Cleavage of individual DNA strands by the different subunits of the heterodimeric restriction endonuclease BbvCI

BbvCI cleaves an asymmetric DNA sequence, 5'-CC downward arrow TCAGC-3'/5'-GC downward arrow TGAGG-3', as indicated. While many Type II restriction enzymes consist of identical subunits, BbvCI has two different subunits: R(1), which acts at GC downward arrow TGAGG; and R(2), which acts at CC downward arrow TCAGC. Some mutants of BbvCI with defects in one subunit, either R(1)(-)R(2)(+) or R(1)(+)R(2)(-), cleave only one strand, that attacked by the native subunit. In analytical ultracentrifugation at various concentrations of protein, wild-type and mutant BbvCI enzymes aggregated extensively, but are R(1)R(2) heterodimers at the concentrations used in DNA cleavage reactions. On a plasmid with one recognition site, wild-type BbvCI cleaved both strands before dissociating from the DNA, while the R(1)(-)R(2)(+) and R(1)(+)R(2)(-) mutants acted almost exclusively on their specified strands, albeit at relatively slow rates. During the wild-type reaction, the DNA is cleaved initially in one strand, mainly that targeted by the R(1) subunit. The other strand is then cleaved slowly by R(2) before the enzyme dissociates from the DNA. Hence, the nicked form accumulates as a transient intermediate. This behaviour differs from that of many other restriction enzymes, which cut both strands at equal rates. However, the activities of the R(1)(+) and R(2)(+) subunits in the wild-type enzyme can differ from their activities in the R(1)(+)R(2)(-) and R(1)(-)R(2)(+) mutants. Each active site in BbvCI therefore influences the other.     

Equilibrium and kinetic parameters for the binding of inhibitors to the QB pocket in bacterial chromatophores: dependence on the state of QA

The equilibrium and kinetic parameters for the binding of various inhibitors to the Q(B) pocket of the bacterial reaction center were investigated in chromatophores from Rhodobacter capsulatus and Rhodobacter sphaeroides. By monitoring the near-IR absorption changes specific to Q(A)(-) and Q(B)(-), we measured the fraction of inhibited centers in the dark and the kinetics and extent of inhibitor displacement after one flash due to the formation of the Q(A)Q(B)(-) state. The inhibitor release rate was much faster for triazines and o-phenanthroline (t(1/2) in the 50 ms to 1 s range) than for stigmatellin (t(1/2) approximately 20 s). For inhibitors with a rapid release rate, the fast phase of P(+) decay observed in the absence of secondary donor reflects the competition between P(+)Q(A)(-) recombination and inhibitor release: it is thus faster than the P(+)Q(A)(-) recombination, and its relative extent is smaller than the fraction of initially inhibited centers. At appropriate inhibitor concentrations, one can have almost total binding in the dark and almost total inhibitor displacement after one flash. Under such conditions, a pair of closely spaced flashes resets the two-electron gate in a single state (Q(A)Q(B)(-)), irrespective of the initial state. The apparent dissociation constant of terbutryn was significantly increased (by a factor of 4-7) in the presence of Q(A)(-), in agreement with the conclusion of Wraight and co-workers [Stein, R. R., et al. (1984) J. Cell. Biochem. 24, 243-259]. We suggest that this effect is essentially due to a tighter binding of ubiquinone in the Q(A)(-) state.     

Single-step nitrification models erroneously describe batch ammonia oxidation profiles when nitrite oxidation becomes rate limiting

