Modulation of carrier-mediated uptake of abscisic acid by methyl jasmonate in Phaseolus coccineus L

The effects of methyl jasmonate and jasmonic acid on uptake of abscisic acid (ABA) by suspension-cultured runner-bean cells and subapical runner-bean root segments have been investigated. Increasing concentrations of methyl jasmonate inhibit ABA uptake by the cultured cells with a K _i of 22±3 M. This is not due to cytoplasmic acidification or to effects on metabolism of ABA, and is not additive with inhibition of radioactive ABA uptake by nonradioactive ABA. Uptake of indol-3-yl acetic acid (IAA) is unaffected by methyl jasmonate. The maximum effect of nonradioactive ABA in inhibiting uptake of radioactive ABA, previously shown to reflect saturation of an ABA carrier, is generally greater than the effect of maximally inhibitory concentrations of methyl jasmonate. Similar results were obtained with root segments, but longer incubation times were necessary to observe inhibitory effects of methyl jasmonate. Demethylation of methyl jasmonate to jasmonic acid does not appear to be required since similar concentrations of jasmonic acid had no observable direct effect on ABA uptake other than that attributable to cytoplasmic acidification. Histidine reagents, a proton ionophore and acidic external pH all affect in parallel the inhibition by methyl jasmonate and nonradioactive ABA of uptake of radioactive ABA by the cultured cells. There is no effect of ABA or nonradioactive methyl jasmonate on uptake of radioactive methyl jasmonate by the cultured cells. It is proposed that methyl jasmonate interacts with the ABA carrier. Various models for this interaction are discussed.Abbreviations ABA abscisic acid - DMO 5,5-dimethyloxazolidine-2,4-dione - IAA indol-3-yl acetic acid     

Mitogen Synergism in low-responding CBA/CaJ mice.

Although neither phytohemagglutinin (PHA) nor concanavalin A (Con A) stimulated blood cultures in vitro from low-responding CBA/CaJ mice effectively, a mixture of PHA and Con A over a range of concentrations stimulated a response from CBA/CaJ mouse blood that was greater than the sum of the responses produced by using PHA or Con A individually. This synergistic effect was expressed as the percentage by which the responses to the PHA and Con A mixture exceeded the sum of the responses to PHA alone and Con A alone. When the mitogen concentrations that gave maximum responses individually were used, the synergistic effect averaged 319% in cultures of blood from low-responding CBA/CaJ mice. Apparently simultaneous exposure to PHA and Con A stimulates DNA synthesis in white blood cells of CBA/CaJ mice that fail to respond to either mitogen alone.     

The SH2 domain containing inositol polyphosphate 5-phosphatase-2: SHIP2

Phosphoinositides are membrane-bound signaling molecules that recruit, activate and localize target effectors to intracellular membranes regulating apoptosis, cell proliferation, insulin signaling and membrane trafficking. The SH2 domain containing inositol polyphosphate 5-phosphatase-2 (SHIP2) hydrolyzes phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) generating phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). Overexpression of SHIP2 inhibits insulin-stimulated phosphoinositide 3-kinase (PI3K) dependent signaling events. Analysis of diabetic human subjects has revealed an association between SHIP2 gene polymorphisms and type 2 diabetes mellitus. Genetic ablation of SHIP2 in mice has generated conflicting results. SHIP2 knockout mice were originally reported to show lethal neonatal hypoglycemia resulting from insulin hypersensitivity, but in addition to inactivating the SHIP2 gene, the Phox2a gene was also inadvertently deleted. Another SHIP2 knockout mouse has now been generated which inactivates the SHIP2 gene but leaves Phox2a intact. These animals show normal insulin and glucose tolerance but are highly resistant to weight gain on high fat diets, exhibiting an obesity-resistant phenotype. Therefore, SHIP2 remains a significant therapeutic target for the treatment of both obesity and type 2 diabetes.     

Identification of a proline-rich inositol polyphosphate 5-phosphatase (PIPP)•collapsin response mediator protein 2 (CRMP2) complex that regulates neurite elongation

