Evaluation of the efficacy of immunocastration vaccine composed of gonadotrophin-releasing hormone conjugated with Salmonella typhimurium flagellin in rats.

Immunocastration is an alternative method to replace surgical castration that is commonly performed in domestic and pet animals. In this study, a new immunocastration vaccine was developed, and its efficacy was evaluated in male rats. Six tandem copies of gonadotrophin-releasing hormone (GnRH) peptide were genetically fused to Salmonella typhimurium flagellin fljB (STF2) that is a ligand of toll-like receptor 5 (TLR5). The recombinant STF2-GnRH protein expressed in Escherichia coli was used as the immunocastration vaccine. Sixteen male rats were equally assigned to four groups. Excluding the control rats, three groups were immunized with 100, 200 and 400 µg of the STF2-GnRH vaccine, respectively. All of the immunized rats developed significantly higher titres of antibodies to GnRH than the control rats. The size and weight of both testes and epididymides from the immunized rats were significantly smaller than those of the control rats. Testicular tissues in the immunized rats demonstrated atrophy of seminiferous tubules and decreased numbers of both spermatogonia and spermatocytes. These data indicate that the newly developed STF2-GnRH vaccine has a potent immunogenicity to GnRH and efficiently suppresses the development of testes in rats.

Characterization of low-pathogenicity H5 and H7 Korean avian influenza viruses in chickens.

To date, all isolated highly pathogenic avian influenza (HPAI) viruses that cause systemic infection with a high mortality rate in poultry species have been known to belong to either the H5 or H7 subtypes. The HPAI viruses may originate because of the insertion of multiple basic amino acids at the cleavage site of the hemagglutinin protein after the low-pathogenic H5 and H7 viruses have been introduced into poultry. In the present study, we investigated the phylogenetic characteristics of the H5 (n = 4) and H7 (n = 3) low-pathogenic avian influenza (LPAI) viruses isolated from wild birds in Korea by using nucleotide sequences of all 8 gene segments of the viral genome. Further, we evaluated the infectivity, transmissibility, and pathogenic potential of these viruses in chickens. Phylogenetic analysis showed that all viruses used in the study clustered in the Eurasian lineage and were similar to the viruses isolated in Asian countries that share the East Asian-Australasian migratory bird flyway. Our H5N2 isolates could not be replicated and transmitted in chickens, but the H7N8 isolates could efficiently be replicated and transmitted to contact-exposure chickens. In addition, because our H7N8 isolates caused watery diarrhea in chickens, these viruses cannot only serve as progenitors of novel HPAI strains but also potentially cause clinical disease in poultry. Although there have been no reports of LPAI mutation to HPAI in these regions, the wild bird surveillance effort should focus on monitoring the introduction and transmission of the HPAI H5N1 and LPAI H5 and H7 viruses.

Anti-influenza virus activity of green tea by-products in vitro and efficacy against influenza virus infection in chickens.

Polyphenolic compounds present in green tea, particularly catechins, are known to have strong anti-influenza activity. The goal of this study was to determine whether green tea by-products could function as an alternative to common antivirals in animals compared to original green tea. Inhibition of viral cytopathic effects ascertained by neutral red dye uptake was examined with 50% effective (virus-inhibitory) concentrations (EC50)determined. Against the H1N1 virus A/NWS/33, we found the anti-influenza activity of green tea by-products (EC50 = 6.36 µg/mL) to be equivalent to that of original green tea (EC50= 6.72 µg/mL). The anti-influenza activity of green tea by-products was further examined in mouse and chicken influenza infection models. In mice, oral administration of green tea by-products reduced viral titers in the lungs in the early phase of infection, but they could not protect these animals from disease and death. In contrast, therapeutic administration of green tea by-products via feed or water supplement resulted in a dose-dependent significant antiviral effect in chickens, with a dose of 10 g/kg of feed being the most effective (P < 0.001). We also demonstrated that unidentified hexane-soluble fractions of green tea by-products possessed strong anti-influenza activity, in addition to ethyl acetate-soluble fractions, including catechins. This study revealed green tea by-product extracts to be a promising novel antiviral resource for animals.

Live attenuated nephropathogenic infectious bronchitis virus vaccine provides broad cross protection against new variant strains.