Nitrification involves the sequential biological oxidation of reduced nitrogen species such as ammonium-nitrogen (NH(4)(+)-N) to nitrite-nitrogen (NO(2)(-)-N) and nitrate-nitrogen (NO(3)(-)-N). The adequacy of modeling NH(4)(+)-N to NO(3)(-)-N oxidation as one composite biochemical reaction was examined at different relative dynamics of NH(4)(+)-N to NO(2)(-)-N and NO(2)(-)-N to NO(3)(-)-N oxidation. NH(4)(+)-N to NO(2)(-)-N oxidation and NO(2)(-)-N to NO(3)(-)-N oxidation by a mixed nitrifying consortium were uncoupled using selective inhibitors allylthiourea and sodium azide. The kinetic parameters of NH(4)(+)-N to NO(2)(-)-N oxidation (q(max,ns) and K(S,ns)) and NO(2)(-)-N to NO(3)(-)-N oxidation (q(max,nb) and K(S,nb)) were determined by a rapid extant respirometric technique. The stoichiometric coefficients relating nitrogen removal, oxygen uptake and biomass synthesis were derived from an electron balanced equation. NH(4)(+)-N to NO(2)(-)-N oxidation was not affected by NO(2)(-)-N concentrations up to 100 mg NO(2)(-)-N L(-1). NO(2)(-)-N to NO(3)(-)-N oxidation was noncompetitively inhibited by NH(4)(+)-N but was not inhibited by NO(3)(-)-N concentrations up to 250 mg NO(3)(-)-N L(-1). When NH(4)(+)-N to NO(2)(-)-N oxidation was the sole rate-limiting step, complete NH(4)(+)-N to NO(3)(-)-N oxidation was adequately modeled as one composite process. However, when NH(4)(+)-N to NO(2)(-)-N oxidation and NO(2)(-)-N to NO(3)(-)-N oxidation were both rate limiting, the estimated lumped kinetic parameter estimates describing NH(4)(+)-N to NO(3)(-)-N oxidation were unrealistically high and correlated. These findings indicate that the use of single-step models to describe batch NH(4)(+) oxidation yields erroneous kinetic parameters when NH(4)(+)-to-NO(2)(-) oxidation is not the sole rate-limiting process throughout the assay. Under such circumstances, it is necessary to quantify NH(4)(+)-N to NO(2)(-)-N oxidation and NO(2)(-)-N to NO(3)(-)-N oxidation, independently.     

Bidirectional electron transfer in photosystem I: determination of two distances between P700+ and A1- in spin-correlated radical pairs

The spin-correlated radical pair [P(700)(+)A(1)(-)] gives rise to a characteristic "out-of-phase" electron spin-echo signal. The electron spin-echo envelope modulation (ESEEM) of these signals has been studied in thylakoids prepared from the wild-type strain of Chlamydomonas reinhardtii and in two site-directed mutants, in which the methionine residue which acts as the axial ligand to the chlorin electron acceptor A(0) has been substituted with a histidine either on the PsaA (PsaA-M684H) or the PsaB (PsaB-M664H) reaction center subunits. The analysis of the time domain ESEEM provides information about the spin-spin interaction in the [P(700)(+)A(1)(-)] radical pair, and the values of the dipolar (D) and the exchange (J) interaction can be extracted. From the distance dependence of the dipolar coupling term, the distance between the unpaired electron spin density clouds of the primary donor P(700)(+) and the phyllosemiquinone A(1)(-) can be determined. The [P(700)(+)A(1)(-)] ESEEM spectrum obtained in wild-type thylakoids can be reconstructed using a linear combination of the spectra measured in the PsaA and PsaB A(0) mutants, demonstrating that electron transfer resulting in charge separation is occurring on both the PsaA and PsaB branches. The [P(700)(+)A(1B)(-)] distance in the point dipole approximation in the PsaA-M684H mutant is 24.27 +/- 0.02 A, and the [P(700)(+)A(1A)(-)] distance in the PsaB-M664H mutant is 25.43 +/- 0.01 A. An intermediate value of 25.01 +/- 0.02 A is obtained in the wild-type membranes which exhibit both spin-polarized pairs.     

Excited-state dynamics of overlapped optically-allowed 1B(u)+ and optically-forbidden 1B(u)- or 3A(g)- vibronic levels of carotenoids: possible roles in the light-harvesting function

