Complete Loss Of Biological Activity Research Articles

Development of a protective lyophilisation medium and conditions to stabilise the erythrocyte diagnostic preparation of tularaemia immunoglobulin

Liquid erythrocyte diagnostic preparations have a practical disadvantage; i.e., long-distance transportation involving possible non-compliance with cold-chain requirements may result in a complete loss of biological activity. A lyophilisation technology is necessary to ensure that the preparations retain their original properties for a long time. The aim of the work was to develop a protective medium and conditions for lyophilisation to stabilise the erythrocyte diagnostic preparation of tularaemia immunoglobulin. Materials and methods: Gelatin, thiourea, trehalose, sucrose, dextran, and Tween 80 were used as excipients for protective media. The authors used nine strains of homologous and heterologous microorganisms of different genera and species to control the lyophilised diagnostic preparation sensitivity and specificity. Evaluation of the main stability-related quality attributes (appearance of the dried preparation, loss on drying, solubility, appearance after reconstitution, appearance after settling, sensitivity, specificity) considered the temperatures specific to the climatic zones where the in vitro diagnostics is intended to be marketed and used. Results: The authors developed protective stabilising media with different compositions, used them in freeze-drying of the preparation and carried out control testing. The most promising was the lyophilisation medium containing a smaller amount of ingredients —6% of dextran, 0.06% of Tween 80 and up to 0.01% of sodium azide—as it was the simplest one to prepare and ensured complete preservation of the quality attributes. The authors carried out practical evaluation of lyophilisation procedures, and the 12–14-hour procedure proved to be the most cost-effective. Conclusions: The results of long-term, or real time, and accelerated stability testing of the lyophilised diagnostic preparation demonstrated the possibility of two-year storage at a labelled temperature of 2–8 °C, as well as at elevated and low temperatures of 30±2 °С and –18 °С, respectively. The tests showed no negative effects of the temperatures on the controlled quality attributes.

Are Smaller Nanoparticles Always Better? Understanding the Biological Effect of Size-Dependent Silver Nanoparticle Aggregation Under Biorelevant Conditions.

PurposeSilver nanoparticles (AgNPs) are one of the most commonly investigated nanomaterials, especially due to their biomedical applications. However, their excellent cytotoxic and antimicrobial activity is often compromised in biological media due to nanoparticle aggregation. In this work, the aggregation behavior and the related biological activity of three different samples of citrate capped silver nanoparticles, with mean diameters of 10, 20, and 50 nm, respectively, were examined.MethodsFollowing nanoparticle synthesis and characterization with transmission electron microscopy, their aggregation behavior under various pH values, NaCl, glucose, and glutamine concentrations, furthermore in cell culture medium components such as Dulbecco’s Modified Eagle’s Medium and fetal bovine serum, was assessed through dynamic light scattering and ultraviolet-visible spectroscopy.ResultsThe results indicated that acidic pH and physiological electrolyte content universally induce micron-scale aggregation, which can be mediated by biomolecular corona formation. Remarkably, larger particles demonstrated higher resistance against external influences than smaller counterparts. In vitro cytotoxicity and antimicrobial assays were performed by treating cells with nanoparticulate aggregates in differing stages of aggregation.ConclusionOur results revealed a profound association between colloidal stability and toxicity of AgNPs, as extreme aggregation led to the complete loss of biological activity. The higher degree of aggregation resistance observed for larger particles had a significant impact on the in vitro toxicity, since such samples retained more of their activity against microbes and mammalian cells. These findings lead to the conclusion that aiming for the smallest possible nanoparticles might not be the best course of action, despite the general standpoint of the relevant literature.

The stabilization of biopharmaceuticals: current understanding and future perspectives

Proteins have many therapeutic advantages over small synthetic drugs in terms of their high specificity and activity. Accordingly, the global biopharmaceutical market grows rapidly and has driven the continuous increase of R&D investment in protein-based drug products, thus the number of approved protein drugs also continuously increases. However, during their manufacture, transport, and storage, proteins are often exposed to various detrimental environments that can cause protein destabilization, resulting in undesirable drug properties such as partial or complete loss of biological activity, altered solubility, and immunogenicity. Therefore, the thorough evaluation/monitoring and optimization of process variables and product formulations are critical to ensure product quality during its appropriate shelf-life. However, because of (i) the complexity of protein structures, (ii) multiple degradation pathways, and (iii) various intrinsic and extrinsic factors that can affect the stability, protein stabilization is not straightforward and remains a big challenge in the clinical development and commercialization of protein-based drug products, although significant progress has been made. Considering that a better understanding of the various instability mechanisms and factors is important for the quality control of protein drug products and their successful clinical usage, this review briefly overviews protein destabilization pathways and deals with general issues such as controlling the factors of protein stabilization, especially during manufacturing, shipping and storage.

