Wild type human (h) interleukin 5 (wt IL5) is composed of two identical peptide chains linked by disulfide bonds. A gene encoding a single chain form of hIL5 dimer was constructed by linking the two hIL5 chain coding regions with Gly-Gly linker. Expression of this gene in COS cells yielded a single chain IL5 protein (sc IL5) having biological activity similar to that of wt IL5, as judged by stimulation of human cell proliferation. Single chain and wt IL5 also had similar binding affinity for soluble IL5 receptor alpha chain, the specificity subunit of the IL5 receptor, as measured kinetically with an optical biosensor. The design of functionally active sc IL5 molecule. Such mutagenesis was exemplified by changes at residues Glu-13, Arg-91, Glu-110, and Trp-111. The receptor binding and bioactivity data obtained are consistent with a model in which residues from both IL5 monomers interact with the receptor alpha chain, while the interaction likely is asymmetric due to the intrinsic asymmetry of folded receptor. The results demonstrate a general route to the further mapping of receptor and other binding sites on the surface of human IL5.

Amino Acid Sequence↗

PubMed1996

The European study of assisted reproduction families: family functioning and child development.

Findings are presented of a European study (conducted in the UK, Italy, Spain and The Netherlands) of family relationships and the social and emotional development of children in families created as a result of the two most widely used reproductive technologies, in-vitro fertilization (IVF) and donor insemination (DI), in comparison with control groups of families with naturally conceived child and adoptive families. Mothers of children conceived by assisted reproduction expressed greater warmth towards their child, were more emotionally involved with their child, interacted more with their child and reported less stress associated with parenting than mothers who conceived their child naturally. Similarly, assisted reproduction fathers were found to interact more with their child and to contribute more to parenting than fathers with a naturally conceived child. With respect to the children themselves, no group differences were found for either the presence of psychological disorder or for children's perceptions of the quality of family relationships. The findings relating to the quality of parenting and the socio-emotional development of the children were similar in each of the four countries studied.

Adoption↗

PubMed1996

Expansion of bronchial epithelial cell populations by in vitro culture of explants from dysplastic and histologically normal sites.

The genetic and phenotypic properties of cells which ultimately give rise to carcinoma of the lung are not well defined in part because of unavailability of preneoplastic cells from well-characterized dysplastic sites. In order to expand bronchial epithelial cell populations from patients at high risk for lung cancer, endobronchial biopsy specimens were explanted onto collagen- and fibronectin-coated dishes and cultured in serum-free, chemically defined media. One hundred forty-nine biopsy pairs were obtained from smokers and from healthy volunteers for culture and histologic evaluation. The histologic appearances of mucosa adjacent to the site of the cultured biopsies ranged from normal through varying degrees of noninvasive squamous dysplasia to invasive carcinoma. Confluent monolayers of pure epithelial cells were obtained from 68% of the cultured explants. Sites exhibiting high-grade dysplasia were 51% more likely to yield successful cultures than sites exhibiting normal histology (13 of 14 cultures successful versus 52 of 83 cultures successful, P < 0.02). Cultures had a maximum proliferative life span of 81 days and none of the cultures spontaneously became immortalized. Immunolabeling studies revealed that all cultured epithelial cells, regardless of the in situ histologic appearances of the mucosa at the biopsy site, strongly expressed keratin and epidermal growth factor receptor, weakly expressed transferrin receptor and human folate receptor, and were negative for neural cell adhesion molecule and human leukocyte antigen DR (HLADR). Ploidy and karyotypic analyses were performed in a limited number of explants from normal and dysplastic sites and all were found to be diploid without karyotypic abnormality. We conclude that pure bronchial epithelial cell populations can be routinely expanded from histologically normal and dysplastic sites by tissue culture of biopsy explants and that the expanded cell populations may represent a library of normal and preneoplastic cells which are suitable for immunophenotypic, ploidy, genetic, or functional analyses.

Bronchi↗

PubMed1996

Correlative assessment of morphologic, immunophenotypic, and genetic changes in bronchial epithelium of tobacco smokers.

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Bronchi↗

PubMed1996

Urine toxicology screens in drivers suspected of driving while impaired from drugs.

