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Biological systems are acknowledged to be robust to perturbations but a rigorous understanding of this has been elusive. In a mathematical model, perturbations often exert their effect through parameters, so sizes and shapes of parametric regions offer an integrated global estimate of robustness. Here, we explore this "parameter geography" for bistability in post-translational modification (PTM) systems. We use the previously developed "linear framework" for timescale separation to describe the steady-states of a two-site PTM system as the solutions of two polynomial equations in two variables, with eight non-dimensional parameters. Importantly, this approach allows us to accommodate enzyme mechanisms of arbitrary complexity beyond the conventional Michaelis-Menten scheme, which unrealistically forbids product rebinding. We further use the numerical algebraic geometry tools Bertini, Paramotopy, and alphaCertified to statistically assess the solutions to these equations at ∼109 parameter points in total. Subject to sampling limitations, we find no bistability when substrate amount is below a threshold relative to enzyme amounts. As substrate increases, the bistable region acquires 8-dimensional volume which increases in an apparently monotonic and sigmoidal manner towards saturation. The region remains connected but not convex, albeit with a high visibility ratio. Surprisingly, the saturating bistable region occupies a much smaller proportion of the sampling domain under mechanistic assumptions more realistic than the Michaelis-Menten scheme. We find that bistability is compromised by product rebinding and that unrealistic assumptions on enzyme mechanisms have obscured its parametric rarity. The apparent monotonic increase in volume of the bistable region remains perplexing because the region itself does not grow monotonically: parameter points can move back and forth between monostability and bistability. We suggest mathematical conjectures and questions arising from these findings. Advances in theory and software now permit insights into parameter geography to be uncovered by high-dimensional, data-centric analysis., Competing Interests: The authors have declared that no competing interests exist.

A common problem when analyzing models, such as mathematical modeling of a biological process, is to determine if the unknown parameters of the model can be determined from given input-output data. Identifiable models are models such that the unknown parameters can be determined to have a finite number of values given input-output data. The total number of such values over the complex numbers is called the identifiability degree of the model. Unidentifiable models are models such that the unknown parameters can have an infinite number of values given input-output data. For unidentifiable models, a set of identifiable functions of the parameters are sought so that the model can be reparametrized in terms of these functions yielding an identifiable model. In this work, we use numerical algebraic geometry to determine if a model given by polynomial or rational ordinary differential equations is identifiable or unidentifiable. For identifiable models, we present a novel approach to compute the identifiability degree. For unidentifiable models, we present a novel numerical differential algebra technique aimed at computing a set of algebraically independent identifiable functions. Several examples are used to demonstrate the new techniques., Competing Interests: The authors have declared that no competing interests exist.

Researchers working with mathematical models are often confronted by the related problems of parameter estimation, model validation and model selection. These are all optimization problems, well known to be challenging due to nonlinearity, non-convexity and multiple local optima. Furthermore, the challenges are compounded when only partial data are available. Here, we consider polynomial models (e.g. mass-action chemical reaction networks at steady state) and describe a framework for their analysis based on optimization using numerical algebraic geometry. Specifically, we use probability-one polynomial homotopy continuation methods to compute all critical points of the objective function, then filter to recover the global optima. Our approach exploits the geometrical structures relating models and data, and we demonstrate its utility on examples from cell signalling, synthetic biology and epidemiology., (© 2016 The Author(s).)

Aims: The authors' aim was to conduct a proof-of-principle study to test whether c-Jun N-terminal kinase (JNK) phosphorylation and Noxa induction occur in peripheral blood chronic lymphocytic leukaemia (CLL) cells in patients receiving a vincristine infusion., Methods: Patients with CLL received 2 mg vincristine by a 5-min intravenous infusion. Blood samples were collected at baseline and up to 6 h after the vincristine infusion, and assayed for JNK activation, Noxa induction and vincristine plasma concentrations., Results: Ex vivo treated peripheral CLL cells activated JNK in response to 10-100 nM vincristine in 6 h. Noxa protein expression, while variable, was also observed over this time frame. In CLL patients, vincristine infusion led to rapid (<1 h) JNK phosphorylation in peripheral blood CLL cells which was sustained for at least 4-6 h after the vincristine infusion. Noxa protein expression was not observed in response to vincristine infusion., Conclusions: This study confirmed that vincristine can activate JNK but not induce Noxa in CLL cells in vivo. The results suggest that novel JNK-dependent drug combinations with vincristine warrant further investigation., (© 2015 The British Pharmacological Society.)

Background: The mulga snake (Pseudechis australis) is the largest terrestrial venomous snake in Australia. It is capable of inflicting severe and occasionally fatal envenoming, but there have been few studies of P. australis bites., Objectives: To highlight and reinforce the main features of P. australis envenoming and to provide a clearer picture of the epidemiology of bites from this species., Methods: Selected case records kept by the Toxinology Dept. (Women's and Children's Hospital, Adelaide, Australia) were reviewed retrospectively to determine definite P. australis bites., Inclusion Criteria: definite cases where the snake was identified by a competent person and/or lab specimens (bite site/urine) tested positive for "black snake" using CSL snake venom detection kit in a locality within the known range of P. australis, but without sympatry with other Pseudechis spp., Exclusion Criteria: where the snake could not be clearly identified under criteria above. Epidemiological and clinical information was recorded and analysed for the definite cases., Results: A total of 27 cases were identified as definite P. australis bites; there were no fatalities. The median age was 35.5 years (IQR 51-23) and 80% of bites occurred in males. More bites occurred in the warmer months (Dec-March) and in those handling/interfering with snakes. Seven people were bitten whilst asleep at night. 21/27 patients developed systemic envenoming (based on signs, symptoms and laboratory results) and 17 cases received antivenom. Local bite site pain (18) and swelling (17) were common as were non-specific generalised symptoms such as nausea, vomiting and headache. Myotoxicity (11) and anticoagulant coagulopathy (10) occurred frequently; haemolysis was seen in fewer cases (3). Two patients developed local tissue injury around the bite site requiring further treatment., Conclusions: This study confirms previous reports about P. australis bites with respect to high rates of envenoming, commonly associated with pain and swelling and systemic effects of rhabdomyolysis and anticoagulant coagulopathy. Systemic envenoming, even severe cases, responds well to antivenom therapy. Compared to other Australian snakes, a high proportion of bites occur in people asleep at night. Medically significant local tissue injury around the bite site may occur and may be associated with inappropriate first-aid, particularly the vascular occlusive type., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

1. Isoeugenol (IEG) has been tested for toxicity and carcinogenicity due to high potential for human exposure and the structural resemblance to known carcinogenic allylbenzenes. In order to support the interpretation of toxicity and carcinogenecity study outcomes, a toxicokinetic study was performed in which both sexes of F344 rats and B6C3F1 mice were given IEG as a single intravenous (IV) or gavage administration. 2. Following IV administration, IEG was rapidly eliminated from systemic circulation in both species and sexes. Gavage administration revealed a rapid absorption of IEG with tmax values ≤20 min for both species and sexes. In rats, AUC increased in a greater than dose-proportional manner and Clapp values decreased with increasing dose in both sexes suggesting saturation of IEG metabolism. On the other hand, Clapp values in male mice increased with increasing dose suggesting induction of IEG metabolism although this was not evident in the females. 3. Absolute bioavailability was greater in female rats (19%) than male rats (10%) (p < 0.0001), but was not different between the sexes for mice (28% males; 31% females) (p = 0.2437). The collective toxicokinetic data supported that low bioavailability following administration of IEG was the result of extensive first-pass metabolism.

