One is enough: in vivo effective population size is dose-dependent for a plant RNA virus

Effective population size (N(e)) determines the strength of genetic drift and the frequency of co-infection by multiple genotypes, making it a key factor in viral evolution. Experimental estimates of N(e) for different plant viruses have, however, rendered diverging results. The independent action hypothesis (IAH) states that each virion has a probability of infection, and that virions act independent of one another during the infection process. A corollary of IAH is that N(e) must be dose dependent. A test of IAH for a plant virus has not been reported yet. Here we perform a test of an IAH infection model using a plant RNA virus, Tobacco etch virus (TEV) variants carrying GFP or mCherry fluorescent markers, in Nicotiana tabacum and Capsicum annuum plants. The number of primary infection foci increased linearly with dose, and was similar to a Poisson distribution. At high doses, primary infection foci containing both genotypes were found at a low frequency (<2%). The probability that a genotype that infected the inoculated leaf would systemically infect that plant was near 1, although in a few rare cases genotypes could be trapped in the inoculated leaf by being physically surrounded by the other genotype. The frequency of mixed-genotype infection could be predicted from the mean number of primary infection foci using the independent-action model. Independent action appears to hold for TEV, and N(e) is therefore dose-dependent for this plant RNA virus. The mean number of virions causing systemic infection can be very small, and approaches 1 at low doses. Dose-dependency in TEV suggests that comparison of N(e) estimates for different viruses are not very meaningful unless dose effects are taken into consideration.     

Quantitative comparison of CFD predicted and MRI measured velocity fields in a carotid bifurcation phantom

Steady flow of a blood mimicking fluid in a physiologically realistic model of the human carotid bifurcation was studied using both magnetic resonance imaging (MRI) and computational fluid dynamics (CFD) modelling techniques. Quantitative comparisons of the 3D velocity field in the bifurcation phantom were made between phase contrast MRI measurements and CFD predictions. The geometry for the CFD model was reconstructed from T(1) weighted MR imaging of the test phantom. It was found that the predicted velocity fields were in fair agreement with MR measured velocities. In both the internal and external carotid arteries, the agreement between CFD predictions and MRI measurements was better along the inner-outer wall axis with a correlation factor C>0.897 (average 0.939) where the velocity profiles were skewed, than along the anterior-posterior axis (average correlation factor 0.876) where the velocity profiles were in M-shape.     

Bone cancer risk of (239)pu in humans derived from animal models

Two-mutation model fits to bone cancer mortality data from mice, rats and beagle dogs injected with (239)Pu or (226)Ra show that (1) it is possible to fit the radiation-related parameters for animals from different strains of the same species together; (2) for every species the same significant parameters are found in the models for (239)Pu and in the models for (226)Ra, and the only difference is in the value of the linear mutation coefficient; and (3) the toxicity ratio, when defined as the ratio of the linear mutation coefficients for (239)Pu over (226)Ra, has a relatively uniform value of approximately 8 for the species considered. This relatively constant ratio enables the development of a (239)Pu model for humans that is based on the radium dial painters and the toxicity ratio for beagles. The model predictions agree well with published risk estimates based on other data and derived using alternative approaches. This has two important implications: (1) The two-mutation model appears to be a useful tool in translating from animal models to humans in a meaningful way; and (2) once a two-mutation model for humans has been derived, radiation risks can be calculated that depend on doses, dose rates and ages at exposure. Such a model therefore supplements published risk estimates that often lack such dependences.     

The radioprotective effect of a hypoxic respiratory mixture (8% O2) for intestinal epithelial stem cells during single and fractionated irradiation of mice]

The method of intestinal "microcolonies" was used to study the radioprotective effect of a gas mixture, containing 8% of O2, on mice subjected to single and fractionated (5 fractions for 30 min) irradiation. The protective effect was indicated by a decreased slope of dose curves of the stem cell injury; the extrapolation number decreased simultaneously. So the values of dose modifying factors (DMF) were higher, when calculated by D0 ratio (where they amounted to 1.76 and 1.39 for single and fractionated exposure respectively), than those determined by equally effective doses (1.19 and 1.26 for single and fractionated effects respectively, which corresponded to LD50/4 when calculated at lg N = 1.9). It is suggested that the radiation response of certain stem cell populations of intestinal epithelium are different: this is attributed to different degrees of hypoxia in cells and to different directions of the hypoxia effects on the injury and the ability of postirradiation repair.     

