Antibody To Synthetic Peptide Research Articles

Synthetic Peptide Antibodies as TNF-α Inhibitors: Molecularly Imprinted Polymer Nanogels Neutralize the Inflammatory Activity of TNF-α in THP-1 Derived Macrophages.

Tumor Necrosis Factor-α (TNF-α) is a cytokine that is normally produced by immune cells when fighting an infection. But, when too much TNF-α is produced as in autoimmune diseases, this leads to unwanted and persistent inflammation. Anti-TNF-α monoclonal antibodies have revolutionized the therapy of these disorders by blocking TNF-α and preventing its binding to TNF-α receptors, thus suppressing the inflammation. Herein, we propose an alternative in the form of molecularly imprinted polymer nanogels (MIP-NGs). MIP-NGs are synthetic antibodies obtained by nanomoulding the 3-dimensional shape and chemical functionalities of a desired target in a synthetic polymer. Using an in-house developed in silico rational approach, epitope peptides of TNF-α were generated and 'synthetic peptide antibodies' were prepared. The resultant MIP-NGs bind the template peptide and recombinant TNF-α with high affinity and selectivity, and can block the binding of TNF-α to its receptor. Consequently they were applied to neutralize pro-inflammatory TNF-α in the supernatant of human THP-1 macrophages, leading to a downregulation of the secretion of pro-inflammatory cytokines. Our results suggest that MIP-NGs, which are thermally and biochemically more stable and easier to manufacture than antibodies, and cost-effective, are very promising as next generation TNF-α inhibitors for the treatment of inflammatory diseases.

Synthetic Peptide Antibodies as TNF‐α Inhibitors: Molecularly Imprinted Polymer Nanogels Neutralize the Inflammatory Activity of TNF‐α in THP‐1 Derived Macrophages

AbstractTumor Necrosis Factor‐α (TNF‐α) is a cytokine that is normally produced by immune cells when fighting an infection. But, when too much TNF‐α is produced as in autoimmune diseases, this leads to unwanted and persistent inflammation. Anti‐TNF‐α monoclonal antibodies have revolutionized the therapy of these disorders by blocking TNF‐α and preventing its binding to TNF‐α receptors, thus suppressing the inflammation. Herein, we propose an alternative in the form of molecularly imprinted polymer nanogels (MIP‐NGs). MIP‐NGs are synthetic antibodies obtained by nanomoulding the 3‐dimensional shape and chemical functionalities of a desired target in a synthetic polymer. Using an in‐house developed in silico rational approach, epitope peptides of TNF‐α were generated and ‘synthetic peptide antibodies’ were prepared. The resultant MIP‐NGs bind the template peptide and recombinant TNF‐α with high affinity and selectivity, and can block the binding of TNF‐α to its receptor. Consequently they were applied to neutralize pro‐inflammatory TNF‐α in the supernatant of human THP‐1 macrophages, leading to a downregulation of the secretion of pro‐inflammatory cytokines. Our results suggest that MIP‐NGs, which are thermally and biochemically more stable and easier to manufacture than antibodies, and cost‐effective, are very promising as next generation TNF‐α inhibitors for the treatment of inflammatory diseases.

Identification of cGAS as an innate immune sensor of extracellular bacterium Pseudomonas aeruginosa.

SummaryCyclic GMP-AMP synthase (cGAS) is reported essential for detecting intracellular bacteria. However, it remains to be determined whether and how cGAS is involved in extracellular bacterial infection. Here, we report that cGAS is essential for mediating type I interferon (IFN) production in infection by multiple extracellular pathogens, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus. In addition, the canonical cGAS-stimulator of interferon gene (STING)-IFN axis is required for protecting mice from P. aeruginosa-induced mouse acute pulmonary infection, confirmed in cGAS pathway-specific gene deficiency mouse models. cGAS−/− and STING−/− mice exhibited reduced type I IFNs production, excessive inflammatory response accompanied with decreased resistance to P. aeruginosa challenge. Unfolded protein response was also modulated by cGAS through IRF3 and type I IFNs under P. aeruginosa infection. Collectively, these findings uncover the importance of cGAS in initiating immune responses against extracellular bacterial infection.

