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Objectives: Phage-gonadotropin-releasing hormone (GnRH) constructs with potential contraceptive properties were generated in our previous study via selection from a phage display library using neutralizing GnRH antibodies as selection targets. In mice, these constructs invoked the production of antibodies against GnRH and suppressed serum testosterone. The goal of this study was to evaluate this vaccine against GnRH for its potential to suppress reproductive characteristics in cats., Methods: Sexually mature male cats were injected with a phage-GnRH vaccine using the following treatment groups: (1) single phage-GnRH vaccine with adjuvant; (2) phage-GnRH vaccine without adjuvant and half-dose booster 1 month later; or (3) phage-GnRH vaccine with adjuvant and two half-dose boosters with adjuvant 3 and 6 months later. Anti-GnRH antibodies and serum testosterone, testicular volume and sperm characteristics were evaluated monthly for 7-9 months., Results: All cats developed anti-GnRH antibodies following immunization. Serum antibody titers increased significantly after booster immunizations. In group 3, serum testosterone was suppressed 8 months after primary immunization. Total testicular volume decreased in group 1 by 24-42% and in group 3 by 15-36% at 7 months after immunization, indicating potential gonadal atrophy. Vacuolation of epididymides was observed histologically. Although all cats produced sperm at the conclusion of the study, normal morphology was decreased as much as 38%. Phage alone produced no local or systemic reactions. Immunization of phage with AdjuVac produced unacceptable injection site reactions., Conclusions and Relevance: Our phage-based vaccine against GnRH demonstrated a potential for fertility impairment in cats. Future research is required to optimize vaccine regimens and identify animal age groups most responsive to the vaccine. If permanent contraception (highly desirable in feral and shelter cats) cannot be achieved, the vaccine has a potential use in zoo animals or pets where multiple administrations are more practical and/or reversible infertility is desirable.

