The insulin hypoglycemia test: Hypoglycemic criteria and reproducibility

The insulin hypoglycemia test (IHT) is widely regarded as the 'gold standard' for dynamic stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. This study aimed to investigate the temporal relationship between a rapid decrease in plasma glucose and the corresponding rise in plasma adenocorticotropic hormone (ACTH), and to assess the reproducibility of hormone responses to hypoglycemia in normal humans. Ten normal subjects underwent IHTs, using an insulin dose of 0.15 U/kg. Of these, eight had a second IHT (IHT2) and three went on to a third test (IHT3). Plasma ACTH and cortisol were measured at 15-min intervals and, additionally, in four IHT2s and the three IHT3s, ACTH was measured at 2.5- or 5-min intervals. Mean glucose nadirs and mean ACTH and cortisol responses were not significantly different between IHT1, IHT2 and IHT3. Combined data from all 21 tests showed the magnitude of the cortisol responses, but not the ACTH responses, correlated significantly with the depth and duration of hypoglycemia. All subjects achieved glucose concentrations of of less than or equal to 1.6 mmol/l before any detectable rise in ACTH occurred. In the seven tests performed with frequent sampling, an ACTH rise never preceeded the glucose nadir, but occurred at the nadir, or up to 15 min after. On repeat testing, peak ACTH levels varied markedly within individuals, whereas peak cortisol levels were more reproducible (mean coefficient of variation 7%). In conclusion, hypoglycemia of less than or equal to 1.6 mmol/l was sufficient to cause stimulation of the HPA axis in all 21 IHTs conducted in normal subjects. Nonetheless; our data cannot reveal whether higher glucose nadirs would stimulate increased HPA axis activity in all subjects. Overall, the cortisol response to hypoglycemia is more reproducible than the ACTH response but, in an individual subject, the difference in peak cortisol between two IHTs may exceed 100 nmol/l.

Response of a scleractinian coral, Stylophora pistillata, to iron and nitrate enrichment

The purpose of this study was to determine whether the addition of iron alone or in combination with nitrate affects growth and photosynthesis of the scleractinian coral, Stylophora pistillata, and its symbiotic dinoflagellates. For this purpose, we used three series of two tanks for a 3-week enrichment with iron (Fe), nitrate (N) and nitrate + iron (NFe). Two other tanks were kept as a control (C). Stock solutions of FeCl3 and NaNO3 were diluted to final concentrations of 6 nM Fe and 2 muM N and continuously pumped from batch tanks into the experimental tanks with a peristaltic pump. Results obtained showed that iron addition induced a significant increase in the areal density of zooxanthellae (ANOVA, p = 0.0013; change from 6.3 +/- 0.7 x 10(5) in the control to 8.5 +/- 0.6 x 10(5) with iron). Maximal gross photosynthetic rates normalized per surface area also significantly increased following iron enrichment (ANOVA, p = 0.02; change from 1.23 +/- 0.08 for the control colonies to 1.81 +/- 0.24 mu mol O-2 cm(-2) h(-1) for the iron-enriched colonies). There was, however, no significant difference in the photosynthesis normalized on a per cell basis. Nitrate enrichment alone (2 muM) did not significantly change the zooxanthellae density or the rates of photosynthesis. Nutrient addition (both iron and nitrogen) increased the cell-specific density of the algae (CSD) compared to the control (G-test, p = 0.3 x 10(-9)), with an increase in the number of doublets and triplets. CSD was equal to 1.70 +/- 0.04 in the Fe-enriched colonies, 1.54 +/- 0.12 in the N- and NFe-enriched colonies and 1.37 +/- 0.02 in the control. Growth rates measured after 3 weeks in colonies enriched with Fe, N and NFe were 23%, 34% and 40% lower than those obtained in control colonies (ANOVA. p = 0.011). (C) 2001 Elsevier Science B.V. All rights reserved.