Механізми стійкості до антибіотиків представників родини Enterobacteriaceae

The paper deals with the basic medical scheme of antibiotics use for treatment of lesions caused by enterobacteria and mechanisms of resistance of Enterobacteriaceae to different classes of antibiotics. It is known that the main mechanisms of resistance to antibiotics are enzymatic inactivation, modification of the target, efflux, violation of conduct through the membrane and formation of metabolic shunt. The most common cases of resistance to beta-lactams among Enterobacteriaceae relate to production of plasmid and chromosomal beta-lactamases, violation of the permeability of the outer membrane, and modification of target penicillin binding proteins. Active release of antibiotics from the cell, or efflux, in Enterobacteriaceae is used for maintaining resistance to tetracyclines, macrolides, carbapenems. Genes of efflux system are localized on plasmids and contribute to rapid spreading among Enterobacteriaceae. Mutations are the basis of resistance to novobiocinum and rifampicinum. Enzymatic inactivation by modifying is typical for resistance to aminoglycosides. Three groups of enzymes are engaged in the process, by adding the molecule of acetic acid, phosphate or adenine. Joining of these groups is irreversible and leads to complete loss of biological activity of the antibiotic. Resistance to aminoglycosides appears also due to inhibition of drug penetration, that is associated with genetically determined mechanisms of electron transport through the membrane. Resistance to quinolones and fluoroquinolones is associated with the modification of topoisomerase II and IV which are targets of these groups of antibiotics. Resistance is possible as a result of changes in the structure of the target, breaching of penetration into the cell, and active release from the cell. The highest level of resistance is develope in the case of two- or three-stage mutations in one or the other, or both, subunits in different genes. At the same time, for breaching of the bacterial cell it is enough to suppress the activity of only one enzyme associated with different functions of both topoisomerases. Another mechanism of resistance to quinolones is the reduction of permeability through bacterial outer membrane, that it’s possible due to decreasing of permeability of porine channels. In this case, decreasing of permeability efficacy takes place not only for quinolones, but also for other classes of antibitics. In addition, reduced sensitivity to quinolones efflux may play the significant role. For clinical strains of microorganisms, cross-resistance to various drugs, associated with simultaneous production of various enzymes that inactivate antibiotics, is typical.

Novel fluorescent cycloheximide derivatives for the imaging of protein synthesis

Cycloheximide (CHX) is one of the most interesting protein synthesis inhibitors. For this reason, fluorescent derivatives of CHX could find useful applications in cell biology. We report the successful synthesis of a set of novel fluorescent derivatives of CHX. The effect of different functional groups on the biological activity of CHX was studied upon their modification through suitable strategies, i.e., acetylation of the hydroxyl group and reductive amination of the ketone group. The first route induced a complete loss of biological activity, while the second approach allowed a retained inhibition of protein synthesis, as demonstrated by in vitro translation assays. Various fluorescent dyes for reductive amination were tested (i.e., ANTS, APTS, and Rhodamine-123), and the success of the syntheses was demonstrated by diverse analytical techniques. Cycloheximide labeling with fluorescent dyes is a promising approach for developing fluorescence reporters for various applications, both in vitro (fluorescence spectroscopy) and in vivo (live imaging).