Neuron polarization is essential for the formation of one axon and multiple dendrites, establishing the neuronal circuitry. Phosphoinositide 3-kinase (PI3K) signaling promotes axon selection and elongation. Here we report in hippocampal neurons siRNA knockdown of the proline-rich inositol polyphosphate 5-phosphatase (PIPP), which degrades PI3K-generated PtdIns(3,4,5)P(3), results in multiple hyperelongated axons consistent with a polarization defect. We identify collapsin response mediator protein 2 (CRMP2), which regulates axon selection by promoting WAVE1 delivery via Kinesin-1 motors to the axon growth cone, as a PIPP-interacting protein by Y2H screening, direct binding studies, and coimmunoprecipitation of an endogenous PIPP, CRMP2, and Kinesin-1 complex from brain lysates. The C-terminal growth cone-targeting domain of PIPP facilitates its interaction with CRMP2. PIPP growth cone localization is CRMP2-dependent. PIPP knockdown in PC12 cells promotes neurite elongation, WAVE1 and Kinesin-1 growth cone localization, whereas knockdown of CRMP2 exhibits the opposite phenotype, with shorter neurites and decreased WAVE1/Kinesin-1 at the growth cone. In contrast, CRMP2 overexpression promotes neurite elongation, a phenotype rescued by full-length PIPP, or expression of the CRMP2-binding PIPP domain. Therefore this study identifies PIPP and CRMP2 exert opposing roles in promoting axon selection and neurite elongation and the complex between these proteins serves to regulate the localization of effectors that promote neurite extension.     

Genetically diverse mice are novel and valuable models of age-associated susceptibility to <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background

Tuberculosis, the disease due to Mycobacterium tuberculosis, is an important cause of morbidity and mortality in the elderly. Use of mouse models may accelerate insight into the disease and tests of therapies since mice age thirty times faster than humans. However, the majority of TB research relies on inbred mouse strains, and these results might not extrapolate well to the genetically diverse human population. We report here the first tests of M. tuberculosis infection in genetically heterogeneous aging mice, testing if old mice benefit from rapamycin.

Findings

We find that genetically diverse aging mice are much more susceptible than young mice to M. tuberculosis, as are aging human beings. We also find that rapamycin boosts immune responses during primary infection but fails to increase survival.

Conclusions

Genetically diverse mouse models provide a valuable resource to study how age influences responses and susceptibility to pathogens and to test interventions. Additionally, surrogate markers such as immune measures may not predict whether interventions improve survival.

     

Rapamycin‐mediated lifespan increase in mice is dose and sex dependent and metabolically distinct from dietary restriction

Rapamycin, an inhibitor of mTOR kinase, increased median lifespan of genetically heterogeneous mice by 23% (males) to 26% (females) when tested at a dose threefold higher than that used in our previous studies; maximal longevity was also increased in both sexes. Rapamycin increased lifespan more in females than in males at each dose evaluated, perhaps reflecting sexual dimorphism in blood levels of this drug. Some of the endocrine and metabolic changes seen in diet‐restricted mice are not seen in mice exposed to rapamycin, and the pattern of expression of hepatic genes involved in xenobiotic metabolism is also quite distinct in rapamycin‐treated and diet‐restricted mice, suggesting that these two interventions for extending mouse lifespan differ in many respects.     

Longer lifespan in male mice treated with a weakly estrogenic agonist,an antioxidant,an α‐glucosidase inhibitor or a Nrf2‐inducer

The National Institute on Aging Interventions Testing Program (ITP) evaluates agents hypothesized to increase healthy lifespan in genetically heterogeneous mice. Each compound is tested in parallel at three sites, and all results are published. We report the effects of lifelong treatment of mice with four agents not previously tested: Protandim, fish oil, ursodeoxycholic acid (UDCA) and metformin – the latter with and without rapamycin, and two drugs previously examined: 17‐α‐estradiol and nordihydroguaiaretic acid (NDGA), at doses greater and less than used previously. 17‐α‐estradiol at a threefold higher dose robustly extended both median and maximal lifespan, but still only in males. The male‐specific extension of median lifespan by NDGA was replicated at the original dose, and using doses threefold lower and higher. The effects of NDGA were dose dependent and male specific but without an effect on maximal lifespan. Protandim, a mixture of botanical extracts that activate Nrf2, extended median lifespan in males only. Metformin alone, at a dose of 0.1% in the diet, did not significantly extend lifespan. Metformin (0.1%) combined with rapamycin (14 ppm) robustly extended lifespan, suggestive of an added benefit, based on historical comparison with earlier studies of rapamycin given alone. The α‐glucosidase inhibitor, acarbose, at a concentration previously tested (1000 ppm), significantly increased median longevity in males and 90th percentile lifespan in both sexes, even when treatment was started at 16 months. Neither fish oil nor UDCA extended lifespan. These results underscore the reproducibility of ITP longevity studies and illustrate the importance of identifying optimal doses in lifespan studies.     

