Biomedical subjects

R K Naz

Publications and source records attributed to R K Naz.

At least 19 recordsLinked to original sources

PubMed2001

Prostate-specific genes: present status and future direction.

Prostate cancer (prostatic adenocarcinoma) is the second highest cause of cancer mortality in men and the benign prostatic hyperplasia (BPH) affects 80% of men by age 80. The current diagnosis of prostate cancer relies on the serum levels of the well-known molecule designated as prostate-specific antigen (PSA). PSA, however, has limited sensitivity and specificity in appropriately detecting the earlier stages of abnormal prostate growth. Additional molecules need to be identified that are prostate-specific and have better sensitivity and specificity that can detect prostate cancer and BPH at an earlier stage for clinical management. Presently, several laboratories are actively engaged in searching for such molecules. The aim of this article is to review the current status of various prostate genes reported in the literature that have been claimed to be prostate-specific with a function in normal and abnormal prostate growth and development. The long-term objective is to define the lacunae that exist in the literature in our search for an ideal antigen.

Gene Expression Profiling↗

PubMed2001

Molecular cloning and sequencing of a novel cDNA encoding for a protein involved in human sperm function.

A cDNA encoding for an antigen, designated as NZ-3, was cloned and sequenced from human testis. The 1481-bp NZ-3 cDNA yielded an open reading frame (ORF) of 231 amino acids (aa) with the first ATG, Met start codon at nucleotide (nt) 104 and the stop codon TGA at nt 797. Extensive computer search indicated it to be a novel cDNA/protein. The ORF of NZ-3 cDNA was subcloned into pGEX-1lambdaT vector and expressed in glutathione S-transferase gene fusion system. The expressed recombinant protein had a molecular size of approximately 25 kDa, and the rabbit antibodies (Ab) against the recombinant antigen recognized a specific protein band of 63 +/- 3 kDa in the human testis extract. The NZ-3 antigen was located on the acrosomal and tail regions of human sperm cell and the NZ-3 Ab significantly (P < 0.001) inhibited human sperm capacitation and/or acrosome reaction. The novel recombinant NZ-3 antigen may find applications in immunocontraception and in specific diagnosis of human infertility.

Amino Acid Sequence↗

PubMed2001

Novel human testis-specific cDNA: molecular cloning, expression and immunobiological effects of the recombinant protein.

A differential display-polymerase chain reaction was employed to obtain a testis-specific cDNA fragment. On screening the human testis-(lambda)gt10-cDNA library with testis-specific cDNA fragment, a novel cDNA encoding for a sperm antigen, designated TSA-1, was obtained. It has a novel open reading frame (ORF) of 471 base pairs encoding for 156 amino acids. The computer generated translated protein has a calculated molecular mass of 17.4 kDa and contains a potential N-glycosylation site at amino acids 122-124. The hydrophilicity analysis of the amino acid sequence suggested that this protein is a membrane-anchored peptide. Extensive analysis for tissue-specificity by Northern blots and RT-PCR-Southern blot procedures using various human tissues indicated that TSA-1 was specifically expressed only in the human testis. Based on the results of in vitro transcription and translation experiments, the TSA-1 (ORF) was subcloned into pGEX-6P-3 vector and expressed using the glutathione S-transferase gene fusion system. Antibodies (Ab) against the purified recombinant protein specifically recognized the approximately 17 kDa recombinant TSA-1, and a approximately 24 kDa band in human sperm extract in the Western blot procedure. The recombinant TSA-1 Ab recognized the acrosomal, equatorial, mid-piece, and tail regions of human sperm cell in indirect immunofluorescence, bound to live human sperm in the immunobeads binding technique (IBT) and caused a significant concentration-dependent inhibition of human sperm acrosome reaction. These findings indicate that the novel sperm-specific recombinant TSA-1 has a role in sperm function and may have applications in the development of a contraceptive vaccine, and in the specific diagnosis and treatment of male infertility.

Acrosome Reaction↗

PubMed2001

Cloning and sequencing of cDNA encoding for a novel human testis-specific contraceptive vaccinogen: role in immunocontraception.