Biological activity of the predicted red pigment-concentrating hormone of Daphnia pulex in a crustacean and an insect

The elucidation of the genome of the waterflea Daphnia pulex made it possible to search for orthologue genes for the crustacean red pigment-concentrating hormone (named Panbo-RPCH after the species Pandalus borealis in which the red pigment-concentrating hormone was first identified); Panbo-RPCH is a member of the adipokinetic hormone (AKH)/red pigment-concentrating hormone (RPCH) peptide family. The information pointed to a putative mature RPCH octapeptide in D. pulex with the primary sequence of pGlu-Val-Asn-Phe-Ser-Thr-Ser-Trp amide (=Dappu-RPCH). Since Panbo-RPCH is endogenous in decapod crustaceans and in the green stink bug Nezara viridula, we assayed Dappu-RPCH in the shrimp Palaemon pacificus and in N. viridula. Here we show that this variant member of the AKH/RPCH family has no activity to concentrate the red, brown, yellow and blue pigments in the epithelium of the shrimp at physiological doses but is effective in mobilising lipids in the green stink bug N. viridula. Moreover, since Panbo-RPCH and Dappu-RPCH differ structurally at three positions, viz. Leu 2 to Val 2; Pro 6 to Thr 6; Gly 7 to Ser 7, we tested other members of the peptide family which have single or dual amino acid substitutions at the appropriate positions, for their chromatophorotropic action at physiological doses. These studies show unequivocally that a single change from Gly 7 to Ser 7 (as in the peptide Corpu-AKH) does not inflict any loss of biological activity, and the same is true for a single change from Pro 6 to Thr 6 (represented by the peptide Schgr-AKH-II). The change from Leu 2 to Val 2 (embodied in Manto-CC), however, is accompanied with a substantial loss of chromatophorotropic activity; combinations of Val 2 and Ser 7 (as in Anaim-AKH) or Val 2 and Thr 6 (as in Grybi-AKH) result in almost complete loss of biological activity. Dappu-RPCH with its three substitutions is not active at all in the shrimp at the tested concentration range of up to 30 pmol.

Existence, uniqueness and stability of the equilibrium points of a SHARON bioreactor model

This paper addresses the dynamics of a SHARON reactor, a promising technology for ammonium removal from concentrated wastewater streams. The contraction mapping theorem is used to determine which operating conditions of a SHARON reactor with pH-control result in a unique equilibrium state. However, this approach only identifies the case of very large dilution rates, in practice corresponding with complete biomass wash-out, i.e. with complete loss of biological activity. Practical operation of a SHARON reactor aims at reaching ammonium conversion to nitrite. To identify such interesting operating points, the equilibrium points are subsequently calculated directly in terms of input variables for a simplified SHARON reactor model. The stability of the obtained equilibrium points is assessed and the corresponding phase portraits are analyzed. The influence of slightly varying parameter and input values is investigated as well.

Bisubstrate Inhibitors of Catechol O‐Methyltransferase (COMT): the Crucial Role of the Ribose Structural Unit for Inhibitor Binding Affinity

Inhibition of the enzyme catechol O-methyltransferase offers a therapeutic handle to regulate the catabolism of catecholamine neurotransmitters, providing valuable assistance in the treatment of CNS disorders such as Parkinson's disease. A series of ribose-modified bisubstrate inhibitors of COMT featuring 2'-deoxy-, 3'-deoxy-, 2'-aminodeoxy-3'-deoxy-, and 2'-deoxy-3'-aminodeoxyribose-derived central moieties and analogues containing the carbocyclic skeleton of the natural product aristeromycin were synthesized and evaluated to investigate the molecular recognition properties of the ribose binding site in the enzyme. Key synthetic intermediates in the ribose-derived series were obtained by deoxygenative [1,2]-hydride shift rearrangement of adenosine derivatives; highlights in the synthesis of carbocyclic aristeromycin analogues include a diastereoselective cyclopropanation step and nucleobase introduction with a modified Mitsunobu protocol. In vitro biological evaluation and kinetic studies revealed dramatic effects of the ribose modification on binding affinity: 3'-deoxygenation of the ribose gave potent inhibitors (IC50 values in the nanomolar range), which stands in sharp contrast to the remarkable decrease in potency observed for 2'-deoxy derivatives (IC50 values in the micromolar range). Aminodeoxy analogues were only weakly active, whereas the change of the tetrahydrofuran skeleton to a carbocycle unexpectedly led to a complete loss of biological activity. These results confirm that the ribose structural unit of the bisubstrate inhibitors of COMT is a key element of molecular recognition and that modifications thereof are delicate and may lead to surprises.

