David Wade

Abstract: The name-to-peptide concept uses the International Union of Pure and Applied Chemistry-International Union of Biochemistry and Molecular Biology, Joint Commission on Biochemical Nomenclature (IUPAC-IUB, JCBN) system for abbreviating the names of amino acids (AAs) with single letters of the English alphabet, and considers the strings of letters in personal and other names as strings of AAs, or peptides. The method can be used to design and create novel peptides of potential medical usefulness. This article reports the results of a theoretical analysis of hundreds of personal names extracted from the 2010 Forbes List of the World�s Billionaires with the goal of finding names containing letter compositions corresponding to AA sequences containing the CendR motif (i.e., R/K-X-X-R/K at the carboxyl terminal end of the peptide, where R is Arginine, K is Lysine, and X can be any AA). The names of US citizens were separated from the Forbes List, and searched for the presence of the CendR motif. Only 10 of the 403 names were found to contain it, and of these only 4 had given and family names composed entirely of letters corresponding to IUPAC-IUB, JCBN abbreviations for gene-encoded AAs: Ty Warner, Stewart Rahr, Randal Kirk, and Nancy Lerner. The given and family names of these four were combined to form letter sequences corresponding to name peptides. Protein databases were searched for the occurrence of these peptides in nature, and portions of each peptide were found in a variety of natural proteins, including proteins of known three dimensional structure. Each name peptide was modeled as two commonly found structures in proteins, the α-helix and the β-strand, and potential properties of these hypothetical peptides are discussed.

Abstract: This article proposes the synthesis and study of a peptide that is composed of 13 amino acids, and that has an amino acid composition and sequence that can be described with International Union of Pure and Applied Chemistry-International Union of Biochemistry and Molecular Biology, Joint Commission on Biochemical Nomenclature single letter abbreviations corresponding to the sequence of letters in the name of Amelia Earhart, the famous aviatrix of the early 20th century: Alanine (A)-Methionine (M)-Glutamic acid (E)-Leucine (L)-Isoleucine (I)-Alanine (A)-Glutamic acid (E)-Alanine (A)-Arginine (R)-Histidine (H)-Alanine (A)-Arginine (R)-Threonine (T). The existence of similar amino acid sequences in nature, and possible structures and uses for the peptide are discussed. The peptide would probably exhibit biological activities.

Abstract: The Temporin-/Vespid Chemotactic Peptide- (or T/V-) like peptides are a group of small [average of 13 amino acids (AAs)], linear (no Cysteine), hydrophobic (avg. 65% hydrophobic AAs), positively charged (avg. net charge at pH 7 = +2), structurally very similar, peptide amides that have been isolated from the skins of frogs and the venoms of insects during the past 30 years. Recently, the number of reports of frog T/V-like peptides has been rapidly increasing. At the time of this writing, 119 unique peptides have been isolated from frogs, 13 from wasps, and 11 from scorpions. Many of these peptides have been subjected to biological testing and found to exhibit defensive types of functions (e.g., antimicrobial). Consensus sequences were first reported for 30 frog T/V-like peptides in 2000 [1], and updated in 2002 (36 peptides) [2] and 2003 (40 peptides) [3] (Table 1). Consensus sequences for wasp peptides were first reported in 2002 (8 peptides) [2], and a combined frog plus wasp consensus sequence was first reported in 2003 (48 peptides) [3]. Table 1 shows these consensus sequences plus several more for 2010. Two 2010 frog consensus sequences were derived from 119 peptides, and neither corresponds to any previously reported, naturally occurring peptide. Two 2010 wasp consensus sequences were derived from 13 peptides. One corresponds to frog peptide, T-1CSb, and the other to wasp peptides, VesCP M and 5h. In addition, there are four 2010 frog plus wasp consensus sequences derived from 132 peptides (119 frog and 13 wasp), and two of these sequences correspond to the frog peptides, T-TOb and T-1CSb, and the wasp peptides, VesCP M and 5h. Biological data has been reported for all four of these naturally occurring peptides. Consensus sequences may represent ancestral sequences from which other T/V-like peptides evolved, or common sequences toward which T/V-like peptides are evolving.

Abstract: This article proposes the chemical synthesis and study of a peptide that has an amino acid (AA) sequence with one letter abbreviations that correspond to the name, PRINCETON: Proline (P)-Arginine (R)-Isoleucine (I)-Asparagine (N)-Cysteine (C)-Glutamic acid (E)-Threonine (T)- Ornithine (O)-Asparagine (N). In this sequence, eight of the nine AAs are represented by official International Union of Pure and Applied Chemistry (IUPAC) one letter abbreviations for AAs, and the letter, O, represents Ornithine. A possible structure for the peptide is described, the existence of similar AA sequences in nature are discussed, and it is concluded that the peptide would probably exhibit biological activities.

Abstract: This article proposes the chemical synthesis and study of a peptide that has an amino acid (AA) sequence with one letter abbreviations that correspond to the name, KAROLINSKA: Lysine (K)-Alanine (A)-Arginine (R)-Ornithine (O)-Leucine (L)-Isoleucine (I)-Asparagine (N)-Serine (S)-Lysine (K)-Alanine (A). In this sequence, nine of the ten AAs are represented by official International Union of Pure and Applied Chemistry (IUPAC) one letter abbreviations for AAs, and the letter, O, represents Ornithine. A possible structure for the peptide is described, the existence of similar AA sequences in nature are discussed, and it is concluded that the peptide would probably exhibit biological activities.