Quaternary Cu-0.7%Cr-0.3%Fe-X alloys

This research is part of a project whose scope was to investigate the engineering properties of new non-commercial alloy formulations based on the Cu rich corner of the Cu-Fe-Cr ternary system with the primary aim of exploring the development of a new cost-effective high-strength, high-conductivity copper alloy. The aim of the present work was to increase the electrical conductivity and strength of the Cu-0.7wt%Cr-0.3wt%Fe alloy through selective minor additions (less than or equal to0.15 wt%) of elements expected to promote precipitation of dissolved Fe: Ti, B, P, Ni & Y. Such quaternary alloys with reduced Fe in solid solution would be expected to have properties equivalent to or better than those of the Cu-1%Cr reference alloy (Alloy Z). The investigation showed that none of the trace element additions significantly improved the size of the age hardening response or the peak aged electrical conductivity of Alloy A, although further work is required on the influence of Ti. Additions of P and B were detrimental. Other trace additions had little or no effect apart from causing some slight changes to the precipitation kinetics. The mechanical properties of the Cu-0.7%Cr-0.3%Fe alloy made with less expensive high carbon ferrochrome were found to be inferior to those of the equivalent alloy made with low carbon ferrochrome. (C) 2001 Kluwer Academic Publishers.

Resolution of natural groups using iterative assignment tests: an example from two species of Australian native rats (Rattus)

Sympatric individuals of Rattus fuscipes and Rattus leucopus, two Australian native rats from the tropical wet forests of north Queensland, are difficult to distinguish morphologically and are often confused in the field. When we started a study on fine-scale movements of these species, using microsatellite markers, we found that the species as identified in the field did not form coherent genetic groups. In this study, we examined the potential of an iterative process of genetic assignment to separate specimens from distinct (e.g. species, populations) natural groups. Five loci with extensive overlap in allele distributions between species were used for the iterative process. Samples were randomly distributed into two starting groups of equal size and then subjected to the test. At each iteration, misassigned samples switched groups, and the output groups from a given round of assignment formed the input groups for the next round. All samples were assigned correctly on the 10th iteration, in which two genetic groups were clearly separated. Mitochondrial DNA sequences were obtained from samples from each genetic group identified by assignment, together with those of museum voucher specimens, to assess which species corresponded to which genetic group. The iterative procedure was also used to resolve groups within species, adequately separating the genetically identified R. leucopus from our two sampling sites. These results show that the iterative assignment process can correctly differentiate samples into their appropriate natural groups when diagnostic genetic markers are not available, which allowed us to resolve accurately the two R. leucopus and R. fuscipes species. Our approach provides an analytical tool that may be applicable to a broad variety of situations where genetic groups need to be resolved.

The filter system for tertiary treatment of sewage effluent by land application - its performance in a subtropical environment

FILTER is an innovative, CSIRO developed system for treating effluent using high rate land application and subsequent effluent recapture via a closely spaced, subsurface drainage network. We report on the summer performance of a FILTER system established in a subtropical environment on a relatively impermeable swelling clay soil underlain by a deep regional water table. Using secondary treated sewage effluent, the FILTER system produced effluent of tertiary nutrient standards (less than or equal to5 mg/L TN; less than or equal to1 mg/L TP), with salinity levels suitable for subsequent irrigation reuse (EC less than or equal to2.5 dS/m). Removal of faecal coliforms was considerably less effective. The hydraulic loading rate achieved was about two and a half times larger than conventional irrigation demand, but this was associated with high deep percolation losses (e 3 mm/day). Comparisons are made with the original FILTER system developed and tested by Jayawardane et al. in temperate Australia. Suggestions are made for modifications to, and further testing of FILTER in a subtropical environment.

Ivermectin, an unconventional agonist of the glycine receptor chloride channel

The effects of the antihelmintic, ivermectin, were investigated in recombinantly expressed human alpha (1) homomeric and alpha (1)beta heteromeric glycine receptors (GlyRs), At low (0.03 muM) concentrations ivermectin potentiated the response to sub-saturating glycine concentrations, and at higher (greater than or equal to0.03 muM) concentrations it irreversibly activated both alpha (1) homomeric and alpha (1)beta heteromeric GlyRs. Relative to glycine-gated currents, ivermectin-gated currents exhibited a dramatically reduced sensitivity to inhibition by strychnine, picrotoxin, and zinc. The insensitivity to strychnine could not be explained by ivermectin preventing the access of strychnine to its binding site. Furthermore, the elimination of a known glycine- and strychnine-binding site by site-directed mutagenesis had little effect on ivermectin sensitivity, demonstrating that the ivermectin- and glycine-binding sites were not identical. Ivermectin strongly and irreversibly activated a fast-desensitizing mutant GlyR after it had been completely desensitized by a saturating concentration of glycine. Finally, a mutation known to impair dramatically the glycine signal transduction mechanism had little effect on the apparent affinity or efficacy of ivermectin, Together, these findings indicate that ivermectin activates the GlyR by a novel mechanism.

