Target identification by image analysis.

Covering: 1997 to the end of 2015Each biologically active compound induces phenotypic changes in target cells that are characteristic for its mode of action. These phenotypic alterations can be directly observed under the microscope or made visible by labelling structural elements or selected proteins of the cells with dyes. A comparison of the cellular phenotype induced by a compound of interest with the phenotypes of reference compounds with known cellular targets allows predicting its mode of action. While this approach has been successfully applied to the characterization of natural products based on a visual inspection of images, recent studies used automated microscopy and analysis software to increase speed and to reduce subjective interpretation. In this review, we give a general outline of the workflow for manual and automated image analysis, and we highlight natural products whose bacterial and eucaryotic targets could be identified through such approaches.

Antiviral drug discovery: broad-spectrum drugs from nature.

Covering: up to April 2014. The development of drugs with broad-spectrum antiviral activities is a long pursued goal in drug discovery. It has been shown that blocking co-opted host-factors abrogates the replication of many viruses, yet the development of such host-targeting drugs has been met with scepticism mainly due to toxicity issues and poor translation to in vivo models. With the advent of new and more powerful screening assays and prediction tools, the idea of a drug that can efficiently treat a wide range of viral infections by blocking specific host functions has re-bloomed. Here we critically review the state-of-the-art in broad-spectrum antiviral drug discovery. We discuss putative targets and treatment strategies, with particular focus on natural products as promising starting points for antiviral lead development.

The chondramides: cytostatic agents from myxobacteria acting on the actin cytoskeleton.

BACKGROUND: Chondramides are cyclodepsipeptides produced by strains of the myxobacterium, Chondromyces crocatus. These peptides, which have been reported to inhibit yeast and mammalian cell proliferation, are related to jasplakinolide, which has been isolated from marine sponges of the genus Jaspis and has been shown to interfere with the actin cytoskeleton (a structural component of cells that helps maintain their shape and is involved in processes, such as cell division and cell locomotion). We studied the effects of the chondramides (A, B, C, and D) on tumor cell growth, on cytoskeletal structure, and on actin polymerization in vitro and compared these effects with those of cytochalasin D and jasplakinolide. METHODS: Cell proliferation was measured by means of tetrazolium salt reduction assays. Effects on the cytoskeleton were studied by use of fluorescence techniques, and actin polymerization in vitro was measured by means of viscosimetry. RESULTS: Proliferation of tested tumor cell lines was inhibited by the chondramides. Concentrations that inhibited proliferation by 50% (IC50 values) ranged from 3 to 85 nM and were of the same order of magnitude as those found for cytochalasin D and jasplakinolide. Fluorescence staining of potoroo cells incubated with chondramides A and B showed that organization of the actin cytoskeleton was disrupted; however, the microtubule system was not affected. Viscosimetric measurement showed that, depending on the experimental conditions, chondramide A induced or accelerated actin polymerization in vitro. CONCLUSION: The chondramides--unlike jasplakinolide--can be produced in large amounts by fermentation, and, similar to jasplakinolide, they appear to have antiproliferative activity against carcinoma cell lines by targeting the actin cytoskeleton.

Epothilone B stabilizes microtubuli of macrophages like taxol without showing taxol-like endotoxin activity.

Epothilones are a new class of potential antitumor compounds that were isolated from the myxobacterium Sorangium cellulosum. Epothilones have effects on the cytoskeleton similar to those of the antineoplastic drug Taxol. Both compounds inhibit cell proliferation by stabilizing microtubuli, and they compete for the same binding site. In addition, Taxol displays endotoxin-like properties in that it activates macrophages to synthesize proinflammatory cytokines and nitric oxide. We measured nitric oxide release by IFN-gamma-treated murine macrophages as an indicator of macrophage activation by epothilone B. Although epothilone B showed the expected effects on the microtubuli, there was no indication of macrophage stimulatory activity by epothilone B, nor did epothilone B inhibit lipopolysaccharide-mediated nitric oxide release. We conclude that, unlike Taxol, epothilone-mediated microtubuli stabilization does not trigger endotoxin-signaling pathways. Moreover, because the endotoxin-like activity of Taxol may be the cause of some nonhematological clinical side effects, it is to be expected that such effects may not occur with epothilones.