Infectious bronchitis virus (IBV) infections cause great economic losses to the poultry industry worldwide, and the emergence of new variant strains complicates disease control. The present study investigated the genetic and protectotypic features of newly emerged Korean IBV strains. A phylogenetic analysis showed that several recent isolates formed 2 different clusters (new cluster 1 and 2), which were distinct from other preexisting clusters. New cluster 1 IBV strains represented recombinants between Korean nephropathogenic strain KM91 and the QXIBV strain. New cluster 2 IBV strains showed low amino acid homology (<58.7%) compared with previous isolates. We evaluated the protective efficacy of commercial IBV vaccines (H120 and K2 strain) against these new isolates. In cross-protection studies, the H120 strain did not provide sufficient protection against these variants. However, highly attenuated nephropathogenic IBV vaccine, K2 strain, provided significantly higher levels of protection against variants compared with chickens vaccinated with H120 (P < 0.05 or better). These results indicate that the K2 vaccine could be helpful for the reduction of economic losses caused by newly evolving IBV recombinants (new cluster 1) and variants (new cluster 2).

Outbreak of gizzard erosion associated with fowl adenovirus infection in Korea.

The pathogenicity of a fowl adenovirus serotype-1 (FAdV-1, K181 strain) isolated from a case of gizzard erosion in layer chickens was investigated in specific-pathogen-free (SPF) chicks. One-week-old SPF chicks were inoculated orally or intramuscularly with the isolate of FAdV-1 and euthanized for necropsy at 7, 14, and 21 d postinoculation. Although there were no clinical signs after inoculation, gizzard erosions were observed grossly and the virus was recovered from the gizzards in the inoculated chickens. Histologically, in the chickens that were infected orally, the lesions found in the gizzard consisted of severe degeneration and necrosis of glandular epitheliums and eosinophilic inclusion bodies. These results indicate that the Korean FAdV-1 isolate could induce gizzard lesions in chickens. Moreover, the present investigation reproduced an outbreak of gizzard erosion caused by FAdV-1 infection and, for the first time, described the isolation of FAdV-1 from chickens in Korea. These findings provide important information on the epidemiology and pathogenesis of FAdV-1 infection in chickens.

Effect of intranasal administration of Lactobacillus fermentum CJL-112 on horizontal transmission of influenza virus in chickens.

The aim of this study was to determine whether intranasal administration of Lactobacillus sp. could prevent horizontal transmission of H9N2 avian influenza virus (AIV) in specific-pathogen-free chickens. Three-week-old chickens received 500 µL of 1.5 × 10(9) cfu of Lactobacillus fermentum CJL-112 strain (CJL) intranasally for 7 d before and 14 d after a challenge. Challenged chickens, each inoculated with H9N2 AIV, were kept in either direct or indirect contact with naive chickens, and morbidity and viral shedding were monitored. We demonstrated that the intranasal administration of CJL significantly decreased the number of chickens with viral shedding from the gastrointestinal tract in the indirect contact chickens (P < 0.001) and also significantly reduced viral shedding from the respiratory tract in the challenged (P < 0.05) and the direct contact chickens (P < 0.001) than those in the control group. Hence, the use of this lactobacilli strain may constitute a novel and effectively plausible alternative to prevent and control H9N2 AIV infection in chickens.

Prevalence and antimicrobial resistance of Salmonella species isolated from chicken meats produced by different integrated broiler operations in Korea.

Prevalence and antimicrobial resistance profiles of Salmonella serotypes isolated from 7 chicken meat brands produced by different integrated broiler operations in Korea were determined. In total, 210 samples were collected from retail supermarkets in Seoul, South Korea, and analyzed for the presence of Salmonella. Of 210 chicken meat samples, overall Salmonella prevalence was 22.4%. Salmonella Enteritidis was the dominant serovar, with an isolation rate of 57.4% from the Salmonella-positive chickens, followed by Salmonella Montevideo. Salmonella isolates frequently were resistant to various antibiotics, including 100% to erythromycin, 87% to cephalothin, 85% to nalidixic acid, and 70% to streptomycin. Of the 47 isolates, 41 (87.2%) isolates were resistant to 3 or more antibiotics. Moreover, the Salmonella profiles of each chicken meat brand were different by broiler operation. Brand A showed the highest prevalence of Salmonella (18 isolates, 60%), whereas brand G showed the lowest prevalence (one isolate, 3.3%). Eight among the 18 isolates of brand A were resistant to 11 antibiotics, whereas 5 of the 6 brand C isolates were resistant to only 2 antibiotics. This study demonstrates that a high proportion of chicken meat in Korea is contaminated with Salmonella and the prevalence and antimicrobial resistance patterns of Salmonella of chicken meat differ significantly according to the integrated broiler operation.

