Regulation of bone mineral density in the grey squirrel, Sciurus carolinensis: Bioavailability of calcium oxalate, and implications for bark stripping.

The damage caused when grey squirrels strip the outer bark off trees and ingest the underlying phloem can result in reduced timber quality or tree death. This is extremely costly to the UK forestry industry and can alter woodland composition, hampering conservation efforts. The calcium hypothesis proposes that grey squirrels ingest phloem to ameliorate a seasonal calcium deficiency. Calcium in the phloem predominantly takes the form of calcium oxalate (CaOx), however not all mammals can utilise CaOx as a source of calcium. Here, we present the results of a small-scale study to determine the extent to which grey squirrels can utilise CaOx. One of three custom-made diets containing calcium in varying forms and quantities (CaOx diet, Low-calcium carbonate (CaCO3 ) diet and Control diet) were fed to three treatment groups of six squirrels for 8 weeks. Bone densitometric properties were measured at the end of this time using peripheral quantitative computed tomography and micro-computed tomography. Pyridinoline-a serum marker of bone resorption-was measured regularly throughout the study. Bone mineral density and cortical mineralisation were lower in squirrels fed the CaOx diet compared to the Control group but similar to that of those on the Low-calcium diet, suggesting that calcium from calcium oxalate was not effectively utilised to maintain bone mineralisation. Whilst no differences were observed in serum pyridinoline levels between individuals on different diets, females had on average higher levels than males throughout the study. Future work should seek to determine if this apparent lack of ability to utilise CaOx is common to a large sample of grey squirrels and if so, whether it is consistent across all areas and seasons.

Developing a contributing factor classification scheme for Rasmussen's AcciMap: Reliability and validity evaluation.

One factor potentially limiting the uptake of Rasmussen's (1997) Accimap method by practitioners is the lack of a contributing factor classification scheme to guide accident analyses. This article evaluates the intra- and inter-rater reliability and criterion-referenced validity of a classification scheme developed to support the use of Accimap by led outdoor activity (LOA) practitioners. The classification scheme has two levels: the system level describes the actors, artefacts and activity context in terms of 14 codes; the descriptor level breaks the system level codes down into 107 specific contributing factors. The study involved 11 LOA practitioners using the scheme on two separate occasions to code a pre-determined list of contributing factors identified from four incident reports. Criterion-referenced validity was assessed by comparing the codes selected by LOA practitioners to those selected by the method creators. Mean intra-rater reliability scores at the system (M = 83.6%) and descriptor (M = 74%) levels were acceptable. Mean inter-rater reliability scores were not consistently acceptable for both coding attempts at the system level (MT1 = 68.8%; MT2 = 73.9%), and were poor at the descriptor level (MT1 = 58.5%; MT2 = 64.1%). Mean criterion referenced validity scores at the system level were acceptable (MT1 = 73.9%; MT2 = 75.3%). However, they were not consistently acceptable at the descriptor level (MT1 = 67.6%; MT2 = 70.8%). Overall, the results indicate that the classification scheme does not currently satisfy reliability and validity requirements, and that further work is required. The implications for the design and development of contributing factors classification schemes are discussed.

What makes a 'good group'? Exploring the characteristics and performance of undergraduate student groups.

Group work forms the foundation for much of student learning within higher education, and has many educational, social and professional benefits. This study aimed to explore the determinants of success or failure for undergraduate student teams and to define a 'good group' through considering three aspects of group success: the task, the individuals, and the team. We employed a mixed methodology, combining demographic data with qualitative observations and task and peer evaluation scores. We determined associations between group dynamic and behaviour, demographic composition, member personalities and attitudes towards one another, and task success. We also employed a cluster analysis to create a model outlining the attributes of a good small group learning team in veterinary education. This model highlights that student groups differ in measures of their effectiveness as teams, independent of their task performance. On the basis of this, we suggest that groups who achieve high marks in tasks cannot be assumed to have acquired team working skills, and therefore if these are important as a learning outcome, they must be assessed directly alongside the task output.

The driver, the road, the rules and the rest? A systems-based approach to young driver road safety.

