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SARS-CoV-2 Omicron subvariants with increased transmissibility and immune evasion are spreading globally with alarming persistence. Whether the mutations and evolution of spike (S) Omicron subvariants alter the viral hijacking of human TMPRSS2 for viral entry remains to be elucidated. This is particularly important to investigate because of the large number and diversity of mutations of S Omicron subvariants reported since the emergence of BA.1. Here we report that human TMPRSS2 is a molecular determinant of viral entry for all the Omicron clinical isolates tested in human lung cells, including ancestral Omicron subvariants (BA.1, BA.2, BA.5), contemporary Omicron subvariants (BQ.1.1, XBB.1.5, EG.5.1) and currently circulating Omicron BA.2.86. First, we used a co-transfection assay to demonstrate the endoproteolytic cleavage by TMPRSS2 of spike Omicron subvariants. Second, we found that N-0385, a highly potent TMPRSS2 inhibitor, is a robust entry inhibitor of virus-like particles harbouring the S protein of Omicron subvariants. Third, we show that N-0385 exhibits nanomolar broad-spectrum antiviral activity against live Omicron subvariants in human Calu-3 lung cells and primary patient-derived bronchial epithelial cells. Interestingly, we found that N-0385 is 10-20 times more potent than the repositioned TMPRSS2 inhibitor, camostat, against BA.5, EG.5.1, and BA.2.86. We further found that N-0385 shows broad synergistic activity with clinically approved direct-acting antivirals (DAAs), i.e., remdesivir and nirmatrelvir, against Omicron subvariants, demonstrating the potential therapeutic benefits of a multi-targeted treatment based on N-0385 and DAAs., Competing Interests: Declaration of competing interest PLB and RL are inventors on patent applications (US9365853B2 and US10988505B2) that cover matriptase and other type II transmembrane serine protease inhibitors for treating and preventing viral infections, respiratory disorders, inflammatory disorders, pain disorders, tissue disorders, hyperproliferative disorders, and disorders associated with iron overload. The remaining authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global public health crisis. The reduced efficacy of therapeutic monoclonal antibodies against emerging SARS-CoV-2 variants of concern (VOCs), such as omicron BA.5 subvariants, has underlined the need to explore a novel spectrum of antivirals that are effective against existing and evolving SARS-CoV-2 VOCs. To address the need for novel therapeutic options, we applied cell-based high-content screening to a library of natural products (NPs) obtained from plants, fungi, bacteria, and marine sponges, which represent a considerable diversity of chemical scaffolds. The antiviral effect of 373 NPs was evaluated using the mNeonGreen (mNG) reporter SARS-CoV-2 virus in a lung epithelial cell line (Calu-3). The screening identified 26 NPs with half-maximal effective concentrations (EC 50 ) below 50 μM against mNG-SARS-CoV-2; 16 of these had EC 50 values below 10 μM and three NPs (holyrine A, alotaketal C, and bafilomycin D) had EC 50 values in the nanomolar range. We demonstrated the pan-SARS-CoV-2 activity of these three lead antivirals against SARS-CoV-2 highly transmissible Omicron subvariants (BA.5, BA.2 and BA.1) and highly pathogenic Delta VOCs in human Calu-3 lung cells. Notably, holyrine A, alotaketal C, and bafilomycin D, are potent nanomolar inhibitors of SARS-CoV-2 Omicron subvariants BA.5 