Isolation of Leu-Pro-Pro-Gly-Pro-Leu-Pro-Arg-Pro-NH2 (Antho-RPamide), an N-terminally protected, biologically active neuropeptide from sea anemones.

Using a radioimmunoassay against the C-terminal sequence Arg-Pro-NH2 (RPamide), we have isolated the peptide Leu-Pro-Pro-Gly-Pro-Leu-Pro-Arg-Pro-NH2 (Antho-RPamide) from an extract of the sea anemone Anthopleura elegantissima. Antho-RPamide is located in neurons of sea anemones. Application of low concentrations of Antho-RPamide to tentacle preparations of sea anemones strongly increased the frequency and duration of spontaneous contractions, suggesting that this peptide is involved in neurotransmission. Antho-RPamide has a free N-terminus, yet its X-Pro-Pro sequence makes it relatively resistant to degradation by nonspecific aminopeptidases. Thus, we have discovered another strategy by which sea anemones protect the N-termini of their bioactive neuropeptides.     

Alternative sources of biologically active substances

The majority of antibiotics and substances with diverse biological activity used in medicine are produced by actinomycetes, nonfilamentous bacteria and fungi. Other microorganisms, such as myxobacteria, pseudomonads, nocardias, basidiomycetes, marine microorganisms, enterobacteria, halobacteria, hyperthermophiles etc. are investigated for new biologically active metabolites.     

Placenta--a source of biologically active substances

In the review there are some data displaying the scientific and patent literature presenting placenta as a rich source of some biologically active substances (BAS): proteins, lipids, enzymes, glycolipids and glycolipoproteins, hormones. This information is capable to be used while developing the techniques of BAS producing from placenta in order of creating the organospecific complex preparations with medically-preventive properties.     

Receptor-based assays in screening for biologically active substances.

Molecular biology has identified new receptors and ligands which are deregulated in diseases such as cancer and autoimmune conditions and which provide rational targets for therapeutic intervention. Advances in instrumentation and methodology make it possible to screen large numbers of samples in simple receptor-ligand binding assays in the search for drug candidates. Caution must be exercised in the interpretation of data derived from such assays. This is particularly pertinent to the recently characterized receptors, such as the cytokine receptors, as we do not fully understand the relationship between the receptor type and the linkage of receptors to the appropriate or inappropriate second messenger systems that are used in the experimental screening protocols and the disease state.     

A new culture system for the cultivation of mammalian cells for the production of several biologically active substances

A maximum cell density of 8×10⁶ viable human fibroblast cells/ml was obtained on microcarriers at a perfusion rate of 0.6 mL/min — a rate which maintained a quasi-steady state. The maximum tPA production was approximately 1.2 g/ml and obtained when the cell density was relatively constant during the later periods of cultivation. Specific UTP and tPA production rates had a correlation factor of 0.85, and cell growth to oxygen consumption had an 0.92 correlation factor. ATP generation was strongly correlated to glutamine consumption, with a lower correlation to oxygen utilization.     

PASS: prediction of activity spectra for biologically active substances

The concept of the biological activity spectrum was introduced to describe the properties of biologically active substances. The PASS (prediction of activity spectra for substances) software product, which predicts more than 300 pharmacological effects and biochemical mechanisms on the basis of the structural formula of a substance, may be efficiently used to find new targets (mechanisms) for some ligands and, conversely, to reveal new ligands for some biological targets. We have developed a WWW interface for the PASS software. A WWW server for the on-line prediction of the biological activity spectra of substances has been constructed.     

Polyelectrolyte microcapsules as the systems for delivery of biologically active substances

Based on the method of the layer-by-layer (LbL) adsorption of oppositely charged polyelectrolytes, sodium alginate (Alg) and poly-L-lysine (PLL), novel biodegradable microcapsules have been prepared for delivery of biological active substances (BAS). Porous spherical CaCO₃ microparticles were used as templates. The template cores were coated with several layers of oppositely charged polyelectrolytes forming shell on the core surface. The core-shell microparticles were converted into hollow microcapsules by means of core dissolution with EDTA. Mild conditions for microcapsules preparation allow to perform incorporation of various biomolecules maintaining their bioactivity. Biocompatibility and biodegradability of the polyelectrolytes give a possibility to use the microcapsules as the target delivery systems. Chymotrypsin entrapped into the microcapsules was used as a model enzyme. The immobilized enzyme retained about 86% of the activity compared to a native chymotrypsin. The resultant microcapsules were stable in acidic medium and could be easily decomposed by trypsin treatment in slightly alkaline medium. Chymotrypsin was shown to be active after its release from the microcapsules decomposed by the trypsin treatment. Thus, the microcapsules prepared by the LbL technique can be used for the development of new type of BAS delivery systems in humans and animals.     