Detection of IgE-positive leukocytes by synthetic peptide and monoclonal antibodies

The aim: to assess the use of the synthetic peptide p137-142FcεRIα, the fragment of the FcεRI active site, in identifying the IgE+ cells, compared to monoclonal antibodies to IgE. Materials and methods. The study involved 39 children, mean age of 11 [10.0; 12.8] years. 105 blood samples were received. For phenotyping blood cells we used a synthetic hexapeptide p137-142FcεRIα, FITC-labeled (NIKP “Resan” Belarus), and monoclonal antibodies to IgE, Mouse Anti-Human (FITC), the production of Thermo Fisher Scientific Inc. USA. To count the eosinophils number general blood analysis was conducted. Results. No significant differences between the number of cells identified with the synthetic peptide and mAb were found (p = 0,2), the correlation between the IgE+-cells number identified by both methods was high (0.8). From the 105 samples in 51 (49%) – normal and in 54 (51%) - increased level of eosinophils (≥6%) was found. IgE+-cells number was higher in children with an increased level of eosinophils, than with normal eosinophils (p = 0,00). Correlation between IgE+-cells and eosinophils increased in samples with eosinophilia. In 68% of cases with a low level of eosinophils (0-3%), the level of IgE+ cells was above half of the medium: 3-3.5% at 30,000 cells. Conclusions. Determining the cells bound IgE by a synthetic peptide p137-142FcεRIα, fragment of the active center of the high affinity receptor for immunoglobulin E, is not worse than standard method of determining, using monoclonal antibodies to IgE. Number of eosinophils in peripheral blood correlates with the amount of IgE+-cells. In children with eosinophilia IgE+ -cell number is higher than in children with normal eosinophils level. There is a higher correlation IgE+-cell number with the eosinophils number in case of eosinophilia. In 14% of cases the other IgE+-cells (except eosinophils) were dominant.

Molecular cloning and characterisation of the methionine sulphoxide reductase A (msrA) gene locus in Campylobacter lari organisms

The methionine sulphoxide reductase A (msrA) gene and its adjacent genetic loci from urease-negative (UN) Campylobacter lari RM2100 and urease-positive thermophilic Campylobacter (UPTC) CF89–12 strains appear to be composed of a msrA structure gene (507 base pairs [bp]) and another five-gene cluster (approximately 6300 bp) in the same strand and direction. A primer pair (F1/R4-msrA) for polymerase chain reaction (PCR) amplification was designed to generate a product of approximately 900 bp of the msrA gene, including its adjacent genetic loci for the thermophilic Campylobacter organisms and generate an amplicon with 16 C. lari isolates (n=4 for UN C. lari; n=12 for UPTC). Following direct nucleotide sequencing, sequence analysis and nucleotide sequence alignment analysis, the putative full-length msrA gene from the 16 C. lari isolates showed high nucleotide sequence similarities (91.8–100%) to each other and relatively low similarity (69.3–71.8%) to three reference C. jejuni and C. coli strains. In addition, the msrA gene was transcribed in both the UPTC CF89–12 and NCTC12893 cells using reverse transcription PCR. An immunoreactively positive signal was identified in the UPTC CF89–12 and NCTC12893 cells with anti-UPTC MsrA synthetic peptide antibodies.

Identification of a Highly Antigenic Linear B Cell Epitope within Plasmodium vivax Apical Membrane Antigen 1 (AMA-1)

