Differential inhibition of the human cell DNA replication complex-associated DNA polymerases by the antimetabolite 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP).

The antimetabolite 1-beta-D-arabinofuranosylcytosine (ara-C) has been used as a highly effective agent for the treatment of leukemia. The active metabolite 1-beta-D-arabinofuranosylcytosine triphosphate (ara-CTP) is a potent inhibitor of DNA polymerases alpha, delta, and epsilon, and is responsible for inhibiting intact cell DNA synthesis. We have shown that a multiprotein complex, exhibiting many of the properties expected of the human cell DNA replication apparatus, can be readily isolated from human cells and tissues and is capable of supporting origin-dependent DNA synthesis in vitro. DNA polymerases alpha, delta, and epsilon are components of this multiprotein complex, termed the DNA synthesome, and we report here that the activities of these DNA synthesome-associated DNA polymerases are inhibited differentially by ara-CTP. Inhibition of the DNA synthesome-associated DNA polymerase alpha increased in a concentration-dependent manner, and was correlated closely with the inhibition of simian virus 40 (SV40) origin-dependent in vitro DNA replication, whereas DNA synthesome-associated DNA polymerase delta activity was not inhibited significantly by ara-CTP at 100 microM. Recent work has shown that the synthesome-associated DNA polymerase epsilon does not function in in vitro SV40 DNA replication, suggesting that only polymerases alpha and delta drive the DNA replication fork. Therefore, our results suggest that inhibition of the activity of the mammalian cell DNA synthesome by ara-CTP is due primarily to the inhibition of the DNA synthesome-associated DNA polymerase alpha. This observation implies that the drug may target specific phases of the DNA synthetic process in human cells.

Identification of multiprotein complexes containing DNA replication factors by native immunoblotting of HeLa cell protein preparations with T-antigen-dependent SV40 DNA replication activity.

Increasing evidence has supported the concept that many of the enzymes and factors involved in the replication of mammalian DNA function together as a multiprotein complex. We have previously reported on the partial purification of a multiprotein form of DNA polymerase from human HeLa cells shown to be fully competent to support origin-specific large T-antigen-dependent simian virus 40 (SV40) DNA replication in vitro. In an attempt to more definitively identify the complex or complexes responsible for DNA replication in vitro, partially purified human HeLa cell protein preparations competent to replicate DNA in vitro were subjected to native polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. The Native Western blots were probed with a panel of antibodies directed against proteins believed to be required for DNA replication in vitro. Apparent complexes of 620 kDa and 500 kDa were identified by monoclonal antibodies directed against DNA polymerase alpha and DNA polymerase delta, respectively. To detect epitopes possibly unexposed within the native multiprotein complexes, blots were also analyzed following denaturation in situ following treatment with detergent and reducing agent. The epitope or access to the epitope recognized by the monoclonal antibody against DNA polymerase alpha was destroyed by exposure of the blots to denaturing conditions. In contrast, an epitope present on a very large complex of approximately 1000 kDa was recognized by a monoclonal antibody against proliferating cell nuclear antigen only following treatment of the native immunoblots with denaturing agents. Identification of these complexes will allow their further purification, characterization, and elucidation of their role in the replication of DNA.

Molecular cloning and expression of SmIrV1, a Schistosoma mansoni antigen with similarity to calnexin, calreticulin, and OvRal1.

Protective immunity against schistosomiasis induced by vaccination of mice with irradiated cercaria can be passively transferred to uninfected mice with sera or IgG. Antigens that are uniquely or more strongly recognized by such protective sera compared with sera from infected unprotected mice have been identified previously. Two genes, SmIrV1 and SmIrV5, were selected from an adult worm cDNA library by screening with antibodies raised against these candidate vaccine proteins. Active immunization with the SmIrV5 protein induces high levels of protection in mice. We report here the molecular cloning and sequencing of SmIrV1 which contains a deduced amino acid sequence of 582 residues with similarity to three proteins: calnexin, calreticulin, and OvRal1, a surface antigen of the filarial nematode Onchocerca volvulus. SmIrV1 can be divided into three regions: a neutral N-terminal region with a putative signal sequence, followed by a proline- and tryptophan-rich P region in which two sets of sequences are repeated four times and a C-terminal region which is highly acidic with an isoelectric point of 4.7. We expressed the P and C regions of SmIrV1 and showed that this polypeptide reacts with sera of immunized as well as chronically infected mice.

