Effects of excluded volume and polydispersity on solution properties of lentinan in 0.1 M NaOH solution

Seven lentinan fractions of various weight-average molecular weights (M(w)), ranging from 1.45 x 10(5) to 1.13 x 10(6) g mol(-1) were investigated by static light scattering and viscometry in 0.1M NaOH solution at 25 degrees C. The intrinsic viscosity [eta] - M(w) and radius of gyration s(2)(z) (1/2) - M(w) relationships for lentinan in 0.1M NaOH solution were found to be represented by [eta] = 5.1 x 10(-3)M(w) (0.81) cm(3) g(-1) and s(2)(z) (1/2) = 2.3 x 10(-1)M(w) (0.58) nm, respectively. Focusing on the effects of the M(w) polydispersity with the Schulz-Zimm distribution function, the data of M(w), s(2)(z) (1/2), and [eta] was analyzed on the basis of the Yoshizaki-Nitta-Yamakawa theory for the unperturbed helical wormlike chain combined with the quasi-two-parameter (QTP) theory for excluded-volume effects. The persistence length, molecular weight per unit contour length, and the excluded-volume strength were determined roughly to be 6.2 nm, 980 nm(-1), and 0.1, respectively. Compared with the theoretical value calculated by the Monte Carlo model, the persistence length is longer than that of the single (1 --> 3)-beta-(D)-glucan chain. The results revealed that lentinan exists as single-stranded flexible chains in 0.1M NaOH solution with a certain degree of expansion due to the electrostatic repulsion from the interaction between the OH(-) anions and lentinan molecules.     

Individual and population penalized regression splines for accelerated longitudinal designs

In an accelerated longitudinal design (ALD), individuals enter the study at different points of their growth trajectory and are observed over a short time span relative to the entire time span of interest. ALD data are combined across independent units to provide an estimate of an overall population curve and predictions of individual patterns of change. As a modest extension of the work of Ruppert et al. (2003, Semiparametric Regression, Cambridge University Press), we develop a computationally efficient procedure for the application of longitudinal semiparametric methods under ALD sampling schemes. We compare balanced and complete longitudinal designs to ALDs using the Berkeley Growth Study data and apply our method to longitudinal magnetic resonance imaging (MRI) brain structure size (volume) measurements from an ongoing developmental study. Potential applications extend beyond growth studies to many other fields in which cost and feasibility constraints impose restrictions on sample size and on the numbers and timings of repeated measurements across subjects.     

Candidate Inhibitor of the Volume-Sensitive Kinase Regulating K-Cl Cotransport: The Myosin Light Chain Kinase Inhibitor ML-7

K-Cl cotransport, KCC, is activated by swelling in many cells types, and promotes volume regulation by a KCl efflux osmotically coupled to water efflux. KCC is probably activated by swelling-inhibition of a kinase, permitting dephosphorylation, and activation of the cotransporter by a phosphatase. The myosin light chain kinase (MLCK) inhibitor ML-7 inhibits transporters activated by shrinkage. In red blood cells from three mammalian species, ML-7 stimulated KCC in a volume-dependent manner. Relative stimulation was greatest in more shrunken cells. Stimulation was reduced by moderate cell swelling and abolished by further swelling. The half-maximal stimulation is at ∼20 μm ML-7, 50-fold greater than the IC₅₀ for inhibition of MLCK in vitro. Stimulation of KCC by ML-7 did not require cell Ca, while MLCK does. Therefore the target of ML-7 in stimulating KCC in red cells is probably not MLCK. The evidence favors stimulation of KCC by ML-7 by inhibiting the volume-sensitive kinase. Qualitatively similar effects of ML-7 on KCC in red cells from three mammalian species suggest a general mechanism. Received: 17 March 2000/Revised: 28 July 2000     

A member of a novel Arabidopsis thaliana gene family of candidate Mg2+ ion transporters complements a yeast mitochondrial group II intron-splicing mutant

Autocatalytic activity of some group II introns has been demonstrated in vitro, but helper functions such as the yeast MRS2 protein are essential for splicing in vivo. In our search for such helper factors in plants, we pursued the cloning of two Arabidopsis thaliana homologues, atmrs2-1 and atmrs2-2. Atmrs2-1, but not atmrs2-2, complements the yeast deletion mutant of mrs2, and this is congruent with the prediction of two adjacent transmembrane stretches in AtMRS2-1 and yeast MRS2 but not in AtMRS2-2. This complementation depends on fusion of the native yeast mitochondrial import sequence to atmrs2-1. A differing, non-mitochondrial, cellular targeting in Arabidopsis is supported by the analysis of green fluorescent protein fusion constructs after transient transformation into plant protoplasts. Further members of what now appears to be a family of 10 mrs2 homologues are identified in the Arabidopsis genome. Similarity searches with the PSI-BLAST algorithm in the protein database fail to identify homologues of this novel gene family in any eukaryotes other than yeasts, but do identify its distant relatedness to the corA group of bacterial magnesium transporters. In line with this observation, intramitochondrial magnesium concentrations are indeed restored to wild-type levels in the yeast mutant on complementation with atmrs2-1.     

