Function of a bacterial activator protein that binds to transcriptional enhancers.

The nitrogen regulatory (NtrC) protein of enteric bacteria, which binds to sites that have the properties of transcriptional enhancers, is known to activate transcription by a form of RNA polymerase that contains the NtrA protein (sigma 54) as sigma factor (referred to as sigma 54-holoenzyme). In the presence of adenosine triphosphate, the NtrC protein catalyzes isomerization of closed recognition complexes between sigma 54-holoenzyme and the glnA promoter to open complexes in which DNA in the region of the transcription start site is locally denatured. NtrC is not required subsequently for maintenance of open complexes or initiation of transcription.

Octamer and SPH motifs in the U1 enhancer cooperate to activate U1 RNA gene expression.

The transcriptional enhancer of a chicken U1 small nuclear RNA gene has been shown to extend over approximately 50 base pairs of DNA sequence located 180 to 230 base pairs upstream of the U1 transcription initiation site. It is composed of multiple functional motifs, including a GC box, an octamer motif, and a novel SPH motif. The contributions of these three distinct sequence motifs to enhancer function were studied with an oocyte expression assay. Under noncompetitive conditions in oocytes, the SPH motif is capable of stimulating U1 RNA transcription in the absence of the other functional motifs, whereas the octamer motif by itself lacks this ability. However, to form a transcription complex that is stable to challenge by a second competing small nuclear RNA transcription unit, both the octamer and SPH motifs are required. The GC box, although required for full enhancer activity, is not essential for stable complex formation in oocytes. Site-directed mutagenesis was used to study the DNA sequence requirements of the SPH motif. Functional activity of the SPH motif is spread throughout a 24-base-pair region 3' of the octamer but is particularly dependent upon sequences near an SphI restriction site located at the center of the SPH motif. Using embryonic chicken tissue as a source material, we identified and partially purified a factor, termed SBF, that binds sequence specifically to the SPH motif of the U1 enhancer. The ability of this factor to recognize and bind to mutant enhancer DNA fragments in vitro correlates with the functional activity of the corresponding enhancer sequences in vivo.

The ruv proteins of Thermotoga maritima: branch migration and resolution of Holliday junctions.

In homologous recombination in bacteria, the RuvAB Holliday junction-specific helicase catalyzes Holliday junction branch migration, and the RuvC Holliday junction resolvase catalyzes formation of spliced or patched structures. RuvAB and RuvC from the hyperthermophile Thermotoga maritima were expressed in Escherichia coli and purified to homogeneity. An inverted repeat sequence with unique termini was produced by PCR, restriction endonuclease cleavage, and head-to-tail ligation. A second inverted repeat sequence was derived by amplification of a second template containing a three-nucleotide insertion. Reassociation products from a mixture of these two sequences were homoduplex linear molecules and heteroduplex heat-stable Holliday junctions, which acted as substrates for both T. maritima RuvAB and RuvC. The T. maritima RuvAB helicase catalyzed energy-dependent Holliday junction branch migration at 70 degrees C, leading to heteroduplex linear duplex molecules with two three-nucleotide loops. Either ATP or ATP gamma S hydrolysis served as the energy source. T. maritima RuvC resolved Holliday junctions at 70 degrees C. Remarkably, the cleavage site was identical to the preferred cleavage site for E. coli RuvC [(A/T)TT(downward arrow)(G/C)]. The conservation of function and the ease of purification of wild-type and mutant thermophilic proteins argues for the use of T. maritima proteins for additional biochemical and structural studies.

Analysis of the structure and expression of the chicken gene encoding a homolog of the human RREB-1 transcription factor.

Ras proteins are involved in a number of signal transduction pathways including the mitogen-activated kinase cascade. Activated MAPKs translocate to the nucleus and phosphorylate transcription factors such as c-myc, TCF and AP-1. Recently, a Ras-responsive element binding transcription factor, RREB-1, was cloned from a human medullary thyroid carcinoma cell line. RREB-1 is a zinc finger protein that binds to a Ras-responsive element in the promoter of the human calcitonin gene. We report the cloning of the chicken homologue to human RREB-1. Amino-acid alignment demonstrates that chicken and human RREB-1 are 53% identical and 69% similar. Genomic southern analysis indicates that chicken rreb-1 is a single-copy gene in the chicken genome. We demonstrate that chicken and human rreb-1 display the same tissue distribution, being expressed in all tissues examined except the brain. Interestingly, chicken RREB-1 has an extended N-terminus and contains 16 zinc fingers of the TFIIIA subclass, in comparison to human RREB-1 which was reported to contain only four zinc fingers. The size discrepancy between the two predicted gene products is further discussed. An unusual structural feature of RREB-1 is the widely spaced arrangement of the zinc fingers.

