Cytoskeletal protein binding kinetics at planar phospholipid membranes.

It has been hypothesized that nonspecific reversible binding of cytoskeletal proteins to lipids in cells may guide their binding to integral membrane anchor proteins. In a model system, we measured desorption rates k(off) (off-rates) of the erythrocyte cytoskeletal proteins spectrin and protein 4.1 labeled with carboxyfluorescein (CF), at two different compositions of planar phospholipid membranes (supported on glass), using the total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP) technique. The lipid membranes consisted of either pure phosphatidylcholine (PC) or a 3:1 mixture of PC with phosphatidylserine (PS). In general, the off-rates were not single exponentials and were fit to a combination of fast, slow, and irreversible fractions, reported both separately and as a weighted average. By a variation of TIR/FRAP, we also measured equilibrium affinities (the ratio of surface-bound to bulk protein concentration) and thereby calculated on-rates, k(on). The average off-rate of CF-4.1 from PC/PS (approximately 0.008/s) is much slower than that from pure PC (approximately 1.7/s). Despite the consequent increase in equilibrium affinity at PC/PS, the on-rate at PC/PS is also substantially decreased (by a factor of 40) relative to that at pure PC. The simultaneous presence of (unlabeled) spectrin tends to substantially decrease the on-rate (and the affinity) of CF-4.1 at both membrane types. Similar experiments for CF-spectrin alone showed much less sensitivity to membrane type and generally faster off-rates than those exhibited by CF-4.1. However, when mixed with (unlabeled) 4.1, both the on-rate and off-rate of CF-spectrin decreased drastically at PC/PS (but not PC), leading to a somewhat increased affinity. Clearly, changes in affinity often involve countervailing changes in both on-rates and off-rates. In many of these studies, the effect of varying ionic strength and bulk concentrations was examined; it appears that the binding is an electrostatic attraction and is far from saturation at the concentrations employed. These results and the techniques implemented carry general implications for understanding the functional role of nonspecific protein binding to cellular lipid membranes.     

ApoC-III content of apoB-containing lipoproteins is associated with binding to the vascular proteoglycan biglycan

Retention of apolipoprotein (apo)B and apoE-containing lipoproteins by extracellular vascular proteoglycans is critical in atherogenesis. Moreover, high circulating apoC-III levels are associated with increased atherosclerosis risk. To test whether apoC-III content of apoB-containing lipoproteins affects their ability to bind to the vascular proteoglycan biglycan, we evaluated the impact of apoC-III on the interaction of [(35)S]SO(4)-biglycan derived from cultured arterial smooth muscle cells with lipoproteins obtained from individuals across a spectrum of lipid concentrations. The extent of biglycan binding correlated positively with apoC-III levels within VLDL (r = 0.78, P < 0.01), IDL (r = 0.67, P < 0.01), and LDL (r = 0.52, P < 0.05). Moreover, the biglycan binding of VLDL, IDL, and LDL was reduced after depletion of apoC-III-containing lipoprotein particles in plasma by anti-apoC-III immunoaffinity chromatography. Since apoC-III does not bind biglycan directly, enhanced biglycan binding may result from a conformational change associated with increased apo C-III content by which apoB and/or apoE become more accessible to proteoglycans. This may be an intrinsic property of lipoproteins, since exogenous apoC-III enrichment of LDL and VLDL did not increase binding. ApoC-III content may thus be a marker for lipoproteins characterized as having an increased ability to bind proteoglycans.     

The male determinant of self-incompatibility in Brassica oleracea is located in the pollen coating

An in vitro bioassay has been developed to explore the role of the pollen coating in the pollen/stigma interaction in Brassica oleracea . In the assay, coating is removed from pollen grains, supplemented with protein fractions isolated from coatings of different S (self incompatibility) haplotypes, and then—using micromanipulation—interposed between individual pollen grains and the stigmatic surface. Normally, the coating used is of the same haplotype as the pollen in the experiment—thus constituting an 'extension' of its own coat—but carrying the supplemented protein fractions. Initial experiments confirmed preliminary data that the pollen coating contained the male determinant of self incompatibility (SI); not only did the addition of 'self' coating (i.e. that with the same S -haplotype as the stigma) prevent the success of a compatible cross pollination, but a 'cross' coating (i.e. that with a different S -haplotype from the stigma) could induce the germination and growth of self pollen. Protein supplementation experiments demonstrated that the pollen-held determinant is contained within the water soluble component of the pollen coat, while further analysis revealed that the active molecular species possesses an M_r10 kDa. More extensive fractionation by gel filtration and reverse phase HPLC was used to isolate a family of basic, cysteine-rich proteins (PCP-A: P ollen C oat P roteins-class A)—one of which is known to bind to stigmatically-expressed components of the S -locus in Brassica . Introduction of the PCP-A protein fraction into the bioassay confirmed the male determinant of SI as a protein, and probably a member of the PCP-A protein family.     