This review summarizes the current state of scientific understanding of the apoptosis pathway, with a focus on the proteins involved in the pathway, their interactions and functions. This forms the rationale for detailing the preclinical and clinical pharmacology of drugs that modulate the pivotal proteins in this pathway, with emphasis on drugs that are furthest advanced in clinical development as anticancer agents. There is a focus on describing drugs that modulate three of the most promising targets in the apoptosis pathway, namely antibodies that bind and activate the death receptors, small molecules that inhibit the anti-apoptotic Bcl-2 family proteins, and small molecules and antisense oligonucleotides that inactivate the inhibitors of apoptosis, all of which drive the equilibrium of the apoptotic pathway towards apoptosis. These structurally different yet functionally related groups of drugs represent a promising novel approach to anticancer therapeutics whether used as monotherapy or in combination with either classical cytotoxic or other molecularly targeted anticancer agents., (© 2013 The British Pharmacological Society.)

Proteins of the BCL2 family provide a survival mechanism in many human malignancies, including chronic lymphocytic leukemia (CLL). The BCL2 inhibitor ABT-263 (navitoclax) is active in clinical trials for lymphoid malignancies, yet resistance is expected on the basis of preclinical models. We recently showed that vinblastine can dramatically sensitize several leukemia cell lines to ABT-737 (the experimental congener of ABT-263). The goal of these experiments was to determine the impact of vinblastine on ABT-737 sensitivity in CLL cells isolated from peripheral blood and to define the underlying mechanism. Freshly isolated CLL cells from 35 patients, as well as normal lymphocytes and platelets, were incubated with various microtubule-disrupting agents plus ABT-737 to assess sensitivity to the single agents and the combination. ABT-737 and vinblastine displayed a range of sensitivity as single agents, and vinblastine markedly sensitized all CLL samples to ABT-737 within six hours. Vinblastine potently induced the proapoptotic protein PMAIP1 (NOXA) in both time- and dose-dependent manner and this was required for the observed apoptosis. Combretastatin A4, which dissociates microtubules by binding to a different site, had the same effect, confirming that interaction of these agents with microtubules is the initial target. Similarly, vincristine and vinorelbine induced NOXA and enhanced CLL sensitivity to ABT-737. Furthermore, vinblastine plus ABT-737 overcame stroma-mediated resistance to ABT-737 alone. Apoptosis was induced with clinically achievable concentrations with no additional toxicity to normal lymphocytes or platelets. These results suggest that vinca alkaloids may improve the clinical efficacy of ABT-263 in patients with CLL.

1. Methyleugenol (MEG) has been used as a flavouring agent in food, as a fragrance in cosmetic products, and as an insect attractant. MEG was carcinogenic in both rats and mice following gavage administration. In this study we investigated plasma toxicokinetics of MEG in F344 rats and B6C3F₁ mice of both sexes following single gavage (37, 75, or 150 mg/kg) and intravenous (IV) (37 mg/kg) administration. 2. Following IV administration, MEG was rapidly distributed and cleared from the systemic circulation in both species and sexes. Absorption of MEG was rapid following gavage administration with secondary peaks in the plasma MEG concentration-versus-time profiles. C(max) and AUC(T) increased and the clearance decreased greater than proportional to the dose in rats and mice of both sexes. In general, rats had higher internal exposure to MEG than mice. 3. The results for AUC(T) and clearance suggest that perhaps the metabolism of MEG is saturated at higher doses tested in this study. Absolute bioavailability following gavage administration of 37 mg/kg was low in both rats (~4%) and mice (7-9%) of both sexes indicating extensive first-pass metabolism. There was no sex difference in plasma toxicokinetics of MEG following gavage administration both in rats and mice.

Naturally occurring nitroalkene fatty acids (NAs) derived from oleic (NO(2)-OA) and linoleic (NO(2)-LA) acids mediate a variety of cellular responses. We examined the signaling pathways involved in NA activation of Nrf2/ARE-dependent versus PPARγ/PPRE-dependent transcription in human MCF7 breast cancer cells. Additionally, we compared the relative potencies of NO(2)-OA and NO(2)-LA in activating these two transcriptional programs. Here it is demonstrated that, in addition to the direct adduct formation of NA with the Nrf2 inhibitory protein, Keap1, shown by others, NA activation of Nrf2/ARE-mediated transcription results from increased nuclear Nrf2 levels and depends upon activation of the PI3K/AKT and PKC, but not ERK and JNK MAPK, signaling pathways. Examination of the relationship between NA stimulation of the Nrf2/ARE versus PPARγ/PPRE transcriptional programs revealed concentration-dependent activation of distinct signaling pathways that were readily distinguished by selective attenuation of Nrf2/ARE-dependent, but not PPARγ-dependent, transcription by inhibitors of PI3K and PKC. Moreover, measurable, statistically significant activation of PPARγ/PPRE-dependent transcription occurred at nanomolar concentrations of NAs-the 12-NO(2) isomer of NO(2)-LA showing the most potent activity-whereas significant activation of Nrf2/ARE-dependent transcription occurred at much higher NA concentrations (≥3 μM) with the NO(2)-OA isomers the most potent. These findings have implications for the physiological roles of NAs, suggesting that, at concentrations likely to be encountered in vivo, their direct activation of PPARγ transcription will dominate over their electrophilic activation of Nrf2 antioxidant/protective responses.

The efficacy of many chemotherapeutic agents can be attenuated by expression of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Mcl-1. Flavopiridol and dinaciclib are cyclin-dependent kinase 7 and 9 inhibitors that transcriptionally inhibit expression of Mcl-1. We have investigated the ability of flavopiridol and dinaciclib to sensitize a panel of leukemia cell lines to vinblastine and paclitaxel. Both drugs acutely sensitized most of the leukemia lines to vinblastine, with 100% apoptosis in 4 h. Furthermore, dinaciclib sensitized freshly isolated chronic lymphocytic leukemia cells to vinblastine. This rapid induction of apoptosis was attributed to vinblastine-mediated activation of JNK because (a) flavopiridol and dinaciclib failed to induce apoptosis when combined with non-JNK activating concentrations of vinblastine; (b) JNK inhibitors suppressed JNK activity and prevented apoptosis; (c) flavopiridol did not potentiate apoptosis induced by paclitaxel which does not activate JNK in these cells; and (d) Jurkat cells failed to activate JNK in response to vinblastine and were not sensitive to combinations of vinblastine and flavopiridol or dinaciclib. The rapid induction of apoptosis by this combination in multiple cell systems but not in normal lymphocytes provides justification for performing a clinical trial to assess the efficacy in patients.

