An international survey of the nutrition management of chylothorax: a time for change.

INTRODUCTION: Although chylothorax is an uncommon complication following paediatric cardiothoracic surgery, it has significant associated morbidities and increased in-hospital mortality, as well as results in higher costs. A lack of prospective evidence or consensus guidelines for management of chylothorax further hinders optimal management. The aim of this survey was to characterise variations in practice in the management of chylothorax and to identify areas for future research. MATERIALS AND METHODS: A descriptive, observational survey investigating conservative management practices of chylothorax was distributed internationally to health-care professionals in paediatric intensive care and cardiology units. The survey investigated five domains: the first providing general information about health-care professionals and four domains focusing on clinical practice including diet composition and duration. RESULTS: In total, sixty-four health-care professionals completed the survey, representing 38 organisations from 16 countries. The respondents were dietitians (80%), physicians (19%), and nurses (1%). In Australia and New Zealand, management was most commonly directed by physicians' preference (67%) as compared to unit protocols in Europe (67%), United States of America (67%), and Other regions (55%). Dietitians in Australia/New Zealand, United Kingdom, and Ireland followed the most restrictive diet therapy recommending <5 g long chain triglyceride fat per day (p < 0.00001). The duration of diet therapy significantly varied between regions: Australia/New Zealand: 4 weeks (36%) and 6 weeks (43%); Europe: 4 weeks (25%) and 6 weeks (57%); and North America: 4 weeks (18%) and 6 weeks (75%) (p < 0.00001). CONCLUSIONS: This survey highlights international variations in practice in the management of chylothorax, particularly with respect to treatment duration and dietary fat restriction. Future research should include a multi-centre randomised controlled trial to inform evidence-based practice and reduce morbidity, particularly poor growth.

Identification of glucokinase mutations in subjects with gestational diabetes mellitus.

Recent studies have shown that mutations in the glucokinase gene on chromosome 7 can cause an autosomal dominant form of NIDDM with a variable clinical phenotype and onset during childhood. The variable clinical phenotype includes mild fasting hyperglycemia (i.e., a plasma glucose value of > 110 mg/dl, a value that is at least 2-3 SDs above normal), impaired glucose tolerance, gestational diabetes mellitus, as well as overt NIDDM as defined using National Diabetes Data Group or World Health Organization criteria. Because gestational diabetes mellitus was a clinical feature associated with glucokinase mutations, we have screened a group of women with gestational diabetes who also had a first-degree relative with diabetes mellitus for the presence of mutations in this gene. Among 40 subjects, we identified two mutations, suggesting a prevalence of approximately 5% in this group. Extrapolating from this result, the prevalence of glucokinase-deficient NIDDM among Americans may be approximately 1 in 2500.

Cortical remodeling following suppression of endogenous estrogen with analogs of gonadotrophin releasing hormone.

The effects of estrogen suppression on osteonal remodeling in young women was investigated using transiliac biopsies (eight paired biopsies + four single pre; three single post biopsies) taken before and after treatment for endometriosis (6 months) with analogs of gonadotrophin releasing hormone (GnRH). Estrogen withdrawal increased the proportion of Haversian canals with an eroded surface (106%, p = 0.047), a double label (238%, p = 0.004), osteoid (71%, p = 0.002), and alkaline phosphatase (ALP) 116%, p = 0.043) but not those showing tartrate-resistant acid phosphatase (TRAP) activity (p = 0.25) or a single label (p = 0.30). Estrogen withdrawal increased TRAP activity in individual osteoclasts in canals with diameters greater than 50 microns (p = 0.0089) and also the number of osteons with diameters over 250 microns (p = 0.049). ALP activity in individual osteoblasts was increased but not significantly following treatment (p = 0.051). Wall thickness was significantly correlated with osteon diameter (p < 0.001). In a separate group of patients (four pairs + one post biopsy) on concurrent treatment with tibolone, there was no significant increase in the osteon density, cortical porosity, median canal diameter, or the markers of bone formation and resorption. Enzyme activities and numbers of active canals were also not increased with the concurrent treatment, but there was still an increase in the osteon diameter. As previously shown for cancellous bone, estrogen withdrawal increased cortical bone turnover. We have now shown that resorption depth within Haversian systems was also increased with treatment. The enhanced TRAP activity in individual osteoclasts supports the concept that osteoclasts are more active following estrogen withdrawal in agreement with theoretical arguments advanced previously. Understanding the cellular and biochemical mechanisms responsible for increased depth of osteoclast resorption when estrogen is withdrawn may allow the development of new strategies for preventing postmenopausal bone loss.