Mark-recapture may reveal more about ecology than about population trends: Demography of a threatened ghost bat (Macroderma gigas) population

Since European settlement in Australia, the geographical range of ghost bats (Macroderma gigas) has contracted northwards. Ghost bats are thought to occur in disjunct populations with little interpopulation migration, raising concerns over the current status and future viability of the southernmost colony, which has also been threatened by mining activity. To address these concerns, demographic parameters of the southernmost colony were estimated from a mark-recapture study conducted during 1975-1981. Female bats gave birth to a single young in late spring, but only 40% (22-70%, 95% CI) of females bred in their second year, increasing to 93% (87-97%, 95% CI) for females greater than or equal to 2 years old. Sixty-five percent of juveniles caught were female. Annual adult survival ranged between 0.57-0.77 for females and 0.43-0.66 for males, and was lowest over winter-spring and greatest in autumn-winter. Juvenile survival for the first year ranged between 0.35-0.46 for females and 0.29-0.42 for males. Adult survival varied among seasons, was negatively associated with rainfall, but was not associated with temperature beyond being lower in late winter. Poor survival may result from the inferior daytime roosts that bats must use if water seepage forces them to leave their normal roosts. Although these age-specific rates of fecundity and survival suggested a declining population, mark-recapture estimates of the population trend indicated stability over the study period. Counts at daytime roosts also suggested a population decline, but were considered unreliable because of an increasing tendency of bats to avoid detection. It is therefore likely that some assumptions in estimating survival were violated. These results provide a caution against the uncritical use of population projections derived from mark-recapture estimates of demographic parameters, and the use of untested indices as the basis for conservation decisions.

Safety and efficacy of low anterior en bloc resection as part of cytoreductive surgery for patients with ovarian cancer

Objective. To examine the feasibility and safety of a low anterior resection of the rectosigmoid plus adjacent pelvic tumour as part of primary cytoreduction for ovarian cancer. Methods. This study included 65 consecutive patients with primary ovarian cancer who had debulking surgery from 1996 through 2000. All patients underwent an en bloc resection of ovarian cancer and a rectosigmoid resection followed by an end-to-end anastomosis. Parameters for safety and efficacy were considered as primary statistical endpoints for the aim of this analysis. Results. Postoperative residual tumour was nil, 1 cm in 14, 34, and 14 patients, respectively. The median postoperative hospital stay was 11 days (range, 6 to 50 days). Intraoperative complications included an injury to the urinary bladder in one patient. Postoperative complications included wound complications (n=14, 21.5%), septicemia (n=9, 13.8%), cardiac complications (n=7, 10.8%), thromboembolic complications (n=5, 7.7%) ileus (n=2, 3.1%) anastomotic leak (n=2, 3.1%) and fistula (n=1, 1.5%). Reasons for a reoperation during the same admission included repair of an anastomotic leak (n=1), postoperative hemorrhage (n=1), and wound debridement (n=1). Wound complications, septicemia, and anastomotic leak formation were more frequent in patients who had a serum albumin level of less than or equal to 30 g/L preoperatively. There was one surgically related mortality in a patient who died from a cerebral vascular accident 2 days postoperatively. Conclusions. An en bloc resection as part of primary cytoreductive surgery for ovarian cancer is effective and its morbidity is acceptably low. (C) 2001 Academic Press.

An improved survival model of hypoxia/ischaemia in the piglet suitable for neuroprotection studies