Quantitative NMR spectroscopy of biologically active substances and excipients

Biologically active ingredients and excipients are the essentials of a drug formulation, such as a tablet, dragee, solution, etc. Quality control of such substances thus plays a pivotal role in the production process of pharmaceutical drugs. Since these agents often exhibit complex structures, consist of multiple components, or lack of a chromophore, traditional means of characterization are often not feasible. Furthermore, substances of small molecular weight or strong polar character generally exhibit poor chromatographic properties, thus, conventional procedures such as high-performance liquid chromatography are often not applicable. Instead, quantitative nuclear magnetic resonance (qNMR) spectroscopy has emerged as an alternative or orthogonal method in drug analysis. In this review, we elaborate on the application of qNMR to three important classes of biological substances, namely polysaccharides, amino acids, and lipids, and demonstrate the benefits of this modern tool in contrast to traditional techniques.     

Biologically active polypeptides of sea anemones: Structure, function, and prospects for application

This paper presents the results of a structure-functional study of neurotoxins, actinoporins, and proteinase inhibitors from the tropical sea anemone Heteractis crispa (= Radianthus macrodactylus) and the low boreal sea anemone Oulactis orientalis. The new data significantly contribute to the knowledge of the mechanisms of action of polypeptides from marine coelenterates and can be useful for solving practical problems of the biotechnological development of medicines.     

Evidence of dietary feedback in a facultative association between deep-sea gastropods and sea anemones

While epibiotic associations between macrobenthic invertebrates are common, the role they play in the feeding ecology of intervening species is often incompletely understood. The diets of epibiotic sea anemones Allantactis parasitica and their gastropod hosts were analyzed using digestive tract contents, lipid biomarkers and observations of live specimens in an attempt to detect dietary feedback from the facultative association. Comparisons were made using symbiotic individuals and asymbiotic counterparts collected at depths of 191-627 m from three neighbouring areas in the northwest Atlantic. Gastropods carrying one or two epibionts had higher stomach indices than those harbouring three epibionts or no epibiont. The diet of symbiotic gastropods was also more diversified based on stomach contents and lipid analysis. Among other things, symbiotic gastropods contained four times more lipids and a greater proportion of Σn−3 fatty acids. Gastrovascular cavity content indices of asymbiotic sea anemones were generally lower than those of symbiotic counterparts. Their cavities were more often empty, and their diet less diversified with fewer benthic items, suggesting that foraging of gastropods through the sediments makes more food available to sea anemones living as epibionts. Lipid analysis showed some disparities between symbiotic and asymbiotic sea anemones at the regional scale, including in percent phospholipids and in the proportion of certain fatty acids. Together these findings indicate that mutual protection against predators leads to prolonged and more efficient foraging for gastropods and increased time spent deployed (feeding) in food-rich areas for sea anemones, thus enabling both partners to fully exploit food resources that may be limited at bathyal depths.     

Isolation of two novel neuropeptides from sea anemones: the unusual, biologically active L-3-phenyllactyl-Tyr-Arg-Ile-NH2 and its des-phenyllactyl fragment Tyr-Arg-Ile-NH2

Using a radioimmunoassay for the carboxyl-terminal sequence Arg-Val-NH₂, two novel peptides were purified from extracts of the sea anemone Anthopleura elegantissima. These peptides were L-3-phenyllactyl-Tyr-Arg-Ile-NH₂ (name: Antho-RIamide I) and its des-phenyllactyl fragment Tyr-Arg-Ile-NH₂ (Antho-RIamide II). Immunocytochemical staining showed that these peptides were localized in neurons of sea anemones. Application of low concentrations (10⁻⁸ M) of Antho-RIamide I inhibited spontaneous contractions in several muscle groups of sea anemones, whereas Antho-RIamide II was inactive. Antho-RIamide I is the second neuropeptide from sea anemones that bears the unusual, amino-terminal L-3-phenyllactyl blocking group. We suggest that this group renders the peptide resistant against degradation by nonspecific aminopeptidases. In addition, the L-3-phenyllactyl residue might also play a role in receptor binding.