Intracapsular hip fracture and the region-specific loss of cortical bone: analysis by peripheral quantitative computed tomography.

Generalized bone loss within the femoral neck accounts for only 15% of the increase in intracapsular hip fracture risk between the ages of 60 and 80 years. Conventional histology has shown that there is no difference in cancellous bone area between cases of intracapsular fracture and age and sex-matched controls. Rather, a loss of cortical bone thickness and increased porosity is the key feature with the greatest change occurring in those regions maximally loaded during a fall (the inferoanterior [IA] to superoposterior [SP] axis). We have now reexamined this finding using peripheral quantitative computed tomography (pQCT) to analyze cortical and cancellous bone areas, density, and mass in a different set of ex vivo biopsy specimens from cases of intracapsular hip fracture (female, n = 16, aged 69-92 years) and postmortem specimens (female, n = 15, aged 58-95 years; male, n = 11, aged 56-86 years). Within-neck location was standardized by using locations at which the ratio of maximum to minimum external diameters was 1.4 and at more proximal locations. Cortical widths were analyzed using 72 radial profiles from the center of area of each of the gray level images using a full-width/half-maximum algorithm. In both male and female controls, cancellous bone mass increased toward the femoral head and the rate of change was gender independent. Cancellous bone mass was similar in cases and controls at all locations. Overall, cortical bone mass was significantly lower in the fracture cases (by 25%; p < 0.001) because of significant reductions in both estimated cortical area and density. These differences persisted at locations that are more proximal. The mean cortical width in the cases was significantly lower in the IA (22.2%;p = 0.002) and inferior regions (19%;p < 0.001). The SP region was the thinnest in both cases and controls. These data confirm that a key feature in the etiology of intracapsular hip fracture is the site-specific loss of cortical bone, which is concentrated in those regions maximally loaded during a fall on the greater trochanter. An important implication of this work is that the pathogenesis of bone loss leading to hip fracture must be by a mechanism that varies in its effect according to location within the femoral neck Key candidate mechanisms would include those involving locally reduced mechanical loading. This study also suggests that the development of noninvasive methodologies for analyzing the thickness and estimated densities of critical cortical regions of the femoral neck could improve detection of those at risk of hip fracture.

Structure of the femoral neck in hip fracture: cortical bone loss in the inferoanterior to superoposterior axis.

Although bone mass is a contributory risk factor for hip fracture, its distribution about the femoral neck is also important. Femoral neck biopsies were obtained from 13 females with intracapsular hip fracture (fracture: mean age 74.3 +/- 2.3 years [SEM]) and 19 cadaveric samples (control: 9 males and 10 females 79.4 +/- 1.7 years) and the areas of cortical and cancellous bone were quantitated in octants. In the control group, although males had larger bones than females, the proportions of cortical and cancellous bone were not different (p > 0.05) between the genders. The total amount of bone, as a proportion of bone + marrow, was significantly reduced in the fractures compared with the female controls (%Tt.Ar: fracture 27.83 +/- 1.18, female control 33.62 +/- 1.47; p = 0.0054). Reductions in cortical bone area occurred in all regions but particularly in the inferior, inferoanterior, and anterior octants (p < 0.05). There were no differences between cases and controls in the regional amount of cancellous bone (all regions, p > 0.178). Marked reductions in mean cortical bone width between the fracture and female control group occurred in the anterior, inferoanterior (31%), and superoposterior (25%) regions. Representing cortical widths as simple Fourier functions of the angle about the center of area (R2adj = 0.79) showed in the cases that there was preservation of the cortical bone in the inferior region, with the proportional loss of cortical bone being greatest in the inferoanterior and superoposterior regions. It is concluded that loss of cortical, rather than cancellous, bone predominates in cases of femoral neck fracture. This loss occurs primarily along the inferoanterior to superoposterior axis. As this axis bears the greatest strain during a fall, it is hypothesized that specific thinning of the cortex in these regions leads to an exaggerated propensity to fracture in those so affected, above that resulting from an equivalent general decrease in bone mass.