Use of the γ-H2AX Assay to Investigate DNA Repair Dynamics Following Multiple Radiation Exposures

Radiation therapy is one of the most common and effective strategies used to treat cancer. The irradiation is usually performed with a fractionated scheme, where the dose required to kill tumour cells is given in several sessions, spaced by specific time intervals, to allow healthy tissue recovery. In this work, we examined the DNA repair dynamics of cells exposed to radiation delivered in fractions, by assessing the response of histone-2AX (H2AX) phosphorylation (γ-H2AX), a marker of DNA double strand breaks. γ-H2AX foci induction and disappearance were monitored following split dose irradiation experiments in which time interval between exposure and dose were varied. Experimental data have been coupled to an analytical theoretical model, in order to quantify key parameters involved in the foci induction process. Induction of γ-H2AX foci was found to be affected by the initial radiation exposure with a smaller number of foci induced by subsequent exposures. This was compared to chromatin relaxation and cell survival. The time needed for full recovery of γ-H2AX foci induction was quantified (12 hours) and the 1:1 relationship between radiation induced DNA double strand breaks and foci numbers was critically assessed in the multiple irradiation scenarios.     

Cancer risk estimates for gamma-rays with regard to organ-specific doses

A previous analysis of the solid cancer mortality data for 1950-1990 from the Japanese life-span study of the A-bomb survivors has assessed the solid cancer risk coefficients for gamma-rays in terms of the low dose risk coefficient ERR/Gy, i.e. the initial slope of the ERR vs. dose relation, and also in terms of the more precisely estimated intermediate dose risk coefficient, ERR(D1)/D1, for a reference dose, D1, which was chosen to be 1 Gy. The computations were performed for tentatively assumed values 20-50 of the neutron RBE against the reference dose and in terms of organ-averaged doses, rather than the traditionally applied colon doses. The resulting risk estimate for a dose of 1 Gy was about half as large as the most recent UNSCEAR estimate. The present assessment repeats the earlier analysis with two major extensions. It parallels computations based on organ-average doses with computations based on organ-specific doses and it updates the previous results by using the cancer mortality data for 1950-1997 which have recently been made available. With an assumed neutron RBE of 35, the resulting intermediate dose estimate of the lifetime attributable risk (LAR) for solid cancer mortality for a working population (ages 25-65 years) is 0.059/Gy with the attained-age model, and 0.044/Gy with the age-at-exposure model. For a population of all ages, 0.055/Gy is obtained with the attained-age model and 0.073/Gy with the age-at-exposure model. These values are up to about 20% higher than those obtained in the previous analysis with the 1950-1990 data. However, considerably more curvature in the dose-effect relation is now supported by the computations. A dose and dose-rate reduction factor DDREF=2 is now much more in line with the data than before. With this factor the LAR for a working population is--averaged over the age-at-exposure and the age-attained model--equal to 0.026/Gy. This is only half as large as the current ICRP estimate which is also based on the assumption DDREF=2.     

