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Dimethandrolone undecanoate (DMAU), an oral investigational male hormonal contraceptive, is a prodrug that is rapidly converted to its active metabolite, dimethandrolone (DMA). Poor and variable oral bioavailability of DMA after DMAU dosing is a critical challenge to develop it as an oral drug. The objective of our study was to elucidate the mechanisms of variable pharmacokinetics of DMA. We first identified DMA metabolites formed in vitro and in vivo in human hepatocyte incubation and serum samples following oral DMAU administration in men, respectively. The metabolite identification study revealed two metabolites, DMA-glucuronide (DMA-G; major) and the androstenedione analog of DMA (minor), in the hepatocyte incubations. After oral DMAU administration, only DMA-G was detected in serum, which was >100-fold compared with DMA levels, supporting glucuronidation as the major elimination mechanism for DMA. Next, 13 clinically relevant UDP-glucuronosyltransferase (UGT) enzymes were tested for their involvement in DMA-G formation, which revealed a major role of UDP-glucuronosyltransferase 2B17 (UGT2B17) isoform with a smaller contribution of UGT1A9 in DMA-G formation. These data were confirmed by dramatically higher DMA glucuronidation rates (>200- and sevenfold) in the high versus the null UGT2B17-expressing human intestinal and liver microsomes, respectively. Since human UGT2B17 is a highly variable enzyme with a 20%–80% gene deletion frequency, the in vitro data suggest a major role of UGT2B17 polymorphism on the first-pass metabolism of DMA. Further, considering DMA is a selective and sensitive UGT2B17 substrate, it could be used as a clinical probe of UGT2B17 activity. SIGNIFICANCE STATEMENT: Dimethandrolone (DMA) is an active metabolite of dimethandrolone undecanoate (DMAU), an investigational male hormonal contraceptive. Previous studies have indicated poor and inconsistent bioavailability of DMAU following oral administration. This study found that UDP-glucuronosyltransferase 2B17–mediated high intestinal first-pass metabolism is the key mechanism of variable DMA bioavailability.

The long and challenging drug development process begins with discovery biology for the selection of an appropriate target for a specific indication. Target is a broad term that can be applied to a range of biological entities such as proteins, genes, and ribonucleic acids (RNAs). Although there are numerous databases available for mining biological entities, publicly available searchable, downloadable databases to aid in target selection for a specific disease or indication (e.g., developing contraceptives and infertility treatments) are limited. We report the development of the Contraceptive and Infertility Target DataBase (https://www.citdbase.org), which provides investigators an interface to mine existing transcriptomic and proteomic resources to identify high-quality contraceptive/infertility targets. The development of similar databases is applicable to the identification of targets for other diseases and conditions.

Ruptured abdominal aortic aneurysms (RAAAs) are surgical emergencies that require immediate and expert treatment. It has been unclear whether presentation during evenings and weekends, when "on call" teams are primarily responsible for patient care, is associated with worse outcomes. Our objective was to evaluate the outcomes of patients presenting with RAAAs after-hours vs during the workday.A retrospective cohort study of all RAAAs in Nova Scotia between 2005 and 2015 was performed through linkage of administrative databases. Patients who had presented to the hospital with RAAAs during the workday (Monday through Friday, 6 am to 6 pm) were compared with those who had presented after-hours (6 pm to 6 am during the week and on weekends). The baseline and operative characteristics were identified for all patients through the available databases and a review of the medical records. Mortality before surgery, 30-day mortality, and operative mortality were compared between groups using multivariable logistic regression, adjusting for factors clinically significant on univariable analysis.A total of 390 patients with RAAAs were identified from 2005 to 2015, of whom 205 (53%) had presented during the workday and 185 (47%) after-hours. The overall chance of survival (OCS) was 45% overall, 49% if admitted to hospital, and 64% if surgery had been performed. During the workday, the OCS was 43% overall, 48% if admitted to hospital, and 67% if surgery had been performed. After-hours, the OCS was 46% overall, 49% if admitted to hospital, and 61% if surgery had been performed. Mortality before surgery was increased for patients who had presented to the hospital during the workday compared with after-hours (36% vs 26%; P = .04). The 30-day mortality (57% vs 54%; P = .62), rates of operative management (63% vs 72%; P = .06), and operative mortality (33% vs 39%; P = .33) were similar between the workday and after-hours groups (57% vs 54%; P = .06). After adjusting for significant clinical variables, the patients who had presented with RAAAs after-hours had had a similar odds of dying before surgery (odds ratio [OR], 0.64; 95% confidence interval [CI], 0.41-1.03), operative management (OR, 1.47; 95% CI, 0.93-2.31), 30-day mortality (OR, 0.98; 95% CI, 0.63-1.51), and operative mortality (OR, 1.33; 95% CI, 0.78-2.26). In the subgroup of patients presenting to a hospital with endovascular capabilities, patients presenting after-hours had had similar odds of 30-day mortality (OR, 1.07; 95% CI, 0.57-2.02), and operative mortality (OR, 1.14; 95% CI, 0.58-2.23).We found that patients presenting to the hospital with RAAAs after-hours did not have increased adjusted odds of mortality before surgery, operative management, 30-day mortality, or operative mortality.