GnRH receptors play vital roles in mammalian reproduction via regulation of gonadotropin secretion, which is essential for gametogenesis and production of gonadal steroids. GnRH receptors for more than 20 mammalian species have been sequenced, including human, mouse, and dog. This study reports the molecular cloning and sequencing of GnRH receptor (GnRHR) cDNA from the pituitary gland of the domestic cat, an important species in biomedical research. Feline GnRHR cDNA is composed of 981 nucleotides and encodes a 327 amino acid protein. Unlike the majority of mammalian species sequenced so far, but similar to canine GnRHR, feline GnRHR protein lacks asparagine in position three of the extracellular domain of the protein. At the amino acid level, feline GnRHR exhibits 95.1% identity with canine, 93.8% with human, and 88.9% with mouse GnRHR. Comparative sequence analysis of GnRHRs for multiple mammalian species led to resequencing of canine GnRHR, which differed from that previously published by a single base change that translates to a different amino acid in position 193. This single base change was confirmed in dogs of multiple breeds. Reverse transcriptase PCR analysis of GnRHR messenger RNA in different tissues from four normal cats indicated the presence of amplicons of varying lengths, including full-length as well as shortened GnRHR amplicons, pointing to the existence of truncated GnRHR transcripts in the domestic cat. This study is the first insight into molecular composition and expression of feline GnRHR and promotes better understanding of receptor organization, and distribution in various tissues of this species., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Multiple phage-peptide constructs, where the peptides mimic sperm epitopes that bind to zona pellucida (ZP) proteins, were generated via selection from a phage display library using a novel approach. Selections were designed to allow for identification of ZP-binding phage clones with potential species-specific properties, an important feature for wildlife oral vaccines as the goal is to control overpopulation of a target species while not affecting non-target species' reproduction. Six phage-peptide antigens were injected intramuscularly into pigs and corresponding immune responses evaluated. Administration of the antigens into pigs stimulated production of anti-peptide antibodies, which were shown to act as anti-sperm antibodies. Potentially, such anti-sperm antibodies could interfere with sperm delivery or function in the male or female genital tract, leading to contraceptive effects. Staining of semen samples collected from different mammalian species, including pig, cat, dog, bull, and mouse, with anti-sera from pigs immunized with ZP-binding phage allowed identification of phage-peptide constructs with different levels of species specificity. Based on the intensity of the immune responses and specificity of these responses in different species, two of the antigens with fusion peptide sequences GEGGYGSHD and GQQGLNGDS were recognized as the most promising candidates for development of contraceptive vaccines for wild pigs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The focus of this study is on development of vaccines using filamentous phage as a delivery vector for immunogenic peptides. The use of phage as a carrier for immunogenic peptides provides significant benefits such as high immunogenicity, low production costs, and high stability of phage preparations. However, introduction of live recombinant phage into the environment might represent a potential ecological problem. This, for example, may occur when vaccines are used in oral or nasal formulations in field conditions for wild and feral animals. To address this issue, comparative studies of antigenic properties of live and inactivated (non-viable) phage were accomplished. Inactivated phage, if released, will not propagate and will degrade as any other protein. In these experiments, a model phage clone that was previously selected from a phage display library and shown to stimulate production of anti-sperm antibodies with contraceptive properties was used. Multiple methods of phage inactivation were tested, including drying, freezing, autoclaving, heating, and UV irradiation. Under studied conditions, heating at 76°C for 3h, UV irradiation, and autoclaving resulted in complete phage inactivation. Phage samples treated by heat and UV were characterized by spectrophotometry and electron microscopy. To test antigenicity, live and inactivated phage preparations were injected into mice and antibody responses assayed by ELISA. It was found that phage killed by heat causes little to no immune responses, probably due to destruction of phage particles. In contrast, UV-inactivated phage stimulated production of IgG serum antibodies at the levels comparable to live phage. Thus, vaccines formulated to include UV-inactivated filamentous phage might represent environmentally safe alternatives to live phage vaccines., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Zona pellucida (ZP) glycoproteins play a central role in sperm-oocyte binding and fertilization. Sperm protein sequences that are involved in sperm-ZP recognition and have an important role in fertilization represent attractive targets for development of contraceptive vaccines, yet are currently unknown. To identify peptide sequences that recognize and bind to ZP proteins, we developed a novel selection procedure from phage display libraries that utilizes intact oocytes surrounded by ZP proteins. The major advantage of this procedure is that ZP proteins remain in their native conformation unlike a selection protocol previously published that utilized solubilized ZP on artificial solid support. Several peptides of 7 and 12 amino acids with binding specificity to canine ZP proteins were identified. Four of them (LNSFLRS, SSWYRGA, YLPIYTIPSMVY, and NNQSPILKLSIH) plus a control ZP-binding peptide (YLPVGGLRRIGG) from the literature were synthesized and tested for antigenic properties in dogs. NNQSPILKLSIH peptide stimulated production of anti-peptide antibodies. These antibodies bind to the acrosomal region of the canine sperm cell, demonstrating ability to act as sperm antibodies. The identified ZP-binding peptides (mimicking sperm cell surface antigens) may be useful in the design of immunocontraceptive agents for dogs., (Copyright 2010 Elsevier B.V. All rights reserved.)

Lysosomal beta-galactosidase is required for the degradation of GM1 ganglioside and other glycolipids and glycoproteins with a terminal galactose moiety. Deficiency of this enzyme leads to the lysosomal storage disorder, GM1 gangliosidosis, marked by severe neurodegeneration resulting in premature death. As a step towards preclinical studies for enzyme replacement therapy in an animal model of GM1 gangliosidosis, a feline beta-galactosidase cDNA was cloned into a mammalian expression vector and subsequently expressed in Chinese hamster ovary (CHO-K1) cells. The enzyme secreted into culture medium exhibited specific activity on two synthetic substrates as well as on the native beta-galactosidase substrate, GM1 ganglioside. The enzyme was purified from transfected CHO-K1 cell culture medium by chromatography on PATG-agarose. The affinity-purified enzyme preparation consisted mainly of the protein with approximate molecular weight of 94 kDa and displayed immunoreactivity with antibodies raised against a 16-mer synthetic peptide corresponding to C-terminal amino acid sequence deduced from the feline beta-galactosidase cDNA.