and BA.2. The pan-SARS-CoV-2 activity of alotaketal C [protein kinase C (PKC) activator] and bafilomycin D (V-ATPase inhibitor) suggest that these two NPs are acting as host-directed antivirals (HDAs). Future research should explore whether PKC regulation impacts human susceptibility to and the severity of SARS-CoV-2 infection, and it should confirm the important role of human V-ATPase in the VOC lifecycle. Interestingly, we observed a synergistic action of bafilomycin D and N-0385 (a highly potent inhibitor of human TMPRSS2 protease) against Omicron subvariant BA.2 in human Calu-3 lung cells, which suggests that these two highly potent HDAs are targeting two different mechanisms of SARS-CoV-2 entry. Overall, our study provides insight into the potential of NPs with highly diverse chemical structures as valuable inspirational starting points for developing pan-SARS-CoV-2 therapeutics and for unravelling potential host factors and pathways regulating SARS-CoV-2 VOC infection including emerging omicron BA.5 subvariants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Breakfast consumption is associated with better diet quality and healthier weights, yet many adolescents miss breakfast. Nationally, 17.1% of students participate in the School Breakfast Program (SBP). Only 10% of high school students participate., Objective: Our aim was to evaluate an environmental intervention to increase SBP participation in high schools., Design: A group randomized trial was carried out from 2012 to 2015., Participants/setting: Ninth- and 10th-grade students enrolled in 16 rural schools in Minnesota (median 387 students) were randomized to intervention or control condition., Intervention: A school-based intervention that included two key components was implemented over a 12-month period. One component focused on increasing SBP participation by increasing student access to school breakfast through changes in school breakfast service practices (eg, serving breakfast from a grab-n-go cart in the atrium; expanding breakfast service times). The other component focused on promoting school breakfast through student-directed marketing campaigns., Main Outcome Measure: Change in school-level participation in the SBP was assessed between baseline (among ninth and tenth graders) and follow-up (among tenth and eleventh graders). School meal and attendance records were used to assess change in school-level participation rates in the SBP., Statistical Analyses: The Wilcoxon test was used for analysis of difference in change in mean SBP participation rate by experimental group., Results: The median change in SBP participation rate between baseline and follow-up was 3% (interquartile range=13.5%) among the eight schools in the intervention group and 0.5% (interquartile range=0.7%) among the eight schools in the control group. This difference in change between groups was statistically significant (Wilcoxon test, P=0.03). The intervention effect increased throughout the intervention period, with change in mean SBP participation rate by the end of the school year reaching 10.3% (95% CI 3.0 to 17.6). However, among the intervention schools, the change in mean SBP participation rates was highly variable (range=-0.8% to 24.8%)., Conclusions: Interventions designed to improve access to the SBP by reducing environmental and social barriers have potential to increase participation among high school students., (Copyright © 2019 Academy of Nutrition and Dietetics. Published by Elsevier Inc. All rights reserved.)