BH3 mimetics are small molecules designed or discovered to mimic the binding of BH3-only proteins to the hydrophobic groove of antiapoptotic BCL2 proteins. The selectivity of these molecules for BCL2, BCL-X(L), or MCL1 has been established in vitro; whether they inhibit these proteins in cells has not been rigorously investigated. In this study, we used a panel of leukemia cell lines to assess the ability of seven putative BH3 mimetics to inhibit antiapoptotic proteins in a cell-based system. We show that ABT-737 is the only BH3 mimetic that inhibits BCL2 as assessed by displacement of BAD and BIM from BCL2. The other six BH3 mimetics activate the endoplasmic reticulum stress response inducing ATF4, ATF3, and NOXA, which can then bind to and inhibit MCL1. In most cancer cells, inhibition of one antiapoptotic protein does not acutely induce apoptosis. However, by combining two BH3 mimetics, one that inhibits BCL2 and one that induces NOXA, apoptosis is induced within 6 h in a BAX/BAK-dependent manner. Because MCL1 is a major mechanism of resistance to ABT-737, these results suggest a novel strategy to overcome this resistance. Our findings highlight a novel signaling pathway through which many BH3 mimetics inhibit MCL1 and suggest the potential use of these agents as adjuvants in combination with various chemotherapy strategies.

Although significant advances have been made in the study of the molecular mechanisms controlling brain aging, post-transcriptional gene regulation in normal brain aging has yet to be explored. Our lab recently reported that predominant microRNA up-regulation is observed in liver during aging, with key microRNAs predicted to target detoxification genes. Here we examine the role of microRNA regulation in brain during the normal aging process. MicroRNA microarrays and global proteomic profiling were used to compare the brain tissues of 10-, 18-, 24-, and 33-month-old mice. Our results suggest that: (1) like liver, during aging the brain exhibits predominant microRNA up-regulation, and this trend starts in mid-life; (2) of the 70 up-regulated microRNAs, 27 are predicted to target 10 genes of mitochondrial complexes III, IV, and F0F1-ATPase, which exhibit inversely correlated expression; (3) mice of extreme longevity (33-month old) exhibit fewer microRNA expression changes from 10-month-old levels than do old adult mice (24-month old). We found unique de-regulated microRNAs shared between aging brain and aging liver, as well as brain- vs. liver-specific microRNAs during normal aging., (Published by Elsevier Inc.)

Chemotherapeutic agents modify intracellular signaling that culminates in the inhibition of Bcl-2 family members and initiates apoptosis. Inhibition of the extracellular signal-regulated kinase by PD98059 dramatically accelerates vinblastine-mediated apoptosis in ML-1 leukemia with cells dying in 4 hours from all phases of the cell cycle. Inhibition of protein synthesis by cycloheximide also markedly accelerated vinblastine-induced apoptosis, showing that the proteins required for this acute apoptosis are constitutively expressed. Vinblastine induced the rapid induction of Mcl-1 that was inhibited by PD98059 and cycloheximide. No change in Bcl-2 or Bcl-X was observed. We hypothesize that ML-1 cells use Mcl-1 for protection from the rapid vinblastine-induced apoptosis. This was confirmed by targeting Mcl-1 with short hairpin RNA. We also investigated the response of 13 other leukemia and lymphoma cell lines and cells from seven chronic lymphocytic leukemia patients. Four cell lines and all chronic lymphocytic leukemia cells were killed in 6 hours by vinblastine alone. Two additional cell lines were sensitized to vinblastine by PD98059, which suppressed Mcl-1. This acute apoptosis either alone or in combination with PD98059 required vinblastine-mediated activation of c-Jun-NH(2)-terminal kinase. PD98059 did not suppress Mcl-1 in other cell lines whereas sorafenib did, but this did not sensitize the cells to vinblastine, suggesting that the acute apoptosis varies depending on which Bcl-2 protein mediates protection. Most of the cell lines were sensitized to vinblastine by cycloheximide, suggesting that inhibition of a short-lived protein in addition to Mcl-1 can acutely sensitize cells. These results suggest several clinical strategies that might provide an effective therapy for selected patients. Mol Cancer Ther; 9(4); 791-802. (c)2010 AACR.

The Ames dwarf mouse is well known for its remarkable propensity to delay the onset of aging. Although significant advances have been made demonstrating that this aging phenotype results primarily from an endocrine imbalance, the post-transcriptional regulation of gene expression and its impact on longevity remains to be explored. Towards this end, we present the first comprehensive study by microRNA (miRNA) microarray screening to identify dwarf-specific lead miRNAs, and investigate their roles as pivotal molecular regulators directing the long-lived phenotype. Mapping the signature miRNAs to the inversely expressed putative target genes, followed by in situ immunohistochemical staining and in vitro correlation assays, reveals that dwarf mice post-transcriptionally regulate key proteins of intermediate metabolism, most importantly the biosynthetic pathway involving ornithine decarboxylase and spermidine synthase. Functional assays using 3'-untranslated region reporter constructs in co-transfection experiments confirm that miRNA-27a indeed suppresses the expression of both of these proteins, marking them as probable targets of this miRNA in vivo. Moreover, the putative repressed action of this miRNA on ornithine decarboxylase is identified in dwarf mouse liver as early as 2 months of age. Taken together, our results show that among the altered aspects of intermediate metabolism detected in the dwarf mouse liver--glutathione metabolism, the urea cycle and polyamine biosynthesis--miRNA-27a is a key post-transcriptional control. Furthermore, compared to its normal siblings, the dwarf mouse exhibits a head start in regulating these pathways to control their normality, which may ultimately contribute to its extended health-span and longevity.

The facultative anaerobe Shewanella oneidensis MR-1 respires a variety of anaerobic electron acceptors, including insoluble Fe(III) oxides. S. oneidensis employs a number of novel strategies for respiration of insoluble Fe(III) oxides, including localization of respiratory proteins to the cell outer membrane (OM). The molecular mechanism by which S. oneidensis adheres to and respires Fe(III) oxides, however, remains poorly understood. In the present study, whole cell fractionation and MALDI-TOF-MS/MS techniques were combined to identify a serine protease (SO3800) associated with the S. oneidensis OM. SO3800 contained predicted structural motifs similar to cell surface-associated serine proteases that function as bacterial adhesins in other gram-negative bacteria. The gene encoding SO3800 was deleted from the S. oneidensis genome, and the resulting mutant strain (DeltaSO3800) was tested for its ability to adhere to and respire Fe(III) oxides. DeltaSO3800 was severely impaired in its ability to adhere to Fe(III) oxides, yet retained wild-type Fe(III) respiratory capability. Laser Doppler velocimetry and cryoetch high-resolution SEM experiments indicated that DeltaSO3800 displayed a lower cell surface charge and higher amount of surface-associated exopolysaccharides. Results of this study indicate that S. oneidensis may respire insoluble Fe(III) oxides at a distance, negating the requirement for attachment prior to electron transfer.

