Evolution of Multilocus Genetic Structure in an Experimental Barley Population

Data from 311 selfed families isolated from four generations (F8, F13, F23, F45) of an experimental barley population were analyzed to determine patterns of change in character expression for seven quantitative traits, and in single-locus allelic frequencies, and multilocus genetic structure, for 16 Mendelian loci that code for discretely recognizable variants. The analyses showed that large changes in single-locus allelic frequencies and major reorganizations in multilocus genetic structure occurred in each of the generation-to-generation transitions examined. Although associations among a few traits persisted over generations, dynamic dissociations and reassociations occurred among several traits in each generation-transition period. Overall, the restructuring that occurred was characterized by gradual decreases in the number of clusters of associated traits and increases in the number of traits within each cluster. The observed changes in single-locus frequencies and in multilocus genetic structure were attributed to interplay among various evolutionary factors among which natural selection acting in a temporally heterogeneous environment was the guiding force.     

Simultaneous recording of cell volume changes and intracellular pH or Ca2+ concentration in single osteosarcoma cells UMR-106-01.

We present a new technique for the simultaneous measurement of cell volume changes and intracellular ionic activities in single cells. The technique uses measurement of changes in the concentration of intracellularly trapped fluorescent dyes to report relative cell volume. By using pH- or Ca(2+)-sensitive dyes and recording at the ion-sensitive and -insensitive (isosbestic) wavelengths, the method can measure both cell volume changes and intracellular ionic activities. The technique was used to study the mechanisms of regulatory volume decrease (RVD) in the osteosarcoma cell line UMR-106-01 grown on cover slips. Swelling cells in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered hypotonic medium was followed by stable cytosolic acidification and a decrease in cell volume back toward normal. The recovery of cell volume could be blocked by depolarization, treatment with ouabain, or depletion of cell Cl-. These suggest the conductive efflux of K+ and Cl- during RVD. The cytosolic acidification that accompanied cell swelling was not blocked by amiloride, bafilomycin A, or removal of Cl- and could not be reproduced by depletion of cellular ATP. These findings exclude Na+/H+ and Cl-/HCO-3 exchange, intracellularly generated acid, or increased metabolism, respectively, as the cause of the acidification. The cell swelling-induced acidification was inhibited by depolarization, suggesting the involvement of an electrogenic pathway. The acidification, as well as RVD, was inhibited by short incubation with deoxyglucose, and these effects could not be reversed by valinomycin. Thus, the anionic pathway(s) participating in RVD and the acidification are sensitive to the cellular level of ATP. Together, these studies indicate that RVD in UMR-106-01 cells in HEPES-buffered medium is mediated by the conductive efflux of K+, Cl-, and OH-.     

Mechanism of human keratinocyte migration on fibronectin: unique roles of RGD site and integrins.

The migration of human keratinocytes over the wound bed plays an important role in the re-epithelialization of cutaneous wounds. Fibronectin, a large glycoprotein matrix component that is abundant within cutaneous wound beds, promotes keratinocyte migration. However, the mechanisms by which keratinocytes migrate over fibronectin are unknown. In this study, we sought to identify specific sites within the fibronectin molecule that induce keratinocyte locomotion and to characterize the cell surface receptors involved. The data show that the domain within the fibronectin molecule that induces human keratinocyte migration is the 120 kD cell-binding domain close to the carboxyl terminus. The 40 kD heparin-binding domain near the carboxyl terminus and the 45 kD gelatin-binding domain near the amino terminus did not promote keratinocyte migration. In addition, keratinocyte migration on both fibronectin and the 120 kD cell-binding domain was completely inhibited by the presence of GRGDSP peptide, suggesting that keratinocyte migration on fibronectin is mediated by recognizing the RGD sequence located within the cell-binding domain of fibronectin. Furthermore, keratinocytes were able to migrate directly on immobilized RGD substratum. Cell migration on fibronectin is mediated by the alpha 5 beta 1 integrin since antibodies blocking the alpha 5 and the beta 1 subunits completely inhibited keratinocyte migration on fibronectin. In addition, we demonstrate that human keratinocytes express alpha 5 beta 1 integrin in culture by flow cytometry.     