Gephyronic acid, a novel inhibitor of eukaryotic protein synthesis from Archangium gephyra (myxobacteria). Production, isolation, physico-chemical and biological properties, and mechanism of action.

A new antibiotic compound, gephyronic acid was isolated from the culture broth of the myxobacterium, Archangium gephyra strain Ar 3895. Up to 3 mg/liter was produced during the logarithmic and stationary growth phase. The compound is an aliphatic acid, which tends to form a hemiacetal. Both forms inhibited growth of yeasts and molds (MIC 1-25 micrograms/ml) and had a cytostatic effect on mammalian cell cultures (IC50 10-60 ng/ml). Gephyronic acid is a specific inhibitor of eukaryotic protein synthesis showing an IC50 of 1-2 x 10(-7) mol/liter in an in vitro translation assay.

Rhizopodin, a new compound from Myxococcus stipitatus (myxobacteria) causes formation of rhizopodia-like structures in animal cell cultures. Production, isolation, physico-chemical and biological properties.

A new cytostatic compound, rhizopodin, was isolated from the culture broth of the myxobacterium, Myxococcus stipitatus. The compound inhibited growth of various animal cell cultures without killing the cells. The ID50, measured by an MTT assay, was 12 approximately 30 ng/ml, depending on the cell line. Especially cells growing fibroblast-like showed typical morphological changes. They became larger and within hour formed long branching and reticular runners. These morphological changes were irreversible. Rhizopodin suppresses bleb formation in K-562 cells, and therefore could act by interacting with protein phosphorylation.

Chondramides A approximately D, new antifungal and cytostatic depsipeptides from Chondromyces crocatus (myxobacteria). Production, physico-chemical and biological properties.

Novel depsipeptides, named chondramides were produced at levels up to 4.3 mg/liter by several myxobacteria of the genus Chondromyces. The compounds are structurally closely related to jaspamide/jasplakinolide from marine sponges of the genus Jaspis. Initially the chondramides were detected in acetone extracts of the biomass of Chondromyces crocatus, strain Cm c2. So far, four structural variants could be characterized, the chondramides A approximately D. They inhibited the growth of a few yeasts and showed high cytostatic activity against cultivated human and animal cells.

Apicularens A and B, new cytostatic macrolides from Chondromyces species (myxobacteria): production, physico-chemical and biological properties.

A novel macrolide, apicularen A, was produced by several species of the genus Chondromyces. Initially it was discovered by bioassay-guided RP-HPLC-fractionation of culture extracts of Chondromyces robustus, strain Cm a13. Apicularen A showed no antimicrobial activity, but was highly cytotoxic for cultivated human and animal cells, with IC50 values ranging between 0.1 and 3 ng/ml. A cometabolite of apicularen A, the N-acetylglucosamine glycoside apicularen B, was distinctly less cytotoxic with IC50 values between 0.2 and 1.2 microg/ml, and showed weak activity against a few Gram-positive bacteria. Apicularen A is chemically closely related to the salicylihalamides A and B from the marine sponge Haliclona sp.

Tubulysins, new cytostatic peptides from myxobacteria acting on microtubuli. Production, isolation, physico-chemical and biological properties.

New cytostatic compounds, tubulysins, were isolated from the culture broth of strains of the myxobacteria Archangium gephyra and Angiococcus disciformis. The compounds are peptides partly consisting of unusual amino acids and are distantly related to the dolastatins. The tubulysins were not active against bacteria and only little against fungi, but showed high cytostatic activity against mammalian cell lines with IC50 values in the picomolar range. An incubation with 50 ng/ml tubulysin A led to a complete disappearance of the microtubuli network of the cells within 24 hours. The more active tubulysin D induced multipolar spindles: At 0.5 ng/ml all mitotic cells showed more than four spindle poles.

Melithiazols, new beta-methoxyacrylate inhibitors of the respiratory chain isolated from myxobacteria. Production, isolation, physico-chemical and biological properties.

