Screening of Living Kidney Donors for Genetic Diseases Using a Comprehensive Genetic Testing Strategy.

Related living kidney donors (LKDs) are at higher risk of end-stage renal disease (ESRD) compared with unrelated LKDs. A genetic panel was developed to screen 115 genes associated with renal diseases. We used this panel to screen six negative controls, four transplant candidates with presumed genetic renal disease and six related LKDs. After removing common variants, pathogenicity was predicted using six algorithms to score genetic variants based on conservation and function. All variants were evaluated in the context of patient phenotype and clinical data. We identified causal variants in three of the four transplant candidates. Two patients with a family history of autosomal dominant polycystic kidney disease segregated variants in PKD1. These findings excluded genetic risk in three of four relatives accepted as potential LKDs. A third patient with an atypical history for Alport syndrome had a splice site mutation in COL4A5. This pathogenic variant was excluded in a sibling accepted as an LKD. In another patient with a strong family history of ESRD, a negative genetic screen combined with negative comparative genomic hybridization in the recipient facilitated counseling of the related donor. This genetic renal disease panel will allow rapid, efficient and cost-effective evaluation of related LKDs.

Critical Risk Results - An Update on International Initiatives.

Direct communication of significant (often life-threatening) results is a universally acknowledged role of the pathology laboratory, and an important contributor to patient safety. Amongst the findings of a recent survey of 871 laboratories from 30 countries by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM), only 3 tests were noted to be common to 90% of alert lists, and only 48% of laboratories consulted clinicians in developing these alert lists despite ISO15189 recommendations to do so. These findings are similar to previous national surveys demonstrating significant variation worldwide in how critical risk results are managed and also in how these protocols are developed. In order to promote "best practice" and harmonization of critical risk results management, guidelines and recommendations have been published, most recently by Clinical and Laboratory Standards Institute (CLSI) and Australasian Association of Clinical Biochemists (AACB). These statements in particular have placed strong emphasis on patient risk and risk assessment in the management of critical risk results. This focus has resulted in recommendations to adopt new terminology, the consideration of risk assessment when compiling alert tables, consultative involvement of laboratory users in setting up protocols, and the need for outcome-based evidence to support our practices. With time it is expected that emerging evidence and technological improvements will facilitate the advancement of laboratories down this path to harmonization, best practice, and improve patient safety.

Designing environmental research for impact.

Transdisciplinary research, involving close collaboration between researchers and the users of research, has been a feature of environmental problem solving for several decades, often spurred by the need to find negotiated outcomes to intractable problems. In 2005, the Australian government allocated funding to its environment portfolio for public good research, which resulted in consecutive four-year programmes (Commonwealth Environmental Research Facilities, National Environmental Research Program). In April 2014, representatives of the funders, researchers and research users associated with these programmes met to reflect on eight years of experience with these collaborative research models. This structured reflection concluded that successful multi-institutional transdisciplinary research is necessarily a joint enterprise between funding agencies, researchers and the end users of research. The design and governance of research programmes need to explicitly recognise shared accountabilities among the participants, while respecting the different perspectives of each group. Experience shows that traditional incentive systems for academic researchers, current trends in public sector management, and loose organisation of many end users, work against sustained transdisciplinary research on intractable problems, which require continuity and adaptive learning by all three parties. The likelihood of research influencing and improving environmental policy and management is maximised when researchers, funders and research users have shared goals; there is sufficient continuity of personnel to build trust and sustain dialogue throughout the research process from issue scoping to application of findings; and there is sufficient flexibility in the funding, structure and operation of transdisciplinary research initiatives to enable the enterprise to assimilate and respond to new knowledge and situations.

Harmonization of critical result management in laboratory medicine.