The DeKAF study was developed to better understand the causes of late allograft loss. Preliminary findings from the DeKAF cross-sectional cohort (with follow-up < 20 months) have been published. Herein, we present long-term outcomes in those recipients (mean follow-up ± SD, 6.6 ± 0.7 years). Eligibility included being transplanted prior to October 1, 2005; serum creatinine ≤ 2.0 mg/dL on January 1, 2006; and subsequently developing new-onset graft dysfunction leading to a biopsy. Mean time from transplant to biopsy was 7.5 ± 6.1 years. Histologic findings and DSA were studied in relation to postbiopsy outcomes. Long-term follow-up confirms and expands the preliminary results of each of 3 studies: (1) increasing inflammation in area of atrophy (irrespective of inflammation in nonscarred areas [Banff i]) was associated with increasingly worse postbiopsy death-censored graft survival; (2) hierarchical analysis based on Banff scores defined clusters (entities) that differed in long-term death-censored graft survival; and (3) C4d-/DSA- recipients had significantly better (and C4d+/DSA+ worse) death-censored graft survival than other groups. C4d+/DSA- and C4d-/DSA+ had similar intermediate death-censored graft survival. Clinical and histologic findings at the time of new-onset graft dysfunction define high- vs low-risk groups for long-term death-censored graft survival, even years posttransplant. These findings can help differentiate groups for potential intervention studies., (© 2018 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Angiotensin II (AngII) type 1 receptor (AT 1 R) is a G protein-coupled receptor known for its role in numerous physiological processes and its implication in many vascular diseases. Its functions are mediated through G protein dependent and independent signaling pathways. AT 1 R has several endogenous peptidic agonists, all derived from angiotensinogen, as well as several synthetic ligands known to elicit biased signaling responses. Here, surface plasmon resonance (SPR) was used as a cell-based and label-free technique to quantify, in real time, the response of HEK293 cells stably expressing the human AT 1 R. The goal was to take advantage of the integrative nature of this assay to identify specific signaling pathways in the features of the response profiles generated by numerous endogenous and synthetic ligands of AT 1 R. First, we assessed the contributions of Gq, G12/13, Gi, Gβγ, ERK1/2 and β-arrestins pathways in the cellular responses measured by SPR where Gq, G12/Rho/ROCK together with β-arrestins and ERK1/2 were found to play significant roles. More specifically, we established a major role for G12 in the early events of the AT 1 R-dependent response, which was followed by a robust ERK1/2 component associated to the later phase of the signal. Interestingly, endogenous AT 1 R ligands (AngII, AngIII and AngIV) exhibited distinct responses signatures with a significant increase of the ERK1/2-like components for both AngIII and AngIV, which points toward possibly distinct physiological roles for the later. We also tested AT 1 R biased ligands, all of which affected both the early and later events. Our results support SPR-based integrative cellular assays as a powerful approach to delineate the contribution of specific signaling pathways for a given cell response and reveal response differences associated with ligands with distinct pharmacological properties., (Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.)