New antibiotic compounds, melithiazols, were isolated from the culture broth of strains of the myxobacteria Melittangium lichenicola, Archangium gephyra, and Myxococcus stipitatus. The compounds belong to the group of beta-methoxyacrylate (MOA) inhibitors and are related to the myxothiazols. The melithiazols show high antifungal activity, but are less toxic than myxothiazol A and its methyl ester in a growth inhibition assay with mouse cell cultures. The melithiazols inhibit NADH oxidation by submitochondrial particles from beef heart. Melithiazol A blocks the electron transport within the bc1-segment (complex III) and causes a red shift in the reduced spectrum of cytochrome b.

Cytotoxic effect of atrazine on murine B-lymphocytes in vitro.

When a stable cell line from mouse connective tissue was used to test the cytotoxicity of atrazine no significant influence on cell proliferation was observed at concentrations of 0.003-200 microM. However, freshly isolated mouse spleen cells reacted very sensitively to the presence of atrazine. During the course of the experiment the spleen cells were pretreated with atrazine (0.05-5 microM) before the proliferation of B-lymphocytes was stimulated with LPS. A reduced number of developing B-blasts was observed in the atrazine pretreated cultures. The B-blasts were fused with myeloma cells and cultivated in HAT (hypoxantine, aminopterin,thymidine)-medium for hybridoma selection. The number of surviving clones was significantly lower on atrazine pretreatment.

Effects of halothane dose and stimulus rate on canine spinal, far-field and near-field somatosensory evoked potentials.

Evidence that canine spinal, far-field and near-field somatosensory evoked potentials resemble those recorded in humans and other species has been presented, and the vulnerability of each component to varying depths of halothane anesthesia is reported. Lumbar spinal peak latencies are not affected by halothane dose, but the negative peak is significantly prolonged by rapid rates of stimulation. Elevated stimulus rates and halothane doses reduce lumbar spinal cord potential amplitudes. Early far-field cephalic components are refractory to halothane. Late far-field components and near-field cortical potentials are substantially altered by increments in halothane dose. Both near-field and far-field responses are more readily identified in vertex-neck than vertex-brow derivations. Early far-field somatosensory evoked potentials recorded from vertex to neck, together with lumbar spinal cord potentials, may be the preferred monitoring technique when the use of halothane anesthesia is desired. Rapid rates of stimulation may facilitate earlier recognition of cord dysfunction, but supplement rather than replace baseline recordings at slow stimulus rates.

Accumulation of beta-Carboline Alkaloids and Serotonin by Cell Cultures of Peganum harmala L: I. CORRELATION BETWEEN PLANTS AND CELL CULTURES AND INFLUENCE OF MEDIUM CONSTITUENTS.

A number of cell cultures of Peganum harmala were initiated to check for a correlation between the harman alkaloid content of seedlings and cell lines derived therefrom. Despite a poor correlation between callus or suspension culture lines and parent plants, the mean alkaloid contents of strains derived from seedlings with higher alkaloid yields were nevertheless higher than the mean contents of strains derived from low yield plants. Generally, alkaloid accumulation decreased with the numbers of transfers. By permanent visual selection for fluorescent areas of the calluses, however, a mean content of 0.1% harman alkaloids and 0.1% serotonin could be maintained, which was 10 times higher than in unselected callus cultures.The effects of medium constituents on harman alkaloid and serotonin accumulation were measured for a low yielding, faster growing suspension culture strain and a slowly growing, but high yielding cell line. This led to the development of a production medium without 2,4-dichlorophenoxyacetic acid and phosphate, and with Ca, Mg, and nitrate as sole macronutrients. When this production medium was used, the accumulation of harman alkaloids and serotonin was increased from 0.1 to 1% in the low yielding cell line and from 1 to 2% in the high yielding strain.

Biotransformation of Tryptamine to Serotonin by Cell Suspension Cultures of Peganum harmala.

