A coupled oscillator model for the origin of bimodality and multimodality.

Perhaps because of the elegance of the central limit theorem, it is often assumed that distributions in nature will approach singly-peaked, unimodal shapes reminiscent of the Gaussian normal distribution. However, many systems behave differently, with variables following apparently bimodal or multimodal distributions. Here, we argue that multimodality may emerge naturally as a result of repulsive or inhibitory coupling dynamics, and we show rigorously how it emerges for a broad class of coupling functions in variants of the paradigmatic Kuramoto model.

Key Matrix Proteins Within the Pancreatic Islet Basement Membrane Are Differentially Digested During Human Islet Isolation.

Clinical islet transplantation achieves insulin independence in selected patients, yet current methods for extracting islets from their surrounding pancreatic matrix are suboptimal. The islet basement membrane (BM) influences islet function and survival and is a critical marker of islet integrity following rodent islet isolation. No studies have investigated the impact of islet isolation on BM integrity in human islets, which have a unique duplex structure. To address this, samples were taken from 27 clinical human islet isolations (donor age 41-59, BMI 26-38, cold ischemic time < 10 h). Collagen IV, pan-laminin, perlecan and laminin-α5 in the islet BM were significantly digested by enzyme treatment. In isolated islets, laminin-α5 (found in both layers of the duplex BM) and perlecan were lost entirely, with no restoration evident during culture. Collagen IV and pan-laminin were present in the disorganized BM of isolated islets, yet a significant reduction in pan-laminin was seen during the initial 24 h culture period. Islet cytotoxicity increased during culture. Therefore, the human islet BM is substantially disrupted during the islet isolation procedure. Islet function and survival may be compromised as a consequence of an incomplete islet BM, which has implications for islet survival and transplanted graft longevity.

Production study of Fr, Ra and Ac radioactive ion beams at ISOLDE, CERN.

The presented paper discusses the production of radioactive ion beams of francium, radium, and actinium from thick uranium carbide (UC x ) targets at ISOLDE, CERN. This study focuses on the release curves and extractable yields of francium, radium and actinium isotopes. The ion source temperature was varied in order to study the relative contributions of surface and laser ionization to the production of the actinium ion beams. The experimental results are presented in the form of release parameters. Representative extractable yields per µ C are presented for 222 - 231 Ac, several Ra and Fr isotopes in the mass ranges 214 ≤ A ≤ 233 and 205 ≤ A ≤ 231 respectively. The release efficiency for several isotopes of each of the studied elements was calculated by comparing their yields to the estimated in-target production rates modeled by CERN-FLUKA. The maximal extraction efficiency of actinium was calculated to be 2.1(6)% for a combination of surface ionization using a Ta ion source and resonant laser ionization using the two-step 438.58 nm, and 424.69 nm scheme.

14-3-3 proteins are essential signalling hubs for beta cell survival.

AIMS/HYPOTHESIS: Diabetes is characterised by pancreatic beta cell death and dysfunction, resulting from unbalanced pro-survival and pro-death signalling. The 14-3-3 proteins are molecular adaptors that integrate numerous signalling pathways, including the v-raf-leukaemia viral oncogene 1 (RAF1)/B cell leukaemia/lymphoma 2 (BCL-2)-associated agonist of cell death (BAD) pathway, which we have previously implicated in insulin-dependent beta cell survival. Nevertheless, the roles of 14-3-3 proteins in beta cell fate and function have not been investigated. METHODS: We examined the abundance, localisation, modulation and roles of 14-3-3 proteins using quantitative RT-PCR, immunoblot or imaging. MIN6 cells or mouse islets cells were manipulated with inhibitors, short interfering RNA (siRNA) or plasmids overexpressing 14-3-3. RESULTS: We first characterised the abundance and subcellular location of all seven 14-3-3 isoforms in mouse and human beta cells. Most isoforms were cytoplasmic, except 14-3-3σ, which appeared to be nuclear. Analysis of 14-3-3 abundance under stress conditions revealed distinct modulation in mouse islets and MIN6 cells. Generalised 14-3-3 inhibition promoted apoptosis and dysfunction, and siRNA-mediated knockdown revealed isoform-specific roles in caspase-3-dependent beta cell apoptosis, with a clear role for 14-3-3ζ. Overabundance of 14-3-3ζ sequestered BAD-BCL2-associated X protein (BAX) from mitochondria, attenuated Dp5 (also known as Hrk) and Puma (also known as Bbc3) induction, and increased survival in response to pro-inflammatory cytokines or thapsigargin. Anti-apoptotic insulin treatment increased the sequestration of BAD/BAX by 14-3-3ζ. BAD mutants that were unable to bind 14-3-3ζ localised to mitochondria and induced apoptosis. CONCLUSIONS/INTERPRETATION: This first study of the 14-3-3 family in beta cells revealed specific regulation, localisation and anti-apoptotic roles among the isoforms. Focus on 14-3-3ζ revealed its importance in preventing BAD-BAX mitochondrial localisation and protecting beta cells from multiple stresses. Thus, some 14-3-3 proteins are pro-survival signalling hubs.

