Modeling and optimization of populations subject to time-dependent mutation.

It has become clear that many organisms possess the ability to regulate their mutation rate in response to environmental conditions. So the question of finding an optimal mutation rate must be replaced by that of finding an optimal mutation schedule. We show that this task cannot be accomplished with standard population-dynamic models. We then develop a "hybrid" model for populations experiencing time-dependent mutation that treats population growth as deterministic but the time of first appearance of new variants as stochastic. We show that the hybrid model agrees well with a Monte Carlo simulation. From this model, we derive a deterministic approximation, a "threshold" model, that is similar to standard population dynamic models but differs in the initial rate of generation of new mutants. We use these techniques to model antibody affinity maturation by somatic hypermutation. We had previously shown that the optimal mutation schedule for the deterministic threshold model is phasic, with periods of mutation between intervals of mutation-free growth. To establish the validity of this schedule, we now show that the phasic schedule that optimizes the deterministic threshold model significantly improves upon the best constant-rate schedule for the hybrid and Monte Carlo models.

Time-dependent vascular regression and permeability changes in established human tumor xenografts induced by an anti-vascular endothelial growth factor/vascular permeability factor antibody

The hyperpermeability of tumor vessels to macromolecules, compared with normal vessels, is presumably due to vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) released by neoplastic and/or host cells. In addition, VEGF/VPF is a potent angiogenic factor. Removal of this growth factor may reduce the permeability and inhibit tumor angiogenesis. To test these hypotheses, we transplanted a human glioblastoma (U87), a human colon adenocarcinoma (LS174T), and a human melanoma (P-MEL) into two locations in immunodeficient mice: the cranial window and the dorsal skinfold chamber. The mice bearing vascularized tumors were treated with a bolus (0.2 ml) of either a neutralizing antibody (A4.6.1) (492 μg/ml) against VEGF/VPF or PBS (control). We found that tumor vascular permeability to albumin in antibody-treated groups was lower than in the matched controls and that the effect of the antibody was time-dependent and influenced by the mode of injection. Tumor vascular permeability did not respond to i.p. injection of the antibody until 4 days posttreatment. However, the permeability was reduced within 6 h after i.v. injection of the same amount of antibody. In addition to the reduction in vascular permeability, the tumor vessels became smaller in diameter and less tortuous after antibody injections and eventually disappeared from the surface after four consecutive treatments in U87 tumors. These results demonstrate that tumor vascular permeability can be reduced by neutralization of endogenous VEGF/VPF and suggest that angiogenesis and the maintenance of integrity of tumor vessels require the presence of VEGF/VPF in the tissue microenvironment. The latter finding reveals a new mechanism of tumor vessel regression—i.e., blocking the interactions between VEGF/VPF and endothelial cells or inhibiting VEGF/VPF synthesis in solid tumors causes dramatic reduction in vessel diameter, which may block the passage of blood elements and thus lead to vascular regression.

Temperature, ordering, and equilibrium with time-dependent confining forces

The concepts of temperature and equilibrium are not well defined in systems of particles with time-varying external forces. An example is a radio frequency ion trap, with the ions laser cooled into an ordered solid, characteristic of sub-mK temperatures, whereas the kinetic energies associated with the fast coherent motion in the trap are up to 7 orders of magnitude higher. Simulations with 1,000 ions reach equilibrium between the degrees of freedom when only aperiodic displacements (secular motion) are considered. The coupling of the periodic driven motion associated with the confinement to the nonperiodic random motion of the ions is very small at low temperatures and increases quadratically with temperature.

A joint hazard and time scaling model to compare survival curves.

To provide a more general method for comparing survival experience, we propose a model that independently scales both hazard and time dimensions. To test the curve shape similarity of two time-dependent hazards, h1(t) and h2(t), we apply the proposed hazard relationship, h12(tKt)/ h1(t) = Kh, to h1. This relationship doubly scales h1 by the constant hazard and time scale factors, Kh and Kt, producing a transformed hazard, h12, with the same underlying curve shape as h1. We optimize the match of h12 to h2 by adjusting Kh and Kt. The corresponding survival relationship S12(tKt) = [S1(t)]KtKh transforms S1 into a new curve S12 of the same underlying shape that can be matched to the original S2. We apply this model to the curves for regional and local breast cancer contained in the National Cancer Institute's End Results Registry (1950-1973). Scaling the original regional curves, h1 and S1 with Kt = 1.769 and Kh = 0.263 produces transformed curves h12 and S12 that display congruence with the respective local curves, h2 and S2. This similarity of curve shapes suggests the application of the more complete curve shapes for regional disease as templates to predict the long-term survival pattern for local disease. By extension, this similarity raises the possibility of scaling early data for clinical trial curves according to templates of registry or previous trial curves, projecting long-term outcomes and reducing costs. The proposed model includes as special cases the widely used proportional hazards (Kt = 1) and accelerated life (KtKh = 1) models.

