Use of superabsorbent polymer gels for surface decontamination of Bacillus anthracis spores.

AIMS: This study evaluated the inactivation of Bacillus anthracis Vollum spores dried on a nonporous surface using a superabsorbent polymer (SAP) gel containing commercially available liquid decontaminants. METHODS AND RESULTS: The first phase determining the availability of the liquid decontaminant within the SAP showed that the SAP gel containing pH-adjusted sodium hypochlorite (NaOCl) inhibited B. anthracis growth while the water control SAP gel had no affect on growth. For testing surface decontamination, B. anthracis spores were dried onto steel coupons painted with chemical agent resistant coating and exposed to SAP containing either pH-adjusted NaOCl, chlorine dioxide (ClO(2)) or hydrogen peroxide/peracetic acid (H(2)O(2)/PA) for 5 and 30 min. At contact times of both 5 and 30 min, all of the SAP gels containing pH-adjusted NaOCl, ClO(2) or H(2)O(2)/PA inactivated B. anthracis spores at levels ranging from 2.2 to > or =7.6 log reductions. CONCLUSIONS: Incorporation of three commercially available decontaminant technologies into a SAP gel promotes inactivation of B. anthracis spores without observable physical damage to the test surface. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides preliminary data for the feasibility of using SAP in inactivating B. anthracis spores on a nonporous surface, supporting the potential use of SAP in surface decontamination.

Structure-activity studies on high affinity NOP-active hexapeptides.

Nociceptin/orphanin FQ (N/OFQ) is a 17 amino acid peptide that is the endogenous ligand for the G-protein coupled receptor ORL1 (NOP), a member of the opioid receptor family. Although it is clear that this receptor system is involved in a variety of physiologic functions, including analgesia, the precise actions of N/OFQ remain largely uncharacterized. One reason for this has been limited number of high-affinity ligands to NOP, and particularly the lack of availability of useful specific antagonists. Herein, we describe the pharmacologic activity of a series of modified amino acid containing modifications of the hexapeptide Ac-RYYRWR-NH2, with high affinity for NOP. These compounds were tested for binding affinity using [3H]N/OFQ binding to human NOP in CHO cells, and functional activity by measuring stimulation of [35S]GTPgammaS-binding in CHO cell membranes. These studies suggest that each Arg of the hexapeptide is required to maintain high-binding affinity. The peptide maintains high affinity if the Tyr2 or Tyr3 are modified, but at least one of these residues must maintain its hydroxyl group or there is a large decrease in intrinsic activity of the peptide.

N-terminal modifications leading to peptide ORL1 partial agonists and antagonists.

Nociceptin/Orphanin FQ (N/OFQ) is a 17 amino acid peptide that is the endogenous ligand for the G protein-coupled receptor (opioid receptor like 1, ORL1), a member of the opioid receptor family. Although it is clear that this receptor system is involved in a variety of physiological functions, including analgesia, the precise actions of N/OFQ remain largely uncharacterized. One reason for this has been limited high affinity ligands to ORL1, and particularly the lack of availability of useful specific antagonists. Herein we describe the pharmacological activity of a series of N-terminally modified hexapeptides with high affinity for ORL1. These compounds were tested for binding affinity using [3H]N/OFQ binding to human ORL1 in CHO cells, and functional activity by measuring stimulation of [35S]GTPgammaS binding in CHO cell membranes. The N-terminal modifications have produced compounds that maintained very high receptor affinity, but led to significant changes in intrinsic activity. One compound, pentanoyl-RYYRWR-NH2, with barely measurable agonist activity was tested in vivo. It was found to possess modest analgesic activity, but it was unable to block the morphine modulatory activity of N/OFQ.

Stabilized analogs of thymopentin. 1. 4,5-Ketomethylene pseudopeptides.

