Split-enzyme fragment as a single affinity tag that enables protein expression,purification, and functional assays

Expressing, isolating, and characterizing recombinant proteins is crucial to many disciplines within the biological sciences. Different molecular tagging technologies have been developed to enable each individual step of protein production, from expression through purification and characterization. Monitoring the entire production process requires multiple tags or molecular interactions, because no individual tag has provided the comprehensive breadth of utility. An ideal molecular tag is small and does not interrupt expression, solubility, folding or function of the protein being purified and can be used throughout the production process. We adapted and integrated a split-luciferase system (NanoBiT®, Promega ®) to perform the range of techniques essential to protein production. We developed a simple method to monitor protein expression in real time to optimize expression conditions. We constructed a novel affinity chromatography system using the split-luciferase system to enable purification. We adapted western blot analysis, enzyme-linked immunosorbent assay, and cell-based bioassay to characterize the expressed proteins. Our results demonstrate that a single-tag can fulfill all aspects needed throughout protein production.     

Karyometric image analysis for intraepithelial and invasive cervical lesions

OBJECTIVE: To derive an objective, numeric measure for the progression of intraepithelial and invasive squamous cell cervical lesions. STUDY DESIGN: Thin-layer cervical cytology preparations from colposcopically confirmed normal cervix, low grade squamous intraepithelial lesions, high grade squamous intraepithelial lesions and carcinoma were identified from a cross-sectional study. Fifty-nine cases representing 4 diagnostic categories were selected, and 2,375 nuclei from epithelial cells representative of the diagnostic category were randomly selected for imaging and measurement from these cases. Additionally, 1,378 visually normal appearing intermediate cells from low and high grade squamous intraepithelial lesions, as well as from carcinoma cases, were identified for analysis. The nuclei were quantitatively characterized, and discriminant analyses were performed to derive a progression curve from normal cytology to carcinoma. RESULTS: The lesion signatures show a clear increase in nuclear abnormality with increasing progression. A progression curve was derived based on mean discriminant function scores for each diagnostic category and on the mean nuclear abnormality values for the nuclei in each category, as expressed by their deviation in feature values from normal reference nuclei. CONCLUSION: A numeric assessment of lesion progression for cervical precancerous and cancerous lesions based on karyometric measurements is possible and may provide an objective, precise characterization of each lesion as well as a basis for improved performance in automated cytology-based cervical cancer screening.     

Developmental evaluation of children born to mothers occupationally exposed to waste anesthetic gases

BACKGROUND: The etiology of developmental delay in children is frequently unknown. Increasing evidence supports the possibility that environmental and occupational factors might be part of the basis for such delays. This study focuses on the development of children born to mothers who were exposed during their pregnancy to waste anesthetic gases. METHODS: The study population included 40 children aged 5-13 years born to female anesthesiologists and nurses working in operating rooms (OpRs) exposed to waste anesthetic gases, and 40 unexposed children born to female nurses and physicians who worked in hospitals during their pregnancy but did not work in OpRs. The unexposed group was matched for children's age and gender and maternal occupation (nurses vs. doctors). By means of standardized developmental tests, the present study population was evaluated for their medical and neurodevelopmental state. Questionnaires were given for the detection of attention and activity levels as perceived by the parents. Additional questionnaires dealt with information concerning developmental milestones, maternal and fetal morbidity, and gynecological history. RESULTS: No differences were noted between the groups as newborns or in developmental milestones at the age of 5-13 years; however, the mean score of gross motor ability was significantly lower in the exposed versus the unexposed group. Additionally, the mean score of the DSM-III-R Parent-Teacher Questionnaire (PTQ) (i.e., measure of inattention/hyperactivity) was higher in the exposed group. The level of exposure, as measured by the number of weekly hours in the OpRs, was significantly and negatively correlated with fine motor ability and the score of IQ performance. CONCLUSIONS: Our study supports the hypothesis that occupational exposure to anesthetic gases might be a risk factor for minor neurological deficits of children born to mothers who work in OpRs and therefore indicates the need for more studies in this area and perhaps more caution among OpR pregnant women and employers.     

An atomic model for actin binding by the CH domains and spectrin-repeat modules of utrophin and dystrophin

Utrophin and dystrophin link cytoskeletal F-actin filaments to the plasmalemma. Genetic strategies to replace defective dystrophin with utrophin in individuals with muscular dystrophy requires full characterization of these proteins. Both contain homologous N-terminal actin-binding motifs composed of a pair of calponin-homology (CH) domains (CH1 and CH2) that are connected by spectrin-repeat modules to C-terminal membrane-binding sequences. Here, electron microscopy and 3D reconstruction of F-actin decorated with utrophin and dystrophin actin-binding constructs were performed using Utr261 (utrophin's CH domain pair), Utr416 (utrophin's CH domains and first spectrin-repeat) and Dys246 (dystrophin's CH domain pair). The lozenge-like utrophin CH domain densities localized to the upper surface of actin subdomain 1 and extended azimuthally over subdomain 2 toward subdomains 3 and 4. The cylinder-shaped spectrin-repeat was located at the end of the CH domain pair and was aligned longitudinally along the cleft between inner and outer actin domains, where tropomyosin is present when on thin filaments. The connection between the spectrin-repeat module and the CH domains defined the orientation of CH1 and CH2 on actin. Resolution of utrophin's CH domains and spectrin-repeats permitted docking of crystal structures into respective EM densities, leading to an atomic model where both CH and spectrin-domains bind actin. The CH domain-actin interaction for dystrophin was found to be more complex than for utrophin. Binding assays showed that Utr261 and Utr416 interacted with F-actin as monomers, whereas Dys246 appeared to associate as a dimer, consistent with a bilobed Dys246 structure observed on F-actin in electron microscope reconstructions. One of the lobes was similar in shape, position and orientation to the monomeric CH domains of Utr261, while the other lobe apparently represented a second set of CH domains in the dimeric Dys246. The extensive contact made by dystrophin on actin may be used in vivo to help muscles dissipate mechanical stress from the contractile apparatus to the extracellular matrix.     