Neuropsychological detection and characterization of preclinical Alzheimer's disease.

We attempted to characterize the changes in cognition associated with the earliest, or preclinical, stages of Alzheimer's disease (AD) by administering a comprehensive neuropsychological test battery to a group of initially nondemented older adults participating in a prospective epidemiologic study of dementia. Using Cox regression analyses, we examined the associations between baseline neuropsychological test scores and subsequent development of AD. Results confirmed preliminary findings that baseline scores on the Boston Naming Test, Immediate Recall on the Selective Reminding Test, and the Similarities subtest of the Wechsler Adult Intelligence Scale-Revised were significantly and independently associated with later diagnosis of AD. Analyses controlled for the effects of age, education, sex, and language of test administration. These results lend support to the notion of a preclinical phase of AD and indicate that this very early stage of AD is characterized by poor word-finding ability, abstract reasoning, and memory.

Cognitive function in nondemented older women who took estrogen after menopause.

Investigations of the effects of estrogen replacement on cognitive function in healthy older women have yielded disparate results. We evaluated the relationship between a history of estrogen use and cognitive test performance in 727 women participating in a large community-based study. Participants were followed longitudinally for an average of 2.5 years. Estrogen use history was obtained at baseline. Standardized tests of memory, language, and abstract reasoning were administered at baseline and at follow-up. Results indicate that women who had used estrogen replacement scored significantly higher on cognitive testing at baseline than nonusers, and their performance on verbal memory improved slightly over time. The effect of estrogen on cognition was independent of age, education, ethnicity, and APOE genotype. Results suggest that estrogen replacement therapy may help to maintain cognitive function in nondemented postmenopausal women.

Clinical characteristics of a family with chromosome 17-linked disinhibition-dementia-parkinsonism-amyotrophy complex.

We studied the clinical features, pathology, and molecular genetics of a family (Mo) with an autosomal dominant disinhibition, frontal lobe dementia, parkinsonism, and amyotrophy. We examined seven affected members and gathered clinical information on another six. The mean onset was at age 45 years. Personality and behavioral changes (disinhibition, withdrawal, alcoholism, hyperphagia) were the first symptoms in twelve. There was early memory loss, anomia, and poor construction with preservation until late of orientation, speech, and calculations. All affected members examined had rigidity, bradykinesia, and postural instability. Mean duration to death was 13 years. We studied the neuropathology of six individuals, five of whom had been examined in life. There was atrophy and spongiform change in the frontotemporal cortex, and neuronal loss and gliosis in the substantia nigra and amygdala. Two individuals, including one with fasciculations and muscle wasting, had anterior horn cell loss. There were no Lewy bodies, neurofibrillary tangles, or amyloid plaques. We call this disorder the "disinhibition-dementia-parkinsonism-amyotrophy complex" (DDPAC), based on the clinical syndrome found in this family and linkage to chromosome 17.

Spatial clustering of remodeling osteons in the femoral neck cortex: a cause of weakness in hip fracture?

