Human antibodies neutralizing diphtheria toxin in vitro and in vivo.

Diphtheria is an infectious disease caused by Corynebacterium diphtheriae. The bacterium primarily infects the throat and upper airways and the produced diphtheria toxin (DT), which binds to the elongation factor 2 and blocks protein synthesis, can spread through the bloodstream and affect organs, such as the heart and kidneys. For more than 125 years, the therapy against diphtheria has been based on polyclonal horse sera directed against DT (diphtheria antitoxin; DAT). Animal sera have many disadvantages including serum sickness, batch-to-batch variation in quality and the use of animals for production. In this work, 400 human recombinant antibodies were generated against DT from two different phage display panning strategies using a human immune library. A panning in microtiter plates resulted in 22 unique in vitro neutralizing antibodies and a panning in solution combined with a functional neutralization screening resulted in 268 in vitro neutralizing antibodies. 61 unique antibodies were further characterized as scFv-Fc with 35 produced as fully human IgG1. The best in vitro neutralizing antibody showed an estimated relative potency of 454 IU/mg and minimal effective dose 50% (MED50%) of 3.0 pM at a constant amount of DT (4x minimal cytopathic dose) in the IgG format. The targeted domains of the 35 antibodies were analyzed by immunoblot and by epitope mapping using phage display. All three DT domains (enzymatic domain, translocation domain and receptor binding domain) are targets for neutralizing antibodies. When toxin neutralization assays were performed at higher toxin dose levels, the neutralizing capacity of individual antibodies was markedly reduced but this was largely compensated for by using two or more antibodies in combination, resulting in a potency of 79.4 IU/mg in the in vivo intradermal challenge assay. These recombinant antibody combinations are candidates for further clinical and regulatory development to replace equine DAT.

A comparison of three strategies for biopanning of phage-scFv library against diphtheria toxin.

The biopanning process is a critical step in phage display for isolating peptides or proteins with specific binding properties. Conventional panning methods are sometimes not so effective and may result in nonspecific or low-yield positive results. In this study, three different strategies including soluble antibody-capturing, pH-stepwise elution, and conventional panning were used for enrichment of specific clones against diphtheria toxoid. The reactivity of the selected clones was evaluated using an indirect enzyme-linked immunosorbent assay. The positive clones were screened using Vero cell viability assay. The neutralizing clones were expressed in HB2151 strain of Escherichia coli and soluble single-chain fragment variable (scFv) fragments were purified by nickel-nitrilotriacetic acid affinity chromatography. Finally, the ability of scFv fragments for neutralizing diphtheria toxin (DT) were evaluated again using Vero cell viability assay. After four rounds of panning, the soluble antibody-capturing method yielded 15 positive phage-scFv clones against diphtheria toxoid. Conventional panning and pH-stepwise elution model resulted from nine and five positive phage-scFv clones, respectively. Among all positive clones, three clones were able to neutralize DT in Vero cell viability assay. Two of these clones belonged to a soluble antibody-capturing method and one of them came from conventional panning. Three neutralizing clones were used for soluble expression and purification of scFvs fragments. It was found that these soluble scFv fragments possessed neutralizing activity ranging from 0.15 to 0.6 µg against two-fold cytotoxic dose 99% of DT. In conclusion, the results of our study indicate that soluble antibody-capturing method is an efficient method for isolation of specific scFv fragments.

Inhibition of Cholera Toxin and Other AB Toxins by Polyphenolic Compounds.

Cholera toxin (CT) is an AB-type protein toxin that contains a catalytic A1 subunit, an A2 linker, and a cell-binding B homopentamer. The CT holotoxin is released into the extracellular environment, but CTA1 attacks a target within the cytosol of a host cell. We recently reported that grape extract confers substantial resistance to CT. Here, we used a cell culture system to identify twelve individual phenolic compounds from grape extract that inhibit CT. Additional studies determined the mechanism of inhibition for a subset of the compounds: two inhibited CT binding to the cell surface and even stripped CT from the plasma membrane of a target cell; two inhibited the enzymatic activity of CTA1; and four blocked cytosolic toxin activity without directly affecting the enzymatic function of CTA1. Individual polyphenolic compounds from grape extract could also generate cellular resistance to diphtheria toxin, exotoxin A, and ricin. We have thus identified individual toxin inhibitors from grape extract and some of their mechanisms of inhibition against CT.