The purpose of this study, was to develop a newborn piglet model of hypoxia/ischaemia which would better emulate the clinical situation in the asphyxiated human neonate and produce a consistent degree of histopathological injury following the insult. One-day-old piglets (n = 18) were anaesthetised with a mixture of propofol (10 mg/kg/h) and alfentinal (5,5.5 mug/kg/h) i.v. The piglets were intubated and ventilated. Physiological variables were monitored continuously. Hypoxia was induced by decreasing the inspired oxygen (FiO(2)) to 3-4% and adjusting FiO(2) to maintain the cerebral function monitor peak amplitude at less than or equal to5 muV. The duration of the mild insult was 20, min while the severe insult was 30 min which included 10 min where the blood pressure was allowed to fall below 70% of baseline. Control piglets (n=4 of 18) were subjected to the same protocol except for the hypoxic/ischaemic insult. The piglets were allowed to recover from anaesthesia then euthanased 72 It after the insult. The brains were perfusion-fixed, removed and embedded in paraffin. Coronal sections were stained by haematoxylin/eosin. A blinded observer examined the frontal and parietal cortex, hippocampus, basal ganglia, thalamus and cerebellum for the degree of damage. The total mean histology score for the five areas of the brain for the severe insult was 15.6 +/-4.4 (mean +/-S.D., n=7), whereas no damage was seen in either the mild insult (n=4) or control groups. This 'severe damage' model produces a consistent level of damage and will prove useful for examining potential neuroprotective therapies in the neonatal brain. (C) 2001 Elsevier Science BY. All rights reserved.

Reduced Red Cell 2,3-Diphosphoglycerate Concentrations in Critical Illness without Decreased In Vivo P50

We investigated whether red cell 2,3-diphosphoglycerate (2,3-DPG) concentrations are reduced in critical illness, whether acidaemia, hypophosphataemia or anaemia influence 2,3-DPG, and whether there is any net effect on in vivo P50. Twenty healthy, non-smoking, male volunteers were compared with 20 male intensive care patients with APACHE 2 scores > 20 on the preceding day. Those transfused in this time were excluded. Venous red cell 2,3-DPG concentrations were measured in both groups. In the patient group, routine multichannel biochemical profile and arterial blood gas analysis were also performed and in vivo P50 calculated. The mean 2,3-DPG concentration was significantly lower in the patient group than in the controls (4.2 +/-1.3 mmoll/l vs 4.9 +/-0.5 mmol/l, P=0.016). The patients were well oxygenated (lowest arterial PO2=75 mm Hg) and showed a tendency to acidaemia (median pH 7.37, range 7.06 to 7.48) and anaemia (median haemoglobin concentration 113 g/l, range 89 to 154 g/l). By linear regression of patient data, pH had a significant effect on 2,3-DPG concentrations (r=0.6, P=0.011). Haemoglobin and phosphate concentrations did not, but there were few abnormal phosphate values. There was no correlation between 2,3-DPG concentrations and in vivo P50 (r(2) less than or equal to 0.08). We conclude that 2,3-DPG concentrations were reduced in a broad group of critically ill patients. Although this would normally reduce the P50, the reduction was primarily linked with acidaemia, which increases the P50. Overall, there was no net effect on the P50 and thus no affinity-related decrease in tissue oxygenation.

Effect of Fusobacterium nucleatum on the T and B cell responses to Porphyromonas gingivalis in a mouse model

T cell cytokine profiles and specific serum antibody levels in five groups of BALB/c mice immunized with saline alone, viable Fusobacterium nucleatum ATCC 25586, viable Porphyromonas gingivalis ATCC 33277, F. nucleatum followed by P. gingivalis and P. gingivalis followed by F nucleatum were determined. Splenic CD4 and CD8 cells were examined for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma and IL-10 by dual colour flow cytometry and the levels of serum anti-F. nucleatum and anti-P. gingivalis antibodies determined by an ELISA. Both Th1 and Th2 responses were demonstrated by all groups, and while there were slightly lower percentages of cytokine positive T cells in mice injected with F. nucleatum alone compared with the other groups immunized with bacteria., F nucleatum had no effect on the T cell production of cytokines induced by P gingivalis in the two groups immunized with both organisms. However, the percentages of cytokine positive CD8 cells were generally significantly higher than those of the CD4 cells. Mice immunized with F nucleatum alone had high levels of serum anti-E nucleatum antibodies with very low levels of P. gingivalis antibodies, whereas mice injected with P gingivalis alone produced anti-P. gingivalis antibodies predominantly. Although the levels of anti-E nucleatum antibodies in mice injected with E nucleatum followed by P. gingivalis were the same as in mice immunized with F nucleatum alone, antibody levels to P. gingivalis were very low. In contrast, mice injected with P. gingivalis followed by F nucleatum produced equal levels of both anti-P. gingivalis and anti-F nucleatum antibodies, although at lower levels than the other three groups immunized with bacteria, respectively. Anti-Actinobacillus actitiomycetemcomitans, Bacteroides forsythus and Prevotella intermedia serum antibody levels were also determined and found to be negligible. In conclusion, F nucleatum immunization does not affect the splenic T cell cytokine response to P. gingivalis. However, F nucleatum immunization prior to that of P. gingivalis almost completely inhibited the production of anti-P gingivalis antibodies while P. gingivalis injection before F. nucleatum demonstrated a partial inhibitory effect by P. gingivalis on antibody production to F. nucleatum. The significance of these results with respect to human periodontal disease is difficult to determine. However, they may explain in part differing responses to P. gingivalis in different individuals who may or may not have had prior exposure to F. nucleatum. Finally, the results suggested that P. gingivalis and F. nucleatum do not induce the production of cross-reactive antibodies to other oral microorganisms.