Intracapsular femoral neck fractures are associated with decreased cortical width and increased proportions of Haversian canals with diameters greater than the normal mean plus 3 SD (i.e., >385 microm). Such canals might be formed if closely associated resorbing osteons merge; a cortical event analogous with the loss of cancellous connectivity. To test this, we investigated the pattern of osteon distribution in the aging femoral neck to determine if remodeling osteons were distributed in anatomical clusters. Femoral neck biopsies from female patients with intracapsular hip fractures (n = 13) were compared with age/gender-matched cadaveric controls (n = 13). Solochrome-stained sections were analyzed for Haversian canal location, canal diameter, and the presence of an osteoid surface. Clustering was investigated using statistical software with a cluster defined as two or more osteoid-bearing osteon centers within 0.75 mm of each other. Clusters occurred more frequently than would be expected by chance (p < 0.001). Fracture cases had more clusters per unit area (3.14 +/- 0.31 clusters/25 mm2 of cortical bone) than controls (1.89 +/- 0.22) (p = 0.002). In fracture cases, the antero-inferior, antero-superior, and infero-anterior regions had more clusters per 25 mm2 than comparable control regions (ant/inf: 4.12 +/- 0.79, 1.70 +/- 0.60,p = 0.025; ant/sup: 5.31 +/- 1.1, 1.80 +/- 0.59,p = 0.013; inf/ant: 3.15 +/- 0.49, 1.27 +/-0.29, p = 0.004). The mean number of clusters per 25 mm2 per region correlated with the mean porosity per region (adjusted r2 = 0.60;p = 0.014), and the total number of giant canals per region correlated with the total number of clusters per region (adjusted r2 = 0.58; p = 0.011). In conclusion, remodeling osteons are clustered or grouped anatomically, and fracture cases have more clusters than controls. Our data suggest that merging of adjacent, clustered osteons during resorption could lead to the rapid development of canals with excessive diameters and focal weakness. Clustering is greatest in those regions that we have previously shown to have the largest relative reductions in bone strength compared with controls and known to be maximally loaded during a sideways fall. This implicates the remodeling process underlying clustering of remodeling osteons in the aetiology of hip fracture.

Cortical and cancellous bone in the human femoral neck: evaluation of an interactive image analysis system.

An interactive image analysis package was developed to examine whole cross-sections from the femoral neck. The package quantifies cortical width (Ct.Wi), cortical porosity (Ct.Po), and proportions of cortical, cancellous bone as a percentage of bone plus marrow area. Segmental analysis was used to quantify circumferential variations in bone distribution within the femoral cross-section. To evaluate reproducibility of data four independent operators analyzed previously prepared femoral neck sections from a 2000 BC population. Differences in total and circumferential distributions of cortical and cancellous bone with respect to gender and age of samples were demonstrated. Reproducibility was assessed using coefficients of variation (CV). Analysis of sections using a variable magnification, giving largest possible image size, rather than a set magnification reduced variation between operators for all measurements. Use of a calculated threshold significantly decreased variation between operators for the proportions of cortical and cancellous bone (p < or = 0.026). Dividing the image into 8 rather than 16 segments also improved reproducibility. There was little agreement between operators in the determination of cortical porosity. The mean CV for the other quantitative indices such as cortical width and proportions of cortical and cancellous bone ranged from 4.87% to 13.52%. The genders showed similar patterns in circumferential distribution of bone. Cortical width was significantly greater in the inferior region compared to the other areas, whereas percent cortical bone was lowest at the superior region. The center of mass (COM) for the younger age group was located anteriorly, whereas in the older samples the COM was located posteriorly of the center of area (p = 0.041). Basic data relating to cortical and cancellous bone of acceptable reproducibility in comparison with current standards in iliac histomorphometry can now be provided at modest cost in operator time and resources.

A novel mechanism for induction of increased cortical porosity in cases of intracapsular hip fracture.

