Structures of active and latent PAI-1: a possible stabilizing role for chloride ions.

Serpins exhibit a range of physiological roles and can contribute to certain disease states dependent on their various conformations. Understanding the mechanisms of the large-scale conformational reorganizations of serpins may lead to a better understanding of their roles in various cardiovascular diseases. We have studied the serpin, plasminogen activator inhibitor 1 (PAI-1), in both the active and the latent state and found that anionic halide ions may play a role in the active-to-latent structural transition. Crystallographic analysis of a stable mutant form of active PAI-1 identified an anion-binding site between the central beta-sheet and a small surface domain. A chloride ion was modeled in this site, and its identity was confirmed by soaking crystals in a bromide-containing solution and calculating a crystallographic difference map. The anion thus located forms a 4-fold ligated linchpin that tethers the surface domain to the central beta-sheet into which the reactive center loop must insert during the active-to-latent transition. Timecourse experiments measuring active PAI-1 stability in the presence of various halide ions showed a clear trend for stabilization of the active form with F(-) > Cl(-) > Br(-) >> I(-). We propose that the "stickiness" of this pin (i.e., the electronegativity of the anion) contributes to the energetics of the active-to-latent transition in the PAI-1 serpin.

Tool-related injuries among amateur and professional woodworkers.

Although woodworking is a popular hobby and the woodworking industry employs thousands of workers nationwide, few studies have examined injuries associated with this activity, especially in relation to woodshop tool use. We conducted a survey of amateur and professional woodworkers (n = 283) in New Mexico to determine histories and rates of tool-specific injuries. Injuries associated with woodshop tool use were reported by 64% of all respondents. Hammers, chisels/gouges, and table saws were most frequently reported in association with injuries, although the highest tool-specific injury rates were associated with use of jointer-planers (4.9 injuries per 1000 person-hours of use), chisels/ gouges (3.3 injuries), and drill presses (3.1 injuries). One third reported tool use-associated injuries that were severe enough to require professional medical attention; 5% of all respondents suffered partial amputations. Courses in the safe use of shop tools may help to reduce rates of injuries among woodworkers.

Dietary micronutrients and cervical dysplasia in southwestern American Indian women.

American Indian women in the Southwest have high rates of cervical cancer and cervical dysplasia in contrast to low rates of cancers for other sites. Despite their high rates of cervical disease, no published information has specifically examined risk factors for cervical cancer or cervical dysplasia among American Indian women. We carried out a pilot case-control study of cervical dysplasia in southwestern American Indian women to examine the relationship of dietary intake of vitamin C, folacin, vitamin E, carotenoids, and retinol with cervical cytological abnormalities. Twenty-four-hour dietary recalls were collected from women with cervical dysplasia (n = 42) and women with normal cervical cytologies (n = 58). Macro- and micronutrient intake was estimated from these recalls utilizing food and nutrient data from the USDA Survey Nutrient Database. Although mean differences between cases and controls were not statistically significant for any of the micronutrients examined, women with low intake of vitamin C, folacin, and vitamin E were at increased risk of having cervical dysplasia when the data were analyzed as stratified for level of intake (low vs. high intake odds ratios were 3.0 for vitamin C, 3.3 for folacin, and 1.7 for vitamin E). The relationship between dietary micronutrients and cervical dysplasia among American Indian women warrants further investigation using more refined measures of dietary micronutrient intake, together with consideration of other risk factors for cervical disease.

Low molecular weight human pulmonary surfactant protein (SP5): isolation, characterization, and cDNA and amino acid sequences.

Pulmonary surfactant is a lipid-protein complex that promotes alveolar stability by lowering the surface tension at the air-fluid interface in the peripheral air spaces. A group of hydrophobic surfactant-associated proteins has been shown to be essential for rapid surface film formation by surfactant phospholipids. We have purified a hydrophobic surfactant protein of approximately 5 kDa that we term SP5 from bronchopulmonary lavage fluid from a patient with alveolar proteinosis and shown that it promotes rapid surface film formation by simple mixtures of phospholipids. We have derived the full amino acid sequence of human SP5 from the nucleotide sequence of cDNAs identified with oligonucleotide probes based on the NH2-terminal sequence of SP5. SP5 isolated from surfactant is a fragment of a much larger precursor protein (21 kDa). The precursor contains an extremely hydrophobic region of 34 amino acids that comprises most of the mature SP5. This hydrophobicity explains the unusual solubility characteristics of SP5 and the fact that it is lipid-associated when isolated from lung.