Inactivated H9N2 avian influenza virus vaccine with gel-primed and mineral oil-boosted regimen could produce improved immune response in broiler breeders.

The frequent economic losses incurred with H9N2 low pathogenic avian influenza viruses (LPAI) infection have raised serious concerns for the poultry industry. A 1-dose regimen with inactivated H9N2 LPAI vaccine could not prevent vaccinated poultry from becoming infected and from shedding wild viruses. A study was conducted to determine whether a 2-dose regimen of inactivated H9N2 LPAI vaccine could enhance the immunologic response in chickens. Such gel-primed and mineral oil-boosted regimen has produced encouraging results associated with improved immune responses to an H9N2 LPAI. This strategy could be cost effective and helpful for preventing avian influenza virus in the poultry industry.

The influence of postnatal development on drug-induced hepatic porphyria and the synthesis of cytochrome P-450. A biochemical and morphological study.

The mitochondrial enzyme delta-aminolevulinate synthetase (ALAS) controls the rate-limiting step in the synthesis of porphyrins and heme. An experimental form of hepatic porphyria can be readily elicited in laboratory animals, such as the rat, by drugs and foreign chemicals which are known to enhance the de novo formation of this enzyme in the liver. The present study shows that there is a striking refractoriness to the induction of ALAS during the perinatal period in the rat. Chemicals which have potent porphyria-inducing activity in adult animals have no significant inducing effect on hepatic ALAS in neonates. The ultrastructural changes which accompany the induction of ALAS by drugs and chemicals in adult liver also fail to take place in the livers of neonates. A progressive capacity for responding to the action of chemical inducers of hepatic ALAS does, however, develop in neonatal animals so that by approximately 5-6 wk of age experimental porphyria can be elicited as effectively in them as in adults. The reasons for the refractoriness of hepatic ALAS to induction in the perinatal period are not known; but the findings of this study make it clear that ALAS belongs to that increasingly large group of liver enzymes in mammals whose appearance, increase of activity, or inducibility is developmentally determined. The occurrence of developmental changes in the indicibility of ALAS in the liver of neonates also provided an opportunity to study the relationship of this enzyme activity to the drug-mediated induction of the hepatic hemoprotein cytochrome P-450. This inducible hemoprotein serves as the terminal oxygenase in the microsomal mixed-function oxidase system in the liver. The results of this study indicate that, in contrast to the refractoriness of ALAS to induction, significant drug-induced changes of hepatic P-450 content and of hemeprecursor incorporation into this cytochrome do take place in neonates. The synthesis of P-450 thus appears to be under a regulatory control different from that of ALAS in neonates, and the relation between ALAS activity and P-450 formation is not therefore a direct one.

Plasma membranes of the rat liver. Isolation and enzymatic characterization of a fraction rich in bile canaliculi.

A method is described for the rapid isolation of a plasma membrane fraction containing a high concentration of intact bile canaliculi from the rat liver. Isolated bile canaliculi retain most of the ultrastructural features exhibited in the intact liver cell. The final fraction contains 5'-nucleotidase activity at approximately the same concentration as that in previous preparations of plasma membranes. In the presence of 0.01 M Mg(++), 5'-nucleotidase exhibits a double pH optimum at pH values of 7.5 and 9.5. The activities of glucose-6-phosphatase and alkaline phosphatase are present in low amounts. Cytochrome P-450 is not detectable. Na(+)-K(+)-activation of ATPase is observed to the extent of 20-36% in about half of the assays. The availability of a method for preparation of intact bile canaliculi should prove useful for studying the biochemical events associated with the transport of bile constituents into canaliculi.

Niacin Reduces Paraquat Toxicity in Rats.

Rats poisoned with paraquat benefited from daily niacin therapy. Niacin-treated rats showed delayed and reduced dyspnea. Deaths began approximately 30 hours later. The time required for niacin-treated rats to reach 50 percent mortality increased from 60 to 120 hours, and the dealth rate was reduced form 75 to 55 percent. The benefit by niacin is consistent with the demonstrated role of niacin in preventing cellular decreases of nicotinamide adenine dinucleotide during poisoning of bacteria by paraquat and by hyperbaric oxygen.

Limited pathogenicity and transmissibility of Korean highly pathogenic avian influenza H5N6 clade 2.3.4.4 in ferrets.

The pathogenicity and transmissibility of a reassortant clade 2.3.4.4 avian influenza A (H5N6) virus were evaluated in ferrets. Virus excretion was detected in the upper respiratory tract, but the ferrets did not show any clinical signs of infection. Transmission did not occur between cohoused or respiratory droplet-contact ferrets.