Inhibition of Na+/K+-ATPase may be one mechanism contributing to potassium efflux and cell shrinkage in CD95-induced apoptosis

To investigate the involvement of K⁺ efflux in apoptotic cell shrinkage, we monitored efflux of the K⁺ congener,⁸⁶ Rb⁺, and cell volume during CD95-mediated apoptosis in Jurkat cells. An anti-CD95 antibody caused apoptosis associated with intracellular GSH depletion, a significant increase in ⁸⁶Rb⁺ efflux, and a decrease in cell volume compared with control cells. Preincubating Jurkat cells with Val-Ala-Asp-chloromethylketone (VAD-cmk), an inhibitor of caspase proteases, prevented the observed ⁸⁶Rb⁺ efflux and cell shrinkage induced by the anti- CD95 antibody. A wide range of inhibitors against most types of K⁺ channels could not inhibit CD95-mediated efflux of⁸⁶ Rb⁺, however, the uptake of⁸⁶ Rb⁺ by Jurkat cells was severely compromised when treated with anti-CD95 antibody. Uptake of⁸⁶ Rb⁺ in Jurkat cells was sensitive to ouabain (a specific Na⁺/K⁺-ATPase inhibitor), demonstrating Na⁺/K⁺-ATPase dependent K⁺ uptake. Ouabain induced significant⁸⁶ Rb⁺ efflux in untreated cells, as well as it seemed to compete with⁸⁶ Rb⁺ efflux induced by the anti-CD95 antibody, supporting a role for Na⁺/K⁺-ATPase in the CD95-mediated⁸⁶ Rb⁺ efflux. Ouabain treatment of Jurkat cells did not cause a reduction in cell volume, although together with the anti-CD95 antibody, ouabain potentiated CD95-mediated cell shrinkage. This suggests that the observed inhibition of Na⁺+/K⁺-ATPase during apoptosis may also facilitate apoptotic cell shrinkage.     

NMR snapshots of a fluctuating protein structure: ubiquitin at 30 bar-3 kbar

Conformational fluctuation plays a key role in protein function, but we know little about the associated structural changes. Here we present a general method for elucidating, at the atomic level, a large-scale shape change of a protein molecule in solution undergoing conformational fluctuation. The method utilizes the intimate relationship between conformation and partial molar volume and determines three-dimensional structures of a protein at different pressures using variable pressure NMR technique, whereby NOE distance and torsion angle constraints are used to create average coordinates. Ubiquitin (pH 4.6 at 20 degrees C) was chosen as the first target, for which structures were determined at 30 bar and at 3 kbar, giving "NMR snapshots" of a fluctuating protein structure at atomic resolution. The result reveals that the helix swings in and out by >3 angstroms with a simultaneous reorientation of the C-terminal segment, providing an "open" conformer suitable for enzyme recognition. Spin relaxation analysis indicates that this fluctuation occurs in the ten microsecond time range with activation volumes -4.2(+/-3.2) and 18.5(+/-3.0) ml/mol for the "closed-to-open" and the "open-to-closed" transitions, respectively.     

Cerebrocellular Swelling in the Presence of Uraemic Guanidino Compounds: Ameliorative Effects of Taurine

