Characterization and cloning of a Tenebrio molitor hemolymph protein with sequence similarity to insect odorant-binding proteins.

The yellow mealworm beetle, Tenebrio molitor, produces a number of moderately abundant low molecular weight hemolymph proteins ( approximately 12 kDa) which behave in a similar manner during purification and share antigenic epitopes. The cDNA sequence of the major component (THP12) was determined and the deduced protein sequence was found to be similar to those of insect odorant-binding proteins. Southern blot analysis suggests that at least some of the diversity in this family of proteins is encoded at the gene level. Both northern and western blot analysis indicate that THP12 is present in a variety of developmental stages and both sexes. THP12 was originally classified as an antifreeze protein, but the lack of antifreeze activity in the recombinant protein, as well as the clear separation of the antifreeze activity from THP12 following HPLC purification, has ruled out this function. The abundance of THP12, the similarity of THP12 to insect odorant-binding proteins, and the presence of hydrophobic cavities inside the protein (Rothemund et al., A new class of hexahelical insect proteins revealed as putative carriers of small hydrophobic ligands. Structure, 7 (1999) 1325-1332.) suggest that THP12 may function to carry non-water soluble compounds in the hemolymph. THP12 is also similar, particularly in structurally important regions, to other insect proteins from non-sensory tissues, suggesting the existence of a large family of carrier proteins which may perform diverse functions throughout the insect.

Childhood exposure to infection and risk of adult onset wheeze and atopy.

BACKGROUND: The prevalence of asthma and allergic diseases in children and young adults is inversely associated with family size. It has been suggested that more frequent exposure to infections in a large family group, particularly those spread by the faecal-oral route, may protect against atopic diseases, although not all published data support this hypothesis. Whether similar considerations apply to adult onset wheeze is unknown. The relationship between adult onset wheezing and atopy measured in adulthood and childhood exposure to a range of infections was investigated. METHODS: A nested case control study of participants in a 30 year follow up survey was conducted. Questionnaire data on childhood infections had been obtained in a 1964 survey. In 1995 a further questionnaire on respiratory symptoms and other risk factors for wheezing illness was administered, total IgE, skin and RAST tests were performed, and serum was stored. In 1999 serological tests for hepatitis A, Helicobacter pylori, and Toxoplasma gondii were performed on the stored samples. Information from the 1964 questionnaires was available for 97 cases and 208 controls and serological tests were obtained for 85 cases and 190 controls. The potential risk factors were examined for all cases, those who reported doctor diagnosed asthma, those who described persistent cough and phlegm with wheeze, and subjects stratified by atopic status. RESULTS: The sibship structure was similar in cases and controls. In univariate analysis of all cases, childhood infections reported by parents as acquired either before or after the age of three years did not influence case:control or atopic status. Seropositivity was also similar for all cases and controls, but cases in the subgroup with chronic cough and phlegm were more likely to be seropositive for hepatitis A and H pylori. Seropositivity was unrelated to atopic status. In multivariate analyses both the effect of having two or more younger siblings (OR 0.1, 95% CI 0.03 to 0.8) and of acquiring measles up to the age of three (OR 0.2, CI 0.03 to 0.8) were significantly related to a lower risk of doctor diagnosed asthma. CONCLUSIONS: In these well characterised subjects, exposure to infections as measured by parental reports obtained at age 10-14 years and by serological tests obtained in adulthood did not influence the development of wheezing symptoms or atopic status in adulthood. However, early exposure to measles and family size may be associated with a lower risk of adult onset doctor diagnosed asthma.

Characterization of the gene encoding human TAFI (thrombin-activable fibrinolysis inhibitor; plasma procarboxypeptidase B).

Thrombin-activable fibrinolysis inhibitor (TAFI) is a recently described human plasma zymogen that is related to pancreatic carboxypeptidase B. The active form of TAFI (TAFIa), which is formed by thrombin cleavage of the zymogen, likely inhibits fibrinolysis by removal from partially degraded fibrin of the carboxyl-terminal lysine residues which act to stimulate plasminogen activation. We have isolated and characterized genomic clones which encompass the entire human TAFI gene from lambda phage and bacterial artificial chromosome genomic libraries. The complete TAFI gene contains 11 exons and spans approximately 48 kb of genomic DNA. The positions of intron/exon boundaries are conserved between the TAFI gene and the rat pancreatic carboxypeptidase A1, A2, and B and the human mast cell carboxypeptidase A genes, indicating that these carboxypeptidases arose from a common ancestral gene. However, the intron lengths diverge significantly among all of these genes. The TAFI promoter lacks a consensus TATA sequence, and transcription is initiated from multiple sites. Transient transfection of reporter plasmids containing portions of the TAFI 5'-flanking region into mammalian cells allowed localization of the promoter and identified a approximately 70 bp region crucial for liver-specific transcription. Sequence analysis of cDNA clones obtained from human liver RNA indicated that the TAFI transcript is polyadenylated at three different sites. Our findings will facilitate the assessment of the regulation of TAFI expression by transcriptional and/or posttranscriptional mechanisms. Furthermore, knowledge of the genomic structure of the TAFI gene will aid in the identification of mutations that may be associated with the tendency to either bleed or thrombose.