Ubiquitin C-terminal hydrolase L1 is required for pancreatic beta cell survival and function in lipotoxic conditions.

AIMS/HYPOTHESIS: Ubiquitin C-terminal hydrolase L1 (UCHL1) is associated with neurodegenerative diseases and has been suggested to have roles in pancreatic beta cells. Our proteomic analysis revealed that UCHL1 was the most increased protein in MIN6 cells exposed to palmitate. The present study used a genetic loss-of-function model to test the hypothesis that UCHL1 is required for normal beta cell function and fate under lipotoxic conditions. METHODS: Human islets, mouse islets and MIN6 cells were used to analyse UCHL1 protein levels and regulation of UCHL1 by palmitate. The levels of free mono-ubiquitin and poly-ubiquitinated proteins were assessed. Gracile axonal dystrophy (GAD) mutant mice lacking UCHL1 were fed a normal or lipotoxic high-fat diet. Glucose tolerance, insulin tolerance and insulin secretion were assessed in vivo. Beta cell death and proliferation were assessed by TUNEL and proliferating cell nuclear antigen (PCNA) staining. Insulin secretion, calcium signalling, endoplasmic reticulum (ER) stress, apoptosis and SNARE protein levels were assessed in vitro. RESULTS: UCHL1 protein, which was highly specific to beta cells, was increased by palmitate at basal glucose, but not in the context of hyperglycaemia associated with frank diabetes. Although islet development and function were initially normal in Uchl1 (-/-) mice, a 4-week high-fat diet caused glucose intolerance and impaired insulin secretion. Uchl1 (-/-) mice had increased ER stress and beta cell apoptosis. The levels of SNARE proteins were dysregulated in Uchl1 (-/-) islets. Palmitate-stimulated vesicle-associated membrane protein 2 (VAMP2) ubiquitination was modulated by a chemical UCHL1 inhibitor. CONCLUSIONS/INTERPRETATION: Together, these data suggest that UCHL1 has essential functional and anti-apoptotic roles in beta cells under stress conditions associated with lipotoxicity.

The effect of using an alternative irrigant between sodium hypochlorite and chlorhexidine to prevent the formation of para-chloroaniline within the root canal system.

AIM: To determine if the formation of para-chloroaniline (PCA) can be avoided by using an alternative irrigant following sodium hypochlorite but before chlorhexidine. METHODOLOGY: Fifty-five single-rooted teeth were decoronated, instrumented to size 40, .06 taper whilst being irrigated with 14% ethylene-diamine-tetra-acetic acid (EDTA) and 6% NaOCl. Samples were then randomly divided into three experimental and two control groups. Group 1 was irrigated with saline followed by 2% chlorhexidine gluconate (CHX). Group 2 was irrigated with 50% citric acid (CA) followed by 2% CHX. Group 3 was irrigated with 14% EDTA followed by 2% CHX. The chemical identity and quantification of the PCA in the formed precipitate was determined using gas chromatography/mass spectrometry (GC/MS). RESULTS: All experimental groups contained PCA. The mean level of PCA for group 1 (sterile saline) was 229 ng mL(-1), group 2 (citric acid) 72 ng mL(-1) and group 3 (EDTA) 400 ng mL(-1), respectively. A significant difference was found between the saline and EDTA groups and the negative control (P < 0.05). Although no statistical significance was found between the negative control and citric acid group, PCA was still present in this experimental group. CONCLUSIONS: Citric acid used as the intermittent irrigant had the least amount of PCA formation in the canal system. Until the threshold required to cause biological damage in humans is determined, the combination of NaOCl and CHX in root canal treatment should be avoided.