Induction of protective immunity in mice using a 62-kDa recombinant fragment of a Schistosoma mansoni surface antigen.

Mice exposed to radiation-attenuated cercariae of Schistosoma mansoni are highly resistant to challenge infection, and sera from these mice can confer partial resistance when transferred to naive recipients. These sera recognize Ag present in schistosomular and adult worms, among them an Ag of 200 kDa. A cDNA encoding a 62-kDa portion of this Ag was cloned; the deduced amino acid sequence of this cDNA clone shares homology with myosins of other species. To assess the immunoprophylactic potential, we carried out vaccination trials in mice using the recombinant polypeptide expressed as a fusion protein with beta-galactosidase presented in the form of proteosome complexes with the outer membrane protein of meningococcus. The level of protection achieved was 32%, and this level could be increased to 75% by removal of those amino acids included in the fusion protein that were derived from the vector to yield a polypeptide, designated rIrV-5. A similar level of protection was achieved when mice were immunized with the same dose of rIrV-5 in the form of protein complexes but without outer membrane protein, suggesting that protection did not require the use of adjuvant. However, at least three immunizations were necessary to achieve protection. Using mAb and sera from mice vaccinated with rIrV-5, we demonstrated that the native protein recognized by antibodies against rIrV-5 is a 200-kDa protein that is expressed on the surface of newly transformed schistosomula. The protection achieved with rIrV-5 in mice encourages additional studies of its potential as a vaccine candidate for the prevention of schistosomiasis.

Characterization of cDNA clones encoding a novel calcium-activated neutral proteinase from Schistosoma mansoni.

To identify and characterize Schistosoma mansoni proteins that are recognized by infected hosts, we have used a pool of sera from infected humans to screen cDNA libraries constructed from poly(A)+ mRNA of adult S. mansoni. The deduced amino acid sequences of the three isolated clones showed a high degree of similarity to the large subunit of calcium-activated neutral proteinase (CANP) from humans and chicken. These overlapping clones, which include a nearly full-length clone with an open reading frame of 758 amino acid residues, together encode the entire large subunit of CANP. The deduced sequence of this S. mansoni protein can be divided into four domains (I-IV) that include the two domains characteristic of other large subunits of CANP: a thiol-protease domain (II) and a calcium-binding domain (IV) containing EF hand motifs. However, the schistosome protein is unique in having only three EF hand motifs in the calcium-binding domain and in having an additional EF hand motif that is shared between domains II and III. We have shown that these EF hand motifs are capable of binding 45Ca2+. Furthermore, the large subunit is S. mansoni contains an NH2-terminal sequence of 28 residues that is absent from the mammalian CANPs and has a high degree of similarity to the presumed receptor binding sequence of colicin Ia and Ib.

Cloning of a cDNA encoding a surface antigen of Schistosoma mansoni schistosomula recognized by sera of vaccinated mice.

Spleen cells of mice vaccinated with radiation-attenuated Schistosoma mansoni cercariae were used to produce monoclonal antibodies directed against newly transformed schistosomular surface antigens. One of these monoclonal antibodies recognized a polypeptide of 18 kDa. This glycoprotein was purified by monoclonal antibody immunoaffinity chromatography and a polyclonal antiserum was prepared against it. Immunofluorescence assays showed that the polyclonal antiserum bound to the surface of newly transformed schistosomula and lung-stage organisms but not to the surface of liver-stage and adult worms. Using this polyclonal antiserum we isolated recombinant clones from an adult worm cDNA expression library constructed in lambda gt11. Clone 654.2 contained an insert of 0.52 kilobase and hybridized to a 1.2-kilobase mRNA species from adult worms. Most importantly, clone 654.2 produced a fusion protein of 125 kDa that was reactive with sera of vaccinated mice that are capable of transferring resistance. This result encourages future vaccination trials with the fusion protein.

Characterization and cloning of Schistosoma mansoni immunogens recognized by protective antibodies.