Sperm-specific antigens are attractive candidates for the development of a contraceptive vaccine. Using the subtractive cDNA hybridization technology, the present study was conducted to obtain a human sperm-specific antigen. The 32P-labeled single stranded cDNA of human testis, subtracted with poly(A)+ RNA of human peripheral white blood cells, was used to screen the human testis cDNA-ZAP II library. The putative positive clones were further screened for binding with the solubilized human oocyte zona pellucida preparation (HZP). After screening 10(7) colonies, one positive clone, designated contraceptive vaccinogen (CV), was obtained. It had an insert of approximately 1.3 kb, that was cloned and sequenced. The sense strand was identified by using the in vitro transcription and translation procedures, and the full-length sequence was obtained by using the 5' rapid amplification of 5' -cDNA ends (5'-RACE) procedure. The full-length CV cDNA has an ORF of 312 amino acids (aa) with the first ATG Met start codon at nucleotide (nt) 35 and the stop codon TAA, at nt 959. The translated protein has a calculated molecular mass of 35.3 kD and four potential N-linked glycosylation and several phosphorylation sites. Hydropathy plot generated from the deduced aa sequence showed it to be a membrane-anchored peptide. Extensive computer search in the database did not find any homology of existing sequences with CV both for nt and aa. Northern blot analysis indicated the human testis-specific expression of CV antigen. The coding region of CV cDNA was subcloned into pET22b(+) vector and expressed. The expressed recombinant (r)CV protein had a molecular size of approximately 44 kD, and it specifically reacted with the ZP3 component of HZP. Rabbit rCV antibodies recognized the rCV, and a cognate antigen of approximately 64 kD in the human sperm extract. The antibodies showed binding with the live and methanol-fixed human sperm, and significantly (P < 0.001) inhibited human sperm penetration of zona-free hamster oocytes, as well as human sperm binding to human oocyte zona pellucida. These findings indicate that the testis/sperm- specific CV antigen has a role in human sperm function and may find clinical applications in the contraceptive vaccine development and in the specific diagnosis and treatment of male infertility.

Adult↗

PubMed2001

Effect of antibodies to sperm-specific recombinant contraceptive vaccinogen (rCV) on murine fertilization: search for an animal model to examine its contraceptive potential.

Recently, we cloned and sequenced a sperm-specific antigen, designated as Contraceptive Vaccinogen (rCV), from human testis (Naz et al., 2001). The present study was conducted to examine its proteomic homologue and function in murine sperm, in order to find out whether or not the mouse can provide a suitable model for examining its immunocontraceptive effects. This was examined by using purified antibodies (Ab) raised against the recombinant (r) human CV antigen of approximately 44 kD. In the Western blot procedure, rCV antibodies recognized a specific protein band of approximately 64 +/- 5 kD in murine testis and murine sperm extracts, the band similar to that found in human testis and human sperm. In the immunoprecipitation procedure, rCV Ab immunoprecipitated a protein band of similar size from murine sperm and murine testis extracts. The immunocytochemical (ICT), immunoscanning electronmicroscopic (ISEM) and the immunobead binding technique (IBT) revealed the subcellular localization of CV antigen on the surface of acrosome and tail regions of the noncapacitated and capacitated murine sperm cell. In functional bioassays, rCV Ab inhibited the acrosome reaction as well as sperm-egg binding in vitro. These data indicate that the CV antigen is expressed in murine sperm and has a biological role in sperm function and sperm-egg binding. In vitro inhibition of capacitation/acrosome reaction and sperm-zona binding suggest that the mouse can provide a suitable model to examine the immunocontraceptive effects of CV antigen in actively-immunized animals.

Animals↗

PubMed2001

Presence of antibodies to sperm YLP(12) synthetic peptide in sera and seminal plasma of immunoinfertile men.

