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Reactive intermediate enzyme complexes are difficult to study directly and the use of physical methods requiring observation periods of more than a second has not been possible heretofore. Here we introduce a simple approach, the "Le Chatelier forcing method" which does for the first time produce significant concentrations of such kinetically competent central intermediates observable for extended periods of time. The method involves only the forcing of the accumulation of intermediate complexes at thermodynamic equilibrium by the use of high reactant concentrations working against a high concentration of a product, combined with a valid and applicable method of analysis. We demonstrate this approach using the glutamate dehydrogenase catalyzed reaction with the reaction product ammonia as a "dam" to oppose the forward driving force of NADP and l-glutamate. We demonstrate the accumulation of substantial amounts measurable amounts of stable enzyme-NADPH-alpha-carbinolamine and alpha-iminoglutarate complexes in three different alpha-amino acid dehydrogenases. We describe the manipulation of such Le Chatelier forced equilibria to increase the prominence of particular species and discuss the implications of these findings for previously unattainable experimental approaches.

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The efficacy and toxicity of sodium stibogluconate (SSG) at a dosage of 20 mg/(kg.d) for either 20 days (for cutaneous disease) or 28 days (for visceral, mucosal, or viscerotropic disease) in the treatment of leishmaniasis is reported. Ninety-six U.S. Department of Defense health care beneficiaries with parasitologically confirmed leishmaniasis were prospectively followed for 1 year. One patient was infected with human immunodeficiency virus; otherwise, comorbidity was absent. Clinical cure occurred in 91% of 83 cases of cutaneous disease and 93% of 13 cases of visceral/viscerotropic disease. Adverse effects were common and necessitated interruption of treatment in 28% of cases, but they were generally reversible. These included arthralgias and myalgias (58%), pancreatitis (97%), transaminitis (67%), headache (22%), hematologic suppression (44%), and rash (9%). No subsequent mucosal leishmaniasis was identified, and there were no deaths attributable to SSG or leishmaniasis.

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Many species of the west African "soap tree" Dracaena are used in traditional medicine for the treatment of a variety of diseases. In continuation of our search for anti-infective agents from plants implicated in traditional medicine, we evaluated the biological activities of saponins from extracts of Dracaena mannii and Dracaena arborea by using a battery of test systems such as radiorespirometry, Cytosensor bioautography, and agar dilution methods and molluscicidal tests. Bioassay-directed fractionation of the methanol extracts of seed pulp using a combination of chromatographic techniques, gel filtration, droplet countercurrent chromatography (DCCC), and low-pressure liquid chromatography (Lobar), led to the isolation and characterization of spiroconazole A, a pennogenin triglycoside [3 beta-O-[(alpha-L-rhamnopyranosyl(1-->2), alpha-L-rhamnopyranosyl(1-->3)-beta-D-glucopyranosyl]-17 alpha-hydroxyl-spirost-5-ene] (Fig. 1). As the active constituent, spiroconazole A exhibited pronounced antileishmanial, antimalarial, and molluscicidal activities. This paper also reports on the fungistatic, fungicidal and bacteriostatic activity of spiroconazole A against 17 species of fungi and 4 of bacteria.

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Extracts of eleven plants used in Nigerian traditional medicine have been evaluated for possible antileishmanial activity using a radiorespirometric microtest technique based on in vitro inhibition of catabolism of 14CO2 from a battery of 14C-substrates by promastigotes. Of 13 methanol extracts tested, 5 from Gongronema latifolia, Dorstenia multiradiata, Picralima nitida, Cola attiensis, and Desmodium gangeticum, were active at concentrations of 50 micrograms/ml or less against a visceral Leishmania isolate.