Unsafe medical care is a major source of disabling injuries and death throughout the world. The failure to notify, follow up, and action critical results, which signify life threatening situations, is of particular concern and may cause avoidable morbidity and mortality. International accreditation standards require pathology laboratories to have a system for the timely and reliable communication of critical results to clinical personnel responsible for patient care. In response, various practices and a number of different terminologies have been described in the literature. Increased attention to patient safety standards and multinational surveys, however, highlighted shortcomings and inefficiencies in existing communication systems. These failures and variations in practice call for clear guidance and harmonization of approaches in order to improve communications and to provide safer patient care. The objectives of this review are to create a harmonized terminology and to learn from international practices by systematically reviewing the best available evidence on existing approaches. Based on literature review findings we highlight key areas where harmonization is necessary and feasible and offer a conceptual framework and methods for designing better and more evidence-based systems for the timely notification of laboratory results that represent potential patient safety hazards.

Risk of water contamination by nitrogen in Canada as estimated by the IROWC-N model.

With increasing amounts of nitrogen (N) being added to farmland in the form of fertilizer and manure to optimize crop yields, and more broadly, to meet the growing demands for food, feed and energy, there are public concerns regarding its possible negative impact on the environment. An optimal balance between N requirements for production versus efficient N use is required, so as to minimize N losses from the agricultural system. An agri-environmental indicator i.e., the Indicator of the Risk of Water Contamination by Nitrogen (IROWC-N) was developed to assess the risk of N moving from agricultural areas into groundwater and/or nearby surface water bodies. The indicator linked the quantity of mineral nitrogen remaining in the soil at harvest, i.e., the Residual Soil Nitrogen (RSN) indicator, and the subsequent climatic conditions during the winter period. The results were assessed in terms of nitrate lost through leaching and nitrate concentration in the drainage water, expressed in five IROWC-N risk classes. Unlike previous versions of the indicator, the current model provided a more complete description of the soil-water balance, including the calculation of rainfall interception by crops, surface runoff, actual evapotranspiration and soil-water contents. Consequently, the current IROWC-N estimates differed markedly from those obtained previously. Between 1981 and 2006, the risk of water contamination by N in Canada was small, and reflected what was happening in the three Prairie provinces where 85% of Canada's farmland is located. However, the aggregated IROWC-N index, which is a combination of all five risk classes, increased steadily by 2.3% per year, from 6.7 in 1981 to 10.6 in 2006. The proportion of farmland in the very low IROWC-N risk class decreased from 88 to 78%; correspondingly, the proportion in the low risk class increased from 2 to 12%. The proportion of farmland in the moderate-, high- and very high-risk classes changed by less than 3% over time. The trends in IROWC-N in the Atlantic provinces were significantly worse than the national trend; for example, in Atlantic Canada, the aggregated IROWC-N index tripled from 27.8 in 1981 to 87.5 in 2006. Increases in fertilizer use (except in British Columbia), increases in livestock numbers in Manitoba and the Atlantic provinces, and an increase in legume crop acreage were the main factors that contributed to the increase in IROWC-N estimates. Climatic factors were also involved, as droughts reduced yields, N uptake and N leaching in many regions of Canada in 2001.

Treating hop plants with (Z)-jasmone increases colonization by Phorodon humuli (Hemiptera: Aphididae) spring migrants.

Hop plants were sprayed with (Z)-jasmone, at a rate of 50 g ha(-1), during the spring migration of the damson-hop aphid Phorodon humuli (Schrank) in 2002 and 2003. Numbers of P. humuli spring migrants colonizing hop plants, Humulus lupulus L., 2-6 and 7-11 days after applying this treatment were assessed in both years. During the first five-day period, significantly more spring migrants were found on hop plants treated with (Z)-jasmone, in comparison with control plants, in 2002. By contrast, no significant difference was evident in the second five-day period. Although the migration in 2003 was much lighter than in 2002, greater numbers of migrants were again removed from treated plants. Indeed, more spring migrants were removed from plants sprayed with (Z)-jasmone in this year during both five-day periods (11 and 44%, respectively) compared with the 23% greater numbers removed in the first five-day period in 2002. Therefore, unlike some other species of aphid, where numbers were consistently lower on plots sprayed with (Z)-jasmone, spraying the secondary host of P. humuli with this compound appears to increase colonization by spring migrants.

Seroprevalence of dengue in Trinidad using rapid test kits: a cord blood survey.