As the molecular mechanisms associated with aging become more understood, it is apparent that the normal processes involved in the development and metabolism of an organism are subject to changes that upset its crucial homeostatic balance, which in turn sets in motion the weakening and disease-prone process of senescence. This imbalance is the result of a variety of effectors, such as environmental insults, endogenous toxins, and genetic mishaps. In addition, it is highly probable that posttranscriptional regulatory events play a large role in the changes associated with aging. The emerging knowledge of posttranscriptional regulation is redefining our understanding of the complexities of cellular systems biology and genetics. The implications of the impact that small regulatory RNAs have on the many facets of developmental and molecular biology should be included as part of our current understanding of the biochemistry involved in these processes. These molecular regulators-along with other epigenetic events-restrict the flow of genetic expression, thus affording the cell an adjustable and tempered homeostatic balance control. Recent findings in the fields of organismal development, cancer, and aging indicate that small noncoding RNA plays a greater role than previously believed in orchestrating the changes associated with these processes. Furthermore, any misappropriations of these regulatory resources could lead to age-related diseases, and are therefore promising targets for prophylactics and therapeutics to combat maladies associated with aging. Here we report a brief overview of noncoding RNA as well as the potential roles of microRNAs in biochemical equilibriums where imbalance contributes to the many phenotypes of aging.

The naturally occurring nitroalkenes, nitrolinoleic (NO(2)-LA) and nitrooleic (NO(2)-OA) acids, are among the most potent endogenous ligand activators of PPARgamma-dependent transcription. In order to understand mechanisms that regulate cellular response to these nitroalkenes, we previously demonstrated that glutathione conjugation of NO(2)-LA and MRP1-mediated efflux of the conjugates were associated with significant attenuation of PPARgamma activation by this nitroalkene [(2006) Biochemistry 45, 7889-7896]. Here we show that NO(2)-OA activation of PPARgamma is similarly affected by nonenzymatic conjugation and MRP1-mediated efflux. Moreover, the roles of glutathione S-transferases (GSTs) in the glutathione conjugation and bioactivities of NO(2)-LA and NO(2)-OA were investigated. While none of the GST isozymes tested (GSTA1-1, A4-4, M1a-1a, and P1a-1a) enhanced the rate of glutathione conjugation, expression of GSTA1-1, M1a-1a, or P1a-1a in MCF7 cells significantly reduced the magnitude of PPARgamma-dependent reporter gene transcription in response to NO(2)-LA and NO(2)-OA treatment, with GSTP1a-1a expression mediating the most potent inhibition of PPARgamma. Although these GSTs failed to catalyze nitroalkene conjugation with glutathione, the nitroalkenes were found to associate avidly with all four GST isozymes as indicated by their ability to inhibit GST activity with K(i)'s in the nanomolar range. Treatment of purified GSTP1a-1a with excess NO(2)-LA and NO(2)-OA resulted in the formation of covalent adducts between GSTP1a monomers and nitroalkenes, although separate experiments indicated that such covalent bond formation was not necessary for avid GST-nitroalkene interactions. These results suggest that GSTs can inhibit the activation of transcription by nitroalkenes via noncatalytic sequestration of these ligands, and their glutathione conjugates, away from their nuclear target, PPARgamma.

MicroRNAs are a major category among the noncoding RNA fraction that negatively regulate gene expression at the post-transcriptional level, by either degrading the target messages or inhibiting their translation. MicroRNAs may be referred to as 'dimmer switches' of gene expression, because of their ability to repress gene expression without completely silencing it. Whether through up-regulating specific groups of microRNAs to suppress unwanted gene expressions, or by down-regulating other microRNAs whose target genes' expression is necessary for cellular function, such as cell proliferation, apoptosis, or differentiation, these regulatory RNAs play pivotal roles in a wide variety of cellular processes. The equilibrium between these two groups of microRNA expressions largely determines the function of particular cell types. Our recent results with several model systems show that upon aging, there is a trend of up-regulation of microRNA expression, with concomitant inverse down-regulation of target genes. This review addresses molecular mechanisms that may provide the underlying control for this up-regulating trend, focusing on activation by various microRNAs' own promoters, through binding with pivotal transcription factors, stress response, methylation of clustered DNA domains, etc. Thus, epigenomic control of aging may be due in part to heightened promoter activation of unwanted microRNA expressions, which in turn down-regulate their target gene products. Overriding and dampening the activation of these noncoding RNAs may prove to be a new frontier for future research, to delay aging and extend healthy life-span.

Bacterial exopolymers perform various roles, including acting as a carbon sink, a protective layer against desiccation or antimicrobial agents, or a structural matrix in biofilms. Despite such varied roles, little is known about the heterogeneity of bacterial exopolymer production under varying growth conditions. Here we describe experiments designed to characterize the quantity and quality of exopolymers produced by two commonly studied members of the widely distributed genus Shewanella. Electrokinetic, spectroscopic, and electron microscopic techniques were employed to demonstrate that cell surfaces of Shewanella oneidensis MR-1 (electrophoretic softness, lambda(-1), range from 0.4 to 2.6 nm) are associated with less extracellular polymeric material than surfaces of Shewanella putrefaciens 200R (lambda(-1) range from 1.6 to 3.0 nm). Both species exhibit similar responses to changes in electron acceptor with nitrate- and fumarate-grown cells producing relatively little exopolymer compared to trimethylamine N-oxide (TMAO)-grown cells. In S. oneidensis, the increase in exopolymers has no apparent effect upon cell-surface fixed charge density (-7.7 to -8.7 mM), but for S. putrefaciens a significant drop in fixed charge density is observed between fumarate/nitrate-grown cells (-43 mM) and TMAO-grown cells (-20.8 mM). For both species, exopolymers produced during growth on TMAO have significant amide functionality, increasing from approximately 20-25% of C-containing moieties in nitrate-grown cells to over 30% for TMAO-grown cells (determined from X-ray photoelectron spectroscopy). The increased exopolymer layer associated with TMAO-grown cells appears as a continuous, convoluted layer covering the entire cell surface when viewed by low-temperature, high-resolution scanning electron microscopy. Such significant changes in cell-surface architecture, dependent upon the electron acceptor used for growth, are likely to influence a variety of cell interactions, including aggregation and attachment to surfaces, and the binding of aqueous metal species.

Recent data has shown that nitrolinoleic acid (LNO(2)), an electrophilic derivative of linoleic acid, has several important bioactivities including antiinflammatory, antiplatelet, vasorelaxation, and-as a novel potent ligand of PPARgamma-transcription regulating activities. Moreover, LNO(2) is formed in abundance in vivo at levels sufficient to mediate these bioactivities. In order to investigate the role of glutathione conjugation and MRP1-mediated efflux in the regulation of PPARgamma-dependent LNO(2) signaling, regioisomers of LNO(2) were synthesized and characterized. Analysis by 1D and 2D (1)H and (13)C NMR revealed that the LNO(2) preparation consisted of four, rather than two, nitrated regioisomers in approximately equal abundance. At physiologic pH and intracellular glutathione levels, LNO(2) was rapidly and quantitatively converted to glutathione conjugates (LNO(2)-SG) via Michael addition. MRP1 mediated efficient ATP-dependent transport of LNO(2)-SG. Using a PPRE-containing reporter gene transiently transfected into MRP-poor MCF7/WT cells, we verified that the LNO(2) mixture was a potent activator of PPARgamma-dependent transcription. However, expression of MRP1 in the stably transduced MCF7 derivative, MCF7/MRP1-10, resulted in strong inhibition of LNO(2)-induced transcription activation. Taken together, these results suggest that glutathione conjugation and MRP1-mediated conjugate transport can attenuate LNO(2) bioactivity and thereby play important roles in the regulation of cellular signaling by LNO(2).