Experimental infection of H5N1 and H5N8 highly pathogenic avian influenza viruses in Northern Pintail (Anas acuta).

The wide geographic spread of Eurasian Goose/Guangdong lineage highly pathogenic avian influenza (HPAI) clade 2.3.4.4 viruses by wild birds is of great concern. In December 2014, an H5N8 HPAI clade 2.3.4.4 Group A (2.3.4.4A) virus was introduced to North America. Long-distance migratory wild aquatic birds between East Asia and North America, such as Northern Pintail (Anas acuta), were strongly suspected of being a source of intercontinental transmission. In this study, we evaluated the pathogenicity, infectivity and transmissibility of an H5N8 HPAI clade 2.3.4.4A virus in Northern Pintails and compared the results to that of an H5N1 HPAI clade 2.3.2.1 virus. All of Northern Pintails infected with either H5N1 or H5N8 virus lacked clinical signs and mortality, but the H5N8 clade 2.3.4.4 virus was more efficient at replicating within and transmitting between Northern Pintails than the H5N1 clade 2.3.2.1 virus. The H5N8-infected birds shed high titre of viruses from oropharynx and cloaca, which in the field supported virus transmission and spread. This study highlights the role of wild waterfowl in the intercontinental spread of some HPAI viruses. Migratory aquatic birds should be carefully monitored for the early detection of H5 clade 2.3.4.4 and other HPAI viruses.

The influence of pyrogen-induced fever on salicylamide metabolism in man.

Salicylamide is metabolized in man by biotransformation to salicylamide glucuronide, salicylamide sulfate, and gentisamide glucuronide. The metabolites are quantitatively and rapidly excreted in urine. Study of the metabolism of this drug in volunteers during episodes of pyrogen-induced fever shows a significant reduction in the half-life (t(1/2)) of the excretion of the drug metabolites. The proportion of the drug transformed to its major metabolite, salicylamide glucuronide, is significantly reduced by fever, with concomitant increase in the proportion of one or both of the other metabolites. Thus, the pattern of urinary metabolites of salicylamide is altered. The shortened t(1/2) of the metabolite excretion is probably due to increased hepatic and renal blood flow known to accompany pyrogen-induced fever. This concept was supported by the observation that when two subjects were placed in a high-temperature environmental chamber, a condition in which hepatic and renal blood flows are known to diminish, the t(1/2) of salicylamide metabolite excretion actually increased. No simple explanation exists to explain the changed metabolite pattern noted during febrile periods. It is most likely to be due to complex interactions between the direct or indirect effects of the pyrogens and the factors affecting the hepatic biotransformation of drugs.

Effects of homologous and heterologous neuraminidase vaccines in chickens against H5N1 highly pathogenic avian influenza.

The 2004 Asian H5N1 epizootic outbreak indicates the urgent need for vaccines against highly pathogenic avian influenza (HPAI) virus. The manufacture of inactivated whole-virus vaccines from HPAI viruses by traditional methods is not feasible for safety reasons as well as technical issues. The low pathogenic avian influenza A/wild bird feces/CSM2/02 (H5N3) virus was used as a heterologous neuraminidase vaccine, and HPAI A/CK/Korea/ES/03 (H5N1) virus was used as a homologous neuraminidase vaccine. Protection efficacy of both vaccines was evaluated by clinical signs, mortality rates, and virus shedding from oropharynx and cloaca of vaccinated chickens after challenge with HPAI A/CK/Korea/ES/03 (H5N1) virus. One dose of 128 hemagglutinin (HA) homologous H5N1 vaccine induced 100% protection in mortality and prevented viral shedding completely after lethal dose virus challenge, whereas one dose of 64 HA unit of heterologous H5N3 vaccine only induced 50% protection in mortality, and it did not prevent viral shedding. However, two doses at a 3-wk interval of 64 HA unit of heterologous H5N3 vaccine as well as one dose of 1024 HA unit of heterologous H5N3 vaccine induced 100% survival rate and could prevent viral shedding completely. Furthermore, we could differentiate the sera of infected birds from those of vaccinated birds by indirect immunofluorescent antibody test. These results suggest that heterologous neuraminidase H5N3 vaccine could be a useful tool for the control of H5N1 HPAI epidemic in poultry.

Increased efficacy of inactivated vaccine candidates prepared with Salmonella enterica serovar Typhimurium strains of predominant genotypes in ducks.