BackgroundThe advent of new methods of male contraception would increase contraceptive options for men and women and advance male contraceptive agency. Pharmaceutical R&D for male contraception has been dormant since the 1990s. The Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) has supported a contraceptive development program since 1969 and supports most ongoing hormonal male contraceptive development. Nonhormonal methods are in earlier stages of development.ContentSeveral hormonal male contraceptive agents have entered clinical trials. Novel single agent products being evaluated include dimethandrolone undecanoate, 11β-methyl-nortestosterone dodecylcarbonate, and 7α-methyl-19-nortestosterone. A contraceptive efficacy trial of Nestorone®/testosterone gel is underway. Potential nonhormonal methods are at preclinical stages of development. Many nonhormonal male contraceptive targets that affect sperm production, sperm function, or sperm transport have been identified.SummaryNICHD supports development of reversible male contraceptive agents. Other organizations such as the World Health Organization, the Population Council, and the Male Contraception Initiative are pursuing male contraceptive development, but industry involvement remains limited.

Objective The objective of this study was to evaluate the safety and effectiveness of single ProGlide use per bilateral access site for endovascular aneurysm repair (EVAR) of abdominal aortic aneurysms. Methods A retrospective cohort study was performed for all elective percutaneous EVARs from November 2015 to December 2017 at the QEII Health Sciences Centre (Halifax, Nova Scotia, Canada). Exposure of interest was number of ProGlides used per access site, dichotomized into bilateral single ProGlide closure vs nonsingle ProGlide closure on at least one femoral arteriotomy. Outcomes included Valve Academic Research Consortium (VARC)-2 and Bleeding Academic Research Consortium (BARC) criteria. Groups were compared with Fisher exact test, analysis of variance, or Wilcoxon rank sum, as appropriate. Logistic regression was used to compare the effect of single ProGlide use on VARC-2 and BARC criteria. Results A total of 131 cases were included, of which 116 had bilateral single ProGlide use for access closure. Baseline characteristics including comorbidities and smoking status were compared between groups. Groups were similar for all characteristics except smoking status, with an increased proportion of former smokers in the nonsingle ProGlide group. There were 119 (90.8%) patients who had single ProGlide use on the right femoral artery and 121 (92.4%) on the left; 16 (12.2%) patients had ProGlide deployment issues. Median maximal right and left femoral sheath diameters were 16F (interquartile range [IQR], 16F-18F) and 14F (IQR, 14F-16F), respectively. Median length of stay was 1 day (IQR, 1-1 day). VARC-2 criteria occurred in 8 of 131 (6.11%) patients, 6 of 116 (5.17%) with bilateral single ProGlides and 2 of 15 (13.3%) with nonsingle ProGlides. BARC criteria occurred in 6 of 131 (4.58%) patients, 5 of 116 (4.31%) with bilateral single ProGlides and 1 of 15 (6.67%) with nonsingle ProGlides. Single ProGlide use was not associated with a difference in VARC-2 (odds ratio, 0.35; 95% confidence interval, 0.64-1.94) or BARC (odds ratio, 0.63; 95% confidence interval, 0.07-6.79) criteria. No patients developed pseudoaneurysms or required repeated intervention for bleeding. Conclusions Single ProGlide use per vascular access site in patients undergoing EVAR is a safe and effective method for access closure with sheath diameters up to and including 16F.

Neutrophils are rapidly recruited from the peripheral blood to the inflammatory site to initiate inflammatory response against pathogenic infections. The process to recruit neutrophils must be properly regulated since the abnormal accumulation of neutrophils can cause organ damage and dysfunction. The acute respiratory distress syndrome (ARDS)/acute lung injury (ALI) is a common cause of respiratory failure that is characterized by the infiltration of neutrophils and epithelial integrity disruption. Indeed, recent studies suggest a pathogenic role of neutrophils in the clinic severity of the coronavirus disease 2019 (COVID-19) ARDS. The chemokine CXCL1, which is rapidly induced by inflammatory stimuli, plays a key role in neutrophil influx during lung inflammation. The molecular basis ofCxcl1induction is not fully understood. Here we report that TET1, a member of the ten eleven translocation (TET) methylcytosine dioxygenase protein family, displays a striking specificity in the regulation of gene expression in macrophages. RNA sequencing (RNA-seq) analysis showed thatTet1disruption significantly altered the expression of only 48 genes that includeCxcl1and several other genes known to be important for cell migration and trafficking in bone marrow derived macrophages (BMDMs) in response to LPS stimulation. TET1 regulates the induction ofCxcl1by facilitating the DNA demethylation of theCxcl1promoter. InTet1−/−mice, the induction ofCxcl1was suppressed, resulting in defective neutrophil recruitment to the lung during LPS-induced acute lung injury. Our results identify a novel epigenetic mechanism that selectively controlsCxcl1induction and neutrophil recruitment during acute lung injury.Key PointsTET1 has a striking specificity in macrophage gene regulation and controlsCxcl1induction by inflammatory stimuli via DNA demethylationNeutrophil recruitment is defective inTet1deficient mice during acute lung injury