Beyond the first posttransplant year, 3% of kidney transplants fail annually. In a prospective, multicenter cohort study, we tested the relative impact of early versus late events on risk of long-term death-censored graft failure (DCGF). In grafts surviving at least 90 days, early events (acute rejection [AR] and delayed graft function [DGF] before day 90) were recorded; serum creatinine (Cr) at day 90 was defined as baseline. Thereafter, a 25% rise in serum Cr or new-onset proteinuria triggered graft biopsy (index biopsy, IBx), allowing comparison of risk of DCGF associated with early events (AR, DGF, baseline serum Cr >2.0 mg/dL) to that associated with later events (IBx). Among 3678 patients followed for 4.7 ± 1.9 years, 753 (20%) had IBx at a median of 15.3 months posttransplant. Early AR (HR = 1.77, P < .001) and elevated Cr at Day 90 (HR = 2.56, P < .0001) were associated with increased risk of DCGF; however, later-onset dysfunction requiring IBx had far greater impact (HR = 13.8, P < .0001). At 90 days, neither clinical characteristics nor early events distinguished those who subsequently did or did not undergo IBx or suffer DCGF. To improve long-term kidney allograft survival, management paradigms should promote prompt diagnosis and treatment of both early and later events., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The apelinergic system is an important player in the regulation of both vascular tone and cardiovascular function, making this physiological system an attractive target for drug development for hypertension, heart failure and ischemic heart disease. Indeed, apelin exerts a positive inotropic effect in humans whilst reducing peripheral vascular resistance. In this study, we investigated the signaling pathways through which apelin exerts its hypotensive action. We synthesized a series of apelin-13 analogs whereby the C-terminal Phe 13 residue was replaced by natural or unnatural amino acids. In HEK293 cells expressing APJ, we evaluated the relative efficacy of these compounds to activate Gα i1 and Gα oA G-proteins, recruit β-arrestins 1 and 2 (βarrs), and inhibit cAMP production. Calculating the transduction ratio for each pathway allowed us to identify several analogs with distinct signaling profiles. Furthermore, we found that these analogs delivered i.v. to Sprague-Dawley rats exerted a wide range of hypotensive responses. Indeed, two compounds lost their ability to lower blood pressure, while other analogs significantly reduced blood pressure as apelin-13. Interestingly, analogs that did not lower blood pressure were less effective at recruiting βarrs. Finally, using Spearman correlations, we established that the hypotensive response was significantly correlated with βarr recruitment but not with G protein-dependent signaling. In conclusion, our results demonstrated that the βarr recruitment potency is involved in the hypotensive efficacy of activated APJ., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Biomarker profiles of acute rejection in liver transplant recipients could enhance the diagnosis and management of recipients. Our aim was to identify diagnostic proteoform signatures of acute rejection in circulating immune cells, using an emergent "top-down" proteomics methodology. We prepared differentially processed and cryopreserved cell lysates from 26 nonviral liver transplant recipients by molecular weight-based fractionation and analyzed them by mass spectrometry of whole proteins in three steps: (i) Nanocapillary liquid chromatography coupled with high-resolution tandem mass spectrometry; (ii) database searching to identify and characterize intact proteoforms; (iii) data processing through a hierarchical linear model matching the study design to quantify proteoform fold changes in patients with rejection versus normal liver function versus acute dysfunction without rejection. Differentially expressed proteoforms were seen in patients with rejection versus normal and nonspecific controls, most evidently in the cell preparations stored in traditional serum-rich media. Mapping analysis of these proteins back to genes through gene ontology and pathway analysis tools revealed multiple signaling pathways, including inflammation mediated by cytokines and chemokines. Larger studies are needed to validate these novel rejection signatures and test their predictive value for use in clinical management., (© 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The number of older adults undergoing kidney transplantation has increased, yet little is known about calcineurin inhibitor (CNI) metabolism in this group. We studied CNI troughs and doses to determine if there were age-related differences in metabolism and dose requirements. We studied 348 young (18-34 years), 1831 middle (35-64 years) and 374 older (65-84 years) adult kidney transplant recipients enrolled in a seven-center prospective study. Troughs were obtained from each patient 2×/week in weeks 1-8 and 2×/month in months 3-6. A multivariable linear-mixed model examined the effect of age on log dose and weight normalized troughs. Older recipients had higher normalized tacrolimus troughs than middle or young age adults despite receiving doses a median of 1-2 mg/day lower. Age and CYP3A5*1 genotype had the largest effect on tacrolimus troughs. Older recipients also had higher normalized cyclosporine troughs than middle or young adults despite receiving median doses 100 mg/day lower. After normalization for dose and weight, CNI troughs were more than 50% higher in older adults than young adults. These data support age-related changes in CNI metabolism. Further studies are needed to determine optimal dosing of CNIs in the elderly., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The Banff scoring schema provides a common ground to analyze kidney transplant biopsies. Interstitial inflammation (i) and tubulitis (t) in areas of viable tissue are features in scoring acute rejection, but are excluded in areas of tubular atrophy (TA). We studied inflammation and tubulitis in a cohort of kidney transplant recipients undergoing allograft biopsy for new-onset late graft dysfunction (N = 337). We found inflammation ('iatr') and tubulitis ('tatr') in regions of fibrosis and atrophy to be strongly correlated with each other (p < 0.0001). Moreover, iatr was strongly associated with death-censored graft failure when compared to recipients whose biopsies had no inflammation, even after adjusting for the presence of interstitial fibrosis (Hazard Ratio = 2.31, [1.10-4.83]; p = 0.0262) or TA (hazard ratio = 2.42, [1.16-5.08]; p = 0.191), serum creatinine at the time of biopsy, time to biopsy and i score. Further, these results did not qualitatively change after additional adjustments for C4d staining or donor specific antibody. Stepwise regression identified the most significant markers of graft failure which include iatr score. We propose that a more global assessment of inflammation in kidney allograft biopsies to include inflammation in atrophic areas may provide better prognostic information. Phenotypic characterization of these inflammatory cells and appropriate treatment may ameliorate late allograft failure., (© 2010 The Authors Journal compilation © 2010 The American Society of Transplantation and the American Society of Transplant Surgeons.)