Different Structural Requirements for Melanin‐Concentrating Hormone (MCH) Interacting with Rat MCH‐R1 (SLC‐1) and Mouse B16 Cell MCH‐R

Melanin‐concentrating hormone (MCH) is a neuropeptide occurring in all vertebrates and some invertebrates and is now known to stimulate pigment aggregation in teleost melanophores and food‐intake in mammals. Whereas the two MCH receptor subtypes hitherto cloned, MCH‐R1 and MCH‐R2, are thought to mediate mainly the central effects of MCH, the MCH‐R on pigment cells has not yet been identified, although in some studies MCH‐R1 was reported to be expressed by human melanocytes and melanoma cells. Here we present data of a structure‐activity study in which 12 MCH peptides were tested on rat MCH‐R1 and mouse B16 melanoma cell MCH‐R, by comparing receptor binding affinities and biological activities. For receptor binding analysis with HEK‐293 cells expressing rat MCH‐R1 (SLC‐1), the radioligand was [125I]–[Tyr13]‐MCH with the natural sequence. For B16 cells (F1 and G4F sublines) expressing B16 MCH‐R, the analog [125I]–[D‐Phe13, Tyr19]‐MCH served as radioligand. The bioassay used for MCH‐R1 was intracellular Ca2+ mobilization quantified with the FLIPR instrument, whereas for B16 MCH‐R the signal determined was MAP kinase activation. Our data show that some of the peptides displayed a similar relative increase or decrase of potency in both cell types tested. For example, linear MCH with Ser residues at positions 7 and 16 was almost inactive whereas a slight increase in side‐chain hydrophilicity at residues 4 and 8, or truncation of MCH at the N‐terminus by two residues hardly changed binding affinity or bioactivity. On the other hand, salmonic MCH which also lacks the first two residues of the mammalian sequence but in addition has different residues at positions 4, 5, 9, and 18 exhibited a 5‐ to 10‐fold lower binding activity than MCH in both cell systems. A striking difference in ligand recognition between MCH‐R1 and B16 MCH‐R was however observed with modifications at position 13 of MCH: whereas L‐Phe13 in [Phe13, Tyr19]‐MCH was well tolerated by both MCH‐R1 and B16 MCH‐R, change of configuration to D‐Phe13 in [D‐Phe13, Tyr19]‐MCH or [D‐Phe13]‐MCH led to a complete loss of biological activity and to a 5‐ to 10‐fold lower binding activity with MCH‐R1. By contrast, the D‐Phe13 residue increased the affinity of [D‐Phe13, Tyr19]‐MCH to B16 MCH‐R about 10‐fold and elicited MAP kinase activation as observed with [Phe13, Tyr19]‐MCH or MCH. These data demonstrate that ligand recognition by B16 MCH‐R differs from that of MCH‐R1 in several respects, indicating that the B16 MCH‐R represents an MCH‐R subtype different from MCH‐R1.

Conformationally restricted analogues of 1N,14N-bisethylhomospermine (BE-4-4-4): synthesis and growth inhibitory effects on human prostate cancer cells.

Twelve analogues of 1N,14N-bisethylhomospermine (BE-4-4-4) with restricted conformations were synthesized in the search for cancer chemotherapeutic agents with higher cytotoxic activities and lower systemic toxicities than BE-4-4-4. The central butane segment of BE-4-4-4 was replaced with a 1,2-substituted cyclopropane ring, a 1,2-substituted cyclobutane ring, and a 2-butene residue. In each case, the cis/trans-isomeric pair was synthesized. Cis-monounsaturation(s) was also introduced at the outer butane segment(s) of BE-4-4-4. The two possible cis-dienes and a cis-triene formally derived from the tetraazaeicosane skeleton of BE-4-4-4 were also prepared. Four cultured human prostate cancer cell lines (LnCap, DU145, DuPro, and PC-3) were treated with the new tetramines to examine their effects on cell growth with a MTT assay. One representative cell line (DuPro) was selected to further study the cellular uptake of the novel tetramines, their effects on intracellular polyamine pools, and their cytotoxicity. All tetramines entered the cells, reduced cellular putrescine and spermidine pools while exerting only a small effect on the spermine pool, inhibited cell growth, and killed 2-3 logs of cells after 6 days of treatment at 10 microM. Four new tetramines, the two cyclopropyl isomers, the trans-cyclobutyl isomer, and the (5Z)-tetraazaeicosene, were more cytotoxic than their saturated counterpart (BE-4-4-4). Their cytotoxicity, however, could not be correlated either with their cellular uptake or with their ability to deplete intracellular polyamine pools. We attribute their cytotoxicity to their specific molecular structures. The cytotoxicity was markedly reduced when the central butane segment was deprived of its rotational freedom by replacing it with a double bond. Introduction of a triple bond or a benzene-1,2-dimethyl residue at the central segment of the polyamine chain, led to complete loss of biological activity. The conformationally restricted alicyclic derivatives were not only more cytotoxic than was the freely rotating BE-4-4-4 by several orders of magnitude but also had much lower systemic toxicities than the latter. Thus, we obtained new tetramines with a wider therapeutic window than BE-4-4-4.