Abstract: This article is a revised version of an article with the same title that was published by the author in 2006, and it corrects errors that are present in the previous version. A method is presented for determining the energy of a name as the acoustical energy of the vocalized name. The names, Madonna and Esther, as spoken by the author and by a computerized text-to-speech program, were used to test the method. Differences between the acoustical energies of the two names depended upon the method used to measure the energies. There was no difference between the energies of the names as spoken by the author, but when the names were vocalized with a computerized text-to-speech program, Madonna had 2.6-fold greater energy than Esther.

Abstract: This article describes a theoretical experiment that uses the International Union of Pure and Applied Chemistry�s one letter system for abbreviating the chemical names of amino acids to interpret the letter sequences, YHWH and YHVH, English transliterations of the Hebrew name of God, as representing the amino acid sequences of two tetrapeptides, and proposes the chemical synthesis and biological testing of the two peptides. Both sequences of letters were found to occur commonly among the nearly 4.7 million amino acid sequences in the protein databases of the National Center for Biotechnology Information, and to be associated with proteins of known biological function. There are no barriers to the chemical synthesis of these two peptides, and they have a high probability of exhibiting biological and �other� activities.

Abstract: Three of the most recognizable corporate names are Microsoft, a manufacturer of computer software, and Google and Yahoo, two internet search providers. Two of the most popular soft drink brand names are Coca-Cola and Pepsi-Cola. This article proposes to use the sequences of English alphabet letters in the names of these companies and products as the basis for creating five new peptides, MICROSOFT, GOOGLE, YAHOO, COCACOLA, and PEPSICOLA. This can be done by using a modified version of the International Union of Pure and Applied Chemistry�s one letter abbreviations for the names of amino acids. In this modification, the unassigned letter, O, is used to represent the amino acid, Ornithine. Precedent for this assignment already exists in the chemical literature. Ornithine does not occur in natural proteins, and, therefore, the letter, O, does not occur among the amino acid sequences of any protein database. However, Ornithine is structurally and chemically very similar to the amino acid, Lysine, which does occur in natural proteins and which is represented by the one letter abbreviation, K, in the IUPAC system. A search of the National Center for Biotechnology Information protein databases for the letter sequences, MICRKSKFT, GKKGLE, YAHKK, CKCACKLA and PEPSICKLA, where the one letter abbreviation, K, for Lysine, replaced the letter, O, returned numerous exact (GKKGLE and YAHKK) and partial (MICRKSKFT, CKCACKLA and PEPSICKLA) sequence matches. This result indicates that all of the search sequences are common in proteins. There are no synthetic barriers to the creation of these Ornithine containing peptides. Consequently, it is predicted that the synthetic peptides, MICROSOFT, GOOGLE, YAHOO, COCACOLA, and PEPSICOLA, will all exhibit biological activities.

Abstract: What is the basis of the preference for one given, or first, name versus another? This article explores that question by analyzing the acoustic, or sound, energy of vocalized names from the US Social Security Administration�s list of the thousand most popular given names for the period of 2000-2005. The 100 most popular, and 100 least popular, male and female given names were vocalized by a computerized text-to-speech program, the vocalizations were recorded and analyzed by the Sound Ruler acoustic analysis program, and the results for the most and least popular groups were compared. Although it was possible to find substantial differences between the acoustic energies of individual names, when the names were considered as groups of 100, there were no significant differences between the average energies of the most popular and least popular name groups, or between male and female names. Consequently, the acoustic energies of vocalized names do not seem to be the basis of given name preferences.

Abstract: A method is presented for determining the energy of a name as the acoustic energy of the spoken name. The names, Madonna and Esther, as spoken by the author, differed by ~0.5 V of acoustic energy. This evidence may support the hypothesis of a difference in the energies of names.

Abstract: The ability of nisin, synthetic temporin analogs, magainins, defensins, and cecropins to inhibit Bacillus anthracis, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, and Bacillus subtilis growth from spore inocula was determined using well diffusion assays. Nisin, magainin II amide, and defensins were inhibitory in screening against B. anthracis Sterne or B. cereus ATCC 7004, but only nisin inhibited virulent B. anthracis strains. The MICs of nisin against the 10 Bacillus strains examined were 0.70 to 13.51 microg/ml. Synthetic temporin analogs also inhibited B. anthracis but were not as potent as nisin. None of the strains examined were appropriate B. anthracis surrogates for testing sensitivity to antimicrobial peptides.

Abstract: Topical antimicrobicides hold great promise in reducing human immunodeficiency virus (HIV) transmission. Amphibian skin provides a rich source of broad-spectrum antimicrobial peptides including some that have antiviral activity. We tested 14 peptides derived from diverse amphibian species for the capacity to inhibit HIV infection. Three peptides (caerin 1.1, caerin 1.9, and maculatin 1.1) completely inhibited HIV infection of T cells within minutes of exposure to virus at concentrations that were not toxic to target cells. These peptides also suppressed infection by murine leukemia virus but not by reovirus, a structurally unrelated nonenveloped virus. Preincubation with peptides prevented viral fusion to target cells and disrupted the HIV envelope. Remarkably, these amphibian peptides also were highly effective in inhibiting the transfer of HIV by dendritic cells (DCs) to T cells, even when DCs were transiently exposed to peptides 8 h after virus capture. These data suggest that amphibian-derived peptides can access DC-sequestered HIV and destroy the virus before it can be transferred to T cells. Thus, amphibian-derived antimicrobial peptides show promise as topical inhibitors of mucosal HIV transmission and provide novel tools to understand the complex biology of HIV capture by DCs.