Control of time-dependent biological processes by temporally patterned input

Temporal patterning of biological variables, in the form of oscillations and rhythms on many time scales, is ubiquitous. Altering the temporal pattern of an input variable greatly affects the output of many biological processes. We develop here a conceptual framework for a quantitative understanding of such pattern dependence, focusing particularly on nonlinear, saturable, time-dependent processes that abound in biophysics, biochemistry, and physiology. We show theoretically that pattern dependence is governed by the nonlinearity of the input–output transformation as well as its time constant. As a result, only patterns on certain time scales permit the expression of pattern dependence, and processes with different time constants can respond preferentially to different patterns. This has implications for temporal coding and decoding, and allows differential control of processes through pattern. We show how pattern dependence can be quantitatively predicted using only information from steady, unpatterned input. To apply our ideas, we analyze, in an experimental example, how muscle contraction depends on the pattern of motorneuron firing.

Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human β-globin in engrafted mice

Transcriptional silencing of genes transferred into hematopoietic stem cells poses one of the most significant challenges to the success of gene therapy. If the transferred gene is not completely silenced, a progressive decline in gene expression as the mice age often is encountered. These phenomena were observed to various degrees in mouse transplant experiments using retroviral vectors containing a human β-globin gene, even when cis-linked to locus control region derivatives. Here, we have investigated whether ex vivo preselection of retrovirally transduced stem cells on the basis of expression of the green fluorescent protein driven by the CpG island phosphoglycerate kinase promoter can ensure subsequent long-term expression of a cis-linked β-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice (n = 7) engrafted with preselected cells concurrently expressed human β-globin and the green fluorescent protein in 20–95% of their RBC for up to 9.5 mo posttransplantation, the longest time point assessed. This expression pattern was successfully transferred to secondary transplant recipients. In the presence of β-locus control region hypersensitive site 2 alone, human β-globin mRNA expression levels ranged from 0.15% to 20% with human β-globin chains detected by HPLC. Neither the proportion of positive blood cells nor the average expression levels declined with time in transplanted recipients. Although suboptimal expression levels and heterocellular position effects persisted, in vivo stem cell gene silencing and age-dependent extinction of expression were avoided. These findings support the further investigation of this type of vector for the gene therapy of human hemoglobinopathies.

hMutSα- and hMutLα-dependent phosphorylation of p53 in response to DNA methylator damage

hMSH2⋅hMSH6 heterodimer (hMutSα) and hMLH1⋅hPMS2 complex (hMutLα) have been implicated in the cytotoxic response of mammalian cells to a number of DNA-damaging compounds, including methylating agents that produce O6-methylguanine (O6MeG) adducts. This study demonstrates that O6MeG lesions, in which the damaged base is paired with either T or C, are subject to excision repair in a reaction that depends on a functional mismatch repair system. Furthermore, treatment of human cells with the SN1 DNA methylators N-methyl-N-nitrosourea or N-methyl-N′-nitro-N-nitrosoguanidine results in p53 phosphorylation on serine residues 15 and 392, and these phosphorylation events depend on the presence of functional hMutSα and hMutLα. Coupled with the previous demonstration that O6MeG⋅T and O6MeG⋅C pairs are recognized by hMutSα, these results implicate action of the mismatch repair system in the initial step of a damage-signaling cascade that can lead to cell-cycle checkpoint activation or cell death in response to DNA methylator damage.

Patch-clamp and amperometric recordings from norepinephrine transporters: Channel activity and voltage-dependent uptake

Transporters for the biogenic amines dopamine, norepinephrine, epinephrine and serotonin are largely responsible for transmitter inactivation after release. They also serve as high-affinity targets for a number of clinically relevant psychoactive agents, including antidepressants, cocaine, and amphetamines. Despite their prominent role in neurotransmitter inactivation and drug responses, we lack a clear understanding of the permeation pathway or regulation mechanisms at the single transporter level. The resolution of radiotracer-based flux techniques limits the opportunities to dissect these problems. Here we combine patch-clamp recording techniques with microamperometry to record the transporter-mediated flux of norepinephrine across isolated membrane patches. These data reveal voltage-dependent norepinephrine flux that correlates temporally with antidepressant-sensitive transporter currents in the same patch. Furthermore, we resolve unitary flux events linked with bursts of transporter channel openings. These findings indicate that norepinephrine transporters are capable of transporting neurotransmitter across the membrane in discrete shots containing hundreds of molecules. Amperometry is used widely to study neurotransmitter distribution and kinetics in the nervous system and to detect transmitter release during vesicular exocytosis. Of interest regarding the present application is the use of amperometry on inside-out patches with synchronous recording of flux and current. Thus, our results further demonstrate a powerful method to assess transporter function and regulation.

