Altered glycosylation, expression of serum haptoglobin and alpha-1-antitrypsin in chronic hepatitis C, hepatitis C induced liver cirrhosis and hepatocellular carcinoma patients.

Liver cirrhosis with hepatitis C viral infection (HCV-LC) causes high risk to develop hepatocellular carcinoma (HCC). Besides diagnosis of liver cirrhosis by biochemical test, imaging techniques, assessment of structural liver damage by biopsy shows several disadvantages. Our aim was to monitor the changes in the expression level of serum proteins and their glycosylation pattern among chronic hepatitis C (HCV-CH), HCV-LC and HCC patients with respect to controls. 2D gel electrophoresis of HCV-CH, HCV-LC and HCC patients' sera showed several protein spots, which were identified by LC-MS. The change in the expression of two prominent protein spots, haptoglobin (Hp) and alpha 1-antitrypsin (AAT) was evaluated by western blot and ELISA. The changes in glycosylation pattern of these serum proteins were assayed using different lectins. Increased level of Hp and AAT was observed in HCV-LC and HCC patients' group whereas those were found to be present less in HCV-CH patient groups with respect to control as determined by ELISA using monoclonal antibodies. Decreased level of sialylation in both Hp and AAT was observed in HCV-LC and HCV-CH patients' group whereas increased level of sialylation was observed in HCC patient groups by ELISA using Sambucus nigra agglutinin. On the other hand increased level of fucosylation in two serum glycoproteins was observed in HCV-LC and HCC patients' group using Lens culinarris agglutinin. High glycan branching was found in HCV-LC and HCC patient groups in Hp but not in HCV-CH as determined by Datura stramonium agglutinin. However, there was no such change observed in glycan branching in AAT of HCV-CH and HCV-LC patients' groups, to the contrary high glycan branching was observed in HCC patients' group. Increased level of exposed galactose in both serum proteins was observed in both HCC patients' group as determined by Ricinus communis agglutinin. The present glycoproteomics study could predict the progression of HCV-CH, HCV-LC and HCC without the need of liver biopsy.

Two Dimensional Effective Electron Mass at the Fermi Level in Quantum Wells of III-V, Ternary and Quaternary Semiconductors.

In this paper we study the influence of strong electric field on the two dimensional (2D)effective electron mass (EEM) at the Fermi level in quantum wells of III-V, ternary and quaternary semiconductors within the framework of k x p formalism by formulating a new 2D electron energy spectrum. It appears taking quantum wells of InSb, InAs, Hg(1-x)Cd(x)Te and In(1-x)Ga(x)As(1-y)P(y) lattice matched to InP as examples that the EEM increases with decreasing film thickness, increasing electric field and increases with increasing surface electron concentration exhibiting spikey oscillations because of the crossing over of the Fermi level by the quantized level in quantum wells and the quantized oscillation occurs when the Fermi energy touches the sub-band energy. The electric field makes the mass quantum number dependent and the oscillatory mass introduces quantum number dependent mass anisotropy in addition to energy. The EEM increases with decreasing alloy composition where the variations are totally band structure dependent. Under certain limiting conditions all the results for all the cases get simplified into the well-known parabolic energy bands and thus confirming the compatibility test. The content of this paper finds three applications in the fields of nano-science and technology.

Threshold collision-induced dissociation of Sr(2+)(H(2)O)(x) complexes (x=1-6): An experimental and theoretical investigation of the complete inner shell hydration energies of Sr(2+).

The sequential bond energies of Sr(2+)(H(2)O)(x) complexes, where x=1-6, are determined by threshold collision-induced dissociation using a guided ion beam tandem mass spectrometer equipped with an electrospray ionization source. The electrospray source produces an initial distribution of Sr(2+)(H(2)O)(x) complexes, where x=6-9. Smaller Sr(2+)(H(2)O)(x) complexes, where x=1-5, are accessed using a recently developed in-source fragmentation technique that takes place in the high pressure region of a rf-only hexapole ion guide. This work constitutes the first experimental study for the complete inner shell of any multiply charged ion. The kinetic energy dependent cross sections are determined over a wide energy range to monitor all possible dissociation products and are modeled to obtain 0 and 298 K binding energies for loss of a single water molecule. These binding energies decrease monotonically for the Sr(2+)(H(2)O) complex to Sr(2+)(H(2)O)(6). Our experimental results agree well with previous literature results obtained by equilibrium and kinetic studies for x=5 and 6. Because there has been limited theory for the hydration of Sr(2+), we also present an in-depth theoretical study on the energetics of the Sr(2+)(H(2)O)(x) systems by employing several levels of theory with multiple effective core potentials for Sr and different basis sets for the water molecules.