Conservation of the DNA binding domain and other properties between porcine and rat glucocorticoid receptors.

Activated glucocorticoid receptor protein (GCR) was partially purified from porcine liver cytosol by sequential chromatography on phosphocellulose and DNA-cellulose using a modification of a protocol developed for purification of rat GCR. This partially purified preparation, when separated by SDS-polyacrylamide gel electrophoresis and immunoblotted, indicated that a Mr = 94,000 protein band cross-reacts with a monoclonal antibody against rat GCR. A nitrocellulose filter binding assay showed that both the partially purified porcine and rat GCRs interact specifically with a cloned synthetic 24 base pair deoxyoligonucleotide containing the GCR binding sequence in the first intron of the human growth hormone (hGH) gene. This specific protein-DNA interaction is blocked by a single base pair change in the binding site. All three putative domains of the GCR molecule: the steroid binding, immunoreactive, and DNA binding have been conserved between two divergent species.

The role of extracellular calcium in corticotropin-stimulated steroidogenesis.

The role of extracellular Ca2+ in the binding of corticotropin (ACTH) to adrenocortical cell receptors as well as in the post-binding events involved in steroidogenesis were investigated. Binding studies using [125I-Tyr23,Phe2,Nle4]ACTH (1-38) peptide showed that extracellular Ca2+ is essential not only for the interaction of ACTH with its receptor, but also for continued occupancy of the receptor. In view of the requirement of Ca2+ for binding the hormone to the receptor, the role of Ca2+ in post-receptor events was investigated by covalently attaching the hormone to its receptor by photoaffinity labeling in the presence of Ca2+. Persistent activation of steroidogenesis induced by photoaffinity labeling in the presence of Ca2+ was depressed when cells were incubated in medium containing EGTA but was unaffected when the cells were merely washed and incubated in Ca2+-free medium. In the presence of EGTA, 8-Br-cAMP partially restored persistent activation of steroidogenesis. The concentration of extracellular Ca2+ required for restoring steroidogenesis was 10-fold lower than the concentration of Ca2+ needed for optimal binding of ACTH to its receptor. These results suggest that the primary role of extracellular Ca2+ in the action of ACTH is to facilitate the association of the hormone with its receptor.

The first intron of the human growth hormone gene contains a binding site for glucocorticoid receptor.

Glucocorticoid receptor (GCR) protein stimulates transcription from a variety of cellular genes. We show here that GCR partially purified from rat liver binds specifically to a site within the first intron of the human growth hormone (hGH) gene, approximately 100 base pairs downstream from the start of hGH transcription. GCR binding is selectively inhibited by methylation of two short, symmetrically arranged clusters of guanine residues within this site. A cloned synthetic 24-base-pair deoxyoligonucleotide containing the predicted GCR binding sequence interacts specifically with GCR. The hGH binding site shares sequence homology with a GCR binding site upstream from the human metallothionein II gene and a subset of GCR binding sites from mouse mammary tumor virus. All of these binding sites for this eukaryotic transcriptional regulatory protein show remarkable similarity in overall geometry to the binding sites for several prokaryotic transcriptional regulatory proteins.

Characterization of adrenocorticotropin receptors that appear when 3T3-L1 cells differentiate into adipocytes.

The binding of an 125I-labeled analog of ACTH, [125I]Tyr23,Phe2,Nle4-ACTH-(1-38), to differentiated 3T3-L1 fat cells was characterized. Time-dependent binding, which was inhibited by saturating concentrations of unlabeled ACTH (0.44 microM), could be demonstrated in the differentiated cells. Using 0.4 nM [125I]ACTH analog and increasing concentrations of ACTH, the half-maximal concentration for inhibition by ACTH was 4.3 nM. Scatchard analysis demonstrated a single class of ACTH binding. There were approximately 3500 binding sites/cell. The binding of [125I]ACTH analog was specific in that it could be displaced by ACTH, ACTH-(1-19), ACTH-(1-17), and N-acetyl-Ser1-ACTH, but not by high concentrations of insulin, beta-endorphin, or polylysine. There was an excellent correlation between the ability of ACTH and its analogs to inhibit [125I]ACTH analog binding and the ability of ACTH and its analogs to stimulate cAMP production. In contrast, no saturable binding could be demonstrated when undifferentiated 3T3-L1 fibroblasts, which are not responsive to ACTH, were studied. Thus, differentiation of 3T3-L1 cells into the adipocyte form is accompanied by the appearance of receptors for ACTH. These receptors allow the adipocytes to respond to ACTH.