Abstract: The ability of five purified amphibian antimicrobial peptides (dermaseptin-1, temporin A, magainin I, and II, PGLa), crude peptide fractions isolated from the skin of Rana pipiens and R. catesbeiana, and four antimicrobial peptides (AMPs) from hybrid striped bass (piscidin-1N, -1H, -2, and -3) were examined for their ability to reduce the infectivity of channel catfish virus (CCV) and frog virus 3 (FV3). All compounds, with the exception of magainin I, markedly reduced the infectivity of CCV. In contrast to CCV, FV3 was 2- to 4-fold less sensitive to these agents. Similar to an earlier study employing two other amphibian peptides, the agents used here acted rapidly and over a wide, physiologically relevant, temperature range to reduce virus infectivity. These results extend our previous findings and strongly suggest that various amphibian and piscine AMPs may play important roles in protecting fish and amphibians from pathogenic viruses.

Abstract: Many mammalian antimicrobial peptides (AMPs) have multiple effects on antimicrobial immunity. We found that temporin A (TA), a representative frog-derived AMP, induced the migration of human monocytes, neutrophils, and macrophages with a bell-shaped response curve in a pertussis toxin-sensitive manner, activated p44/42 MAPK, and stimulated Ca(2+) flux in monocytes, suggesting that TA is capable of chemoattracting phagocytic leukocytes by the use of a G(ialpha) protein-coupled receptor. TA-induced Ca(2+) flux in monocytes was cross-desensitized by an agonistic ligand MMK-1 specific for formyl peptide receptor-like 1 (FPRL1) and vice versa, suggesting that TA uses FPRL1 as a receptor. This conclusion was confirmed by data showing that TA selectively stimulated chemotaxis of HEK 293 cells transfected with human FPRL1 or its mouse ortholog, murine formyl peptide receptor 2. In addition, TA elicited the infiltration of neutrophils and monocytes into the injection site of mice, indicating that TA is also functionally chemotactic in vivo. Examination of two additional temporins revealed that Rana-6 was also able to attract human phagocytes using FPRL1, but temporin 1P selectively induced the migration of neutrophils using a distinct receptor. Comparison of the chemotactic and antimicrobial activities of several synthetic analogues suggested that these activities are likely to rely on different structural characteristics. Overall, the results demonstrate that certain frog-derived temporins have the capacity to chemoattract phagocytes by the use of human FPRL1 (or its orthologs in other species), providing the first evidence suggesting the potential participation of certain amphibian antimicrobial peptides in host antimicrobial immunity.

Abstract: A peptide was designed so that the one letter abbreviations for its amino acid sequence corresponded to the joined first and last names of the current United States Secretary of State, Colin Powell. Peptide COLINPOWELL (i.e., Cys-Orn-Leu-Ile-Asn-Pro-Orn-Trp-Glu-Leu-Leu) was synthesized as a C-terminal amide, and found to be inactive against bacteria [methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE)] and herpes simplex virus (HSV-1), and it had no effect on plasma coagulation. However, it was capable of inducing migration of human monocytes and neutrophils, and inhibiting the proliferation of human breast cancer (T47D) cells. Molecular modeling indicated that the peptide has the potential to form intramolecular hydrogen-bonded structures, such as short helices or a small β-sheet, but circular dichroism analysis indicated that the reduced form of the peptide has very little structure in aqueous solutions of 0-90% trifluoroethanol. In such a hydrophobic environment, the structure of COLINPOWELL apparently resembles the relatively nonstructured conformations of similar amino acid sequences that are found in four proteins of known 3D structure (pancreatic kallikrein A, matrix metalloproteinase 8/neutrophil collagenase, Cre recombinase, and hexulose reductase). This study demonstrates the potential for designing bioactive peptides based solely on words or names constructed from the letters of the English alphabet. In addition, the bioactivity test results indicate that peptide, COLINPOWELL, might have potentially interesting biomedical uses.

Abstract: Temporin A and structurally related peptides produced in amphibian dermal granular glands and in wasp venom were tested for growth inhibition of Batrachochytrium dendrobatidis, a pathogen associated with global amphibian declines. Two natural amphibian temporins, a wasp temporin, and six synthetic analogs effectively inhibited growth. Differences in potency due to amino acid substitution suggest that ability to penetrate membranes and form an alpha-helical structure is important for their effectiveness against this pathogen.