Paracrine signalling loops in adult human and mouse pancreatic islets: netrins modulate beta cell apoptosis signalling via dependence receptors.

AIMS/HYPOTHESIS: Adult pancreatic islets contain multiple cell types that produce and secrete well characterised hormones, including insulin, glucagon and somatostatin. Although it is increasingly apparent that islets release and respond to more secreted factors than previously thought, systematic analyses are lacking. We therefore sought to identify potential autocrine and/or paracrine islet growth factor loops, and to characterise the function of the netrin family of islet-secreted factors and their receptors, which have been previously unreported in adult islets. METHODS: Gene expression databases, islet-specific tag sequencing libraries and microarray datasets of FACS purified beta cells were used to compile a list of secreted factors and receptors present in mouse or human islets. Netrins and their receptors were further assessed using RT-PCR, Western blot analysis and immunofluorescence staining. The roles of netrin-1 and netrin-4 in beta cell function, apoptosis and proliferation were also examined. RESULTS: We identified 233 secreted factors and 234 secreted factor receptors in islets. The presence of netrins and their receptors was further confirmed. Downregulation of caspase-3 activation was observed when MIN6 cells were exposed to exogenous netrin-1 and netrin-4 under hyperglycaemic conditions. Reduction in caspase-3 cleavage was linked to the decrease in dependence receptors, neogenin and unc-5 homologue A, as well as the activation of Akt and extracellular signal-regulated protein kinase (ERK) signalling. CONCLUSIONS/INTERPRETATION: Our results highlight the large number of potential islet growth factors and point to a context-dependent pro-survival role for netrins in adult beta cells. Since diabetes results from a deficiency in functional beta cell mass, these studies are important steps towards developing novel therapies to improve beta cell survival.

Time-dependent mediators of HPA axis activation following live Escherichia coli.

The hypothalamus-pituitary-adrenal (HPA) axis is activated during an immune challenge to liberate energy and modulate immune responses via feedback and regulatory mechanisms. Inflammatory cytokines and prostaglandins are known contributors to HPA activation; however, most previous studies only looked at specific time points following LPS administration. Since whole bacteria have different immune stimulatory properties compared with LPS, the aim of the present studies was to determine whether different immune products contribute to HPA activation at different times following live Escherichia coli challenge. Sprague-Dawley rats were injected intraperitoneally with E. coli (2.5 × 10(7) CFU) and a time course of circulating corticosterone, ACTH, inflammatory cytokines, and PGE(2) was developed. Plasma corticosterone peaked 0.5 h after E. coli and steadily returned to baseline by 4 h. Plasma PGE(2) correlated with the early rise in plasma corticosterone, whereas inflammatory cytokines were not detected until 2 h. Pretreatment with indomethacin, a nonselective cyclooxygenase inhibitor, completely blocked the early rise in plasma corticosterone, but not at 2 h, whereas pretreatment with IL-6 antibodies had no effect on the early rise in corticosterone but attenuated corticosterone at 2 h. Interestingly, indomethacin pretreatment did not completely block the early rise in corticosterone following a higher concentration of E. coli (2.5 × 10(8) CFU). Further studies revealed that only animals receiving indomethacin prior to E. coli displayed elevated plasma and liver cytokines at early time points (0.5 and 1 h), suggesting prostaglandins suppress early inflammatory cytokine production. Overall, these data indicate prostaglandins largely mediate the early rise in plasma corticosterone, while inflammatory cytokines contribute to maintaining levels of corticosterone at later time points.