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Quantitative in vitro drug sensitivity of 32 Leishmania isolates (16 from patients failing pentavalent antimony [SbV] therapy) was determined using a radiorespirometric microtechnique (RAM). Of 30 isolates with histories, 22 (73%) RAM tests agreed with patient history; the remaining 8 (27%) did not. There was no difference in RAM drug sensitivity: clinical correlation between 15 isolates tested blindly and 15 with known clinical history (4 did not agree with clinical history in both). Test sensitivity appeared to be limited only by the sensitivity of the Leishmania to SbV and could be detected at 2 micrograms/ml Sb (about 10% of serum drug level). An isolate from a patient with untreated self-healing cutaneous disease was drug resistant. Using RAM, parasite drug sensitivity can be quantified apart from patient physiologic and immunologic variables intrinsic to clinical data. Potency differed a maximum of 100% (weight% Sb:weight% Sb) among drug lots and also between Glucantime and Pentostam. Potency changes between drug lots were not explainable based on Sb content or test-to-test variability. This microtest offers a rapid method for evaluating the drug sensitivity of patient isolates and for determining of the activity of pentavalent antimonials and other candidate anti-leishmanials prior to the initiation of therapy.

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We describe an in vitro microtest which is quantitative, rapid, and readily applicable to parasites isolated from all major forms of human leishmaniasis. It is based on drug-mediated inhibition of promastigote catabolism of a battery of simple 14C-substrates to 14CO2. Each test requires less than 1 microCi. The test is conducted in a serum-free, chemically defined medium containing 120 micrograms protein/ml, minimizing the possible interference of drug: serum protein interaction. Prior adaptation is not necessary to cultivate "difficult-to-grow" species. Leishmania sensitivity to pentavalent antimonials is detectable at micrograms levels below concentrations achievable in patients' sera.

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Football helmets have become safer in the past few years, but liability claims have increased. Manufacturers and school districts are considering solutions such as new designs, insurance coverage, and education programs.

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Electron cytochemical localizations of acid phosphatase, aryl sulfatase, deoxyribonuclease, adenylate cyclase, and c-AMP phosphodiesterase activity sites in thin sections of cells of the two growth phases of the zoopathogenic Histoplasma capsulatum are described and illustrated by transmission electron micrographs. Various activity sites of these enzymes included the cytomembranes of the nucleus, mitochondria, and endoplasmic reticulum. At the same time, electron opaque reaction products were sequestered within membrane-bound, vacuolar regions of the cytosol. These vacuoles may be ontogenically related to membranous or vesicular inclusions commonly seen in thin sections of glutaraldehyde osmium tetroxide-fixed cells. These enzymatically-active vacuoles are believed consistent with previous descriptions of fungal lysosomal-like structures found in certain other fungi. Lysosomal-like vacuoles of H. capsulatum may provide a means of compartmentalization of various hydrolytic enzymes involved in catabolism and mobilization of storage reserves, and perhaps to function as well in other aspects of the life cycle of this important pathogenic dimorphic fungus.

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Aspects of the fine structure as well as electron cytochemical localization studies of certain hydrolytic enzymes were examined by electron microscopy of ultrathin sections of the vegatative hyphae and conidia of the phycomycetous fungus, Entomophthora coronata. This entomogenous fungal organism is of interest since it has been increasingly implicated as the etiologic agent of phycomycosis of man and animals. On thin section, hyphal cells were frequently observed with septa while the cytoplasm was multinucleate. The conidium was bound by a multilayered cell wall. The cytoplasm of ungerminated conidia characteristically contained large numbers of a class of cytoplasmic organelle found in loose aggregates with lipid storage bodies. Similar organelles were observed in the cytoplasm of hyphal cells from 7-day old cultures. This round to oval to slightly reniform structure was bound by a single limiting membrane and composed of an electron dense, slightly granular matrix without evidence of crystalloid formation. The limiting membrane of the lipid storage bodies was observed to be intimately associated with that of one or more of these microbody-like organelles. This intimate association of the two cytoplasmic organelles suggests that the microbody-like organelle may be involved in some manner with lipid metabolism during the life cycle of the fungus. Cautious interpretations of electron cytochemical localization studies suggested that lipase, nonspecific esterase, and possibly aryl sulfatase were associated with the microbody-like organelles. Neither peroxidatic nor acid phosphatase activity could be demonstrated with these organelles of the conidial cytoplasm.

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