In this report we have shown that mice vaccinated twice with radiation-attenuated cercariae elicit a much enhanced or unique response against six adult worm glycoproteins with molecular sizes of 200, 160, 140, 94, 58-56, and 43 kDa. In the case of the schistosomulum, vaccinated mice showed an enhanced or unique response to antigens of 200, 58, 46, 43, 25, and several glycoproteins in the range 65 to 50 kDa. That some or all of these antigens may be important for immunoprophylaxis against schistosomiasis is supported by the observations that 1. polyclonal antiserum (anti-IrV) prepared against these antigens also reacts with the major schistosomular surface antigens, and 2. this antiserum reacts with epitopes exposed on the surface of both newly transformed schistosomula and lung-stage schistosomula. In this study we also observed that the majority of the surface-iodinated antigens recognized by the anti-IrV serum were also recognized by sera from both vaccinated and patently infected mice. Simpson et al. (1985) have also shown that sera from vaccinated and infected mice recognized the same schistosomular surface antigens. It is possible, however, that the immune response of vaccinated mice is directed against different carbohydrate or peptide epitopes on these molecules, and that recognition of such epitopes is important for immune protection. Towards this goal we have cloned several schistosoma proteins reactive with the anti-IrV serum to identify peptide epitopes relevant for immunoprotection.

Fasciola hepatica: comparison of immature and mature immunoreactive glycoproteins.

A comparison of the 35S-methionine metabolically labelled immunoreactive glycoproteins of immature and mature F. hepatica was carried out by one-and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sera of rabbits infected for 3 weeks reacted much more strongly with glycoproteins of immature flukes than with glycoproteins of mature flukes as compared to sera of rabbits infected for 9 weeks. Several of the immunoreactive glycoproteins were also released by immature F. hepatica into the culture medium. At least one was a component of the T1 type granules. Analysis of the in vitro translation products of mature F. hepatica indicated that the initial humoral immune response of rabbit hosts may be directed against carbohydrate moieties.

Specificity, efficacy, and toxicity of radioimmunotherapy in erythroleukemic mice.

Radioimmunotherapy with 131I-labeled monoclonal immunoglobulins was studied using the Rauscher murine erythroleukemia. Tumor-specific monoclonal antibody, nonrelevant monoclonal antibody, F(ab')2 fragments, polyclonal gamma-globulin, and serum albumin were used as carriers of 131I. Therapeutic effects as measured by the reduction in splenomegaly were seen with all the radiolabeled proteins tested, but not with 131I-tyrosine. Dose-response curves showed that about 90% reduction in spleen size occurred at 80 muCi injected per animal, irrespective of whether specific or nonrelevant monoclonal antibody was used. Therapeutic efficacy was affected by the size of the 131I-carrier and could be correlated with half-life of carrier protein in vivo. As expected, increase in the serum concentration of circulating antigen decreased the targeting of the tumor-specific monoclonal antibody and also contributed to a shorter half-life for the tumor-specific monoclonal antibody in leukemic animals compared to uninfected controls. This study showed that there was no therapeutic advantage to the use of tumor-specific monoclonal antibody over nonrelevant immunoglobulin as a carrier for 131I in the treatment of murine erythroleukemia and that, although it was extremely effective, radioimmunotherapy with 131I was not specific in this system.

Diazepam reverses bombesin-induced gastric spasms and food intake reduction in the rat.

Bombesin (2-16 micrograms/kg, i.p.) produced abnormally large gastric contractions in intact rats consisting of increases in gastric pressure and motility. The effect was antagonized by diazepam (5 mg/kg, i.p.). The same dosage of diazepam abolished bombesin-induced food intake reduction. Diazepam by itself did not increase food intake above control levels. The time course of this behavioral antagonism was followed. There was no significant difference in the intake time course of diazepam-alone and diazepam-bombesin treatments, while there was a significant difference between the saline control and the bombesin-alone treatments. Intake of the saline control, the diazepam-alone and the diazepam-bombesin treatment terminated at the same level, while the bombesin-alone remained significantly different. Additionally, swift aversion to 8 micrograms/kg bombesin was obtained when flavored nutrient was substituted for flavored water, suggesting that the aversion developed as a consequence of interaction with the ingested diet. Abnormalities that result from intake reduction is due to the bombesin-produced intragastric abnormalities, and not by satiety.