Using an enzyme-linked immunosorbent assay (ELISA), sera (n = 15) and seminal plasma (n = 30) from antisperm antibody-positive immunoinfertile men (n = 45) and from fertile men (n = 45), were tested for the immunoreactivity with the synthetic YLP(12) sperm peptide. Of the 15 immunoinfertile sera tested, 46% were positive for immunoglobulin (Ig)M, 73% for IgG, and 40% for IgA. Of the 30 samples of immunoinfertile seminal plasma tested, 10% were positive for IgM, 20% for IgG, and 43% for IgA. None of the sera or seminal plasma from fertile men showed a positive reaction. There was no significant correlation between the sperm immobilization technique (SIT) or tray agglutination technique (TAT) titres or percentage binding in immunobead binding technique (IBT) and the antibody reactivity for any class in the ELISA. The YLP(12) peptide conjugated to bovine serum albumin-Sepharose 4B beads pulled out IgG antibodies from the serum of the immunoinfertile, but not the fertile, men. The beads pulled out IgA antibodies from the immunoinfertile, but not the fertile, seminal plasma. The immuno-affinity purified antipeptide antibodies reacted with a specific band of 72 +/- 5 kDa in the human testis and with a specific band of approximately 50 +/- 5 kDa in the human sperm extracts. The YLP(12) peptide may have applications in the specific diagnosis and treatment of male infertility and in contraceptive vaccine development.

Adult↗

PubMed2001

Inhibition of murine sperm-oolemma binding by antibodies to an oocyte membrane (OM) antigen: implication in contraceptive vaccine development.

PROBLEM: The present study was conducted to investigate the oocyte membrane protein(s) that is involved in sperm binding in the mouse, and whether or not it can be used for the development of a contraceptive vaccine. METHOD OF STUDY: The zona-free oocytes were treated with Triton X-100 and the extract was analyzed for homogeneity in the sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) after staining with silver nitrate. The appropriate band of 50+/-4 kD, designated as OM antigen, was excised from the gel and used for the immunization. The female rabbits were immunized with the excised band per se and the female mice were immunized with the OM antigen after conjugation to keyhole limpet hemocyanin (KLH). The affinity-purified antibodies were analyzed in the enzyme-linked immunosorbent assay (ELISA), immunoprecipitation procedure, western blot procedure, indirect immunofluorescence technique (IFT), and spermoolemma binding assay. Actively-immunized mice were analyzed for in vivo fertility. RESULTS: The Triton X-100 extract of zona-free oocytes predominantly showed a single protein band of 50+/-4 kD in the SDS-PAGE. Active immunization of female rabbits and of female mice with OM antigen raised high antibody titers (ELISA titer > 1:4096) that specifically recognized the OM antigen in the immunoprecipitation and Western blot procedures, and reacted with the oocyte in the IFT. These antibodies demonstrated a significant (P < 0.05) up to a complete block of sperm-oolemma binding in the in vitro binding assay. Binding of both the acrosome-intact and acrosome-reacted sperm was inhibited. Mice actively immunized with OM antigen also showed a significant reduction in vivo fertility as seen by the 9-day implants in uteri. Preliminary data indicate that the antibodies to OM antigen were tissue-specific and did not react with the specific band in any tissue extract in the western blot procedure. CONCLUSIONS: These results indicate that the OM antigen is involved in spermoolemma binding and constitute an attractive molecule that needs further investigation for examining its utility in the contraceptive vaccine development.

Animals↗

PubMed2000

Expression of alpha and gamma interferon receptors in the sperm cell.

The present study was conducted to examine whether or not the sperm cell has the expression of receptors for interferon (IFN) -alpha and -gamma. This was investigated using specific antibodies. Antibody to IFN-alpha receptor reacted with the acrosomal and tail regions of the murine sperm cell in the indirect immunofluorescence technique (IFT) and immunoscanning electron microscopic procedure (ISEP). In the immunoprecipitation and Western blot procedures, this antibody specifically recognized a protein band of approximately 100 kD, which corresponds to the molecular weight of IFN-alpha receptor present in other cell types. Antibody to IFN-gamma receptor specifically reacted with the posterior head, midpiece, and tail regions of sperm cell in IFT and ISEP, and recognized a band of approximately 85 kD in the immunoprecipitation and Western blot procedures, corresponding to the IFN-alpha receptor. Similar bands of approximately 100 kD and approximately 85 kD molecular identities were also detected in the testes extracts and sperm extracts of other mammalian species namely human, rabbit, and pig, the species tested. These findings indicate that the mammalian sperm cell has expression of IFN-alpha and IFN-gamma receptors, which seem to develop during spermatogenesis in the testes. These findings may have implications in male infertility and antisperm contraceptive vaccine development.