The persistent overrepresentation of young drivers in road crashes is universally recognised. A multitude of factors influencing their behaviour and safety have been identified through methods including crash analyses, simulated and naturalistic driving studies, and self-report measures. Across the globe numerous, diverse, countermeasures have been implemented; the design of the vast majority of these has been informed by a driver-centric approach. An alternative approach gaining popularity in transport safety is the systems approach which considers not only the characteristics of the individual, but also the decisions and actions of other actors within the road transport system, along with the interactions amongst them. This paper argues that for substantial improvements to be made in young driver road safety, what has been learnt from driver-centric research needs to be integrated into a systems approach, thus providing a holistic appraisal of the young driver road safety problem. Only then will more effective opportunities and avenues for intervention be realised.

The role of protein kinase C in regulating equine eosinophil adherence and superoxide production.

OBJECTIVE: To determine if protein kinase C (PKC) regulates equine eosinophil function. MATERIAL OR SUBJECTS: Blood eosinophils were obtained from healthy ponies. METHODS: IL-5- and histamine-induced adherence to serum-coated plastic was measured as the eosinophil peroxidase content of adherent cells and serum treated zymosan (STZ)-and IL-5-induced superoxide production by the reduction of cytochrome C. Eosinophil PKC activity was quantitated as the rate of transfer of (32)P from ATP to substrate. The effects of Ro31-8220 (isotype non-selective PKC inhibitor), Go6976 (conventional PKC inhibitor), and rottlerin (PKCdelta inhibitor) were determined by ANOVA and Bonferroni's or Dunnett's test. RESULTS: Ro31-8220 and Go6976 reduced superoxide production whereas only Go6976 inhibited adherence. Rottlerin inhibited histamine-induced adherence and increased STZ-induced superoxide production. Ro31-8220 and Go6976, but not rottlerin, inhibited PKC activity. CONCLUSIONS: PKC is involved in regulating equine eosinophil adherence and superoxide production. The role of PKCdelta appears to depend upon the stimulus used and response measured.

Biochemical and functional assessment of equine lymphocyte phosphodiesterases and protein kinase C.

Lymphocytes play an important role in allergic inflammation and have been implicated in the pathogenesis of equine allergic skin and respiratory disease. Targeting intracellular signalling pathways in human lymphocytes has demonstrated a role for both phosphodiesterase and protein kinase C in cell activation. The aim of this study was to measure total cyclic nucleotide hydrolysing phosphodiesterase activity and to identify the phosphodiesterase and protein kinase C isoenzymes present in equine lymphocytes. The functional significance of these isoenzymes was then investigated by examining their role in peripheral blood mononuclear cell proliferation using isoenzyme selective inhibitors. Total cyclic adenosine monophosphate hydrolysing phosphodiesterase activity was double that of cyclic guanosine monophosphate (30+/-2 pmol/min mg versus 16+/-3 pmol/min mg for cyclic adenosine and cyclic guanosine monophosphate phosphodiesterase activity, respectively). Evidence for the presence of PDE1, 3, 4 and 5 was obtained and PKCalpha, beta, delta, eta, iota, theta and zeta were identified. Selective inhibitors of PDE4, PKCdelta and conventional PKCs alpha and beta caused significant inhibition of mitogen-induced peripheral blood mononuclear cell proliferation. This study demonstrates a functional role for specific signalling isoenzymes and suggests that, in the context of allergic inflammation, targeting inflammatory cells involved in disease pathogenesis with relevant isoenzyme inhibitors may have therapeutic potential.

Protein kinase C (PKC) isotype profile in eosinophils from ponies with sweet itch and role in histamine-induced eosinophil activation.