An ERP Analysis of Recognition and Categorization Decisions in a Prototype-Distortion Task

Background

Theories of categorization make different predictions about the underlying processes used to represent categories. Episodic theories suggest that categories are represented in memory by storing previously encountered exemplars in memory. Prototype theories suggest that categories are represented in the form of a prototype independently of memory. A number of studies that show dissociations between categorization and recognition are often cited as evidence for the prototype account. These dissociations have compared recognition judgements made to one set of items to categorization judgements to a different set of items making a clear interpretation difficult. Instead of using different stimuli for different tests this experiment compares the processes by which participants make decisions about category membership in a prototype-distortion task and with recognition decisions about the same set of stimuli by examining the Event Related Potentials (ERPs) associated with them.

Method

Sixty-three participants were asked to make categorization or recognition decisions about stimuli that either formed an artificial category or that were category non-members. We examined the ERP components associated with both kinds of decision for pre-exposed and control participants.

Conclusion

In contrast to studies using different items we observed no behavioural differences between the two kinds of decision; participants were equally able to distinguish category members from non-members, regardless of whether they were performing a recognition or categorisation judgement. Interestingly, this did not interact with prior-exposure. However, the ERP data demonstrated that the early visual evoked response that discriminated category members from non-members was modulated by which judgement participants performed and whether they had been pre-exposed to category members. We conclude from this that any differences between categorization and recognition reflect differences in the information that participants focus on in the stimuli to make the judgements at test, rather than any differences in encoding or process.     

The inositol polyphosphate 5-phosphatase, PIPP, Is a novel regulator of phosphoinositide 3-kinase-dependent neurite elongation

The spatial activation of phosphoinositide 3-kinase (PI3-kinase) signaling at the axon growth cone generates phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P₃), which localizes and facilitates Akt activation and stimulates GSK-3β inactivation, promoting microtubule polymerization and axon elongation. However, the molecular mechanisms that govern the spatial down-regulation of PtdIns(3,4,5)P₃ signaling at the growth cone remain undetermined. The inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P₂) and/or PtdIns(3,4,5)P₃. We demonstrate here that PIPP, an uncharacterized 5-phosphatase, hydrolyzes PtdIns(3,4,5)P₃ forming PtdIns(3,4)P₂, decreasing Ser473-Akt phosphorylation. PIPP is expressed in PC12 cells, localizing to the plasma membrane of undifferentiated cells and the neurite shaft and growth cone of NGF-differentiated neurites. Overexpression of wild-type, but not catalytically inactive PIPP, in PC12 cells inhibited neurite elongation. Targeted depletion of PIPP using RNA interference (RNAi) resulted in enhanced neurite differentiation, associated with neurite hyperelongation. Inhibition of PI3-kinase activity prevented neurite hyperelongation in PIPP-deficient cells. PIPP targeted-depletion resulted in increased phospho-Ser473-Akt and phospho-Ser9-GSK-3β, specifically at the neurite growth cone, and accumulation of PtdIns(3,4,5)P₃ at this site, associated with enhanced microtubule polymerization in the neurite shaft. PIPP therefore inhibits PI3-kinase-dependent neurite elongation in PC12 cells, via regulation of the spatial distribution of phospho-Ser473-Akt and phospho-Ser9-GSK-3β signaling.     

Numbers and functions of transplantable primitive immunohematopoietic stem cells. Effects of age

This report introduces a new method in immunology, a use of the binomial formula with covariance to estimate numbers and proliferative patterns of the most primitive lymphoid precursors. We studied the primitive stem cells (PSC) from which most circulating lymphocytes and erythrocytes were descended during 300 to 400 days in recipients of genetically distinguishable marrow mixtures in competitive repopulation. Equivalent PSC concentrations (Eq. PSC Conc. or Conc.) were estimated, with the notion of common PSCs contributing equally in lymphoid and myeloid compartments. Similar estimation was done for common PSCs from which lymphocytes (and erythrocytes) drawn at successive sampling times about 100 days apart were descended. The percentages of lymphocyte and erythrocyte types, P1 and Pe, measured in each recipient were closely correlated, especially after 6 months and later. Close correlations were also found in cells sampled at successive one hundred day intervals, especially after the first. Apparently a few PSCs or their direct descendents produced most of the blood lymphocytes and E, and this production continued for many months. Concentrations of these PSCs (Equivalent PSC concentrations) were about one per 10(5) marrow cells from young donors. This is much lower than previous estimates, probably because our methods focus only on the most interesting precursors, those from which most of the circulating cells were descended. Equivalent PSC concentrations were about two-fold higher in old donors; old marrow produced correspondingly higher P1 and Pe values, but these declined with time. There were also small increases with time in the P1 and Pe values with young donors. To explain the temporal trends, we suggest that excess concentrations of precursors less primitive than PSC are present in old marrow, and their contribution to the differentiated cell population gradually declines. Possibly such precursors, as well as true PSC, proliferate in old donors to compensate for deficiencies that develop with age.