Relative affinity of 17 alpha- and/or 21-esters and 17 alpha, 21-diesters of cortisol for a glucocorticoid receptor from rat thymocytes.

The affinity, relative to cortisol (1), of 17 alpha- and 21-esters and 17 alpha,21-diesters of cortisol for the glucocorticoid receptor of rat thymus cytosol was determined by a competitive binding assay which used [3H]dexamethasone. Esterification of the 21-hydroxy group of cortisol caused a loss of relative affinity to 0.046 for acetate and 0.32 for valerate. Esterification of the 17 alpha-hydroxy group resulted in an increase in relative affinity to 1.14 for acetate, 12.4 for butyrate, and 11.5 for valerate. Diesters had relative affinities which reflected both trends. Thus, the 21-acetate, 21-propionate, 21-butyrate, and 21-valerate of cortisol 17-acetate had relative affinities of 0.036, 0.093, 0.152, and 0.272. The 21-acetate, 21-propionate, and 21-butyrate of cortisol 17-valerate had relative affinities of 0.76, 1.17, and 1.33.

Benzophenothiazine and benzoporphyrin derivative combination phototherapy effectively eradicates large murine sarcomas.

The tumoricidal effects of photochemotherapy with two photosensitizers, 5-ethylamino-9-diethylaminobenzo[a] phenothiazinium chloride (EtNBS) and benzoporphyrin derivative monoacid ring A (BPD-MA), were evaluated separately and in combination against the EMT-6 fibrosarcoma implanted subcutaneously in BALB/c mice. Animals carrying tumors 8-10 mm in diameter were divided into eight different groups (approximately 20/group) and subjected to various photoirradiation and drug conditions. The tumor response to photodynamic therapy (PDT) was measured as the mean tumor wet weight 2 weeks post-PDT. The combination treatment with 5.25 mg/kg EtNBS and 2.5 mg/kg BPD-MA followed by photoirradiation with 100 J/cm2 at 652 nm and then by 100 J/cm2 at 690 nm resulted in a 95% reduction in the average tumor weights compared to controls (no light, no drugs) with 76% of the mice being tumor free 2 weeks post-PDT. Because treatment with EtNBS or BPD-MA at twice the light dose and drug concentration resulted in either no significant reduction in tumor weights or increased the lethality of treatment, respectively, the data suggest that the enhanced PDT effect observed with the combination of drugs is synergistic rather than additive. Histology of tumors 24 h post-PDT with the combination of drugs showed nearly complete destruction of the tumor mass with little or no damage to the vasculature and no extravasation of red blood cells. There was no damage to the normal skin adjacent to the tumor. Fluorescence microscopy of EMT-6 cells incubated in vitro with the two photosensitizers revealed that they were localized to different intracellular compartments. The fluorescence pattern from frozen tumor tissue slices following the in vivo administration of the photosensitizers indicated a greater intracellular localization for EtNBS vs BPD-MA.

Theophylline bioavailability in the dog.

The bioavailability of theophylline following single oral doses of a theophylline capsule, a theophylline tablet, and an aminophylline tablet in beagle dogs was compared against an intravenous standard. Plasma theophylline levels after oral and intravenous drug administration were described by the one-compartment open model. The onset of theophylline absorption from the oral products was rapid. While the theophylline tablet showed a slower absorption rate than the capsule or the aminophylline tablet, all three products appeared to be completely bioavailable.

Effect of caffeine on circulating theophylline levels in beagle dogs.

The pharmacokinetic interactions of caffeine with theophylline were examined in two beagle dogs by administering 100 mg of aminophylline intravenously, 3 weeks before and immediately after repeated oral doses of caffeine. Serial plasma samples were analyzed for caffeine and theophylline simultaneously by high-performance liquid chromatography. Upon multiple oral dosing, 100 mg every 12 hr for 7 days, the caffeine half-life increased slightly in one dog but decreased in the other. As predicted from single-dose data, caffeine accumulation in plasma after repeated doses was slight, while plasma levels of the N-demethylated metabolite, theophylline, rose to about three times the initial values. After rapid intravenous doses of aminophylline, the theophylline half-life was 5-7 hr, which decreased slightly when the drug was administered concomitantly with caffeine during steady state of caffeine. The theophylline volume of distribution (0.75 liter/kg) was unaffected by caffeine. On the other hand, an acute aminophylline injection prolonged the elimination half-life and increased the apparent volume of distribution of caffeine, causing little overall change in its plasma clearance. The results suggested that interactions between theophylline and caffeine may be attributed to changes in drug distribution and drug elimination characteristics.