BACKGROUNDDevelopment of new methods of male contraception would address an unmet need for men to control their fertility and could increase contraceptive options for women. Pharmaceutical research and development for male contraception was active in the 1990s but has been virtually abandoned. The Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) has supported a contraceptive development program since 1969 and supports the majority of hormonal male contraceptive development. Nonhormonal methods are also in development but are at earlier stages.CONTENTSeveral hormonal male contraceptive agents have entered clinical trials. Single-agent products being evaluated include dimethandrolone undecanoate, 11β-methyl-nortestosterone dodecyl carbonate, and 7α-methyl-19-nortestosterone. A contraceptive efficacy trial of Nestorone® gel and testosterone gel in a single application will begin in 2018. Potential nonhormonal methods are at preclinical stages of development. Many nonhormonal male contraceptive targets that affect either sperm production or sperm function have been identified. Targeted pathways include the retinoic acid pathway, bromodomain and extraterminal proteins, and pathways for Sertoli cell–germ cell adhesion or sperm motility. Druggable targets include CatSper, the sperm Na+/K+-exchanger, TSSK, HIPK4, EPPIN, and ADAMs family proteins. Development of a procedure to reversibly block the vas deferens (initially developed in India in the 1980s) is undergoing early stage research in the US under the trade name Vasalgel™.SUMMARYNICHD has supported the development of reversible male contraceptive agents. Other organizations such as the World Health Organization and the Population Council are pursuing male contraceptive development, but industry involvement remains dormant.

Clouds and aerosols increase the fraction of global solar irradiance that is diffuse light. This phenomenon is known to increase the photosynthetic light use efficiency (LUE) of closed-canopy vegetation by redistributing photosynthetic photon flux density (400–700 nm) from saturated, sunlit leaves at the top of the canopy, to shaded leaves deeper in the canopy. We combined a process-based carbon cycle model with 10 years of eddy covariance carbon flux measurements and other ancillary data sets to assess 1) how this LUE enhancement influences interannual variation in carbon uptake, and 2) how errors in modeling diffuse fraction affect predictions of carbon uptake. Modeled annual gross primary productivity (GPP) increased by ≈0.94% when observed levels of diffuse fraction were increased by 0.01 (holding total irradiance constant). The sensitivity of GPP to increases in diffuse fraction was highest when the diffuse fraction was low to begin with, and lowest when the diffuse fraction was already high. Diffuse fraction also explained significantly more of the interannual variability of modeled net ecosystem exchange (NEE), than did total irradiance. Two tested radiation partitioning models yielded over- and underestimates of diffuse fraction at our site, which propagated to over- and underestimates of annual NEE, respectively. Our findings highlight the importance of incorporating LUE enhancement under diffuse light into models of global primary production, and improving models of diffuse fraction.

CONTEXT: 11β-Methyl-19-nortestosterone-17β-dodecylcarbonate (11β-MNTDC) is an orally bioavailable prodrug of 11β-methyl-19-nortestosterone (11β-MNT) with androgenic and progestational activity. OBJECTIVES: (i) Quantify 11β-MNT binding to androgen and progesterone receptors. (ii) Evaluate safety, tolerability, and serum gonadotropin and testosterone suppression by 11β-MNTDC in men. DESIGN AND SETTING: (i) In vitro receptor binding and transactivation studies and (ii) randomized, double-blind, placebo-controlled single-dose, dose-escalating phase I study at two academic medical centers. PARTICIPANTS AND INTERVENTION: Twelve healthy male volunteers were randomized (five active, one placebo) to escalating single oral doses (100, 200, 400, and 800 mg) of 11β-MNTDC or placebo given with or without food. MAIN OUTCOME MEASURES: (i) In vitro 11β-MNT/11β-MNTDC human receptor binding and transactivation and (ii) safety and tolerability, pharmacokinetics, and quantification of serum gonadotropin and testosterone concentrations for 24 hours following dosing. RESULTS: 11β-MNT avidly binds and activates human androgen and progesterone receptors, but 11β-MNTDC has minimal activity. Single oral doses of 11β-MNTDC were well tolerated without serious adverse events. Administration of 11β-MNTDC with food markedly increased average 11β-MNTDC and 11β-MNT serum concentrations (P < 0.001 for all doses) compared with fasting with a significant dose-related effect on average serum drug concentrations (P < 0.0001). The 200-, 400-, and 800-mg doses significantly suppressed average serum testosterone concentrations (P < 0.05). CONCLUSIONS: A single, oral dose of 11β-MNTDC up to 800 mg administered with food is safe and well tolerated in healthy men. The active drug 11β-MNT has androgenic and progestational activity, rapidly suppresses serum testosterone, and is a promising candidate for an effective once-daily oral male hormonal contraceptive.