Chemical and biological characterization of salmon melanocyte-stimulating hormones.

Ten peptides related to melanocyto-stimulating hormone (MSH) have been identified in an acid acetone extract of the chum salmon pituitary. All these peptides are related to the alpha-MSH and beta-MSH families, but no peptide related to gamma-MSH has been found. This result is in accordance with the finding that the gamma-MSH segment is deleted from the N-terminal peptide of salmon pro-opiocortin (NPP I). Based on the structures of newly isolated peptides, the maturation process of MSH is discussed. The major components of salmon MSH were tested for biological activities. In the lipolytic assay with rabbit fat cells, alpha-MSH I and alpha-MSH II were equipotent, but beta-MSH I and NPP I exhibited very low or no activity. On the other hand, the des-acetyl-alpha-MSH I was found to be four times as potent as alpha-MSH I in this assay. The steroidogenic activities of alpha-MSH I and N-des-acetyl-alpha-MSH I were approximately 0.05% of the potency of ovine ACTH. All other peptides exhibited less than 0.01% potency. Salmon alpha-MSHs were found to be somewhat more potent melanophore-stimulating agents than the beta-MSHs.

Characterization of corticotropin receptors on adrenocortical cells.

The binding of corticotropin (ACTH) to receptors on isolated rat adrenocortical cells was investigated with the aid of [[125I]ITyr23, Phe2, Nle4]ACTH-(1-38) (125I-ACTH analog) which retained full biological potency and had a specific radioactivity of 1800 +/- 75 Ci/mmol. Binding was highly specific to adrenocortical cells, and the radioactive peptide was displaced by low concentrations of ACTH but not by other basic peptides. Binding was rapid, reversible, and linearly related to the number of cells. 125I-ACTH analog was not significantly degraded by incubation with the cells at 23 degrees C for 1 hr. Scatchard analysis of the binding was compatible with a single class of binding sites with Kd = 1.41 +/- 0.21 nM, and the number of sites was estimated to be 3840 +/- 1045 per cell. The binding curve was superimposable on the concentration-response curve for cyclic AMP. Small, but significant amounts of 125I-ACTH analog were bound at concentrations sufficient for maximal stimulation of steroidogenesis. For a series of ACTH analogs, the concentrations of the peptides required for half-maximal stimulation of cyclic AMP production were in excellent agreement with the concentration required for half-maximal inhibition of binding. These results suggest that the adrenocortical cells contain only one class of ACTH receptors and that stimulation of a small fraction of these receptors (less than 3%) is sufficient for maximal steroidogenesis.

A sensitive radioimmunoassay for corticotropin using a fully biologically active 125I-labeled ligand.

The human corticotropin (ACTH) analog, Phe2,Nle4-ACTH-(1-38) was iodinated by the chloramine-T procedure and the product was purified by reverse phase high performance liquid chromatography. The specific radioactivity of [125I]Tyr23,Phe2,Nle4-ACTH-(1-38) was determined by comparing the antiserum binding curves of the iodinated peptide and [3H]ACTH of known specific activity. This method gave a value of 1800 +/- 75 Ci/mmol, which is close to the theoretical radioactivity expected for the introduction of a single 125I atom into the peptide. [125I]Tyr23,Phe2,Nle4-ACTH-(1-38) was as potent as ACTH in stimulating corticosterone production in isolated rat adrenocortical cells. The concentrations for half-maximal steroidogenesis were 36.5 +/- 6.1 pM for the 125I derivative and 37.6 +/- 6.7 pM for ACTH. By the use of this 125I-labeled ligand, a highly sensitive RIA capable of detecting 1 pg aCTH was developed.l The antiserum employed in this study appeared to be directed against residues 11-13 of ACTH.

Synthesis of a corticotropin analog that retains full biological activity after iodination.