GTP-Binding-Defective ARL4D Alters Mitochondrial Morphology and Membrane Potential

ARL4D, ARL4A, and ARL4C are closely related members of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of GTPases. All three ARL4 proteins contain nuclear localization signals (NLSs) at their C-termini and are primarily found at the plasma membrane, but they are also present in the nucleus and cytoplasm. ARF function and localization depends on their controlled binding and hydrolysis of GTP. Here we show that GTP-binding-defective ARL4D is targeted to the mitochondria, where it affects mitochondrial morphology and function. We found that a portion of endogenous ARL4D and the GTP-binding-defective ARL4D mutant ARL4D(T35N) reside in the mitochondria. The N-terminal myristoylation of ARL4D(T35N) was required for its localization to mitochondria. The localization of ARL4D(T35N) to the mitochondria reduced the mitochondrial membrane potential (ΔΨm) and caused mitochondrial fragmentation. Furthermore, the C-terminal NLS region of ARL4D(T35N) was required for its effect on the mitochondria. This study is the first to demonstrate that the dysfunctional GTP-binding-defective ARL4D is targeted to mitochondria, where it subsequently alters mitochondrial morphology and membrane potential.     

Structure of gel phase saturated lecithin bilayers: temperature and chain length dependence.

Systematic low-angle and wide-angle x-ray scattering studies have been performed on fully hydrated unoriented multilamamellar vesicles of saturated lecithins with even chain lengths N = 16, 18, 20, 22, and 24 as a function of temperature T in the normal gel (L beta') phase. For all N, the area per chain Ac increases linearly with T with an average slope dAc/dT = 0.027 A2/degree C, and the lamellar D-spacings also increase linearly with an average slope dD/dT = 0.040 A/degree C. At the same T, longer chain length lecithins have more densely packed chains, i.e., smaller Ac's, than shorter chain lengths. The chain packing of longer chain lengths is found to be more distorted from hexagonal packing than that of smaller N, and the distortion epsilon of all N approaches the same value at the respective transition temperatures. The thermal volume expansion of these lipids is accounted for by the expansion in the hydrocarbon chain region. Electron density profiles are constructed using four orders of low-angle lamellar peaks. These show that most of the increase in D with increasing T is due to thickening of the bilayers that is consistent with a decrease in tilt angle theta and with little change in water spacing with either T or N. Because of the opposing effects of temperature on area per chain Ac and tilt angle 0, the area expansivity alpha A is quite small. A qualitative theoretical model based on competing head and chain interactions accounts for our results.     

Thiazolium C(2)-proton exchange: structure-reactivity correlations and the pKa of thiamin C(2)-H revisited

Rate constants for C(2)-proton exchange from thiamin, N(1')-methylthiamin, and several 3-substituted-4-methylthiazolium ions catalyzed by D2O and deuterioxide ion were determined by 1H NMR at 30 degrees C and ionic strength 2.0 M. Values of pKa for the thiazolium ions, including thiamin itself, were found to be in the range pKa = 17-19; the pKa values for N(1')-protonated thiamin and free thiamin C(2)-H in H2O are 17.7 and 18.0, respectively. The pKa value for N(1')-protonated thiamin was calculated from the observed rate constant for the pD-independent reaction with D2O after correction for a secondary solvent deuterium isotope effect of kH2O/kD2O = 2.6. The pKa value for free thiamin was calculated from the rate constant for catalysis by OD- after correction by a factor of 3.3 = 8/2.4 for an 8-fold negative deviation of kOD from the Br?nsted plot of slope 1.0 for general base catalysis and a secondary solvent isotope effect of kOD/kOH = 2.4. Values of k-a = 2 X 10(10) and 3 X 10(9) M-1 s-1 were assumed for diffusion-controlled protonation of the C(2) ylide in the reverse direction by H3O+ and H2O, respectively. The Hammett rho I value for the exchange reaction catalyzed by deuterioxide ion or D2O is 8.4 +/- 0.2. There is no positive deviation of the rate constants for free or N(1')-substituted thiamin analogues in either Hammett correlation. This shows that the aminopyrimidinyl group does not provide significant intramolecular catalysis of nonenzymic C(2)-proton removal in the coenzyme.     

How might spatial nonuniformity of dose in a homogeneous biological system affect its total response?