Objective To compare postoperative morphine equivalent intake after open abdominal aortic aneurysm (AAA) repair among analgesic modalities: systemic analgesia (SA) only with no regional anesthesia, surgically positioned paravertebral catheter (PVC), and thoracic epidural analgesia (TEA). Methods This retrospective cohort study included patients undergoing elective open AAA at the Queen Elizabeth II Health Science Center, Halifax, Nova Scotia. Demographics, morphine equivalents, methods of analgesia administration, and outcomes data were collected on all patients from 2005 to 2016. Total morphine equivalent (MEQ) on postoperative days (PODs) 1, 2, and 3 were compared among patients with SA, PVC, and TEA. A multivariable zero-inflated log-linear regression was used to determine the association between analgesic modality and MEQ. Multivariable logistic regression models were used to determine associations between analgesic modality and postoperative pain, rates of discharge from intensive care within 1 day and opioid-related adverse events. Results The study cohort included 355 patients: 177 retroperitoneal and 178 transperitoneal repairs; 173 patients underwent SA, 117 PVC, and 65 TEA. On POD1, median MEQs were 984 (interquartile range [IQR], 342-1525) for SA, 89 (33-246) for PVC, and 49 (0-90) for TEA. On POD2, the median MEQs were 105 (IQR, 57-210) for SA, 45 (15-99) for PVC, and 30 (0-64) for TEA. On POD3, the median MEQs were 45 (IQR, 15-120) for SA, 30 (0-60) for PVC, and 10 (0-45) for TEA. On multivariable log-linear regression, compared with SA, PVC and TEA were associated with increased odds of receiving no opioids on POD1 (odds ratio [OR], 66.85; 95% confidence interval [CI], 17.49-255.57; and OR, 214.68; 95% CI, 60.20-766.38; respectively), POD 2 (OR, 6.97; 95% CI, 3.61-13.46; and OR, 28.73; 95% CI, 15.68-52.62; respectively), and POD 3 (OR, 3.93; 95% CI, 2.72-5.67; and OR, 4.68; 95% CI, 3.20-6.86; respectively). If patients did receive opioids, compared with SA, PVC and TEA were associated with decreased consumption on POD1 (RR, 0.22; 95% CI, 0.18-0.27; and RR, 0.16; 95% CI, 0.12-0.20; respectively), POD2 (RR, 0.50; 95% CI, 0.42-0.58; and RR, 0.46; 95% CI, 0.37-0.56; respectively), and POD3 (RR, 0.78; 95% CI, 0.66-0.93; and RR, 0.76; 95% CI, 0.63-0.93; respectively). Compared with SA, PVC was associated with earlier discharge from intensive care (OR, 2.75; 95% CI, 1.17-6.45) and TEA was not (OR, 1.12; 95% CI, 0.56-2.2). Compared with TEA, PVC was not associated with increased rate of opioid-related adverse events (OR, 0.44; 95% CI, 0.08-2.44). Conclusions PVC and TEA are associated with decreased MEQ compared with SA. PVC is associated with earlier discharge from intensive care compared with SA and similar rates of opioid-related adverse events compared with TEA. Paravertebral analgesia appears to be a safe and effective analgesic modality in patients undergoing retroperitoneal approach for abdominal aneurysm repair.

Paravertebral catheters are a well-established analgesic modality in thoracic surgery but have not been described in abdominal aortic surgery. We describe a simple, safe, and effective technique of paravertebral catheter insertion by the operative surgeon after a retroperitoneal abdominal aortic aneurysm repair. Once the aneurysm repair is complete, an extrapleural plane between the parietal pleura and the twelfth rib is created through blunt dissection. A catheter is advanced into the space percutaneously under direct vision, and a continuous infusion of local anesthetic is administered. Paravertebral catheters typically remain in place for 3 to 5 days and provide excellent postoperative non-narcotic analgesia.

Asunaprevir (ASV; BMS-650032) is a hepatitis C virus (HCV) NS3 protease inhibitor that has demonstrated efficacy in patients chronically infected with HCV genotype 1 when combined with alfa interferon and/or the NS5A replication complex inhibitor daclatasvir. ASV competitively binds to the NS3/4A protease complex, with K i values of 0.4 and 0.24 nM against recombinant enzymes representing genotypes 1a (H77) and 1b (J4L6S), respectively. Selectivity was demonstrated by the absence of any significant activity against the closely related GB virus-B NS3 protease and a panel of human serine or cysteine proteases. In cell culture, ASV inhibited replication of HCV replicons representing genotypes 1 and 4, with 50% effective concentrations (EC 50 s) ranging from 1 to 4 nM, and had weaker activity against genotypes 2 and 3 (EC 50 , 67 to 1,162 nM). Selectivity was again demonstrated by the absence of activity (EC 50 , >12 μM) against a panel of other RNA viruses. ASV exhibited additive or synergistic activity in combination studies with alfa interferon, ribavirin, and/or inhibitors specifically targeting NS5A or NS5B. Plasma and tissue exposures in vivo in several animal species indicated that ASV displayed a hepatotropic disposition (liver-to-plasma ratios ranging from 40- to 359-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥110-fold above the inhibitor EC 50 s observed with HCV genotype-1 replicons. Based on these virologic and exposure properties, ASV holds promise for future utility in a combination with other anti-HCV agents in the treatment of HCV-infected patients.

Asunaprevir (BMS-650032) is a potent hepatitis C virus (HCV) NS3 protease inhibitor demonstrating efficacy in alfa interferon-sparing, direct-acting antiviral dual-combination regimens (together with the NS5A replication complex inhibitor daclatasvir) in patients chronically infected with HCV genotype 1b. Here, we describe a comprehensive in vitro genotypic and phenotypic analysis of asunaprevir-associated resistance against genotypes 1a and 1b using HCV replicons and patient samples obtained from clinical studies of short-term asunaprevir monotherapy. During genotype 1a resistance selection using HCV replicons, the primary NS3 protease substitutions identified were R155K, D168G, and I170T, which conferred low- to moderate-level asunaprevir resistance (5- to 21-fold) in transient-transfection susceptibility assays. For genotype 1b, a higher level of asunaprevir-associated resistance was observed at the same selection pressures, ranging from 170- to 400-fold relative to the wild-type control. The primary NS3 protease substitutions identified occurred predominantly at amino acid residue D168 (D168A/G/H/V/Y) and were associated with high-level asunaprevir resistance (16- to 280-fold) and impaired replication capacity. In asunaprevir single-ascending-dose and 3-day multiple-ascending-dose studies in HCV genotype 1a- or 1b-infected patients, the predominant pre-existing NS3 baseline polymorphism was NS3-Q80K. This substitution impacted initial virologic response rates in a single-ascending-dose study, but its effects after multiple doses were more ambiguous. Interestingly, for patient NS3 protease sequences containing Q80 and those containing K80, susceptibilities to asunaprevir were comparable when tested in an enzyme assay. No resistance-associated variants emerged in these clinical studies that significantly impacted susceptibility to asunaprevir. Importantly, asunaprevir-resistant replicons remained susceptible to an NS5A replication complex inhibitor, consistent with a role for asunaprevir in combination therapies.

