0%. Acetate production from glycerol increased over 4-fold when the pO2 was reduced from 95% to 4%, and then declined with further reduction in pO2. No succinate was formed from either substrate until complete anaerobiosis. This pattern of response, while differing from that when glucose was sole exogenous carbon source, is also consistent with the regulation of metabolism by a high and a low affinity O2 sensor. Cells from cultures in early stationary phase, before the appearance of metacyclic forms, consumed glucose at about the same rate as log phase promastigotes, but did not show a Pasteur effect. Stationary cells also consumed glycerol at the same rate as did log phase promastigotes, but consumed alanine at a much lower rate. Reduction of pO2 affected product formation from each of these substrates differently than for log phase promastigotes, demonstrating the sensitivity of several pathways of intermediary metabolism to regulation by pO2 during the transition from log to stationary phase.