Synthetic Polyamino Acids Research Articles

ECM components are essential for proper in vitro myogenesis

Objective: In vitro models of skeletal muscle often utilize primary myoblast cells or myoblast cell lines. Myoblasts require adhesion to the extracellular matrix (ECM) to grow, proliferate, migrate, and differentiate in their natural environments in vivo. To meet the adhesion needs of adhesive cells under in vitro conditions, culture surfaces are coated with various biological or synthetic compounds. Within the scope of the study, the differentiation potential of H2K myoblasts, a cell line resembling primary myoblasts, were comparatively evaluated through morphological analysis on culture surfaces coated with various ECM and synthetic materials. Methods: The culture surfaces were coated with fibronectin and laminin, the major adhesion proteins of ECM; gelatin, a molecular derivative of collagen; matrigel, an ECM extract; and PLL, a synthetic poly-amino acid. Cells were allowed to differentiate in each culture medium for 4 days and their capacity to adhere to the surface and differentiation rates from myoblast to myotube were evaluated by morphological analysis. Results: In the uncoated culture environment, cells could only attach to 30-50% of the culture surface and myotube development was limited and not aligned with each other. On surfaces coated with PLL, no myotube development was observed and cells could only attach to 30-40% of the culture surface. Myotube development and alignment were similar on all surfaces coated with ECM components. On surfaces coated with ECM components laminin, fibronectin and matrigel, cells covered the entire culture surface and exhibited similar myotube development. However, on surfaces coated with gelatin, both cell adhesion to the surface and myotube development were limited compared to other ECM components. The mean myotube diameters of fibronectin, laminin, matrigel, PLL+laminin and gelatin were 49.71µm (±16.3µm), 52.31µm (±15.7µm), 51.9µm (±15.3µm), 53.06µm (±14.2µm) and 35.25µm (±11.4µm), respectively. Conclusion: Within the scope of the study, it was revealed that coating the culture surface with only a cationic material such as PLL does not support myogenesis and ECM components are needed for cell viability and differentiation.

Macromolecular Prodrugs Based on Synthetic Polyaminoacids: Drug Delivery and Drug Targeting in Antitumor Therapy

In the last twenty years a depth study on potential pharmaceutical applications of synthetic polymers at proteinlike structure as carrier for macromolecular prodrug production has been performed in academia and in industry. In particular α,β-poly(N-2-hydroxyethyl)-DL-aspartamide (PHEA), α,β-polyaspartylhydrazide (PAHy), poly(glutamic acid) (PGA), poly(aspartic acid) (PAA) and polylysine (PLL) have been extensively studied in this field. In the present review, the use of PHEA, PAHy, PGA as starting materials to prepare macromolecular prodrugs is reported and drug delivery and targeting aspects have been considered.

Proliferation capacity of cord blood derived neural stem cell line on different micro-scale biofunctional domains.

Physical interactions of cells with the adhesive substrates of the microenvironment as well as the presence of the soluble growth factors are important for the proliferation capacity of neural stem cells. We have used biofunctionalized surface domains microcontact printed with either synthetic polyaminoacid poly-L-lysine or extracellular matrix (ECM) component such as fibronectin, to study the proliferation capacity of human umbilical cord blood-derived neural stem cells (HUCB-NSC). The proliferation measured by the expression of Ki-67 protein was accompanied by the investigation of the cell morphology under the transmission and scanning electron microscopy in different culture time, plating densities of cells and medium condition (serum-free or 2 percent of FBS). The poly-L-lysine domains of defined micro-scale area promoted the presence of round, loosely attached Ki-67-positive cells, while fibronectin domains of the same size allowed appearance of flattened, strongly attached cells with more differentiated phenotype. These results were in agreement with the non-specific, electrostatic type of interaction between cell and substrate on poly-L-lysine and integrin receptor-mediated specific adhesion on fibronectin. In this report we have described in vitro culture conditions, which allow for immobilization of the non-differentiated and highly proliferating population of neural stem/progenitor cells to the biofunctionalized surface. The microarrays with bioactive domains allocating non-differentiated and proliferating neural stem/progenitor cells may find application for drug and chemicals toxicology screening of diverse factors influencing neural development.