A phenotypic high-throughput cell culture screen was performed to identify compounds that prevented proliferation of the human Papilloma virus type 16 (HPV-16) transformed cell line Ca Ski. A series of quinoxaline compounds exemplified by Compound 1 was identified. Testing against a panel of cell lines demonstrated that Compound 1 selectively inhibited replication of all HPV-16, HPV-18, and HPV-31 transformed cell lines tested with 50% Inhibitory Concentration (IC50) values of 2 to 8 μM relative to IC50 values of 28 to 73 μM in HPV-negative cell lines. Treatment with Compound 1 resulted in a cascade of multiple apoptotic events, including selective activation of effector caspases 3 and 7, fragmentation of cellular DNA, and PARP (poly(ADP-ribose) polymerase) cleavage in HPV-positive cells relative to HPV-negative cells. Unregulated proliferation of HPV transformed cells is dependent on the viral oncogenes, E6 and E7. Treatment with Compound 1 resulted in a decrease in HPV E7 protein in Ca Ski cells. However, the timing of this reduction relative to other effects of compound treatment suggests that this was a consequence, rather than a cause, of the apoptotic cascade. Likewise, compound treatment resulted in no obvious effects on the E6- and E7- mediated down regulation of p53 and Rb, or their downstream effectors, p21 or PCNA. Further investigation of apoptotic signals induced by Compound 1 revealed cleavage of Caspase-8 in HPV-positive cells as early as 2 hours post-treatment, suggesting the compound initiates apoptosis through the extrinsic, death receptor-mediated, pathway of cell death. These studies provide proof of concept that cells transformed by oncogenic Papillomaviruses can be selectively induced to undergo apoptosis by compound treatment.

Thylakoid soluble phosphoprotein of 9 kDa (TSP9) has been identified as a plant-specific protein in the photosynthetic thylakoid membrane (Carlberg et al. (2003) Proc. Natl. Acad. Sci. 100, 757-762). Nonphosphorylated TSP9 is associated with the membrane, whereas, after light-induced phosphorylation, a fraction of the phosphorylated TSP9 is released into the aqueous stroma. By NMR spectroscopy, we have determined the structural features of nonphosphorylated TSP9 both in aqueous solution and in membrane mimetic micelles. The results show that both wild type nonphosphorylated TSP9 and a triple-mutant (T46E + T53E + T60E) mimic of the triphosphorylated form of TSP9 are disordered under aqueous conditions, but adopt an ordered conformation in the presence of detergent micelles. The micelle-induced structural features, which are similar in micelles either of SDS or dodecylphosphocholine (DPC), consist of an N-terminal alpha-helix, which may represent the primary site of interaction between TSP9 and binding partners, and a less structured helical turn near the C-terminus. These structured elements contain mainly hydrophobic residues. NMR relaxation data for nonphosphorylated TSP9 in SDS micelles indicated that the molecule is highly flexible with the highest order in the N-terminal alpha-helix. Intermolecular NOE signals, as well as spin probe-induced broadening of NMR signals, demonstrated that the SDS micelles contact both the structured and a portion of the unstructured regions of TSP9, in particular, those containing the three phosphorylation sites (T46, T53, and T60). This interaction may explain the selective dissociation of phosphorylated TSP9 from the membrane. Our study presents a structural model for the role played by the structured and unstructured regions of TSP9 in its membrane association and biological function.

The structure of At3g04780.1-des15, an Arabidopsis thaliana ortholog of the C-terminal domain of human thioredoxin-like protein, was determined by NMR spectroscopy. The structure is dominated by a beta-barrel sandwich. A two-stranded anti-parallel beta-sheet, which seals off one end of the beta-barrel, is flanked by two flexible loops rich in acidic amino acids. Although this fold often provides a ligand binding site, the structure did not reveal an appreciable cavity inside the beta-barrel. The three-dimensional structure of At3g04780.1-des15 provides an entry point for understanding its functional role and those of its mammalian homologs.

Treatment of rat pituitary GH4C1 cells with estradiol, insulin, and epidermal growth factor induces secretory granule accumulation, PRL storage, and stabilization of ICA512, a membrane protein associated with secretory granules. In these investigations we found that the same treatment induced accumulation over 2-fold of other proteins in the secretory pathway, including synaptosomal-associated protein of 25 kDa (SNAP-25), synaptotagmin III, synaptobrevin, synaptophysin, and cyclophilin B, and did not affect accumulation of others, including synaptotagmin I, calnexin, and glucose-regulated protein 94. The induction of proteins was not a coordinate event, because epidermal growth factor alone maximally stimulated SNAP-25 accumulation, but not that of synaptotagmin III. Induction of SNAP-25 accumulation occurred without an increase in its synthesis, and induction of cyclophilin B occurred without an increase in its messenger RNA accumulation, suggesting that accumulation may be caused by stabilization of the proteins. SNAP-25 immunofluorescence was located in the cytoplasm and on the plasma membrane and sometimes was heavily concentrated in protrusions from the cell surface, especially in hormone-treated cells. Frequenin immunofluorescence was also sometimes concentrated in intense patches, but did not colocalize with SNAP-25. Growth hormone and prolactin immunofluorescence was not found in the protrusions and sometimes did not colocalize with each other when they were present in the same cell. Hormone treatment of GH4C1 cells therefore induces accumulation of specific proteins in all parts of the secretory pathway and causes morphological changes in addition to accumulating secretory granules.

