A binding assay for serine hydroxymethyltransferase

A sensitive assay for measuring serine hydroxymethyltransferase activity has been developed, based on the binding of N5,N10-[14C]methylene tetrahydrofolate (THF) to DEAE-cellulose paper. The complete assay requires THF, pyridoxal 5'-phosphate, [14C]serine, and enzyme. The reaction is stopped by streaking an aliquot of the reaction mixture onto a square of DEAE-cellulose paper, washing the paper with water to remove unreacted serine, drying the paper, and counting the bound N5,N10-[14C]methylene-THF. To determine that the labeled product was N5,N10-methylene-THF, unlabeled formaldehyde, which exchanges with the labeled methylene carbon, was added after the product had accumulated; 2 min after the addition of formaldehyde the amount of labeled product was reduced by 50%, and by 85% after 10 min. In addition, glycine, which reverses the reaction, and hydroxylamine, which reacts with the methylene carbon, reduced the number of counts bound to the paper. Binding of product to the filter is proportional to both enzyme concentration and assay time. No counts were retained on phosphocellulose filters. This assay represents a new and simple method for measuring serine hydroxymethyltransferase activity, which can be used to measure enzyme activity in tissue homogenates and for screening large numbers of samples.     

Implications of the human genome initiative for the primary care physician

... Despite the need for physicians to be knowledgeable about and open to advances in genetic technology, little is known about the level of preparedness of primary care physicians to offer new genetic tests. Evidence suggests that several barriers exist to physicians adopting genetic tests. These include lack of knowledge, inability to interpret probabilistic information, low tolerance for uncertainty, negative attitudes about their responsibility for genetic counseling and testing, lack of confidence in their clinical skills, and unfamiliarity with ethical issues raised by testing. This paper will explore some of these barriers in further depth, discuss the ethical impact of physician unpreparedness on both patient care and the diffusion of genetic tests, and describe a study that is currently underway to investigate some of these issues.     

Filter mesh size and food particle uptake by Daphnia

Food size selection of four Daphnia, species (D. magna, D. hyalina, D. galeata, D. pulicaria) was investigated using spherical plastic beads as artificial food and with small bacteria. The size of the particles ranged from 0.1 to 35 m with special emphasis to the particle diameters between 0.1 and 1 m. In one set of experiments a mixture of differently sized particles was offered as food suspension and the selectivity of filtering was determined by comparing the size spectrum of the particles found in the gut contents with the spectrum in the food suspension. In a second series of experiments suspensions of uniformly sized particles were offered to single animals and their feeding activity was observed directly. In both types of experiments the mesh sizes of the filtering apparatus of the respective animals studied were measured after the experiments by, scanning electron microscopy. The mean sizes of the filter meshes were about 0.4–0.7 m. In all experiments the size of the particles found in the gut or those which caused high feeding activities were larger than the smallest mesh sizes of the filters. As a consequence simple mechanical sieving provides a sufficient explanation for the mechanism of particle retention of the filtering process in Daphnia. D. magna was found to feed with high efficiency on suspended freshwater bacteria, the residual species investigated showed low filtering efficiencies when bacteria were offered as food.The present study was supported by Deutscher Akademischer Austauschdients     

The bacteriophage kh receptor of Lactococcus lactis subsp. cremoris KH is the rhamnose of the extracellular wall polysaccharide

A receptor for bacteriophages of lactic acid bacteria, including Lactococcus lactis subsp. cremoris KH, was found on the cell wall and not on the cell membrane, as determined by a phage-binding assay of sodium dodecyl sulfate- and mutanolysin-treated cell walls. The cell wall carbohydrates of L. lactis subsp. cremoris KH were analyzed by gas chromatography and mass spectrometry and found to contain rhamnose, galactose, glucose and N-acetylglucosamine. Similar analysis of mutants that were reduced in the ability to bind phages kh, 643, c2, ml3, and 1 indicated that galactose was essential for binding all phages. In addition, rhamnose was required for binding phages kh and ml3. Inhibition studies of phage binding by using two different lectins with a specificity for galactose indicated that phage kh may not bind directly to galactose. Rather, galactose may be an essential structural component located in the vicinity of the receptor. Incubation of any of the five phages with rhamnose or of phage kh with purified cell walls inactivated the phages. Inactivation required divalent cations and was irreversible. Inactivation of phages was stereospecific for rhamnose, as neither L-(+)- nor D-(-)-fucose (the stereoisomers of rhamnose) inhibited the phage. Furthermore, phage infection of a culture was completely inhibited by the addition of rhamnose to the medium. Therefore, the receptor for phage kh appears to be a rhamnose component of the extracellular wall polysaccharide.     