A cross-sectional sero-epidemiological study was conducted to determine the prevalence of dengue in Trinidad. Two commercial rapid test kits, PanBio Dengue Duo IgM and IgG Rapid Strip Test and the Bio-Check Plus Dengue G/M Cassette Test (Brittney) were used. The immunosorbent assay (ELISA) (FOCUS Technologies, California) was used as the control. One hundred and twenty five cord blood samples were collected (46 from Mt. Hope Women's Hospital (MH) and 79 from the San Fernando General Hospital (SF)). All blood samples were tested in accordance with the two rapid kits and ELISA assay manufacturer's instructions. From 125 cord blood samples, the IgG FOCUS ELISA results showed 93.5 and 95% infections at MH and SF, respectively. Whereas the Brittney and PanBio kits showed 10.9 and 5.1%, and 26.1 and 50.6% for MH and SF, respectively. Based on the FOCUS ELISA (control) assays, the combined seroprevalence rate from north and south Trinidad was 94.4%. IgG and IgM sensitivity and specificity levels were higher in the PanBio than Brittney test kits. The high seroprevalence rates observed in Trinidad are discussed to stimulate more research to explain this phenomenon and to prevent the Southeast Asian scenario from developing in the Americas.

Linkage of otosclerosis to a third locus (OTSC3) on human chromosome 6p21.3-22.3.

Clinical otosclerosis (OMIM 166800/605727) has a prevalence of 0.2-1% among white adults, making it the single most common cause of hearing impairment in this group. It is caused by abnormal bone homeostasis of the otic capsule with the consequent development of sclerotic foci that invade the stapedio-vestibular joint (oval window) interfering with free motion of the stapes. Impaired ossicular chain mobility results in a conductive hearing loss. We identified the first locus for otosclerosis (OTSC1) on chromosome 15 in 1998 and reported a second locus (OTSC2) on chromosome 7 last year. Here we present results of a genome wide linkage study on a large Cypriot family segregating otosclerosis. Results of this study exclude linkage to OTSC1 and OTSC2 and identify a third locus, OTSC3, on chromosome 6p. The defined OTSC3 interval covers the HLA region, consistent with reported associations between HLA-A/HLA-B antigens and otosclerosis.

Increased cortical expression of the orexin-1 receptor following permanent middle cerebral artery occlusion in the rat.

The orexins (hypocretins) have recently been implicated in neurodegeneration associated with narcolepsy. Therefore, the current study was designed to investigate changes in the expression of prepro-orexin and the orexin receptors, OX1R and OX2R following permanent middle cerebral artery occlusion (MCAO) in the rat. Six and twenty-four hours following MCAO, increased OX1R mRNA and protein expression (as assessed by Western blotting and immunohistochemistry) was detected in the ischaemic cortex compared with control tissue. In contrast, however, no increase in OX2R mRNA was detected at any time-point and prepro-orexin levels in the cortex were below assay detection levels. This study shows that orexin receptor localization is altered following cerebral ischaemia. The development of selective orexin receptor antagonists will be crucial in establishing a role for this family of novel peptides in the mechanisms underlying ischaemic cell death.

Receptor-mediated endocytosis of Neisseria gonorrhoeae into primary human urethral epithelial cells: the role of the asialoglycoprotein receptor.

Urethral epithelial cells are invaded by Neisseria gonorrhoeae during gonococcal infection in men. To understand further the mechanisms of gonococcal entry into host cells, we used the primary human urethral epithelial cells (PHUECs) tissue culture system recently developed by our laboratory. These studies showed that human asialoglycoprotein receptor (ASGP-R) and the terminal lactosamine of lacto-N-neotetraose-expressing gonococcal lipooligosaccharide (LOS) play an important role in invasion of PHUECs. Microscopy studies showed that ASGP-R traffics to the cell surface after gonococcal challenge. Co-localization of ASGP-R with gonococci was observed. As ASGP-R-mediated endocytosis is clathrin dependent, clathrin localization in PHUECs was examined after infection. Infected PHUECs showed increased clathrin recruitment and co-localization of clathrin and gonococci. Preincubating PHUECs in 0.3 M sucrose or monodansylcadaverine (MDC), which both inhibit clathrin-coated pit formation, resulted in decreased invasion. N. gonorrhoeae strain 1291 produces a single LOS glycoform that terminates with Gal(beta1-4)GlcNac(beta1-3)Gal(beta1-4)Glc (lacto-N-neotetraose). Invasion assays showed that strain 1291 invades significantly more than four isogenic mutants expressing truncated LOS. Sialylation of strain 1291 LOS inhibited invasion significantly. Preincubation of PHUECs in asialofetuin (ASF), an ASGP-R ligand, significantly reduced invasion. A dose-response reduction in invasion was observed in PHUECs preincubated with increasing concentrations of NaOH-deacylated 1291 LOS. These studies indicated that an interaction between lacto-N-neotetraose-terminal LOS and ASGP-R allows gonococcal entry into PHUECs.

