Synthesis,degradation, and subcellular localization of proteins encoded by the primary response genes TIS7/PC4 and TIS21/PC3

Murine TIS7 and TIS21 cDNAs were cloned from phorbol ester-induced Swiss 3T3 cells. The cognate rat cDNAs. PC4 and PC3, were cloned from nerve growth factor (NGF)-treated PC12 pheochromocytoma cells. The TIS7/PC4 and TIS21/PC3 primary response genes are rapidly and transiently induced in response to serum, phorbol esters, and polypeptide growth factors in quiescent Swiss 3T3 cells and by NGF and other ligands in PC12 cells. In both 3T3 and PC12 cells the appearance of the TIS21/PC3 message precedes that of TIS7/PC4 message following ligand stimulation, suggesting that the TIS21/PC3 protein is likely to be synthesized more rapidly than the TIS7/PC4 protein. Using antisera prepared against recombinant TIS21 and TIS7 proteins, we find that the TIS21/PC3 protein is, indeed, synthesized more rapidly than the TIS7/PC4 protein following stimulation in both 3T3 and PC12 cells. In addition, “pulse-chase” experiments demonstrate that the TIS21/PC3 protein is degraded much more rapidly than the TIS7/PC4 protein. The sequences of the predicted PC3 and PC4 proteins have lead to the speculation that these two proteins may both be secreted from cells following stimulation. The PC4 protein is reported to have some sequence similarity to interferons. The TIS21/PC3 protein contains a presumptive leader sequence. Using our antisera to the recombinant proteins, however, we cannot detect secretion of radiolabelled TIS7/PC4 or TIS21/PC3 protein. Immunohistochemical and subcellular fractionation experiments suggest that the TIS7 protein is a membrane associated, non-nuclear intracellular protein. The TIS21 protein, in contrast, is' a non-nuclear, soluble intracellular protein. © 1994 Wiley-Liss, Inc.     

Wound-induced and developmental activation of a poplar tree chitinase gene promoter in transgenic tobacco

Wounding hybrid poplar (Populus trichocarpa × P. deltoides) trees results in the expression of novel wound-inducible (win) mRNAs thought to encode proteins involved in defense against pests and pathogens. Members of thewin6 gene family encode acidic multi-domain chitinases, with combined structure and charge characteristics that differ from previously described chitinases.Win6 expression has been shown to occur in pooled unwounded leaves of a wounded (on multiple leaves) poplar plant. Here we demonstrate that wounding a single leaf induceswin6 expression locally, in the wounded leaf, and remotely, in specific unwounded leaves with strong vascular connections to the wounded leaf. We also demonstrate that awin6 promoter--glucuronidase (GUS) gene fusion (win6-GUS) responds to wounding locally and remotely in transgenic tobacco. These data indicate that the poplarwin6 promoter has regulatory elements that are responsive to wound signals in the heterologous host. In addition,win6-GUS is developmentally activated in unwounded young leaves and floral tissues of transgenic tobacco. Similar developmental expression patterns are found to occur forwin6 in poplar trees, demonstrating that a herbaceous plant can serve as a host for woody tree transgene analysis and can accurately predict expression patterns in tree tissues (e.g. flowers) that would be difficult to study in free-living trees.     

“Macrophage” nitric oxide synthase is a glucocorticoid-inhibitable primary response gene in 3T3 cells