Sorting Fermionization from Crystallization in Many-Boson Wavefunctions.

Fermionization is what happens to the state of strongly interacting repulsive bosons interacting with contact interactions in one spatial dimension. Crystallization is what happens for sufficiently strongly interacting repulsive bosons with dipolar interactions in one spatial dimension. Crystallization and fermionization resemble each other: in both cases - due to their repulsion - the bosons try to minimize their spatial overlap. We trace these two hallmark phases of strongly correlated one-dimensional bosonic systems by exploring their ground state properties using the one- and two-body density matrix. We solve the N-body Schrödinger equation accurately and from first principles using the multiconfigurational time-dependent Hartree for bosons (MCTDHB) and for fermions (MCTDHF) methods. Using the one- and two-body density, fermionization can be distinguished from crystallization in position space. For N interacting bosons, a splitting into an N-fold pattern in the one-body and two-body density is a unique feature of both, fermionization and crystallization. We demonstrate that this splitting is incomplete for fermionized bosons and restricted by the confinement potential. This incomplete splitting is a consequence of the convergence of the energy in the limit of infinite repulsion and is in agreement with complementary results that we obtain for fermions using MCTDHF. For crystalline bosons, in contrast, the splitting is complete: the interaction energy is capable of overcoming the confinement potential. Our results suggest that the spreading of the density as a function of the dipolar interaction strength diverges as a power law. We describe how to distinguish fermionization from crystallization experimentally from measurements of the one- and two-body density.

Acute undifferentiated human diarrhea in the tropics. I. Alterations in intestinal micrflora.

The microflora of the small and large intestine was determined in 17 adults with acute undifferentiated diarrhea in Calcutta, India. On the basis of bacteriologic findings, the patients could be divided into two groups: those with a predominant flora of Escherichia coli (eight patients) and those with a mixed coliform flora (nine patients). In the former group, E. coli were distributed throughout the small and large bowel. Broth filtrates of these isolates contained an enterotoxin which caused fluid accumulation in the rabbit intestinal loop model. Toxigenic E. coli were cleared rapidly from the small bowel during the acute period; some patients only had the "hot" strains in their fecal effluent. During convalescence, the serotypes of E. coli changed and the new strains did not elaborate enterotoxin. Only one of the eight patients had a serotype previously associated with diarrhea. Acute undifferentiated diarrhea in the remaining cases was apparently caused by untypable E. coli or by typable strains not generally considered pathogenic. Small bowel and fecal cultures from the mixed flora group revealed a heterogeneous mixture of Gram-negative enteric bacilli and a distinct pattern could not be discerned. Further study will be needed to elucidate the cause of diarrhea in these cases.

Non-canonical NFκB mutations reinforce pro-survival TNF response in multiple myeloma through an autoregulatory RelB:p50 NFκB pathway.