Electronic Health Records and Pulmonary Function Data: Developing an Interoperability Roadmap. An Official American Thoracic Society Workshop Report.

A workshop "Electronic Health Records and Pulmonary Function Data: Developing an Interoperability Roadmap" was held at the American Thoracic Society 2019 International Conference. "Interoperability" is defined as is the ability of different information-technology systems and software applications to directly communicate, exchange data, and use the information that has been exchanged. At present, pulmonary function test (PFT) equipment is not required to be interoperable with other clinical data systems, including electronic health records (EHRs). For this workshop, we assembled a diverse group of experts and stakeholders, including representatives from patient-advocacy groups, adult and pediatric general and pulmonary medicine, informatics, government and healthcare organizations, pulmonary function laboratories, and EHR and PFT equipment and software companies. The participants were tasked with two overarching Aobjectives: 1) identifying the key obstacles to achieving interoperability of PFT systems and the EHR and 2) recommending solutions to the identified obstacles. Successful interoperability of PFT data with the EHR impacts the full scope of individual patient health and clinical care, population health, and research. The existing EHR-PFT device platforms lack sufficient data standardization to promote interoperability. Cost is a major obstacle to PFT-EHR interoperability, and incentives are insufficient to justify the needed investment. The current vendor-EHR system lacks sufficient flexibility, thereby impeding interoperability. To advance the goal of achieving interoperability, next steps include identifying and standardizing priority PFT data elements. To increase the motivation of stakeholders to invest in this effort, it is necessary to demonstrate the benefits of PFT interoperability across patient care and population health.

Reciprocal modulation of adult beta cell maturity by activin A and follistatin.

AIMS/HYPOTHESIS: The functional maturity of pancreatic beta cells is impaired in diabetes mellitus. We sought to define factors that can influence adult beta cell maturation status and function. METHODS: MIN6 cells labelled with a Pdx1 monomeric red fluorescent protein-Ins1 enhanced green fluorescent protein dual reporter lentivirus were used to screen candidate growth and/or differentiation factors using image-based approaches with confirmation by real-time RT-PCR and assays of beta cell function using primary mouse islets. RESULTS: Activin A strikingly decreased the number of mature beta cells and increased the number of immature beta cells. While activins are critical for pancreatic morphogenesis, their role in adult beta cells remains controversial. In primary islets and MIN6 cells, activin A significantly decreased the expression of insulin and several genes associated with beta cell maturity (e.g. Pdx1, Mafa, Glut2 [also known as Slc2a2]). Genes found in immature beta cells (e.g. Mafb) tended to be upregulated by activin A. Insulin secretion was also reduced by activin A. In addition, activin A-treated MIN6 cells proliferated faster than non-treated cells. The effects of endogenous activin A on beta cells were completely reversed by exogenous follistatin. CONCLUSIONS/INTERPRETATION: These results suggest that autocrine and/or paracrine activin A signalling exerts a suppressive effect on adult beta cell maturation and function. Thus, the maturation state of adult beta cells can be modulated by external factors in culture. Interventions inhibiting activin or its signalling pathways may improve beta cell function. Understanding of maturation and plasticity of adult pancreatic tissue has significant implications for islet regeneration and for in vitro generation of functional beta cells.

Suggested mechanism for the selective excretion of glucosylated albumin. The effects of diabetes mellitus and aging on this process and the origins of diabetic microalbuminuria.