Polyaminoacid-Induced Growth of Metal Nanoparticles on Layer-by-Layer Templates

We report on preparation of redox-active nanoscale layer-by-layer (LbL) films with polyaminoacid-decorated surfaces that serve for both nucleation and growth of uniformly distributed gold nanoparticles at ambient conditions. We found that a poly-l-tyrosine (pTyr), a synthetic polyaminoacid, was able to direct nanoparticle formation to solid, flexible, and patterned surfaces preventing particle agglomeration. The gold particles were 8 ± 2 nm in diameter, surrounded by 3−6 nm polyaminoacid shell, and confined to the topmost polyaminoacid layer. The reported results on bioinspired gold formation can be readily expanded to any inorganic-selective surface and provide a simple, robust, and nontoxic method to obtain nonaggregated inorganic nanoparticles at ambient conditions.

Antitumor activity of 4-( N-hydroxyphenyl)retinamide conjugated with poly( l-glutamic acid) against ovarian cancer xenografts

Objective. Natural and synthetic retinoids such as N-(4-hydroxyphenyl)retinamide (4HPR) have been used for prevention and treatment of a variety of cancers; however, relapse usually occurs after treatment is stopped. Furthermore, the retinoid analogues are insoluble in water, making it difficult for systemic administration. The purpose of this study was to develop and evaluate a water-soluble polymeric formulation of 4HPR that can release 4HPR over a period of a few days. Methods. 4HPR was bound to a synthetic polyamino acid poly( l-glutamic acid) (PG). PG–4HPR was evaluated for its release kinetics and in vitro anti-proliferative and in vivo antitumor activities against ovarian cancer cell lines. Results. The release profile of PG–4HPR in phosphate buffered saline at 37 °C followed a first order kinetic, with a rate constant of 8.8 × 10 − 3 h − 1 . Approximately 60% of 4HPR was released over a period of 100 h. In vitro, both 4HPR and PG–4HPR inhibited proliferation of three ovarian cancer cells lines (SKOV3, OVCA420, and OVCA433) and an immortalized human ovarian epithelium cell line (IOSE) in a time- and dose-dependent manner. Increasing the exposure time of SKOV3 cells to both agents from 1 to 5 days resulted in an increased apoptotic response. In vivo, PG–4HPR demonstrated significantly enhanced antitumor activities compared to 4HPR in both early treatment and later treatment protocols. Treatments with PG–4HPR suppressed the expression of VEGF and reduced blood flow into the tumor. Conclusions. PG–4HPR may have potential applications in the prevention and therapy of ovarian cancers.

Synthesis and characterisation of novel chemical conjugates based on α,β-polyaspartylhydrazide and β-cyclodextrins

A new family of supramolecular systems based on a synthetic polyaminoacid and cyclic oligosaccharides such as β-cyclodextrins (β-CDs) was synthesised. The pharmaceutical potential of these systems arises from the proper combination between the complexing properties of cyclodextrins and the particular pharmacokinetic profile that can be obtained by using macromolecular conjugates with a biocompatible backbone. Five supramolecular conjugates were synthesised by using α,β-polyaspartylhydrazide (PAHy) as a polymeric component and various amounts of two β-CD derivatives. In particular, by reaction of PAHy with β-CD monoaldehyde, samples named as A 1, A 2 and A 3, bearing, respectively, 4.0, 7.5 and 10.7 mol% of β-CDs were obtained. The reaction of PAHy with 6-[aminoethyl(4′-carboxybutanamide)]-β-CD afforded samples named as B 1 and B 2, bearing, respectively, 1.8 and 2.6 mol% of β-CDs linked to the polymer. The occurrence of the conjugation reactions as well as the evaluation of the amount of oligosaccharides conjugated to the polymeric backbone were confirmed by FT-IR, 1H NMR, DSC, SEC analyses and viscosimetric measurements. Molecular weight values obtained by SEC analysis were in good agreement with the theoretical increase of molecular weight of PAHy due to the β-CD moieties linked to the polymeric backbone. Fluorescence studies on the conjugate A 3 evidenced an interaction of a probe molecule with β-CDs linked to PAHy greater than that found with β-CDs alone and even in both cases the formation of a 1:1 host–guest complex occurs.