Pump-probe spectroscopy after selective excitation of all-trans Cars (n = 9-13) in nonpolar solvent identified a symmetry selection rule of diabatic electronic mixing and diabatic internal conversion, i.e., '1B(u)(+)-to-1B(u)(-) is allowed but 1B(u)(+)-to-3A(g)(-) is forbidden'. Kerr-gate fluorescence spectroscopy showed that this selection rule breaks down, due to the symmetry degradation when the Car molecules are being excited, and, as a result, the 1B(u)(+)-to-3A(g)(-) diabatic electronic mixing and internal conversion become allowed. On the other hand, pump-probe spectroscopy after coherent excitation of the same set of Cars in polar solvent identified three stimulated-emission components (generated by the quantum-beat mechanism), consisting of the long-lived coherent cross term from the 1B(u)(+) + 1B(u)(-) or 1B(u)(+) + 3A(g)(-) diabatic pair and incoherent short-lived 1B(u)(+) and 1B(u)(-) or 3A(g)(-) split incoherent terms. The same type of stimulated-emission components were identified in Cars bound to LH2 complexes, their lifetimes being substantially shortened by the Car-to-BChl singlet-energy transfer. Each diabatic pair and its split components appeared with high intensities in the first component. The low-energy shifts of the 1B(u)(+)(0), 1B(u)(-)(0) and 3A(g)(-)(0) levels and efficient triplet generation were also found.     

The Ross-Macdonald model in a patchy environment

We generalize to n patches the Ross-Macdonald model which describes the dynamics of malaria. We incorporate in our model the fact that some patches can be vector free. We assume that the hosts can migrate between patches, but not the vectors. The susceptible and infectious individuals have the same dispersal rate. We compute the basic reproduction ratio R(0). We prove that if R(0)1, then the disease-free equilibrium is globally asymptotically stable. When R(0)>1, we prove that there exists a unique endemic equilibrium, which is globally asymptotically stable on the biological domain minus the disease-free equilibrium.     

The null distribution of the heterogeneity lod score does depend on the assumed genetic model for the trait.

It is well known that the asymptotic null distribution of the homogeneity lod score (LOD) does not depend on the genetic model specified in the analysis. When appropriately rescaled, the LOD is asymptotically distributed as 0.5 chi(2)(0) + 0.5 chi(2)(1), regardless of the assumed trait model. However, because locus heterogeneity is a common phenomenon, the heterogeneity lod score (HLOD), rather than the LOD itself, is often used in gene mapping studies. We show here that, in contrast with the LOD, the asymptotic null distribution of the HLOD does depend upon the genetic model assumed in the analysis. In affected sib pair (ASP) data, this distribution can be worked out explicitly as (0.5 - c)chi(2)(0) + 0.5chi(2)(1) + cchi(2)(2), where c depends on the assumed trait model. E.g., for a simple dominant model (HLOD/D), c is a function of the disease allele frequency p: for p = 0.01, c = 0.0006; while for p = 0.1, c = 0.059. For a simple recessive model (HLOD/R), c = 0.098 independently of p. This latter (recessive) distribution turns out to be the same as the asymptotic distribution of the MLS statistic under the possible triangle constraint, which is asymptotically equivalent to the HLOD/R. The null distribution of the HLOD/D is close to that of the LOD, because the weight c on the chi(2)(2) component is small. These results mean that the cutoff value for a test of size alpha will tend to be smaller for the HLOD/D than the HLOD/R. For example, the alpha = 0.0001 cutoff (on the lod scale) for the HLOD/D with p = 0.05 is 3.01, while for the LOD it is 3.00, and for the HLOD/R it is 3.27. For general pedigrees, explicit analytical expression of the null HLOD distribution does not appear possible, but it will still depend on the assumed genetic model.     

Regulation of the high-affinity NO3- uptake system by NRT1.1-mediated NO3- demand signaling in Arabidopsis

The NRT2.1 gene of Arabidopsis thaliana encodes a major component of the root high-affinity NO(3)(-) transport system (HATS) that plays a crucial role in NO(3)(-) uptake by the plant. Although NRT2.1 was known to be induced by NO(3)(-) and feedback repressed by reduced nitrogen (N) metabolites, NRT2.1 is surprisingly up-regulated when NO(3)(-) concentration decreases to a low level (<0.5 mm) in media containing a high concentration of NH(4)(+) or Gln (>or=1 mm). The NRT3.1 gene, encoding another key component of the HATS, displays the same response pattern. This revealed that both NRT2.1 and NRT3.1 are coordinately down-regulated by high external NO(3)(-) availability through a mechanism independent from that involving N metabolites. We show here that repression of both genes by high NO(3)(-) is specifically mediated by the NRT1.1 NO(3)(-) transporter. This mechanism warrants that either NRT1.1 or NRT2.1 is active in taking up NO(3)(-) in the presence of a reduced N source. Under low NO(3)(-)/high NH(4)(+) provision, NRT1.1-mediated repression of NRT2.1/NRT3.1 is relieved, which allows reactivation of the HATS. Analysis of atnrt2.1 mutants showed that this constitutes a crucial adaptive response against NH(4)(+) toxicity because NO(3)(-) taken up by the HATS in this situation prevents the detrimental effects of pure NH(4)(+) nutrition. It is thus hypothesized that NRT1.1-mediated regulation of NRT2.1/NRT3.1 is a mechanism aiming to satisfy a specific NO(3)(-) demand of the plant in relation to the various specific roles that NO(3)(-) plays, in addition to being a N source. A new model is proposed for regulation of the HATS, involving both feedback repression by N metabolites and NRT1.1-mediated repression by high NO(3)(-).     