The nonspecific diagnoses 'chronic rejection''CAN', or 'IF/TA' suggest neither identifiable pathophysiologic mechanisms nor possible treatments. As a first step to developing a more useful taxonomy for causes of new-onset late kidney allograft dysfunction, we used cluster analysis of individual Banff score components to define subgroups. In this multicenter study, eligibility included being transplanted prior to October 1, 2005, having a 'baseline' serum creatinine < or =2.0 mg/dL before January 1, 2006, and subsequently developing deterioration of graft function leading to a biopsy. Mean time from transplant to biopsy was 7.5 +/- 6.1 years. Of the 265 biopsies (all with blinded central pathology interpretation), 240 grouped into six large (n > 13) clusters. There were no major differences between clusters in recipient demographics. The actuarial postbiopsy graft survival varied by cluster (p = 0.002). CAN and CNI toxicity were common diagnoses in each cluster (and did not differentiate clusters). Similarly, C4d and presence of donor specific antibody were frequently observed across clusters. We conclude that for recipients with new-onset late graft dysfunction, cluster analysis of Banff scores distinguishes meaningful subgroups with differing outcomes.

We are studying two cohorts of kidney transplant recipients, with the goal of defining specific clinicopathologic entities that cause late graft dysfunction: (1) prevalent patients with new onset late graft dysfunction (cross-sectional cohort); and (2) newly transplanted patients (prospective cohort). For the cross-sectional cohort (n = 440), mean time from transplant to biopsy was 7.5 +/- 6.1 years. Local pathology diagnoses included CAN (48%), CNI toxicity (30%), and perhaps surprisingly, acute rejection (cellular- or Ab-mediated) (23%). Actuarial rate of death-censored graft loss at 1 year postbiopsy was 17.7%; at 2 years, 29.8%. There was no difference in postbiopsy graft survival for recipients with versus without CAN (p = 0.9). Prospective cohort patients (n = 2427) developing graft dysfunction >3 months posttransplant undergo 'index' biopsy. The rate of index biopsy was 8.8% between 3 and 12 months, and 18.2% by 2 years. Mean time from transplant to index biopsy was 1.0 +/- 0.6 years. Local pathology diagnoses included CAN (27%), and acute rejection (39%). Intervention to halt late graft deterioration cannot be developed in the absence of meaningful diagnostic entities. We found CAN in late posttransplant biopsies to be of no prognostic value. The DeKAF study will provide broadly applicable diagnostic information to serve as the basis for future trials.

Death with function causes half of late kidney transplant failures, and cardiovascular disease (CVD) is the most common cause of death in these patients. We examined the use of potentially cardioprotective medications in a prospective observational study at seven transplant centers in the United States and Canada. Among 935 patients, 87% received antihypertensive medications at both 1 and 6 months after transplantation. Similar antihypertensive regimens were used for patients with and without diabetes and CVD, but with wide variability among centers. In contrast, while 44% of patients were on angiotensin converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB) at the time of transplantation, the proportion taking these agents dropped to 12% at month 1, then increased to 24% at 6 months. Fewer than 30% with CVD or diabetes received ACEI/ARB therapy 6 months posttransplant. Aspirin use was uncommon (<40% of patients). Even among those with diabetes and/or CVD, fewer than 60% received aspirin and only half received a statin at 1 and 6 months. This study demonstrates marked variability in the use of cardioprotective medications in kidney transplant recipients, a finding that may reflect, among several possible explanations, clinical uncertainty due the lack of randomized trials for these medications in this population.

ADAMTS5 (aggrecanase-2), a key metalloprotease mediating cartilage destruction in arthritis, is synthesized as a zymogen, proADAMTS5. We report a detailed characterization of the propeptide excision mechanism and demonstrate that it is a major regulatory step with unusual characteristics. Using furin-deficient cells and a furin inhibitor, we found that proADAMTS5 was processed by proprotein convertases, specifically furin and PC7, but not PC6B. Mutagenesis of three sites containing basic residues within the ADAMTS5 propeptide (RRR(46), RRR(69) and RRRRR(261)) suggested that proADAMTS5 processing occurs after Arg(261). That furin processing was essential for ADAMTS5 activity was illustrated using the known ADAMTS5 substrate aggrecan, as well as a new substrate, versican, an important regulatory proteoglycan during mammalian development. When compared to other ADAMTS proteases, proADAMTS5 processing has several distinct features. In contrast to ADAMTS1, whose furin processing products were clearly present intracellularly, cleaved ADAMTS5 propeptide and mature ADAMTS5 were found exclusively in the conditioned medium. Despite attempts to enhance detection of intracellular proADAMTS5 processing, such as by immunoprecipitation of total ADAMTS5, overexpression of furin, and secretion blockade by monensin, neither processed ADAMTS5 propeptide nor the mature enzyme were found intracellularly, which was strongly suggestive of extracellular processing. Extracellular ADAMTS5 processing was further supported by activation of proADAMTS5 added exogenously to HEK293 cells stably expressing furin. Unlike proADAMTS9, which is processed by furin at the cell-surface, to which it is bound, ADAMTS5 does not bind the cell-surface. Thus, the propeptide processing mechanism of ADAMTS5 has several points of distinction from those of other ADAMTS proteases, which may have considerable significance in the context of osteoarthritis.