Female F344 rats and B6C3F1 mice were exposed to vanadium pentoxide (V2O5) at concentrations of 0, 0.5, 1, or 2 mg/m3 (rats) and 0, 1, 2, or 4 mg/m3 (mice) for 6 h/day, 5 days/week (for up to 18 months), by whole-body inhalation. Lung weights and lung burdens of vanadium were determined for exposed animals after 1, 5, and 12 days and after 1, 2, 6, 12, and 18 months of V2O5 exposure. Blood vanadium concentrations were determined at 1, 2, 6, 12, and 18 months for all animals including controls. A model that assumed a first-order deposition rate and a first-order elimination rate for vanadium was employed to fit the lung burden data. Comparisons between exposed groups indicated a progressive increase in lung weight with exposure concentration and time on exposure for both species. The vanadium lung burdens appeared to reach steady state in the lowest exposure groups (0.5 and 1 mg/m3 for rats and mice, respectively) but showed a decline in the higher exposure groups. This deposition pattern was similar between rats and mice but the maximum lung burdens were observed at different times (1 or 2 months in mice vs. 6 months in rats). The vanadium deposition rate decreased faster in mice, while the elimination half-lives of vanadium lung burdens were about six- to nine-fold shorter in mice than in rats at 1 and 2 mg/m3. Thus, the retention of vanadium in the lungs at 18 months was lower in mice (approximately 2% retained) compared with rats (13-15% retained) at the common exposure concentrations of 1 and 2 mg/m3. The lung burden data were approximately proportional to the exposure concentration in both species, likely due to concomitant decreases in deposition and elimination to a similar extent with increasing exposure. The area under the lung burden versus time curves and the area under the blood concentration (control-normalized) versus time curves were also proportional to exposure concentration. The progression of pathological changes in the lung with exposure and time is thought to affect the pattern and/or extent of vanadium deposition in the lungs following repeated exposures to V2O5.

Recent epidemiologic investigations of the relationship between residential radon gas exposure and lung cancer relied on contemporary radon gas measurements to estimate past radon gas exposures. Significant uncertainties in these exposure estimates can arise from year-to-year variation of indoor radon concentrations and subject mobility. Surface implanted 210Po has shown potential for improving retrospective radon gas exposure estimates. However, in previous studies, the ability of implanted 210Po activity to reconstruct cumulative radon gas exposure was not tested because glass was not available from homes with known radon-gas concentration histories. In this study, we tested the validity of the retrospective radon gas reconstruction using implanted 210Po surface activity by measuring glass surfaces from homes whose annual-average radon gas concentrations had been measured almost every year during two decades. Regression analysis showed a higher correlation between measured surface activity and cumulative radon gas exposure in these homes (R2>0.8) than was observed in homes where only contemporary radon gas measurements were available. The regression slope (0.57 ky m(-1)) was consistent with our earlier retrospective results. Surface activity measurements were as reliable for retrospective radon gas exposure reconstruction as yearlong gas measurements. Both methods produced estimates that were within 25% of the long-term average radon gas concentrations in a home. Surface measurements can be used for home screening tests because they can provide rapid, reliable estimates of past radon gas concentrations. Implanted 210Po measurements are also useful in retrospective epidemiologic studies that include participants who may have been exposed to highly variable radon concentrations in previously occupied or structurally modified homes.

Impaired respiratory function has been found frequently in ex-premature children, but it is unclear which specific factors influence this impairment the most. The aim of this study was to determine the importance of the contributions of birth weight, gestational age, neonatal respiratory disease, and its treatment on subsequent childhood lung function at age 11 years in a cohort of children of very low birth weight (VLBW; 2,000 g) of similar age. VLBW children were shorter and lighter than controls (P < 0.0001) at 11 years of age, and had reduced expiratory flows (P < 0.00001) and forced vital capacities (P < 0.001). The residual volume to total lung capacity ratio (RV/TLC ratio) was increased (P < 0.00001), while total lung capacity (TLC) remained unchanged. Those with bronchopulmonary dysplasia (BPD) had the lowest mean expiratory flows. Males had lower expiratory flows than females. On univariate analysis, gestational age by itself accounted for 8.8% of the explained variance in FEV(1) at 11 years of age, but birth weight accounted for 16% on its own; both together accounted for a further 0.2% (16.2%), suggesting that the latter was the dominant factor. On multivariate analysis, the contribution of birth weight and gestational age was small, and the best predictors at 11 years of age, which together explained 43.4% of the total variance in FEV(1), were log days of supplemental oxygen (9.6%) and a reported history of asthma (10.8%). For FEF(25-75), these predictors explained 7.2% and 13.4%, respectively, of the total explained variance of 40.6%. The relation between neonatal oxygen supplementation and childhood FEV(1) was such that up to 20 days of supplemental oxygen had little effect on subsequent FEV(1) at 11 years of age, but each additional week of supplemental oxygen after that time was associated with a progressive reduction in FEV(1) of 3%. These data confirm the significant role of supplemental oxygen in the neonatal period and a history of asthma on the subsequent reduction of expiratory flows in VLBW children. Birth weight was a more important prenatal factor than gestational age, but both were of lesser predictive significance than either supplemental oxygen or a reported history of asthma., (Copyright 2000 Wiley-Liss, Inc.)

2-Butoxyethanol (2BE) is used extensively in the production of cleaning agents and solvents. It is primarily metabolized in the liver to 2-butoxyacetic acid (2BAA), which is believed to be responsible for 2BE toxicities associated with hemolysis of red blood cells. The objective of the study was to characterize the systemic disposition of 2BE and 2BAA in rats and mice during 2-year 2BE inhalation toxicity studies. Male and female F344 rats and B6C3F1 mice (6-7 weeks old) were exposed to target 2BE concentrations of 0, 31.2, 62.5, or 125 ppm (rats), or 0, 62.5, 125, or 250 ppm (mice), by whole-body inhalation for 6 h/day, 5 days/week for up to 18 months. Postexposure blood samples were collected after 1 day, 2 weeks, and 3, 6, 12, and 18 months of exposure. Postexposure 16-h urine samples were collected after 2 weeks and 3, 6, 12, and 18 months of exposure. A separate set of mice was kept in the control chamber and exposed to 2BE for 3 weeks when they were approximately 19 months old. Postexposure blood samples were collected after 1 day and 3 weeks of exposure and 16-h urine samples were collected after 2 weeks of exposure from these aged mice. Blood samples were analyzed for both 2BE and 2BAA and urine samples were analyzed for 2BAA using GC/MS, and their kinetic parameters were estimated through the curve-fitting method using SAS. Systemically absorbed 2BE was rapidly cleared from blood (t1/2-RAT < 10 min; t1/2-MOUSE < 5 min after the 1-day exposure) independent of exposure concentration. Proportional increases in AUC2BE relative to increases in exposure concentration indicated linear 2BE kinetics. In contrast, the rate of 2BAA elimination from blood decreased as the exposure concentration increased. Nonproportional increases in AUC2BAA also indicated that 2BAA is eliminated following dose-dependent, nonlinear kinetics. Overall, mice eliminated both 2BE and 2BAA from blood faster than rats. Sex-related differences in 2BAA elimination were most significant with rats, in that females were less efficient in clearing 2BAA from the blood. Differences in renal excretion of 2BAA are possibly responsible for the sex-related difference in the 2BAA blood profiles in rats. As exposure continued, the rates of elimination for both 2BE and 2BAA decreased in both species, resulting in longer residence times in the blood. When 19-month-old naive mice were exposed to 125 ppm, 2BE was rapidly cleared from the systemic circulation, exhibiting clearance profiles similar to young mice. However, old mice eliminated 2BAA from blood > 10 times slower than young mice after 1-day of exposure. This delayed elimination of 2BAA in old mice was less obvious after 3 weeks of exposure, suggesting that there might be other factors in addition to the age of animals that could influence the apparent difference in 2BAA kinetics between old and young mice. It was concluded that the elimination kinetics of 2BE and 2BAA following repeated 2BE exposure appear to be dependent on species, sex, age, time of exposure, as well as the exposure concentration., (Copyright 1998 Academic Press.)