Apical membrane antigen 1 (AMA-1) is considered to be a major candidate antigen for a malaria vaccine. Previous immunoepidemiological studies of naturally acquired immunity to Plasmodium vivax AMA-1 (PvAMA-1) have shown a higher prevalence of specific antibodies to domain II (DII) of AMA-1. In the present study, we confirmed that specific antibody responses from naturally infected individuals were highly reactive to both full-length AMA-1 and DII. Also, we demonstrated a strong association between AMA-1 and DII IgG and IgG subclass responses. We analyzed the primary sequence of PvAMA-1 for B cell linear epitopes co-occurring with intrinsically unstructured/disordered regions (IURs). The B cell epitope comprising the amino acid sequence 290–307 of PvAMA-1 (SASDQPTQYEEEMTDYQK), with the highest prediction scores, was identified in domain II and further selected for chemical synthesis and immunological testing. The antigenicity of the synthetic peptide was identified by serological analysis using sera from P. vivax-infected individuals who were knowingly reactive to the PvAMA-1 ectodomain only, domain II only, or reactive to both antigens. Although the synthetic peptide was recognized by all serum samples specific to domain II, serum with reactivity only to the full-length protein presented 58.3% positivity. Moreover, IgG reactivity against PvAMA-1 and domain II after depletion of specific synthetic peptide antibodies was reduced by 18% and 33% (P = 0.0001 for both), respectively. These results suggest that the linear epitope SASDQPTQYEEEMTDYQK is highly antigenic during natural human infections and is an important antigenic region of the domain II of PvAMA-1, suggesting its possible future use in pre-clinical studies.

A novel polyclonal antibody against human cytomegalovirus: General characteristics and potential application in diagnosis

There is no vaccine available for human cytomegalovirus (HCMV) infection and the treatment is very limited; therefore, it is still an important cause of morbidity and occasional mortality in transplant recipients. An HCMV peptide was selected based on the GenBank sequence M60929. The peptide was conjugated with keyhole limpet haemocyanin and used to immunise New Zealand rabbits to prepare polyclonal antibody. After immunisation, the serum of the rabbits was obtained and purified. ELISA, immunoprecipitation and immunocytochemical staining were used to identify antibodies and antibody titre. The identification of the synthetic peptide antibody was confirmed by ELISA, immunoprecipitation and immunofluorescence against HCMV Towne and AD169 strains. The antibody can be used to detect the HCMV infection by immunocytochemical staining. The synthetic peptide showed favourable immunogenicity. The serum from immunised rabbits could be used to identify HCMV AD169 and Towne strains. Future research should be directed to epitope screening of synthetic HMCV peptides, which could help to understand HCMV infection and virus-neutralising antibodies more fully and to prepare HCMV vaccines and antiviral drugs.

Assessment of Specific Antibodies to F Protein in Serum Samples from Chinese Hepatitis C Patients Treated with Interferon plus Ribavarin

The hepatitis C virus (HCV) alternate reading frame protein or F protein of the HCV 1b genotype is a double-frameshift product of the HCV core protein. In order to assess the presence of antibodies specific for F protein and their clinical relevance in sera from HCV patients, we produced recombinant F protein and core protein of the HCV 1b genotype in Escherichia coli. An enzyme-linked immunosorbent assay was developed using purified recombinant HCV core, F protein, and a 99-residue synthetic F peptide (F99). The seroprevalences of anticore, anti-F protein, and anti-F99 synthetic peptide were 95%, 68%, and 36%, respectively, in 168 HCV patients. The prevalence of anti-F antibodies did not correlate with viral load, genotype, or alanine aminotransferase level. Interferon combination therapy induced a decline in the level of anti-F antibodies in 55 responders (P < 0.01). Thirteen responders (24%) lost their anti-F recombinant protein antibodies, and 17 (31%) lost their anti-F synthetic peptide antibodies, whereas no decrease was observed for the 17 nonresponders. These changes were significant between responders and nonresponders (P < 0.05). Meanwhile, no change was found in the anticore antibody titer of the 72 treated patients. The percentage of anti-F-protein-negative patients (15/15 [100%]) who achieved a sustained virological response (SVR) was higher than that of the anti-F-positive patients (70%) (P < 0.05). Based on these findings, HCV F protein elicits a specific antibody response other than the anticore protein response. Our data also suggest that the presence and level of anti-F antibody responses might be influenced by the treatment (interferon plus ribavirin) and associated with an SVR in Chinese hepatitis C patients.