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Abstract: Several naturally occurring antimicrobial peptides, from mammals and insects, have previously been shown to be chemotactic for human inflammatory cells. Based on this evidence, ten synthetic analogs of naturally occurring antibiotic peptides from the skin secretions of three species of Ranid frogs and the venom of one species of Vespid wasp (i.e., T/V-like peptides) were tested for their abilities to induce migration of human neutrophils and monocytes. These included temporin A (TA from Rana temporaria), temporin 1P (T1P from R. pipens), ranateurin 6 (Rana-6 from R. catesbeiana)], three TA analogs [all D-amino acids (D-TA), reversed sequence (Rev-TA), and Pro3�Gly (G3-TA)], two frog skin-related T/V-like peptide consensus sequences (I4S10-Con and I4G10-Con), VesCP-M (VCP-M from Vespa mandarinia), and a hybrid peptide composed of portions of the insect antibiotic peptide, cecropin A (CA), and TA (CATA). TA, T1P, Rana-6, VCP-M, G3-TA, I4S10-Con, I4G10-Con, and CATA all induced cell migration at micromolar concentrations. D-TA and Rev-TA did not induce cell migration, suggesting that this process involves a chiral interaction, such as receptor binding, and also depends on the order of amino acids within TA. The results demonstrate, for the first time, that certain T/V-like antibiotic peptides are capable of inducing chemotaxis of human phagocytes and suggest that these peptides are multifunctionalmolecules with antimicrobial, hemolytic, and chemotactic capabilities.

Abstract: A group of 34 peptides, that have been isolated from the skins of frogs and the venom of wasps, are structurally similar to 10 antibiotic peptides, called temporins, isolated from the skin of the European red frog, Rana temporaria. These temporin-like peptides have antibiotic and neutrophil stimulating properties. A preliminary consensus sequence derived from 30 frog skin peptides was a good predictor of the sequences of recently isolated temporin-like peptides, but it contained ambiguities at 2 of 13 positions. The discovery of new frog skin temporins, and temporin-like peptides from wasp venom, enabled the derivation of two new and unambiguous consensus sequences, one for frog skin peptides and the other for wasp venom peptides. This article describes some of the properties of temporin-like peptides, their phylogenic relationships, and the derivation of the two new consensus sequences and some of their biological properties.

Abstract: A computerized database for synthetic antibiotic peptides, the SAPD, has been created and is available on the Internet at web address, http://oma.terkko.helsinki.fi:8080/~SAPD. The SAPD is modelled on two pre-existing computer databases for naturally occurring peptide antibiotics, the Antimicrobial Sequences Database and the Peptaibol Database, and it will contain both chemical and biological information on all published synthetic antibiotic peptides.

Abstract: A group of 34 peptides, that have been isolated from the skins of frogs and the venom of wasps, are structurally similar to 10 antibiotic peptides, called temporins, isolated from the skin of the European red frog, Rana temporaria. These temporin-like peptides have antibiotic and neutrophil stimulating properties. A preliminary consensus sequence derived from 30 frog skin peptides was a good predictor of the sequences of recently isolated temporin-like peptides, but it contained ambiguities at 2 of 13 positions. The discovery of new frog skin temporins, and temporin-like peptides from wasp venom, enabled the derivation of two new and unambiguous consensus sequences, one for frog skin peptides and the other for wasp venom peptides. This article describes some of the properties of temporin-like peptides, their phylogenic relationships, and the derivation of the two new consensus sequences and some of their biological properties.

Abstract: Temporin A, 18 analogs, and a cecropin A-temporin A hybrid peptide were tested with antibiotic sensitive and resistant bacteria, fungi, human erythrocytes, and in clotting assays.Several peptides were active in these assays, and some analogs (D-TA, W1-TA, and Con-L4,G10) may be useful lead compounds for further antibiotics development.The activity of temporin A was found to be dependent upon several of its structural features, including amino acid composition and sequence, chirality, helicity, and positive charge.

Abstract: Temporin A is one of a group of 31 structurally similar antibiotic peptides isolated from the skins of anurans, 10 of which were isolated from the European red frog, Rana temporaria. The synthesis and bacteriological study of temporin A and 8 analogs were reported previously, and this article describes the synthesis and analyses of 11 new temporin A analogs, and a cecropin A-temporin A hybrid peptide.

Abstract: CAMELs are a group of novel synthetic antibiotic peptides that may be effective substitutes for ciprofloxacin in the treatment of anthrax infections.

Abstract: Temporin A (TA) and a cecropin A-temporin A hybrid peptide (CATA) were synthesized and assayed for their hemolytic, anticoagulant, and antifungal properties. CATA retains significant antifungal activity, is less hemolytic than TA, and inhibits blood coagulation. These results recommend further studies of the biological activities of CATA.

Abstract: Cysteine proteinase found in the spinal cord of rat, called nociceptin-converting enzyme (NCE), is competitively inhibited by dynorphin A and its fragment des-[Tyr(1)]-DYN A. This proteinase converts orphanin FQ/nociceptin (OFQ/N) to two major fragments: OFQ/N(1-11) and further OFQ/N(1-6) with analgesic properties. Dynorphin A at the concentration of 10 microM increases K(M) from 15.0 to 55.9 microM. The calculated K(i) for this interaction was estimated at 3.7 microM. This observation may suggest an interaction between opioid and nociceptive systems which may be affected by the balance between opioid and antiopioid systems. This balance between particular OFQ/N sequences that are derived from the same precursor and regulated by proteinases may play an important role in pain. Interestingly, dynorphin B does not reveal a similar action on the NCE.

