David A. Rintoul

Major intrinsic polypeptide (MIP26K) from lens membrane: Reconstitution into vesicles and inhibition of channel forming activity by peptide antiserum

Bovine and human lens membrane, when reconstituted into lipid vesicles containing oxidized cytochrome C, will mediate the transmembrane passage of ascorbate into the vesicles, where the reduction of cytochrome C is measured spectrophotometrically. This channel forming activity is specifically inhibited by antiserum made against a synthetic octapeptide near the C-terminus of MIP26K. Together, these studies describe a direct and more sensitive assay system for measurement of channel-forming activity of MIP26K, and suggest that the C-terminus of this molecule may be particularly important in the regulation of channel formation.

Early events in polyomavirus infection: fusion of monopinocytotic vesicles containing virions with mouse kidney cell nuclei☆

The entry of polyomavirus enclosed in monopinocytotic vesicles into mouse kidney cell nuclei was studied and evidence for a fusion mechanism was obtained. In vivo studies using the fluorescent lipophilic dye diI-C16(3) as a plasma membrane label showed that polyomavirus-infected nuclei accumulate plasma membrane, while uninfected or polyoma capsid-infected nuclei do not. Further evidence for fusion was obtained with electron microscopy of thin sections of infected mouse kidney cells. These specimens showed accumulation of plasma membrane in the outer nuclear membrane as well as evidence of recent fusion events. The polyoma virions (capsid proteins) were seen to accumulate on the inner nuclear membrane and in the nucleus and were identified by immunogold staining of the thin sections. The combined results of the in vivo dye studies and thin section immunoelectron microscopy studies provide evidence for a fusion mechanism for polyomavirus entry into mouse kidney cell nuclei.

Evidence for reduced lipid order in plasma membranes from cataractous human lenses

Membrane preparations from normal and cataractous human lenses were prepared by a procedure which minimally disrupts membrane lipid structure. Fluorescence depolarization of cis and trans-parinaric acid probes was measured as a function of temperature in membranes, lipid extracts, and phospholipids. These measurements indicated that membranes from cataractous lenses were less ordered (more fluid) than equivalent membranes from normal, age-matched control lenses. No significant differences could be detected between normal and cataractous membrane total lipid extracts, or between normal and cataractous membrane phospholipids. These observations imply that membrane lipid-protein interactions play a significant role in determination of lens membrane structure, and that these interactions are altered in cataractous lens membranes.

Ganglioside GM1 sensitizes tumor cells to growth inhibitory glycopeptides

Abstract We have purified and characterized a glycopeptide from surfaces of brain cortex cells that inhibits cellular protein synthesis in normal but not transformed cells. Data are presented that correlate the ability of C3H fibrosarcoma 1316 cells to avoid the inhibitors activity with a reduced cellular level of the ganglioside GM1. When 1316 cells were incubated with GM1 under conditions in which the ganglioside is incorporated into the cells, the cells became sensitive to the inhibitor, Similar experiments with ceramide resulted in no change in 1316 sensitivity. In addition, when 1316 cells were incubated with GM1 for 18 h, they apparently metabolized GM1 to simpler gangliosides and lost their sensitivity to the glycopeptide inhibitor.

Reconstitution of MIP26 from single human lenses into artificial membranes. I. Differences in pH sensitivity of cataractous vs. normal human lens fiber cell proteins

Reconstitution of the lens fiber cell protein known as MIP26 into liposomes composed of heterologous phospholipids was achieved; this protein renders the liposomes permeable to low molecular weight compounds. MIP26 from either bovine or human lenses was capable of forming channels in artificial membranes. The assay technique was sufficiently sensitive to allow reconstitution of MIP26 from single human lenses, enabling us to examine the function of channels from either cataractous or age-matched normal lenses. Decreases in pH can cause these channels to close, analogous to the hypothesized channel closing in the in vivo situation. The pH optimum of reconstituted channels in liposomes containing MIP26 from bovine lenses or normal human lenses is very sharp; but is substantially broadened if the liposomes contain MIP26 from cataractous human lenses. This latter result suggests a functional alteration in human lens membranes which is correlated with the development of human senile cataract.