Familial isolated GH deficiency type II is an autosomal dominant form of short stature, associated in some families with mutations that result in missplicing to produce del32–71-GH, a protein that cannot fold normally. The mechanism by which this mutant suppresses the secretion of wild-type GH encoded by the normal allele is not known. Coexpression of del32–71-GH with wild-type human GH in transient transfections of the neuroendocrine cell lines GH4C1 and AtT20 suppressed accumulation of wild-type GH. The suppression of wild-type GH accumulation by del32–71-GH was a posttranslational effect on wild-type GH caused by decreased stability, rather than decreased synthesis, of wild-type GH. Coexpression of del32–71-GH with human PRL did not suppress accumulation of PRL, indicating that there was not a general suppression of secretory pathway function. Accumulation of del32–71-GH protein was not necessary for the suppression of wild-type GH, because del32–71-GH did not accumulate in the neuroendocrine cell line...

Treatment with 1 nM estradiol, 300 nM insulin, and 5 nM epidermal growth factor induces secretory granule accumulation and prolactin storage in GH4C1 rat pituitary tumor cells. The same triple treatment induced more than 6-fold accumulation of both the precursor (100 kDa, pro-ICA512) and the mature forms (60-70 kDa, ICA512 transmembrane fragment) of ICA512, a receptor protein tyrosine phosphatase-like protein that is preferentially localized in secretory granule membranes. Accumulation of ICA512 resembles that of prolactin storage, for the combination of all three, estradiol, insulin, and epidermal growth factor, gave the greatest induction, which was maximal at 4 days. This effect was specific, as the levels of the small GTP-binding protein Rab3, which is also associated with secretory granule membranes, were unaffected by the triple hormone/growth factor treatment. Increased transcription and translation of ICA512 could only partially account for its 6-fold accumulation, as ICA512 messenger RNA and ICA512 synthesis levels were 1.8 +/- 0.35- and 1.6 +/- 0.17-fold in triple treated GH4C1 cells compared with those in untreated cells, respectively. Pulse-chase procedures showed that pro-ICA512 was more stable in treated cells. These results indicate that the enlargement of the secretory granule compartment results in the stabilization of ICA512 and raise the possibility that trafficking of secretory granules may affect ICA512's function and vice versa.

Traditionally, CD has been used extensively for peptides in secondary structure analysis. In recent years, NMR chemical shifts and nuclear Overhauser enhancements have been widely used in conjunction with CD to assess the secondary structures of peptides and proteins; however, there are many instances where the estimation of secondary structure contents differs significantly between the two methods. In order to elucidate the perceived differences between the two methods, secondary structure estimations by CD and 1H NMR chemical shifts were compared for over 50 peptides. The linear peptides investigated were largely unstructured, approximately 15-50 residues in size, and lacked stable tertiary conformation. These peptides were studied in different solvent systems including water, alcohol-water, micelles and urea. A strong correlation exists for secondary structure assessment by CD and NMR chemical shifts; however, an interesting trend of higher estimation of helical contents by NMR was observed for peptide fragments from globular proteins studied in water. This may be a result of associative properties of these peptides in water. Additionally, a new method of quantitating secondary structure contents based on 1H NMR chemical shifts is reported.

The three-dimensional solution structure of the DNA binding domain (DBD) of the retinoid X receptor alpha (RXR alpha) was determined by nuclear magnetic resonance spectroscopy. The two zinc fingers of the RXR DBD fold to form a single structural domain that consists of two perpendicularly oriented helices and that resembles the corresponding regions of the glucocorticoid and estrogen receptors (GR and ER, respectively). However, in contrast to the DBDs of the GR and ER, the RXR DBD contains an additional helix immediately after the second zinc finger. This third helix mediates both protein-protein and protein-DNA interactions required for cooperative, dimeric binding of the RXR DBD to DNA. Identification of the third helix in the RXR DBD thus defines a structural feature required for selective dimerization of the RXR on hormone response elements composed of half-sites (5'-AGGTCA-3') arranged as tandem repeats.

Zinc fingers of the transcription factor IIIA (TFIIIA) type, in which zinc is co-ordinated by two cysteine and two histidine ligands (Cys2/His2), contain a length of helix packed against a β-hairpin. These zinc fingers comprise the wider range of structurally homologous proteins for which 1H chemical shifts are available. A number of key resonances have chemical shifts that are highly sensitive to tertiary structure and are conserved between these peptides. The high conservation of these fingerprint chemical shifts is correlated with the common global fold of Cys2/His2 zinc fingers. These chemical shifts are largely independent of primary structure and should facilitate NMR assignments for future zinc finger proteins, as well as provide a diagnostic signature for the characteristic Cys2/His2 zinc finger fold.

