A SENSITIVE RADIOIMMUNOASSAY FOR 7S NERVE GROWTH FACTOR ANTIGENS IN SERUM AND TISSUES

A radioimmunoassay was developed which can measure accurately concentrations of mouse 7S nerve growth factor antigens (NGFA) as low as 3·0 ng/ml in serum or tissue homogenates. Extremely large amounts of presumed nerve growth factor were found in the submaxillary gland; but considerable quantities were also present in mouse serum, kidney, adrenal gland and vas deferens. Heart, spleen, liver and muscle contained less of the presumed nerve growth factor, and only small amounts were recovered from brain. Rat adrenal gland and serum from rats, guinea pigs and man contained much less immunologically reactive material. The level of presumed nerve growth factor in the mouse heart was highest at birth and decreased slowly during maturation. In the mouse submaxillary gland the content of presumed nerve growth factor increased rapidly after 2 weeks of postnatal age, with higher levels found in male animals. Destruction with 6-hydroxydopamine of the sympathetic nerves in the hearts of newborn or adult mice did not significantly alter the amount of presumed nerve growth factor recovered in the heart.     

Phosphorylation by cAMP-dependent protein kinase inhibits the degradation of tau by calpain.

The effects of cAMP-dependent protein kinase (cAMP-PK) phosphorylation on the degradation of the microtubule-associated protein tau by calpain were studied. Purified bovine brain tau that had been phosphorylated by cAMP-PK had a slower migration pattern on sodium dodecyl sulfate-polyacrylamide gels and a more acidic, less heterogeneous pattern on two-dimensional, nonequilibrium pH gradient electrophoresis (NEPHGE) gels compared with untreated tau. Phosphorylation of tau by cAMP-PK significantly inhibited its proteolysis by calpain compared with untreated tau. To our knowledge this is the first demonstration that phosphorylation of tau by a specific kinase results in increased resistance to hydrolysis by calpain. Tau dephosphorylated by alkaline phosphatase migrated more rapidly on sodium dodecyl sulfate-polyacrylamide gels and also showed an altered two-dimensional NEPHGE pattern. Dephosphorylation of tau had no effect on its susceptibility to calpain proteolysis, indicating that regulation of the susceptibility to calpain hydrolysis is due to the phosphorylation of a specific site(s). These results suggest a role for phosphorylation in regulating the degradation of tau. Abnormal phosphorylation could result in a protease-resistant tau population which may contribute to the formation of paired helical filaments in Alzheimer's disease.     

Promoting Smoke-Free Homes: A Novel Behavioral Intervention Using Real-Time Audio-Visual Feedback on Airborne Particle Levels

Interventions are needed to protect the health of children who live with smokers. We pilot-tested a real-time intervention for promoting behavior change in homes that reduces second hand tobacco smoke (SHS) levels. The intervention uses a monitor and feedback system to provide immediate auditory and visual signals triggered at defined thresholds of fine particle concentration. Dynamic graphs of real-time particle levels are also shown on a computer screen. We experimentally evaluated the system, field-tested it in homes with smokers, and conducted focus groups to obtain general opinions. Laboratory tests of the monitor demonstrated SHS sensitivity, stability, precision equivalent to at least 1 µg/m³, and low noise. A linear relationship (R² = 0.98) was observed between the monitor and average SHS mass concentrations up to 150 µg/m³. Focus groups and interviews with intervention participants showed in-home use to be acceptable and feasible. The intervention was evaluated in 3 homes with combined baseline and intervention periods lasting 9 to 15 full days. Two families modified their behavior by opening windows or doors, smoking outdoors, or smoking less. We observed evidence of lower SHS levels in these homes. The remaining household voiced reluctance to changing their smoking activity and did not exhibit lower SHS levels in main smoking areas or clear behavior change; however, family members expressed receptivity to smoking outdoors. This study established the feasibility of the real-time intervention, laying the groundwork for controlled trials with larger sample sizes. Visual and auditory cues may prompt family members to take immediate action to reduce SHS levels. Dynamic graphs of SHS levels may help families make decisions about specific mitigation approaches.     

Magnetic circular dichroism and electron paramagnetic resonance studies of hydrogenases I and II from Clostridium pasteurianum

The two iron-only hydrogenases (I and II) from Clostridium pasteurianum have been investigated by variable temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies. Samples were studied both reduced with dithionite under an atmosphere of H2 and after oxidation with thionine. The results are consistent with four and two [4Fe-4S]1+,2+ (F)-clusters in hydrogenases I and II, respectively. All four F-clusters are reduced and paramagnetic in reduced hydrogenase I, with up to one exhibiting an S = 3/2 ground state and the remainder having conventional S = 1/2 ground states. Both F-clusters have S = 1/2 ground states in reduced hydrogenase II; however, one appears to be only partially reduced under the conditions used for reduction. MCD studies of the oxidized enzymes show no temperature-dependent features in the visible region which can be attributed to the EPR-active S = 1/2 hydrogen-activating cluster, suggesting predominantly oxygen and nitrogen coordination for the iron atoms of this center. However, temperature-dependent MCD transitions arising from a hitherto undetected S greater than 1/2 Fe-S clusters are apparent in both oxidized hydrogenases. Detailed EPR studies of oxidized hydrogenase I revealed resonances from an S = 3/2 species, however, spin quantitation reveals this to be a trace component that is unlikely to be responsible for the observed low temperature MCD spectrum. The nature and origin of these S greater than 1/2 Fe-S clusters are discussed in light of the available spectroscopic data for these and other iron-only hydrogenases.     

Ruptured fission yeast walls

Distributions of rupture sites of fission yeast cells ruptured by glass beads have been related to a new morphometric analysis. As shown previously (Johnson et al.,Cell Biophysics, 1995), ruptures were not randomly distributed nor was their distribution dictated by geometry, rather, ruptures at the extensile end were related to cell length just as the rate of extension is related to cell length. The extension patterns of early log, mid-log, late log, and stationary phase cells from suspension cultures were found to approximate the linear growth patterns of Kubitschek and Clay (1986). The median length of cells was found to decline through the log phase in an unbalanced manner.