Kisspeptin treatment induces gonadotropic responses and rescues ovulation in a subset of preclinical models and women with polycystic ovary syndrome.

STUDY QUESTION: Can kisspeptin treatment induce gonadotrophin responses and ovulation in preclinical models and anovulatory women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Kisspeptin administration in some anovulatory preclinical models and women with PCOS can stimulate reproductive hormone secretion and ovulation, albeit with incomplete efficacy. WHAT IS KNOWN ALREADY: PCOS is a prevalent, heterogeneous endocrine disorder, characterized by ovulatory dysfunction, hyperandrogenism and deregulated gonadotrophin secretion, in need of improved therapeutic options. Kisspeptins (encoded by Kiss1) are master regulators of the reproductive axis, acting mainly at GnRH neurons, with kisspeptins being an essential drive for gonadotrophin-driven ovarian follicular maturation and ovulation. Altered Kiss1 expression has been found in rodent models of PCOS, although the eventual pathophysiological role of kisspeptins in PCOS remains unknown. STUDY DESIGN, SIZE, DURATION: Gonadotrophin and ovarian/ovulatory responses to kisspeptin-54 (KP-54) were evaluated in three preclinical models of PCOS, generated by androgen exposures at different developmental windows, and a pilot exploratory cohort of anovulatory women with PCOS. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three models of PCOS were generated by exposure of female rats to androgens at different periods of development: PNA (prenatal androgenization; N = 20), NeNA (neonatal androgenization; N = 20) and PWA (post-weaning androgenization; N = 20). At adulthood (postnatal day 100), rats were subjected to daily treatments with a bolus of KP-54 (100 µg/kg, s.c.) or vehicle for 11 days (N = 10 per model and treatment). On Days 1, 4, 7 and 11, LH and FSH responses were assessed at different time-points within 4 h after KP-54 injection, while ovarian responses, in terms of follicular maturation and ovulation, were measured at the end of the treatment. In addition, hormonal (gonadotrophin, estrogen and inhibin B) and ovulatory responses to repeated KP-54 administration, at doses of 6.4-12.8 nmol/kg, s.c. bd for 21 days, were evaluated in a pilot cohort of anovulatory women (N = 12) diagnosed with PCOS, according to the Rotterdam criteria. MAIN RESULTS AND THE ROLE OF CHANCE: Deregulated reproductive indices were detected in all PCOS models: PNA, NeNA and PWA. Yet, anovulation was observed only in NeNA and PWA rats. However, while anovulatory NeNA rats displayed significant LH and FSH responses to KP-54 (P < 0.05), which rescued ovulation, PWA rats showed blunted LH secretion after repeated KP-54 injection and failed to ovulate. In women with PCOS, KP-54 resulted in a small rise in LH (P < 0.05), with an equivalent elevation in serum estradiol levels (P < 0.05). Two women showed growth of a dominant follicle with subsequent ovulation, one woman displayed follicle growth but not ovulation and desensitization was observed in another patient. No follicular response was detected in the other women. LIMITATIONS, REASONS FOR CAUTION: While three different preclinical PCOS models were used in order to capture the heterogeneity of clinical presentations of the syndrome, it must be noted that rat models recapitulate many but not all the features of this condition. Additionally, our pilot study was intended as proof of principle, and the number of participants is low, but the convergent findings in preclinical and clinical studies reinforce the validity of our conclusions. WIDER IMPLICATIONS OF THE FINDINGS: Our first-in-rodent and -human studies demonstrate that KP-54 administration in anovulatory preclinical models and women with PCOS can stimulate reproductive hormone secretion and ovulation, albeit with incomplete efficacy. As our rat models likely reflect the diversity of PCOS phenotypes, our results argue for the need of personalized management of anovulatory dysfunction in women with PCOS, some of whom may benefit from kisspeptin-based treatments. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by research agreements between Ferring Research Institute and the Universities of Cordoba and Edinburgh. K.S. was supported by the Wellcome Trust Scottish Translational Medicine and Therapeutics Initiative (STMTI). Some of this work was undertaken in the MRC Centre for Reproductive Health which is funded by the MRC Centre grant MR/N022556/1. M.T.-S. is a member of CIBER Fisiopatología de la Obesidad y Nutrición, which is an initiative of Instituto de Salud Carlos III. Dr Mannaerts is an employee of Ferring International PharmaScience Center (Copenhagen, Denmark), and Drs Qi, van Duin and Kohout are employees of the Ferring Research Institute (San Diego, USA). Dr Anderson and Dr Tena-Sempere were recipients of a grant support from the Ferring Research Institute, and Dr Anderson has undertaken consultancy work and received speaker fees outside this study from Merck, IBSA, Roche Diagnostics, NeRRe Therapeutics and Sojournix Inc. Dr Skorupskaite was supported by the Wellcome Trust through the Scottish Translational Medicine and Therapeutics Initiative 102419/Z/13/A. The other authors have no competing interest.