In previous studies in the Sprague-Dawley rat, Williams and coworkers reported the phenomenon of selective urinary excretion of glucosylated albumin (editing, i.e., the percent glucosylation of urinary albumin is more than that of plasma albumin) by the mammalian kidney. Ghiggeri and coworkers subsequently found that the extent of editing is reduced in human diabetics. Moreover, the reduction in editing in diabetes correlates inversely with levels of microalbuminuria. We also find reduction in the extent of editing in diabetic humans. We find a striking inverse correlation not only with the magnitude of microalbuminuria but also with the extent of plasma albumin glucosylation. In contrast, we found little correlation between the reduction in editing and the duration of diabetes in human subjects. Stz induced diabetes in the Sprague-Dawley rat is associated with a striking and rapid reduction in editing which develops virtually with the same kinetics exhibited by the appearance of hyperglycemia. This loss of editing is rapidly reversed by daily administration of insulin but not by aldose reductase inhibitors. Mannitol infusion in anesthetized Wistar rats resulted in an increase in urine volume, GFR, and microalbuminuria, and was also accompanied by a marked reduction in editing. This reduction was rapidly reversed by a cessation of mannitol infusion. We propose here that glucosylated albumin (in contrast to unmodified albumin) is not reabsorbed by the proximal tubule, and thus, is preferentially excreted in the urine. We postulate that the increase in GFR which emerges as a consequence of increased plasma osmolality in diabetes mellitus delivers more albumin to the proximal tubule than can be reabsorbed. This results in a dilution of excreted glucosylated albumin molecules by excreted unmodified albumin, which appears as the early microscopic albuminuria of diabetes. Paradoxically, the fall in apparent editing is accompanied by an absolute increase in the total quantity of glucosylated albumin excreted. In contrast, we found that editing of glucosylated albumin by the normal kidney is found to gradually decline as a function of age without the appearance of microalbuminuria. This suggests that a different mechanism operates to produce the loss of editing seen with aging in man, and as clearly (but in a shorter absolute time intervals) in the Fischer-344 rat.

Oils and rubber from arid land plants.

In this article the economic development potentials of Cucurbita species (buffalo gourd and others), Simmondsia chinensis (jojoba), Euphorbia lathyris (gopher plant), and Parthenium argentatum (guayule) are discussed. All of these plants may become important sources of oils or rubber.

Pulp revascularization of necrotic bilateral bicuspids using a modified novel technique to eliminate potential coronal discolouration: a case report.

AIM: To present a case report in which the pulp of two bilateral mandibular premolars with dens evaginatus were revascularized using a modified novel technique to avoid undesired crown discolouration. SUMMARY: Recently, regeneration of necrotic pulps has become an alternative conservative treatment option for young permanent teeth with immature roots and is a subject of great interest in the field of endodontics. This novel procedure exploits the full potential of the pulp for dentine deposition and produces a stronger mature root that is better able to withstand the forces than can result in fracture. However, the current protocol has potential clinical and biological complications. Amongst them, crown discolouration, development of resistant bacterial strains and allergic reaction to the intracanal medication. In the case presented, a modified technique to avoid undesired crown discolouration was applied sealing the dentinal tubules of the chamber, thus avoiding any contact between the tri-antibiotic paste and the dentinal walls. KEY LEARNING POINTS: * Sealing the dentinal tubules of the chamber prevents the undesirable crown discolouration produced by tri-antibiotic medication whilst maintaining the revascularization potential of the pulp. * Further research is warranted to seek an alternative infection control protocol capable of preventing possible allergic reactions and development of resistant strains of bacteria, as well as a biological material capable of inducing angiogenesis and allow a more predictable scaffold and tissue regeneration.

Evaluation of Total Hydrocarbon Contents in the Leaves of Selected Crops Grown on Crude Oil Polluted Agricultural Soil