Environmental drug resistance constitutes a serious impediment for therapeutic intervention in multiple myeloma. Tumor-promoting cytokines, such as tumor necrosis factor (TNF), induce nuclear factor-κB (NFκB)- driven expression of pro-survival factors, which confer resistance in myeloma cells to apoptotic insults from TNF-related apoptosis-inducing ligand (TRAIL) and other chemotherapeutic drugs. It is thought that RelA:p50 dimer, activated from IκBα-inhibited complex in response to TNF-induced canonical NFκB signal, mediates the pro-survival NFκB function in cancerous cells. Myeloma cells additionally acquire gain-of-function mutations in the non-canonical NFκB module, which induces partial proteolysis of p100 into p52 to promote RelB:p52/NFκB activation from p100-inhibited complex during immune cell differentiation. However, role of non-canonical NFκB signaling in the drug resistance in multiple myeloma remains unclear. Here we report that myeloma-associated non-canonical aberrations reinforce pro-survival TNF signaling in producing a protracted TRAIL-refractory state. These mutations did not act through a typical p52 NFκB complex, but completely degraded p100 to reposition RelB under IκBα control, whose degradation during TNF signaling induced an early RelB:p50 containing NFκB activity. More so, autoregulatory RelB synthesis prolonged this TNF-induced RelB:p50 activity in myeloma cells harboring non-canonical mutations. Intriguingly, TNF-activated RelB:p50 dimer was both necessary and sufficient, and RelA was not required, for NFκB-dependent pro-survival gene expressions and suppression of apoptosis. Indeed, high RelB mRNA expressions in myeloma patients correlated with the augmented level of pro-survival factors and resistance to therapeutic intervention. In sum, we provide evidence that cancer-associated mutations perpetuate TNF-induced pro-survival NFκB activity through autoregulatory RelB control and thereby exacerbate environmental drug resistance in multiple myeloma.

Ficus cunia agglutinin for recognition of bacteria.

Interaction of bacteria with lectin using anti-lectin antibody by ELISA is an established method. In the present study, we have devised a simple ELISA using a biotinylated lectin and antibiotin-HRP. Ficus cunia agglutinin (FCA), which has shown the specificity towards alpha/beta anomers of GlcNAc and other-NAc containing sugars like LacNAc and GlcNAcbeta(1-4/6)GlcNAc, was used as a model lectin for the study of interaction with immobilized microorganisms on ELISA plate. The bacterial cells of E. coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus subtilis and Staphylococcus aureus showed binding with FCA and the degree of binding was dependent on the bacterial surface antigen. This method is considered a simple technique to study the lectin-bacteria interaction.

Cardiovascular phenotyping of fetal mice by noninvasive high-frequency ultrasound facilitates recovery of ENU-induced mutations causing congenital cardiac and extracardiac defects.

As part of a large-scale noninvasive fetal ultrasound screen to recover ethylnitrosourea (ENU)-induced mutations causing congenital heart defects in mice, we established a high-throughput ultrasound scanning strategy for interrogating fetal mice in utero utilizing three orthogonal imaging planes defined by the fetus' vertebral column and body axes, structures readily seen by ultrasound. This contrasts with the difficulty of acquiring clinical ultrasound imaging planes which are defined by the fetal heart. By use of the three orthogonal imaging planes for two-dimensional (2D) imaging together with color flow, spectral Doppler, and M-mode imaging, all of the major elements of the heart can be evaluated. In this manner, 10,091 ENU-mutagenized mouse fetuses were ultrasound scanned between embryonic days 12.5 and 19.5, with 324 fetuses found to die prenatally and 425 exhibiting cardiovascular defects. Further analysis by necropsy and histology showed heart defects that included conotruncal anomalies, obstructive lesions, and shunt lesions as well as other complex heart diseases. Ultrasound imaging also identified craniofacial/head defects and body wall closure defects, which necropsy revealed as encephalocele, holoprosencephaly, omphalocele, or gastroschisis. Genome scanning mapped one ENU-induced mutation associated with persistence truncus arteriosus and holoprosencephaly to mouse chromosome 2, while another mutation associated with cardiac defects and omphalocele was mapped to mouse chromosome 17. These studies show the efficacy of this novel ultrasound scanning strategy for noninvasive ultrasound phenotyping to facilitate the recovery of ENU-induced mutations causing congenital heart defects and other extracardiac anomalies.