Abstract: Temporin A (TA) is a small, basic, highly hydrophobic, antimicrobial peptide amide (FLPLIGRVLSGIL-NH2) found in the skin of the European red frog, Rana temporaria. It has variable antibiotic activities against a broad spectrum of microorganisms, including clinically important methicillin-sensitive and -resistant Staphylococcus aureus as well as vancomycin-resistant Enterococcus faecium strains. In this investigation the antimicrobial activity and structural characteristics of TA synthetic analogs were studied. For antibacterial activity against S. aureus and enterococcal strains, the hydrophobicity of the N-terminal amino acid of TA was found to be important as well as a positive charge at amino acid position 7, and bulky hydrophobic side chains at positions 5 and 12. Replacing isoleucine with leucine at amino acid positions 5 and 12 resulted in the greatest enhancement of antibacterial activity. In addition, there was little difference between the activities of TA and its all-D enantiomer, indicating that the peptide probably exerts its effect on bacteria via non-chiral interactions with membrane lipids.

Abstract: The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. One line of investigation is the synthesis of antimicrobial hybrid peptides. The aim of the present investigation was to determine the in vitro activities of 16 cecropin-melittin hybrid peptides (CAMEL analogues) against 60 anaerobic bacterial strains, to compare their activities with those of seven clinically used antimicrobial agents, and to compare different methods for anaerobic susceptibility testing of these peptides. The stability of one of the peptides, temporin B, with different stereoisomeric configurations was investigated in a fecal milieu. The CAMEL analogues showed antimicrobial activity against the anaerobic bacteria, with MICs ranging from 0.125 to 32 microg/ml. The overall activities (the MICs at which 90% of isolates are inhibited) of the CAMEL analogues against anaerobic bacteria were mainly inferior to those of imipenem, clindamycin, and piperacillin but were equal to or superior to those of metronidazole, cefoxitin, ciprofloxacin, and chloramphenicol. The agarose dilution method was found to be an accurate method for the testing of large numbers of bacterial strains. The D isomer of temporin B was inactivated more slowly in feces than the L isomer. This study shows that the CAMEL analogues are potential agents for the treatment of anaerobic infections.

Abstract: The temporins are a group of small, linear, basic, highly hydrophobic, antibiotic, peptide amides that were originally isolated from the skin of the European red frog, Rana temporaria. During the past decade, 30 temporin or temporin-like peptides have been isolated from the skins of Anurans. This article presents a brief review of the temporin literature, an analysis of the amino acid sequences of temporins and temporin-like peptides, and the derivation of a composite or consensus sequence, [FLP(I/L)IASLL(S/G)KLL-am].

Abstract: The topic of deuterium isotope effects is usually concerned with the effects on chemical reactions that are caused by the substitution of deuterium atoms for protium, or hydrogen, atoms in a molecule. These effects include changes in the rate of cleavage of covalent bonds to deuterium, or to an atom located adjacent to deuterium, in a reactant molecule. Deuterium isotope effects on other, noncovalent, interactions between molecules are known to occur, but they are generally considered to be insignificant, especially in biological experiments where deuterium substituted molecules are used as tracers. Noncovalent interactions between molecules include hydrogen bonding, and ionic and van der Waals interactions. This article reviews evidence for deuterium isotope effects on noncovalent interactions, with an emphasis on binding interactions between molecules of biological interest, but also including examples of nonbiological molecules in order to demonstrate the generality of these effects. The reality of this effect relies on the assumption that the only difference between the isotopomers considered is the presence of deuterium or hydrogen; there are no impurities present. The physical basis of the effect may be due to differences in the polarities and/or sizes of deuterated versus nondeuterated isomers, and the extent of a deuterium isotope effect on a noncovalent interaction depends on the site of deuteration within a biomolecule. The presence of this effect requires careful interpretation of results obtained in experiments with deuterium labeled compounds.

Abstract: Temporin A is a small, basic, highly hydrophobic, antibacterial peptide found in the skin of the European red frog, Rana temporaria. It was synthesized twice by the FastMoc solid phase method using amino acids protected at the N(alpha)-position with either 9-fluorenylmethoxycarbonyl or 2-(4-nitrophenylsulfonyl)ethoxycarbonyl. The syntheses of temporin A demonstrates the difference between 2-(4-nitrophenylsulfonyl)ethoxycarbonyl and 9-fluorenylmethoxycarbonyl amino acids. The purified peptide showed also antibacterial activity against clinically important gram-positive bacteria. It was found to have a moderately good activity against both methicillin resistant and sensitive strains of Staphylococcus aureus, but a weaker activity against vancomycin resistant strains of Enterococcus faecium.

Abstract: Efb (previously Fib) is a fibrinogen-binding protein secreted by Staphylococcus aureus. It has previously been shown that it plays a role in a wound infection model in the rat and that antibodies against Efb reduce the number of recovered bacteria from the mammary glands in a mouse mastitis model. Efb binds to the alpha-chain of fibrinogen and does not participate in bacterial adherence to fibrinogen. The binding of Efb to fibrinogen is divalent, with one binding site within the two repeat regions in Efb at the N terminus and one binding site at the C terminus. The divalent binding nature leads to precipitation of Efb-fibrinogen complex when the proteins are added to each other at a 1:1 molar ratio. The interaction between Efb and fibrinogen is strongly enhanced by Ca2+ or Zn2+ but not by Mg2.