Aim: The Total Hydrocarbon Content (THC) levels in the leaves of three edible plants (Allium cepa, Telfairia occidentalis and Zea mays) grown on soil polluted with 100 ml of crude oil were examined. Study Design: This experiment was conducted in two groups where the soil samples were polluted before planting (PB) and polluted 2 weeks after planting (PA). Place and Duration of Study: The research was carried out at the Federal University of Technology, Owerri (FUTO), Imo State, Nigeria, within a period of 4 weeks based on each group’s pollution time. Methodology: The tests for total hydrocarbon content (THC) were analyzed at different wavelengths for polluted soil and leaf samples using UV-Vis spectrophotometric method. Soil THC tests were determined on days 0, 14 and 28 respectively while THC tests on leaves of study plants were assessed on day 14 and 28. Results: The total hydrocarbon content values of the unpolluted soil (UPS) and the non-planted polluted soil (PS) subjected to this analysis on day zero were (UPS = 96.38 mg/kg; PS = 1082.80 mg/kg). However, results obtained on day 14 and 28 showed significant difference (P < 0.05) between the unpolluted soil sample (UPS) and all the planted polluted soil for Telfairia occidentalis (TOPB, TOPA), Allium cepa (ACPB, ACPA), Zea mays (ZMPB, ZMPA) as well as, the non-planted polluted soil (PS). For the leaf samples, TOPB had the highest THC value of 14.47mg/kg and 36.73 mg/kg for day 14 and 28 respectively while ZMPB had the lowest value of 5.38mg/kg at day 14 and ZMPA, the least THC value of 7.76mg/kg at day 28. Conclusion: Based on the varying THC levels observed in the leaves of crops used for this study; it was connoted that, bioaccumulation of hydrocarbons depends on the plants phytoremediation capability and the mode of pollution as observed in TOPB.

Transcriptional synergy between LIM-homeodomain proteins and basic helix-loop-helix proteins: the LIM2 domain determines specificity.

LIM-homeodomain proteins direct cellular differentiation by activating transcription of cell-type-specific genes, but this activation requires cooperation with other nuclear factors. The LIM-homeodomain protein Lmx1 cooperates with the basic helix-loop-helix (bHLH) protein E47/Pan-1 to activate the insulin promoter in transfected fibroblasts. In this study, we show that two proteins originally called Lmx1 are the closely related products of two distinct vertebrate genes, Lmx1.1 and Lmx1.2. We have used yeast genetic systems to delineate the functional domains of the Lmx1 proteins and to characterize the physical interactions between Lmx1 proteins and E47/Pan-1 that produce synergistic transcriptional activation. The LIM domains of the Lmx1 proteins, and particularly the second LIM domain, mediate both specific physical interactions and transcriptional synergy with E47/Pan-1. The LIM domains of the LIM-homeodomain protein Isl-1, which cannot mediate transcriptional synergy with E47/Pan-1, do not interact with E47/Pan-1. In vitro studies demonstrate that the Lmx1.1 LIM2 domain interacts specifically with the bHLH domain of E47/Pan-1. These studies provide the basis for a model of the assembly of LIM-homeodomain-containing complexes on DNA elements that direct cell-type-restricted transcription in differentiated tissues.

The homeodomain of PDX-1 mediates multiple protein-protein interactions in the formation of a transcriptional activation complex on the insulin promoter.

Activation of insulin gene transcription specifically in the pancreatic beta cells depends on multiple nuclear proteins that interact with each other and with sequences on the insulin gene promoter to build a transcriptional activation complex. The homeodomain protein PDX-1 exemplifies such interactions by binding to the A3/4 region of the rat insulin I promoter and activating insulin gene transcription by cooperating with the basic-helix-loop-helix (bHLH) protein E47/Pan1, which binds to the adjacent E2 site. The present study provides evidence that the homeodomain of PDX-1 acts as a protein-protein interaction domain to recruit multiple proteins, including E47/Pan1, BETA2/NeuroD1, and high-mobility group protein I(Y), to an activation complex on the E2A3/4 minienhancer. The transcriptional activity of this complex results from the clustering of multiple activation domains capable of interacting with coactivators and the basal transcriptional machinery. These interactions are not common to all homeodomain proteins: the LIM homeodomain protein Lmx1.1 can also activate the E2A3/4 minienhancer in cooperation with E47/Pan1 but does so through different interactions. Cooperation between Lmx1.1 and E47/Pan1 results not only in the aggregation of multiple activation domains but also in the unmasking of a potent activation domain on E47/Pan1 that is normally silent in non-beta cells. While more than one activation complex may be capable of activating insulin gene transcription through the E2A3/4 minienhancer, each is dependent on multiple specific interactions among a unique set of nuclear proteins.