Effect of Rubber Polarity on Cluster Formation in Rubbers Cross-Linked with Diels-Alder Chemistry.

Diels-Alder chemistry has been used for the thermoreversible cross-linking of furan-functionalized ethylene/propylene (EPM) and ethylene/vinyl acetate (EVM) rubbers. Both furan-functionalized elastomers were successfully cross-linked with bismaleimide to yield products with a similar cross-link density. NMR relaxometry and SAXS measurements both show that the apolar EPM-g-furan precursor contains phase-separated polar clusters and that cross-linking with polar bismaleimide occurs in these clusters. The heterogeneously cross-linked network of EPM-g-furan contrasts with the homogeneous network in the polar EVM-g-furan. The heterogeneous character of the cross-links in EPM-g-furan results in a relatively high Young's modulus, whereas the more uniform cross-linking in EVM-g-furan results in a higher tensile strength and elongation at break.

Bone-site-specific responses to zoledronic acid.

OBJECTIVES: Bisphosphonates are widely used to treat bone diseases such as osteoporosis. However, they may cause osteonecrosis of the jaw. Here, we investigated whether in vivo exposure to bisphosphonates has a different effect on long bone and jaw osteoclasts, and on the turnover of these different bones. MATERIALS AND METHODS: Zoledronic acid (0.5 mg kg-1 weekly) was administered intraperitoneally to 3-month-old female mice for up to 6 months. The effects on the number of osteoclasts, bone mineralization and bone formation were measured in the long bones and in the jaw. RESULTS: Long-term treatment with zoledronic acid reduced the number of jaw bone marrow cells, without affecting the number of long bone marrow cells. Zoledronic acid treatment did not affect the number of osteoclasts in vivo. Yet, the bisphosphonate increased bone volume and mineral density of both long bone and jaw. Interestingly, 6 months of treatment suppressed bone formation in the long bones without affecting the jaw. Unexpectedly, we showed that bisphosphonates can cause molar root resorption, mediated by active osteoclasts. CONCLUSIONS: Our findings provide more insight into bone-site-specific effects of bisphosphonates and into the aetiology of osteonecrosis of the jaw. We demonstrated that bisphosphonates can stimulate osteoclast activity at the molar roots.

RPL5 on 1p22.1 is recurrently deleted in multiple myeloma and its expression is linked to bortezomib response.

Chromosomal region 1p22 is deleted in ⩾20% of multiple myeloma (MM) patients, suggesting the presence of an unidentified tumor suppressor. Using high-resolution genomic profiling, we delimit a 58 kb minimal deleted region (MDR) on 1p22.1 encompassing two genes: ectopic viral integration site 5 (EVI5) and ribosomal protein L5 (RPL5). Low mRNA expression of EVI5 and RPL5 was associated with worse survival in diagnostic cases. Patients with 1p22 deletion had lower mRNA expression of EVI5 and RPL5, however, 1p22 deletion status is a bad predictor of RPL5 expression in some cases, suggesting that other mechanisms downregulate RPL5 expression. Interestingly, RPL5 but not EVI5 mRNA levels were significantly lower in relapsed patients responding to bortezomib and; both in newly diagnosed and relapsed patients, bortezomib treatment could overcome their bad prognosis by raising their progression-free survival to equal that of patients with high RPL5 expression. In conclusion, our genetic data restrict the MDR on 1p22 to EVI5 and RPL5 and although the role of these genes in promoting MM progression remains to be determined, we identify RPL5 mRNA expression as a biomarker for initial response to bortezomib in relapsed patients and subsequent survival benefit after long-term treatment in newly diagnosed and relapsed patients.