Abstract: OBJECTIVE: Several small, 15-residue peptides that contain portions of the amino acid sequences of both cecropin A and melittin have previously been shown to have broad-spectrum antibacterial activities against aerobic microorganisms, with no undesirable hemolytic properties. It would also be useful to know what effect these hybrid peptides have on anaerobic bacteria. METHODS: The minimum inhibitory concentrations of one hybrid, CA(1--7)M(2--9)NH2, were compared with those of seven other antimicrobial agents against 111 clinical anaerobic strains; Bacteroides fragilis, 24 strains; other Bacteroides fragilis group, 14 strains; other Bacteroides species, 13 strains; Fusobacterium nucleatum, six strains; Clostridium difficile, 22 strains; Clostridium perfringens, 10 strains, Propionibacterium spp., nine strains; and anaerobic cocci, 13 strains. RESULTS: Ninety per cent of strains belonging to the B. fragilis group, fusobacteria, propionibacteria and peptostreptococci were inhibited by 4 mg/L CA(1--7)M(2--9)NH2, and the antimicrobial activity was approximately in the same range as that of chloramphenicol. CONCLUSION: This investigation showed that the antimicrobial spectrum of this cecropin---melittin hybrid also includes anaerobic organisms.

Abstract: Staphylococcus aureus produces and secretes a small, basic protein, designated Efb, that binds to fibrinogen and seems to be required for virulence of the organism. A 3D model of Efb was developed, and it predicts that the C-terminal half of the protein contains substantial a-helical content. CD analysis of Efb yielded a value of 40-41% a-helix. Calcium and zinc both influence the interactions between Efb and fibrinogen, and an analysis of the amino acid sequence of Efb revealed the presence of consensus sequences for the binding of both metals.

Abstract: Staphylococcus aureus produces and secretes a protein, Efb, that binds to fibrinogen, seems to be required for virulence, and may benefit the microorganism by delaying wound healing. Interactions of Efb with fibrinogen are influenced by divalent metal cations, including Ca2+. Increasing concentrations of Ca2+ increased the binding of fibrinogen to immobilized Efb, whereas binding of Efb to immobilized fibrinogen was decreased with increasing Ca2+ concentration. Studies with synthetic peptides showed that peptides from the carboxyl terminal half of Efb bound to soluble fibrinogen and enhanced the binding of fibrinogen to Efb. A peptide corresponding to a repeated sequence in the amino terminal half of the protein also bound fibrinogen and inhibited binding of fibrinogen to Efb. These results may provide clues to the biological function of Efb and aid in the rational design of agents to block the Efb fibrinogen interaction.

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Abstract: The last decade has seen the rapid development of research investigating the mechanisms of apoptosis in a variety of experimental systems. Among the multitude of changes observed in apoptotic cells, chromatin cleavage is considered a biochemical hallmark of apoptosis. Chromatin fragmentation is an enzymatic process which depends on the activity of endogenous nuclease(s) and the susceptibility of chromatin to endonuclease activity. The characteristics of some nucleases of potential importance in apoptosis and their possible role in the regulation of this process are discussed in this paper.

Abstract: Melittin, a 26-residue bee venom peptide, is known to induce murine Abs specific for its hydrophilic C-terminus of residues 20-26 and T cell responses specific for its hydrophobic mid-region of residue 11-19. Synthetic melittin analogues with transposed sequences of Ac(21-26) (1-20) and Ac(26-21) (1-20) are found to induce murine Abs specific for the transposed peptide segment and to induce T cell responses that are cross-reactive with melittin. Compared with melittin, the transposed melittin analogues are weaker immunogens and have lower hemolytic activities, lower helical contents, and a lower degree of association in micelles. A melittin analogue with a lactoside group at its C-terminus was found to induce lactoside-specific murine Abs. Present studies show that another analogue with a lactoside group at its N-terminus induces only Abs specific for the C-terminal region of melittin, and no lactoside-specific Abs are detected. These immunochemical observations suggest that the immunogenicity of melittin or its analogues is a consequence of its binding to cell membranes with subsequent oligomer formation in lipid bilayers. Apparently melittin or its analogues bind to cell membrane in an asymmetric manner with the exposed and the buried segments functioning as B and T cell epitopes, respectively. D-melittin is non-immunogenic in mice, although D-melittin has the same hemolytic activity as melittin. This finding may be correlated with the known resistance of D-melittin to proteolysis and hence to processing for Ag presentation to T lymphocytes.

Abstract: Isolated rat liver nuclei were incubated in the presence of divalent cations, and the mechanisms underlying the subsequent chromatin fragmentation were investigated. Either of the two cations, Ca2+ or Mg2+ was sufficient to produce chromatin fragments with sizes between 700 and 300 kbp. The formation of chromatin fragments of 50 kbp as well as the following internucleosomal DNA cleavage--which are characteristic of apoptosis--were markedly stimulated in the presence of Ca2+. Chromatin degradation to 50 kbp and smaller (oligonucleosome-size) fragments was prevented by inhibitors of endonucleases and serine proteases. We suggest a mechanism whereby the concerted activity of both proteases and endonucleases results in the widespread chromatin cleavage observed in cells undergoing apoptosis.