My Contributions to Science and Society

My Contributions to Science and Society

Silicification and biosilicification - Part 7: Poly-L-arginine mediated bioinspired synthesis of silica

Precipitated silica is synthesized commercially by neutralizing sodium silicate solution under harsh conditions of pH and temperature. In contrast, the formation of ornate silica structures in biological systems (biosilicification) occurs at (or close to) pH 7 under ambient conditions and is thought to be mediated by proteins. Determination of the primary sequences of these proteins has led to the identification of various amino acids that have been proposed to be important in biosilicification. The corresponding synthetic polyamino acids are now being successfully used in bioinspired materials chemistry for developing new materials and processes. Here we report the formation of well-defined silica in vitro as facilitated by poly-L-arginine (PLAr) under ambient conditions and at neutral pH. Two different silica precursors were used in this investigation; tetramethoxysilane (TMOS) and water glass. Scanning Electron Microscopy (SEM) was used for studying the silica morphology and it was revealed that the silica spheres had typical diameters in the range 300–500nm. The PLAr is a cationically charged macromolecule at neutral pH and is believed to act as a catalyst/template/scaffold for the formation of silica in vitro in analogous fashion to certain biomacromolecules that are able to facilitate silicification/biosilicification. These results are discussed here in the context of the role(s) of (bio)macromolecules that facilitate (bio)mineralization.

The role of bacteriolysis in the pathophysiology of inflammation, infection and post-infectious sequelae.

The literature dealing with the biochemical basis of bacteriolysis and its role in inflammation, infection and in post-infectious sequelae is reviewed and discussed. Bacteriolysis is an event that may occur when normal microbial multiplication is altered due to an uncontrolled activation of a series of autolytic cell-wall breaking enzymes (muramidases). While a low-level bacteriolysis sometimes occurs physiologically, due to "mistakes" in cell separation, a pronounced cell wall breakdown may occur following bacteriolysis induced either by beta-lactam antibiotics or by a large variety of bacteriolysis-inducing cationic peptides. These include spermine, spermidine, bactericidal peptides defensins, bacterial permeability increasing peptides from neutrophils, cationic proteins from eosinophils, lysozyme, myeloperoxidase, lactoferrin, the highly cationic proteinases elastase and cathepsins, PLA2, and certain synthetic polyamino acids. The cationic agents probably function by deregulating lipoteichoic acid (LTA) in Gram-positive bacteria and phospholipids in Gram-negative bacteria, the presumed regulators of the autolytic enzyme systems (muramidases). When bacteriolysis occurs in vivo, cell-wall- and -membrane-associated lipopolysaccharide (LPS (endotoxin)), lipoteichoic acid (LTA) and peptidoglycan (PPG), are released. These highly phlogistic agents can act on macrophages, either individually or in synergy, to induce the generation and release of reactive oxygen and nitrogen species, cytotoxic cytokines, hydrolases, proteinases, and also to activate the coagulation and complement cascades. All these agents and processes are involved in the pathophysiology of septic shock and multiple organ failure resulting from severe microbial infections. Bacteriolysis induced in in vitro models, either by polycations or by beta-lactams, could be effectively inhibited by sulfated polysaccharides, by D-amino acids as well as by certain anti-bacteriolytic antibiotics. However, within phagocytic cells in inflammatory sites, bacteriolysis tends to be strongly inhibited presumably due to the inactivation by oxidants and proteinases of the bacterial muramidases. This might results in a long persistence of non-biodegradable cell-wall components causing granulomatous inflammation. However, persistence of microbial cell walls in vivo may also boost innate immunity against infections and against tumor-cell proliferation. Therapeutic strategies to cope with the deleterious effects of bacteriolysis in vivo include combinations of autolysin inhibitors with combinations of certain anti-inflammatory agents. These might inhibit the synergistic tissue- and- organ-damaging "cross talks" which lead to septic shock and to additional post-infectious sequelae.