Immunoregulatory effects of vitamin D3 in experimental type 1 diabetes.

The mechanisms of diabetes-associated impairment of cellular immune defense and its regulation by vitamin D3 are not fully elucidated. The study was devoted to investigating the functional state of T-cell immunity as well as humoral immune activity in response to artificial immunization in experimental diabetes and after prolonged administration of vitamin D3. It was established that diabetes is characterized by a 2.3 times decrease in blood serum 25OHD3 content. Vitamin D3 deficiency was accompanied by the failures in proliferative activity of T-lymphocytes and alterations of the regulatory (CD4+-postive lymphocytes) and cytotoxic (CD8+-positive lymphocytes) cell subpopulations. It was found an increase in the content of phosphorylated p65 subunit of nuclear factor κB in total lysates of spleen T lymphocytes and its enhanced translocation to the nucleus. In addition, it was shown intensification of humoral IgG response to administration of recombinant diphtheria toxin subunit B. Revealed impairments in the cellular link of the immune system were associated with an increase in splenocytes apoptosis, which was detected by Annexin V-GFP ability to bind phosphatidyl serine that is specifically located on the outer surface of plasmalemma in apoptosis. Prolonged vitamin D3 treatment (within 2 months) in a dose of 20 IU/animal leads to normalization of the proliferative activity and the ratio of T-cell subpopulations, reduces the formation of phosphorylated subunit of NF-κB - p65 and contributes to a balanced secretion of IgG against artificial antigen. These changes were accompanied by a decrease in apoptotic events in the total population of splenocytes. Our findings suggest an important role of vitamin D3 in the regulation of the immune system abnormalities related to type 1 diabetes.

Resolving self-association of a therapeutic antibody by formulation optimization and molecular approaches.

A common challenge encountered during development of high concentration monoclonal antibody formulations is preventing self-association. Depending on the antibody and its formulation, self-association can be seen as aggregation, precipitation, opalescence or phase separation. Here we report on an unusual manifestation of self-association, formation of a semi-solid gel or "gelation." Therapeutic monoclonal antibody C4 was isolated from human B cells based on its strong potency in neutralizing bacterial toxin in animal models. The purified antibody possessed the unusual property of forming a firm, opaque white gel when it was formulated at concentrations >30 mg/mL and the temperature was <6°C. Gel formation was reversible with temperature. Gelation was affected by salt concentration or pH, suggesting an electrostatic interaction between IgG monomers. A comparison of the C4 amino acid sequences to consensus germline sequences revealed differences in framework regions. A C4 variant in which the framework sequence was restored to the consensus germline sequence did not gel at 100 mg/mL at temperatures as low as 1°C. Additional genetic analysis was used to predict the key residue(s) involved in the gelation. Strikingly, a single substitution in the native antibody, replacing heavy chain glutamate 23 with lysine (E23K), was sufficient to prevent gelation. These results indicate that the framework region is involved in intermolecular interactions. The temperature dependence of gelation may be related to conformational changes near glutamate 23 or the regions it interacts with. Molecular engineering of the framework can be an effective approach to resolve the solubility issues of therapeutic antibodies.