RNA interference of PFUS results in inhibition of secretory vesicle biogenesis

In protistan evolution, gene duplication gives rise to phosphoglucomutase (PGM) superfamily members which had virtually no PGM activity, including Paramecium parafusin (PFUS) and its Toxoplasma and mammalian orthologs, phosphoglycoproteins whose role in Ca2+ ‐regulated exocytosis has been controversial. We show that RNAi knockdown of PFUS leads to an exo− phenotype. Three different regions of the PFUS sequence were targeted. As a control we used the empty RNAi vector. Exocytosis was scored after stimulation. Overall, exocytosis becomes inhibited with all three RNAi constructs, compared to the control. In order to examine if inhibition correlated with a reduction of PFUS, we performed western blot analysis using a polyclonal antibody which recognizes all Paramecium proteins in the PGM superfamily (pan‐PGM antibody) and a synthetic peptide antibody, PFUS antibody, which recognizes PFUS specifically. Cells exposed to RNAi for 72 hr show that PFUS decreases, whereas no decrease is seen in other PGM isoforms. Double labeling in confocal microscopy for PFUS and secretory content (T‐8) shows a corresponding decrease in dense core secretory vesicles (DCSVs) in the cells. These changes are reversed by re‐feeding in the absence of PFUS RNAi. These data demonstrate that knockdown of PFUS inhibits exocytosis, probably by blocking DCSV packaging.

Prediction of linear B-cell epitopes using amino acid pair antigenicity scale

Identification of antigenic sites on proteins is of vital importance for developing synthetic peptide vaccines, immunodiagnostic tests and antibody production. Currently, most of the prediction algorithms rely on amino acid propensity scales using a sliding window approach. These methods are oversimplified and yield poor predicted results in practice. In this paper, a novel scale, called the amino acid pair (AAP) antigenicity scale, is proposed that is based on the finding that B-cell epitopes favor particular AAPs. It is demonstrated that, using SVM (support vector machine) classifier, the AAP antigenicity scale approach has much better performance than the existing scales based on the single amino acid propensity. The AAP antigenicity scale can reflect some special sequence-coupled feature in the B-cell epitopes, which is the essence why the new approach is superior to the existing ones. It is anticipated that with the continuous increase of the known epitope data, the power of the AAP antigenicity scale approach will be further enhanced.

Experimental model of IgA nephropathy using KM mouse (second report): Possible protective effect of anti‐IgA1 synthetic hinge peptide antibody on glomerular deposition of underglycosylated IgA1

Experimental model of IgA nephropathy using KM mouse (second report): Possible protective effect of anti‐IgA1 synthetic hinge peptide antibody on glomerular deposition of underglycosylated IgA1

Accuracy of Testing for Antibodies to Synthetic Gliadin–Related Peptides in Celiac Disease

Our aim was to explore the diagnostic value of a newly developed synthetic peptide antibody assay addressing specific synthetic gliadin-derived deamidated peptides (AGA II) for the diagnosis of celiac disease (CD). We assayed serum samples obtained prospectively at diagnosis from a population of 92 consecutive adult patients with CD and 113 non-CD controls. Patients were reevaluated after 6 months (n = 56) and 1 year (n = 20) of treatment. All patients and controls underwent intestinal biopsy and a set of CD-related serology tests. A newly developed enzyme-linked immunosorbent assay (ELISA) for detecting IgA and IgG antibodies against synthetic deamidated gliadin epitopes was used. At diagnosis, sensitivity and specificity were 94.6% and 99.1% for AGA II IgA and 92.4% and 100% for AGA II IgG. Absolute values and the proportion of positive samples for both antibodies were significantly reduced at 6 months (P < .0000) and 1 year (P < .001) after initiation of a gluten-free diet. Compared with conventional AGA, the peptide antibodies had greater sensitivity, specificity, positive and negative predictive values, accuracy, and likelihood ratios. Compared with antitissue transglutaminase antibodies, AGA II had similar sensitivity but greater specificity and predictive values, better likelihood ratios, and an excellent agreement (kappa statistic = .92). This study assessed the value of an ELISA assay in detecting antibodies to gliadin-related peptides. This assay appears to be a reliable tool for diagnosing CD and suggests promising accuracy that may be very useful in clinical practice.