Picosecond transient absorption spectroscopy in the blue spectral region of photosystem I

Picosecond transient absorption difference spectroscopy in the blue wavelength region (380-500 nm) was used to study the early electron acceptors in photosystem I. Samples were photosystem I core particles with about 100 chlorophylls per reaction center isolated from the cyanobacterium Synechocystis sp. PCC 6803. After excitation at 590 nm at room temperature, decay-associated spectra (DAS) were determined from global analysis in the blue region, yielding two transient components and one nondecaying component. A 3 ps decay phase is interpreted as primarily due to antenna excited-state redistribution. A 28 ps decay phase is interpreted as due to overall excited-state decay by electron transfer. The nondecaying component is ascribed to the difference spectrum of P(700) and the quinone or A(1) electron acceptor (P(700)(+)A(1)(-) - P(700)A(1)). Decay curves on the millisecond time scale at different wavelengths were measured with an autoxidizable artificial electron acceptor, benzyl viologen, and the (P(700)(+) - P(700)) difference spectrum was constructed. The (A(1)(-) - A(1)) difference spectrum was obtained by taking the difference between the above two difference spectra. A parallel picosecond experiment under strongly reducing conditions was also done as a control experiment. These conditions stabilize the electron on an earlier acceptor, A(0). The nondecaying component of the DAS at low potential was assigned to (P(700)(+)A(0)(-) - P(700)A(0)) since the electron-transfer pathway from A(0) to A(1) was blocked. The [(P(700)(+)A(0)(-) - P(700)A(0)) - (P(700)(+) - P(700))] subtraction gives a spectrum, interpreted as the (A(0)(-) - A(0)) difference spectrum of a chlorophyll a molecule, consistent with previous studies. The (A(1)(-) - A(1)) spectrum resolved on the picosecond time scale shows significant differences with similar spectra measured on longer time scales. These differences may be due to electrochromic effects and spectral evolution.     

Bacterial immobilization and remineralization of N at different growth rates and N concentrations

An experiment was designed to resolve two largely unaddressed questions about the turnover of N in soils. One is the influence of microbial growth rate on mobilization and remineralization of cellular N. The other is to what extent heterotrophic immobilization of NO(3)(-) is controlled by the soil concentration of NH(4)(+). Bacteria were extracted from a deciduous forest soil and inoculated into an aqueous medium. Various N pool dilution/enrichment experiments were carried out to: (1) calculate the gross N immobilization and remineralization rates; (2) investigate their dependence on NH(4)(+)and NO(3)(-) concentrations; (3) establish the microbial preference for NH(4)(+)and NO(3)(-) depending on the NH(4)(+)/NO(3)(-) concentration ratio. Remineralization of microbial N occurred mainly at high growth rates and NH(4)(+) concentrations. There was a positive correlation between NH(4)(+) immobilization and remineralization rates, and intracellular recycling of N seemed to be an efficient way for bacteria to withstand low inorganic N concentrations. Thus, extensive remineralization of microbial N is likely to occur only when environmental conditions promote high growth rates. The results support previous observations of high NO(3)(-) immobilization rates, especially at low NH(4)(+) concentrations, but NO(3)(-) was also immobilized at high NH(4) concentrations. The latter can be understood if part of the microbial community has a preference for NO(3)(-) over NH(4)(+).     