Construção, clonagem e expressão do fragmento B da toxina diftérica de Corynebacterium diphtheriae (cepa PW- 8) em Mycobacterium bovis BCG sub-cepa Moreau / Construction, cloning and expression of the fragment B of diphtheria toxin from Corynebacterium diphtheriae (strain PW-8) in Mycobacterium bovis BCG Moreau sub-strain

A vacina anti-diftérica de uso corrente no Brasil (DTP), embora de alta eficácia na prevenção da difteria, está associada com episódios de toxicidade e reatogenicidade no recipiente vacinal, resultantes de proteínas residuais derivadas do processo de produção ou detoxificação. Estratégias para o desenvolvimento de vacinas menos reatogênicas e ao mesmo tempo mais eficazes e economicamente viáveis contra a difteria têm sido alvo de intensa investigação. A alternativa proposta por nosso grupo é a utilização da vacina contra a tuberculose (Mycobacterium bovis BCG sub-cepa Moreau), como vetor do gene que codifica o fragmento B da toxina diftérica (dtb) de 58,3 kDa. Neste trabalho o dtb foi clonado no vetor micobacteriano bifuncional (pUS977) de expressão citoplasmática e os clones recombinantes (pUS977dtbPW8), após a transformação do BCG, foram testados com relação a expressão do DTB em BCG e quanto a antigenicidade frente a anticorpos policlonais anti-toxóide diftérico por Immunobloting. A integridade do gene dtb e a identidade das sequências de DNA da construção plasmidial pUS977dtbPW8 foram confirmadas por sequenciamento de DNA e análise de similaridade. A imunogenicidade do BCGr pUS977dtbPW8 expressando o DTB foi investigada em camundongos BALB/c, os resultados obtidos revelaram uma soroconversão específica (IgG). A infectividade e atividade microbicida do BCGr pUS977dtbPW8 no ambiente intracelular foi avaliada através da infecção de linhagens de células de monócitos humano (THP-1), os dados obtidos indicaram que houve sobrevivência intracelular em até 12 dias. Nesse contexto, esplenócitos dos camundongos imunizados com 30 e 60 dias foram extraídos, mostrando que o BCGr pUS977dtbPW8 persistiu até 60 dias na ausência de pressão seletiva e a viabilidade celular não sofreu alteração significativa durante o período testado...

The diphtheria vaccine currently used in Brazil (DTP), despite its history of high efficacy in the prevention of diphtheria, is associated with episodes of toxicity and vaccine reactogenicity in the vaccinee, resulting from the presence in the vaccine of residual proteins derived from the production process or detoxification. Strategies for the development of new vaccines more effective and economically viable against diphtheria have been the subject of intense investigation. The alternative proposed by our group is the use of the vaccine against tuberculosis (Mycobacterium bovis BCG Moreau sub strain) as a vector for the gene that encodes the 58.3 kDa fragment B of the diphtheria toxin (DTB). In our project the dtb gene was cloned into the bifunctional vector pUS977 for cytoplasmic expression and recombinant BCG (rBCG) clones, selected after transformation of BCG, were tested for expression of the DTB polypeptide and antigenicity against polyclonal antibodies anti- diphtheria toxoid by immunoblotting. The integrity and identity of the DNA sequence encoding the dtb gene carried by the plasmid construct pUS977dtbPW8 was confirmed by DNA Sequencing and Analysis of Similarity. The immunogenicity of the rBCG expressing the DTB was investigated in BALB/c mice and the results revealed a specific seroconversion (IgG)...

Identification of a human monoclonal antibody to replace equine diphtheria antitoxin for treatment of diphtheria intoxication.

Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 µg of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic.

Pneumococcal Surface Protein A does not affect the immune responses to a combined diphtheria tetanus and pertussis vaccine in mice.