PHOX2B Regulates Its Own Expression by a Transcriptional Auto-regulatory Mechanism

The specification of neuronal identity is a result of interactions between the following two distinct classes of determinants: extrinsic factors that include secreted or cell membrane-associated signals in the local environment, and intrinsic factors that generally consist of ordered cascades of transcription factors. Little is known about the molecular mechanisms underlying the interplay between these extrinsic and intrinsic factors and the transcriptional processes that establish and maintain a given neuronal phenotype. Phox2b is a vertebrate homeodomain transcription factor and a well established intrinsic factor in developing autonomic ganglia, where its expression is triggered by the bone morphogenic proteins secreted by the dorsal aorta. In this study we characterized its proximal 5'-regulatory region and found that it contained five putative DNA sites that potentially bind homeodomain proteins, including PHOX2B itself. Chromatin immunoprecipitation assays showed that PHOX2B could bind its own promoter in vivo, and electromobility gel shift assays confirmed that four of the five sites could be involved in PHOX2B binding. Functional experiments demonstrated that 65% of the transcriptional activity of the PHOX2B promoter in neuroblastoma cells depends on this auto-regulatory mechanism and that all four sites were required for full self-transactivation. Our data provide a possible molecular explanation for the maintenance of PHOX2B expression in developing ganglia, in which initially its expression is triggered by bone morphogenic proteins, but may become independent of external stimuli when it reaches a certain nuclear concentration and sustains its own transcription.

Identification of a novel cellular transcriptional repressor interacting with the latent nuclear antigen of Kaposi's sarcoma-associated herpesvirus.

The latent nuclear antigen (LNA) of Kaposi's sarcoma-associated herpesvirus (KSHV) has an essential role in viral latent infection. LNA maintains the stability of KSHV episomes and modulates the expression of cellular genes. A novel cellular protein KLIP1 was identified to interact with LNA through yeast two-hybrid screening, and confirmed by a glutathione S-transferase pull down assay. Domain mapping showed that KLIP1 interacted with the N-terminal domain of LNA. Northern blot hybridization with a KLIP1 probe identified a major transcript of 1.8 kb and a minor transcript of 2.8 kb. cDNA library screening and 5'-RACE revealed that the major transcript encoded an open-reading-frame of 1,257 bp and had a 5'-untranslated region of 73 nucleotides. The major KLIP1 transcript was ubiquitously present in different cell types examined. A KLIP1 synthetic peptide antibody detected a doublet of 58-kDa and 63-kDa proteins in a Western blot assay. KLIP1 had two putative nuclear localization signals and showed punctate nuclear localization when expressed as a GFP-fusion protein. KLIP1 interacted with LNA in vivo, as demonstrated by coimmunoprecipitation using KSHV-infected cells and colocalization when they were expressed as GFP- and DsRed-fusion proteins, respectively. Consistent with its interaction with LNA, nuclear localization, and possession of two leucine zipper motifs, KLIP1 behaved like a transcriptional factor and repressed herpes simplex virus thymidine kinase (TK) promoter activity in a mammalian one-hybrid assay. In addition, cotransfection with LNA alleviated the transcriptional repression effect of KLIP1 on TK promoter activity. These results suggest that KLIP1 is a new member of cellular transcriptional repressors, and that LNA is involved in deregulating cellular transcription process.