Essentially complete assignments have been obtained for the 1H and protonated 13C NMR spectra of the zinc finger peptide Xfin-31 in the presence and absence of zinc. The patterns observed for the 1H and 13C chemical shifts of the peptide in the presence of zinc, relative to the shifts in the absence of zinc, reflect the zinc-mediated folding of the unstructured peptide into a compact globular structure with distinct elements of secondary structure. Chemical shifts calculated from the 3D solution structure of the peptide in the presence of zinc and the observed shifts agree to within ca. 0.2 and 0.6 ppm for the backbone C alpha H and NH protons, respectively. In addition, homologous zinc finger proteins exhibit similar correlations between their 1H chemical shifts and secondary structure.

A synthetic peptide corresponding to zinc finger 31 of the Xenopus protein Xfin adopts a folded conformation in the presence of zinc. The same peptide in the absence of zinc is not folded in a stable tertiary conformation, as determined by NMR. Binding experiments have shown that the peptide binds non-specifically to DNA only in the presence of zinc. Moreover, competitive DNA binding experiments indicate interaction with 3.9 +/- 0.4 base pairs.

We describe X-ray crystal and NMR solution structures of the protein coded for by Arabidopsis thaliana gene At1g77540.1 (At1g77540). The crystal structure was determined to 1.15 Å with an R factor of 14.9% (Rfree = 17.0%) by multiple-wavelength anomalous diffraction using sodium bromide derivatized crystals. The ensemble of NMR conformers was determined with protein samples labeled with 15N and 13C+15N. The X-ray structure and NMR ensemble were closely similar with r.m.s.d 1.4 Å for residues 8–93. At1g77540 was found to adopt a fold similar to that of GCN5-related N-acetyltransferases. Enzymatic activity assays established that At1g77540 possesses weak acetyltransferase activity against histones H3 and H4. Chemical shift perturbations observed in 15N-HSQC spectra upon the addition of CoA indicated that the cofactor binds and identified its binding site. The molecular details of this interaction were further elucidated by solving the X-ray structure of the At1g77540–CoA complex. This work establishes that the domain family COG2388 represents a novel class of acetyltransferase and provides insight into possible mechanistic roles of the conserved Cys76 and His41 residues of this family.

We report the three-dimensional structure of a late embryogenesis abundant (LEA) protein from Arabidopsis thaliana gene At1g01470.1. This protein is a member of Pfam cluster PF03168, and has been classified as a LEA14 protein. LEA proteins are expressed under conditions of cellular stress, such as desiccation, cold, osmotic stress, and heat. The structure, which was determined by NMR spectroscopy, revealed that the At1g01470.1 protein has an alphabeta-fold consisting of one alpha-helix and seven beta-strands that form two antiparallel beta-sheets. The closest structural homologs were discovered to be fibronectin Type III domains, which have

We have used NMR spectroscopy to determine the solution structure of protein AAH26994.1 from Mus musculus and propose that it represents the first three-dimensional structure of a ubiquitin-related modifier 1 (Urm1) protein. Amino acid sequence comparisons indicate that AAH26994.1 belongs to the Urm1 family of ubiquitin-like modifier proteins. The best characterized member of this family has been shown to be involved in nutrient sensing, invasive growth, and budding in yeast. Proteins in this family have only a weak sequence similarity to ubiquitin, and the structure of AAH26994.1 showed a much closer resemblance to MoaD subunits of molybdopterin synthases (known structures are of three bacterial MoaD proteins with 14%-26% sequence identity to AAH26994.1). The structures of AAH26994.1 and the MoaD proteins each contain the signature ubiquitin secondary structure fold, but all differ from ubiquitin largely in regions outside of this fold. This structural similarity bolsters the hypothesis that ubiquitin and ubiquitin-related proteins evolved from a protein-based sulfide donor system of the molybdopterin synthase type.

Rat prolactin in the dense cores of secretory granules of the pituitary gland is a Lubrol-insoluble aggregate. In GH(4)C(1) cells, newly synthesized rat prolactin and growth hormone were soluble, but after 30 min about 40% converted to a Lubrol-insoluble form. Transport from the endoplasmic reticulum is necessary for conversion to Lubrol insolubility, since incubating cells with brefeldin A or at 15 degrees C reduced formation of insoluble rat (35)S-prolactin. Formation of Lubrol-insoluble aggregates has protein and cell specificity; newly synthesized human growth hormone expressed in AtT20 cells underwent a 40% conversion to Lubrol insolubility with time, but albumin did not, and human growth hormone expressed in COS cells underwent less than 10% conversion to Lubrol insolubility. del32-46 growth hormone, a naturally occurring form of growth hormone, and P89L growth hormone underwent conversion, although they were secreted more slowly, indicating that there is some tolerance in structural requirements for aggregation. An intracellular compartment with an acidic pH is not necessary for conversion to Lubrol insolubility, because incubation with chloroquine or bafilomycin slowed, but did not prevent, the conversion. GH(4)C(1) cells treated with estradiol, insulin, and epidermal growth factor accumulate more secretory granules and store more prolactin, but not more growth hormone, than untreated cells; Lubrol-insoluble aggregates of prolactin and growth hormone formed to the same extent in hormone-treated or untreated GH(4)C(1) cells, but prolactin was retained longer in hormone-treated cells. These findings indicate that aggregation alone is not sufficient to cause retention of secretory granule proteins, and there is an additional selective process.