Targeting of Polyelectrolyte RNA Complexes to Cell Surface Integrins as an Efficient Cytoplasmic Transfection Mechanism

This is the first demonstration of receptor-mediated delivery of mRNA and establishes a new approach to gene therapy. Messenger RNA (mRNA) provides a promising alternative to plasmid DNA as a genetic material for delivery in non-viral gene therapy strategies. Since it does not require access to the nucleus and is less dependent on the cell cycle for expression, mRNA delivered using cationic lipids or short cationic polymers can be effectively translated within target cells. In this study, mRNA formed discrete nanoparticles following self assembly with a range of cationic polymers. Based on transfection activities, the low molecular weight polycations were more efficient than high molecular weight, while protamine and polyethylenimine were far more efficient than poly(L-lysine). Receptor-mediated delivery of mRNA was demonstrated using the synthetic polyamino acid (K)16GACDCRGDCFCA designed to promote cell entry following interaction with cell surface αυ integrins. RGD-bearing mRNA complexes showed very high levels of expression, reaching over 60% transduction of B16F10 cells.

Kinetic properties of the C-terminal Src kinase, p50 csk

Csk is an important regulator of tyrosine kinases of the Src family. In this paper, we have characterised the kinetics and catalytic properties of a highly active and stable enzyme obtained in milligram amounts by expressing the enzyme as a fusion protein with glutathione- S-transferase (GST) in Escherichia coli. Using the synthetic polyamino acid poly(Glu,Tyr) as substrate, phosphotransferase activity was linear for 7–8 min with Mg 2+ and 5 min with Mn 2+. With Mg 2+ and Mn 2+, respectively, K m(ATP) was 56.9±6.2 and 5.4±0.6 μM and V max was 293±52 and 217±38 pmol phosphate transferred ( μg Csk) −1 min −1. Optimal concentrations of Mg 2+ and Mn 2+ were 4–10 mM and 2–3 mM, respectively, and higher concentrations of both cations were inhibitory. The Csk activity was highly sensitive to monovalent (Na +, K +) and divalent (Ca 2+) cations, the sensitivity being 2–5-fold higher with Mg 2+ than Mn 2+. Physiological concentrations of Ca 2+ (less than 10 μM) were without effect. Autophosphorylation of Csk was demonstrated in vitro, but did not influence the catalytic activity. Addition of inorganic phosphate above 100 μM strongly inhibited Csk catalytic activity towards poly(Glu,Tyr) in the presence of Mn 2+, but not in the presence of Mg 2+. Phosphorylation of a physiological substrate (Lck) and autophosphorylation of Csk was not inhibited by phosphate, indicating that the phosphate-dependent inhibition of Csk activity was substrate specific.

The pH dependence of the emitting properties of ruthenium polypyridyl complexes bound to poly-l-lysine

The synthetic poly-amino acid, poly-l-lysine has been covalently coupled to a ruthenium polypyridyl complex, a long-lived fluorescent species, via a thiourea linkage between the amino side groups of the lysine residues and the isothiocyanate group of the label. The pH dependence of the emission lifetime of the label is reported whereby the behavior observed is explained in terms of the conformational changes of the bound biomolecules due to the label's sensitivity to its local environment.

Scientist and Statesman: Ephraim Katchalski-Katzir at 80.