Pharmacogenomics and chemical library screens reveal a novel SCFSKP2 inhibitor that overcomes Bortezomib resistance in multiple myeloma.

While clinical benefit of the proteasome inhibitor (PI) bortezomib (BTZ) for multiple myeloma (MM) patients remains unchallenged, dose-limiting toxicities and drug resistance limit the long-term utility. The E3 ubiquitin ligase Skp1-Cullin-1-Skp2 (SCFSkp2) promotes proteasomal degradation of the cell cycle inhibitor p27 to enhance tumor growth. Increased SKP2 expression and reduced p27 levels are frequent in human cancers and are associated with therapeutic resistance. SCFSkp2 activity is increased by the Cullin-1-binding protein Commd1 and the Skp2-binding protein Cks1B. Here we observed higher CUL1, COMMD1 and SKP2 mRNA levels in CD138+ cells isolated from BTZ-resistant MM patients. Higher CUL1, COMMD1, SKP2 and CKS1B mRNA levels in patient CD138+ cells correlated with decreased progression-free and overall survival. Genetic knockdown of CUL1, COMMD1 or SKP2 disrupted the SCFSkp2 complex, stabilized p27 and increased the number of annexin-V-positive cells after BTZ treatment. Chemical library screens identified a novel compound, designated DT204, that reduced Skp2 binding to Cullin-1 and Commd1, and synergistically enhanced BTZ-induced apoptosis. DT204 co-treatment with BTZ overcame drug resistance and reduced the in vivo growth of myeloma tumors in murine models with survival benefit. Taken together, the results provide proof of concept for rationally designed drug combinations that incorporate SCFSkp2 inhibitors to treat BTZ resistant disease.

Loss of CYLD expression unleashes Wnt signaling in multiple myeloma and is associated with aggressive disease.

Deletion or mutation of the gene encoding the deubiquitinating enzyme CYLD is a common genomic aberration in multiple myeloma (MM). However, the functional consequence of CYLD loss and the mechanism underlying its putative role as a tumor suppressor gene in the pathogenesis of MM has not been established. Here, we show that CYLD expression is highly variable in myeloma cell lines and primary MMs and that low CYLD expression is associated with disease progression from monoclonal gammopathy of undetermined significance to MM, and with poor overall and progression free-survival of MM patients. Functional assays revealed that CYLD represses MM cell proliferation and survival. Furthermore, CYLD acts as a negative regulator of NF-κB and Wnt/ß-catenin signaling and loss of CYLD sensitizes MM cells to NF-κB-stimuli and Wnt ligands. Interestingly, in primary MMs, low CYLD expression strongly correlated with a proliferative and Wnt signaling-gene expression signature, but not with an NFκB target gene signature. Altogether, our findings identify CYLD as a negative regulator of NF-κB and Wnt/ß-catenin signaling in MM and indicate that loss of CYLD enhances MM aggressiveness through Wnt pathway activation. Thus, targeting the Wnt pathway could be a promising therapeutic strategy in MM with loss of CYLD activity.

(18)F-fluoride-PET for dynamic in vivo monitoring of bone formation in multiple myeloma.

BACKGROUND: Bone disease in multiple myeloma is characterized by reduced bone formation. The gold standard of bone formation is the mineral apposition rate (MAR), an invasive technique reflecting bone formation at a single site. We compared (18)F-fluoride-PET with the MAR in myeloma patients. METHODS: Bone formation was measured before and after bortezomib treatment by determination of the MAR in iliac bone marrow biopsies and the measurement of (18)F-uptake. RESULTS: The inter- and intra-individual variations in (18)F-uptake (SUVA50%) were pronounced as 33.50 (range 4.42 to 37.92) and 27.18 (range 4.00 to 31.18), respectively. A significant correlation between the MAR and (18)F-uptake was found (r = 0.80, p = 0.017). There was a heterogeneous response after treatment varying from -2.20 to 4.53. CONCLUSIONS: Iliac (18)F-uptake was associated with the local MAR in myeloma patients. Furthermore, (18)F-fluoride-PET demonstrated the heterogeneity of in vivo bone formation, enabling monitoring during treatment.