Purification and characterization of staphylococcin BacR1, a broad-spectrum bacteriocin.

The bacteriocin BacR1 was purified from culture supernatant of Staphylococcus aureus UT0007 by sequential ammonium sulfate precipitation, cation-exchange chromatography, and C4 reverse-phase chromatography steps. Mass spectrographic analysis indicated that the purified peptide has a molecular mass of 3,338 Da. It is resistant to environmental conditions, retaining full biological activity after exposure to pH extremes (pHs 3 to 11), heating at 95 degrees C for 15 min, and exposure to strong chaotropic agents. BacR1 was destroyed with a complete loss of biological activity after digestion with trypsin and proteinase K. Amino acid sequence analysis revealed a high concentration of Asx, Gly, and Pro residues and a high proportion of hydrophobic amino acids. The peptide is bactericidal and kills in a dose-dependent manner, but it does not lyse log-phase cells of Corynebacterium renale, the routine indicator organism for bacteriocin assay. A specific receptor for binding was detected on sensitive cells but not on insensitive cells. Competition assays showed that UV-inactivated cells could protect susceptible cells from antibacterial action. A partial inhibitory spectrum revealed that organisms from the following genera are susceptible: Staphylococcus, Streptococcus, Corynebacterium, Haemophilus, Bordetella, Moraxella, Pasteurella, Neisseria, and Bacillus.

Unstable proteins: how to subject them to chromatographic separations for purification procedures

The chromatographic separation of an unstable protein is often a challenge to the scientist working in the field of life sciences. Especially for the purification of sensitive enzymes, making use of conventional chromatographic techniques is difficult and frequently results in a complete loss of biological activity of the target protein. This report summarizes some general strategies that may help to keep unstable proteins in their native conformation during the rather harsh conditions of a purification procedure. In this context, a recently developed hollow fiber membrane module, suitable for performing on-line dialysis, is introduced and examples of its application to liquid column chromatography are given. Many innovative separation techniques, characterized by dramatic improvements in both performance and separation time, have recently been developed. Since the chromatographic separation of unstable proteins requires the use of modern state-of-the-art equipment and technology, emphasis is given to newly developed separation techniques such as expanded bed adsorption, perfusion chromatography, protein free flow electrophoresis and the use of tentacle gels. In addition, examples of recently published purifications of unstable proteins are discussed with respect to strategies ensuring the preservation of the native protein structure during chromatographic separation.

Identification of residues in the putative TolA box which are essential for the toxicity of the endonuclease toxin colicin E9.

E colicins are plasmid-coded, protein antibiotics which bind to the BtuB outer membrane receptor of Escherichia coli cells and are then translocated either to the outer surface of the cytoplasmic membrane in the case of the pore-forming colicin E1, or to the cytoplasm in the case of the enzymic colicins E2-E9. Translocation has been proposed to be dependent on a putative TolA box; a pentapeptide (DGSGW) located in the N-terminal 39 residues of several Tol-dependent colicins. In this study, site-directed mutagenesis was used to change each of the residues of the putative TolA box of colicin E9 to alanines. In the case of the two glycine residues, the resulting mutant proteins were indistinguishable from the native colicin E9 protein in a biological assay; whereas the other three residues when mutated to alanines resulted in a complete loss of biological activity. Mutagenesis of two serine residues flanking the putative TolA box, Ser34 and Ser40, to alanines did not abolish the biological activity of the mutant colicin E9, although the zones of growth inhibition were hazy and slow to form. The size of the zone of inhibition was significantly smaller than the control in the case of the Ser40Ala mutant. The ColE9/Im9 complex was isolated from the three biologically inactive TolA box alanine mutants. In competition assays all three mutant protein complexes were capable of protecting sensitive E. coli cells against killing by the native ColE9/Im9 complex. On removal of the Im9 protein from the three mutant ColE9/Im9 complexes, all three mutant colicins exhibited DNase activity. These results confirm the importance of the putative TolA box in the biological activity of colicin E9, and suggest that the TolA box has a role in the translocation of colicin E9.