Animals↗

PubMed2000

Identification of human sperm peptide sequence involved in egg binding for immunocontraception.

Development of a vaccine based on sperm antigens represents a promising approach to contraception. The sperm-zona pellucida (ZP) interaction constitutes the most important event in the fertilization process, and the molecular sequences involved at this site may provide the most attractive candidates for immunocontraception. In the present study, using the phase peptide display technique, a novel dodecamer sequence, designated as YLP(12), was identified that is involved in sperm-ZP recognition/binding. The synthetic 12-mer peptide based on this sequence and its monovalent Fab' antibodies specifically and significantly (P < 0.05) inhibited human sperm-ZP binding. In Western blot and immunoprecipitation procedures, the YLP(12) peptide recognized the ZP3 component of solubilized human ZP proteins. In the Western blot procedure involving 10 different human tissue extracts, the anti-YLP(12) Fab' antibodies recognized a protein band of approximately 72 +/- 2 kDa only in the testis lane. The peptide sequence was localized on the acrosomal region of the human sperm cell. These findings indicate that the novel testis-specific 12-mer YLP(12) that is present in the acrosomal region and is involved in human sperm-ZP interaction may find applications in contraceptive vaccine development, as well as in diagnosis and treatment of male infertility mediated through sperm dysfunction.

Antibodies↗

PubMed2000

Fertilization-related sperm antigens and their immunocontraceptive potentials.

Vaccine(s) for contraception will provide an attractive alternative to contraception. There has been a renewed interest in defining fertilization-related sperm antigens that can be used for the development of a contraceptive vaccine. The sperm-zona pellucida (ZP) recognition and binding constitutes the most important event in the fertilization process, and the molecules involved at this site are attractive candidates for immunocontraception. Extensive research indicates that there are four sperm surface proteins that bind to oocyte ZP3 in humans and they belong to the four molecular regions of 95, 63, 51, and 14-18 kDa, respectively. Our laboratory is actively engaged in cloning and sequencing of cDNAs encoding for these sperm proteins and investigating the applications of recombinant (r) proteins in immunocontraception and infertility. Presently, the cDNAs encoding for the 14-18-kDa proteins, designated the NZ-1 and NZ-2, respectively, and the 51-kDa protein, designated fertilization antigen, have been cloned and sequenced. Active immunization with the rFA-1 antigen caused a reversible block/inhibition in fertility of female mice by raising a sperm/testis-specific immune response. FA-1 antigen is also involved in human immunoinfertility, and a recent clinical trial indicates that it will have a clinical application in the treatment of immunoinfertile men. The long-term goal is to employ various recombinant fertilization antigens and/or their bioeffective peptide epitopes in a single formulation, to generate an anti-sperm vaccine that will be strongly immunogenic and highly efficacious for regulation of fertility.

Animals↗

PubMed2000

Immunobiologic implication of RANTES in seminal plasma of fertile, infertile and immunoinfertile men.

PROBLEM: To investigate the presence of Regulated on Activation, Normal T Expressed and Secreted (RANTES) in the seminal plasma of fertile, infertile, and immunoinfertile men in order to determine whether it has any role in fertility/infertility and in the modulation of immunogenicity of the sperm cell. METHOD OF STUDY: The enzyme-linked immunosorbent assay was performed by using a commercially available immunoassay kit to measure RANTES levels in seminal plasma of fertile (n = 28), male-factor infertile (n = 35), and immunoinfertile (n = 21) men. The levels were correlated with various seminal parameters. RESULTS: RANTES was detected in the seminal plasma of fertile, male-factor infertile, and immunoinfertile men. Its levels differed significantly (P<0.05) between the fertile and immunoinfertile groups, with the immunoinfertile group showing an overall decrease of 19.9% in mean values. When RANTES levels were correlated with sperm concentration, total sperm number, and motility, only the total number of the immunoinfertile group correlated (r = 0.86) significantly (P = 0.03) with the RANTES levels, and none of the other seminal parameters of any other group correlated (r = -0.37-0.30) significantly (P>0.05). CONCLUSIONS: The findings of this study indicate that RANTES may have a role in immunomodulation of antigenicity of sperm cells in the male genital tract before ejaculation, and perhaps also in the female genital tract after intercourse. The exact physiologic, pathophysiologic, and molecular mechanism(s) involved in these processes require further study.