It has been suggested that, in hip fracture, the cortex on the inferoanterior (IA) to superoposterior (SP) axis is thinned and shows increased porosity. This is dependent on the presence of giant canals (i.e., diameter >385 microm), which are related to clusters of remodeling osteons. To investigate further the relationship between remodeling and bone loss, osteonal diameter (On.Dm), wall thickness (W.Th), osteoid width (O.Wi), and extent (OS) were measured in femoral neck biopsies from 12 female intracapsular hip fracture cases and 11 age- and gender-matched controls. Over 83% of giant canals were "composite" osteonal systems in which a single canal was surrounded by multiple packets of osteonal bone. Among smaller canals, over 80% of systems had a canal encircled by a single cement line containing one packet of bone ("simple"). Composites were nearly twice as prevalent in fractures (fracture cases 9.8 +/- 0.7/25 mm(2), controls 5.3 +/- 0.4/25 mm(2), p < 0. 0001), and were dependent (R(2) = 0.52) on femoral neck region (p = 0.0008) and the regional distribution of clusters of remodeling osteons (p = 0.0045). Both the inferior (I) and anterior (A) regions had an elevated number of composites (I: 263% of control values, p = 0.0054; A: 202% of control values, p = 0.0092). On.Dm was similar in fracture cases and controls (simple: fracture cases 183 +/- 3 microm, controls 191 +/- 4 microm; composites: fracture cases 446 +/- 13 microm, controls 460 +/- 13 microm). W.Th in simples was similar in fracture cases and controls (fracture cases 51 +/- 0.8 microm, controls 49 +/- 0.7 microm), but composites had significantly (p < 0. 0001) thinner walls, with the reduction in fracture cases (31%) being twice that of controls (12%, p < 0.0001). There were no differences in O.Wi. It was unusual for osteoid to fully surround the composite canal surface; OS was 38% lower in composite than simple canals (p < 0.0001). This study indicates that, in the femoral neck cortex, the principal remodeling deficit in hip fracture is specific to composite osteons. Hip fracture cases had zonal increases in composite osteon density with reduced bone formation. The data suggest that generation of composite osteons is a plausible mechanism leading to increasing porosity and trabecularization of the cortex, thus weakening the cortex in regions maximally loaded on fall impact.

The effects of hormone replacement therapy on cortical bone in postmenopausal women. A histomorphometric study.

Investigations of the actions of estrogen on the skeleton have mainly focused on cancellous bone and there are no reported histomorphometric studies of the effects of oestrogen on cortical bone in humans. The aim of this study was to investigate the effects of both conventional hormone replacement therapy (HRT) and high-dose oestradiol on cortical bone in postmenopausal women. Transiliac biopsies were obtained from nine postmenopausal women aged 54-71 yr before and after 2 yr (mean, 23.5 months) of conventional HRT and in seven postmenopausal women aged 52-67 yr after long-term, high-dose oestradiol implant therapy (at least 14 yr). Indices of bone turnover, remodeling, and cortical structure were assessed by image analysis. Cortical width was highest in the women treated with high-dose oestrogen therapy (2.29 +/- 0.78 mm; mean +/- SD) and lowest in untreated women (1.36 +/- 0.60 mm; P=0.014). The proportion of canals with an eroded surface was significantly lower in the high-dose oestrogen group than in women before or after conventional HRT (3.03 +/- 3.7% vs. 11.1 +/- 7.1% and 10.5 +/- 8.6%; P=0.017 and 0.05, respectively). Bone formation rate (microm2/microm/day) in untreated women was significantly higher than in the high-dose oestrogen group (0.121 +/- 0.072 vs. 0.066 +/- 0.045, respectively; P=0.05), values in women treated with conventional HRT being intermediate. Our results provide the first histomorphometric evidence in postmenopausal women of dose-dependent oestrogen-induced suppression of bone turnover in iliac crest cortical bone. There was also a trend toward higher wall width with increasing dose of oestrogen, consistent with the previously reported anabolic effect in cancellous bone.

Regional differences in cortical porosity in the fractured femoral neck.