Gene expression profile alone is inadequate in predicting complete response in multiple myeloma.

With advent of several treatment options in multiple myeloma (MM), a selection of effective regimen has become an important issue. Use of gene expression profile (GEP) is considered an important tool in predicting outcome; however, it is unclear whether such genomic analysis alone can adequately predict therapeutic response. We evaluated the ability of GEP to predict complete response (CR) in MM. GEP from pretreatment MM cells from 136 uniformly treated MM patients with response data on an IFM, France led study were analyzed. To evaluate variability in predictive power due to microarray platform or treatment types, additional data sets from three different studies (n=511) were analyzed using same methods. We used several machine learning methods to derive a prediction model using training and test subsets of the original four data sets. Among all methods employed for GEP-based CR predictive capability, we got accuracy range of 56-78% in test data sets and no significant difference with regard to GEP platforms, treatment regimens or in newly diagnosed or relapsed patients. Importantly, permuted P-value showed no statistically significant CR predictive information in GEP data. This analysis suggests that GEP-based signature has limited power to predict CR in MM, highlighting the need to develop comprehensive predictive model using integrated genomics approach.

A gene expression signature for high-risk multiple myeloma.

There is a strong need to better predict the survival of patients with newly diagnosed multiple myeloma (MM). As gene expression profiles (GEPs) reflect the biology of MM in individual patients, we built a prognostic signature based on GEPs. GEPs obtained from newly diagnosed MM patients included in the HOVON65/GMMG-HD4 trial (n=290) were used as training data. Using this set, a prognostic signature of 92 genes (EMC-92-gene signature) was generated by supervised principal component analysis combined with simulated annealing. Performance of the EMC-92-gene signature was confirmed in independent validation sets of newly diagnosed (total therapy (TT)2, n=351; TT3, n=142; MRC-IX, n=247) and relapsed patients (APEX, n=264). In all the sets, patients defined as high-risk by the EMC-92-gene signature show a clearly reduced overall survival (OS) with a hazard ratio (HR) of 3.40 (95% confidence interval (CI): 2.19-5.29) for the TT2 study, 5.23 (95% CI: 2.46-11.13) for the TT3 study, 2.38 (95% CI: 1.65-3.43) for the MRC-IX study and 3.01 (95% CI: 2.06-4.39) for the APEX study (P<0.0001 in all studies). In multivariate analyses this signature was proven to be independent of the currently used prognostic factors. The EMC-92-gene signature is better or comparable to previously published signatures. This signature contributes to risk assessment in clinical trials and could provide a tool for treatment choices in high-risk MM patients.

Characterization of the morphology of co-extruded, thermoplastic/rubber multi-layer tapes.

Tapes with alternating semi-crystalline thermoplastic/rubber layers with thicknesses varying from 100 nm up to several microm were prepared by multi-layer co-extrusion. The variation in layer thickness was obtained by varying the thermoplastic/rubber feed ratio. A systematic study on the use of various microscopy techniques to visualize the morphology of the layered systems is presented. The relatively large length scales and the sample preparation make optical microscopy (OM) unsuitable to study the morphology of the multi-layer tapes. Although excellent contrast between the thermoplastic and rubber layers can be obtained, the usually applied, relatively large magnifications limit the use of transmission electron microscopy (TEM) and atomic force microscopy (AFM) to small sample areas. The large range of applicable magnifications makes scanning electron microscopy (SEM) the most suitable technique to study the morphology of the multi-layer tapes. The sample preparation for SEM with a secondary electron (SE) detector is often based on the removal of one of the components, which may induce changes in the morphology. SEM with a back-scattered electron (BSE) detector is a very convenient method to study the morphology over a wide range of length scales, where the contrast between the different layers can be enhanced by chemical staining. Finally, the nucleation behavior (homogeneous versus heterogeneous) of the semi-crystalline layers, as probed by differential scanning calorimetry (DSC), provides valuable information on the layered morphology. The use of relatively straightforward DSC measurements shows a clear advantage with respect to the discussed microscopy techniques, since no sample preparation is required and relatively large samples can be studied, which are more representative for the bulk.