Physical Properties of Membranes and Membrane Lipids from the Fiber Cell of the U18666A-Cataractous Rat

Steady state fluorescence anisotropy of parinaric acid probes was used to examine lipid motion in membranes and membrane lipids from U18666A-induced cataractous rat lenses. Cortical and nuclear fractions were examined separately. The drug treatment resulted in an increase in the sterol/phospholipid ratio in the nucleus and a decrease in this ratio in the cortex. Fluorescence anisotropy of trans parinaric acid (tPnA) suggested that membranes from the cortex of cataractous lenses were more ordered than the cortical membranes from control or treated-but-clear lenses. Membranes from the nucleus of control lenses were more ordered than membranes from the cortex of controls. Nuclear membranes from cataractous lenses were slightly less ordered than nuclear membrane from control or treated-but-clear lenses. Similar experiments using liposomes prepared with membrane lipid from lens cortex showed that membrane lipids from treated lenses, control lenses, and treated-but-clear lenses had similar fluorescence anisotropy profiles, suggesting that cortical lipids had similar order. Conversely, fluorescence anisotropy of parinaric acid probes in liposomes prepared from nuclear fractions showed a slight increase in lipid order from control to treated-but-clear to cataractous preparations. These results are interpreted to indicate the presence of an alteration in lipid-protein interactions in cortical membranes in the cataractous lenses; this results in more ordered membranes at the physiological temperature in these lenses. The increase in the order of cortical membranes from cataractous lenses is dependent on the presence of membrane proteins rather than lipids, since it is not seen in the liposome preparations (which are protein-free).(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of the Effectiveness of the Studio Format in Introductory Undergraduate Biology

Kansas State University converted its introductory biology course, previously taught as an audio-tutorial (A-T), to a studio format in 1997. We share with others information about the process involved and present assessment data for the studio format course that address 1) student exam performance in A-T and studio; 2) student course grades in A-T and studio; 3) student and instructor perceptions and attitudes for A-T and studio; 4) student performance in subsequent biology courses for A-T and studio; and 5) gains in student learning for the studio course and other traditional lecture/lab courses. Collectively, these measures demonstrate that the studio format is as effective as or more effective (for some measures) than the A-T approach and traditional approaches in providing an effective learning environment. We discuss the issues involved in comparing course formats.

Phase behavior and arrangement of molecular species in mixtures of a mixed chain and a symmetric phosphatidylethanolamine in the gel and fluid phases

1-Octadecanoyl, 2-decanoylphosphatidylethanolamine (C(18:0)C(10:0)PE) has been reported to exhibit mixed interdigitated gel-phase packing of the phospholipid acyl chains (Mason, J.T. and Stephenson, F.A. (1990) Biochemistry 29, 590-598). In contrast, ditetradecanoylphosphatidylethanolamine (C(14:0)C(14:0)PE) packs without significant interdigitation of the phospholipid acyl chains across the bilayer center. In this report, the gel-fluid transition temperatures of C(18:0)C(10:0)PE and C(14:0)C(14:0)PE in multilamellar dispersions were determined by fluorescence anisotropy of cis-parinaric acid and trans-parinaric acid with a descending temperature scan rate of 0.67 degrees C/min. The transition mid-points detected for C(18:0)C(10:0)PE with cis-parinaric acid were 19 degrees C in water, 18 degrees C at pH 8.1, and 14 degrees C at pH 10. The phase diagram for C(14:0)C(14:0)PE and C(18:0)C(10:0)PE at pH 10 suggests complete mixing in the fluid phase and considerable immiscibility in the gel phase. Cross-linking of equimolar mixtures of C(14:0)C(14:0)PE and C(18:0)C(10:0)PE with dimethylsuberimidate at pH 10 revealed a random arrangement of the two species in the fluid phase, confirming the notion that C(18:0)C(10:0)PE and C(14:0)C(14:0)PE are miscible in the fluid phase, as determined from the phase diagram. In contrast, cross-linking of the equimolar mixture of C(18:0)C(10:0)PE and C(14:0)C(14:0)PE in the gel phase at 0 degrees C revealed a non-random arrangement, demonstrating and confirming immiscibility in the gel phase.