The total response of a homogeneous biological system to a fixed total dose of a biological agent is modeled by dividing the system into N cubical voxels, each of which can be associated with an individual dose D(n) and an individual response R(n) =F(D(n)). Among the results shown are the following: A. (Voxel Theorem). Let the average dose D(avg) be held fixed as the dose distribution is shifted from uniform u to arbitrary a. Then, if F' > or = 0 over [D(min), D(max)] and R = summation operator (n = 1)(N) R(n), a sufficient condition that NF(D(avg)) = R(u) < or = R(a) is that F be a concave-upwards function of dose; that is, F" > or = 0 over [D(min), D(max)]. B.If F' is constant over [D(min), D(max)], then R(a) = R(u). That is, the total response is a function of D(avg) only. The applications of these (and other) results are illustrated by examples from bioelectromagnetics.     

Meal and oral glucose tests for assessment of beta -cell function: modeling analysis in normal subjects

We investigated beta-cell function and its relationship to insulin sensitivity in 17 normal volunteers. For insulin secretion (derived by C-peptide deconvolution), a mathematical model was applied to 24-h triple-meal tests (MT) as well as oral glucose tolerance tests (OGTT); insulin sensitivity was assessed by the euglycemic insulin clamp technique. The beta-cell model featured a glucose concentration-insulin secretion dose response (characterized by secretion at 5 mM glucose and slope), a secretion component proportional to the glucose concentration derivative, and a time-dependent potentiation factor (modulating the dose response and accounting for effects of sustained hyperglycemia and incretins). The beta-cell dose-response functions estimated from the whole 24-h MT, the first 2 h of the MT, and the OGTT differed systematically, because a different potentiation factor was involved. In fact, potentiation was higher than average during meals (1.6 +/- 0.1-fold during the first meal) and had a different time course in the MT and OGTT. However, if potentiation was accounted for, the 24- and 2-h MT and the OGTT yielded similar dose responses, and most beta-cell function parameters were intercorrelated (r = 0.50-0.86, P < or = 0.05). The potentiation factor was found to be related to plasma glucose-dependent insulin-releasing polypeptide concentrations (r = 0.49, P < 0.0001). Among beta-cell function parameters, only insulin secretion at 5 mM glucose from MT correlated inversely with insulin sensitivity (24-h MT: r = -0.74, P < 0.001; 2-h MT: r = -0.52, P < 0.05), whereas the dose-response slope and the OGTT parameters did not. In nine other subjects, reproducibility of model parameters was evaluated from repeated MTs. Coefficients of variation were generally approximately 20%, but the derivative component was less reproducible. We conclude that our model for the multiple MT yields useful information on beta-cell function, particularly with regard to the role of potentiation. With cautious interpretation, a 2-h MT or a standard OGTT can be used as surrogates of 24-h tests in assessing spontaneous beta-cell function.     

Decay of γ-H2AX foci correlates with potentially lethal damage repair and P53 status in human colorectal carcinoma cells

The influence of p53 status on potentially lethal damage repair (PLDR) and DNA double-strand break (DSB) repair was studied in two isogenic human colorectal carcinoma cell lines: RKO (p53 wild-type) and RC10.1 (p53 null). They were treated with different doses of ionizing radiation, and survival and the induction of DNA-DSB were studied. PLDR was determined by using clonogenic assays and then comparing the survival of cells plated immediately with the survival of cells plated 24 h after irradiation. Doses varied from 0 to 8 Gy. Survival curves were analyzed using the linear-quadratic formula: S(D)/S(0) = exp-(αD+βD²). The γ-H2AX foci assay was used to study DNA DSB kinetics. Cells were irradiated with single doses of 0, 0.5, 1 and 2 Gy. Foci levels were studied in non-irradiated control cells and 30 min and 24 h after irradiation. Irradiation was performed with gamma rays from a ¹³⁷Cs source, with a dose rate of 0.5 Gy/min. The RKO cells show higher survival rates after delayed plating than after immediate plating, while no such difference was found for the RC10.1 cells. Functional p53 seems to be a relevant characteristic regarding PLDR for cell survival. Decay of γ-H2AX foci after exposure to ionizing radiation is associated with DSB repair. More residual foci are observed in RC10.1 than in RKO, indicating that decay of γ-H2AX foci correlates with p53 functionality and PLDR in RKO cells.     

