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.     

A putative GDP–GTP exchange factor is required for development of the excretory cell in Caenorhabditis elegans

The Caenorhabditis elegans excretory cell extends tubular processes, called canals, along the basolateral surface of the epidermis. Mutations in the exc-5 gene cause tubulocystic defects in this canal. Ultrastructural analysis suggests that exc-5 is required for the proper placement of cytoskeletal elements at the apical epithelial surface. exc-5 encodes a protein homologous to guanine nucleotide exchange factors and contains motif architecture similar to that of FGD1, which is responsible for faciogenital dysplasia. exc-5 interacts genetically with mig-2, which encodes Rho GTPase. These results suggest that EXC-5 controls the structural organization of the excretory canal by regulating Rho family GTPase activities.     

Effect of various methods of immobilization on the stability of a microbial biosensor for surfactants based onPseudomonas rathonis T

The operating and storage stability of a receptor element of an amperometric biosensor based on thePseudomonas rathonis strain T capable of degrading surfactants was tested. Microbial cells were immobilized by incorporation in gels (agar, agarose, and calcium-alginate), polyvinyl alcohol membrane, adhesion to Chromatographic paper GF/A, or by cross-linking induced by glutaric aldehyde. Incorporation of microbial cells in agar gel provides long-standing conservation of their activity and viability during measurements of high concentrations of surfactants and allows the receptor element of the biosensor to be rapidly recovered after measurements.     

Savignin, a potent thrombin inhibitor isolated from the salivary glands of the tick Ornithodoros savignyi (Acari: Argasidae).

A thrombin (E.C. 3.4.21.5) inhibitor, savignin, was isolated from the salivary glands of Ornithodoros savignyi by a combination of size exclusion, anion-exchange, and reversed-phase chromatography. The inhibitor has a molecular mass of 12,430.4 Da as determined by electrospray mass spectrometry. The behavior of savignin during anion-exchange chromatography indicated that it has an acidic pI. The available N-terminal sequence (residues 1-11) differed from that of ornithodorin with only one residue. Savignin inhibits thrombin-induced platelet aggregation, but has no effect on ADP- or collagen-induced aggregation. Kinetic studies indicated that savignin is a competitive, slow-, tight-binding inhibitor of alpha-thrombin (K(i) = 4.89 +/- 1.39 pM). Tight-binding kinetics showed that the inhibitor has a lower affinity for gamma-thrombin (K(i) = 22.3 +/- 5.9 nM). Plasmin, factor Xa, and trypsin are not inhibited by savignin.     

Lung tissue mechanics and extracellular matrix composition in a murine model of silicosis.

The dynamic mechanical properties of lung tissue and its contents of collagen and elastic fibers were studied in strips prepared from mice instilled intratracheally with saline (C) or silica [15 (S15) and 30 days (S30) after instillation]. Resistance, elastance, and hysteresivity were studied during oscillations at different frequencies on S15 and S30. Elastance increased from C to silica groups but was similar between S15 and S30. Resistance was augmented from C to S15 and S30 and was greater in S30 than in S15 at higher frequencies. Hysteresivity was higher in S30 than in C and S15. Silica groups presented a greater amount of collagen than did C. Elastic fiber content increased progressively along time. This increment was related to the higher amount of oxytalan fibers at 15 and 30 days, whereas elaunin and fully developed elastic fibers were augmented only at 30 days. Silicosis led not only to pulmonary fibrosis but also to fibroelastosis, thus assigning a major role to the elastic system in the silicotic lung.