Engineered eglin c variants inhibit yeast and human proprotein processing proteases, Kex2 and furin

We engineered eglin c, a potent subtilisin inhibitor, to create inhibitors for enzymes of the Kex2/furin family of proprotein processing proteases. A structural gene was synthesized that encoded "R(1)-eglin", having Arg at P(1) in the reactive site loop in place of Leu(45). Ten additional variants were created by cassette mutagenesis of R(1)-eglin. These polypeptides were expressed in Escherichia coli, purified to homogeneity, and their interactions with secreted, soluble Kex2 and furin were examined. R(1)-eglin itself was a modest inhibitor of Kex2, with a K(a) of approximately 10(7) M(-)(1). Substituting Arg (in R(4)R(1)-eglin) or Met (in M(4)R(1)-eglin) for Pro(42) at P(4) created potent Kex2 inhibitors exhibiting K(a) values of approximately 10(9) M(-)(1). R(4)R(1)-eglin inhibited furin with a K(a) of 4.0 x 10(8) M(-)(1). Introduction of Lys at P(1), in place of Arg in R(4)R(1)-eglin reduced affinity only approximately 3-fold for Kex2 but 15-fold for furin. The stabilities of enzyme-inhibitor complexes were characterized by association and dissociation rate constants and visualized by polyacrylamide gel electrophoresis. R(4)R(1)-eglin formed stable 1:1 complexes with both Kex2 and furin. However, substitution of Lys at P(2) in place of Thr(44) resulted in eglin variants that inhibited both Kex2 and furin but which were eventually cleaved (temporary inhibition). Surprisingly, R(6)R(4)R(1)-eglin, in which Arg was substituted for Gly(40) in R(4)R(1)-eglin, exhibited stable, high-affinity complex formation with Kex2 (K(a) of 3.5 x 10(9) M(-)(1)) but temporary inhibition of furin. This suggests that enzyme-specific interactions can alter the conformation of the reactive site loop, converting a permanent inhibitor into a substrate. Eglin variants offer possible avenues for affinity purification, crystallization, and regulation of proprotein processing proteases.     

Cicaprost inhibits metastases of animal tumors.

Since an involvement of platelet aggregation in the metastatic process has been found, platelet activation inhibitors were investigated for their potential to reduce tumor metastases. Recent in-vitro and in-vivo investigations showed an antimetastatic effect of prostacyclin (PGI2) and stable prostacyclin analogues. This study concentrates on the effect of the stable prostacyclin analogue Cicaprost (Schering AG) on tumor metastases in two metastasizing tumors of rodents. C57BL/6 mice bearing s.c.-implanted M5076 reticulum sarcoma were treated with Cicaprost in doses of 0.1-1.0 mg/kg throughout the experiment. Cicaprost in all doses tested reduced the number of liver metastases in a statistically significant manner. The 1.0 mg/kg dose, which decreases the median number of liver metastases to more than 93% compared to the control, was most effective. Cicaprost in the 0.5 mg/kg dose reduced the number of liver metastases in mice bearing i.v.-implanted M5076 reticulum sarcoma. In Cop-Fisher rats bearing s.c.-implanted spontaneously metastasizing R3327 MAT Lu prostate carcinoma, Cicaprost in a dose of 1.0 mg/kg p.o. daily strongly reduced the number of lung metastases. These results indicate that Cicaprost is a potent inhibitor of tumor metastases in different tumor models in rodents.     

Absorption changes induced by the binding of triazines to the QB pocket in reaction centers of Rhodobacter capsulatus

Inhibitors which block electron transfer from the primary (Q(A)) to the secondary (Q(B)) quinone of the bacterial reaction center are competing with the pool ubiquinones for binding at the Q(B) pocket. Due to the much greater stability of the semiquinone state Q(B)(-) compared with fully oxidized or reduced quinone, a displacement of the inhibitors takes place after one flash from state Q(A)(-)I to state Q(A)Q(B)(-). This process can be monitored from near-IR absorption changes which reflect local absorption shifts specific to Q(A)(-) and Q(B)(-). An anomalous behavior was observed when using triazines in chromatophores of R. capsulatus: the IR absorption change reflecting the formation of Q(B)(-) after one flash was absent. A normal transient decay of this signal was, however, triggered by a second flash, followed by a rapid return to the baseline. We show that this phenomenon is due to an absorption change induced by inhibitor binding (thus present in the dark baseline), with a spectrum close to that of Q(B)(-), so that the Q(B)(-) changes are canceled out during the inhibitor displacement process. On the second flash, one monitors the destruction of the semiquinone, leading transiently to the Q(A)Q(B) state, followed by inhibitor rebinding. This allows a direct measurement of the binding kinetics. This behavior was observed both in chromatophores and in isolated reaction centers from R. capsulatus, but not in R. sphaeroides.     