Cell suspension cultures of P. HARMALA, unable to form serotonin DE-NOVO, hydroxylated fed tryptamine to serotonin independently of the growth cycle up to 20 mg per g dry mass per day. The best biotransformation yield of 80% was obtained with a high cell density (30 g fresh mass/100 ml) and a 5 mM tryptamine concentration in the transformation medium. Under these conditions the cells accumulated serotonin up to 7.5% per unit dry mass, and a maximum yield of 800 mg serotonin/l was achieved. Other indoleethylamines such as alpha-methyltryptamine, N-methyltryptamine, and 6-fluorotryptamine were also transformed to the corresponding 5-hydroxyl compounds, while N,N-dimethyltryptamine, 7-methyltryptamine, tyramine, and phenylethylamine were not hydroxylated.

Automatic control of anesthesia using two feedback variables.

A new controller of an ON/OFF type was implemented for halothane anesthesia. A proportional-plus-integral controller with time-delay compensation proved not to be robust enough for the known clinical situation, as shown both in computer simulations and in animal trials. The ON/OFF controller proved to be less sensitive to parameter mismatches, and repeated animal trials showed a short response time and acceptable steady-state tracking. A method for switching the controlled effect of the drug was also developed, since anesthetic agents have multiple effects. Mean arterial blood pressure and a measure of EEG frequency were chosen as controlled variables, both being depressed by halothane. A coordinator forces the system state as near the desired values of these variables as possible, given that only one drug is used.

Formation of beta-peltatin-A methyl ether and coniferin by root cultures of Linum flavum.

Extracts of root cultures of Linum flavum contained high cytotoxic activity due to the presence of 1% beta-peltatin-A methyl ether of the dry mass. During chromatographic analysis of the cell extracts, coniferin was identified as the major uv-absorbing but noncytotoxic constituent with levels of up to 3% of the dry mass. Growth, culture appearance, and product accumulation varied greatly with the 2,4-D concentration in the medium.

Effects of hypocapnia on canine spinal, subcortical, and cortical somatosensory-evoked potentials during isoflurane anesthesia.

Although hyperventilation with hypocapnia is frequently used in the management of neurosurgical patients in whom sensory-evoked potentials may be monitored, the effects of hypocapnia on evoked potentials have not been described with precision. In the present experiment, the effects of randomized arterial carbon dioxide tensions of 20, 25, 30, and 35 mm Hg on spinal, subcortical, and cortical somatosensory-evoked potentials (SEPs) were measured in dogs anesthetized with 1.40% isoflurane. Other variables known to affect the SEP (temperature, blood pressure, and arterial oxygen tension) were stable throughout the experiment. Hypocapnia caused reductions in the latencies of the early peaks of the spinal and subcortical SEPs. These differences were small, consisting of a 2% shortening of latency at 20 mm Hg carbon dioxide tension when compared with 35 mm Hg. No changes were detected in the later subcortical and cortical latencies. SEP amplitudes were also unchanged. These results in a controlled animal study corroborate the direction and magnitude of changes due to hypocapnia observed by other investigators in surgical patients. The magnitude of the changes indicates that SEP monitoring sensitivity is not compromised by clinically useful levels of induced hypocapnia during isoflurane anesthesia. Because hypocapnia may produce small SEP changes, baseline recordings should be acquired prior to initiation of hyperventilation. It is not warranted, however, to impute a severe deterioration of the SEP to hypocapnia alone, and causes must be sought elsewhere in a patient's status and management.

Use of positive airway pressure without endotracheal intubation.

Continuous positive airway pressure (CPAP) and expiratory positive airway pressure (E-PAP) may be used safely without endotracheal intubation in patients with acute respiratory failure when strict selection criteria are adhered to. The therapy should be titrated to reduce intrapulmonary shunting, improve PaO2, and reduce FIO2. Other considerations include balancing oxygen consumption against cardiac output and oxygen transport. Absolute or relative indications for abandoning the technique and using endotracheal intubation with mechanical ventilatory support include unrelenting hypoxia, patient exhaustion, rising PaCO2, development of metabolic acidosis, presence of ventricular arrhythmias, and inability to protect the airway.