OBJECTIVE: Police departments, in conjunction with the National Highway Traffic Safety Administration, have developed a standardized evaluation aimed at identifying drivers impaired by drugs other than ethanol. These evaluations are performed by specially trained police officers known as Drug Recognition Experts. METHODS: We retrospectively reviewed the evaluations of 242 drivers detained for driving while impaired in the City and County of Denver from January 1, 1988 to June 30, 1990. RESULTS: All drivers had urine toxicology screens performed, which were positive for a mean 1.2 +/- 0.9 SD (range zero to four) for drugs having the potential for causing driving impairment. The 193/242 urine screens (79.8%) testing positive showed the following drugs: cannabis 162 (66.9%), stimulants (including cocaine metabolites) 80 (33.1%), depressants (benzodiazepines and barbiturates) 24 (9.9%), narcotics 12 (5.0%), inhalants (toluene) 1 (0.4%), hallucinogens (LSD) 1 (0.4%), and other 3 (1.2%). Drug Recognition Experts, based on their initial evaluation, were able to predict correctly some or all of the drugs found on the urine screens in 178/242 (73.6%) of cases. Overall agreement between the Drug Recognition Experts opinions and urine screen results had a kappa value (p < 0.05) of 0.41. CONCLUSIONS: There was a high rate (79.8%) of positive urine toxicology screens in drivers suspected of nonethanol drug impairment. In most cases, Drug Recognition Experts were able to reliably predict the results of these screens.

Adolescent↗

PubMed1995

Mutagenesis in the C-terminal region of human interleukin 5 reveals a central patch for receptor alpha chain recognition.

Cassette mutagenesis was used to identify side chains in human interleukin 5 (hIL-5) that mediate binding to hIL-5 receptor alpha chain (hIL-5R alpha). A series of single alanine substitutions was introduced into a stretch of residues in the C-terminal region, including helix D, which previously had been implicated in receptor alpha chain recognition and which is aligned on the IL-5 surface so as to allow the topography of receptor binding residues to be examined. hIL-5 and single site mutants were expressed in COS cells, their interactions with hIL-5R alpha were measured by a sandwich surface plasmon resonance biosensor method, and their biological activities were measured by an IL-5-dependent cell proliferation assay. A pattern of mutagenesis effects was observed, with greatest impact near the interface between the two four-helix bundles of IL-5, in particular at residues Glu-110 and Trp-111, and least at the distal ends of the D helices. This pattern suggests the possibility that residues near the interface of the two four-helix bundles in hIL-5 comprise a central patch or hot spot, which constitutes an energetically important alpha chain recognition site. This hypothesis suggests a structural explanation for the 1:1 stoichiometry observed for the complex of hIL-5 with hIL-5R alpha.

Amino Acid Sequence↗

PubMed1995

Identification of three proteins in the eye of Aplysia, whose synthesis is altered by serotonin (5-HT). Possible involvement of these proteins in the ocular circadian system.

Previous results using translation inhibitors in the ocular circadian system of Aplysia suggest that protein synthesis may be involved in the light and serotonin (5-HT) entrainment pathways or perhaps in the circadian oscillator. Proteins have been previously identified whose synthesis was altered by treatments of light capable of perturbing the phase of the circadian rhythm in the eye of Aplysia. We extended these studies by investigating the effects of other treatments that perturb the ocular circadian rhythm on protein synthesis. 5-HT altered the synthesis of nine proteins. Interestingly, five of the proteins affected by treatments with 5-HT were previously shown to be affected by treatments with light. Four of the proteins affected by treatments with 5-HT were also affected by treatments with analogs of cAMP, a treatment which mimics the effects of 5-HT on the ocular circadian rhythm. To identify the cellular function of some of these proteins, we obtained their partial amino acid sequences. Based on these sequences and additional characterizations, a 78-kDa, pI 5.6 Aplysia protein appears to be glucose-regulated protein 78/binding protein, and a 36-kDa, pI 5.7 Aplysia protein appears to be porin/voltage-dependent anion channel. Heat shock experiments on Aplysia eyes revealed that yet another one of the Aplysia proteins (70 kDa) affected by 5-HT appears to be a heat-inducible member (heat shock protein 70) of the family of heat shock proteins. These findings suggest that these three identified proteins, together or individually, may be involved in some way in the regulation of the timing of the circadian oscillator in the eye of Aplysia.