During May 13-16, 1996, the Weizmann Institute of Science in Rehovot was the host to an ititernational symposium to mark the 80th birthday of Professor Ephraim Katzir, better known to older biochemists as Ephraim Katchalski. The symposium, entitled“Perspectives in Molecular Biology and Biotechnology,” included five Nobelists-Max Perutz, Paul Berg, Arthur Kornberg, Manfred Eigen, and Edmond Fischer-with other scientists from North America and Europe and strong participation in the program by Israeli scientists. The symposium was a tribute from the Israel Academy of Sciences and Humanities and the Weizmann Institute to one of Israel’s most distinguished scientists. Born in Poland on May 16, 1916, Katchalski emigrated to Palestine in 1922 and with his brotherAharon decided, after joining the Hebrew University of Jerusalem, to pursue careers in research and teaching. This they did against a background of strife with the Arab population, in which they were soon involved by their joining the illegal Jewish defense organization, the Haganah. Katchalski has described in an autobiographical article (1) secretly manufacturing war materials at night in the research laboratory (for example, tear gas) while in his overt daytime research he was preparing and studying synthetic polypeptides, one of the contributionsforwhich he is best known. The Weizmann Institute, the successor to the Sieff Institute, was founded in 1949. Itwas named after Chaim Weizmann, himself an organic chemist, who became the first President of the State of Israel and whose career Katchalski’s has paralleled. When the State of Israel was established in 1948, it was invaded by the Arab armies and Katchaiski was placed in charge of the Israeli army science corps. Because, as Katchalski writes (1), “some of the American scientists who were (at that time) supposed to take charge of departments at the Institute were jittery about coming to Israel, Aharon was asked to be temporary head of the Department of Polymers and I was made acting head of the Department of Biophysics. These two appointments soon became permanent.” Reference should be made to Katchalski’s memoirs (1,2) for a detailed account and bibliography of his many contributions to protein chemistry, beginning with the synthetic polyaminoacids and including the preparationof immobilized enzymes, something that at the time was not recognized to be of significant industrial importance and from which the Weizmann Institute gained only a small sum from a patent, but which is now used on a large scale in industry. The most notable example is the manufacture of high fructose corn syrup, the productionof which in the United States alone in 1995 exceeded 10 million tons. Scientific recognition of this achievement came to Katchalski from the country that first exploited immobilized enzymes for industrial use. In 1985 lie was the first recipient of the Japan Prize. Katchalski continued as head of the Weizmann Department of Biophysics through 1973. He was again involved in the wars with the Arab countries, servimig during the Six Day War in 1967 as the chief scientist of the Defense Ministry and then for the five years from 1973 to 1978 was swept up completely into the affairs of his country by accepting the invitation of Prime Minister Golda Meir to be nominated forthe presidency,as a result of which he became the fourth President of the State of Israel. He has defined his presidency as beginning with the Yom Kippur War in October 1973 and ending with the surprise visit to Jerusalem by Anwar Sadat in November 1977 that opened

Activation of an 85 kDa ribosomal S6 kinase during serotonin-induced oocyte maturation.

Oocytes from the Japanese clam Ruditapes philippinarum are naturally blocked at the prophase-I stage of meiosis. Following physiological activation by the neurohormone serotonin (5HT), oocytes undergo germinal vesicle breakdown (GVBD) and reach a second cell cycle arrest in metaphase-I. To identify the kinases activated during meiosis reinitiation, we used a phosphorylation assay following sodium dodecyl sulphate-polyacrylamide gel electrophoresis and in situ renaturation. A soluble 85-kDa serine/threonine kinase (PK85) was highly and consistently activated (up to 17-fold) within 5 minutes following addition of the hormone. This activation occurred 5 to 10 minutes before GVBD and only when 5HT concentration was sufficient to induce meiosis reinitiation. The calcium ionophore A23187 and NH4Cl, two compounds known to induce GVBD by increasing intracellular calcium concentration, also activate PK85. In crude oocyte extracts, the presence of beta-glycerophosphate, NaF, okadaic acid, calyculin A or microcystin, prevented inactivation of PK85, suggesting that it is activated by phosphorylation. Partial purification of PK85 followed by Western blotting showed that this kinase is related to the ribosomal S6 kinase pp90rsk. PK85 phosphorylates the peptides LRRASLG (kemptide) and PLARTLSVAGLPGGK (syntide-2), and to a lesser extent the synthetic polyamino acids poly(R3:S1) while myelin basic protein (MBP), histone III-S, casein, the peptides pEKRPSQRSKYL ((pGlu4)-MBP 4-14), GTFRASIRRLAARRR (NIMA kinase substrate), the protein kinase C (PKC) substrate LRTLRR and the synthetic polyaminoacids poly(R1:P1:T1) were poor substrates. 5HT-induced GVBD and PK85 activation are both inhibited by the phorbol ester 12-myristate 13-acetate (PMA) and this inhibition can be reversed by 5 microM of the bisindolyl-maleimide GF109203X, a potent PKC inhibitor. PMA inhibitory action appears to take place between 5HT binding to its receptor and the intracellular calcium surge since it has no effect on GVBD induced by calcium ionophore A23187 and thapsigargin. Taken together, these results suggest that serotonin-induced activation of PK85 occurs after the intracellular calcium surge in a PKC-independent pathway.