A novel method for polypeptide design to prepare specific antibody of the peptide and applied to immunoassay

Peptide was polymerized by a novel method: compound containing double bond between two carbons such as acdryloyl chloride was introduced into peptide during peptide synthesis, then it was transformed to a polymer which has a poly propionyl core matrix with peptide branches by radical polymerization either after being cleaved from resin or before being cleaved from resin according to the peptide physical–chemical character. According to this design, the macromolecules with average MW about 40 kD for poly-Osteogenic Growth Peptide (poly-OGP), 25 KD for poly-penetratin could be produced. Poly-OGP was further applied for antibody preparation and immunoassays. Immunizing New Zealand rabbits, we obtained the antiserum with titer of 2.5×10 4, examined by enzyme-linked immunosorbent assay (ELISA), without cross-reaction with bovine serum albumin (BSA), while the antiserum produced by using BSA as peptide carrier strongly reacted with BSA (with titer of more than 50×10 4 for BSA). Based on competitive ELISA, the anti-poly-OGP antibody showed much better immunoreactive sensitivity than anti-BSA–OGP antibody by comparing their IC 50 toward OGP. The antigen determinant of OGP and the OGP content in the serum of mice, determined by anti-poly-OGP antibody, showed that the anti-polypeptide antibody can be used as tools for immunoassay. Thus, the polypeptide system is not only a new approach for preparing synthetic peptide antibody for immunoassays but also provided the prospect for preparing synthetic peptide-based vaccine.

Localization and functional importance of a conserved zona pellucida 2 protein domain in the human and bovine ovary using monoclonal anti-ZP2 peptide antibodies

In mammals, gamete recognition and sperm binding to the oocyte are mediated by the zona pellucida (ZP), an acellular coat surrounding the plasma membrane of the oocyte that consists of particular ZP proteins. The ZP2 protein mediates secondary sperm binding to the ZP. Its primary structures are highly conserved as revealed by cDNA cloning. In the present study, we investigated the localization of ZP2 in human and bovine ovaries and oocytes and the influence of monoclonal anti-ZP2 peptide antibodies upon bovine sperm–egg interactions. We generated a monoclonal anti-ZP2 synthetic peptide antibody, mAb ZP2–20, against a sequence that is strongly conserved in the mammalian ZP2 amino acid sequence. Specificity of mAb ZP2–20 was determined by ELISA and immunoblotting, respectively. Our results show that mAb ZP2–20 specifically detected the peptide used as an antigen and reacted with its corresponding protein antigen in human and bovine ovaries. In order to elucidate effects of mAb ZP2–20 upon bovine sperm–ZP binding, we used the competitive hemizona assay (cHZA) and found that the antibodies clearly inhibit sperm binding to the ZP. We conclude that (i) monoclonal antibodies against ZP2 peptides react with ZP proteins present in bovine and human ovaries and can be used as a specific marker for ZP2; and that (ii) mAb ZP2–20 detects a ZP2 epitope that is of functional relevance for sperm–ZP interactions.

Synthetic peptide vaccine and antibody therapeutic development: prevention and treatment of Pseudomonas aeruginosa.

Pseudomonas aeruginosa and Pseudomonas maltophilia account for 80% of opportunistic infections by pseudomonads. Pseudomonas aeruginosa is an opportunistic pathogen that causes urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, and a variety of systemic infections, particularly in patients with severe burns, and in cancer and AIDS patients who are immunosuppressed. Pseudomonas aeruginosa is notable for its resistance to antibiotics, and is therefore a particularly dangerous pathogen. Only a few antibiotics are effective against Pseudomonas, including fluoroquinolones, gentamicin, and imipenem, and even these antibiotics are not effective against all strains. The difficulty treating Pseudomonas infections with antibiotics is most dramatically illustrated in cystic fibrosis patients, virtually all of whom eventually become infected with a strain that is so resistant that it cannot be treated. Since antibiotic therapy has proved so ineffective as a treatment, we embarked on a research program to investigate the development of a synthetic peptide consensus sequence vaccine for this pathogen. In this review article we will describe our work over the last 15 years to develop a synthetic peptide consensus sequence anti-adhesin vaccine and a related therapeutic monoclonal antibody (cross-reactive to multiple strains) to be used in the prevention and treatment of P. aeruginosa infections. Further, we describe the identification and isolation of a small peptide structural element found in P. aeruginosa strain K (PAK) bacterial pili, which has been proven to function as a host epithelial cell-surface receptor binding domain. Heterologous peptides are found in the pili of all strains of P. aeruginosa that have been sequenced to date. Several of these peptide sequences have been used in the development of an consensus sequence anti-adhesin vaccine targeted at the prevention of host cell attachment and further for the generation of a monoclonal antibody capable of prevention and treatment of existing infections.