Cell volumes (equilibrium non-inulin spaces) have been measured in slices of rat cerebral cortex incubated in the presence of uraemic guanidino compounds. Of 5 guanidino compounds tested, all but one caused significant cell swelling. This was most pronounced for guanidinosuccinic acid (GSA, 40 μmol/l)(+22%) and guanidine hydrochloride (G, 3 μmol/l)(+13%). Swelling was reduced by taurine in a dose-dependent manner, being completely abolished at 20 mmol/l. Swelling was also abolished by the antioxidants ascorbic acid (0.4 mmol/l) and butylated hydroxytoluene (0.5 mmol/l), the free radical scavenger N-acetyl-l-cysteine (10 mmol/l) and the lipid peroxidase inhibitor desmethyl tirilazad (100 μmol/l). The remission of swelling by 20 mmol/l taurine was reduced by 50% by the taurine transport inhibitor guanidinoethylsulphonate (GES, 1 mmol/l). This figure was not significantly altered when the concentration of GES was increased to 10 mmol/l. It was also reduced by 45% by the GABA_a receptor antagonist bicuculline (100 μmol/l). It was completely abolished when both GES and bicuculline were present. It is suggested that guanidino compounds result in cells undergoing oxidative-nitrosative stress, and that taurine protects against the resultant cell swelling by 2 mechanisms One (intracellular) requires taurine transport and depends on its role as an antioxidant, with lipid peroxidation being probably a significant factor. The other (extracellular) is associated with activation of GABA_a receptors. Special issue dedicated to Simo S. Oja     

Epidermal Growth Factor Receptor is a Common Element in the Signaling Pathways Activated by Cell Volume Changes in Isosmotic,Hyposmotic or Hyperosmotic Conditions

Changes in external osmolarity, including both hyper- or hyposmotic conditions, elicit the tyrosine phosphorylation of a number of tyrosine kinase receptors (TKR). We show here that the epidermal growth factor receptor (EGFR) is activated by both cell swelling (hyposmolarity, isosmotic urea, hyperosmotic sorbitol) or shrinkage (hyperosmotic NaCl or raffinose) and discuss the mechanisms by which these apparently opposed conditions come to the same effect, i.e., EGFR activation. Evidence suggests that this results from early activation of integrins, p38 and tyrosine kinases of the Src family, which are all activated in the two anisosmotic conditions. TKR transactivation by integrins and p38 is likely occurring via an effect on the metalloproteinases. Information discussed in this review, points to TKR as elements in osmotransduction as a useful mechanism to amplify and diversify the initial response to anisosmolarity and cell volume changes, due to their privileged situation as convergence point for numerous intracellular signaling pathways. The variety of effector pathways connected to TKR is advantageous for the cell to cope with the changes in cell volume including adaptation to stress, cytoskeleton remodeling, adhesion reactions, cell survival and the adaptive mechanisms to ultimately restore the original cell volume. Special Issue dedicated to Dr. Simo S. Oja     

Mitogen-activated protein kinases (p38 and c-Jun NH2-terminal kinase) are differentially regulated during cardiac volume and pressure overload hypertrophy

Chronic pressure overload (PO) and volume overload (VO) result in morphologically and functionally distinct forms of myocardial hypertrophy. However, the molecular mechanism initiating these two types of hypertrophy is not yet understood. Data obtained from different cell types have indicated that the mitogen-activated protein kinases (MAPKs) comprising c-Jun NH₂-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 play an important role in transmitting signals of stress stimuli to elicit the cellular response. We tested the hypothesis that early induction of MAPKs differs in two types of overload on the heart and associates with distinct expression of hypertrophic marker genes, namely ANF, α-myosin heavy chain (α-MHC), and β-MHC. In rats, VO was induced by aortocaval shunt and PO by constriction of the abdominal aorta. The PO animals were further divided into two groups depending on the severity of the constriction, mild (MPO) and severe pressure overload (SPO), having 35 and 85% aortic constriction, respectively. Early changes in MAPK activity (2–120 min and 1 to 2 d) were analyzed by the in vitro kinase assay using kinase-specific antibodies for p38, JNK, and ERK2. The change in expression of hypertrophy marker genes was examined by Northern blot analysis. In VO hypertrophy, the activity of p38 was markedly increased (10-fold), without changing the activity of ERK and JNK. However, during PO hypertrophy, the activity of JNK was significantly increased (two-to sixfold) and depended on the severity of the load. The activity of p38 was not changed in MPO hypertrophy, whereas it was slightly elevated (50%) in hearts with SPO. Similarly, ERK activity was not changed in hearts with MPO, but a transient rise in activity was observed in hearts with SPO. The expression of ANF and β-MHC genes was elevated in both PO and VO hypertrophy; however, this change was much greater in hearts subjected to PO than VO hypertrophy. α-MHC expression was downregulated in PO but remained unchanged in VO hypertrophy hearts. Thus, these results demonstrate differential activation of MAPKs in two types of cardiac hypertrophy and this, in part, may contribute to differential expression of cardiac muscle gene expression, giving rise to unique cardiac phenotype associated with different hemodynamic overloads.