The inhibition of a plant proteinase by lysine copolymers is modulated by the hydrophobicity of the interposed amino acid side chains

One Lys/Phe copolymer and two series of copolymers of lysine with either alanine or tyrosine have been used as inhibitors of a plant proteinase that is known to be inhibited by polycationic inhibitors. The copolymers differ in the hydrophobicity of the non-lysine amino acid residue and the single members of each series differ from each other in their degree of polymerization and in their charge density, i.e., the frequency of occurrence of the lysine residue in the synthetic polyamino acid chain. All the tested copolymers show cooperative inhibition, with a Hill coefficient higher than 1. CD measurements indicate that the inhibition is realized through a conformational change of the enzyme molecule. Both the enzyme inhibition and the conformational change are supported by aspecific electrostatic binding between the positively charged groups of the lysine moiety and the negatively charged groups of the enzyme surface. In each series the inhibitory power increases with the charge density, while at the same charge density the inhibitory efficiency depends on the hydrophobicity of the side chain of the non-lysine amino acid in the order Phe>Tyr>Ala.

Use of polyamino acid analogs of biomineral proteins in dispersion of inorganic particulates important to water treatment

Synthetic polyamino acids (peptides) based on the structure and activity of matrix proteins isolated from oyster shell and other biomineral structures have been identified for use in the prevention of mineral scaling. Matrix proteins are polyanionic and are thought to act as regulators of crystallization during the development of skeletal and other mineral structures. These proteins and their synthetic analogs also contain hydrophobic regions that may enhance their surface-active properties. The dispersion activity of a variety of polyamino acids that are matrix protein analogs has been evaluated in bench-top tests using inorganic mineral particles. Dispersion activities were measured using particles of iron oxide, kaolin, calcium carbonate, and calcium phosphate (hydroxyapatite). The activity was measured by increases in the spectrophotometric absorbance of test particle suspensions in the presence of dispersants. The increases in absorbance were due to turbidity resulting from the production of smaller particle sizes or slower rates of settling. The results suggest that biopolymers composed of polyanionic polyamino acids may be effective as dispersants. Polyamino acids containing a hydrophobic or phosphorylated domain attached to a polyaspartate backbone demonstrate enhanced activity over polyaspartate. These polyamino acids display comparable activity to commercially available hydrocarbon-based polymeric dispersants. An economical process for the manufacture of polyamino acids by thermal polycondensation is under development. Clearly, non-toxic and biodegradable polyamino acids present a desirable alternative to toxic non-biodegradable polymers in a number of applications such as detergents and cooling tower additives.

Biomimetic properties of simulated protomembranes

Experimental data of the concept of a pregenetic stage of the origin of a spherical protomembrane are reported. The protomembrane is considered as a result of transition from the phase separation to a structural-cellular membrane with primordial channel-like pores controlling ionic permeability. Research, based on a synthetic polyamino acid (Przybylski et al., 1982) and polyamino acid-lipid model (Ishima et al., 1981) of the membrane, demonstrated that the model reveals membrane and action potentials. The observed membrane of this model displays also: electrical conductance, ion permeability, current-voltage characteristics, hysteresis and negative resistance. The electrical phenomena were observed both in planar and spherical membranes. The pH effect upon electrical phenomena of synthetic membranes was observed. It is known that lipid-only models of the membrane do not reveal permeability and excitability. These properties are present in such models only after additon of proteins or polypeptides (Mueller and Rudin, 1968). It has been found that a deposition of thermal polyamino acids on the phosphatidylcholine membrane also results in electrical activity. (Przybylski, 1985). Membranes made from proteinoids-only display electrical activity as well. Electrical polarization, discharges, selective permeability, osmotic properties, and black films support electron micrograph evidence that structures made from thermal proteins have a double membrane resembling biological ones (Fox et al. 1978).