Oxidative interactions of synthetic lung epithelial lining fluid with metal-containing particulate matter

Epidemiology studies show association of morbidity and mortality with exposure to ambient air particulate matter (PM). Metals present in PM may catalyze oxidation of important lipids and proteins present in the lining of the respiratory tract. The present study investigated the PM-induced oxidation of human bronchoalveolar lavage (BAL) fluid (BALF) and synthetic lung epithelial lining fluid (sELF) through the measurement of oxygen incorporation and antioxidant depletion assays. Residual oil fly ash (ROFA), an emission source PM that contains approximately 10% by weight of soluble transition metals, was added (0-200 microg/ml) to BALF or sELF and exposed to 20% (18)O(2) (24 degrees C, 4 h). Oxygen incorporation was quantified as excess (18)O in the dried samples after incubation. BALF and diluted sELF yielded similar results. Oxygen incorporation was increased by ROFA addition and was enhanced by ascorbic acid (AA) and mixtures of AA and glutathione (GSH). AA depletion, but not depletion of GSH or uric acid, occurred in parallel with oxygen incorporation. AA became inhibitory to oxygen incorporation when it was present in high enough concentrations that it was not depleted by ROFA. Physiological and higher concentrations of catalase, superoxide dismutase, and glutathione peroxidase had no effect on oxygen incorporation. Both protein and lipid were found to be targets for oxygen incorporation; however, lipid appeared to be necessary for protein oxygen incorporation to occur. Based on these findings, we predict that ROFA would initiate significant oxidation of lung lining fluids after in vivo exposure and that AA, GSH, and lipid concentrations of these fluids are important determinants of this oxidation.     

How does time since feeding affect the fuels pigeons use during flight?

Time between meals can vary from multiple hours to days within and among species. We investigated the effects of time since feeding on lipid, protein, and carbohydrate oxidation in flying pigeons (Columba livia) by interpreting changes in blood plasma metabolite concentrations and mass during flight. Five pigeons were flown or rested for 4 h after food deprivations of 2, 12, 24, and 48 h. After flight, blood plasma concentrations of uric acid and beta-hydroxybutyrate were elevated over control and preflight values, indicating elevated protein and lipid catabolism during flight. Lipid oxidation, as indicated by changes in beta-hydroxybutyrate concentration, increased more in unfed flying pigeons compared with recently fed flying pigeons and with resting controls. Protein oxidation, as indicated by changes in uric acid concentrations, also positively covaried with feeding time; the covariation was mostly caused by increases in 48-h food-deprived pigeons. Unfed birds lost less mass during a 4-h flight than recently fed birds. We reasoned that recently fed pigeons oxidized more glycogen in flight than pigeons not recently fed; calculated glycogen stores explained 72%-117% of mass loss differences between 2- and 48-h-fed pigeons. Thus, time since feeding was an important determinant of the fuels pigeons used in flight.     

Population differentiation in the red-legged kittiwake (Rissa brevirostris) as revealed by mitochondrial DNA

Population decline in red-legged kittiwakes(Rissa brevirostris) over recent decadeshas necessitated the collection of informationon the distribution of genetic variation withinand among colonies for implementation ofsuitable management policies. Here we present apreliminary study of the extent of geneticstructuring and gene flow among the threeprincipal breeding locations of red-leggedkittiwakes using the hypervariable Domain I ofthe mitochondrial control region. Geneticvariation was high relative to other species ofseabirds, and was similar among locations.Analysis of molecular variance indicated thatpopulation genetic structure was statisticallysignificant, and nested clade analysissuggested that kittiwakes breeding on BeringIsland maybe genetically isolated from thoseelsewhere. However, phylogeographic structurewas weak. Although this analysis involved onlya single locus and a small number of samples,it suggests that red-legged kittiwakes probablyconstitute a single evolutionary significantunit; the possibility that they constitute twomanagement units requires furtherinvestigation.     

Lysophosphatidylethanolamine acyltransferase activity is elevated during cardiac cell differentiation

This work attempts to explain several aspects of the response of plasminogen to 6-aminohexanoate (6-AH). These responses include the overall fluorescent changes that occur when plasminogen binds the ligand, the changes shown by the individual domains when they bind the ligand, and the changes in structure shown by the holoprotein when it binds 6-AH. The results have implications for understanding the physicochemical behavior of all kringle based proteins.