Binding of the non-typeable Haemophilus influenzae lipooligosaccharide to the PAF receptor initiates host cell signalling.

Non-typeable Haemophilus influenzae (NTHi) invades host cells by binding of the platelet-activating factor (PAF) receptor via lipooligosaccharide (LOS) glycoforms containing phosphorylcholine (ChoP). The effect of NTHi infection on host cell signalling and its role in NTHi invasion was examined. The infection of human bronchial epithelial cells with NTHi 2019 increased cytosolic Ca2+ levels, and the invasion of bronchial cells by NTHi 2019 was inhibited by pretreatment with the cell-permeant intracellular Ca2+ chelator BAPTA-AM (P = 0.022) or thapsigargin (P = 0.016). Cytosolic inositol phosphate (IP) levels were also increased after infection with NTHi 2019 (P < 0.001), but not after infection with isogenic mutants expressing altered LOS glycoforms lacking ChoP. PAF receptor antagonist reduced NTHi 2019-stimulated IP production in a dose-dependent manner. NTHi 2019 invasion was inhibited by pertussis toxin (PTX) and the phosphatidylinositol-3-kinase inhibitors wortmannin and LY294002. The less invasive strain NTHi 7502 also initiated IP production, but was unaffected by PAF receptor antagonist or PTX. These data demonstrate that the binding of the PAF receptor by NTHi initiates receptor coupling to a PTX-sensitive heterotrimeric G protein complex, resulting in a multifactorial host cell signal cascade and bacterial invasion. Moreover, the data suggest that NTHi strains initiate cell signalling and invade by different mechanisms, and that invasion mediated by PAF receptor activation is more efficient than macropinocytosis.

Ischaemic preconditioning in the rat brain: a longitudinal magnetic resonance imaging (MRI) study.

Ischaemic preconditioning in rats was studied using MRI. Ischaemic preconditioning was induced, using an intraluminal filament method, by 30 min middle cerebral artery occlusion (MCAO), and imaged 24 h later. The secondary insult of 100 min MCAO was induced 3 days following preconditioning and imaged 24 and 72 h later. Twenty-four hours following ischaemic preconditioning most rats showed small sub-cortical hyperintense regions not seen in sham-preconditioned rats. Twenty-four hours and 72 h following the secondary insult preconditioned animals showed significantly smaller lesions (24 h = 112 +/- 31 mm(3), mean +/- standard error; 72 h = 80 +/- 35 mm(3)), which were confined to the striatum, than controls (24 h = 234 +/- 32 mm(3), p = 0.026; 72 h = 275 +/- 37 mm(3), p = 0.003). In addition during lesion maturation from 24 to 72 h post-secondary MCAO, preconditioned rats displayed an average reduction in lesion size as measured by MRI whereas sham-preconditioned rats displayed increases in lesion size; this is the first report of such differential lesion volume evolution in cerebral ischaemic preconditioning.

Caspase mRNA expression in a rat model of focal cerebral ischemia.