Conformational changes during proteolytic processing of a picornavirus capsid proteins.

We have used synthetic peptide antibodies to probe conformational changes that occur during the cleavage cascade which generates the capsid proteins of a picornavirus. The initial translation product of 97 kDa, the precursor of all four structural proteins, is cleaved to form a 63 kDa fragment which, we show, has significantly different folding characteristics to both its larger parent and its products. We demonstrate that proteolytic cleavages as distant as 520 residues from epitopes confer sufficiently large conformational changes as to render them unrecognisable. To our knowledge, this is the first demonstration of this phenomenon in the picornavirus system.

Pneumocystis carinii uses a functional cdc13 B-type cyclin complex during its life cycle.

Pneumocystis carinii causes severe pneumonia in immunocompromised patients. Recent studies indicate that P. carinii uses a Cdc2 cyclin-dependent kinase to control its proliferation. To further study the regulation of the life cycle of P. carinii, we characterized the P. carinii B-type cyclin termed Cdc13, whose binding to Cdc2 is necessary for kinase activity. Antibodies to B-type cyclins (Cdc13) specifically immunoprecipitated Cdc2/ Cdc13 complexes with associated kinase activity from P. carinii extracts. To clone P. carinii cdc13, degenerate polymerase chain reaction was undertaken using primers generated from amino-acid motifs conserved in fungal Cdc13 proteins. This amplicon was used to obtain full-length genomic and complementary DNA (cDNA) clones. A specific synthetic peptide antibody generated to P. carinii Cdc13 further demonstrated differential Cdc2/Cdc13 activity over the life cycle of P. carinii, with greater activity in cysts compared with trophic forms of the organism. Finally, P. carinii cdc13 cDNA was used to rescue mutant Schizosaccharomyces pombe strains containing temperature-sensitive deficiencies of endogenous Cdc13 activity, thus verifying function of the P. carinii Cdc13 protein. Therefore, P. carinii contains a Cdc13 cyclin, which is variably active over its life cycle and which promotes fungal proliferation.

Species specificity of human and murine anti-ZP3 synthetic peptide antisera and use of the antibodies for localization and identification of ZP3 or ZPC domains of functional significance.

The mammalian zona pellucida has an important function in the fertilization process. The zona pellucida protein 3 (ZP3 or ZPC) is the ligand for primary sperm binding and induces the acrosome reaction. In various species, ZP3 primary structures are highly conserved as revealed by cDNA cloning. The objective of these studies was to localize ZP3 protein using antisera generated against defined synthetic peptides that are specific for mouse or for human ZP3. Immunohistochemistry and transmission electron microscopy were applied to murine and human ovary sections. Immunochemical studies were performed in hemizonae pellucidae from microbisected human oocytes. Using the competitive hemizona assay and various anti-ZP3 antibodies, we further intended to identify human ZP3 epitopes of functional significance. Our results showed that antiserum AS ZP3-9 (mouse specific) detected mouse ZP3 protein in mouse oocytes and in immunoblots, whereas AS ZP3-14 (human specific) detected human ZP3 protein in human ovary sections, native hemizonae pellucidae and in immunoblots. ZP3 material was also detected in cumulus cells by immunohistochemistry. Ultrastructural studies showed an equal distribution of ZP3 throughout the zona pellucida. The human competitive hemizona assay revealed that none of the anti-ZP3 synthetic peptide antisera affected sperm binding suggesting that those epitopes are not involved in primary sperm binding. Anti-porcine ZP3 beta protein antibodies (polyclonal) blocked human sperm-zona pellucida binding. In summary, these anti-ZP3 synthetic peptide antibodies specifically reacted with intact ZP3 protein (murine and human) but did not inhibit human sperm-zona pellucida binding; anti-ZP3 antibodies can therefore be used as biomarkers for ZP3 localization and function.