Na(+) interaction with the pore of Shaker B K(+) channels: zero and low K(+) conditions.

The Shaker B K(+) conductance (G(K)) collapses (in a reversible manner) if the membrane is depolarized and then repolarized in, 0 K(+), Na(+)-containing solutions (Gómez-Lagunas, F. 1997. J. Physiol. 499:3-15; Gómez-Lagunas, F. 1999. Biophys. J. 77:2988-2998). In this work, the role of Na(+) ions in the collapse of G(K) in 0-K(+) solutions, and in the behavior of the channels in low K(+) was studied. The main findings are as follows. First, in 0-K(+) solutions, the presence of Na(+) ions is an important factor that speeds the collapse of G(K). Second, external Na(+) fosters the drop of G(K) by binding to a site with a K(d) = 3.3 mM. External K(+) competes, in a mutually exclusive manner, with Na(o)(+) for binding to this site, with an estimated K(d) = 80 microM. Third, NMG and choline are relatively inert regarding the stability of G(K); fourth, with [K(o)(+)] = 0, the energy required to relieve Na(i)(+) block of Shaker (French, R.J., and J.B. Wells. 1977. J. Gen. Physiol. 70:707-724; Starkus, J.G., L. Kuschel, M. Rayner, and S. Heinemann. 2000. J. Gen. Physiol. 110:539-550) decreases with the molar fraction of Na(i)(+) (X(Na,i)), in an extent not accounted for by the change in Delta(mu)(Na). Finally, when X(Na,i) = 1, G(K) collapses by the binding of Na(i)(+) to two sites, with apparent K(d)s of 2 and 14.3 mM.     

Dynamic and steady-state responses of inorganic nitrogen pools and NH(3) exchange in leaves of Lolium perenne and Bromus erectus to changes in root nitrogen supply

Short- and long-term responses of inorganic N pools and plant-atmosphere NH(3) exchange to changes in external N supply were investigated in 11-week-old plants of two grass species, Lolium perenne and Bromus erectus, characteristic of N-rich and N-poor grassland ecosystems, respectively. A switch of root N source from NO(-)(3)to NH(4)(+) caused within 3 h a 3- to 6-fold increase in leaf apoplastic NH(4)(+) concentration and a simultaneous decrease in apoplastic pH of about 0.4 pH units in both species. The concentration of total extractable leaf tissue NH(4)(+) also increased two to three times within 3 h after the switch. Removal of exogenous NH(4)(+) caused the apoplastic NH(4)(+) concentration to decline back to the original level within 24 h, whereas the leaf tissue NH(4)(+)concentration decreased more slowly and did not reach the original level in 48 h. After growing for 5 weeks with a steady-state supply of NO(-)(3)or NH(4)(+), L. perenne were in all cases larger, contained more N, and utilized the absorbed N more efficiently for growth than B. erectus, whereas the two species behaved oppositely with respect to tissue concentrations of NO(-)(3), NH(4)(+), and total N. Ammonia compensation points were higher for B. erectus than for L. perenne and were in both species higher for NH(4)(+)- than for NO(-)(3)-grown plants. Steady-state levels of apoplastic NH(4)(+), tissue NH(4)(+), and NH(3) emission were significantly correlated. It is concluded that leaf apoplastic NH(4)(+) is a highly dynamic pool, closely reflecting changes in the external N supply. This rapid response may constitute a signaling system coordinating leaf N metabolism with the actual N uptake by the roots and the external N availability.     