Salmonella enterica serovar Typhimurium has been a major causative agent of food-borne human disease, mainly due to consumption of contaminated food animal products. In particular, ducks serve as a reservoir of serovar Typhimurium, and are one of the common sources of human infection. To prevent infection of ducks, and therefore minimize human infection, it is critical to control the persistent epidemic strains in ducks. Here, we analyzed the genetic diversity and virulence of serovar Typhimurium isolates from ducks in Korea to identify the predominant strains that might be used as efficient vaccine candidates for ducks. Among the isolates, 2 representative isolates (ST26 and ST76) of predominant genotypes were selected as vaccine strains on the basis of genotypic analysis by pulsed-field gel electrophoresis and DNA microarrays. Two-week-old ducks were then injected intramuscularly with inactivated vaccine candidates prepared using ST26 or ST76 (10(8) cfu/0.5 mL/duck or 10(9) cfu/0.5 mL/duck), and oral challenge with a highly virulent serovar Typhimurium strain (10(9) cfu/0.5 mL/duck) was carried out 2 wk later. Shedding of the challenge strain was significantly decreased in group 2 after vaccination. The antibody levels by enzyme-linked immunosorbent assay in all vaccinated groups were enhanced significantly (P < 0.05) compared to the unvaccinated control group. Overall, vaccination with ST26 or ST76 reduced bacterial shedding and colonization in internal organs, and induced elevated antibody response. In particular, serovar Typhimurium ST26 (10(8) cfu/0.5 mL/duck) was the most effective vaccine candidate, which can provide efficient protection against serovar Typhimurium in ducks with higher effectiveness compared to a commercial vaccine currently used worldwide.

Molecular cloning and sequence analysis of the rat liver carnitine octanoyltransferase cDNA, its natural gene and the gene promoter.

The full-length cDNA and the natural gene for rat peroxisomal carnitine octanoyltransferase (COT) have been isolated and sequenced. The 2681 bp long cDNA contains an open reading frame for 613 amino acids, resulting in a protein with a deduced molecular weight of 70,301, and a C-terminal peroxisomal targeting sequence (Ala-His-Leu). The isolated COT cDNA has 51 bp of the 5' untranslated region (UTR), 791 bp of 3' UTR, two putative polyadenylation sites, and a poly(A19-23) tail. Screening of a rat genomic DNA library in the lambda phage with the COT cDNA probe resulted in the isolation of seven overlapping clones, together containing the complete COT gene with seventeen exons. All of the exon-intron boundary sequences conform to the GT-AG rule. The COT gene appears to spread over 40 to 60 kbp region of the rat genome. The transcription initiation site of the COT gene was determined through primer extension, and the promoter sequence up to the position -1140 was established. The promoter lacks the canonical TATA box and a promoter-reporter construct containing the sequence encompassing -1140 to +84 base positions and the firefly luciferase reporter cDNA yielded about 100-fold increase in promoter activity in transfected hepatoma cells. Some of the consensus sequences for putative cis elements present in the promoter sequence are: the two CCAAT motifs for CTF/NF1/CBP binding (at -284 and -93), two GC boxes for Sp1 binding (at -160 and -68), two AP2 sites (at -359 and -25), a half site (TGACCT) for the peroxisome proliferator activated receptor (PPAR) binding at -737 within a partial palindromic sequence region. Potential regulatory elements, such as several palindromes and repeat motifs for five different sequence segments, are also identified.

Negative regulation of the androgen receptor gene promoter by NFI and an adjacently located multiprotein-binding site.

The upstream promoter of the rat androgen receptor (AR) gene contains a strong negative regulatory region located at the -388 to -340 nucleotide position. The distal part (-388/-373) of this regulatory region binds NFI, a ubiquitous transcription factor, while the proximal portion (-372/-340) contains an overlapping binding site for two nuclear proteins. This composite regulatory region (-388/-340) was initially defined by deoxyribonuclease I footprinting as the continuous stretch of a nuclease-protected site. NFI specificity of the distal portion (-388/-373) of the footprint was established through cross-competition in electrophoretic mobility shift assay (EMSA) using the well characterized NFI element of the adenovirus major late promoter and by immunoreactivity to the NFI antibody. EMSA with oligonucleotide duplexes corresponding to the proximal domain (-372/-340) indicated multiple retarded bands with at least two major DNA-protein complexes. Further analysis with truncated oligonucleotide duplexes showed that these two major proteins bind to this domain in an overlapping manner. Within this overlapping area, the position spanning -359 to -347 is essential for the formation of either of these two complexes. Substitution of four G with T residues in the overlapping area totally abolished all protein binding at the downstream -372/-340 site. Point mutations that abolish specific binding at either the NFI or immediately downstream multiprotein-binding site caused about a 10-fold increase in AR promoter activity in transfected HepG2 cells. Double mutation involving both the NFI and proximal overlapping protein-binding sites failed to cause any additional increase in promoter function. From these results we conclude that the AR promoter contains a composite negative regulatory region at -388/-340, and the repressor function may involve a coordinate interaction between NFI and at least two other nuclear factors.