The Pneumococcal Surface Protein A (PspA) is a promising candidate for the composition of a protein vaccine against Streptococcus pneumoniae. We have previously shown that the whole cell Bordetella pertussis vaccine (wP) is a good adjuvant to PspA, inducing protective responses against pneumococcal infection in mice. In Brazil, wP is administered to children, formulated with diphtheria and tetanus toxoids (DTPw) and aluminum hydroxide (alum) as adjuvant. A single subcutaneous dose of PspA5-DTPlow (a formulation containing PspA from clade 5 and a new generation DTPw, containing low levels of B. pertussis LPS and Alum) induced high levels of systemic anti-PspA5 antibodies in mice and conferred protection against respiratory lethal challenges with two different pneumococcal strains. Here we evaluate the mucosal immune responses against PspA5 as well as the immune responses against the DTP antigens in mice vaccinated with PspA5-DTPlow. Subcutaneous immunization of mice with PspA5-DTPlow induced high levels of anti-PspA5 IgG in the airways but no IgA. In addition, no differences in the influx of cells to the respiratory mucosa, after the challenge, were observed in vaccinated mice, when compared with control mice. The levels of circulating anti-pertussis, -tetanus and -diphtheria antibodies were equivalent in mice vaccinated with DTPlow or PspA5-DTPlow. Antibodies induced by DTPlow or PspA5-DTPlow showed similar ability to neutralize the cytotoxic effects of the diphtheria toxin on Vero cells. Furthermore, combination with PspA5 did not affect protection against B. pertussis and tetanus toxin challenges in mice. Our results support the proposal for a combined PspA-DTP vaccine.

A possible clinical adaptation of CRM197 in combination with conventional chemotherapeutic agents for ovarian cancer.

AIM: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a promising target for cancer therapy. We have already started a phase I study of CRM197, a specific HB-EGF inhibitor, for advanced ovarian cancer. In this study, we evaluated possible clinical adaptations of CRM197 in combination with conventional chemotherapeutic agents. MATERIALS AND METHODS: CRM197, bevacizumab, and paclitaxel were intraperitoneally administered either alone or in combination with mice xenografted with ES2 human ovarian cancer cells. The tumor volumes and microvessel densities (MVD) were determined. RESULTS: Enhanced antitumor effects were observed when paclitaxel was used in combination with bevacizumab or CRM197. The antitumor effect of paclitaxel/CRM197 was significantly higher than that of paclitaxel/bevacizumab. The tumor MVD of mice treated with paclitaxel/CRM197 was significantly lower than that of mice treated with paclitaxel/bevacizumab. CONCLUSION: CRM197 in combination with paclitaxel significantly blocked tumor formation and angiogenesis. These results suggest that paclitaxel is a suitable candidate for CRM197 combination therapy.

Quantitative high-throughput screening identifies inhibitors of anthrax-induced cell death.

Here, we report the results of a quantitative high-throughput screen (qHTS) measuring the endocytosis and translocation of a beta-lactamase-fused-lethal factor and the identification of small molecules capable of obstructing the process of anthrax toxin internalization. Several small molecules protect RAW264.7 macrophages and CHO cells from anthrax lethal toxin and protected cells from an LF-Pseudomonas exotoxin fusion protein and diphtheria toxin. Further efforts demonstrated that these compounds impaired the PA heptamer pre-pore to pore conversion in cells expressing the CMG2 receptor, but not the related TEM8 receptor, indicating that these compounds likely interfere with toxin internalization.

Behavior of diphtheria toxin T domain containing substitutions that block normal membrane insertion at Pro345 and Leu307: control of deep membrane insertion and coupling between deep insertion of hydrophobic subdomains.

Diphtheria toxin T domain aids the translocation of toxin A chain across membranes. T domain has two hydrophobic layers/subdomains that can insert deeply into membranes: helices TH8 and 9, which form a transmembrane hairpin, and helices TH5-7, which form a nonclassical, nontransmembrane structure. Substitutions were made at Pro345, a residue located near the turn between TH8 and 9. P345 is critical for toxicity and pore formation by the T domain. Fluorescence methods showed that hairpin-disrupting Gly or Glu substitutions at 345 did not insert into lipid bilayers as deeply as the wild-type protein, and consistent with previous studies, these mutations reduced pore formation activity as assayed by a novel biotin-streptavidin-based influx assay. Introducing Pro at positions 347 or 353 not only failed to compensate for substitutions at P345, but also they further disrupted deep insertion and/or pore formation. Substitution of P345 with Asn, a residue that promotes helical hairpin formation almost as well as Pro, resulted in somewhat more normal insertion and pore formation than other substitutions. Importantly, a P345E substitution disrupted deep insertion of TH5-7. This suggests that TH8 and 9 and TH5-7 undergo some sort of coordinated insertion into the lipid bilayer and/or that the membrane-inserted T domain has a distinct tertiary structure in which TH5-7 interact with TH8 and 9 instead of consisting of noninteracting hydrophobic segments. Intriguingly, a L307R substitution in TH6, which disrupted deep insertion of TH7, had only a weak effect on pore formation and deep insertion of TH8 and 9. This suggests that the TH8 and 9 region can insert independently of TH5-7 to some degree and that TH8 and 9 insertion may occur early in T-domain insertion.