Eosinophils have been implicated in the pathogenesis of the seasonal equine allergic skin disease, sweet itch. Protein kinase C (PKC) is involved in regulating eosinophil function and antigen challenge has been reported to alter PKC isotype expression in blood eosinophils from allergic human subjects. Here we have compared the pattern of PKC isotype expression in eosinophils from sweet itch ponies with that in cells from normal ponies both during the active and inactive phases of the disease. A role for PKC in histamine-induced eosinophil activation was also investigated. Conventional PKCs alpha and beta, novel PKCs delta and epsilon and atypical PKCs iota and zeta were identified in eosinophils pooled from four allergic ponies during the inactive phase, when no clinical signs were evident. The PKC isotypes, like those in eosinophils from normal ponies, were located primarily in the particulate fraction of the cell. Isotype expression in cells from normal and allergic animals did not appear to be different. In contrast, during the active phase of the disease, when the sweet itch ponies had clinical signs, the expression of PKCs beta, epsilon and iota in eosinophils from these animals appeared to be increased relative to that in cells from normal ponies. When PKC expression in eosinophils from five individual normal and sweet itch ponies was compared, small, but statistically significant, increases in PKC epsilon and PKCdelta expression were evident in eosinophils from the sweet itch ponies during the active and inactive phases, respectively. The non-selective PKC inhibitors, staurosporine and Ro31-8220, significantly reduced histamine-induced superoxide production. Use of Gö6976, an inhibitor of conventional PKCs, suggested that PKCalpha and/or beta were involved and that there was significantly greater inhibition of the response in eosinophils obtained from sweet itch ponies during the active phase. There was no significant difference in histamine-induced superoxide production by eosinophils from allergic and normal ponies and the functional significance of the increased PKC isotype expression in eosinophils from sweet itch ponies relative to that in cells from healthy animals remains to be established.

Cloning, expression and biological activity of equine interleukin (IL)-5.

The cytokine, interleukin (IL)-5 stimulates eosinophil differentiation, activation and survival and can prime these cells, increasing the response to other mediators. In view of its many effects on eosinophils, IL-5 has been implicated in the pathogenesis of allergic disease in man. Here we report the cloning of equine IL-5 and expression of the recombinant protein by transfection of Chinese hamster ovary (CHO) cells. The cloned cDNA sequence consisted of 405 nucleotides and encoded a protein of 135 amino acids. There is >85% identity with feline, bovine, ovine, canine, and human IL-5 sequences at the nucleotide and protein level. Supernatants containing equine IL-5 were also examined for biological activity. CHO supernatant containing equine recombinant (eqr) IL-5, like the human ortholog (hrIL-5), induced concentration dependent equine eosinophil adherence to autologous serum-coated plastic (9.7+/-1.5% with a 1:100 dilution of eqrIL-5 and 9.1+/-1.6% adherence with 1 nM hrIL-5; n = 4). The eqr protein also caused concentration dependent superoxide production (11.9+/-2.4 nmol (reduced cytochrome (cyt) C)/10(6) cells at a 1:50 dilution, n = 4). In contrast, hrIL-5 only caused significant superoxide production when diluted in conditioned CHO medium, an effect that was inhibited by the anti-human mAb, TRFK5 (4.4+/-0.3 versus 0.3+/-0.4 nmol/10(6) cells for 0.5 nM hrIL-5 in the presence of the isotype matched IgG1 control (10 microM) and TRFK5 (10 microM), respectively). TRFK5 also significantly inhibited hrIL-5 induced adherence at concentrations of 0.3 microg/ml and above but had no significant inhibitory effect on either superoxide or adherence caused by eqrIL-5. These results demonstrate that equine IL-5 expressed by CHO cells stimulates equine eosinophils, suggesting that this cytokine could play a role in eosinophil recruitment and activation in equine allergic disease. The anti-human and murine moAb TRFK5 does not appear to recognise the equine protein.

The bradykinin response and early hypotension at the introduction of continuous renal replacement therapy in the intensive care unit.

We assessed the relationship of certain clinical variables (including bradykinin [BK] release and dialysis membrane) to initial mean arterial pressure (MAP) reduction in 47 patients requiring continuous renal replacement therapy (CRRT) in an intensive care unit. The pretreatment MAP was 84 +/- 14 mm Hg for the group as a whole. The initial MAP reduction was 11.5 (7-20) mm Hg, occurring 4 to 8 min after connection. MAP reduction was 9 (6-15) mm Hg with polyacryonitrile (PAN) membranes versus 14 (5-19) mm Hg with polysulfone (PS) (not significant). There were positive correlations between MAP reduction and BK concentration at 3 (BK3; r = 0.58, p < 0.01) and 6 (BK6; r = 0.67, p < 0.001) min with PAN but not with PS. A greater reduction in MAP was seen in patients who were not receiving inotropic support (Mann-Whitney test, p < 0.01). BK3 and BK6 values for the PAN and PS groups were not significantly different. However, BK concentrations greater than 1,000 pg/ml were only seen with PAN (6 patients, MAP reduction 27 [17-31] mm Hg). There were positive (albumin) and negative (age; acute physiology, age, and chronic health evaluation score; C-reactive protein [CRP]; calcium) correlations with BK3/BK6 in the PAN and PS groups, some of which (albumin, CRP) reached statistical significance. In summary, MAP reduction at the start of CRRT correlates with BK concentration. The similarity of response with PAN and PS suggests an importance for other clinical factors. In this study, hemodynamic instability was more likely in patients with evidence of a less severe inflammatory or septic illness.