Catalytic cleavage of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme.

Androgen receptor (AR) plays a key role in cell growth both in the normal prostate and in prostate cancer. Androgen ablation and prolonged antiandrogen therapy can give rise to AR-dependent prostate tumors, which nonetheless can grow in the androgen-deprived milieu. Here we describe the ribozyme approach to selectively degrading the AR mRNA and thereby inhibiting AR function. A trans-acting hammerhead ribozyme was designed to cleave the rat AR mRNA at the position +1827/ 1828, a region predicted to be minimally involved in generating stable secondary structures. Using AR mRNA fragments as substrates, it was established that this ribozyme can specifically cleave the RNA target in a sequence-specific manner. Kinetic experiments determined a Km for the substrate of 77 nM and a kcat/Km value of 1.8 x 10(7) M(-1) x min(-1), suggesting a catalytic efficiency similar to that of protein enzymes such as the relatively nonspecific ribonuclease A and a sequence-specific endonuclease EcoRI. Transient cotransfections of prostate-derived PC3 cells with three plasmids, an AR-inducible chloramphenicol acetyltransferase (CAT) reporter, an AR expression vector, and a ribozyme expression vector, showed that the ribozyme was capable of reducing the functional activity of AR. At an equimolar ratio of the AR expression plasmid to ribozyme expression plasmid, androgen-inducible CAT activity was inhibited 70%. Similar extents of inhibition were also observed at the cellular mRNA level using ribonuclease protection assays, indicating that the ribozyme functioned as an AR mRNA cleaving enzyme in cellulo. Immunocytochemical examination revealed a decline of AR immunoreactivity in ribozyme-transfected cells. In addition, no morphologically detectable cellular abnormalities were associated with ribozyme expression, indicating the absence of deleterious side effects. These results offer a new avenue for the control of AR function and cell growth, especially in the case of androgen-resistant, but AR-dependent, prostate cancer cells.

Functional role of a conformationally flexible homopurine/homopyrimidine domain of the androgen receptor gene promoter interacting with Sp1 and a pyrimidine single strand DNA-binding protein.

The androgen receptor (AR) gene promoter does not contain the TATA or CAAT box, but it contains a long (approximately 90-bp) homopurine/homopyrimidine (pur/ pyr) stretch immediately upstream of the Sp1-binding GC box site. This pur-pyr stretch is conserved at the same proximal position in the rat, mouse, and human AR gene promoters. Mutation of this region results in a 3-fold decline in promoter activity, indicating an important regulatory function. Examination of the conformational state of the AR pur/pyr region with the single-strand-specific S1 nuclease showed that it is capable of forming a non-B DNA structure involving unpaired single strands. Fine mapping of the S1-sensitive site revealed an unsymmetric cleavage pattern indicative of an intramolecular triple helical H-form DNA conformation. Electrophoretic mobility shift analyses showed that the pur/pyr region of the AR promoter can bind a novel pyrimidine single-strand-specific protein (ssPyrBF) and also a double-strand DNA-binding protein. Both oligonucleotide cross-competition and antibody supershift experiments established that the double-strand binding protein is equivalent to Sp1. Deoxyribonuclease I (DNase I) footprinting analysis showed multiple Sp1-binding to the pur/pyr site and a weaker Sp1 interaction to this region compared with the adjacently located GC box, where Sp1 functions to recruit the TFIID complex. These results suggest that the pur/pyr domain of the AR gene can serve to attract additional Sp1 molecules when it exists in the double-stranded B-DNA conformation. However, binding of ssPyrBF and the resultant stabilization of the non-B DNA structure is expected to prevent its interaction with Sp1. We speculate that in the TATA-less AR gene promoter, multiple weak Sp1 sites at the pur/pyr region adjacent to the GC box can provide a readily available source of this transcription factor to the functional GC box, thereby facilitating the assembly of the initiation complex.