Case-Control Studies↗

PubMed1999

Comparison of ZP3 protein sequences among vertebrate species: to obtain a consensus sequence for immunocontraception.

The deduced ZP3 amino acid (aa) sequences of 13 vertebrate species namely mouse, hamster, rabbit, pig, porcine, cow, dog, cat, human, bonnet, marmoset, carp, and frog were compared using the PILEUP and PRETTY alignment programs (GCG, Wisconsin, USA). The published aa sequences obtained from 13 vertebrate species indicated the overall evolutionarily conservation in the N-terminus, central region, and C-terminus of the ZP3 polypeptide. More variations of ZP3 polypeptide sequences were seen in the alignments of carp and frog from the 11 mammalian species making the leader sequence more prominent. The canonical furin proteolytic processing signal at the C-terminus was found in all the ZP3 polypeptide sequences except of carp and frog. In the central region, the ZP3 deduced aa sequences of all the 13 vertebrate species aligned well, and six relatively conserved sequences were found. There are 11 conserved cysteine residues in the central region across all species including carp and frog, indicating that these residues have longer evolutionary history. The ZP3 aa sequence similarities were examined using the GAP program (GCG). The highest aa similarities are observed between the members of the same order within the class mammalia, and also (95.4%) between pig (ungulata) and rabbit (lagomorpha). The deduced ZP3 aa sequences per se may not be enough to build a phylogenetic tree.

Amino Acid Sequence↗

PubMed1999

Fertilization antigen-1 removes antisperm autoantibodies from spermatozoa of infertile men and results in increased rates of acrosome reaction.

OBJECTIVE: To determine if fertilization antigen (FA)-1 will remove autoantibodies from the surface of sperm cells of immunoinfertile men by immune adsorption and permit an increased acrosome reaction (AR). DESIGN: Prospective analytic study. SETTING: University medical center. PATIENT(S): Men from 18 infertile couples with autoantibodies present on their spermatozoa. INTERVENTION(S): Sperm samples after processing were examined for antibody binding and AR before and after adsorption with control medium or FA-1. MAIN OUTCOME MEASURE(S): Sperm-bound antibody was assessed by the immunobead assay (immunoglobulin [Ig] A and IgG) and the AR by induction with ionophore A23187. RESULT(S): Adsorption with FA-1 compared with control medium increased immunobead-free swimming sperm an average of 50% and 76% for IgA and IgG antisperm antibodies, respectively, with 78% and 100% of the 18 semen specimens increasing significantly. The AR rate increased an average of 10.3% compared with control medium and showed improvement in 78% of the sperm samples after FA-1 adsorption. CONCLUSION(S): The FA-1 sperm antigen appears to significantly free sperm cells coated with autoantibodies in the semen of most infertile men examined. Reducing sperm-bound antibodies that inhibited the AR allowed the sperm cells to undergo successful AR induction by calcium ionophore.

Acrosome Reaction↗

PubMed1999

Involvement of protein serine and threonine phosphorylation in human sperm capacitation.

The involvement of serine and threonine phosphorylation in human sperm capacitation was investigated. Anti-phosphoserine monoclonal antibody (mAb) recognized six protein bands in the 43-55-kDa, 94 +/- 2-kDa, 110-kDa, and 190-kDa molecular regions, in addition to a faint band each in the 18-kDa and 35-kDa regions. Anti-phosphothreonine mAb recognized protein bands in six similar regions, except that the 18-kDa, 35-kDa, and 94 +/- 2-kDa protein bands were sharper and thicker, and an additional band was observed in the 110-kDa molecular region. In the 43-55-kDa molecular region, there was a well-characterized glycoprotein, designated fertilization antigen, that showed a further increase in serine/threonine phosphorylation after exposure to solubilized human zona pellucida. In a cell-free in vitro kinase assay carried out on beads or in solution, four to eight proteins belonging to similar molecular regions, namely 20 +/- 2 kDa, 43-55 kDa, 94 +/- 2 kDa, and 110 +/- 10 kDa, as well as in 80 +/- 4 and 210 +/- 10 kDa regions, were phosphorylated at dual residues (serine/tyrosine and threonine/tyrosine). Capacitation increased the intensity of serine/threonine phosphorylation per sperm cell, increased the number of sperm cells that were phosphorylated, and induced a subcellular shift in the serine/threonine-specific fluorescence. These findings indicate that protein serine/threonine phosphorylation is involved and may have a physiological role in sperm capacitation.