The pentapeptide, thymopentin (Arg1-Lys2-Asp3-Val4-Tyr5) is known for its activity as an immunomodulating drug, but with limited half-life in plasma. In this first paper of a series of three studies, the synthesis of analogs stabilized at the peptide bond between the C-terminal amino acids via insertion of a ketomethylene moiety is described. N-Blocked pseudopeptides containing Val(k)Phe, Ala(k)Phe, and Val(k)Val units were prepared and attached to chloromethyl Merrifield resin via the carboxy terminal. Removal of the N-BOC group by trifluoroacetic acid was followed by sequential coupling with N-BOC dipeptides of aspartic acid to yield resin-bound N-BOC pseudotetrapeptides. Removal of N-BOC and coupling with N-BOC-r-N-tosylarginine followed by total cleavage of blocking groups and resin by HF afforded the target pseudopentapeptides. The analogs were found to compete favorably with thymopentin for binding to CEM cells, but binding was reduced by about 20-30% on average. All analogs showed significant enhancement of half-life versus thymopentin in mouse serum, but most showed only modest improvement in human serum. Insertion of proline or norleucine at position 2 in the chain caused a substantial increase in half-life (3-4-fold), while N-methylnorleucine conferred complete stability in the analogs.

Stabilized analogs of thymopentin. 2. 1,2- and 3,4-ketomethylene pseudopeptides.

In this second paper in a series of three studies of stable analogs of thymopentin (Arg1-Lys2-Asp3-Val4-Tyr5), the synthesis of analogs stabilized at peptide bonds 1,2 and 3,4 via insertion of ketomethylene units is described. A tris(carbobenzyloxy)arginyl(k)norleucine pseudopeptide was synthesized and coupled to Asp-Val-Phe-resin units followed by HF cleavage to prepare Arg(k)Nle-Asp-Val-Phe analogs. Preparation of N-BOC Asp(k)Val and N-BOC Asp(k)Ala units followed by coupling to Phe- or Tyr-resin units provided resin-bound pseudotripeptide substrates for attachment of various arginyl dipeptides. Cleavage from the resin afforded 3,4-ketomethylene-stabilized pseudopeptide analogs of thymopentin. The Arg-Lys-Asp(k)Val-Phe and Arg-Lys-Asp(k)Val-Tyr analogs were more strongly bound to CEM cells than thymopentin itself. There was significant enhancement of stability in serum for the analogs, especially those containing Arg(k)Nle or Arg-NMeLys moieties at the 1,2-peptide bond.

In vivo anti-influenza virus activity of a zinc finger peptide.

Matrix protein (M1) is a major structural protein of influenza virus, and it inhibits its own polymerase. A 19-amino-acid peptide, corresponding to a zinc finger region of the M1 sequence of influenza virus strain A/PR/8/34 (H1N1), centered around amino acids 148 to 166, was synthesized. This peptide, designated peptide 6, represents a zinc finger which includes a 7-amino-acid loop or finger and a 4-amino-acid tail at the carboxyl terminus, in addition to the 8 amino acids involved in the coordination of Zn. Three experiments were run to evaluate the activity of peptide 6 on infections induced in mice by influenza A/PR/8/34 and A/Victoria/3/75 (H3N2) viruses. Intranasal (i.n.) treatment of the H1N1 virus infection with 30 or 60 mg/kg of body weight/day, three times daily for 5 days, beginning 4 h pre-or 8 h post-virus exposure, was effective in preventing death, reducing the arterial oxygen decline, and inhibiting lung consolidation. Virus titers in the lungs determined on day 5 were reduced by up to 1.5 log10 in treated groups, but considerable variation in the titers of the recovered virus was seen. The H3N2 virus infection was treated i.n. with 30, 60, or 120 mg of peptide 6/kg/day by using the above-mentioned delayed initiation treatment schedule, and similar protection was seen, although lung virus titers were not reduced in the day-5 assay. Peptide 6 was well tolerated at doses up to 60 mg/kg/day. This zinc finger peptide may provide a new class of antivirals effective against influenza virus.

Antiviral activity of influenza virus M1 zinc finger peptides.