Proteins of the caspase family are involved in the signalling pathway that ultimately leads to programmed cell death (apoptosis), which has been reported to occur in some experimental models of stroke. In a previous paper we used quantitative reverse transcription and polymerase chain reaction (RT-PCR) to characterise changes in the mRNA expression of one member of this family, caspase-3, in a rat model of permanent focal ischemia. Here we have used this technique to study the expression of a further three caspases which are involved in different aspects of caspase signalling. Caspase-8, involved in Fas-mediated apoptosis, was upregulated in the cortex of ischemic rats. Caspase-11, which leads to the synthesis of the functional form of the cytokine interleukin-1 beta, also showed increased expression, but with a different temporal profile from caspase-8. In contrast, caspase-9, which forms part of the pathway signalling through the mitochondria, showed a decrease in expression. The expression of a further four caspases (1, 2, 6 and 7) has also been characterised in a simpler experiment. These caspases all showed distinctive patterns of expression following the induction of ischemia. These data lead us to conclude that caspase expression as a whole is under very strict transcriptional control in this model. Certain elements of caspase signalling, such as the Fas-induced pathway and the events upstream of IL-1 beta processing, are upregulated, while others are not. This may be due to some form of genetic program activated in response to ischemia in the brain and may highlight which biological pathways are modulated.

Distance learning in the health professions: on the verge of collapse or poised to soar?

Distance education provides universal access to education. While the issue of access to education is seemingly resolved, the question "what is the best way to teach?" remains. Thomas Jefferson espoused the creation of the academic village to foster broad, intensive scholarship. John Dewey, Ralph W. Tyler, Malcolm S. Knowles, and Alexander W. Astin echo Jefferson's ideals. To ensure excellence in distance learning, a disciplined rethinking of teaching and a reordering of academic priorities is essential. If consumer confidence in the academy is to be restored, there must be a return to collegial leadership in: defining institutional purpose and resource commitment, improving teacher competence, honing curricular content, perfecting interactive learning, selecting students, and designing outcome measures for both teaching and learning. Then, and only then, will distance learning be ready for "prime time."

Further characterisation of a thromboembolic model of stroke in the rat.

We have used magnetic resonance imaging (MRI) techniques to characterise a rat model of thromboembolic stroke. The consequences of acute perfusion deficit associated with a middle cerebral artery occlusion (MCAo) by a newly formed thrombus was mapped by interrogation of the tissue oxygenation status using gradient echo methods and production of T2* maps. Final infarct size was subsequently assessed at 24-h post-ischaemia by histology with 2,3,5-triphenyltetrazolium chloride (TTC) staining. Animals displayed an infarct volume of 178.7+/-84.2 mm(3) (mean+/-S.D.) with a large coefficient of variation (47%) and range of values (85.6--265.5 mm(3)). This variability provided us with an opportunity to assess the relationships between early imaging observations and eventual infarct size. For a single cerebral slice, at the centre of the MCA territory, a relationship between the area of reduced T2* at 1 and 2 h post MCAo correlated highly with final lesion area (Spearman rank correlation, r=0.98, P<0.01, n=9). Lesion volumes in the thromboembolic MCAo model were compared with a 120-min occlusion, 22-h reperfusion protocol using an intraluminal thread MCAo approach. For the thromboembolic model, the total lesion volume was found to be smaller (178.7+/-84.2 vs. 243.3+/-50.1 mm(3), mean+/-S.D., Student's t-test P=0.046) and showed a greater variability (coefficient of variations: 47% vs. 21%). These data underline the relative variability of this embolic model and provide important preliminary information regarding the value of early changes in T2* in predicting eventual infarct size.

Pseudomonas aeruginosa in cystic fibrosis: cross-infection and the need for segregation.

Evidence-based reasons for segregation of patients colonized with Pseudomonas aerugionsa in the outpatient setting are unclear. To clarify local decisions, Pseudomonas genotyping of the local environment, patients and patient contacts was undertaken in 1993. The hospital environment was re-swabbed in 1997. Pseudomonas genotyping of old and new patients attending the North Staffordshire cystic fibrosis clinic has subsequently been undertaken and more recently been repeated on an annual basis to assess whether the same Pseudomonas genotypes can be found in both the environment and in patients, and whether the same Pseudomonas genotype can be transferred from one patient to another. No Pseudomonas genotype found in the local environment in 1993 or in 1997 has been found in any of our patients. Nine children attending the same special school for many years and sharing the same physiotherapy facilities showed no evidence of cross-infectivity. Except for siblings living in the same household our cross-infectivity rate is very low and where cross-infection has potentially occurred the level of contact between these patients has been minimal. This study does not support the suggestion that patients with cystic fibrosis attending the North Staffordshire clinic and colonized with Pseudomonas aeruginosa should be segregated from non-colonized patients.