Amino Acid Sequence↗

PubMed1995

Paramedics and technicians are equally successful at managing cardiac arrest outside hospital.

OBJECTIVE: To examine the effect on survival of treatment by ambulance paramedics and ambulance technicians after cardiac arrest outside hospital. DESIGN: Prospective study over two years from 1 April 1992 to 31 March 1994. SETTING: Accident and emergency department of university teaching hospital. SUBJECTS: 502 consecutive adult patients with out of hospital cardiopulmonary arrest of cardiac origin. INTERVENTIONS: Treatment by ambulance technicians or paramedics both equipped with semiautomatic defibrillators. MAIN OUTCOME MEASURES: Rate of return of spontaneous circulation, hospital admission, and survival to hospital discharge. RESULTS: Rates of return of spontaneous circulation, hospital admission, and survival to hospital discharge were not significantly different for patients treated by paramedics as opposed to ambulance technicians. Paramedics spent significantly longer at the scene of the arrest than technicians (P < 0.0001). CONCLUSIONS: The response of ambulance paramedics to patients with cardiopulmonary arrest outside hospital does not provide improved outcome when compared with ambulance technicians using basic techniques and equipped with semi-automatic defibrillators.

Adult↗

PubMed1995

Binding interactions of human interleukin 5 with its receptor alpha subunit. Large scale production, structural, and functional studies of Drosophila-expressed recombinant proteins.

Human interleukin 5 (hIL5) and soluble forms of its receptor alpha subunit were expressed in Drosophila cells and purified to homogeneity, allowing a detailed structural and functional analysis. B cell proliferation confirmed that the hIL5 was biologically active. Deglycosylated hIL5 remained active, while similarly deglycosylated receptor alpha subunit lost activity. The crystal structure of the deglycosylated hIL5 was determined to 2.6-A resolution and found to be similar to that of the protein produced in Escherichia coli. Human IL5 was shown by analytical ultracentrifugation to form a 1:1 complex with the soluble domain of the hIL5 receptor alpha subunit (shIL5R alpha). Additionally, the relative abundance of ligand and receptor in the hIL5.shIL5R alpha complex was determined to be 1:1 by both titration calorimetry and SDS-polyacrylamide gel electrophoresis analysis of dissolved cocrystals of the complex. Titration microcalorimetry yielded equilibrium dissociation constants of 3.1 and 2.0 nM, respectively, for the binding of hIL5 to shIL5R alpha and to a chimeric form of the receptor containing shIL5R alpha fused to the immunoglobulin Fc domain (shIL5R alpha-Fc). Analysis of the binding thermodynamics of IL5 and its soluble receptor indicates that conformational changes are coupled to the binding reaction. Kinetic analysis using surface plasmon resonance yielded data consistent with the Kd values from calorimetry and also with the possibility of conformational isomerization in the interaction of hIL5 with the receptor alpha subunit. Using a radioligand binding assay, the affinity of hIL5 with full-length hIL5R alpha in Drosophila membranes was found to be 6 nM, in accord with the affinities measured for the soluble receptor forms. Hence, most of the binding energy of the alpha receptor is supplied by the soluble domain. Taken with other aspects of hIL5 structure and biological activity, the data obtained allow a prediction for how 1:1 stoichiometry and conformational change can lead to the formation of hIL5.receptor alpha beta complex and signal transduction.

Amino Acid Sequence↗

PubMed1995

Disclosure of donor insemination: parental attitudes.

Disclosures by parents with a child conceived by donor insemination were compared to those of parents with adopted children and parents with children conceived by in vitro fertilization. None of the donor-insemination parents had told their child. The factors found to create the greatest difficulties for disclosure were the father's infertility, the timing and method of telling, and the lack of genetic information for the child.

Adoption↗

PubMed1995

A survey of semen donation: phase II--the view of the donors.