The behavior of R-ovalbumin and its individual components A1, A2, and A3 in urea solution: kinetics and equilibria

Procedures are described for the isolation of the individual components A₁, A₂, and A₃ of native R-ovalbumin from freshly laid domestic hen eggs. Because heavy metal ion contaminants result in spurious irreproducible kinetics, particularly at high pH, considerable care is taken to avoid their presence. Kinetics studies are made of the behavior of whole R-ovalbumin and its individual components in urea solution over the pH range 3.7–9.6 following the reaction by determining absorbance differences at 233, 287, and 293 nm and ORD and CD changes at 350 and 221 nm, respectively. Reaction is rapid at low pH, slowing with increasing pH. Except under limited conditions, the reaction is not simple first order. Equations are presented for describing the reactions, and the nature of the reaction products is considered. Unfolding equilibrium profiles were also determined by ORD at several wavelengths and were not stigmoidal in shape and the normalized curves were not superimposed.Deceased December 8, 2001     

Assessing Changes in Biomass of Riverbed Periphyton

Periphytic biomass in a downriver riffle of the Grand River in Southern Ontario, Canada was measured with concrete-block glass slide samplers from May to December 1971. Average rate of accumulation of periphyton on glass slides was 266.2 mg/m²/day ash-free weight, 120.9 mg/m²/day carbon, 20.4 mg/m²/day nitrogen, or 1.11 mg/m²/day chlorophyll a and on concrete blocks was 590.0 mg/m²/day ash-free weight, 202.9 mg/m²/day carbon, 22.8 mg/m²/day nitrogen, or 2.95 mg/m²/day chlorophyll a. The standing biomass of periphyton on glass slides varied from a maximum of 21.6 g/m² between May 28 and July 15 to a minimum of 0.7 g/m² in December with an average of 8.6 g/m². The biomass on concrete blocks ranged from a high of 132.5 g/m² in August to a low of 29.9 g/m² in October with a mean of 66.1 g/m². The well established periphyton on concrete blocks towards the end of the study period was similar to that from native rocks with respect to biomass, carbon and nitrogen contents, and ratios of biomass to chlorophyll a. This suggests that concrete blocks are a better substrate than glass slides for measuring riverbed periphyton. By comparing biomass of periphyton on slides and on concrete blocks, the average rate of loss of periphyton from concrete blocks was estimated to be 2.9 g/m²/day, representing 63% of the mean total accumulation rate of the periphyton. The magnitude of the estimated total loss and the high standing biomass of the periphyton on concrete blocks manifest the importance of the periphyton as a source of organic matter in the river.     

Comparative effects of sodium pyrithione evoked intracellular calcium elevation in rodent and primate ventral horn motor neurons

Oral administration of sodium pyrithione (NaP) causes hindlimb weakness in rodents, but not in primates. Previous work using Aplysia neurons has demonstrated that NaP produces a persistent influx of Ca²⁺ ions across the plasma membrane. To determine whether this also occurs in mammalian neurons and whether this could underlie the inter-species difference between rodents and primates, we have tested the effects of NaP on intracellular Ca²⁺ levels ([Ca²⁺]_i) in rat and monkey motor neurons in vitro. Motor neurons present in spinal cord slices from rhesus monkey embryos (E37 and 56) and from rat E16 were dissected and cultured on glass coverslips. Following 2 weeks (rhesus) or 2-3 days (rat) in culture, neurons were loaded with fura-PE3/AM, and examined for [Ca²⁺]_i changes in response to NaP. Rhesus motor neurons were identified by immunostaining for Islet-1 (MN specific antigen) and neuron specific enolase (NSE). Motor neurons from both species exhibited dose-dependent NaP-evoked increases in [Ca²⁺]_i However, the dose-response curve for the Rhesus motor neurons was significantly shifted to the right of the rat dose-response curve, whereas the overall amplitude of the Ca²⁺ rise was similar in both species. As shown previously for the Aplysia neurons, the action of NaP is attenuated by SKF 96365, an inhibitor of store-operated calcium entry. In contrast the action of NaP is unaffected by nifedipine and tetrodotoxin, blockers of voltage-dependent Ca²⁺ and Na⁺ channels, respectively, or by ouabain, an inhibitor of the plasma membrane Na⁺/K⁺ ATPase. Our results indicate that the NaP-induced increase in [Ca²⁺]_i is conserved across species and suggest that the toxicological sensitivity of rodent over primate to pyrithione could be due to the enhanced sensitivity of rodent motor neurons to NaP-evoked intracellular Ca²⁺ elevation.     

Investigation into the intracellular fates,speciation and mode of action of selenium-containing neuroprotective agents using XAS and XFM

Background

A variety of selenium compounds have been observed to provide protection against oxidative stress, presumably by mimicking the mechanism of action of the glutathione peroxidases. However, the selenium chemistry that underpins the action of these compounds has not been unequivocally established.

Determination of albuterol in plasma after aerosol inhalation by gas chromatography-mass spectrometry with selected-ion monitoring

Albuterol is a β₂-adrenergic agonist commonly used as a bronchdilator for the treatment of patients with asthma. We have developed an assay to determine plasma levels as low as 50 pg/ml of albuterol by gas chromatography-mass spectrometry (GC-MS). This assay utilizes isotopically labeled albuterol ([¹³C]albuterol) as an internal standard. In this assay albuterol and the internal standard are recovered from 1 ml of plasma using solid-phase extraction. The samples are then derivatized to trimethylsilyl ethers using N,O-bis(trimethylsilyl)trifluoro-acetamide with 1% trimethylchlorosilane. The samples are then analyzed by GC-MS with selected-ion monitoring (SIM) for the ions m/z 369.15 and 370.15. The method has been validated for a concentration range of 50–10000 pg/ml in plasma.