Low-molecular-weight model study of peroxide cross-linking of ethylene-propylene-diene rubber using gas chromatography and mass spectrometry II. Addition and combination reactions.

The dicumyl-peroxide-initiated addition and combination reactions of mixtures of alkanes (n-octane, n-decane) and alkenes [5,6-dihydrodicyclopentadiene (DCPDH), 5-ethylidene-2-norbornane (ENBH) and 5-vinylidene-2-norbornane (VNBH)] were studied to mimic the peroxide cross-linking reactions of terpolymerised ethylene, propylene and a diene monomer (EPDM). The reaction products of the mixtures were separated by both gas chromatography (GC) and comprehensive two-dimensional gas chromatography (GCxGC). The separated compounds were identified from their mass spectra and their GC and GCxGC elution pattern. Quantification of the various alkyl/alkyl, alkyl/allyl and allyl/allyl combination products shows that allylic-radicals comprise approximately 60% of the substrate radicals formed. The total concentration of the products formed by combination is found to be independent of the concentration and the type of alkene. The total concentration of the products formed by addition to the alkene increases with increasing concentration of alkene. In addition, the total concentration of the formed addition products depends strongly on the type of the alkene used, viz. VNBH>ENBH approximately DCPDH, which is a consequence of differences in steric hindrance of the unsaturation. The peroxide curing efficiency, defined as the number of moles of cross-linked products formed per mol of peroxide, is 173% using 9% (w/w) 5-vinylidene-2-norbornane (VNBH). This indicates that the addition reaction is recurrent. All these findings are consistent with experimental studies on peroxide curing of EPDM rubber. In addition, the present results provide more-detailed structural information, increasing the understanding of the mechanism of peroxide curing of EPDM. The described approach to use low-molecular-weight model compounds followed by GC-mass spectrometry (MS) and GCxGC-MS analysis is proven to be a very powerful tool to study the cross-linking of EPDM.

Low-molecular-weight model study of peroxide cross-linking of ethylene-propylene (-diene) rubber using gas chromatography and mass spectrometry I. Combination reactions of alkanes.

The combination reaction of linear and branched alkanes, initiated by dicumylperoxide, has been studied as a model for the combination cross-linking reaction of peroxide-cured terpolymerised ethylene, propylene and diene monomer. Both gas chromatography-mass spectrometry (GC-MS) and comprehensive two-dimensional GC-MS (GCxGC-MS) analyses have been employed to analyse the isomeric reaction products. The identification of these products based on their MS fragmentation patterns is quite complex, due to the high tendency of random rearrangements. Careful elucidation of the high-mass ions at optimised ionisation energy (55eV) has resulted in proposed structures for the different isomeric reaction products. The structure assignment by MS is in agreement with the GCxGC elution pattern and with the result of a theoretical model to predict the boiling points and, thus, the GC retention times. In addition, a model that provided a direct correlation between chemical structure and retention times was developed and this was found to provide a useful fit. Quantification of the identified reaction products by GC separation and flame ionization detection allows classification according to the hydrogen abstraction sites for the alkanes by dicumylperoxide. The selectivity for hydrogen abstraction generally follows the expected order, but a higher reactivity was observed for the methylene group next to a primary methyl group, while a reduced reactivity of the methylene group next to ethyl and to methyl groups was observed. The used approach proved to be a very powerful tool to enhance our understanding of the mechanism of peroxide cross-linking of (branched) alkanes.

Molecular dissection of the chromosome band 7q21 amplicon in gastroesophageal junction adenocarcinomas identifies cyclin-dependent kinase 6 at both genomic and protein expression levels.