Synthesis and processing of the dimorphic forms of rat alpha 2u-globulin.

alpha 2u-Globulin the androgen-dependent male rat urinary protein, can be resolved into two distinct molecular forms by SDS-polyacrylamide slab gel electrophoresis. These two forms designated as alpha 2u-A (M, 18,800) and alpha 2u (Mr 18,100) are found both in urine and in the liver cells. Translation of rat liver mRNA in the rabbit reticulocyte lysate produced two preprotein forms of alpha 2u-globulin, designated as alpha 2uA' (Mr 20,300) and alpha 2uB' (Mr 19,600). Cell-free translation of rat liver mRNA in the presence of dog pancreas microsomal membrane or in Xenopus oocytes produced the two processed forms of alpha 2u-globulin (alpha 2uA and alpha 2uB). Quantitation of alpha 2uA and alpha 2uB within the in vitro translation products of the hepatic mRNA from albino rats of Yale, Sprague-Dawley and Fischer strains showed genetic differences in the proportion of translatable mRNA for alpha 2uA and alpha 2uB. The ratio of alpha 2uA: alpha 2uB in the translation products of liver mRNA from Yale rats was found to be 1:2.5 while in the case of both Sprague-Dawley and Fischer rats, the ratio was 1:4. A small portion of the alpha 2uA and alpha 2uB synthesized in the cultured hepatocytes, in the Xenopus oocytes or in the membrane-supplemented cell-free system appeared as two additional forms, designated as alpha 2uA" (Mr 21,200) and alpha 2uB" (Mr 20,600). Unlike alpha 2uA and alpha 2uB both alpha 2uA" and alpha 2uB" were found to bind to Con A-Sepharose, suggesting their glycoprotein nature.

The senescence marker protein (SMP-2) of the rat liver: purification, immunochemical characterization and age-dependent regulation.

In vitro translation of total rat hepatic mRNAs has identified a 31 kilodalton senescence marker protein (SMP-2) which is present in higher amounts in prepubertal and senescent males than in the post-pubertal adult male (more than 10-fold). SMP-2 is an androgen-repressible protein. The negative regulation of the SMP-2 gene activity by androgen accounts for its increased expression during the androgen insensitive states of the prepubertal and senescent livers, and its constitutive expression in the female liver. A combination of separation procedures including salt fractionation, chromatofocusing, ion-exchange chromatography and preparative gel electrophoresis have led to the purification of SMP-2 to apparent homogeneity. The purified protein showed the same electrophoretic mobility as the sex- and age-specific in vitro translation product of hepatic mRNAs. The polyclonal antibody to SMP-2 was produced in the rabbit. The antibody selectively reacted with the 31 kDa sex- and age-specific translation product of hepatic mRNAs. Western blot analysis of the liver cytosol confirms monospecificity of the antiserum, as well as age- and sex-dependent changes in the tissue level of SMP-2. Histochemical staining of liver sections with the antiserum reveals a preferential periportal localization of SMP-2 in the hepatocytes. This finding is in marked contrast to the androgen-inducible alpha 2u globulin which is preferentially synthesized and localized in the pericentral hepatocytes. Thus, the zonal distribution of SMP-2 correlates with polarized androgen sensitivity of the hepatocytes within the liver lobule.

Changes in transcriptional activity and matrix association of alpha 2u-globulin gene family in the rat liver during maturation and aging.

Hepatic synthesis of alpha 2u-globulin in the male rat begins at puberty (about 40 days), reaches a peak level at about 80 days, and ceases at about 750-800 days of age. The age-dependent changes in alpha 2u-globulin synthesis are correlated with both the steady-state level of the hepatic mRNA for this protein and the rate of transcription of the alpha 2u-globulin gene family. Transcriptional activation of the alpha 2u-globulin gene family at puberty and cessation of transcription at senescence correlate with the association and dissociation of this gene domain with the nuclear matrix. Unlike the alpha 2u-globulin gene, the albumin gene in the liver shows preferential association with the nuclear matrix throughout the life. From these results we conclude that the age-dependent changes in alpha 2u-globulin synthesis are due to the alteration in the rate of transcription of the alpha 2u-globulin gene, and that the association of this gene domain to the nuclear matrix is a prerequisite to its transcriptional activation.

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.

Acute elevation of blood carboxyhemoglobin to 6% impairs exercise performance and aggravates symptoms in patients with ischemic heart disease.