Although bone mass is a contributory risk factor for intracapsular hip fracture, its distribution and porosity within the femoral neck is also important for bone strength. In femoral neck biopsies from 13 women with intracapsular hip fracture (mean +/- SEM: 75.4+/-2.1 years, OP) and 19 cadaveric samples (9 men and 10 women [control], aged 79.4+/-1.7 years), a segmental analysis was used to quantify circumferential variations in the characteristics of cortical bone haversian systems. In female control femoral necks, there was an increasing porosity gradient between the inferior (I) (7.7+/-0.6%) and superior regions (S) (16.05+/-1.8%,p < 0.005). In walking, these regions undergo compression and tension, respectively. In men, a similar trend was observed, but the differences were not significant (I: 11.1+/-1.2%; S: 14.1+/-1.7%; p = 0.133). This porosity gradient was not maintained in the fracture group (I: 10+/-1%; S: 12.65+/-1.2%). In contrast, porosity in the fracture group was greatest in the anterior cortex, being 41% higher in that quadrant than in controls (p = 0.06). The areal density of haversian canals ranged from 16.7 to 21.3 canals/mm2 with no significant differences between fractures and controls. In the control women, mean canal diameter was highest in the superior region (60+/-2.8 microm), and these canals were significantly larger than those in the inferior region (49.4+/-1.4 microm, p < 0.05). This difference was less marked in the fracture cases (I: 53.21+/-2.5 microm; S: 59.1+/-2.8 microm; p = 0.0878). Although the mean canal diameter in the anterior quadrant of the fracture cases was higher than in the control women this did not reach significance (OP/F: 59.5+/-3 microm; control/F: 52.7+/-2.6 microm; p = 0.106). However, the proportion of "giant" canals with diameters >385 microm (defined as the top 0.5% in the controls) was doubled in the anterior region of the fracture cases (OP/F: 1.28%; control/F: 0.69%; p < 0.005). Porosity is related to the square of the canal radius; therefore, such canals make a substantial contribution to cortical porosity. Previous work has shown that the elastic modulus of bone decreases approximately as the square root of porosity. Therefore, the increased porosity and the higher prevalence of "giant" canals have a markedly negative influence on the ability of the cortical shell to withstand stresses associated with a fall. The mechanisms responsible for the localized generation of "giant" haversian canals, and ultimately the "trabecularization" of the cortex, require further investigation.

Effect of estrogen suppression on the mineralization density of iliac crest biopsies in young women as assessed by backscattered electron imaging.

The effects of estrogen suppression on bone mineralization in young women were studied by quantitative backscattered electron (BSE) imaging of transiliac biopsies taken before and after treatment for endometriosis. Treatment (6 months) was with analogs of gonadotrophin releasing hormone (GnRH) given either alone (six paired biopsies), which resulted in a marked reduction in the levels of circulating estrogen, or in conjunction with tibolone, a synthetic steroid with estrogenic, progestrogenic, and androgenic properties (four paired biopsies). Estrogen withdrawal increased (p < 0.01) and concomitant tibolone treatment decreased (p < 0.05) the overall mean bone density. Estrogen withdrawal increased the fraction of bone with a high mineralization density [pretreatment: 0.236+/-0.007; GnRH: 0.279+/-0.009, mean +/- standard error of the mean (SEM); p < 0.01]. The concomitant addition of tibolone reversed these effects and increased the proportion of bone with a low mineralization density (pretreatment: 0.198 +/- 0.005; tibolone: 0.230 +/-0.008, p < 0.01). Using previously published data, the mean bone density was inversely correlated with mean wall thickness in cancellous bone (p = 0.030) and with the percentage of active osteons (p = 0.023) in cortical bone. Although treatment had similar effects on the mean bone mineralization density of cortical and cancellous bone, there were different distributions of mineralization between the two sites, with cancellous bone having more skewed and kurtotic distributions both before and after estrogen withdrawal. This study indicates that a short-term estrogen suppression results in the accumulation of bone with a higher mineralization density. As bone with a high mineral content has a decreased impact resistance, this might increase fracture risk. Understanding the cellular and biochemical mechanisms responsible for the local distribution of bone mineral when estrogen is withdrawn may allow the development of new strategies for maintaining bone quality after menopause.

Increased femoral neck cancellous bone and connectivity in coxarthrosis (hip osteoarthritis).