Selective antagonism of endothelin-A-receptors improves outcome in both head trauma and focal stroke in rat.

Increased levels of endothelin (ET) have been demonstrated in the ischemic brain, and ET receptor antagonism has been shown to improve outcome in cerebral ischemia. However, no previous work has been carried out evaluating the role of ET and its antagonism in brain trauma as compared to experimental stroke. In this study, we evaluated changes in brain ET levels following closed head injury (CHI) and the effects of SB 234551, an endothelin-A- (ET(A)) selective antagonist, and SB 209670, a mixed endothelin-A- and -B- (ET(A)/ET(B)) antagonist, on outcome in CHI and focal stroke. Male Sabra rats were subjected to CHI (weight drop model). Male Sprague Dawley rats were subjected to focal stroke (intraluminal suture model). Motor function(s) were assessed and immunoreactive ET (irET) and the degree of cerebral edema were measured for 24 h after CHI. Brain swelling (edema), neurological deficits and forebrain infarct volumes were measured 24 h after focal stroke. Antagonists (total doses of 7.5, 15, 30 or 60 mg/kg) were administered intravenously for 6-24 h (beginning 15 min after injury). Control rats were infused with vehicle. CHI resulted in increased ET levels in the directly contused hemisphere at 12 and 24 h. In addition, SB 234551 significantly reduced neurological deficits (decreased 30%) and brain edema (decreased 40%) following CHI (p < 0.05 at 60 mg/kg dose). SB 209670 had no effects on CHI outcome. Focal stroke studies yielded similar results. SB 234551 reduced focal stroke-induced neurological deficits by 50%, brain swelling by 54% and the degree of infarction by 36% (p < 0.05 at 30 mg/kg). SB 209670 did not provide any neuroprotection in focal stroke. These data indicate that ET plays a significant role in the pathophysiology of CHI, and that selectively targeting ET(A)-receptors similarly in both CHI and stroke might be a therapeutic opportunity.

Characterisation of SB-221420-A - a neuronal Ca(2+) and Na(+) channel antagonist in experimental models of stroke.

For progression to clinical trials in stroke, putative neuroprotective compounds should show robust efficacy post-ischaemia in several experimental models of stroke. This paper describes the characterisation of (+)(1S, 2R)-cis-1-[4-(1-methyl-1-phenylethyl)phenoxy]-2-methylamino indane hydrochloride (SB-221420-A), a Ca(2+) and Na(+) channel antagonist. SB-221420-A inhibited (IC(50)=2.2 microM) N-type voltage-operated Ca(2+) channel currents in cultured superior cervical ganglion neurons, which were pretreated with 10 microM nimodipine to block L-type voltage-operated Ca(2+) channel currents. In dorsal root ganglion neurons pretreated with 1 microM omega-conotoxin GVIA to block N-type voltage-operated Ca(2+) channel currents, SB-221420-A inhibited the residual Ca(2+) current with an IC(50) of 7 microM. SB-221420-A also inhibited Na(+) currents in dorsal root ganglion neurons with an IC(50) of 8 microM. In rats, the pharmacokinetic profile of SB-221420-A shows that it has a half-life of 6.4 h, a high volume of distribution, is highly brain penetrating, and has no persistent metabolites. Following bilateral carotid artery occlusion in gerbils, SB-221420-A significantly reduced the level of ischaemia-induced hyperlocomotor activity and the extent of hippocampal CA1 cell loss compared to the ischaemic vehicle-treated group. SB-221420-A was also effective in focal models of ischaemia. In the mouse permanent middle cerebral artery occlusion model, SB-221420-A (10 mg/kg) administered intravenously, post-ischaemia significantly (P<0.05) reduced lesion volume compared to the ischaemic vehicle-treated group. In the normotensive rat permanent middle cerebral artery occlusion model, SB-221420-A (10 mg/kg) administered intravenously over 1 h, beginning 30 min postmiddle cerebral artery occlusion, significantly (P<0.05) reduced lesion volume from 291+/-16 to 153+/-30 mm(3), compared to ischaemic vehicle-treated controls when measured 24 h postmiddle cerebral artery occlusion. Efficacy was maintained when the same total dose of SB-221420-A was infused over a 6-h period, beginning 30 min postmiddle cerebral artery occlusion. SB-221420-A also significantly (P<0.05) reduced lesion volume following transient middle cerebral artery occlusion in normotensive rats and permanent middle cerebral artery occlusion in spontaneously hypertensive rats (SHR). Investigation of the side effect profile using the Irwin screen in mice revealed that, at neuroprotective doses, there were no overt behavioural or cardiovascular changes. These data demonstrate that robust neuroprotection can be seen post-ischaemia with SB-221420-A in both global and focal ischaemia with no adverse effects at neuroprotective doses, and indicate the potential utility of a mixed cation blocker to improve outcome in cerebral ischaemia.