Matrix protein (M1) of influenza virus inhibits its own polymerase; this suggested that a peptide segment of M1 with inhibitory properties could serve as an antiviral agent. A peptide synthesized to the Zn2+ finger region of the M1 sequence of influenza virus strain A/PR/8/34 centered around amino acids residues 148 to 166 was shown earlier to be 1,000-fold more effective as a polymerase inhibitor than M1. This peptide, designated peptide 6, represents a Zn2+ finger which includes a 7-residue "loop" and a 4-residue "tail" in addition to the 4 residues on either side of the loop involved in coordination of Zn2+. We have now demonstrated antiviral activity for this peptide in microassays measuring inhibition of the viral cytopathic effect. When the peptide was introduced into tissue culture 5 min after viral challenge with A/PR/8/34, antiviral activity was seen at levels as low as 0.1 nM; on a molar basis, the peptide was shown to be 1,000- to 2,500-fold more effective than ribavirin or amantadine. Antiviral activity was seen with addition of the peptide up to 1 h after viral infection; however, little or no activity was seen at later times, suggesting that viral replication is inhibited at an early stage, possibly at the level of transcription. Reduction in the finger loop or tail length reduced antiviral activity; reduction in the number of residues involved in coordination of Zn2+ abolished antiviral activity. In addition to A/PR/8/34, peptide 6 was shown to have antiviral activity against other type A influenza viruses, including those representing H1N1, H2N2, and H3N2 subtypes. Antiviral activity against type B influenza viruses was also seen. A low level of activity against vesicular stomatitis virus was observed. Zn2+ finger peptides or analogs of Zn2+ finger peptides may provide a new class of antiviral agents effective against influenza virus and possibly other viruses.

Use of repetitive sequences and the polymerase chain reaction technique to classify genetically related Bradyrhizobium japonicum serocluster 123 strains.

We have determined that repetitive (repetitive extragenic palindromic [REP] and enterobacterial repetitive intergenic consensus [ERIC]) sequences used in conjunction with the polymerase chain reaction technique (REP and ERIC PCR) provide an effective means of differentiating between and classifying genetically related Bradyrhizobium japonicum serocluster 123 strains. Analysis of REP and ERIC PCR-generated dendrograms indicated that this technique can effectively differentiate between closely related strains which were indistinguishable by using other classification methods. To maximize the genomic differences detected by REP and ERIC PCR fingerprint patterns, the REP and the ERIC data sets were combined for statistical analyses. REP-plus-ERIC PCR fingerprints were also found to provide a method to differentiate between highly diverse strains of Bradyrhizobium spp., but they did not provide an effective means for classifying these strains because of the relatively low number of REP and ERIC consensus sequences found in some of the bradyrhizobia. Our results also suggest that there is a relationship between nodulation phenotypes and the distribution of REP and ERIC consensus sequences within the genomes of B. japonicum serogroup 123 and 127 strains. Results obtained by restriction fragment length polymorphism hybridization analyses were correlated with the phylogenetic classification of B. japonicum serocluster 123 strains obtained by using REP and ERIC PCR.

Ligand-based histochemical localization and capture of cells expressing heat-stable enterotoxin receptors.

The heat stable enterotoxins (ST) of enterotoxigenic Escherichia coli (ETEC) cause diarrhoea by binding specific intestinal receptors. Precise histochemical localization of ST receptors could provide more information about the pathophysiology of secretory diarrhoea and the role of ST receptors in normal biology. To accomplish this, we quantitatively coupled biotin to the N-terminus of ST1b using biotin-X-X-N-hydroxysuccinimide ester. The derivatized toxin (BST) has an apparent Kd of 11.7 +/- 10 nM for rat brush border receptors. We used BST in an affinity panning cell-capture system, to validate its ability to discriminate between receptor-positive and receptor-negative cells. Cell lines expressing ST receptors (human colon carcinoma T84, and COS cells transfected with guanylyl cyclase-C (GC-C) ST receptor cDNA) were captured to streptavidin and anti-biotin-coated plates with high efficiency and specificity. This system provides a novel approach to screening cells for the presence of unique ST-binding proteins. BST was then used with streptavidin-gold to demonstrate the cellular topography of ST receptors at the light microscopic level. Villus enterocytes were intensely stained, but only a faint signal was observed in upper crypts of rat small intestine. Thus, a gradient of increasing receptor density was seen as upper crypt cells matured into villus enterocytes. Higher magnification revealed that ST receptors are concentrated at the apical aspect of villus enterocytes. Recently, guanylin, a putative endogenous ligand for ST receptors, has been localized to Paneth cells, at the base of intestinal crypts. Thus, ST receptors are concentrated in villus enterocytes, while guanylin appears to be produced at the base of the crypts.(ABSTRACT TRUNCATED AT 250 WORDS)

The Bradyrhizobium japonicum serocluster 123 hyperreiterated DNA region, HRS1, has DNA and amino acid sequence homology to IS1380, an insertion sequence from Acetobacter pasteurianus.