Immunoreceptor tyrosine-based activation motif phosphorylation during engulfment of Neisseria gonorrhoeae by the neutrophil-restricted CEACAM3 (CD66d) receptor

Gonorrhea is characterized by a purulent urethral or cervical discharge consisting primarily of neutrophils associated with Neisseria gonorrhoeae. These interactions are facilitated by gonococcal colony opacity-associated (Opa) protein binding to host cellular CEACAM receptors. Of these, CEACAM3 is restricted to neutrophils and contains an immunoreceptor tyrosine-based activation motif (ITAM) reminiscent of that found within certain phagocytic Fc receptors. CEACAM3 was tyrosine phosphorylated by a Src family kinase-dependent process upon infection by gonococci expressing CEACAM-specific Opa proteins. This phosphorylation was necessary for efficient bacterial uptake; however, a less efficient uptake process became evident when kinase inhibitors or mutagenesis of the ITAM were used to prevent phosphorylation. Ligated CEACAM3 was recruited to a cytoskeleton-containing fraction, intense foci of polymerized actin were evident where bacteria attached to HeLa-CEACAM3, and disruption of polymerized actin by cytochalasin D blocked all bacterial uptake by these cells. These data support a model whereby CEACAM3 can mediate the Opa-dependent uptake of N. gonorrhoeae via either an efficient, ITAM phosphorylation-dependent process that resembles phagocytosis or a less efficient, tyrosine phosphorylation-independent mechanism.     

烟草潜叶蛾幼虫空间分布型及其应用研究

本文就烟草潜叶蛾幼虫空间分布型及其垂直分布规律进行了探讨,结果表明：烟草潜叶蛾幼虫在田间呈聚集分布,聚集强度不因种群密度的改变而改变,幼虫主要聚集分布在烟草下部第一段（4片叶）上;降集或随机分布在第二段上;随机分布在第三段上,此外,应用虫株率进行田间种群密度的估计,其中Wilson模型和Gerrard模型所配理论曲线的预测值与实测值显著适合,但Gerrard模型的抽样估计误差较Wilson模型的小,最后本文用Taylor式中的参数a,b确定理论抽样数及序贯抽样,其模型分别为。     

Protection against diethylnitrosoamine-induced hepatocarcinogenesis by an indigenous medicine comprised of Nigella sativa, Hemidesmus indicus and Smilax glabra: a preliminary study

BACKGROUND: A decoction comprised of Nigella sativa seeds, Hemidesmus indicus root and Smilax glabra rhizome is used to treat cancer patients in Sri Lanka. However, the anti-carcinogenic properties of this decoction have not been experimentally confirmed. The purpose of this study was to determine whether the above decoction could protect against chemically induce hepatocarcinogenesis. METHODS: The effects of this decoction on diethylnitrosamine (DEN) induced hepatocarcinogenesis were examined in male Wistar rats using the medium term bioassay system of Ito, based on a 2-step model of hepatocarcinogenesis. Rats were randomly divided into 6 groups of 10 each. Groups 1 to 4 were injected with DEN (200 mg/kg) to initiate carcinogenesis. Twenty-four hours later groups 1 and 2 were administered the decoction at 4 g/kg body weight/day (dose 1) and 6 g/kg body weight/day (dose 2), respectively. Group 3 and group 4 were given distilled water instead of the decoction and a suspension of garlic powder (20 g/kg body weight/day) in distilled water (positive control), respectively. Group 5 and 6 were injected with normal saline and twenty-four hours later group 5 was given distilled water (normal control) while group 6 was given decoction dose 2 (decoction control). Oral feeding continued for two weeks after which all rats were subjected to 2/3 partial hepatectomy to promote carcinogenesis. Oral feeding continued for eight more weeks. At the end of the 10th week, rats were sacrificed and samples of livers taken for immunohistochemical studies.Carcinogenic potential was scored by comparing the number, area and staining intensity of glutathione S-transferase placental form (GST-P) positive foci and the number of cells/cm2 of the positive foci in the livers of the six groups of rats. RESULTS: The number and area of DEN-mediated GST-P positive foci, number of cells/cm2 of foci and staining intensity of the foci were significantly (P > 0.001) reduced by the decoction and garlic in the order dose 2 = garlic >dose 1. CONCLUSION: Overall results indicate that the decoction comprised of N. sativa, S. glabra and H. indicus has the potential to protect rat liver against DEN induced hepatocarcinogenesis     