Molecular basis for amyloidosis related to hereditary brain hemorrhage

The aim of the project has been to elucidate molecular events leading to amyloidosis in Hereditary Cystatin C Amyloid Angiopathy (HCCAA) patients, to enable simple diagnosis of the disease and with the ultimate goal to understand the amyloid formation process in detail, in order to develop inhibitors to the process. At the DNA level, a point mutation segregating with HCCAA was identified in the cystatin C gene on chromosome 20, after basic characterization of cDNA and gene for the wildtype protein. The mutation results in the amino acid substitution Leu-68-Gin (L68Q) and abolishes a recognition site for Alu I. This information was used to design a PCR based assay for simple and rapid mutation detection in DNA from blood samples to allow routine diagnosis of HCCAA. Studies at the protein level, allowed through E. coli expression of wildtype and L68Q mutated cystatin C genes, revealed that both protein variants effectively inhibit the cysteine proteinase cathepsin B (equilibrium constants for dissociation: 0.4 and 0.3 nM, respectively), but differ considerably in their tendency to dimerize and form aggregates. The initial dimerization of L68Q-cystatin C results in complete loss of biological activity and is highly temperature-dependent, with a rise in incubation temperature from 37 to 40 degrees C resulting in a 150% increase in dimerization rate. This result might be of clinical relevance, since medical intervention to abort febrile periods of carriers of the disease trait may reduce the in vivo formation of L68Q-cystatin C aggregates. The three-dimensional structure of normal cystatin C, crystallized in a complex with cathepsin B, was elucidated by X-ray analysis and subsequent refinement of the structure to 3.0 A resolution. Besides pinpointing the cystatin C structures resulting in efficient target enzyme inhibition, the results demonstrated that the Leu-68 residue is buried in the hydrophobic core of the protein. Studies of the three-dimensional solution structure of wildtype cystatin C by NMR spectroscopy revealed that cystatin C dimers can be formed as a result of slight, localized structural changes under conditions preceding complete defolding and denaturation of the protein. Dimers of L68Q-cystatin C are likely similar but are formed at temperatures nearly 30 degrees C lower than needed for the wildtype protein, indicating that the Leu-68-Gln substitution lowers the transition temperature for unfolding. Thus, the results presented suggest that cystatin C provides a system where decreased stability of a mutant protein correlates with its amyloidogenic nature. The NMR results furthermore imply that the hydrophobic proteinase-binding region of cystatin C is directly involved in dimer formation and that compounds designed to interact with this region could serve as inhibitors to the dimerization, and likely also the subsequent amyloid formation process, of cystatin C in HCCAA patients.

Syntheses of a Regioisomer of N-{4-[2-(2-Amino-4(3H)-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-L-glutamic Acid (LY231514), an Active Thymidylate Synthase Inhibitor and Antitumor Agent

Two independent routes to N-(4-[2-(2-amino-4(3H)-oxo-7H-pyrrolo[2, 3-d]pyrimidin-6-yl)ethyl]benzoyl)-L-glutamic acid, a regioisomer of the potent TS inhibitor and antitumor agent LY231514, are described. Preliminary in vitro cell culture evaluation has shown that attachment of the ethanobenzoylglutamate moiety of LY2 31514 to position 6 of the pyrrolopyrimidine ring system rather than to position 5 results in complete loss of biological activity

Structure-activity relationship of analogues of endothelin-1: dissociation of hypotensive and pressor actions

The structural requirements of endothelin-1 to evoke depressor and pressor responses were studied in conscious rats. Formylation of the C-terminal Trp eliminated the depressor but not the pressor activity of endothelin-1. Oxidation of Met 7 in this analogue restored the hypotensive activity. Destruction of the Cys 1-Cys 15 disulphide bridge led to a weak agonist with both depressor and pressor activities. Formylalion of Trp 21 in this analogue resulted in a complete loss of biological activity. These results indicate that Met 7 and the indole moiety of Trp 21 are important for the expression of the depressor activity of endothelin-1 whereas the intramolecular loop structure is less important. The results also provide further evidence that the depressor and pressor effects of endothelin-1 are mediated through different receptors.