We have sequenced and analyzed the hyperreiterated DNA region, HRS1, from Bradyrhizobium japonicum USDA 424. The 2.1-kb HRS1 fragment is closely linked to the B. japonicum common and genotype-specific nodulation genes in serogroup 123 and 127 strains. Southern hybridization analyses indicated that one copy of HRS1 is also located next to the fixRnifA locus in B. japonicum USDA 424. Nucleotide sequence analysis revealed the presence of a 4-bp target site duplication in HRS1 which is identical to a terminal repeat found in the B. japonicum USDA 110 repeated sequence RS alpha. Computer searches of the PIR (Protein Identification Resource) protein data base revealed a high degree of amino acid sequence homology between a putative 329-amino-acid polypeptide from HRS1 and a large polypeptide from IS1380, an insertion sequence from Acetobacter pasteurianus. RNA slot blot hybridizations suggest that transcripts showing homology to HRS1 are constitutively produced in strains USDA 424 (serogroup 127) and USDA 438 (serogroup 123).

Hyperreiterated DNA regions are conserved among Bradyrhizobium japonicum serocluster 123 strains.

We have identified and cloned two DNA regions which are highly reiterated in Bradyrhizobium japonicum serocluster 123 strains. While one of the reiterated DNA regions, pFR2503, is closely linked to the B. japonicum common and genotype-specific nodulation genes in strain USDA 424, the other, pMAP9, is located next to a Tn5 insertion site in a host-range extension mutant of B. japonicum USDA 438. The DNA cloned in pFR2503 and pMAP9 are reiterated 18 to 21 times, respectively, in the genomes of B. japonicum serocluster 123 strains. Gene probes from the reiterated regions share sequence homology, failed to hybridize (or hybridized poorly) to genomic DNA from other B. japonicum and Bradyrhizobium spp. strains, and did not hybridize to DNA from Rhizobium meliloti, Rhizobium fredii, Rhizobium leguminosarum biovars trifolii, phaseoli, and viceae, or Agrobacterium tumefacians. The restriction fragment length polymorphism hybridization profiles obtained by using these gene probes are useful for discriminating among serologically related B. japonicum serocluster 123 strains.

Interaction of the pertussis toxin peptide containing residues 30-42 with DR1 and the T-cell receptors of 12 human T-cell clones.

The interaction of the immunodominant pertussis toxin peptide containing residues 30-42 (p30-42) with soluble DR1 molecules and the T-cell receptor (TCR) of 12 DR1-restricted human T-cell clones has been analyzed. Peptide analogues of p30-42 containing single alanine substitutions were used in DR1-binding and T-cell proliferation assays to identify the major histocompatibility complex and TCR contact residues. Each T-cell clone was found to recognize p30-42 with a different fine specificity. However, a common core comprising amino acids 33-39 was found to be important for stimulation of all T-cell clones. Within this core two residues, Leu33 and Leu36, interact with the DR1 molecule, whereas Asp34, His35, Thr37, and Arg39 are important for TCR recognition in most of the clones. Computer modeling of the structure of p30-42 showed that an alpha-helical conformation is compatible with the experimental data. The analysis of TCR rearrangement revealed that the peptide was recognized by T-cell clones expressing different variable region alpha (V alpha) and variable region beta (V beta) chains, although a preferential use of V alpha 8-V beta 13 and V alpha 11-V beta 18 combinations was found in clones from the same donor. Understanding the details of the interaction of antigenic peptides with the major histocompatibility complex and TCR molecules should provide the theoretical basis to design T-cell epitopes and obtain more immunogenic vaccines.

Isolation and characterization of a new atrial peptide-degrading enzyme from bovine kidney.

An endopeptidase isolated from bovine kidney displays high affinity and selectivity for the Ser-Phe bond located in the C-terminal region of atrial peptides. Enzymatic activity converts APIII and APII to the less active peptide API. This peptidase is inhibited by both metal chelators and sulfhydryl-reactive agents, suggesting both a tightly bound metal and a cysteine residue are important for enzymatic activity. This enzyme may be important for the processing and/or degradation of atrial peptides.

Human T cells recognize multiple epitopes of an immediate early/tegument protein (IE62) and glycoprotein I of varicella zoster virus.