Biosensors based on landscape phages immobilized by physical adsorption on the surface of a quartz crystal microbalance was used for detection of beta-galactosidase from Escherichia coli. The sensor had a detection limit of a few nanomoles and a response time of a approximately 100 s over the range of 0.003-210 nM. The binding dose-response curve had a typical sigmoid shape and the signal was saturated at the beta-galactosidase concentration of about 200 nM. A marked selectivity for beta-galactosidase over BSA was observed in mixed solutions even when the concentration of BSA exceeded the concentration of beta-galactosidase by a factor of approximately 2000. The apparent value of the dissociation constant (K(d)) of the interaction of free phage with beta-galactosidase (9.1+/-0.9 pM) was smaller compared with the one calculated for the bound phage (1.7+/-0.5 nM). The binding was specific with three binding sites needed to bind a single molecule of beta-galactosidase. The K(d) obtained from the enzyme-linked immunosorbent assay (ELISA) for the phage and the monoclonal anti-beta-galactosidase antibodies were 21+/-2 and 26+/-2 nM, respectively. Although the method of physical adsorption is simpler and more economical in comparison with Langmuir-Blodgett and molecular assembling methods the performances of the sensors made by these technologies compare well. This work provides evidence that phage can be used as a recognition element in biosensors using physical adsorption method for immobilization of phage on the sensor surface.

Patented signal analytic algorithms applied to hydrophobically transformed, numerical amino acid sequences have previously been used to design short, protein-targeted, L or D retro-inverso peptides. These peptides have demonstrated allosteric and/or indirect agonist effects on a variety of G-protein and tyrosine kinase coupled membrane receptors with 30% to over 80% hit rates. Here we extend these approaches to a globular protein target. We designed eight peptide ligands targeting an ELISA antibody responsive protein, beta-galactosidase, betaGAL. Three of the eight 14mer peptides allosterically activated betaGAL with ELISA methodology. Using Bayesian statistics, this 38% hit rate would have occurred 2 x 10(-9) by chance. These peptides demonstrated binding site competitive or noncompetitive interactions, suggesting allosteric site multiplicity with respect to their betaGAL binding-mediated ELISA signal. Kinetic studies demonstrated the temperature dependence of the betaGAL peptide binding functions. Using the van't Hoff relation, we found evidence for enthalpy-entropy compensation. This relation is often found for hydrophobic interactions in aqueous media, and is consistent with the postulated hydrophobic series encoding underlying our protein-targeted, peptide design methods. It appears that our algorithmic, hydrophobic autocovariance eigenvector template approach to the design of allosteric peptides targeting membrane receptors may also be applicable to the design of peptide ligands targeting nonmembrane involved globular proteins., (Copyright 2006 Wiley Periodicals, Inc.)

Unfolding and subsequent aggregation of proteins is a common phenomenon that is linked to many human disorders. Misfolded hemoglobin is generally manifested in various autoimmune, infectious and inherited diseases. We isolated micrometer and submicrometer particles, termed proteons, from human and animal blood. Proteons lack nucleic acids but contain two major polypeptide populations with homology to the hemoglobin alpha-chain. Proteons form by reversible seeded aggregation of proteins around proteon nucleating centers (PNCs). PNCs are comprised of 1- to 2-nm metallic nanoclusters containing 40-300 atoms. Each milliliter of human blood contained approximately 7 x 10(13) PNCs and approximately 3 x 10(8) proteons. Exposure of isolated blood plasma to elevated temperatures increased the number of proteons. When an aliquot of this heated plasma was introduced into untreated plasma that was subsequently heated, the number of proteons further increased, reaching a maximum after a total of three such iterations. Small concentrations of PNCs were lethal to cultured cancer cells, whereas noncancerous cells were much less affected.