Differential localization of protein kinase C isotypes in equine eosinophils and neutrophils.

Phorbol esters, which activate protein kinase C (PKC), stimulate equine eosinophil superoxide production and adherence. After showing that superoxide production could be inhibited by the nonselective PKC inhibitors, staurosporine and bisindolymaleimide I, the PKC isotypes in equine eosinophils were characterized, because evidence suggests that individual isotypes may play distinct roles in regulating eosinophil function. Western blots demonstrated that equine eosinophils expressed PKC alpha, beta, delta, epsilon, iota, and zeta. However, unlike the equine neutrophil, the majority of the PKC was detected in the particulate fraction of the cell. Despite this unusual location, the PKC in equine eosinophils was activatable, suggesting that it is functionally competent. The regulatory role of PKC in equine eosinophils may reflect the association of activity with the particulate fraction and the profile of isotype expression.

Expression of an antigen associated with basal bodies of human ciliated epithelial cells.

The process and regulation of ciliogenesis in human epithelia is little understood and many components of the cilium and associated structures have not been characterised. We have identified a monoclonal antibody, LhS28, which recognises a 44,000-45,000 M(r) protein specifically associated with human ciliated epithelial cells. Immunoperoxidase labelling of formalin-fixed paraffin wax-embedded human tissues showed that LhS28 was expressed in the sub-apical zone of ciliated epithelial cells of the Fallopian tube and upper respiratory tract, but not ciliated ependyma, non-ciliated epithelia or testis containing developing spermatozoa. Immunoelectron microscopy demonstrated that the antigen recognised by LhS28 was associated with the basal body structure of the cilium and specifically with the 9 + 0 microtubule arrays. LhS28 should be a useful tool in the identification of ciliated cells in pathological specimens and for investigating mechanisms of ciliogenesis.

Isoform specificity of activators and inhibitors of protein kinase C gamma and delta.

Expression of certain mammalian protein kinase C (PKC) isoforms inhibits the proliferation of Schizosaccharomyces pombe (Goode et al., Mol. Biol. Cell 5 (1994) 907-920). We have taken advantage of this fact to determine the in vivo isoform preference of a number of PKC inhibitors, using a microtitre plate assay which allows rapid screening. This in vivo model has revealed previously unreported preferences; calphostin C is a more efficient inhibitor of the novel PKCS than chelerythrine chloride whereas the efficiencies are reversed for inhibition of the classical PKCgamma. We have also shown that the anti-leukaemic agent bryostatin 1 inhibits or activates in vivo in an isoform-specific manner.

Alterations of glomerular basement membrane charge and structure in diabetic nephropathy.

We examined glomerular basement membrane anionic site distribution identified by cationic gold in seven patients with insulin-dependent and four patients with non-insulin-dependent diabetes mellitus, presenting a spectrum of clinical and glomerular changes. Anionic sites were investigated by pretreatment of tissue with glycosaminoglycan-degrading enzymes prior to cationic gold staining. The distribution of chondroitin sulphate proteoglycans--a previously unrecognized glomerular basement membrane component--and type IV collagen was examined by immunoelectron microscopy to identify structural changes in the basement membrane. Findings were compared with those of non-diabetic patients showing minor proteinuria and morphologically normal glomerular basement membranes. Two patients, originally diagnosed as having diabetic nephropathy were also examined at 19 weeks and 5 years after renal transplantation. Characteristic redistribution of type IV collagen and chondroitin sulphate proteoglycans was noted in thickened glomerular basement membrane segments (> 400 nm) of diabetic patients and those with renal transplants. Extension of anionic sites deep into the glomerular basement membrane at pH 2.5, together with loss of interna sites at pH 5.8 is unique to diabetic nephropathy. Reduced charge density was apparent in some patients due to thickening of the glomerular basement membrane, although the number of anionic sites per unit length of membrane was actually increased. Thus, charge aberration in diabetic nephropathy is due to displacement rather than loss of anionic sites. Removal of more than 90% of these sites by heparitinase, confirms their association with heparan sulphate proteoglycans. Similar derangement of anionic sites in all patients with diabetic nephropathy irrespective of the degree of proteinuria, suggests that a heparan sulphate proteoglycan-related charge barrier plays a minor role in controlling permeability of the diabetic glomerular basement membrane.