Infection with varicella zoster virus (VZV) elicits persistent cell-mediated immunity directed against the immediate early (IE62) protein and the glycoprotein I (gp I) in most healthy subjects. In these experiments, synthetic peptides corresponding to residues of the IE62 protein and gp I were used to identify linear amino acid sequences of these immunogenic VZV proteins that were recognized by peripheral blood T lymphocytes from VZV-immune individuals of known major histocompatibility complex (MHC) type. All of 12 VZV-immune donors had T-cell proliferative responses, defined as a stimulation index (SI) greater than or equal to 2.0, to at least two of ten synthetic IE62 peptides; the mean number of IE62 peptides recognized by T cells from VZV-immune donors was seven. Five of the ten IE62 peptides stimulated T cells from 75% to 83% of the VZV-immune donors; the other five IE62 peptides were recognized by T cells from 42% to 67% of the subjects. All VZV-immune donors also had T proliferation responses to at least two of ten synthetic gp I peptides; the mean number of peptides recognized was six. Six of the ten gp I peptides were recognized by T cells from 67% to 92% of the VZV-immune donors; the frequency of donors responding to the other gp I peptides ranged from 42% to 58%. None of five nonimmune donors demonstrated T-cell proliferation to any of the IE62 or gp I peptides. A combination of two IE62 peptides provided epitopes that could be recognized by T cells from all twelve VZV-immune donors, regardless of DR type. Similarly, one gp I peptide in combination with either of two other gp I peptides induced proliferation of T cells from all immune subjects. Memory T cells with specificity for multiple short amino acid sequences of the IE62 protein and gp I were detected in subjects who had had primary VZV infection more than 20 years earlier. These observations indicate that natural VZV infection elicits a diverse cell-mediated immune response to viral proteins that is not restricted to only one or two immunodominant regions. Although the usefulness of peptide vaccines remains to be established, multiple epitopes of the IE62 protein and gp I were identified that could be presented by antigen-presenting cells (APC) and recognized by T cells from most subjects in an "outbred" human population.

Multiple T and B cell epitopes in the S1 subunit ("A"-monomer) of the pertussis toxin molecule.

The immunogenicity and reactogenicity of Bordetella pertussis vaccine are mediated in part by the S1 subunit of pertussis toxin (PT). To identify the immune epitopes in the S1 subunit of PT, synthetic peptides were prepared and tested for their capacity to induce antibodies in mice with different MHC genotypes. In BALB/c mice, peptides corresponding to sequences 1-17, 70-82 and 189-199 generate T cell proliferative responses, induce the production of antibodies capable of neutralization of the toxin in the Chinese hamster ovary-cell assay, and protect mice from a shock-like syndrome caused by alternate injections of BSA and PT. Protection and neutralization correlated with the ability of these peptides to elicit high anti-PT titers. Different B cell epitopes were detected in other inbred mouse strains. The antibody reactivity against synthetic peptides from two infants vaccinated with pertussis vaccine was tested. These infants had antibodies reactive to a variety of epitopes in the S1 subunit, including peptides 1-17, 70-82, 99-112, 135-145, and 189-199. Thus, it appears that there are multiple T and B cell epitopes in the S1 subunit of PT.

MHC-restricted recognition of immunogenic T cell epitopes of pertussis toxin reveals determinants in man distinct from the ADP-ribosylase active site.

The S1 subunit of Pertussis toxin (PT) is responsible for the reactogenicity and in part the immunogenicity of Bordetella pertussis vaccine. The critical residues associated with the immunomodulatory effects of PT were located around Glu140 in the S1 subunit. In man, T cell responses to PT are directed at S1 peptides distinct from Glu140. Two such epitopes, p64-75 and p151-161, are immunogenic in a panel of individuals covering a wide range of HLA genotypes. The response to PT peptides is HLA class II restricted. The response to p64-75 is blocked by an anti-HLA-DQ mAb, while that to p151-161 is blocked by an anti-HLA-DR mAb. These findings may allow for the development of a B. pertussis vaccine free from reactogenicity.

Synthesis and biological activity of ketomethylene-containing nonapeptide analogues of snake venom angiotensin converting enzyme inhibitors.