Amplification of chromosome band 7q21 has been frequently detected in various types of cancer including gastroesophageal junction (GEJ) adenocarcinomas. At present, no gene has been disclosed that can explain this frequent amplification of 7q21 in GEJ carcinomas. Therefore, a detailed genomic analysis of the 7q21 region was performed on a selected series of GEJ adenocarcinomas, i.e., 14 primary adenocarcinomas and 10 cell lines, by array comparative genomic hybridization (aCGH) with a 7q11.22-q31.2 contig array. A distinct peak of amplification was identified at 92.1 Mb in 7q21.2, precisely comprising cyclin-dependent kinase 6 (CDK6), a gene involved in cell cycle regulation. A smaller peak was seen at 116.2 Mb in 7q31.2, the locus of the MET proto-oncogene. No distinct peak was detected for the hepatocyte growth factor (HGF) at 81.3 Mb in 7q21.11. An immunoprofile of HGF, CDK6 and MET revealed a strong correlation between aCGH and immunohistochemical protein expression for CDK6 (P = 0.002). Furthermore, immunohistochemistry did not show expression of CDK6 in Barrett's dysplasia and carcinoma in situ, correlating expression of CDK6 with a malignant phenotype. We conclude that high-resolution genomic analysis and immunoprofiling identify CDK6 as the main candidate target for the recurrent amplification of 7q21 in GEJ adenocarcinomas.

High-resolution array comparative genomic hybridization of chromosome 8q: evaluation of putative progression markers for gastroesophageal junction adenocarcinomas.

Amplification of 8q is frequently found in gastroesophageal junction (GEJ) cancer. It is usually detected in high-grade, high-stage GEJ adenocarcinomas. Moreover, it has been implicated in tumor progression in other cancer types. In this study, a detailed genomic analysis of 8q was performed on a series of GEJ adenocarcinomas, including 22 primary adenocarcinomas, 13 cell lines and two xenografts, by array comparative genomic hybridization (aCGH) with a whole chromosome 8q contig array. Of the 37 specimens, 21 originated from the esophagus and 16 were derived from the gastric cardia. Commonly overrepresented regions were identified at distal 8q, i.e. 124-125 Mb (8q24.13), at 127-128 Mb (8q24.21), and at 141-142 Mb (8q24.3). From these regions six genes were selected with putative relevance to cancer: ANXA13, MTSS1, FAM84B (alias NSE2), MYC, C8orf17 (alias MOST-1) and PTK2 (alias FAK). In addition, the gene EXT1 was selected since it was found in a specific amplification in cell line SK-GT-5. Quantitative RT-PCR analysis of these seven genes was subsequently performed on a panel of 24 gastroesophageal samples, including 13 cell lines, two xenografts and nine normal stomach controls. Significant overexpression was found for MYC and EXT1 in GEJ adenocarcinoma cell lines and xenografts compared to normal controls. Expression of the genes MTSS1, FAM84B and C8orf17 was found to be significantly decreased in this set of cell lines and xenografts. We conclude that, firstly, there are other genes than MYC involved in the 8q amplification in GEJ cancer. Secondly, the differential expression of these genes contributes to unravel the biology of GEJ adenocarcinomas.

Mechanotransduction of bone cells in vitro: mechanobiology of bone tissue.

Mechanical force plays an important role in the regulation of bone remodelling in intact bone and bone repair. In vitro, bone cells demonstrate a high responsiveness to mechanical stimuli. Much debate exists regarding the critical components in the load profile and whether different components, such as fluid shear, tension or compression, can influence cells in differing ways. During dynamic loading of intact bone, fluid is pressed through the osteocyte canaliculi, and it has been demonstrated that fluid shear stress stimulates osteocytes to produce signalling molecules. It is less clear how mechanical loads act on mature osteoblasts present on the surface of cancellous or trabecular bone. Although tissue strain and fluid shear stress both cause cell deformation, these stimuli could excite different signalling pathways. This is confirmed by our experimental findings, in human bone cells, that strain applied through the substrate and fluid flow stimulate the release of signalling molecules to varying extents. Nitric oxide and prostaglandin E2 values increased by between two- and nine-fold after treatment with pulsating fluid flow (0.6 +/- 0.3 Pa). Cyclic strain (1000 microstrain) stimulated the release of nitric oxide two-fold, but had no effect on prostaglandin E2. Furthermore, substrate strains enhanced the bone matrix protein collagen I two-fold, whereas fluid shear caused a 50% reduction in collagen I. The relevance of these variations is discussed in relation to bone growth and remodelling. In applications such as tissue engineering, both stimuli offer possibilities for enhancing bone cell growth in vitro.

Genomics in target and drug discovery.