In order to circumvent the drastic decrease in biological activity that accompanies iodination of corticotropin, we have synthesized an analog of the human hormone containing phenylalanine in place of tyrosine in position 2 and norleucine in place of methionine in position 4 to give [Phe2,Nle4]ACTH-(1-38). This analog, referred to as Phe2,Nle4-ACTH-(1-38), was purified by ion exchange chromatography and partition chromatography. Phe2,Nle4-ACTH-(1-38) was found to be indistinguishable from synthetic human ACTH in its ability to stimulate corticosterone production in isolated rat adrenocortical cells. Iodination of Phe2,Nle4-ACTH-(1-38) resulted in the formation of monoiodo-Tyr23,Phe2,Nle4-ACTH-(1-38) and diiodo-Tyr23,Phe2,Nle4-ACTH-(1-38), which were completely separated from each other and unmodified Phe2,Nle4-ACTH-(1-38) by reverse phase high performance liquid chromatography. Monoiodo-Tyr23,Phe4,Nle4-ACTH-(1-38) was also found to be as potent as ACTH in stimulating steroidogenesis. These results show that Phe2,Nle4-ACTH-(1-38) can be used for the preparation of the radioligand with full biological activity for studying physiologically relevant corticotropin receptors and for use in RIA.

Isolation of alpha-melanotropin and N, O-diacetylserine1-alpha-melanotropin from porcine pituitary extracts.

In the course of isolation of alpha-melanotropin (alpha-MSH) from porcine pituitary extracts, a peptide with identical amino acid composition but slightly less polar properties compared to alpha-MSH was detected and isolated by partition chromatography. The new peptide was identified as N, O-diacetyl-Ser1-alpha-MSH by high resolution nuclear magnetic resonance spectroscopy. N, O-diacetyl-Ser1-alpha-MSH was readily converted to alpha-MSH by incubation at pH 9.5. The O-acetyl analog of alpha-MSH was found to be as active as alpha-MSH in stimulating lipolysis in isolated rabbit adipocytes.

Preparation of tritiated alpha-melanotropin with high specific radioactivity.

alpha-Melanotropin (alpha-MSH) was iodinated and 3,5-diiodotyr2-alpha-MSH was isolated by reverse phase high performance liquid chromatography (HPLC). Catalytic dehalogenation of the diiodo derivative in the presence of tritium resulted in the formation of 3,5-ditritiotyr2-alpha-MSH. The tritiated peptide was purified by ion exchange and partition chromatography. The radioactive peptide was found to be homogeneous and identical to alpha-MSH by paper electrophoresis and HPLC. The tritiated alpha-MSH stimulated lipolysis in rabbit adipocytes nearly as well as alpha-MSH. The specific radioactivity of tritiated alpha-MSH was 42 Ci/mmol or 73% of the theoretical value.

Photoaffinity labeling of corticotropin receptors.

A photoaffinity label for corticotropin (ACTH) receptors was prepared by selective chemical modification of the single tryptophan residue in the hormone by reaction with 2-nitro-5-azidophenylsulfenyl chloride. The photoreactive derivative, [(2-nitro-5-azidophenylsulfenyl)-Trp9]ACTH (2,5-NAPS-ACTH), stimulated corticosterone synthesis to 60% of the maximal rate induced by ACTH in isolated rat adrenocortical cells. 2.5-NAPS-ACTH caused only a marginal stimulation of cyclic AMP production compared to the unmodified hormone. Stimulation of corticosterone production and cyclic AMP accumulation induced by ACTH were both inhibited in a competitive manner by 2,5-NAPS-ACTH. Photolysis of adrenocortical cells in the presence of 2,5-NAPS-ACTH resulted in a 40% inactivation of ACTH receptors mediating steroidogenesis, as shown by the decrease in response to subsequent stimulation with ACTH. No loss of function was observed when photolysis was conducted in the presence of the photoresistant analog [(2,4-dinitrophenylsulfenyl)-Trp9]ACTH. Covalent attachment of the hormone to the receptors was also demonstrated by photolyzing adrenocortical cells in the presence of tritiated 2,5-NAPS-ACTH of high specific radioactivity (90 Ci/mmol) and analyzing the cell proteins by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. A protein with an approximate molecular weight of 100,000 was specifically labeled by this procedure. The unique labeling of an adrenocortical cell protein and the concomitant loss of ACTH responsiveness suggest that physiologically relevant receptors are photolabeled by this method.