Most G protein-coupled receptors (GPCRs) are N-glycosylated proteins but the role of this post-translational modification in GPCR biosynthesis has not been extensively studied. We previously showed that the non-glycosylated AT(1) receptor is inefficiently expressed at the cell surface. In this study, we addressed whether AT(1) interacts with elements of the ER-based quality control processes. Interestingly, non-glycosylated AT(1) receptors associated with the molecular chaperones calnexin and HSP70, suggesting the importance of protein-based interactions between these partners. We also demonstrate that ER mannosidase I participates in the acquisition of mature glycoforms and in the targeting of the AT(1) receptor to the membrane. Taken together, these results indicate that decreased cell-surface expression of the non-glycosylated receptor cannot be attributed to diminished interactions with molecular chaperones and that mannose trimming of the wild-type AT(1) receptor by ER mannosidase I plays a critical role in its cell-surface expression.

Notch is a conserved cell surface receptor that is activated through direct contact with neighboring ligand-expressing cells. The primary 300-kDa translation product of the Notch1 gene (p300) is cleaved by a furin-like convertase to generate a heterodimeric, cell-surface receptor composed of 180- (p180) and 120- (p120) kDa polypeptides. Heterodimeric Notch is thought to be the only form of the receptor which is both present on the cell surface and able to generate an intracellular signal in response to ligand. Consistent with previous reports, we found that disruption of furin processing of Notch1, either by coexpression of a furin inhibitor or by mutation of furin target sequences within Notch1 itself, perturbed ligand-dependent signaling through the well-characterized mediator of Notch signal transduction, CSL (CBF1, Su(H), and LAG-1). Yet contrary to these reports, we could detect the full-length p300 Notch1 product on the cell surface. Moreover, this uncleaved form of Notch1 could suppress the differentiation of C2C12 myoblasts in response to ligand. Taken together, these data support our previous studies characterizing a CSL-independent Notch signaling pathway and identify this uncleaved isoform of Notch as a potential mediator of this pathway. Our results suggest a novel paradigm in signal transduction, one in which two isoforms of the same cell-surface receptor could mediate two distinct signaling pathways in response to ligand., (Copyright 2001 Academic Press.)

Transforming growth factor (TGF)-beta1 plays an essential role in cell growth and differentiation. It is also considered as a gatekeeper of immune homeostasis with gene disruption leading to autoimmune and inflammatory diseases. TGF-beta1 is produced as an inactive precursor polypeptide that can be efficiently secreted but correct proteolytic cleavage is an essential step for its activation. Assessment of the cleavage site has revealed a unique R-H-R-R sequence reminiscent of proprotein convertase (PC) recognition motifs and has previously demonstrated that this PC-like cleavage site is correctly cleaved by furin, a member of the PC family. Here we report that among PC members, furin more closely satisfies the requirements needed to fulfill the role of a genuine TGF-beta1 convertase. Even though six members of the PC family have the ability to cleave TGF-beta1, ectopic expression of alpha(1)-antitrypsin Portland (alpha(1)-AT-PDX), a potent furin inhibitor, blocked 80% of TGF-beta1 processing mediated by endogenous enzymes as demonstrated in an in vitro digestion assay. Genetic complementation of a furin-deficient LoVo cell line with the wild-type gene restores the production of mature and bioactivable TGF-beta1. Moreover, both furin and TGF-beta are coordinately expressed and regulated in vitro and in vivo in the hematopoietic and immune system, an important tissue target. These results demonstrate for the first time that furin is an authentic and adaptive TGF-beta1-converting enzyme whereas other members of the PC family might substitute or supplement furin activity. Our study advances our comprehension of the complexity of the TGF-beta system and should facilitate the development of therapeutically useful TGF-beta inhibitors.

A Prostaglandin endoperoxide synthase isoenzyme was recently identified in several cell lines. Osteoblasts possess Prostaglandin endoperoxide synthase activity, but it is not known which isoenzymes are present in these cells. Our objective was to identify these isoenzymes in human osteoblasts. Resting cells in culture did not produce measurable amounts of PGE2 and did not express Prostaglandin endoperoxide synthase-1 or Prostaglandin endoperoxide synthase-2 mRNAs detectable by Northern blot. Treatment with rhIL-1 alpha or rhTNF alpha induced both the expression of Prostaglandin endoperoxide synthase-2 mRNA and the synthesis of PGE2, rhIL-1 alpha being more potent on an equimolar basis than rhTNF alpha. Dexamethasone inhibited the increase in Prostaglandin endoperoxide synthase-2 mRNA and the production of PGE2 induced by both cytokines. These results suggest that Prostaglandin endoperoxide synthase-2 may be the relevant isoenzyme for prostanoid production in human osteoblasts in culture.