The diffusion of medical practice patterns in a community hospital.

There has been some evidence which suggests that HMOs alter the hospitalization rate of the fee for service medical sector. If true, this spillover effect would have important implications for reducing hospital utilization, because HMO affiliated physicians use substantially less resource intensive in-patient services. This research investigates one possible mechanism, whether there is a diffusion of medical practice patterns from HMO affiliated physicians to non-HMO physicians when both groups share admitting privileges in the same hospital. Intervention analysis was used to test this hypothesis by comparing the time series of in-patient utilization, in a community hospital, before and after the hospital became affiliated with a HMO. The results of this analysis did not substantiate the theorized HMO spillover effect.

Program used by a national home infusion therapy provider to prepare for Joint Commission site surveys.

A process developed by a national provider of home infusion therapy to prepare branch pharmacy centers for site surveys by the Joint Commission on Accreditation of Healthcare Organizations (JCAHO) is described. Each of the 20 branches to be surveyed by JCAHO was visited three times by a corporate quality assurance team, which consisted of a pharmacy quality assurance manager and a nursing quality assurance manager. During the initial visit, the team performed a baseline quality assurance audit based on pre-established audit criteria. During the second visit, the team conducted a mock survey that replicated the official JCAHO survey. The final visit was scheduled to coincide with the JCAHO survey; its purpose was to provide support to the branch. Results from each of the visits were documented and reviewed with branch and corporate management. As of January 1990, 19 branches and the corporate service had been surveyed. Sixteen branches received a three-year JCAHO accreditation; the JCAHO accreditation status of the remaining three branches is pending. The JCAHO surveyor made only minor recommendations during the oral summary of the survey results. A program for preparing the staff at branch facilities of a major home infusion therapy provider for JCAHO surveys was successful.

P-OTX: a PIT-1-interacting homeodomain factor expressed during anterior pituitary gland development.

A novel OTX-related homeodomain transcription factor has been identified on the basis of its ability to interact with the transactivation domain of the pituitary-specific POU domain protein, Pit-1. This factor, referred to as P-OTX (pituitary OTX-related factor), is expressed in primordial Rathke's pouch, oral epithelium, first bronchial arch, duodenum, and hindlimb. In the developing anterior pituitary, it is expressed in all regions from which cells with distinct phenotypes will emerge in the mature gland. P-OTX is able to independently activate and to synergize with Pit-1 on pituitary-specific target gene promoters. Therefore, P-OTX may subserve functions in generating both precursor and specific cell phenotypes in the anterior pituitary gland and in several other organs.

Pit-1 binding to specific DNA sites as a monomer or dimer determines gene-specific use of a tyrosine-dependent synergy domain.

Transcriptional activation of the prolactin and growth hormone genes, occurring in a cell-specific fashion, requires short-range synergistic interactions between the pituitary-specific POU domain factor Pit-1 and other transcription factors, particularly nuclear receptors. Unexpectedly, we find that these events involve the gene-specific use of alternative Pit-1 synergy domains. Synergistic activation of the prolactin gene by Pit-1 and the estrogen receptor requires a Pit-1 amino-terminal 25-amino-acid domain that is not required for analogous synergistic activation of the growth hormone promoter. The action of this Pit-1 synergy domain is dependent on the presence of two of three tyrosine residues spaced by 6 amino acids and can be replaced by a comparable tyrosine-dependent trans-activation domain of an unrelated transcription factor (hLEF). The gene-specific utilization of this tyrosine-dependent synergy domain is conferred by specific Pit-1 DNA-binding sites that determine whether Pit-1 binds as a monomer or a dimer. Thus, the critical DNA site in the prolactin enhancer, where this domain is required, binds Pit-1 as a monomer, whereas the Pit-1 sites in the growth hormone gene, which do not utilize this synergy domain, bind Pit-1 as a dimer. The finding that the sequence of specific DNA sites dictates alternative Pit-1 synergy domain utilization based on monomeric or dimeric binding suggests an additional regulatory strategy for differential target gene activation in distinct cell types.

Pitx2 regulates lung asymmetry, cardiac positioning and pituitary and tooth morphogenesis.