The specific and selective detection of Salmonella typhymurium based on the use of a polyclonal antibody immobilized by the Langmuir-Blodgett method on the surface of a quartz crystal acoustic wave device was demonstrated in liquid samples. These biosensors were selective to S. typhymurium in the presence of large concentrations of Escherichia coli O157:H7. They were also specific to S. typhymurium since bacteria preincubated with free antibody produced no signal. Dark-field and electron microscopy showed that two different antibodies, polyvalent somatic O and flagellar H7, were immobilized on the sensor surface producing two distinct attachments of bacteria at the liquid-solid interface. The somatic O antibody exhibits a rigid, binding, while the flagellar H7 antibody forms a flexible connection allowing a large degree of freedom. When the attachment of bacteria was rigid and strong, the responses of the acoustic wave sensors correlated with changes in the mass of bacteria present at the liquid-solid interface. In contrast, when attachment was flexible, the sensor signals were inversely proportional to the additional mass of bound bacteria. This difference is probably determined by the interfacial viscoelasticity and by acoustic and electromagnetic coupling. The signals of environmentally aged sensors with either predominantly rigid or flexible positioning of bacteria were correlated with changes in mass at the liquid-solid interface. Sensors with O or H type of binding could be used for analytical purposes.

Biosensors based on phage display-derived peptides as biorecognition molecules were used for the detection of cell surface cross-species markers in tissue homogenates. The peptide selected for murine myofibers was immobilized onto the surface of an acoustic wave sensor by biotin-streptavidin coupling. To detect peptide-receptor interaction, the sensors were exposed to muscle and control (kidney, liver, brain) tissue homogenates. The sensor showed a strong response to murine muscle. The amplitudes of the responses to the feline muscle homogenates were lower compared to those of the murine muscle, while the same K(d) indicated that the peptide has cross-species affinity. In contrast, murine kidney, liver and brain homogenates produced insignificant responses. Specificity of the sensor was shown in a blocking experiment, as reduced signal was detected when muscle preparations were preincubated with free peptide. Additionally, when muscle-specific peptide was replaced with two different random control peptides, the sensors produced no response to murine muscle. Suitability of peptide ligands for a variety of species can be evaluated using this technology., (Copyright 2002 John Wiley & Sons, Ltd.)

Screening with a 7-mer phage display peptide library, a panel of cell-targeting peptides for the murine microglial cell line, EOC 20, was recognized. A number of similar, but not identical, sets of sequences representing more than 75% of all the cell line-binding clones were identified. Comparative analysis indicated that motif S/(T) F T/(X) Y W is present in the vast majority of the binding sequences. The selectivity and specificity of the dominant peptide sequence identified for microglia was confirmed using both phage displaying the peptide and the synthetic peptide alone.

ASSLNIA, a peptide selected for murine myofibers using phage display technology, was immobilized onto an acoustic wave sensor. The sensor responded to murine and feline muscle homogenates indicating crosspieces interactions. Kidney, liver, and brain preparations produced insignificant responses.

Virulence mechanisms of six isolates of Mycoplasma synoviae (MS), previously classified as pathogenic (K1968), moderately pathogenic (WVU 1853, K1858, 92D8034, and F10-2AS), and mildly pathogenic (FMT) in chickens, were examined. The most virulent isolate, K1968, had been found to invade systematically and produce lesions following eye-drop inoculation. In the present study, all isolates were evaluated for presence of a possible cytadhesin and for functional attachment to host cells as indicated by hemagglutination and hemadsorption. Three representative isolates, K1968, 92D8034, and FMT, were evaluated for attachment and colonization in cultured chick tracheal rings and tendon cell monolayers by direct transmission electron microscopic examination and by quantitative polymerase chain reaction assay. Ciliostasis was compared in tracheal organ culture. Previously found differences in pathogenicity of these isolates for chickens could not be explained as differences in attachment and were only partially explained by differences in colonization. Pathogenicity of the most virulent isolate of MS was suspected to be multifactorial, involving attachment and colonization of the upper respiratory tract plus additional unidentified factors associated with systemic invasion and lesion production.

Mitochondrial (mt) DNA, isolated from different sugar beet populations, was analyzed using BamHI and EcoRI restriction enzymes. It was shown that plants possessing the new mtDNA types are revealed among O-type fertilizers quite frequently. Among cytoplasmic male sterile (cms) plants, which evolved during cultivation of O-type fertilizers, plants with altered mt genome were found.