We aimed to determine the natural history of borderline increases in albuminuria in adolescents with insulin-dependent (Type 1) diabetes mellitus (IDDM) and factors which are associated with progression to persistent microalbuminura. Fifty-five normotensive adolescents with IDDM and intermittent microalbuminura (overnight albumin excretion ratte of 20-200 micrograms min-1 on one of three consecutive timed collections, n = 29) or borderline albuminura (mean overnight albumin excretion rate of 7.2-20 micrograms min-1 on one of three consecutive timed collections, n = 30) were followed prospectively at 3 monthly intervals. The endpoint was persistent microalbuminuria defined as a minimum of three of four consecutive overnight albumin excretion rates of greater than 20 micrograms min-1. One hundred and forty-two adolescents with IDDM and normoalbuminura were also followed prospectively. Fifteen of the 59 patients (25.4%) with intermittent (9/29) or borderline (6/30) albuminura progressed to persistent microalbuminura (progressors) over 28 (15-50) months [median (range)] in comparison with two of the 142 patients with normoalbuminuria at entry (relative risk = 12.6; p = 0.001). Progressors to persistent microalbuminura were pubertal and had higher systolic (p = 0.02) and diastolic (p = 0.02) blood pressure, and HbA1c (p = 0.004) than non-progressors. All patients remained normotensive. Glomerular filtration rate, apolipoproteins, dietary phosphorus, protein and sodium intakes, and prevalence of smoking did not differ between progressors and non-progressors. Total renin was higher in the diabetic patients without a difference between progressors and non-progressors. In conclusion there is a relatively high rate of progression to persistent microalbuminuria in pubertal adolescents with borderline increases in albuminura and duration greater than 3 years. These patients require attention to minimize associated factors of poor metabolic control and higher blood pressure in the development of incipient nephropathy.

Objective: To investigate power spectral analysis (PSA) of heart rate variability (HRV) in children and adolescents with IDDM, its relationship with other measures of HRV and standard cardiovascular responses, and factors associated with reduced HVR., Research Design and Methods: A total of 130 subjects with IDDM aged 12.8 +/- 3.2 years and 108 healthy control subjects were studied. Power spectra were analyzed from supine electrocardiograph (ECG) recordings by processing into consecutive R-R intervals and analysis using fast Fourier transformation. Standard cardiovascular responses to deep breathing and standing were performed., Results: IDDM subjects had a reduction in total power including both low-frequency (0.05-0.14 Hz; P = 0.0001) and high-frequency (0.14-0.40 Hz; P = 0.0002) components. These changes were seen from diagnosis. Other measures of HRV, coefficient of variation (CV) and standard deviation (SD) of mean resting heart rate, were also significantly lower in IDDM. All 20 (15%) of the 130 IDDM subjects with total power less than the 5th percentile in control subjects also had reduced HRV when measured by CV of heart rate. There was an independent relationship between age and the high-frequency component in IDDM subjects and control subjects. Total power correlated with mean heart rate (r = -0.56; P < 0.0001), CV of heart rate (r = 0.90; P < 0.00001), SD of heart rate (r = 0.91; P < 0.00001), heart rate response to deep breathing (r = 0.45; P < 0.0001), and duration in IDDM subjects. There was no correlation with short-term or long-term metabolic control. Retesting of 27 subjects showed a variability in total power and its components comparable to other measures of HRV and standard heart rate responses., Conclusions: Changes in HRV are a sensitive and reproducible measure of early autonomic dysfunction in childhood. In this age-group, PSA appears no more sensitive a measure of reduced HRV than other closely correlated measures of HRV.

A humanized antihuman IgE antibody, rhuMAb-E25, was designed to form complexes with free IgE, blocking its interaction with mast cells and basophils and thereby preventing the initiation of the allergic cascade. To characterize the rhuMAb-E25: IgE complexes formed in vivo and to examine the disposition of the antibody in a relevant animal model, 125I-rhuMAb-E25 was administered as an intravenous bolus dose to cynomolgus monkeys that have high levels of IgE. The pharmacokinetic values of unlabeled and radiolabeled antibody were similar, which indicated that the disposition of 125I-rhuMAb-E25 reflected that of rhuMAb-E25. Size-exclusion chromatography of serum samples showed that the rhuMAb-E25:IgE complexes were of limited size and were similar to the small complexes formed in vitro with human IgE. Pharmacokinetic analysis revealed that both rhuMAb-E25 and rhuMAb-E25:IgE complexes cleared the serum compartment, albeit slowly. No specific uptake of radioactivity was seen in any of the tissues collected from the cynomolgus monkeys at 1 hr and 96 hr postadministration; no association was observed between 125I-rhuMAb-E25, or the complexes, and blood cells. Urinary excretion was the primary route of elimination of radioactivity; > 90% of the radioactivity found in urine was not associated with protein. The lack of specific tissue uptake and blood cell association and the slow clearance of rhuMAb-E25:IgE complexes were consistent with low-avidity interaction of small complexes with Fc gamma receptors of leukocytes and the reticuloendothelial system.

Historic releases of key radionuclides were estimated as a first step in determining the radiation doses that resulted from Hanford Site operations. The Hanford Site was built in southcentral Washington State during World War II to provide plutonium for the U.S. nuclear weapons program. As part of the Hanford Environmental Dose Reconstruction (HEDR) Project, releases to the Columbia River of 24Na, 32P, 46Sc, 51Cr, 56Mn, 65Zn, 72Ga, 76As, 90Y, 131I, 239Np, and nonvolatile gross beta activity from operation of eight Hanford single-pass production reactors were estimated. Releases of 90Sr, 103Ru, 106Ru, 131I, 144Ce, and 239Pu to the atmosphere from operation of chemical separation facilities were also estimated. These radionuclides and the atmospheric and Columbia River pathways were selected for study because scoping studies showed them to be the largest contributors to dose from Hanford operations. The highest doses resulted from releases to the atmosphere of 131I from chemical separations plants in the pre-1950 period. Prior to 1950, the technology for limiting iodine releases had not been developed. Hence, a very detailed reconstruction of the hourly 131I release history was achieved for 1944-1949 using Monte Carlo methods. Atmospheric releases of the other radionuclides were estimated on a monthly basis for 1944-1972 using deterministic calculations. Monthly releases to the Columbia River for 1944-1971 were based on Monte Carlo methods.