Clinical features and outcome of patients with thin and ultrathin glomerular membranes.

There is considerable disagreement regarding the natural history of renal disease associated with thin glomerular basement membranes (TGBM). We followed 43 patients (19 male), mean age 41.6 years (range 19-73) for a mean of 88 months (48-140). TGBM was recognized in adults when glomerular basement membrane thickness, measured from multiple sites in electronmicrographs of renal biopsy tissue as the harmonic mean, was < 320 nm. At presentation, 95% had microscopic haematuria, 12% macroscopic haematuria, 14% loin pain, 28% proteinuria, and 14% hypertension. There was no difference in GBM width between the sexes (male 258 nm vs. female 251 nm) but there was a significant negative correlation between age and GBM width (r = -0.53, p < 0.001), with older patients having the thinnest membranes. Twenty six patients had ultrathin GBM (< 270 nm), of whom 54% had 3+ haematuria vs. 12% of the group with BM > 270 nm (p < 0.01). In the ultrathin group, 71% had loss of anionic charge from the GBM, vs. 17% in those with membranes which were thin but > 270 nm (p < 0.05). Proteinuria occurred more frequently in those with GBM > 270 nm, 65% vs. 8% in the ultrathin group (p < 0.01). Thin GBM were associated with a benign prognosis, as after a mean follow-up of 85 months (48-140), there was no significant change in either serum creatinine or mean arterial blood pressure. Patients with ultrathin GBM had greater loss of GBM anionic charge, which might result in both an alteration of flow characteristics within the glomerular capillaries and also increased fragility of the glomerular basement membrane with likelihood of rupture and resultant macroscopic haematuria.

Protein kinase C (PKC)-induced PKC down-regulation. Association with up-regulation of vesicle traffic.

Phorbol esters cause long term activation of protein kinase C (PKC) and frequently the down-regulation of PKC protein levels in mammalian cells. Mammalian PKC-gamma, -delta, and -eta down-regulated in response to phorbol esters when expressed in Schizosaccharomyces pombe. However, PKC-epsilon does not down-regulate in S. pombe, in contrast to the behavior of this isotype in mammalian cells. Co-expression of PKC-gamma or -delta with PKC-epsilon in S. pombe renders PKC-epsilon susceptible to down-regulation. A protein kinase defective form of PKC-delta does not down-regulate efficiently in S. pombe but, like PKC-epsilon, is susceptible when co-expressed with PKC-gamma or full-length PKC-delta. Thus, down-regulation is a consequence of the catalytic function of certain PKC isotypes with other isotypes being affected in trans. PKC down-regulation parallels a striking accumulation of vesicles in S. pombe, suggesting a direct relationship between these events.

Expression of mammalian protein kinase C in Schizosaccharomyces pombe: isotype-specific induction of growth arrest, vesicle formation, and endocytosis.

Mammalian protein kinase C (PKC) isotypes elicit a number of effects on expression in Schizosaccharomyces pombe. A small decrease in growth rate results from PKC-gamma expression, and treatment of these cells with phorbol esters leads to marked growth inhibition and vesicle formation. PKC-delta and -eta expression causes growth inhibition and vesiculation, and the magnitude of both of these effects is increased by phorbol esters. In contrast, PKC-epsilon expression produces growth inhibition but no vesicle accumulation, and this effect is not responsive to phorbol ester. Finally, PKC-zeta has no observable effect. Thus, isotype-specific biological effects are observed. The accumulation of vesicles correlates with phorbol ester-dependent growth inhibition and occurs only with expression of those isotypes that down-regulate in response to phorbol esters in these cells. Antibodies against mammalian clathrin light chain 1a identified clathrin-coated vesicles and up-regulation of clathrin expression in those cells where vesicles accumulate; the increased vesicular traffic includes an element of endocytosis. Thus expression of specific mammalian PKC isotypes up-regulates endocytosis in S. pombe, providing a likely explanation for PKC-mediated receptor internalization in higher eukaryotes.