Mechanistic aspects of the deoxyribonuclease activity of diphtheria toxin.

Here we examined the intrinsic nuclease activity of diphtheria toxin (DTx) to determine the mechanism by which it catalyzes DNA degradation. Results show that DTx degrades double-stranded DNA (dsDNA) by non-processive, endonucleolytic attack, without apparent specificity for nucleotide sequence. Moreover, divalent cation composition determines whether supercoiled dsDNA is cleaved by the introduction of single-strand nicks or double-strand breaks. Circular single-stranded DNA (ssDNA) is also a substrate for endonucleolytic attack. Pre-incubation of DTx with a 2000-fold excess of NAD, the natural substrate for the toxin's ADP-ribosyltransferase (ADPrT) activity, inhibited the transfer of radiolabeled ADP-ribose to elongation factor 2 but had no effect on the degradation of radiolabeled DNA. Based on this result and the fact that compounds known to inhibit the ADPrT activity of DTx had no effect on its nuclease activity and pre-incubation of DTx with DNA had no effect on ADPrT activity, we conclude that the ADPrT and nuclease active sites of DTx are functionally and spatially distinct. Moreover, studies with an ADPrT-inactivated form of DTx indicate that nuclease activity alone can lead to target cell lysis.

Molecular characterization of diphtheria toxin repressor (dtxR) genes present in nontoxigenic Corynebacterium diphtheriae strains isolated in the United Kingdom.

Nontoxigenic strains of Corynebacterium diphtheriae represent a potential reservoir for the emergence of toxigenic C. diphtheriae strains if they possessed functional diphtheria toxin repressor (dtxR) genes. We studied the predominant strain of nontoxigenic C. diphtheriae circulating in the United Kingdom to see if they possessed dtxR genes and ascertain whether they were functional. A total of 26 nontoxigenic C. diphtheriae strains isolated in the United Kingdom during 1995 and 4 nontoxigenic strains isolated in other countries were analyzed by PCR and direct sequencing to determine the presence and intactness of the dtxR genes. The functionality of the DtxR proteins was assayed by testing for the production of siderophore in medium containing high and low concentrations of iron. PCR amplification and sequence analysis of the dtxR genes revealed four variants of the predicted DtxR protein among the nontoxigenic strains isolated in the United Kingdom. Production of siderophore in medium containing a low concentration of iron and repression of siderophore production in medium containing a high concentration of iron demonstrated that in all the strains the dtxR genes were functional. These findings demonstrate that, if lysogenised by a bacteriophage, nontoxigenic strains circulating in the United Kingdom could produce toxin and therefore represent a potential reservoir for toxigenic C. diphtheriae.

Actin--an inhibitor of eukaryotic elongation factor activities.

An inhibitor of diphtheria toxin- and endogenous transferase-dependent ADP-ribosylation of eukaryotic elongation factor 2 (eEF2) has been found in the cytoplasmic fraction from rat liver. We provide evidence that this cytoplasmic inhibitor corresponds to actin, which gives rise also to inhibition of polyphenylalanine (polyPhe) synthesis. Both globular monomeric (G-actin) and filamentous (F-actin) forms of actin appear to be inhibitory on the action of elongation factors 1 and 2 (eEF1 and eEF2) in polyPhe synthesis with the inhibitory effect of G-actin proving to be stronger. Some component(s) in the postribosomal supernatant (S-130) fraction and also DNase I prevent actin-promoted inhibition of polyPhe synthesis.