Molecular cloning of mouse type 2 and type 3 inositol 1,4,5-trisphosphate receptors and identification of a novel type 2 receptor splice variant

We isolated cDNAs encoding type 2 and type 3 inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)R2 and IP(3)R3, respectively) from mouse lung and found a novel alternative splicing segment, SI(m2), at 176-208 of IP(3)R2. The long form (IP(3)R2 SI(m2)(+)) was dominant, but the short form (IP(3)R2 SI(m2)(-)) was detected in all tissues examined. IP(3)R2 SI(m2)(-) has neither IP(3) binding activity nor Ca(2+) releasing activity. In addition to its reticular distribution, IP(3)R2 SI(m2)(+) is present in the form of clusters in the endoplasmic reticulum of resting COS-7 cells, and after ATP or Ca(2+) ionophore stimulation, most of the IP(3)R2 SI(m2)(+) is in clusters. IP(3)R3 is localized uniformly on the endoplasmic reticulum of resting cells and forms clusters after ATP or Ca(2+) ionophore stimulation. IP(3)R2 SI(m2)(-) does not form clusters in either resting or stimulated cells. IP(3) binding-deficient site-directed mutants of IP(3)R2 SI(m2)(+) and IP(3)R3 fail to form clusters, indicating that IP(3) binding is involved in the cluster formation by these isoforms. Coexpression of IP(3)R2 SI(m2)(-) prevents stimulus-induced IP(3)R clustering, suggesting that IP(3)R2 SI(m2)(-) functions as a negative coordinator of stimulus-induced IP(3)R clustering. Expression of IP(3)R2 SI(m2)(-) in CHO-K1 cells significantly reduced ATP-induced Ca(2+) entry, but not Ca(2+) release, suggesting that the novel splice variant of IP(3)R2 specifically influences the dynamics of the sustained phase of Ca(2+) signals.     

Mathematical analysis of an age-structured population model with space-limited recruitment

In this paper, we investigate structured population model of marine invertebrate whose life stage is composed of sessile adults and pelagic larvae, such as barnacles contained in a local habitat. First we formulate the basic model as an Cauchy problem on a Banach space to discuss the existence and uniqueness of non-negative solution. Next we define the basic reproduction number R0 to formulate the invasion condition under which the larvae can successfully settle down in the completely vacant habitat. Subsequently we examine existence and stability of steady states. We show that the trivial steady state is globally asymptotically stable if R0 < or = 1, whereas it is unstable if R0 > 1. Furthermore, we show that a positive (non-trivial) steady state uniquely exists if R0 > 1 and it is locally asymptotically stable as far as absolute value of R0 - 1 is small enough.     

The novel synthetic inhibitor of matrix metalloproteinases Ro 28-2653 induces apoptosis in Dunning tumor cells

The effect of synthetic inhibitors of matrix metalloproteinases (MMP) has been shown against a variety of tumors in preclinical models. Ro 28-2653, a novel synthetic MMP inhibitor, is able to reduce tumor growth in orthotopic prostatic cancer in rats (R3327 Dunning tumor). However, at present this inhibitory mechanism in tumor inhibition in vivo can only be partly explained by the inhibition of the catalytic activity of MMPs overexpressed in cancereous tissue. Using the flow cytometric method, we have investigated the effect of various concentrations of Ro 28-2653 on the Dunning tumor cells with regard to the staining of F-actin and DNA as markers of apoptosis. In combination with fluorescence microscopy we detected the loss of F-actin and the degradation of internucleosomal DNA. This effect of Ro 28-2653 on apoptosis was dose- and time-dependent increasing with concentration between 10 and 100 g/ml as well as with time of treatment between 24 and 48 h.     

Enzyme inhibition assays using fluorescence correlation spectroscopy: a new algorithm for the derivation of kcat/KM and Ki values at substrate concentrations much lower than the Michaelis constant

A new mathematical formalism is deduced which allows for the calculation of the k(cat) over K(M) ratio based on measurements of the enzyme kinetics with substrate concentrations much lower than K(M). The equations are also applied on the action of an inhibitor on enzyme activity yielding the binding constant, K(i), of an inhibitor molecule. For practical evaluation of the new theoretical approach, the enzymatic reaction of CD45 phosphatase was used as a well-characterized model system with known inhibitors for testing the K(i) value determination scheme. The k(cat)/K(M) ratio was calulated to be 4.7 x 10(5) M(-)(1) s(-)(1), the K(i) of the inhibitor molecule PKF52-524 was estimated to be (1-2) x 10(-)(7) M and the association rate of the inhibitor PKF52-524 to CD45 phosphatase was estimated to be 59 M(-)(1) s(-)(1).