Antibodies, Monoclonal↗

PubMed1999

Vaccine for contraception targeting sperm.

Development of a vaccine(s) based on sperm antigens represents a promising approach to contraception. The utility of a sperm antigen in immunocontraception is contingent upon its tissue specificity, involvement in fertility and on raising high antibody titer, especially locally in the genital tract, that is capable of inducing reversible infertility. Several sperm antigens, such as lactate dehydrogenase C4, PH-20, sperm protein (SP)-10, fertilization antigen (FA)-1, FA-2, cleavage signal (CS)-1, NZ-1, and NZ-2 have been proposed as potential candidates for the vaccine development. Spermzona pellucida (ZP) binding is a pivotal tissue- and mostly species-specific event in the fertilization process, and the molecules involved in this site constitute the most exciting candidates for immuno-contraception. FA-1 is a sperm-specific glycoprotein having receptor activity for ZP recognition and binding. Complementary DNA encoding for FA-1 antigen has been cloned and sequenced. Active immunization of animals with recombinant FA-1 antigen causes a long-lasting reversible inhibition in fertility by raising a sperm-specific immune response. This antigen is also involved in human immunoinfertility. The exciting findings from the recent trial in immunoinfertile couples indicate that the FA-1 antigen may have clinical application in the treatment of male infertility. A vaccine having most appropriate tissue-specific and effective recombinant and/or synthetic epitopes of various sperm antigens, such as the FA-1 antigen, in a single formulation may provide a highly immunogenic and efficacious antisperm vaccine for contraception. The advances made during the last 5 years suggest that it may be a realistic proposition.

Animals↗

PubMed1998

The role of carbohydrates in mammalian sperm-egg interactions: how important are carbohydrate epitopes?

Evidence implicating the involvement of carbohydrates in fertilization has been reported for decades in species which span the phylogenetic scale. The exact nature and role of these ligands in fertilization, however, has eluded investigators. Here, such investigations are reviewed as they relate to mammalian fertilization, with the principle focus on reviewing the role of carbohydrates involved in the primary binding event between sperm cell and egg.

Animals↗

PubMed1998

Expression of a novel isoform of cyclin I in human testis.

A new isoform of cyclin I, designated cyclin ITI, was cloned from the human testis cDNA-lambda gt10 library by using a approximately 0.5 kb PCR fragment that was obtained by using primers based on the published cyclin I sequence from the human brain. The cyclin ITI cDNA is 1443-bp long and has an ORF of 178 aa with the first ATG Met start codon at nt 1 and has the well-conserved sequence of cyclin box. The 5' flanking sequence of 728-bp has a high similarity (99.6%) with the 5'-intron of the human beta-polymerase gene. The nt sequence of cyclin ITI cDNA has high similarity (99.3%) with the cyclin I gene sequence. The cyclin ITI was found to have three amino acids mutations at nt 25, 172, 223, respectively, including one in the well-conserved sequence of cyclin box. Northern blot analysis indicated that the cyclin ITI is expressed in human testis at increased level compared to other tissues. The in vitro translated protein of the cyclin ITI cDNA was recognized specifically by the antibodies raised against the human sperm proteins, indicating its presence in human sperm. This new isoform may have a physiological role in spermatogenesis and/or human sperm cell function--especially in capacitation and/or acrosome reaction.

Amino Acid Sequence↗

PubMed1998

Testis-specific proteins and their role in contraceptive vaccine development.

Development of a vaccine(s) based on sperm antigens represents a promising approach for contraception. The utility of an antigen in immunocontraception is contingent upon its testis/sperm specificity and involvement in spermatogenesis and/or fertilization. The aim of the present article is to review the information regarding the proteins that have been reported to be testis/sperm-specific and may have an important function in spermatogenesis and/or fertilization. The potential role of these proteins in the development an antisperm contraceptive vaccine(s) is discussed.

Contraception, Immunologic↗