We aimed to examine the longitudinal relationship between lipoprotein(a) and haemoglobin A1c, albumin excretion rate, and puberty in peripubertal children with insulin-dependent diabetes. A total of 114 patients aged 11.5 +/- 3.6 years (mean (SD)) were followed prospectively for 15.2 +/- 2.8 months. Lipoprotein(a), apolipoproteinB-100, haemoglobin A1c, mean overnight albumin excretion rate and Tanner stage were determined at the beginning and end of the study period. Lipoprotein(a) and apolipoproteinB-100 were measured using nephelometry. This method was correlated with radioimmunoassay and there was no significant change in mean bias during the study. Lipoprotein(a) fell significantly over time (214, (152, 276); 160 (84, 236) mg l-1 geometric mean (0.95 confidence intervals), p < 0.001); apolipoproteinB-100 did not change. Lipoprotein(a) and apolipoproteinB-100 did not differ in 233 cross-sectional controls of similar age. The change in lipoprotein(a) did not correlate with a small fall in haemoglobin A1c or with overnight albumin excretion rate, Tanner stage or insulin dose. Separate analysis of male and female patients and prepubertal and pubertal patients continued to show a significant fall in lipoprotein(a) independent of change in haemoglobin A1c or albumin excretion rate. Likewise, 53 patients with a change in haemoglobin A1c of greater than 1%, and 20 patients who progressed from normal albumin excretion rate to albumin excretion rate above the 95th centile, showed no relationship between lipoprotein(a) and haemoglobin A1c or albumin excretion rate. In conclusion, longitudinal changes in lipoprotein(a) do not relate to metabolic control or early changes in albuminuria in young patients with insulin-dependent diabetes.

Silicone nerve regeneration chambers were implanted between the cut ends of the sciatic nerve of adult rats. Neurotrophic activities in cell-free fluids collected from the chambers were determined using bioassays for survival of embryonic chick ciliary and sympathetic neurons in culture. Separation by molecular exclusion HPLC of the components of fluids collected 1, 2 or 3 days after implantation revealed the presence of a multitude of neurotrophic factors differing in their molecular weights, specificity towards the two types of neurons, and time course. Antiserum to nerve growth factor partially blocked sympathetic activity of fluids collected at 1 day. Affinity purified antibody was also effective and completely eliminated bioactivity of HPLC fractions corresponding to the molecular weight of nerve growth factor. The presence in the fluids of 13-18 and 20-32 kD components active towards ciliary neurons is consistent with the release of fibroblast growth factor and ciliary neurotrophic factor respectively. The stimulation of sympathetic neurons by the 13-18 kD material, and also by 4-6 and 7-11 kD components cannot be entirely accounted for by known factors. This study demonstrates that a number of neurotrophic factors, which differ in their specificity towards sympathetic and parasympathetic neurons, are made available to the region of axonal regrowth over the first few days of regeneration. Contrary to earlier reports, nerve growth factor-like activity was shown to be present in nerve regeneration chambers.

Objective: To measure pedestrian traffic volumes and noise levels in paediatric open bay areas and discuss their impact on the care of sick children., Methods: Between August and October 1992, we recorded the number and duration of entrances to two open bay areas comprising eight and ten beds respectively in a ward for infants and a ward for older children. Eight 24-hour periods (1200 to 1200) Friday to Saturday were assessed. Noise levels in decibels dB(A) were measured at 15-minute intervals., Results: In an average 24-hour period, 5.5 (SD +/- 1.3) patients in the infants' ward and 9.5 (SD +/- 0.6) patients in the children's ward received 617 (SD +/- 85) and 683 (SD +/- 64) visits by 104 (SD +/- 20) and 110 (SD +/- 2) individuals respectively. The maximum numbers of visits per hour were 57 (SD +/- 14) and 54 (SD +/- 8) visits between 1500 and 1600 hours on Friday for each ward. Visits tended to be brief; 225 (SD +/- 23) and 217 (SD +/- 34) visits were of less than one minute's duration. The maximum noise levels of 57.3 dB(A) (SD +/- 6.3) and 64.6 dB(A) (SD +/- 3.5) occurred at 1000 Saturday and 1900 Friday and coincided with peak traffic volumes., Conclusions: Open bay areas generate high traffic volumes and coincident noise. Consideration should be given to either modifying or abolishing open bay areas and to general noise control measures.

Objective: To determine serum lipoprotein(a) in a large sample of IDDM and control children and to examine a possible association with puberty., Research Design and Methods: Serum lipoprotein(a), apoB-100, and apoA-I were measured under identical conditions in 170 Caucasian children with IDDM aged 12.3 +/- 3.59 yr and 233 Caucasian control children aged 13.6 +/- 1.12 yr. Patients with persistent microalbuminuria were excluded. Lipoprotein(a), apoB-100, and apoA-I were measured by nephelometry using a specific monoclonal antibody. Pubertal assessment was performed using Tanner staging and testicular volume measurement., Results: Lipoprotein(a) was higher in the IDDM than control group (geometric mean 237 mg/L, 25-75th percentile 134-465 vs. 172 [99-316] mg/L, P = 0.0008). When analyzed according to pubertal stage, only pubertal and postpubertal patients had higher levels than control subjects (265 [148-560] vs. 174 [101-320] mg/L, P = 0.0001), with prepubertal patients showing no difference. Pubertal and postpubertal patients showed both higher lipoprotein(a) (P = 0.01) levels and higher albumin excretion rates (P = 0.02) than prepubertal patients, correcting for the other variable. Lipoprotein(a) was not related to HbA1c, albumin excretion rate, duration, age, sex, mean arterial pressure, or a family history of premature coronary artery disease in the IDDM group. Lipoprotein(a) was not higher in patients with overnight albumin excretion rate above the 95th percentile but below the microalbuminuric range. ApoB-100 did not differ between IDDM and control children. ApoA-I was significantly lower in the IDDM group (1.04 [0.94-1.17] vs. 1.21 [1.10-1.31] g/L; P < 0.0001)., Conclusions: Pubertal and postpubertal IDDM patients have higher serum lipoprotein(a) than Caucasian control subjects. Our findings suggest a rise in lipoprotein(a) may occur during puberty in IDDM. Longitudinal studies are required to clarify the relationship between lipoprotein(a), albumin excretion rate, and puberty.