Two ketomethylene-containing nonapeptide analogues were synthesized to determine if ketomethylene analogues of the nonapeptide venom inhibitors of angiotensin converting enzyme (ACE) would have oral ACE inhibition activity. Both ketomethylene-containing nonapeptides 18 and 19 were potent inhibitors of rabbit lung ACE with I50s of 3.4 and 8.0 nM, respectively, compared to 340 nM for their parent nonapeptide and 450 nM for captopril. Peptide 18 was rapidly cleaved by trypsin, but 19 was reasonably stable to all enzyme degradation systems tested with maximum degradation of 50% by pepsin in 3 h. Both 18 and 19 when given iv to normotensive rats were between 3 and 10 times more potent than captopril in inhibiting an angiotensin I induced blood pressure increase. Peptide 19 showed no ACE inhibition activity in unanesthetized normotensive rats when administered orally at doses of 10 or 100 mg/kg. Experiments were conducted to determine whether 19 is adsorbed from the gastrointestinal track following oral administration. These experiments indicated that 19 is adsorbed. It is concluded that the lack of oral activity of 19 is probably due to its rapid excretion, probably into the bile.

Importance of disulfide bridges in the structure and activity of Escherichia coli enterotoxin ST1b.

A 13-amino acid sequence of the Escherichia coli heat-stable enterotoxin ST1b encodes its receptor-binding and diarrheal functions. This sequence includes six cysteines involved in three intramolecular disulfide bridges. To determine the importance of disulfide bridges to the biological activity of ST1b, we synthesized 15 analogues of the tridecapeptide representing all possible replacements of two of the six cysteines by alanines. Only 2 analogues--namely, A6,11ST1b-(6-18) and A10,18ST1b(6-18)--could inhibit the binding of a radiolabeled analogue of ST1b to rat intestinal cells. The purified peptides were, respectively, 4200 and 130 times less effective as inhibitors than ST1b(6-18), the sequence that includes all six cysteines. In addition, both peptides produce diarrhea when given orally to suckling mice. These analogues share in common only two cysteines (Cys-7 and Cys-15), suggesting that four cysteines, two of which are Cys-7 and Cys-15, are necessary for activity. A pattern of disulfide linkages is proposed where Cys-7 is paired to Cys-15, Cys-6 to Cys-11, and Cys-10 to Cys-18, the preceding disulfide bridges being ranked in descending order of importance in terms of their respective contribution to the activity of the enterotoxin. Using this disulfide bridge arrangement and constraints derived from NMR spectroscopy, we propose a folding pattern for the toxic domain of ST1b.

Novel mu-selective Met-enkephalinamide analogs with antagonist activity. Synthesis, receptor binding, analgesic properties and conformational studies.

Four novel mu-selective peptide antagonists have been synthesized and examined for receptor binding, analgesic agonist and antagonist activity and energy conformational properties. These peptides were designed by analogy to results of molecular modeling of 3-phenyl piperidines which led to incorporating four modified tyrosine residues, m-Tyr, beta-methyl-m-Tyr, N-phenethyl-m-Tyr and alpha, beta-dimethyl-m-Tyr into D-Ala2-Met5-enkephalinamide. Peptides were synthesized by stepwise solution synthesis using an active ester coupling procedure. Receptor binding assays were performed on rat brain homogenates and data were analyzed by a modified version of the program LIGAND. Analgesic agonist and antagonist activity was evaluated by the mouse tail-flick test. Energy-optimized conformations were obtained using a program called Molecule-AIMS. The results demonstrate that relative ratios of in vivo agonist and antagonist potencies in D-Ala2-Met5-enkephalinamides can be modulated by chemical modification of the tyrosine residue. A shift in the phenolic-OH position from para to meta significantly enhances relative antagonist versus agonist activity; addition of a beta-CH3 group to the m-Tyr enhances mu-selectivity and leads to nearly equal agonist/antagonist activity. Energy conformational studies indicate that all analogs with high mu-receptor affinity examined have a common energy accessible B'II 2-3 turn conformation similar to that previously identified for high mu-affinity binding in peptides, lending further support to this candidate conformer. This conformer also has tyrosine side-chain angles which allowed total overlap with the amine and phenolic groups of a known structure of 3-(m-OH phenyl)-piperidine. This structural similarity together with the observation of mixed agonist antagonist activity in both types of opioids confirms the rationale upon which design of these peptides was based.