Abstract: Eight new analogs of cecropin A, two new analogs of melittin and 30 hybrid peptides containing sequences from cecropins and melittin have been synthesized. The lengths of the peptides have varied from 37 residues (the length of cecropin A) to 18 residues. The peptides have been assayed for lysis of sheep red blood cells and for antibacterial activity against two Gram negative and three Gram positive bacteria. The best analogs of cecropin A maintained the anti-Escherichia coli activity of the parental peptide, and were not lytic for red blood cells. Melittin and its replacement analogs were all lytic for red blood cells, but an analog with transposed segments was not. Several of the hybrid peptides were found to be both non-hemolytic and highly active against all test bacteria. The data were used to define the structural requirements for antibacterial activity.

Abstract: We have earlier reported two 26-residue antibacterial peptides made up from different segments of cecropin A (CA) and melittin (M). We now report a substantial reduction in size at the C-terminal section of the highly active hybrid CA(1-8)M(1-18), leading to a series of 20-, 18- and 15-residue analogs with antibiotic properties similar to the larger molecule. In particular, the 15-residue hybrids CA(1-7)M(2-9), CA(1-7)M(4-11) and CA(1-7)M(5-12) are the shortest cecropin-based peptide antibiotics described so far, with antibacterial activity and spectra similar or better than cecropin A and a 60% reduction in size. Their reduced size and highly alpha-helical structure require an alternative mechanism for their interaction with bacterial membranes.

Abstract: Deuteration of N-nitrosodimethylamine (NDMA) decreases its carcinogenicity and produces an isotope effect on its metabolism. Our previous results showed that deuteration causes a 5-fold increase in the apparent Km, but not the Vmax, for the demethylation and denitrosation of NDMA in microsomes. In the present work, we studied the nature of this deuterium isotope effect with several compounds using acetone-induced microsomes as a source of cytochrome P-450IIE1. In the microsomal N-nitrosodiethylamine deethylase reaction, NDMA and [2H6]NDMA were competitive inhibitors and displayed apparent Ki values of 59 and 441 mM, respectively, showing an isotope effect of 0.13. Similarly, in the p-nitrophenol hydroxylase reaction, a deuterium isotope effect of 0.21 on the Ki was observed. With acetone as an inhibitor for p-nitrophenol hydroxylase, the isotope effect on the Ki was 0.11. Similar deuterium isotope effects were also observed with acetone and dimethylformamide as competitive inhibitors for NDMA demethylase. When the oxidation of ethanol, [1,1-2H2]ethanol, [2,2,2-2H3]ethanol, and [2H6]ethanol was compared, an isotope effect of about 5 was found in the Vmax/Km due to the deuteration of the methylene group (carbon 1) but not due to the methyl group. However, the Vmax was not affected. A corresponding deuterium isotope effect was observed in the Ki when these compounds were used as competitive inhibitors for the NDMA demethylase reaction. The results demonstrate that deuteration of NDMA, ethanol, and related compounds results in an increase in the Km or Ki with little change in the Vmax of P-450IIE1-catalyzed reactions. The molecular basis of this isotope effect is discussed.

Abstract: The D enantiomers of three naturally occurring antibiotics--cecropin A, magainin 2 amide, and melittin--were synthesized. In addition, the D enantiomers of two synthetic chimeric cecropin-melittin hybrid peptides were prepared. Each D isomer was shown by circular dichroism to be a mirror image of the corresponding L isomer in several solvent mixtures. In 20% hexafluoro-2-propanol the peptides contained 43-75% alpha-helix. The all-D peptides were resistant to enzymatic degradation. The peptides produced single-channel conductances in planar lipid bilayers, and the D and L enantiomers caused equivalent amounts of electrical conductivity. All of the peptides were potent antibacterial agents against representative Gram-negative and Gram-positive species. The D and L enantiomers of each peptide pair were equally active, within experimental error. Sheep erythrocytes were lysed by both D- and L-melittin but not by either isomer of cecropin A, magainin 2 amide, or the hybrids cecropin A-(1-13)-melittin-(1-13)-NH2 or cecropin A-(1-8)-melittin-(1-18)-NH2. The infectivity of the bloodstream form of the malaria parasite Plasmodium falciparum was also inhibited by the D and L hybrids. It is suggested that the mode of action of these peptides on the membranes of bacteria, erythrocytes, plasmodia, and artificial lipid bilayers may be similar and involves the formation of ion-channel pores spanning the membranes, but without specific interaction with chiral receptors or enzymes.

Abstract: Solid phase synthesis was used to produce 5 hybrid peptides containing sequences from the antibacterial peptide, cecropin A, and from the bee venom toxin, melittin. Four of these chimeric peptides showed good antibacterial activity against representative Gram-negative and Gram-positive bacterial species. The best hybrid, cecropin A(1-13)-melittin(1-13) was 100-fold more active than cecropin A against Staphylococcus aureus. It was also a 10-fold better antimalarial agent than cecropin B or magainin 2. Sheep red cells were lysed by melittin at low concentrations, but not by the hybrid molecules, even at 50 times higher concentrations.