Human PRL binds Zn2+, but the function of the binding is not known. We investigated the effect on PRL production in pituitary cells by obtaining clones of GH4C1 cells stably transfected with human H27A-PRL, a mutant that does not bind Zn2+. Unexpectedly, clones transfected with the mutant human PRL made little rat PRL. Untransfected GH4C1 cells made between 0.5 to 10 μg rat PRL/105 cells in 24 h. Clones transfected with vector alone (four of four), wild type human PRL (six of six), or with human K69A-PRL (two of two) made amounts of rat PRL in the same range. Clones transfected with human H27A-PRL (five of five) made 0.003–0.1 μg rat PRL/105 cells in 24 h, and the production of rat PRL mRNA was reduced. Human H27A-PRL was not efficiently secreted; 20–40% newly synthesized H27A-PRL was degraded by 60 min, and there was usually a delay in release of newly synthesized H27A-PRL. Reduction of rat PRL production is not mediated through the PRL receptor, because no sequences for the receptor in GH4C1 cells were detected by RT-PCR. Proteins involved in folding, such as BiP, were not specifically elevated in the H27A-PRL clones. In transient transfections, in which cells have not undergone selection, we found no evidence for disulfide-bonded aggregates of the mutant protein. The results indicate that Zn2+ binding stabilizes PRL in the secretory pathway; the instablility of the mutant protein may trigger effects that suppress rat PRL production directly or that indirectly result in selection of clones with low rat PRL production.

The retinoid X receptor (RXR) is a member of the nuclear hormone receptor superfamily and has recently been shown to function in a variety of hormonal signaling pathways by virtue of its ability to heterodimerize with other nuclear hormone receptors. Here we describe resonance assignments, the secondary structural elements and the global folding pattern of the DNA-binding domain (residues 130-223) of human RXR alpha, as determined by multidimensional nuclear magnetic resonance spectroscopy. Its overall structure is similar to those reported for the glucocorticoid, estrogen, and retinoic acid receptors, in that the two zinc fingers of RXR fold to form a single structural domain containing two helices, which are located at the carboxy terminal of the two zinc fingers. There is also a short antiparallel beta-sheet formed between two residues in the amino-terminal base of the first finger and two residues in the carboxy terminal of that same finger just before the first helix. However, in contrast to the other nuclear hormone receptor DNA-binding domains, the RXR domain contains a third helix immediately after the conserved Gly-Met sequence that signals the termination of the second helix. The second and third helices lie orthogonal to and wrap around the first helix, generating an extended hydrophobic core. Since helices two and three are separated by only two residues, the backbone flexibility afforded by the presence of the conserved glycine residue between them may be crucial for the proper positioning of the third helix relative to the first helix. A 12-amino-acid region termed the 'T-box', which includes this third helix, was recently shown to be required for homodimeric binding of RXR to its cognate response element [Wilson, T. E., Paulsen, R. E., Padgett, K. A.Milbrandt, J. (1992) Science 256, 107-110].

Two mutants of the zinc finger peptide Xfin-31 (Ac-YKCGLCERSFVEKSALSRHQRVHKN-CONH2) containing alterations to the conserved hydrophobic core have been constructed and their zinc-bound structures investigated by 1H NMR techniques. In the first (Xfin-31B) a double mutation R8F/F10G places the conserved core aromatic residue at position 8 rather than position 10. In the second (Xfin-31C), Phe-10 is replaced by Leu. A qualitative analysis of 1H chemical shifts, NOE connectivities and coupling constants indicates that the global folds of both mutants are similar to that of the wild-type protein. However, amide exchange rates suggest that the F10L mutant is much less stable than either the wild-type or the R8F/F10G mutant.

A large number of protein-protein interactions involve turn or loop regions. The excised linear peptides from these regions reveal complex conformational averaging. To circumvent this motional averaging and to stabilize the β-turn conformation, extensive effort has been devoted to the design of constrained peptidomimetics. Here, we report the three-dimensional solution structure of a 12-membered cyclic peptidomimetic. The structures were calculated from NMR studies performed in chloroform and in water at 263 and 278K, respectively. This 12-membered cyclic scaffolding is part of a program to design and to construct conformationally stable β-turn peptidomimetics. The impact of the surrounding environment on the conformation of this constrained peptidomimetic is discussed. The general structural features of the cyclic mimetic are retained in both environments; however, the formation of a hydrophobic patch in the aqueous solvent is evident.

A 25-residue synthetic peptide corresponding to zinc finger 31 of the Xenopus protein Xfin adopts a compact, folded conformation in the presence of zinc. Complete 1H resonance assignments have been made. The peptide contains a helix, beginning as an α-helix and ending as a 310-helix, that extends from residue 12 to 23. Several positively charged and polar residues located on this helix are likely to be involved in interactions with DNA. Residues 1–10 appear to adopt a hairpin-like structure.

The three-dimensional solution structure of a zinc finger nucleic acid binding motif has been determined by nuclear magnetic resonance (NMR) spectroscopy. Spectra of a synthetic peptide corresponding to a single zinc finger from the Xenopus protein Xfin yielded distance and dihedral angle constraints that were used to generate structures from distance geometry and restrained molecular dynamics calculations. The zinc finger is an independently folded domain with a compact globular structure in which the zinc atom is bound by two cysteine and two histidine ligands. The polypeptide backbone fold consists of a well-defined helix, starting as alpha and ending as 3(10) helix, packed against two beta strands that are arranged in a hairpin structure. A high density of basic and polar amino acid side chains on the exposed face of the helix are probably involved in DNA binding.