Patients with coxarthrosis (cOA) have a reduced incidence of intracapsular femoral neck fracture, suggesting that cOA offers protection. The distribution of bone in the femoral neck was compared in cases of coxarthrosis and postmortem controls to assess the possibility that disease-associated changes might contribute to reduced fragility. Whole cross-section femoral neck biopsies were obtained from 17 patients with cOA and 22 age- and sex-matched cadaveric controls. Densitometry was performed using peripheral quantitated computed tomography (pQCT) and histomorphometry on 10-microm plastic-embedded sections. Cortical bone mass was not different between cases and controls (P > 0.23), but cancellous bone mass was increased by 75% in cOA (P = 0.014) and histomorphometric cancellous bone area by 71% (P < 0.0001). This was principally the result of an increase of apparent density (mass/vol) of cancellous bone (+45%, P = 0.001). Whereas cortical porosity was increased in the cases (P < 0.0001), trabecular width was also increased overall in the cases by 52% (P < 0.001), as was cancellous connectivity measured by strut analysis (P < 0.01). Where osteophytic bone was present (n = 9) there was a positive relationship between the amount of osteophyte and the percentage of cancellous area (P < 0.05). Since cancellous bone buttresses and stiffens the cortex so reducing the risk of buckling, the increased cancellous bone mass and connectivity seen in cases of cOA probably explain, at least in part, the ability of patients with cOA to resist intracapsular fracture of the femoral neck during a fall.

Functional and electrophysiological effects of a novel imidazoline-based K(ATP) channel blocker, IMID-4F.

1. The functional and electrophysiological effects of IMID-4F (2-[N-(2, 6-dichlorophenyl)-N-(4-flurorobenzyl)amino]imidazoline), a fluoro-benzyl derivative of clonidine, on vascular K(ATP) channels were investigated. In pig coronary artery, IMID-4F inhibited the vasorelaxation response to the K(ATP) channel opener levcromakalim with a pK(B) value of approximately 7.1. IMID-4F (30 microM) did not affect the vasorelaxation response to sodium nitroprusside (SNP). 2. In rat mesenteric artery smooth muscle cells IMID-4F (1 - 10 microM) caused a concentration-dependent depolarization of membrane potential. IMID-4F (10 microM) abolished the hyperpolarizing effects of levcromakalim (10 microM). 3. In patch clamp experiments using rat mesenteric artery smooth muscle cells, K(ATP) channel currents induced by levcromakalim (10 microM) were inhibited by IMID-4F (0.3 - 3 microM) in a concentration-dependent manner. The calculated IC(50) for IMID-4F inhibiting K(ATP) channel current was approximately 0.8 microM. 4. Radioligand binding studies using bovine aortic smooth muscle cell membranes showed that IMID-4F (30 microM) did not displace binding to the K(ATP) channel opener [(3)H]-P1075. However, both levcromakalim (10 microM) and glibenclamide (10 microM) caused significant displacement of [(3)H]-P1075. 5. These studies show that the imidazoline compound IMID-4F is one of the most potent antagonists of arterial K(ATP) channels identified. Vasorelaxation, hyperpolarization and K(+) currents through K(ATP) channels were all inhibited by IMID-4F at micromolar concentrations. Radioligand binding studies indicate that IMID-4F does not bind to the same site as levcromakalim or as glibenclamide. Considering other evidence, it is likely that IMID-4F acts by interacting directly with the pore of the K(IR) channel, rather than through the sulphonylurea subunit of the K(ATP) channel complex.

In vitro superoxide production by hyaline cells of the shore crab Carcinus maenas (L.).

This study examines the phagocytic hyaline cells of the shore crab, Carcinus maenas, for a respiratory burst in vitro. Following stimulation of the cells with phorbol myristate acetate (PMA), ferricytochrome c was reduced at 550 nm. Addition of superoxide dismutase (SOD) to the reaction mixture decreased this reduction, confirming that superoxide ions are produced by the stimulated hemocytes. Phytohemagglutinin, con A, and LPS were also shown to stimulate the cells although laminarin failed to elicit a burst. This is the first demonstration of a respiratory burst for crustacean hemocytes.

Hydrolysis of organophosphorus insecticides by in vitro modified carboxylesterase E3 from Lucilia cuprina.