Sox5 regulates beta-cell phenotype and is reduced in type 2 diabetes.

Type 2 diabetes (T2D) is characterized by insulin resistance and impaired insulin secretion, but the mechanisms underlying insulin secretion failure are not completely understood. Here, we show that a set of co-expressed genes, which is enriched for genes with islet-selective open chromatin, is associated with T2D. These genes are perturbed in T2D and have a similar expression pattern to that of dedifferentiated islets. We identify Sox5 as a regulator of the module. Sox5 knockdown induces gene expression changes similar to those observed in T2D and diabetic animals and has profound effects on insulin secretion, including reduced depolarization-evoked Ca2+-influx and ß-cell exocytosis. SOX5 overexpression reverses the expression perturbations observed in a mouse model of T2D, increases the expression of key ß-cell genes and improves glucose-stimulated insulin secretion in human islets from donors with T2D. We suggest that human islets in T2D display changes reminiscent of dedifferentiation and highlight SOX5 as a regulator of ß-cell phenotype and function.

Sexual dimorphism of the intracellular heat shock protein 72 response.

The majority of previous work examining stress responses has been done in males. Recently, it has become clear that the impact of stressor exposure is modulated by sex. One stress response that may be affected by sex is the induction of intracellular heat shock protein (HSP) 72, which is a stress- responsive molecular chaperone that refolds denatured proteins and promotes cellular survival. The following study compared HSP72 in males and females and also examined whether the estrous cycle altered HSP72 induction in females. We hypothesized that females compared with males would have a constrained HSP72 response after an acute stressor and that the stress-induced HSP72 response in females would fluctuate with the estrous cycle. Male and female F344 rats were either left in their home cage or exposed to acute tail-shock stress (8-10/group). Immediately following stressor, trunk blood was collected and tissues were flash frozen. Vaginal smear and estrogen enzyme immunoassay were used to categorize the phase of estrous. Results show that female rats had a greater corticosterone response than males, that both males and females exhibit a stress-induced release of progesterone, and that males and females had equal levels of stress-induced circulating norepinephrine. Sexual dimorphism of the HSP72 (ELISA) response existed in pituitary gland, mesenteric lymph nodes, and liver such that female rats had an attenuated HSP72 response compared with males after stress. The adrenal glands, spleen, and heart did not exhibit sexual dimorphism of the HSP72 response. The estrous cycle did not have a significant effect on basal or stress-induced HSP72 in females.

The objective assessment of international collaboration between pathology laboratories.

Collaboration between clinical laboratories in different countries is widespread but rarely subject to critical analysis. We developed a management tool for assessing the quality of an international pathology laboratory collaboration. Key areas of activity were identified and relevant performance indicators were established. There was no evidence of improved staff morale arising from the project but research, education, equipment, protocols and ethics activities all had demonstrable outputs. Identification of suboptimal performance in areas of the project allowed effective improvement planning. International collaboration may consume significant resources. Objective assessment should be used to measure performance and to enhance quality.

Inhibition of cyclic nucleotide phosphodiesterase and calcineurin by spermine, a calcium-independent calmodulin antagonist.

Spermine binding to calmodulin and its effects on two calmodulin-dependent enzymes were studied. Spermine bound to dansylated calmodulin with an apparent Ki of 0.7 mM, and to native calmodulin with a Kd of 1.1 mM in equilibrium dialysis experiments. Its binding was found to be independent of calcium. Spermine inhibited calmodulin-activated cyclic nucleotide phosphodiesterase noncompetitively with respect to calcium (Ki = 1.1 mM). Calmodulin activation of calcineurin was inhibited at similar concentrations (Ki = 1.2 mM). Spermine had little effect on basal phosphodiesterase activity or nickel-activated calcineurin activity. Inhibition of both enzymes correlated well with spermine binding to dansylcalmodulin. These findings suggest that spermine might modulate calcium-dependent events in the cell by inactivation of calmodulin via a novel calcium-independent mechanism.