Superoxide-mediated clastogenesis and anticlastogenic effects of exogenous superoxide dismutase

Superoxide-mediated clastogenesis is characteristic for various chronic inflammatory diseases with autoimmune reactions and probably plays a role in radiation-induced clastogenesis and in the congenital breakage syndromes. It is consistently prevented by exogenous superoxide dismutase (SOD), but not by heat-inactivated SOD, indicating that the anticlastogenic effect is related to the catalytic function of the enzyme. Increased superoxide production by activated monocytes/macrophages is followed by release of more long-lived metabolites, so-called clastogenic factors, which contain lipid peroxidation products, unusual nucleotides of inosine, and cytokines such as tumor necrosis factor α. Since these components are not only clastogenic, but can stimulate further superoxide production by monocytes and neutrophils, the genotoxic effects are self-sustaining. It is shown here that anticlastogenic effects of exogenous SOD are preserved despite extensive washing of the cells and removal of all extracellular SOD. Using flow cytometry and confocal laser microscopy, rapid adherence of the fluorescently labeled enzyme to the cell surface could be observed with slow uptake into the cell during the following hours. The degree of labeling was concentration and time dependent. It was most important for monocytes, compared with lymphocytes, neutrophils, and fibroblasts. The cytochrome c assay showed significantly diminished O2− production by monocytes, pretreated with SOD and washed thereafter. The preferential and rapid binding of SOD to monocytes may be of importance not only for the superoxide-mediated genotoxic effects, described above, but also from a therapeutic standpoint. It can explain the observation that beneficial effects of injected SOD lasted for weeks and months despite rapid clearance of the enzyme from the blood stream according to pharmacodynamic studies.

Anti-idiotype RNA selected with an anti-nuclear export signal antibody is actively transported in oocytes and inhibits Rev- and cap-dependent RNA export

The anti-idiotype approach is based on the assumption that an antibody specific for a receptor-binding domain of a ligand could be structurally related to the receptor. Therefore, a structural mimic of a receptor-binding domain, selected with an anti-ligand antibody, might be a functional substrate for the receptor. This hypothesis was addressed here by generating antibodies recognizing the Rev-nuclear export signal (NES). A functional NES is required for active export, presumably by interacting directly or indirectly with the nuclear pore complex. Anti-NES antibodies were used to isolate RNA mimics of the NES peptide from combinatorial RNA libraries. The RNA-mimics are exported actively, block Rev-dependent export of a reporter RNA, and inhibit cap-dependent U1 snRNA export in Xenopus oocytes, properties previously reported for NES-peptide conjugates.

The N terminus of the Drosophila Numb protein directs membrane association and actin-dependent asymmetric localization

Drosophila Numb is a membrane associated protein of 557 amino acids (aa) that localizes asymmetrically into a cortical crescent in mitotic neural precursor cells and segregates into one of the daughter cells, where it is required for correct cell fate specification. We demonstrate here that asymmetric localization but not membrane localization of Numb in Drosophila embryos is inhibited by latrunculin A, an inhibitor of actin assembly. We also show that deletion of either the first 41 aa or aa 41–118 of Numb eliminates both localization to the cell membrane and asymmetric localization during mitosis, whereas C-terminal deletions or deletions of central portions of Numb do not affect its subcellular localization. Fusion of the first 76 or the first 119 aa of Numb to β-galactosidase results in a fusion protein that localizes to the cell membrane, but fails to localize asymmetrically during mitosis. In contrast, a fusion protein containing the first 227 aa of Numb and β-galactosidase localizes asymmetrically during mitosis and segregates into the same daughter cell as the endogenous Numb protein, demonstrating that the first 227 aa of the Numb protein are sufficient for asymmetric localization.

Modes of action of aspirin-like drugs: Salicylates inhibit Erk activation and integrin-dependent neutrophil adhesion

The anti-inflammatory effects of high-dose salicylates are well recognized, incompletely understood and unlikely due entirely to cyclooxygenase (COX) inhibition. We have previously reported a role for activation of the kinase Erk in CD11b/CD18 integrin-dependent adhesiveness of human neutrophils, a critical step in inflammation. We now report the effects of salicylates on neutrophil Erk and adhesion. Exposure of neutrophils to aspirin or sodium salicylate (poor COX inhibitor) inhibited Erk activity and adhesiveness of formylmethionyl-leucyl-phenylalanine- and arachidonic acid-stimulated neutrophils, consistent with anti-inflammation but not COX inhibition (IC50s = 1–8 mM). In contrast, indomethacin blocked neither Erk nor adhesion. Inhibition of Mek (proximal activator of Erk) also blocked stimulation of Erk and adhesion by formylmethionyl-leucyl-phenylalanineand arachidonic acid. Salicylate inhibition of Erk was independent of protein kinase A activation and generation of extracellular adenosine. These data are consistent with a role for Erk in stimulated neutrophil adhesion, and suggest that anti-inflammatory effects of salicylates may be mediated via inhibition of Erk signaling required for integrin-mediated responses.

Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens

The endogenous plant hormones salicylic acid (SA) and jasmonic acid (JA), whose levels increase on pathogen infection, activate separate sets of genes encoding antimicrobial proteins in Arabidopsis thaliana. The pathogen-inducible genes PR-1, PR-2, and PR-5 require SA signaling for activation, whereas the plant defensin gene PDF1.2, along with a PR-3 and PR-4 gene, are induced by pathogens via an SA-independent and JA-dependent pathway. An Arabidopsis mutant, coi1, that is affected in the JA-response pathway shows enhanced susceptibility to infection by the fungal pathogens Alternaria brassicicola and Botrytis cinerea but not to Peronospora parasitica, and vice versa for two Arabidopsis genotypes (npr1 and NahG) with a defect in their SA response. Resistance to P. parasitica was boosted by external application of the SA-mimicking compound 2,6-dichloroisonicotinic acid [Delaney, T., et al. (1994) Science 266, 1247–1250] but not by methyl jasmonate (MeJA), whereas treatment with MeJA but not 2,6-dichloroisonicotinic acid elevated resistance to Alternaria brassicicola. The protective effect of MeJA against A. brassicicola was the result of an endogenous defense response activated in planta and not a direct effect of MeJA on the pathogen, as no protection to A. brassicicola was observed in the coi1 mutant treated with MeJA. These data point to the existence of at least two separate hormone-dependent defense pathways in Arabidopsis that contribute to resistance against distinct microbial pathogens.

Roles of SecG in ATP- and SecA-dependent protein translocation

SecA, the translocation ATPase in Escherichia coli, undergoes cycles of conformational changes (insertion/deinsertion) in response to ATP and a preprotein. The membrane-embedded portion of protein translocase, SecYEG, has crucial roles in the SecA-driven preprotein translocation reaction. We previously identified a secY mutation (secY205) that did not allow an ATP- and preprotein-dependent (productive) insertion of SecA as well as secA mutations that suppressed the secY205 translocation defect. One of the suppressor mutations, secA36, also suppressed the cold-sensitive phenotype of the secG deletion mutant. In vitro experiments at 20°C showed that inverted membrane vesicles lacking SecG were almost inactive in combination with the wild-type SecA protein in translocation of proOmpA as well as in the accompanying ATP hydrolysis. In contrast, the SecA36 mutant protein was found to be able to execute the translocation activity fully at this temperature, even in the absence of SecG. A SecG requirement and its alleviation by the SecA36 alteration also were shown for the SecA insertion reaction. The finding that the SecA36 protein no longer requires assistance from SecG in its insertion and in its catalysis of protein translocation agrees with the idea that SecG normally assists in the functioning of SecA. In agreement with this notion, when the intrinsic SecA function was compromised by a lowered ATP concentration, SecG became essential even at 37°C and even for the SecA36 protein. We propose that in the normal translocase, SecG cooperates with SecA to facilitate efficient movement of preprotein in each catalytic cycle of SecA.

Thrombospondin-1 Induces Tyrosine Phosphorylation of Adherens Junction Proteins and Regulates an Endothelial Paracellular Pathway

Thrombospondin-1 (TSP) induces endothelial cell (EC) actin reorganization and focal adhesion disassembly and influences multiple EC functions. To determine whether TSP might regulate EC–EC interactions, we studied the effect of exogenous TSP on the movement of albumin across postconfluent EC monolayers. TSP increased transendothelial albumin flux in a dose-dependent manner at concentrations ≥1 μg/ml (2.2 nM). Increases in albumin flux were observed as early as 1 h after exposure to 30 μg/ml (71 nM) TSP. Inhibition of tyrosine kinases with herbimycin A or genistein protected against the TSP-induced barrier dysfunction by >80% and >50%, respectively. TSP-exposed monolayers exhibited actin reorganization and intercellular gap formation, whereas pretreatment with herbimycin A protected against this effect. Increased staining of phosphotyrosine-containing proteins was observed in plaque-like structures and at the intercellular boundaries of TSP-treated cells. In the presence of protein tyrosine phosphatase inhibition, TSP induced dose- and time-dependent increments in levels of phosphotyrosine-containing proteins; these TSP dose and time requirements were compatible with those defined for EC barrier dysfunction. Phosphoproteins that were identified include the adherens junction proteins focal adhesion kinase, paxillin, γ-catenin, and p120Cas. These combined data indicate that TSP can modulate endothelial barrier function, in part, through tyrosine phosphorylation of EC proteins.