A new mode for heme-heme interactions in hemoglobin associated with distal perturbations.

The distal side of the heme pocket, known to regulate ligand affinity, is shown to be directly involved in subunit interactions. Valency hybrids with oxygen or carbon monoxide bound to the reduced chain are used to model R-state hemoglobin with different distal perturbations. Electron paramagnetic resonance of the oxidized chains shows that the carbon monoxide perturbation is transmitted between subunits to the distal histidine and the oxidized iron center. A comparison of hybrids with only one type of chain oxidized and hybrids with a single alpha beta dimer oxidized is consistent with this perturbation being transmitted across the alpha 1 beta 1 interface. This represents a new mode of subunit interactions in hemoglobin.     

Magnesium regulates intracellular free ionized calcium concentration and cell geometry in vascular smooth muscle cells.

Regulatory effects of extracellular magnesium ions ([Mg2+]o) on intracellular free ionized calcium ([Ca2+]i) were studied in cultured vascular smooth muscle cells (VSMCs) from rat aorta by use of the fluorescent indicator fura-2 and digital imaging microscopy. With normal Mg2+ (1.2 mM)-containing incubation media, [Ca2+]i in VSMCs was 93.6 +/- 7.93 nM with a heterogeneous cellular distribution. Lowering [Mg2+]o to 0 mM or 0.3 mM (the lowest physiological range) resulted in 5.8-fold (579.5 +/- 39.99 nM) and 3.5-fold (348.0 +/- 31.52 nM) increments of [Ca2+]i, respectively, without influencing the cellular distribution of [Ca2+]i. Surprisingly, [Mg2+]o withdrawal induced changes of cell geometry in many VSMCs, i.e., the cells rounded up. However, elevation of [Mg2+]o up to 4.8 mM only induced slight decrements of [Ca2+]i (mean = 72.0 +/- 4.55 nM). The large increment of [Ca2+]i induced by [Mg2+]o withdrawal was totally inhibited when [Ca2+]o was removed. The data suggest that: (1) [Mg2+]o regulates the level of [Ca2+]i in rat aortic smooth muscle cells, and (2) [Mg2+] acts as an important regulatory ion by modulating cell shapes in cultured VSMc and their metabolism to control vascular contractile activities.     

Monte Carlo simulation studies on the prediction of protein folding types from amino acid composition.

In the methodology development for statistical prediction of protein structures, the founders of different methods usually selected different sets of proteins to test their predicted results. Therefore, it is hard to make a fair comparison according to the results they reported. Even if the predictions by different methods are performed for the same set of proteins, there is still such a problem: a method better that the other for one set of proteins would not necessarily remain so when applied to another set of proteins. To tackle this problem, a Monte Carlo simulation method is proposed to establish an objective criterion to measure the accuracy of prediction for the protein folding type. Such an objective accuracy is actually corresponding to the asymptotical limit genereated during the Monte Carlo simulation process. Based on that, it has been found that the average objective accuracy for predicting the all-alpha, all-beta, alpha + beta, and alpha/beta proteins by the least Euclid's distance method (Nakashima, H., K. Nishikawa, and T. Ooi. 1986. J. Biochem. 99:152-162) is 73.0% and that by the least Minkowski's distance method (Chou, P.Y. 1989. Prediction in Protein Structure and the Principles of Protein Conformation. Plenum Press. New York. 549-586) is 70.9%, indicating that the former is better than the latter. However, according to the original reports, the latter claimed a rate of correct prediction with 79.7% but the former with only 70.2%, leading to a completely opposite conclusion. This indicates the necessity of establishing an objective criterion, and a comparison is meaningful only when it is based on the objective criterion. The simulation method and the idea developed here also can be applied to examine any other statistical prediction methods.