Both nitric oxide and prostaglandins are potent paracrine mediators of intercellular communication. An endotoxin-lipopolysaccharide (LPS) inducible form of nitric oxide synthase (mac-NOS) has recently been cloned from murine macrophages. An inducible prostaglandin synthase (TIS1O/PGS-2), cloned from 3T3 cells, is also induced in LPS-activated macrophage. Because of the wide range of ligands that induce primary response genes in 3T3 cells, the ease of studying chimeric promoter constructs in 3T3 cells, and the importance of both nitric oxide and prostaglandins as paracrine mediators, we examined expression of mac-NOS in 3T3 cells. Tetradecanoyl phorbol-13 acetate (TPA), forskolin, platelet-derived growth factor, fibroblast growth factor, and serum all induce mac-NOS expression in Swiss 3T3 cells. Thus the mac-NOS gene can respond to a far wider range of inducers than previously suspected. mac-NOS is a primary response gene; cycloheximide does not block induction. TPA-induced mac-NOS and TIS10/PGS-2 mRNA accumulation patterns are similar. LPS is a potent inducer of mac-NOS in Swiss 3T3 cells but cannot induce TIS10/PGS-2. In contrast, v-src expression induces TIS10/PGS-2 message, but not iNOS message in a BALB/c 3T3 cell line containing a temperature-sensitive v-src gene. Dexamethasone (DEX) prevents induction of TIS10/PGS-2, but not most other primary response genes. DEX also blocks mac-NOS induction in Swiss 3T3 cells. The inducible TIS10/PGS-2 and mac-NOS genes, responsible for the production of two distinct paracrine agents, appear to share many regulatory features in 3T3 cells. © 1993 Wiley-Liss, Inc.     

Development of a random amplification of polymorphic DNA typing method for Listeria monocytogenes.

The 10-mer primer OPM-01 (5'-GTT GGT GGC T-3') was used to generate random amplification of polymorphic DNA (RAPD) profiles by polymerase chain reaction for 91 strains of Listeria monocytogenes from raw milk, food, and veterinary, medical, and food-environmental sources. The profiles obtained contained 1 to 10 bands within the molecular size range of 0.5 to 5.0 kbp. Reproducibility was enhanced by annealing at low stringency and introducing a 1-min ramp time between annealing and extension temperatures. Thirty-three RAPD profiles were observed, with specific profiles being observed for strains from each source. RAPD profiles allowed discrimination within serogroups, although five RAPD profiles which were not confined to one serotype were found. Within food strains, one RAPD profile was more common than others, suggesting this to be a common type among strains from this source.     

Conformational transitions using molecular dynamics with minimum biasing

The molecular dynamics algorithm (MD), which simulates intramolecular motions on the subnanosecond timescale, has been modified to allow the investigation of slow conformational transitions that do not necessarily occur spontaneously in MD simulations. The method is designated CONTRA MD (CONformational TRAnsitions by Molecular Dynamics with minimum biasing). The method requires the prior definition of a single conformational variable that is required to vary monotonically from an initial conformation to a final target conformation. The simulation is broken up into a series of short free MD segments, and we determine, after each segment of MD, whether or not the system has evolved toward the final conformation. Those segments that do not move the system in that direction are deleted. Those that do move it toward the final conformation are patched together sequentially to generate a single representative trajectory along the transition pathway. The CONTRA MD method is demonstrated first by application to the simultaneous C2′-endo to C3′-endo repucker and anti to syn N-glycosidic torsion transitions in 2′-deoxyadenosine and then to the large-scale bending in phenylalanine transfer RNA. © 1993 John Wiley & Sons, Inc.     

Causal inference and large-scale expert validation shed light on the drivers of SDM accuracy and variance

Aim

To develop a causal understanding of the drivers of Species distribution model (SDM) performance.

Location

United Kingdom (UK).

Methods

We measured the accuracy and variance of SDMs fitted for 518 species of invertebrate and plant in the UK. Our measure of variance reflects variation among replicate model fits, and taxon experts assessed model accuracy. Using directed acyclic graphs, we developed a causal model depicting plausible effects of explanatory variables (e.g. species' prevalence, sample size) on SDM accuracy and variance and quantified those effects using a multilevel piecewise path model.

Results

According to our model, sample size and niche completeness (proportion of a species' niche covered by sampling) directly affect SDM accuracy and variance. Prevalence and range completeness have indirect effects mediated by sample size. Challenging conventional wisdom, we found that the effect of prevalence on SDM accuracy is positive. This reflects the facts that sample size has a positive effect on accuracy and larger sample sizes are possible for widespread species. It is possible, however, that the omission of an unobserved confounder biased this effect. Previous studies, which reported negative correlations between prevalence and SDM accuracy, conditioned on sample size.