Adjuvant activity of Mycobacterium bovis BCG expressing CRM197 on the immune response induced by BCG expressing tetanus toxin fragment C.

In order to develop a combined recombinant Mycobacterium bovis BCG (rBCG) vaccine against diphtheria, pertussis and tetanus (DPT), we have constructed different strains of rBCG expressing tetanus toxin fragment C (FC), driven by the up-regulated M. fortuitum beta-lactamase promoter, pBlaF*. Tetanus toxin FC was expressed in comparable levels in native form or in fusion with the beta-lactamase exportation signal sequence; however, in both constructs it was localized to the cytosol. Immunization of mice with rBCG-FC or its combination with rBCG expressing CRM197, induced anti-tetanus toxin antibodies with a Th2 immunoglobulin profile. Administration of a subimmunizing dose of the diphtheria-tetanus toxoid vaccine showed that rBCG-FC primed mice for production of an intense humoral response. Interestingly, the combination of rBCG-FC and rBCG-CRM197 reduced the time required for maturation of the immune response and increased anti-tetanus toxin antibody levels, suggesting adjuvant properties for rBCG-CRM197; this combination induced 75% protection in mice challenged with 100 minimum lethal doses (MLD) of tetanus toxin. Antisera from guinea pigs immunized with this combination were shown to neutralize tetanus toxin and diphtheria toxin. Our results suggest reciprocal adjuvant effects of rBCG-FC and rBCG-CRM197, which may contribute to induction of a more effective immune response against both diseases.

Diphtheria toxin catalyzed hydrolysis of NAD(+): molecular dynamics study of enzyme-bound substrate, transition state, and inhibitor.

The mechanism of the diphtheria toxin-catalyzed hydrolysis of NAD(+) was investigated by quantum chemical calculations and molecular dynamics simulations. Several effects that could explain the 6000-fold rate acceleration (Delta Delta G(++) approximately 5 kcal/mol) by the enzyme were considered. First, the carboxamide arm of the enzyme-bound NAD(+) adopts a trans conformation while the most stable conformation is cis. The most stable conformation for the nicotinamide product has the amide carbonyl trans. The activation energy for the cleavage of the ribosidic bond is reduced by 2 kcal/mol due to the relaxation of this ground state conformational stress in the transition state. Second, molecular dynamics simulations to the nanosecond time range revealed that the carboxylate of Glu148 forms a hydrogen bond to the substrate's 2' hydroxyl group in E.S (approximately 17% of the time) and E.TS (approximately 57% of the time) complexes. This interaction is not seen in crystal structures. The ApUp inhibitor is held more tightly by the enzyme than the transition state and the substrate. Analysis of correlated motions reveals differences in the pattern of anticorrelated motions for protein backbone atoms when the transition state occupies the active site as compared to the E.NAD(+) complex.

Photoaffinity labeling of diphtheria toxin fragment A with 8-azidoadenosyl nicotinamide adenine dinucleotide.