Endogenous expression of phosphorylated histone H2AX in tumors in relation to DNA double-strand breaks and genomic instability

Microscopically visible gammaH2AX foci signify the presence of DNA double-strand breaks (dsbs) in irradiated cells. However, large foci are also observed in untreated tumour cells, and high numbers reduce the sensitivity for detecting drug or radiation-induced DNA breaks. SW756 cervical carcinoma cells that express about 50 gammaH2AX foci per cell (i.e., equivalent to the number of breaks produced by about 2Gy) showed similar numbers of dsbs as C33A cells that exhibit fewer than three foci per cell. The possibility that differences in numbers of these endogenous foci could be explained by genomic instability perhaps related to misrepair was examined. For 17cell lines selected from the panel of NCI-60 tumor cells previously characterized for karyotypic complexity [A.V. Roschke, G. Tonon, K.S. Gehlhaus, N. McTyre, K.J. Bussey, S. Lababidi, D.A. Scudiero, J.N. Weinstein, I.R. Kirsch, Karyotypic complexity of the NCI-60 drug-screening panel, Cancer Res. 63 (2003) 8634-8647], there was a significant trend (r=0.6) for cell lines with greater numbers of structural or numerical chromosomal rearrangements to show a higher background expression of gammaH2AX. Moreover, cells from this panel with wild-type p53 showed a significantly lower background level of gammaH2AX than cells with mutant p53. To confirm the importance of p53 expression, endogenous and radiation-induced gammaH2AX expression were analyzed using four isogenic SKOV3 cell lines varying in p53 function. Again, higher gammaH2AX expression was found in SKOV3 cell lines expressing mutant p53 compared to wild-type p53. HFL-1 primary lung fibroblasts showed a progressive increase in gammaH2AX as they moved towards senescence, confirming the importance of telomere instability in the development of at least some gammaH2AX foci. Therefore, the explanation for high endogenous levels of gammaH2AX in some tumor cells appears to be multifactorial and may be best described as a consequence of chromatin instability.     

A decision tree approach to modelling nitrogen fertiliser use efficiency in New Zealand pastures

Quantifying nitrogen (N) fertiliser use efficiency (NFUE) in pastoral systems has important implications for fertiliser management from both economic and environmental points of view. The potential of a decision tree approach for modelling NFUE in New Zealand pastures was investigated. The decision tree model suggested that the time of applying N fertiliser was the most important factor influencing NFUE, with August or September (early spring in New Zealand) being the best time of application. The interaction of rainfall and temperature, rainfall, phosphorus (P) fertiliser history, soil Olsen P and slope were other important factors influencing NFUE. The model was validated for 11 of the 16 trials tested with a predictive accuracy of 69%. The mechanisms by which these factors influenced NFUE and the uncertainty associated with the model prediction were discussed. It was concluded that this type of modelling approach can be used to predict NFUE and thereby to assist decisions on when and where to apply N fertiliser in pastures for increasing productivity while reducing the environmental impact.     

Interrelations of M-intermediates in bacteriorhodopsin photocycle.