Genomics-based discovery of novel therapeutic drug targets requires the design of well-controlled biological or pharmacological experiments with experimental questions and hypotheses that relate to the therapeutic area of interest. This will aid the validation level of differentially expressed genes and hence facilitate the de-selection of the genes that are identified in microarray experiments. We here provide an example of how this approach is followed in the manipulation of human macrophage foam cells towards the discovery of novel drug targets for treatment of atherosclerosis.

Toxic fear: the management of uncertainty in the wake of the Amsterdam air crash.

This paper examines the management of uncertainty among emergency responders, the media, and the public following the crash of an Israeli cargo plane carrying apparently hazardous cargo in Amsterdam's Bijlmermeer area. While the authorities' management of the emergency created by the initial crash was effective, the long-term crisis management performance was considerably less effective. It is argued that, particularly in hazardous materials emergencies, considerable management attention is required in the long-term aftermath rather than seeking a quick declaration of "all clear" or determination that the crisis is over. This paper examines the roles of all actors in the crisis and addresses the nature of communications in the "disaster after the disaster". The evolution of a "toxic fear" among citizens is documented and the social psychology of crisis management in the aftermath is examined.

Production of soluble human alpha3-fucosyltransferase (FucT VII) by membrane targeting and in vivo proteolysis.

The rational design of fucosyltransferase (FucT VII) inhibitors as potential medication in the treatment of rheumatoid arthritis requires the three-dimensional structure of this member of the glycosyltransferase family. Structure determination by X-ray diffraction analysis needs purified, soluble enzyme protein. For this purpose we developed a novel method for the high-yield production of soluble FucT VII by in vivo proteolysis. To obtain a soluble form of FucT VII a mammalian expression construct was made encoding an N-terminal portion of FucT VI (amino acids 1-63) fused with the stem region and catalytic domain of FucT VII (amino acids 39-342). Chinese hamster ovary cells stably transfected with this construct produced FucT activity in the supernatant, which has the same catalytic properties as wild-type FucT VII. This soluble form of FucT VII can be obtained in high amounts (1 mg/L) and can be efficiently purified by GDP-hexanolamine affinity chromatography. In conclusion, it was demonstrated that the intrinsic properties of FucT VII could be transferred to secreted FucT VII constructs, which may open possibilities for production of soluble forms of other members of the glycosyltransferase family as well.

Cloning and characterization of the cyclic guanosine monophosphate-inhibited phosphodiesterase PDE3A expressed in mouse oocyte.

In the preovulatory follicle, oocyte meiotic resumption occurs soon after the LH surge and is associated with a decrease in cAMP. Inhibition of cAMP degradation blocks germinal vesicle breakdown as well as activation of meiotic promoting factor, both hallmarks of reentry into the cell cycle. In situ and pharmacological analysis of rodent ovaries suggested the presence of a phosphodiesterase 3 (PDE3) in the germ cell but not the somatic cell compartment. Here we have investigated the structure and properties of the PDE form expressed in mouse oocytes. Polymerase chain reactions using a mouse oocyte cDNA library as a template, and primers based on the conserved sequence of rat and human PDE3As, yielded partial fragments corresponding to mouse PDE3A. Further screening of the mouse oocyte cDNA library and subsequent ligation of individual cDNA clones yielded PDE3A cDNA containing the entire coding region of mouse PDE3A. To determine the kinetic properties of this PDE, the cDNAs encoding the full-length PDE3A and NH(2)-truncation forms Delta 1 (Delta346aa) and Delta 2 (Delta608aa) were expressed in mouse Leydig tumor cells. Whereas the full-length recombinant protein was always found in the particulate fraction, the Delta 1 and Delta 2 truncated PDE3As were recovered mostly in the soluble fraction. The Michaelis constant values for hydrolysis of cAMP of PDE3A Delta 1 and PDE3A Delta 2 were similar to those of intact full-length PDE3A or oocyte PDE (0.2-0.5 microM). More importantly, there was good correlation between the rank of potency of selective and nonselective compounds in inhibiting recombinant PDE3A or PDE activity derived from cumulus-oocyte complexes and in blocking resumption of meiosis. These data provide evidence that the PDE expressed in the oocyte is a soluble form of PDE3A and that activity of this enzyme is involved in the control of resumption of meiosis.