Regulation of actin polymerization by villin, a 95,000 dalton cytoskeletal component of intestinal brush borders

A 95,000 dalton actin-binding polypeptide, villin, has been purified to 98% homogeneity from brush border cytoskeletons of chicken intestinal epithelial cells. In vitro, this protein exerts control over the polymerization of actin. In the presence of villin, the lag phase preceding detectable actin polymerization is shortened and the steady state equilibrium viscosity is reduced in proportion to the amount of villin present. A molar ratio of villin:actin of 1:40 results in a 70% reduction of the Ostwald viscosity. Significant effects can be detected at a ratio of 1:600. These ratios are physiologically relevant because the ratio of villin:actin in brush borders is 1:13 and in isolated microvilli is 1:9-12. Reduction of viscosity is mirrored by an increase in the amount of protein which fails to sediment at 150,000 X g for 60 min. An assay of the nonsedimentable protein for actin monomers by the inhibition of DNAase I showed that the concentration of monomer was not significantly altered by the presence of villin. Electron microscopic examination of negatively stained, nonsedimentable actin demonstrated that the presence of villin during actin polymerization results in the production of short oligomers which cannot anneal with each other to form long filaments. Villin is also effective in reducing the viscosity of F-actin when it is added to a fuly polymerized actin sample. In view of these striking properties, villin is likely to be an important in vivo regulator of cytoskeletal structure and, by implication, of cell shape and motility.     

Do we understand the evolution of genomic imprinting?

Voltage-gated calcium channels can be classified into high voltage activated (HVA) and low voltage activated (LVA or T-type) subtypes. The molecular diversity of HVA channels primarily results from different genes encoding their pore-forming alpha1 subunits. These channels share a common structure with an alpha1 subunit associated with at least two regulatory subunits (beta, alpha2-delta). Any of the six alpha1-related channels identified to date are regulated in their functional properties through an interaction with the ancillary beta-subunit. By contrast, the diversity and the molecular identity of LVA or T-type calcium channels have yet to be defined. Whether LVA channels are modulated by a beta-subunit, like HVA channels, is unknown. To address this issue, we have used an antisense strategy to inhibit beta-subunit expression in the NG 108-15 neuroblastoma cell line. Differentiated NG 108-15 cells express both LVA and HVA channels. We found that LVA currents were unaffected when cells were incubated with beta-antisense, while HVA currents were drastically decreased. Since LVA Ca channel currents in NG 108-15 cells are not regulated by beta-subunits, it is reasonable to postulate that the pore-forming subunit(s) of these channels lacks an interaction domain with a beta-subunit (AID). This molecular feature, which is common to various T-type channels, indicates further that LVA calcium channels belong to a channel family structurally distant from HVA channels.     

Incomplete recovery of working heart function after twenty-four-hour preservation with a modified University of Wisconsin solution

BACKGROUND AND METHODS: This study was designed to determine the function of isolated rabbit hearts after static preservation with modified University of Wisconsin solution for 24 hours. Commercially available University of Wisconsin solution, modified with CaCl2 1 mmol/L and 2,3-butanedione monoxime 30 mmol/L, was used as the preservative. After flushing the coronary vasculature with medium, hearts were submersion stored at 1 degree C to 4 degrees C. After preservation, isolated heart function at 37 degrees C was quantified for 30 minutes in a non-ejecting mode and for 4 hours ejecting at a physiologic workload. Fresh control hearts (n = 5) and University of Wisconsin solution-preserved hearts (n = 6) were studied. RESULTS: Nonworking (non-ejecting) left ventricular function of the two groups did not differ, except for peak rate of left ventricular pressure development which was higher for the University of Wisconsin solution hearts than for controls. When the hearts were subjected to a physiologic workload, however, left ventricular function of the two groups differed significantly. Three of the six University of Wisconsin solution hearts failed before the 4-hour perfusion end point, whereas all five control hearts maintained stable working function for the full 4 hours. The University of Wisconsin solution hearts, while in the ejecting mode, exhibited significantly impaired function. Mean values were as follows (p < 0.05): left ventricular systolic pressure (in millimeters of mercury), control 105 +/- 1, University of Wisconsin solution 86 +/- 4; peak rate of left ventricular pressure development (in millimeters of mercury per millisecond), control 3.33 +/- 0.11, University of Wisconsin solution 2.39 +/- 0.24; cardiac output (in milliliters per minute per gram), control 400 +/- 25, University of Wisconsin solution 288 +/- 26; stroke work (in milliJoules per gram), control 20.1 +/- 1.3, University of Wisconsin solution 11.9 +/- 1.1; left ventricular end-diastolic pressure (in millimeters of mercury), control 5.4 +/- 0.3, University of Wisconsin solution 10.2 +/- 1.3; peak aortic flow rate (in milliliters per minute), control 946 +/- 9, University of Wisconsin solution 659 +/- 44; millimoles of lactate produced in 30 min/Joule stroke work, control 0.50 +/- 0.06, University of Wisconsin solution 6.99 +/- 0.37. CONCLUSIONS: These results indicate that (1) hypothermic storage in this modified University of Wisconsin solution does not preserve hearts sufficiently to support a physiologic workload for an extended period and (2) assessment of post-preservation function with a non-ejecting heart model does not accurately predict the ability of the preserved heart to support a physiologic workload.