Diphtheria toxin fragment A (DT-A) is an important enzyme in the class of mono(ADP-ribosyl)transferases. To identify peptides and amino acid residues which form the NAD(+) binding site of DT-A using a photoaffinity approach, the photoprobes nicotinamide 8-azidoadenine dinucleotide (8-N(3)-NAD) and nicotinamide 2-azidoadenine dinucleotide (2-N(3)-NAD) were synthesized. Binding studies gave an IC(50) of 2.5 microM for 8-N(3)-NAD and 5.0 microM for 2-N(3)-NAD. Irradiation of DT-A and low concentrations of [alpha-(32)P]-8-N(3)-NAD with short-wavelength UV light resulted in rapid covalent incorporation of the photoprobe into the protein. The photoincorporation was shown to be specific for the active site with a stoichiometry of photoincorporation of 75-80%. After proteolytic digestion of photolabeled DT-A, derivatized peptides were isolated using immobilized boronate affinity chromatography followed by reversed phase HPLC. Radiolabeled peptides originating from two regions of the protein were identified. Chymotryptic digestion produced labeled peptides corresponding to His(21)-Gln(32) and Lys(33)-Phe(53). Lys-C digestion gave overlapping peptides Ser(11)-Lys(33) and Ser(40)-Lys(59). Tyr(27) was identified as the site of photoinsertion within the peptide His(21)-Gln(32) on the basis of the absence of PTH-Tyr at the predicted cycle during sequence analysis and by the lack of predicted chymotryptic cleavage at Tyr(27). Within the second modified peptide Ser(40)-Lys(59), Trp(50) is the most probable site of modification. Identification of Tyr(27) as a site of photoinsertion is in agreement with its placement in the NAD binding site of the X-ray structure of the proenzyme DT-NAD complex [Bell, C. E., and Eisenberg, D. (1996) Biochemistry 35, 1137]. Trp(50) is far from the adenine ring in the crystallographic model; however, site-directed mutagenesis studies suggest that Trp(50) is a major determinant of NAD binding affinity [Wilson, B. A., Blanke, S. R., Reich, K. A., and Collier, R. J. (1994) J. Biol. Chem. 269, 23296-23301].

Inhibition of the cytotoxicity of protein toxins by a novel plant metabolite, mansonone-D.

We have studied the effect of several structurally related mansonones on the cytotoxicity of plant and bacterial toxins in Vero and BER-40, a brefeldin A-resistant mutant of Vero cells. Mansonone-D (MD), a sesquiterpenoid ortho-naphthoquinone, inhibited the cytotoxicity of ricin, modeccin, Pseudomonas toxin, and diphtheria toxin in Vero cells to different extents. The inhibition of ricin cytotoxicity was dose dependent and reversed upon removal of the drug. Protection of ricin cytotoxicity was also observed in the presence of cycloheximide, indicating that de novo protein synthesis is not required for the protective effect. Although MD inhibited the degradation and excretion of ricin, the binding and internalization of ricin was not affected. In contrast, MD strongly reduced the specific binding of diphtheria toxin in Vero cells. Fluorescence microscopic studies show that MD treatment dramatically alters the morphology of the Golgi apparatus in Vero cells. The kinetic studies reveal that the protection of ricin cytotoxicity is the consequence of decreased toxin translocation to the cytosol in MD-treated cells. The reactive ortho-quinone moiety of MD is important for the protective effect as thespesone, a para-naphthoquinone with a heterocyclic ring structure identical to that of MD, did not inhibit the cytotoxicity of toxins. Thespone, a dehydromansonone-D, lacking two hydrogens from the heterocyclic dihydrofuran ring of MD, inhibited the cytotoxicity of ricin, but was albeit less potent than MD. Neither mansonone-E nor mansonone-H with reactive ortho-quinone moiety, but with a different heterocyclic structure, had any effect on the cytotoxicity of ricin indicating that the protective effect of MD is specifically related to the overall structure of the metabolite.

Potency assay of diphtheria antitoxin in Vero cell microcultures.

Laboratory conditions were established for the titration of diphtheria toxin and antitoxin in Vero Cell Cultures (CCT) and a comparison was made with the intradermal test (IDT) as currently used throughout the world. Working dilutions and cut off values were established by reading both the change in color of phenol red used as pH indicator and changes in cell viability in the microscope. CCT shows high reproducibility and higher detectability than IDT in the titration of both WHO Reference Standard and high titer horse antisera. In sera of guinea pigs immunized for potency testing of diphtheria toxoid the titre was approximately 10 times lower than in the IDT. The explanation is a subject of speculation. The Vero cell titration might be adopted as such for titration of diphtheria antitoxin. In the case of the toxoid potency test it could be used if the limit for the titration is adjusted to 0.2 IU considering the equivalence obtained between the two tests, by taking into account a ratio of 1:10 between CCT and IDT.