This report presents the findings of phase II of the survey of semen donation commissioned by the Human Fertilization and Embryology Authority (HFEA), the purpose of which was to obtain information about the motivations and attitudes of semen donors in comparison with a matched group of non-donors. A total of 14 centres licensed by the HFEA to conduct donor insemination participated in the investigation. At each centre, all men who attended for their first or second appointment between 1st November 1993 and 31st January 1994 were asked to complete a questionnaire about the factors which prompted them to attend the centre and about their knowledge of, and attitudes towards, semen donation. In spite of the recommendation by the HFEA that attempts should be made to recruit as semen donors older men in stable relationships who already have children of their own and who wish to donate for altruistic reasons, it remains the case that the large majority of men in the UK who donate semen are young single students who are largely motivated by payment. Views on oocyte donation of women who had never donated oocytes were also obtained, and compared with views on semen donation of a matched group of men who had never donated semen.

Attitude↗

PubMed1995

Pericardial and mediastinal involvement from bronchogenic carcinoma demonstrated by Tl-201 imaging.

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Carcinoma, Squamous Cell↗

PubMed1995

Families created by the new reproductive technologies: quality of parenting and social and emotional development of the children.

The creation of families by means of the new reproductive technologies has raised important questions about the psychological consequences for children, particularly where gamete donation has been used in the child's conception. Findings are presented of a study of family relationships and the social and emotional development of children in families created as a result of the 2 most widely used reproductive technologies, in vitro fertilization (IVF) and donor insemination (DI), in comparison with control groups of families with a naturally conceived child and adoptive families. The quality of parenting was assessed using a standardized interview with the mother, and mothers and fathers completed questionnaire measures of stress associated with parenting, marital satisfaction, and emotional state. Data on children's psychiatric state were also obtained by standardized interview with the mother, and by questionnaires completed by the mothers and the children's teachers. The children were administered the Separation Anxiety Test, the Family Relations Test, and the Pictorial Scale of Perceived Competence and Social Acceptance. The results showed that the quality of parenting in families with a child conceived by assisted conception is superior to that shown by families with a naturally conceived child. No group differences were found for any of the measures of children's emotions, behavior, or relationships with parents. The findings are discussed in terms of their implications for understanding the role of genetic ties in family functioning and child development.

Adoption↗

PubMed1995

The lesions of rotavirus infection in 1- and 10-day-old gnotobiotic calves.

Age-related resistance to rotavirus disease has been described with some rotaviruses. In the present study, we investigated age-related resistance to rotavirus disease by defining extent of intestinal infection, virus replication, and severity of intestinal lesions in groups of three 1- and 10-day-old gnotobiotic calves of mixed breed inoculated orally with a cloned bovine rotavirus of low virulence for calves (strain C3-160) and in two groups of three uninoculated control calves of mixed breed. One-day-old calves inoculated with rotavirus developed diarrhea 26 hours after inoculation, and their feces contained 10(8.5)-10(9.2) TCID50/g feces; inoculated 10-day-old calves did not develop diarrhea, virus excretion commenced on the second or third day after inoculation, and peak concentrations of virus in feces were 10(5.7)-10(7.9) TCID50/g feces. Calves were euthanatized within 8-30 hours after the attainment of peak virus shedding while they were still shedding virus at peak levels. The mean percentage of small intestinal epithelium that was immunostained for rotavirus was three times greater in 1-day-old calves than in 10-day-old calves, and the large intestine was infected more extensively in 1-day-old calves. Immunostaining for rotavirus was maximal in the mid small intestine. Staining of mucin was substantially less in the epithelium of the small intestines and colon of rotavirus-inoculated 1-day-old calves than in age-matched controls. The mean height of villi was reduced to approximately half that of controls in the mid and distal small intestine of rotavirus-inoculated 1-day-old calves and was unchanged in 10-day-old calves. Mean crypt cell production rates were greater than that in controls in both groups of rotavirus-inoculated calves, indicating increased enterocyte loss. Age-related resistance to disease was not due to an inability of rotavirus to infect and replicate in enterocytes with lethal effects but appeared to be associated with a slowing of the pathogenic process, which occurred because insufficient enterocytes became infected and destroyed for lesions to develop.

Aging↗