Resistance of the blowfly, Lucilia cuprina, to organophosphorus (OP) insecticides is due to mutations in LcalphaE7, the gene encoding carboxylesterase E3, that enhance the enzyme's ability to hydrolyse insecticides. Two mutations occur naturally, G137D in the oxyanion hole of the esterase, and W251L in the acyl binding pocket. Previous in vitro mutagenesis and expression of these modifications to the cloned gene have confirmed their functional significance. G137D enhances hydrolysis of diethyl and dimethyl phosphates by 55- and 33-fold, respectively. W251L increases dimethyl phosphate hydrolysis similarly, but only 10-fold for the diethyl homolog; unlike G137D however, it also retains ability to hydrolyse carboxylesters in the leaving group of malathion (malathion carboxylesterase, MCE), conferring strong resistance to this compound. In the present work, we substituted these and nearby amino acids by others expected to affect the efficiency of the enzyme. Changing G137 to glutamate or histidine was less effective than aspartate in improving OP hydrolase activity and like G137D, it diminished MCE activity, primarily through increases in Km. Various substitutions of W251 to other smaller residues had a broadly similar effect to W251L on OP hydrolase and MCE activities, but at least two were quantitatively better in kinetic parameters relating to malathion resistance. One, W251G, which occurs naturally in a malathion resistant hymenopterous parasitoid, improved MCE activity more than 20-fold. Mutations at other sites near the bottom of the catalytic cleft generally diminished OP hydrolase and MCE activities but one, F309L, also yielded some improvements in OP hydrolase activities. The results are discussed in relation to likely steric effects on enzyme-substrate interactions and future evolution of this gene.

Some pharmacological studies of venom from the inland taipan (Oxyuranus microlepidotus).

The present study was designed to obtain a basic pharmacological profile of venom from the inland taipan (Oxyuranus microlepidotus). Venom (0.05-50 micrograms/ml) produced dose-dependent contractions in guinea-pig ileum, which could not be reproduced upon second administration. The cyclooxygenase inhibitor indomethacin (1 microM), a preceding anaphylactic response induced by egg albumin and inactivation of phospholipase A2 (PLA2) by incubation with 4-bromophenacyl bromide (1.8 mM) all significantly inhibited responses to venom (0.5 micrograms/ml). Venom (0.5 micrograms/ml) caused inhibition of stimulation-induced contractions in the prostatic segment of rat vas deferens which was not significantly affected by the alpha 2-adrenoceptor antagonist idazoxan (0.3 microM). Venom (10 micrograms/ml) caused time-dependent inhibition of the rat electrically stimulated phrenic nerve-diaphragm preparation, positive inotropic and chronotropic responses in rat isolated atria and relaxation in rat endothelium-denuded and -intact isolated aortae. In endothelium-intact aortae, the nitric oxide synthase inhibitor N-nitro-L-arginine (NOLA, 0.1 mM) significantly inhibited the response to venom (10 micrograms/ml). Venom (50 micrograms/kg, i.v.) caused an immediate drop in blood pressure followed by cardiovascular collapse in anaesthetised rats. Venom (10 micrograms/kg, i.v.) caused a gradual fall in blood pressure which was sometimes accompanied by a temporary cessation of respiration. A PLA2 assay detected the presence of PLA2 in the venom. These results suggest that the venom contains a component capable of causing the synthesis of arachidonic acid metabolites and a component capable of relaxing vascular smooth muscle. The inhibitory effect on the phrenic nerve-diaphragm is probably due to the previously identified neurotoxin (paradoxin).

The smooth muscle relaxant effects of venom from the inland taipan (Oxyuranus microlepidotus).

Venom (10 microg/ml) relaxed phenylephrine-precontracted aortae. This relaxation was unaffected by removal of the endothelium or a combination of N(G)-nitro-L-arginine (L-NOARG; 0.1 mM), oxyhaemoglobin (10 microM) and indomethacin (10 microM). 4-BPB (0.78 mM), propranolol (1 microM), or a combination of apamin (0.1 microM), charybdotoxin (0.1 microM) and glibenclamide (10 microM) did not effect endothelium-independent relaxation, suggesting a lack of PLA2 activity or an effect at beta-adrenoceptors or K+ channels. Venom (10 microg/ml) reversed Bay K 8644 (0.1 microM)-induced contraction indicating the venom may have an effect on L-type Ca2+ channels.