The photocycles of the wild-type bacteriorhodopsin and the D96N mutant were investigated by the flash-photolysis technique. The M-intermediate formation (400 nm) and the L-intermediate decay (520 nm) were found to be well described by a sum of two exponents (time constants, tau 1 = 65 and tau 2 = 250 microseconds) for the wild-type bR and three exponents (tau 1 = 55 microseconds, tau 2 = 220 microseconds and tau 3 = 1 ms) for the D96N mutant of bR. A component with tau = 1 ms was found to be present in the photocycle of the wild-type bacteriorhodopsin as a lag-phase in the relaxation of photoresponses at 400 and 520 nm. In the presence of Lu3+ ions or 80% glycerol this component was clearly seen as an additional phase of M-formation. The azide effect on the D96N mutant of bR suggests that the 1-ms component is associated with an irreversible conformational change switching the Schiff base from the outward to the inward proton channel. The maximum of the difference spectrum of the 1-ms component of D96N bR is located at 404 nm as compared to 412 nm for the first two components. We suggest that this effect is a result of the alteration of the inward proton channel due to the Asp96-->Asn substitution. Proton release measured with pyranine in the absence of pH buffers was identical for the wild-type bR and D96N mutant and matched the M-->M' conformational transition. A model for M rise in the bR photocycle is proposed.     

Early detection of carcinogenic substances and modifiers in rats

Over the past 20 years, we have been developing in vivo medium-term bioassay systems in rats for detecting carcinogenic and modifying effects of test compounds. The systems are based on the two-step hypothesis of carcinogenesis. In a liver model, male F344 rats are initially given a single dose of diethylnitrosamine (DEN, 200 mg/kg, i.p.) and starting 2 weeks later are treated with test compounds for 6 weeks and then killed, all rats being subjected to two-thirds partial hepatectomy at week 3. Carcinogenic potential is scored by comparing the numbers and areas per cm(2) of induced glutathione S-transferase placental form (GST-P) positive foci in the livers of groups of about 15 rats with those of corresponding control groups given DEN alone. A positive response is defined as a significant increase in the quantitative values of GST-P-positive foci, such a negative response as no change or a decrease. The results obtained have been compared with reported Salmonella/microsome and long-term carcinogenicity test findings for the same compounds. Of the liver carcinogens, 30 out of 31 (97%) mutagenic and 29 out of 33 (88%) non-mutagenic compounds gave positive results. Carcinogens other than hepatocarcinogens gave a lower proportion of positive results (9 out of 42, 21%). This bioassay also provides information concerning inhibitory potential. The practical utility and benefits of a multi-organ medium-term experimental protocol for early detection of carcinogenic agents and modifiers acting at sites other than the liver are also discussed.     

The slow Wallerian degeneration protein, WldS, binds directly to VCP/p97 and partially redistributes it within the nucleus

Slow Wallerian degeneration (Wld(S)) mutant mice express a chimeric nuclear protein that protects sick or injured axons from degeneration. The C-terminal region, derived from NAD(+) synthesizing enzyme Nmnat1, is reported to confer neuroprotection in vitro. However, an additional role for the N-terminal 70 amino acids (N70), derived from multiubiquitination factor Ube4b, has not been excluded. In wild-type Ube4b, N70 is part of a sequence essential for ubiquitination activity but its role is not understood. We report direct binding of N70 to valosin-containing protein (VCP; p97/Cdc48), a protein with diverse cellular roles including a pivotal role in the ubiquitin proteasome system. Interaction with Wld(S) targets VCP to discrete intranuclear foci where ubiquitin epitopes can also accumulate. Wld(S) lacking its N-terminal 16 amino acids (N16) neither binds nor redistributes VCP, but continues to accumulate in intranuclear foci, targeting its intrinsic NAD(+) synthesis activity to these same foci. Wild-type Ube4b also requires N16 to bind VCP, despite a more C-terminal binding site in invertebrate orthologues. We conclude that N-terminal sequences of Wld(S) protein influence the intranuclear location of both ubiquitin proteasome and NAD(+) synthesis machinery and that an evolutionary recent sequence mediates binding of mammalian Ube4b to VCP.