Polycations as possible substitutes for protamine in heparin neutralization

Protamine has long been used for the neutralization of heparin excess in clinical situations, but it does have several drawbacks such as being anticoagulatory itself, when in excess, or at times causing heparin rebound, when at verge of neutralization. In an attempt to find better substitutes for protamine, synthetic polyamino-acids and various types of histone were considered and their activity as heparin antagonists was compared to that of protamine. While protamine was found superior to histones in neutralizing heparin under all the test conditions, poly-DL-lysine showed a wider range of neutralization than did protamine. This particular substance, however, is unsuitable because of its toxicity, and it is recommended to search for additional synthetic polymers. Some of the properties to be desired in a heparin neutralizer are enumerated.

The influence of natural and synthetic cationic substances on phagocytic activity of human polymorphonuclear cells. An alternative pathway of phagocytic enhancement.

Purified human lysozyme (LZM) in concentration 300–500 μg/ml significantly enhanced (p < 0.01) phagocytosis and the phagocytic index of normal human granulocytes (PMNs). However, LZM's enhancing activity was selective and did not correlate with susceptibility of microorganisms to its lytic activity. LZM enhanced phagocytic activity of PMN's from patients with collagen diseases, hypogammaglobulinemia or recurrent infections but did not influence immature cells from acute or chronic granulocytic leukemia. Rabbit band-2, another lysosomal cationic protein, also enhanced phagocytosis. Human cationic leukemia antigen (CLA) and lysosomal proteins with less cationic properties, namely RNAse and lactoferrin had no significant enhancing activity. Nuclear cationic proteins, histone and protamine and synthetic polyamino acids containing arginine or lysine enhanced phagocytosis and phagocytic index and increased intracellular killing (p < 0.01) of Gram (−)ve and Gram (+)ve bacteria. They also enhanced phagocytosis and phagocytic index of latex particles. Mastocytotropic compound 4880 enhanced phagocytosis and phagocytic index of various microorganisms but not of latex particles. The enhancing activity of cationic substances was abolished by washing PMNs after exposure to these substances. Preincubation of microorganisms with various cationic substances lead to their death and lysis to various degrees depending on the type and concentration of the substance added. When the phagocytic assays were performed at 40 °C, further enhancement of phagocytosis by nuclear but not by lysosomal cationic proteins was observed. To the contrary, reduction of pH to 4.4 enhanced phagocytosis by lysosomal but not by nuclear cationic proteins. Anionic substances such as heparin, DNA and endotoxin abolished enhancing activity of lysosomal, nuclear and synthetic cationic substances but not of the mastocytotropic compound 4880. Cytochalasin B (CB) reduced, but not abolished, phagocytic activity of human PMNs and suppressed enhancing activity of cationic proteins. The most pronounced action of CB was inhibition of the intracellular killing of microorganisms by PMNs with or without cationic substances. CB did not influence viability of PMNs or of microorganisms and did not impair activity of lysostaphin. Phagocytic activity of human peripheral blood monocytes was not significantly influenced by cationic substances.

The Use of Synthetic Polyamino Acids for the Detection and Purification of Novel Proteolytic Enzymes

AbstractPolyamino acids were used as substrates for the detection and purification of new exopeptidases from bacteria. The enzymes, which were obtained in pure form and were characterized, include: aminopeptidase P, clostridial aminopeptidase, dipeptidocarboxypeptidase and a nonspecific dipeptidase. The possible application for sequential hydrolysis by the combined action of the two aminopeptidases was demonstrated by accomplishing complete hydrolysis of a proline‐rich nonapeptide, bradykinin. The dipeptidocarboxypeptidase, which produces dipeptides from the carboxyl end, and the dipeptidase, which then hydrolyses the dipeptides formed, indicate an efficient mechanism by which polypeptides are hydrolyzed from the carboxyl end of the molecule. The unique nature of the peptide bond, in which proline is involved through its nitrogen, is pointed out and the possible role of proline as a residue that protects terminal regions of polypeptides against the hydrolytic action of exopeptidases is discussed.