Acute exposure to carbon monoxide has the potential to impair exercise capacity in patients with ischemic heart disease. The effect of sufficient inhalation of this compound to gradually produce a level of 6% carboxyhemoglobin was studied in 30 nonsmoking patients with obstructive coronary artery disease and evidence of exercise-induced ischemia. After an initial training session, subjects were exposed to air or carbon monoxide on successive days in a randomized double-blind crossover fashion. Cardiac function and exercise capacity were assessed during symptom-limited supine radionuclide ventriculography. On the carbon monoxide day, mean postexposure carboxyhemoglobin was 5.9 +/- 0.1% compared with 1.6 +/- 0.1% (p less than 0.01) after air exposure. The mean duration of exercise was significantly longer after air compared with carbon monoxide exposure (626 +/- 50 s for air versus 585 +/- 49 s for carbon monoxide, p less than 0.05). Actuarial methods suggested that subjects were likely to experience angina earlier during exercise on the day of carbon monoxide exposure (p less than 0.05). Both the level (62 +/- 2.4 versus 60 +/- 2.4%, p = 0.05) and change in left ventricular ejection fraction at submaximal exercise (1.6 +/- 1.6 versus -1.2 +/- 1.6%, p = 0.05) were greater on the air exposure day compared with the carbon monoxide day. The peak exercise left ventricular ejection fraction was not different for the two exposures (57 +/- 2.5% for both). These results demonstrate earlier onset of ventricular dysfunction, angina and poorer exercise performance in patients with ischemic heart disease after acute carbon monoxide exposure sufficient to increase blood carboxyhemoglobin to 6%.

Chromosomal recombination and breakage associated with instability in mouse centrometric satellite DNA.

A mouse L cell line containing the centromeric insertion of herpes thymidine kinase genes (tk) was previously shown to undergo a high frequency of DNA rearrangement at the site of tk insertion. Analysis of TK- revertants had demonstrated that DNA rearrangements were usually associated with DNA deletion and were always mediated by intrachromosomal recombinations. In this study, we further analyzed several TK+ subclones to examine the mode of DNA rearrangements in the absence of negative selection pressure. In two clones, LC2-3F and LC2-3E17, rearrangements were accompanied by DNA amplification and were mediated by intrachromosomal recombination. In subclone LC2-3E17-19, we further detected perturbations in the pattern of centromeric heterochromatization. This was associated with chromosome instability, as evidenced by chromosome breakage at the centromere. The analysis of three other sibling clones, LC2-3, LC2-6 and LC2-15, further suggests that reciprocal recombination events may play a role in such centromeric rearrangements. These results suggest that DNA rearrangements in the centromere may be mediated by a number of different mechanisms, and generally do not affect chromosome stability except when accompanied by changes in the pattern of heterochromatization.

Molecular characterization of Pax6(2Neu) through Pax6(10Neu): an extension of the Pax6 allelic series and the identification of two possible hypomorph alleles in the mouse Mus musculus.

Phenotype-based mutagenesis experiments will increase the mouse mutant resource, generating mutations at previously unmarked loci as well as extending the allelic series at known loci. Mapping, molecular characterization, and phenotypic analysis of nine independent Pax6 mutations of the mouse recovered in mutagenesis experiments is presented. Seven mutations result in premature termination of translation and all express phenotypes characteristic of null alleles, suggesting that Pax6 function requires all domains to be intact. Of major interest is the identification of two possible hypomorph mutations: Heterozygotes express less severe phenotypes and homozygotes develop rudimentary eyes and nasal processes and survive up to 36 hr after birth. Pax6(4Neu) results in an amino acid substitution within the third helix of the homeodomain. Three-dimensional modeling indicates that the amino acid substitution interrupts the homeodomain recognition alpha-helix, which is critical for DNA binding. Whereas cooperative dimer binding of the mutant homeodomain to a paired-class DNA target sequence was eliminated, weak monomer binding was observed. Thus, a residual function of the mutated homeodomain may explain the hypomorphic nature of the Pax6(4Neu) allele. Pax6(7Neu) is a base pair substitution in the Kozak sequence and results in a reduced level of Pax6 translation product. The Pax6(4Neu) and Pax6(7Neu) alleles may be very useful for gene-dosage studies.