Psychological perspectives of ecosystem health

Behavior-based and person-based perspectives are integrated to summarize approaches for dealing with the human element of ecosystem health. Behavior is selected by its consequences, and therefore interventions are needed to decrease the natural reinforcing consequences (e.g., convenience, efficiency) of environment-destructive behaviors and to increase the reinforcing consequences of environment-protective behaviors. An ABC Model (for Activator-Behavior-Consequence) can be applied effectively to design intervention programs for benefiting the human behavior aspects of ecosystem health. However, intervention agents are needed to implement positive behavior-change techniques, and this requires people to actively care enough to emit other-directed (or altruistic) behaviors for the health of the ecosystem. Person factors which influence one's propensity to actively care include: self-esteem, belongingness, and empowerment, influenced by perceptions of self-efficacy (I can do it), personal control (I am in control), and optimism (I expect the best). Strategies are reviewed for increasing these person states or expectancies. The health of our ecosystem depends upon people changing their behaviors and attitudes regarding the environment. Behavior-based psychology offers the technology for changing behaviors and attitudes in desirable directions; person-based psychology offers the states or expectancies needed in people to increase their willingness to actively care for the environment. By applying the theory and principles from these subdisciplines of applied psychology, the critical human element of our ecosystem can be accounted for and managed effectively.     

Evaluating treatment effects in group sequential multivariate longitudinal studies with covariate adjustment

Jeffries et al. (2018) investigated testing for a treatment difference in the setting of a randomized clinical trial with a single outcome measured longitudinally over a series of common follow-up times while adjusting for covariates. That paper examined the null hypothesis of no difference at any follow-up time versus the alternative of a difference for at least one follow-up time. We extend those results here by considering multivariate outcome measurements, where each individual outcome is examined at common follow-up times. We consider the case where there is interest in first testing for a treatment difference in a global function of the outcomes (e.g., weighted average or sum) with subsequent interest in examining the individual outcomes, should the global function show a treatment difference. Testing is conducted for each follow-up time and may be performed in the setting of a group sequential trial. Testing procedures are developed to determine follow-up times for which a global treatment difference exists and which individual combinations of outcome and follow-up time show evidence of a difference while controlling for multiplicity in outcomes, follow-up, and interim analyses. These approaches are examined in a study evaluating the effects of tissue plasminogen activator on longitudinally obtained stroke severity measurements.     

Kallikrein activation of a high molecular weight atrial peptide

Mammalian atrial extracts contain bioactive peptides that exert profound effects upon renal function and isolated smooth muscle preparations. Gel filtration chromatography of rat atrial extract separates the activity into two peaks having apparent molecular weights of 20,000 to 30,000 and less than 10,000. Mild proteolytic treatment (trypsin 1 U/ml) of the high molecular weight fraction enhances the smooth muscle relaxant activity of this fraction and concomitantly reduces the apparent molecular weight of this fraction to less than 10,000. In this report we show that urinary and submaxillary kallikrein enhances the activity of rat atrial extracts in a similar fashion. Pretreatment of the high molecular weight fraction with either kallikrein (1 microgram/ml) enhances the smooth muscle relaxant activity of this fraction. Similar treatment of the low molecular weight fraction had no effect. The enhancement of the bioactivity of the high molecular weight substance(s) by the kallikreins was abolished by aprotinin but was unaffected by soybean trypsin inhibitor. These results suggest that exogenous addition of tissue kallikrein activates a high molecular weight peptide by limited proteolysis. Analysis of the kallikrein-treated high molecular weight peptide fraction by gel filtration indicates that the biological activity comigrates with the low molecular weight peptides present in the original atrial extract.