Redundant and selective roles for erythropoietin receptor tyrosines in erythropoiesis in vivo

Cytokine receptors have been shown in cell culture systems to use phosphotyrosine residues as docking sites for certain signal transduction intermediates. Studies using various cellular backgrounds have yielded conflicting information about the importance of such residues. The present studies were undertaken to determine whether or not tyrosine residues within the erythropoietin receptor (EPOR) are essential for biologic activity during hematopoiesis in vivo. A variant of the EPOR was constructed that contains both a substitution (R129C) causing constitutive receptor activation as well as replacement of all eight cytoplasmic tyrosines by phenylalanines (cEPORYF). A comparison between animals exposed to recombinant retroviruses expressing cEPOR and cEPORYF showed that efficient red blood cell (RBC) development in vivo is dependent on the pressence of tyrosine residues in the cytoplasmic domain of the EPOR. In addition, an inefficient EPOR tyrosine independent pathway supporting RBC development was detected. Tyrosine add-back mutants showed that multiple individual tyrosines have the capacity to restore full erythropoietic potential to the EPOR as determined in whole animals. The analysis of primary erythroid progenitors transduced with the various cEPOR tyrosine mutants and tyrosine add-backs showed that only tyrosine 343 (Y1) and tyrosine 479 (Y8) were capable of supporting immature burst-forming unit-erythroid progenitor development. Thus, this receptor is characterized by striking functional redundancy of tyrosines in a biologically relevant context. However, selective tyrosine residues may be uniquely important for early signals supporting erythroid development.     

Best estimate of the magnitude of mortality due to occupational exposure to hazardous substances

OBJECTIVES: With a proportionate attributable risk approach, to estimate the magnitude of premature mortality induced by exposure to hazardous substances in the Australian workforce. METHODS: Estimates of the magnitude of mortality induced by exposure to hazardous substances in the Australian work-force were calculated by the proportionate attributable risk approach after careful consideration of options for methodological refinements. The main data sources used were unit mortality datasets (individual deidentified death records), and tabulations when these were unavailable. RESULTS: The estimated number of deaths that occurred in Australia each year from occupational exposure to hazardous substances was 2290; 78% of these were men. The rate of mortality attributable to occupational exposure to hazardous substances was three to four times greater in male workers than in female workers. Male (productive) person-years of life lost (PYLL) were generally eight times higher than female PYLL. Cancer was the prime cause of death, followed by renal, cardiovascular, neurological, and chronic respiratory disease. Acute toxic episodes accounted for a small proportion of mortality but yielded a much larger proportion of PYLL, reflecting the relatively young ages of those who experienced fatal effects. CONCLUSIONS: Although national estimates of the proportions of mortality attributable to occupational exposure to hazardous substances seemed to be validly derived, uncertainties remain associated with the lack of an empirical basis for derivation of proportionate risk fractions used in the calculations. The finding of an appreciable burden of mortality that is attributable to past occupational exposure to hazardous substances emphasises the necessity for occupational health and safety authorities to measure and reduce worksite exposures. There is also an incentive to encourage the construction of appropriately designed cohort studies across industries and occupational groupings so that, ultimately, risk estimates may be directly calculated and applied to total workforce data for the definitive estimation of the magnitude of harm induced by occupational exposure to hazardous substances.     

Young children use motive information to make trait inferences

The present study investigates children's capacity to understand traits in a psychologically meaningful way. Participants included 18 individuals in each of 4 age groups: kindergarten (ages 5-6), 2nd grade (ages 7-8), 5th grade (ages 10-11), and adult. They heard a series of 6 short stories in which a main character performs an action based on a particular motive (positive, negative, or incidental) that results in either a positive or a negative emotional consequence for another character. Participants evaluated each main character and predicted the character's behavior and mental states in different social contexts. Participants in all age groups, even the 5- to 6-year-olds, made trait inferences that were influenced by motive information. These results provide evidence that young children are capable of more sophisticated reasoning about traits than has been suggested previously.     

Inhibition kinetics and affinity labeling of bacterial squalene:hopene cyclase by thia-substituted analogues of 2, 3-oxidosqualene

Five sulfur-containing analogues of 2,3-oxidosqualene (OS) were evaluated as inhibitors of squalene:hopene cyclase (SHC) from Alicyclobacillus acidocaldarius. In these analogues, sulfur replaces carbons at C-6, C-10, C-14, C-18, or C-19 of OS. Each analogue was a submicromolar inhibitor of SHC with IC50 values ranging from 60 to 570 nM. Enzyme inhibition kinetic analysis was performed using homogeneous recombinant A. acidocaldarius SHC. While analogues 9 (S-14, Ki = 109 nM, kinact = 0.058 min-1) and 11 (S-19, Ki = 83 nM, kinact = 0.054 min-1) were time-dependent inhibitors of SHC, analogues 7 (S-6, Ki = 127 nM) and 8 (S-10, Ki = 971 nM) showed no time dependency with SHC. Analogue 10 (S-18) was the most potent inhibitor and showed time-dependent irreversible inhibition (Ki = 31 nM, kinact = 0.071 min-1). Kinetic analysis for the five analogues with purified rat liver OSLC was conducted to compare the vertebrate and prokaryotic enzymes. Affinity labeling experiments, using either [17-3H]10 or [22-3H]10 with crude and with pure recombinant SHC, clearly showed specific labeling. A single major radioactive band at 72 kDa on SDS-PAGE indicated that irreversible covalent modification of SHC had occurred. These results suggest that the presence of sulfur at C-18 of OS can interrupt the cyclization and that an intermediate partially cyclized cation may be captured by a nucleophilic residue of the SHC active site.     

Dependence of the driving force of the sodium pump on rate of transport

The driving force for active transport of Na+ in the isolated toad bladder, ENa, was measured as the reciprocal slope of the change in conductance with change in short-circuit current after stimulation with antidiuretic hormone. The base-line short-circuit current was altered by change in ambient Na+ concentration or addition of amiloride, maneuvers which alter availability of Na+ at the site of active transport. In the absence of a chemical gradient for Na+ across the bladder, ENa was found to be inversely related to the rate of Na+ transport, a finding incompatible with the simple electrical analogue that has been proposed for the system. The results provide additional support for the view that ENa measured in this way has both energetic and kinetic components.     

Surface markers of human eosinophils

Peripheral blood eosinophils from patients with eosinophilia and from healthy subjects were studied for surface immunoglobulins, receptors for the Fc region of IgG, complement receptors, and spontaneous rosette formation with sheep and mouse erythrocytes. Eosinophils were found to have receptors for complement and for aggregated IgG, and to have the same two types of complement receptors as do lymphocytes and monocytes. Immune adherence type receptors were specific for C4 or C3b, while C3d receptors were specific for C3d but unreactive with C4. Eosinophils differed from fully mature neutrophils in that the former had C3d receptors and relatively weak immune adherence (C4 or C3b) receptors, while the later did not have the C3d receptors and had strong immune adherence receptors. Eosinophil phagocytosis of complement-receptor bound erythrocytes was dependent on the presence of IgG in the antibody coating the red blood cells; this requirement for IgG resembled that found in neutrophil phagocytosis. No surface Ig or spontaneous erythrocyte rosette formation was observed with eosinophils.