Human furin, a member of a recently discovered family of cellular endoproteases, has been identified as a membrane bound protein localized in the Golgi apparatus. Here, we report the presence of a secreted form of furin in the media of cells infected with a vaccinia virus recombinant containing the furin gene. Using the fluorogenic substrate boc-Arg-Val-Arg-Arg-MCA, endoproteolytic activity was detected in the media of infected BSC40 cells. Immunoprecipitations of [35S]-labeled proteins from infected cells revealed that the media contained a lower molecular form of furin than the cellular furin or than a previously characterized soluble furin mutant, hFUR713t. By using the direct linear plot representation of the Michaelis-Menten equation the results demonstrate that the soluble furin exhibited similar kinetics to the hFUR713t enzyme. Thus, our results suggest that membrane-bound furin undergoes post-translational processing to produce a soluble form of the enzyme that can be secreted.

Three AERAS low pressure 11 stage cascade impactors with rotatable collecting plates (LPCR) were installed at the Duchesnay forest station near Québec City and four low pressure inertial collectors (LPIC) were installed in the forest. The analysis of the aerosol deposits was performed by static fast atom beam mass spectroscopy (FABMS); SIMS and ESCA analyses were also made. The morphological characterization and the elemental volumetric composition of the aerosols were established by a scanning electron microscope equipped with an X-ray dispersive energy analyser. Micro-weighting of the aluminium substrate before and after the sampling by the AERAS impactor enabled the study of the mass-size-distribution of the aerosols according to their aerodynamic equivalent diameter. Example of the results obtained are presented in relation to air parcels' trajectories. This study has shown that it is possible to characterize the aerosols according to the origin and trajectories of the air mass with which they travel; sources along the tracks have been shown to contribute to the aerosol loading of the air masses.

Chromogranin A has been reported to be processed in vivo by an as yet undefined proteinase(s) suggesting that it is a precursor of biologically active peptides such as pancreastatin. In this study, plasma kallikrein was used as a model proteinase to identify the cleavage sites exposed in bovine parathyroid chromogranin A. Purified bovine parathyroid chromogranin A was digested with human plasma kallikrein. The proteolytic fragments produced were isolated by HPLC and chemically characterized by amino acid composition and sequence analysis. The combined results indicate that the enzyme has preference for specific single Arg residues, cutting C-terminal to this amino acid, although certain pairs of basic sites were also cleaved. The characterized fragments were released in a selective manner from the whole molecule with rapid production of the fragments covering positions 1-247 and 352-358.

Carbofuran, and non-conjugated 3-hydroxycarbofuran and 3-ketocarbofuran were extracted from carrots, corn and potatoes with acetone and partitioned into hexane-methylene chloride. The organic extract was evaporated to a small volume for clean-up on a 2% deactivated Florisil column. All three carbamates were eluted with 15% acetone in hexane. The pesticide residues were hydrolysed to their corresponding phenols with 0.1 M sodium carbonate followed by derivatization with dansyl chloride in acetone. The derivatives were extracted and analysed by high-pressure liquid chromatography with fluorescence detection (excitation, 360 nm; emission, greater than 400 nm). Absolute recoveries for all three compounds were between 50 and 65% for spiked samples by the extraction method used. Detection limits approached 0.01 ppm in the foods studied.

During the course of reverse-phase high pressure liquid chromatography (RP-HPLC) purification of a postulated big ACTH (1) from human pituitary gland extracts, a highly purified peptide bearing no resemblance to any known polypeptide was isolated. The complete sequence of this 74 amino acid polypeptide, called GAWK, has been determined. Search on a computer data bank on the possible homology to any known protein or fragment, using a mutation data matrix, failed to reveal any homology greater than 30%. An antibody produced against a synthetic fragment allowed us to detect several immunoreactive forms. The antisera also enabled us to localize the polypeptide, by immunocytochemistry, in the anterior lobe of the pituitary gland.