Purpose: To evaluate the frequency and nature of spinal pathology, the frequency of clinically silent lesions, and the potential benefit of screening spinal MR in neurofibromatosis patients., Patients and Methods: 28 neurofibromatosis type-1 (NF-1) patients and nine neurofibromatosis type-2 (NF-2) patients were studied with postcontrast spinal MR imaging., Results: NF-1: One patient had a biopsy-proven low-grade glioma; five patients, intradural, extramedullary masses (N = 23); one patient, extradural masses (N = 2) (neurofibromas); 16 patients had bony abnormalities; and three patients thecal sac abnormalities. NF-2: Five patients demonstrated intramedullary masses (five/eight ependymomas); nine patients, intradural, extramedullary masses (meningiomas, schwannomas); and four patients, bony abnormalities. Eight/10 NF-1 and four/nine NF-2 patients had asymptomatic masses., Conclusion: Intradural disease is common, often asymptomatic, and often presents at a young age in NF-1 and NF-2 patients. Because of the propensity to develop significant asymptomatic as well as symptomatic intradural disease, screening of the entire spine with MR is recommended in both NF-1 and NF-2 patients.

The fluid accumulating in silicone nerve regeneration chambers implanted between the cut ends of rat sciatic nerve contains neuronotrophic activities towards embryonic chick ciliary and sympathetic neurons. The blot and culture technique of Carnow et al. was used to determine if part of the neuronotrophic activities is due to ciliary neuronotrophic factor, which supports the survival of both types of neurons in vitro. The technique involves separating the fluid proteins by SDS-polyacrylamide gel electrophoresis, Western transfer, and then culturing of purified neurons on the nitrocellulose blots. After 24 hr surviving neurons are restricted to regions of the blot where neuronotrophic factor is present. Analysis of 1 and 2 day fluids showed that a multitude of factors are present, particularly in the 19-30 kD molecular weight range, with discrete peaks of activity at molecular weights consistent with those reported for ciliary neuronotrophic factor. There were several other peaks of activity present in the fluids in addition to these.

Oxygen consumption (VO2), carbon dioxide production (VCO2), and respiratory quotient were measured in rats given a high-fat cafeteria diet of the type that is said to promote diet-induced thermogenesis. No significant difference in the measurements as compared with controls was found at room temperature, at 5 degrees C, or in animals exposed to cold for several weeks. The result was the same whether open- or closed-circuit methods were used. The stimulatory effect of norepinephrine on the VO2 was identical in each dietary group. These results cast doubt on the alleged identity of diet-induced and nonshivering thermogenesis and may reflect the change in body composition of the animals rather than a primary response to dietary variation.

Using 3H-labeled rat brain mature RNA as substrate, substantial ribonuclease activity was detected in homogenates of rat superior cervical ganglia with acidic (pH 5.5) and neutral (pH 7.0-7.5) optima. Very little activity could be measured at greater than pH 8. The acidic and neutral activities differed in the optimal conditions required for assay, and showed differential sensitivity to the sulfhydryl blocking agent, N-ethylmaleimide. Only the neutral activity was stimulated, optimally by 2 mM N-ethylmaleimide, and the magnitude of stimulation indicated that the contributing ribonucleases exist largely in a latent form in the ganglion. Ribonucleases in other tissues with neutral pH dependence, known usually as "alkaline" ribonucleases, are subject to an N-ethylmaleimide-sensitive endogenous inhibitor protein. The existence of a similar inhibitor in rat superior cervical ganglia was indicated by the latency of neutral ribonuclease activity and confirmed by observing the effect of a soluble fraction from the ganglia on the activity of pancreatic ribonuclease A.

Langendorff perfused rat hearts show synchronous, statistically significant, systematic variations in ATP and ADP. Here we show that AMP and IMP also vary in register with ATP and ADP and we suggest that the synchronizing trigger for these oscillations may be ischaemia. Oscillations in the ATP/ADP ratio were found to be significantly correlated with creatine phosphate content but by contrast these quantities vary quite differently from the GTP/GDP ratio. Cyclic GMP oscillations showed a significant negative correlation with variations in ADP. Epinephrine raised mean cyclic AMP content and stabilized cyclic GMP oscillations, but had little other effect on the purine nucleotide variations.

Radioactivity from [14C] adenosine was linearly incorporated into tissue nucleotides in perfused rat hearts. All the TCA-extractable 14C was confined to the purine nucleoside phosphates for up to 1 h of perfusion. Radioactivity was also incorporated linearly into the TCA-insoluble fraction, which by 40 min accounted for 24% of the tissue 14 C. Estimates based on precursor specific radioactivity suggest that at least 0.6 micro mol/g of the mononucleotide is in this stable insoluble form. Following 2 min total ischaemia, the tissue nucleotide content and soluble radioactivity decreased while the insoluble radioactivity showed a corresponding increase to account now for 35% of the tissue radiolabel. This redistribution was rapidly reversed by post-ischaemic reperfusion. A possible function for the rapid reversible sequestration of adenine nucleotides in ischaemia is proposed.

1. The oxygen consumption (VO2) of unrestrained rats given a 'cafeteria' (high energy, high fat) or control diet was studied. The resting values of VO2 were the same in each dietary group, whether maintained at 26 degrees C or 6 degrees C. This negative finding suggests that cafeteria feeding is not an important cause of diet-induced thermogenesis (DIT). 2. The response of each group of rats to injected noradrenaline or dopamine was also studied. Each catecholamine could increase VO2 values but the response was much less in cold-adapted rats measured at 6 degrees C. In all experimental circumstances the dopamine response exceeded that of noradrenaline. There was no evidence that the cafeteria diet consistently increased the response to either catecholamine. 3. These results suggest that DIT cannot be equated with non-shivering thermogenesis (NST). Furthermore, it is suggested that dopamine would be a better agent for measuring the oxygen equivalent of NST, since it would stimulate the dopamine receptors as well as the alpha- and beta-adrenoreceptors of brown fat.

An in vitro procedure for labeling of RNA in the excised rat nodose ganglion was used to evaluate the changes in incorporation of [3H]uridine into ganglionic RNA following transection of the abdominal vagus nerves. Significant increases in the incorporation into 28S, 18S and 4S RNA were observed at 1 day after injury, which were maximal at 4 days before returning to unoperated control level by 7 days. A second transient increase in the labelling of these RNA species occurred between 9 and 11 days after injury. Comparison of the time course of these increases with those seen previously following cervical vagus nerve crush injury indicate that the time of onset of the increase in incorporation in independent of the site of injury, but that the maximal response is delayed by 1 day with the more distal lesion. These data are consistent with the existence of separate signals for initiating and modulating the cell body response to axon injury, which are transported retrogradely from the site of injury at rates exceeding the slow component of axoplasmic transport.

Nuclei were isolated from homogenates of rat superior cervical ganglion by a conventional differential centrifugation technique with approximately 60% recovery. Ribonuclease activity at pH 7.1 (neutral ribonuclease) was associated with the "nuclei fraction" and represented 19% of the overall activity in normal ganglia. Ribonuclease in the "nuclei fraction" was stimulated variably by the sulfhydryl blocker N-ethylmaleimide indicating that a proportion was bound to the endogenous ribonuclease inhibitor present in these ganglia. The total activity of nuclear ribonuclease was increased 2-6 days after postganglionic nerve injury, such that the inhibitor-bound form of the enzyme increased maximally by 600% at day 4. The percentage of the total ganglionic activity in the "nuclei fraction" decreased in injured ganglia as a result of a rise in the activity of non-nuclear components. The changes in nuclear ribonuclease activity were distinct from those in the 850 g supernatant indicating that specific nuclear enzymes are being affected during regeneration.