New roles for cis-jasmone as an insect semiochemical and in plant defense.

cis-jasmone, or (Z)-jasmone, is well known as a component of plant volatiles, and its release can be induced by damage, for example during insect herbivory. Using the olfactory system of the lettuce aphid to investigate volatiles from plants avoided by this insect, (Z)-jasmone was found to be electrophysiologically active and also to be repellent in laboratory choice tests. In field studies, repellency from traps was demonstrated for the damson-hop aphid, and with cereal aphids numbers were reduced in plots of winter wheat treated with (Z)-jasmone. In contrast, attractant activity was found in laboratory and wind tunnel tests for insects acting antagonistically to aphids, namely the seven-spot ladybird and an aphid parasitoid. When applied in the vapor phase to intact bean plants, (Z)-jasmone induced the production of volatile compounds, including the monoterpene (E)-beta-ocimene, which affect plant defense, for example by stimulating the activity of parasitic insects. These plants were more attractive to the aphid parasitoid in the wind tunnel when tested 48 h after exposure to (Z)-jasmone had ceased. This possible signaling role of (Z)-jasmone is qualitatively different from that of the biosynthetically related methyl jasmonate and gives a long-lasting effect after removal of the stimulus. Differential display was used to compare mRNA populations in bean leaves exposed to the vapor of (Z)-jasmone and methyl jasmonate. One differentially displayed fragment was cloned and shown by Northern blotting to be up-regulated in leaf tissue by (Z)-jasmone. This sequence was identified by homology as being derived from a gene encoding an alpha-tubulin isoform.

Fractalkine cleavage from neuronal membranes represents an acute event in the inflammatory response to excitotoxic brain damage.

Fractalkine is a recently identified chemokine that exhibits cell adhesion and chemoattractive properties. It represents a unique member of the chemokine superfamily because it is located predominantly in the brain in which it is expressed constitutively on specific subsets of neurons. To elucidate the possible role of neuronally expressed fractalkine in the inflammatory response to neuronal injury, we have analyzed the regulation of fractalkine mRNA expression and protein cleavage under conditions of neurotoxicity. We observed that mRNA encoding fractalkine is unaffected by experimental ischemic stroke (permanent middle cerebral artery occlusion) in the rat. Similarly, in vitro, levels of fractalkine mRNA were unaffected by ensuing excitotoxicity. However, when analyzed at the protein level, we found that fractalkine is rapidly cleaved from cultured neurons in response to an excitotoxic stimulus. More specifically, fractalkine cleavage preceded actual neuronal death by 2-3 hr, and, when evaluated functionally, fractalkine represented the principal chemokine released from the neurons into the culture medium upon an excitotoxic stimulus to promote chemotaxis of primary microglial and monocytic cells. We further demonstrate that cleavage of neuron-derived, chemoattractive fractalkine can be prevented by inhibition of matrix metalloproteases. These data strongly suggest that dynamic proteolytic cleavage of fractalkine from neuronal membranes in response to a neurotoxic insult, and subsequent chemoattraction of reactive immune cells, may represent an early event in the inflammatory response to neuronal injury.