HAM: A new epitope-tag for in vivo protein labeling

The ability to metabolically label proteins with ³⁵S-methionine is critical for the analysis of protein synthesis and turnover. Despite the importance of this approach, however, efficient labeling of proteins in vivo is often limited by a low number of available methionine residues, or by deleterious side-effects associated with protein overexpression. To overcome these limitations, we have created a methionine-rich variant of the widely used HA tag, called HAM, for use with ectopically expressed proteins. Here we describe the development of a series of vectors, and corresponding antisera, for the expression and detection of HAM-tagged proteins in mammalian cells. We show that the HAM tag dramatically improves the sensitivity of ³⁵S-methionine labeling, and permits the analysis of Myc oncoprotein turnover even when HAM-tagged Myc is expressed at levels comparable to that of the endogenous protein. Because of the improved sensitivity provided by the HAM tag, the vectors and antisera described here should be useful for the analysis of protein synthesis and destruction at physiological levels of protein expression.     

Auranofin affects early events in human polymorphonuclear neutrophil activation by receptor-mediated stimuli

Auranofin, a new oral antirheumatic gold compound, in concentrations achieved therapeutically, inhibits neutrophil phagocytosis, chemotaxis, chemiluminescence, reduction of cytochrome c, and release of lysosomal enzymes. To further characterize the mechanism by which auranofin affects neutrophils, we studied the effects of auranofin on unstimulated properties and functions of neutrophils as well as on rapidly stimulated functions. When examined by electron microscopy, 4 micrograms/ml of auranofin significantly decreased the number of visualized centriole-associated microtubules in resting cells. Furthermore, auranofin inhibited neutrophil spreading on glass and caused a decrease in negative surface charge (electrophoretic mobility). In addition, auranofin inhibited several fmet-leu-phe-stimulated responses such as shape change, increases in centriole-associated microtubules, decreases in surface charge, and elicited membrane potential changes (di-O-C5(3) dye response). Auranofin (1 micrograms/ml) inhibited fmet-leu-phe-stimulated superoxide and hydrogen peroxide production by 80% (p less than 0.05), and also increased the affinity of receptors for fmet-leu-phe (from Ka 0.035 to Ka 0.48, p less than 0.001). Auranofin also affected neutrophil responses to phorbol myristic acetate (PMA). The total amount of PMA-stimulated superoxide production was suppressed by as little as 0.4 micrograms/ml of auranofin, but the lag time for activation was shortened by low concentrations of auranofin (0.5 to 1 microgram/ml). Four micrograms per milliliter of auranofin suppressed the decrease in surface charge induced by PMA. However, auranofin did not influence superoxide production elicited by the ionophore A23187. The results indicate that auranofin affects the earliest detected responses in neutrophil activation by certain receptor-mediated stimuli.     

5-Hydroxytryptamine stimulates Rb efflux from pancreatic β-cells

The effects of 5-hydroxytryptamine and 5-hydroxytryptophan on ⁸⁶Rb⁺ efflux from prelabelled ob/ob-mouse islets were studied to better understand the cellular mechanisms underlying the effects of 5-hydroxytryptamine and 5-hydroxytryptophan on insulin release. 5-Hydroxytryptophan (4 mM) had no effect on ⁸⁶Rb⁺ efflux either at a low (3 mM) or at a high (20 mM) d-glucose concentration, whereas 5-hydroxytryptamine (4 mM) stimulated ⁸⁶Rb⁺ efflux at both glucose concentrations. These results indicate that 5-hydroxytryptamine may reduce glucose-induced insulin release by inhibiting early steps in the β-cell stimulus-secretion coupling.     

Some dasyclad algae of the sinemurian from the north-western iberian chains (Spain)

In a section of the Liassic southwest of the village of Préjano (Prov. Logroño) Sinemurian Dasyclad algae were found for the first time in the “Bankkalk-Series” of the “Carniolas-Formation” which is not dated up to that time. Six species,Dissocladella lucasi (Cros & Lemoine),Dissocladella iberica nov. sp.,Dissocladella ebroensis nov. sp.,Sestrosphaera liasina Pia,Gyroporella retica (Zanin) andMacroporella nov. sp. aff. sturi Bystricky are described.