Pitx1 and Pitx2 are highly homologous, bicoid-related transcription factors. Pitx2 was initially identified as the gene responsible for the human Rieger syndrome, an autosomal dominant condition that causes developmental abnormalities. Pitx2 is asymmetrically expressed in the left lateral-plate mesoderm, and mutant mice with laterality defects show altered patterns of Pitx2 expression that correlate with changes in the visceral symmetry (situs). Ectopic expression of Pitx2 in the right lateral-plate mesoderm alters looping of the heart and gut and reverses body rotation in chick and Xenopus embryos. Here we describe the phenotype of Pitx2 gene-deleted mice, characterized by defective body-wall closure, right pulmonary isomerism, altered cardiac position, arrest in turning and, subsequently, a block in the determination and proliferation events of anterior pituitary gland and tooth organogenesis. Thus, Pitx2 is a transcription factor that encodes 'leftness' of the lung.

Multistep signaling requirements for pituitary organogenesis in vivo.

During development of the mammalian pituitary gland specific hormone-producing cell types, critical in maintaining homeostasis, emerge in a spatially and temporally specific fashion from an ectodermal primordium. We have investigated the molecular basis of generating diverse pituitary cell phenotypes from a common precursor, providing in vivo and in vitro evidence that their development involves three sequential phases of signaling events and the action of a gradient at an ectodermal boundary. In the first phase, the BMP4 signal from the ventral diencephalon, expressing BMP4, Wnt5a, and FGF8, represents a critical dorsal neuroepithelial signal for pituitary organ commitment in vivo. Subsequently, a BMP2 signal emanates from a ventral pituitary organizing center that forms at the boundary of a region of oral ectoderm in which Shh expression is selectively excluded. This BMP2 signal together with a dorsal FGF8 signal, appears to create opposing activity gradients that are suggested to generate overlapping patterns of specific transcription factors underlying cell lineage specification events, whereas Wnt4 is needed for the expansion of ventral pituitary cell phenotypes. In the third phase, temporally specific loss of the BMP2 signal is required to allow terminal differentiation. The consequence of these sequential organ and cellular determination events is that each of the hormone-producing pituitary cell types-gonadotropes, thyrotropes, somatotropes, lactotropes, corticotropes, and melanotropes-appear to be determined, in a ventral-to-dorsal gradient, respectively.

Hedgehog signaling is required for pituitary gland development.

Pituitary gland development serves as an excellent model system in which to study the emergence of distinct cell types from a common primordium in mammalian organogenesis. We have investigated the role of the morphogen Sonic hedgehog (SHH) in outgrowth and differentiation of the pituitary gland using loss- and gain-of-function studies in transgenic mice. Shh is expressed throughout the ventral diencephalon and the oral ectoderm, but its expression is subsequently absent from the nascent Rathke's pouch as soon as it becomes morphologically visible, creating a Shh boundary within the oral epithelium. We used oral ectoderm/Rathke's pouch-specific 5' regulatory sequences (Pitx1(HS)) from the bicoid related pituitary homeobox gene (Pitx1) to target overexpression of the Hedgehog inhibitor Hip (Huntingtin interacting protein) to block Hedgehog signaling, finding that SHH is required for proliferation of the pituitary gland. In addition, we provide evidence that Hedgehog signaling, acting at the Shh boundary within the oral ectoderm, may exert a role in differentiation of ventral cell types (gonadotropes and thyrotropes) by inducing Bmp2 expression in Rathke's pouch, which subsequently regulates expression of ventral transcription factors, particularly Gata2. Furthermore, our data suggest that Hedgehog signaling, together with FGF8/10 signaling, synergizes to regulate expression of the LIM homeobox gene Lhx3, which has been proved to be essential for initial pituitary gland formation. Thus, SHH appears to exert effects on both proliferation and cell-type determination in pituitary gland development.

Multistep signaling and transcriptional requirements for pituitary organogenesis in vivo.