CD4(81–92)-based peptide derivatives: Structural requirements for blockade of HIV infection, blockade of HIV-induced syncytium formation, and virostatic activity in vitro

CD4(81–92) peptides block human immunodeficiency virus (HIV) infection, virus-induced cell fusion, and antigen production by HIV-1-infected cells when derivatized on specific amino acid residues. An extensive series of structural variants of 1,4,5-tribenzyl-10-acetyl-CD4(81–92) were tested as anti-viral agents in an attempt to define the sequence and derivatization requirements for antiviral activity, and to maximize potency and stability for use as potential therapeutic agents. Alteration of the primary amino acid sequence of the sterm compound 1,4,5-tribenzyl-CD4(81–92) diminished or abolished in parallel all three indices of anti-viral activity in a series of altered sequence compounds. Replacement of d- for l-amino acid residues at positions 1, 2, 3, 4, 5, or 6 but not position 10 decreased anti-viral potency, again with parallel effects on infection, synctium formation, and virostatic activity. Omission of the glutamine residue at position 9 did not affect anti-viral potency, while removal of the glutamic acids at positions 11 and 12 resulted in virtually complete loss of biological activity. Changes in the derivatization pattern of the CD4(81–92) peptide backbone also affected anti-viral potency and efficacy. Optimal activity was obtained with benzyl residues at positions 1, 4, and 5, whereas the 1,4,7-tribenzyl-CD4(81–92) compound was without activity in all assays tested. Replacement of one of the benzyl groups with an acetamidomethyl moiety resulted in complete loss of biological activity. The previously reported (Nara et al., Proc Natl Acad Sci USA 86: 7139–7143, 1989) virostatic activity of 1,4,5-tribenzyl-10-acetyl-CD4(81–92) (peptide #18) is apparently due to the acetylation, since the desacetyl stem compound shows much less virostatic activity while still possessing full anti-infective and anti-syncytial activity, and acetylation of the N-terminus rather than the lysine of 1,4,5-tribenzyl-CD4(81–92) yields a virostatic compound equipotent to peptide #18. Cyclization of the tribenzyl peptide to further conformatinally restrict the molecule resulted in a compound with anti-infection, anti-syncytial, and virostatic activity at submicromolar concentrations.

Site-directed mutagenesis reveals the importance of disulfide bridges and aromatic residues for structure and proliferative activity of human Interleukin-4

Mutant proteins (muteins) of human Interleukin-4 (IL4) were constructed by means of in vitro mutagenesis. The muteins were expressed in E. coli, submitted to a renaturation and purification protocol and analysed for biological activity. Exchange of the cysteines at either position 46 or 99 which form one of the three disulfide bridges resulted in a nearly complete loss of biological activity and an unstable protein. The exchange of tyrosine 124 also inactivated the protein, while a mutation of tyrosine 56 left some residual activity. Exchange of the other four cysteines or of the single tryptophane had smaller effects.

Role of the C-terminus in the biological activity of human interleukin 5

Alterations of the C-terminal amino acid sequence of recombinant human interleukin 5 (rhIL-5) caused significant changes in its biological activity. Removal of eight amino acids from the C-terminus of rhIL-5 by CNBr treatment led to a complete loss of biological activity as determined by the BCL 1 cell IgM-inducing assay. Oxidation of Met residue located at C-terminus also resulted in a loss of activity. These results suggest that the C-terminal amino acids of rhIL-5 are crucial for its biological actions.

Structure-function analysis of human interleukin-6: Evidence for the involvement of the carboxy-terminus in function

C-terminally deleted analogs of human interleukin-6 (IL-6) have been constructed at the cDNA level, and after cell-free transcription and translation their biological activity was analyzed. Removal of only 4 amino acids resulted in complete loss of biological activity as determined by the B9 cell proliferation assay. Secondary structure prediction of human IL-6 resulted in 58% helix, 14% β-structure, and 28% turn and coil (average of 3 independent methods). The circular dichroism of recombinant human IL-6 was measured in the near and far UV. Evaluation of the latter in terms of secondary structures gave 67% helix, 15% β-structure, and 18% turn and coil.