Abstract: With the goal of identifying the organic amine product(s) of enzymatic N-nitrosodimethylamine (NDMA) denitrosation, 4 mM NDMA was incubated with liver microsomes from ethanol-treated rats. The concentrations of dimethylamine and methylamine were determined by derivatization with 2,4-dinitrofluorobenzene followed by gas chromatography-mass spectrometry. There was no net increase in the concentration of dimethylamine during incubation, but the yield of methylamine was equimolar with that of nitrite. Additional incubations of NDMA using acetone-induced microsomes, 1 mM and 0.1 mM substrate, gave methylamine/nitrite ratios of 0.9 and 0.7, respectively, confirming the quantitative linkage between these two products. Control incubations conducted with pure methylamine or dimethylamine indicated that the secondary amine is not a significant intermediate in the metabolic generation of the primary amine. Experiments with 15N-labeled NDMA showed that the methylamine nitrogen came from the amino moiety of the nitrosamine. The results suggest that NDMA metabolism is best viewed as a competition between at least two important pathways, demethylation and the presumably deactivating denitrosation route, a formulation which seems to account for the previously reported detection of methylamine as a urinary metabolite of NDMA and for the production of less than theoretical yields of labeled dinitrogen gas during NDMA metabolism.

Abstract: The present study was undertaken to examine the nature of the low Km (KmI) form of rat liver microsomal N-nitrosodimethylamine demethylase (NDMAd) and its inhibition by organic compounds which are commonly present in the assay mixture. Using radiometric and colorimetric assay methods with an NADPH-generating system consisting of 0.4 mM NADP, 10 mM glucose-6-phosphate, and glucose-6-phosphate dehydrogenase (0.4 units/ml), Km values of 40-50 microM were obtained. These Km values were lower than the values of 60-80 microM reported previously. This decrease was due to the elimination of inhibitors such as glycerol in the assay mixture. Glycerol was a competitive inhibitor, and this observation explained in part why purified P-450ac (acetone-inducible form of P-450), displayed a higher Km value in a reconstituted NDMAd system, which contained glycerol, than in microsomes. Semi-carbazide which had been used in many previous assays of NDMAd was also found to be a competitive inhibitor of this enzyme. Other inhibitors studied include the commonly used solvents dimethylsulfoxide, acetone, ethylene glycol, dimethylformamide, ethyl acetate, benzene, and hexane as well as thiol compounds dithiothreitol and mercaptoethanol. Although very low Km values (10-20 microM) for N-nitrosodimethylamine metabolism were reported in studies with perfused liver, liver slices, and isolated liver cells, we believe that the KmI form of liver NDMAd is responsible for the metabolism and activation of N-nitrosodimethylamine in the rat liver.

Abstract: In an attempt to elucidate the molecular basis for the decrease in rat liver carcinogenicity and DNA-alkylating ability that accompanies deuteration of N-nitrosodimethylamine (NDMA), NDMA and its fully deuterated analogue ([2H6]NDMA) were incubated with acetone-induced rat liver microsomes. Rates for the competing metabolic routes, denitrosation and demethylation, were determined from colorimetric data on nitrite and formaldehyde generation, respectively. The Vmax calculated for demethylation of NDMA was 7.9 nmol/min/mg, while that for denitrosation was 0.83 nmol/min/mg. Deuteration of NDMA did not significantly change the Vmax for either pathway, but it did increase the Km for demethylation from 0.06 to 0.3 mM. The Km for denitrosation was also increased from 0.06 to 0.3 mM on deuteration, as determined by incubating an equimolar mixture of amino-15N-labeled NDMA with [2H6]NDMA and measuring the methyl[15N]amine:[2H3]methylamine ratio by derivatization-gas chromatography-mass spectrometry. The fact that the Km values for denitrosation were so similar to those for demethylation suggested that the two pathways were catalyzed by the same enzyme. The isotope effects calculated from these data [VmaxH/VmaxD approximately 1 and (Vmax/Km)H/(Vmax/Km)D approximately 5] show that microsomal metabolism of NDMA is not significantly shifted from demethylation to denitrosation on deuteration of substrate and may indicate a low commitment to catalysis for the enzyme. The results are consistent with the view that the metabolism of NDMA is initiated by formation of an alpha-nitrosamino radical which either combines with a hydroxyl radical to form the alpha-hydroxynitrosamine as the initial product of the demethylation pathway or fragments to nitric oxide and N-methylformaldimine as the first products of denitrosation.

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Abstract: The cecropins are a family of small, basic peptides which have been isolated from insects and mammals, and shown to possess broad-spectrum antibacterial activities. This article reviews recent research into the structures and functions of cecropins, and concentrates on work involving the solid-phase synthesis and study of cecropin analogs, model peptides, and hybrids of cecropin and melittin, the toxic peptide of bee venom. Cecropins contain an N-terminal amphipathic α-helix linked to a C-terminal hydrophobic α-helix by a flexible segment of amino acids. All of these secondary structure elements were shown to be required for antibacterial activity. Their disruption of liposomes, and effects on conductivities of lipid bilayers suggested that cecropins act by forming pores in the bacterial cell membrane. Melittin resembles the cecropins in size, components of secondary structure, and antibacterial activity. However, unlike the cecropins, it lyses red blood cells. A synthetic hybrid of cecropin and melittin was developed which exhibited enhanced antibacterial activity, greatly reduced red cell lytic activity, and potent antimalarial activity.

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Abstract: Antibiotically and/or antimalarially active D-peptides of naturally occurring antibiotics such as cecropin A, B and D, melittin, and Magainin I and II and their addition, deletion and replacement analogs including homologous and heterologous analogs thereof.

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