A twist in anti-inflammation: annexin 1 acts via the lipoxin A4 receptor.

The inflammatory response is a life-saving protective process mounted by the body to overcome pathogen infection and injury; however, in chronic inflammatory pathologies this response can become deregulated. The existence of specialized anti-inflammatory pathways/mediators that operate in the body to down-regulate inflammation have now emerged. Thus, persistence of inflammation leading to pathology could be due to malfunctioning of one or more of these counter-regulatory pathways. Here we focus on one of them, the anti-inflammatory mediator annexin 1, and provide an update on its inhibitory effects upon the leukocyte trafficking process. In particular, recent evidence that receptors of the formyl-peptide family, which includes also the lipoxin A4 receptor, could be the annexin 1 receptor(s) in the context of anti-inflammation might provide new avenues for exploiting this pathway for drug discovery.

Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells.

An expression construct containing the cDNA encoding a modified aequorea green fluorescent protein (GFP) ligated to the 5'-end of the rat androgen receptor (AR) cDNA (GFP-AR) was used to study the intracellular dynamics of the receptor movement in living cells. In three different cell lines, ie. PC3, HeLa, and COS1, unliganded GFP-AR was seen mostly in the cytoplasm and rapidly (within 15-60 min) moved to the nuclear compartment after androgen treatment. Upon androgen withdrawal, the labeled AR migrated back to the cytoplasmic compartment and maintained its ability to reenter the nucleus on subsequent exposure to androgen. Under the condition of inhibited protein synthesis by cycloheximide (50 microg/ml), at least four rounds of receptor recycling after androgen treatment and withdrawal were recorded. Two nonandrogenic hormones, 17beta-estradiol and progesterone at higher concentrations (10(-7)/10(-6) M), were able to both transactivate the AR-responsive promoter and translocate the GFP-AR into the nucleus. Similarly, antiandrogenic ligands, cyproterone acetate and casodex, were also capable of translocating the cytoplasmic AR into the nucleus albeit at a slower rate than the androgen 5alpha-dihydrotestosterone (DHT). All AR ligands with transactivation potential, including the mixed agonist/antagonist cyproterone acetate, caused translocation of the GFP-AR into a subnuclear compartment indicated by its punctate intranuclear distribution. However, translocation caused by casodex, a pure antagonist, resulted in a homogeneous nuclear distribution. Subsequent exposure of the casodex-treated cell to DHT rapidly (15-30 min) altered the homogeneous to punctate distribution of the already translocated nuclear AR. When transported into the nucleus either by casodex or by DHT, GFP-AR was resistant to 2 M NaCl extraction, indicating that the homogeneously distributed AR is also associated with the nuclear matrix. Taken together, these results demonstrate that AR requires ligand activation for its nuclear translocation where occupancy by only agonists and partial agonists can direct it to a potentially functional subnuclear location and that one receptor molecule can undertake multiple rounds of hormonal signaling; this indicates that ligand dissociation/inactivation rather than receptor degradation may play a critical role in terminating hormone action.

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.

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.

Ribozyme-mediated cleavage of the estrogen receptor messenger RNA and inhibition of receptor function in target cells.

Estrogen receptor (ER) functions as a ligand-activated transcription factor for estrogen-regulated genes. Because of the critical role of the ER in the proliferation of certain estrogen-dependent cancer cell types such as the mammary tumor, inhibitors of estrogen action at the level of receptor function are of major clinical interest. Here we describe developments of two ribozymes that can selectively degrade the human ER mRNA and inhibit trans-activation of an artificial promoter containing the estrogen response element. Two ribozymes, designated RZ-1 and RZ-2, cleave the human ER alpha mRNA at nucleotide positions +956 and +889, respectively. These cleavage sites lie within the coding sequence for the DNA-binding domain of the receptor protein. Both RZ-1 and RZ-2 were also effective in inhibiting the progression of quiescent MCF-7 breast cancer cells to the S phase of the cell cycle after their exposure to 17beta-estradiol (10(-9) M). These results provide a new avenue for inhibition of estrogen action by selective mRNA degradation with its potential therapeutic application through targeted gene delivery vectors.