Main conclusions

Our model explicates the causal basis of previously reported correlations between SDM performance and species/data characteristics. It also suggests that niche completeness has similarly large effects on SDM accuracy and variance as sample size. Analysts should consider niche completeness, or proxies thereof, in addition to sample size when deciding whether modelling is worthwhile.     

Oxidative metabolism and protoplast culture

Summary Barley leaf blade protoplasts accumulate malonaldehyde, a product of lipid peroxidation, during culture. In addition, glutathione levels fall after protoplast isolation and the proportion of glutathione in the oxidized state rises. These data indicate oxidative stress after protoplast isolation and during culture. The cause of this phenomenon is revealed by data showing that the activities of enzymes associated with antioxidative processes including glutathione reductase and ascorbate peroxidase decrease after barley protoplast isolation. In contrast, protoplasts isolated from suspension cultured cells of bromegrass and soybean exhibit little evidence for oxidative stress and increased activities of glutathione reductase and ascorbate peroxidase. We suggest that an antioxidative response is associated with mitosis and colony formation from protoplasts, as exhibited by bromegrass and soybean. Conversely, failure of an antioxidative response is associated with low viability and absence of mitosis, as in barley. Increased viability of barley leaf protoplasts cultured on feeder layer cells is correlated with increased glutathione content and higher glutathione reductase activity.     

Soil legacy effects of plants and drought on aboveground insects in native and range-expanding plant communities

Soils contain biotic and abiotic legacies of previous conditions that may influence plant community biomass and associated aboveground biodiversity. However, little is known about the relative strengths and interactions of the various belowground legacies on aboveground plant–insect interactions. We used an outdoor mesocosm experiment to investigate the belowground legacy effects of range-expanding versus native plants, extreme drought and their interactions on plants, aphids and pollinators. We show that plant biomass was influenced more strongly by the previous plant community than by the previous summer drought. Plant communities consisted of four congeneric pairs of natives and range expanders, and their responses were not unanimous. Legacy effects affected the abundance of aphids more strongly than pollinators. We conclude that legacies can be contained as soil ‘memories’ that influence aboveground plant community interactions in the next growing season. These soil-borne ‘memories’ can be altered by climate warming-induced plant range shifts and extreme drought.     

The stimulatory effect of chronic lithium treatment on basal thyrotropin secretion in rats: In vivo antagonism by methylparaben

Chronic treatment of rats with lithium chloride was examined in order to determine its effect on hypothalamic monoamine and metabolite content, basal thyrotropin (TSH) secretion and thyroid function. The hypothalamic concentrations of noradrenaline (NA), dopamine (DA) and its metabolites, dihydroxyphenylacetic acid. (DOPAC) and homovanillic acid (HVA) in the lithium treated rats remained unaltered when compared to control levels. NA turnover and the NA metabolite, 3-methoxy-4-hydroxyphenylglycol (total MHPG), were significantly lower (p<0.01), whereas both serotonin (5-HT) and its metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), were significantly higher (p<0.01 and p<0.02, respectively) in the lithium treated rat hypothalami than in controls. Chronic lithium treatment significantly elevated basal TSH levels (p<0.05). This effect was antagonized by methylp-hydroxybenzoate (methylparaben, p<0.01), which did not itself affect basal TSH levels. Free serum T₃ and T₄ levels were not significantly affected by chronic lithium treatment, although T₄ tended to be slightly lower than control levels. The monoamine changes observed in the hypothalamus of lithium treated rats did not appear to account for the elevated TSH levels observed in these rats since NA activity which is generally regarded as stimulatory was decreased and 5-HT which has an inhibitory effect on TSH secretion, was increased. The elevated TSH levels may have been due to a reduced negative feedback inhibition of TSH release by the mildly reduced circulating T₄ levels caused by chronic lithium treatment. A further possibility is that the pituitary cGMP (and hence TSH) response to TRH may have been enhanced by chronic lithium treatment and methylparaben may have antagonized this effect.