During development of the mammalian pituitary gland, specific hormone-producing cell types, critical in maintaining homeostasis, emerge in a spatially and temporally specific fashion from an ectodermal primordium. We have investigated the molecular basis of generating diverse cell phenotypes from a common precursor, providing in vivo and in vitro evidence that development of these cell types involves at least four sequential phases of signaling events and the action of a gradient at an ectodermal boundary. In the first phase, we hypothesize that this notochord induces invagination of Rathke's pouch from the oral ectoderm. This is followed by appearance of an ectodermal boundary, formed with exclusion of Shh from the nascent pouch. Next, signals from the ventral diencephalon--expressing BMP4, Wnt5a, FGF10, and FGF8--in concert with Shh represent critical in vivo signals for pituitary determination. Subsequently, a dorsal-ventral BMP2 signal gradient emanates from a ventral pituitary organizing center, forming at the boundary to oral ectoderm region from which Shh expression is selectively excluded. In concert with a dorsal FGF8 signal, this creates opposing gradients that generate overlapping patterns of specific transcription factors that underlie cell lineage specification events. The mechanisms by which these transient gradients of signaling molecules lead to the appearance of four ventral pituitary cell types appear to involve the reciprocal interactions of two transcription factors, Pit-1 and GATA-2, which are epistatic to the remainder of the cell type-specific transcription programs and serve as a molecular memory of the transient signaling events. Unexpectedly, this program includes a DNA-binding-independent function of Pit-1, suppressing the ventral GATA-2-dependent gonadotrope program by inhibiting GATA-2 binding to gonadotrope- but not thyrotrope-specific genes. This indicates that both DNA-binding-dependent and-independent actions of abundant determining factors contribute to generate distinct cell phenotypes. In the fourth phase, temporally specific loss of the BMP2 signal is required to allow terminal differentiation. The consequence of these sequential organ and cellular determination events is that each of the pituitary cell types--gonadotropes, thyrotropes, somatotropes, lactotropes, corticotropes, and melanotropes appears to be determined, in a ventral to dorsal gradient, respectively, apparently based on a combinatorial code of transcription factors induced by the gradient of specific signaling molecules.

Role of the Bicoid-related homeodomain factor Pitx1 in specifying hindlimb morphogenesis and pituitary development.

Pitx1 is a Bicoid-related homeodomain factor that exhibits preferential expression in the hindlimb, as well as expression in the developing anterior pituitary gland and first branchial arch. Here, we report that Pitx1 gene-deleted mice exhibit striking abnormalities in morphogenesis and growth of the hindlimb, resulting in a limb that exhibits structural changes in tibia and fibula as well as patterning alterations in patella and proximal tarsus, to more closely resemble the corresponding forelimb structures. Deletion of the Pitx1 locus results in decreased distal expression of the hindlimb-specific marker, the T-box factor, Tbx4. On the basis of similar expression patterns in chick, targeted misexpression of chick Pitx1 in the developing wing bud causes the resulting limb to assume altered digit number and morphogenesis, with Tbx4 induction. We hypothesize that Pitx1 serves to critically modulate morphogenesis, growth, and potential patterning of a specific hindlimb region, serving as a component of the morphological and growth distinctions in forelimb and hindlimb identity. Pitx1 gene-deleted mice also exhibit reciprocal abnormalities of two ventral and one dorsal anterior pituitary cell types, presumably on the basis of its synergistic functions with other transcription factors, and defects in the derivatives of the first branchial arch, including cleft palate, suggesting a proliferative defect in these organs analogous to that observed in the hindlimb.

Reciprocal interactions of Pit1 and GATA2 mediate signaling gradient-induced determination of pituitary cell types.

The mechanisms by which transient gradients of signaling molecules lead to emergence of specific cell types remain a central question in mammalian organogenesis. Here, we demonstrate that the appearance of four ventral pituitary cell types is mediated via the reciprocal interactions of two transcription factors, Pit1 and GATA2, which are epistatic to the remainder of the cell type-specific transcription programs and serve as the molecular memory of the transient signaling events. Unexpectedly, this program includes a DNA binding-independent function of Pit1, suppressing the ventral GATA2-dependent gonadotrope program by inhibiting GATA2 binding to gonadotrope- but not thyrotrope-specific genes, indicating that both DNA binding-dependent and -independent actions of abundant determining factors contribute to generate distinct cell phenotypes.

Regulation of somatic growth by the p160 coactivator p/CIP.

A family of p160 coactivators was initially identified based on ligand-dependent interactions with nuclear receptors and thought to function, in part, by recruiting CREB-binding protein/p300 to several classes of transcription factors. One of the p160 factors, p/CIP/AIB1, often amplified and overexpressed in breast cancer, also exhibits particularly strong interaction with CREB-binding protein/p300. In this manuscript, we report that p/CIP, which exhibits regulated transfer from cytoplasm to nucleus, is required for normal somatic growth from embryonic day 13.5 through maturity. Our data suggest that a short stature phenotype of p/CIP gene-deleted mice reflect both altered regulation of insulin-like growth factor-1 (IGF-1) gene expression in specific tissues and a cell-autonomous defect of response to IGF-1, including ineffective transcriptional activities by several classes of regulated transcription factors under specific conditions. The actions of p/CIP are therefore required for full expression of a subset of genes critical for regulating physiological patterns of somatic growth in mammals.