H-Ras signaling and K-Ras signaling are differentially dependent on endocytosis

dEndocytosis is required for efficient mitogen-activated protein kinase (MAPK) activation by activated growth factor receptors. We examined if H-Ras and K-Ras proteins, which are distributed across different plasma membrane microdomains, have equal access to the endocytic compartment and whether this access is necessary for downstream signaling. Inhibition of endocytosis by dominant interfering dynamin-K44A blocked H-Ras but not K-Ras-mediated PC12 cell differentiation and selectively inhibited H-Ras- but not K-Ras-mediated Raf-1 activation in BHK cells. H-Ras- but not K-Ras-mediated Raf-1 activation was also selectively dependent on phosphoinositide 3-kinase activity. Stimulation of endocytosis and endocytic recycling by wildtype Rab5 potentiated H-Ras-mediated Raf-1 activation. In contrast, Rab5-Q79L, which stimulates endocytosis but not endocytic recycling, redistributed activated H-Ras from the plasma membrane into enlarged endosomes and inhibited H-Ras-mediated Raf-1 activation. Rab5-Q79L expression did not cause the accumulation of wild-type H-Ras in enlarged endosomes. Expression of wild-type Rab5 or Rab5-Q79L increased the specific activity of K-Ras-activated Raf-1 but did not result in any redistribution of K-Ras from the plasma membrane to endosomes. These results show that H-Ras but not K-Ras signaling though the Raf/MEK/MAPK cascade requires endocytosis and enclocytic recycling. The data also suggest a mechanism for returning Raf-1 to the cytosol after plasma membrane recruitment.

Alterations in gene expression and activity during squamous cell carcinoma development

This study focuses on characterizing the genetic and biological alterations associated with squamous cell carcinoma development. Normal human epidermal keratinocytes (HEKs), cells isolated from a preneoplastic lesion (IEC-1), and two neoplastic cell lines, SCC-25 and COLD-16, were grown as raft cultures, and their gene expression profiles were screened using cDNA arrays. Our data indicated that the expression levels of at least 37 genes were significantly (P less than or equal to 0.05; 1.9% of genes screened) altered in neoplastic cells compared with normal cells. Of these genes, 10 genes were up-regulated and 27 genes were down-regulated in the neoplastic cells. In addition, 51% of the genes altered in the neoplastic cells were already altered in the preneoplastic IEC-1 cells. Immunohistochemical staining of patient tumors was used to verify the cDNA array analysis. Our analysis indicated that alterations in genes associated with extracellular matrix production and apoptosis are disrupted in preneoplastic cells, whereas later stages of neoplasia are associated with alterations in gene expression for genes involved in DNA repair or epidermal growth factor (EGF) receptor/mitogen-activated protein kinase kinase (MAPKK)/MAPK/activator protein-1 (AP-1) signaling. Subsequent functional analysis of the alterations in expression of the EGF receptor/MAPKK/MAPK/AP-1 genes suggested they did not contribute to the neoplastic phenotype.

Determining when the absolute state complexity of a Hermitian code achieves its DLP bound

Let g be the genus of the Hermitian function field H/F(q)2 and let C-L(D,mQ(infinity)) be a typical Hermitian code of length n. In [Des. Codes Cryptogr., to appear], we determined the dimension/length profile (DLP) lower bound on the state complexity of C-L(D,mQ(infinity)). Here we determine when this lower bound is tight and when it is not. For m less than or equal to n-2/2 or m greater than or equal to n-2/2 + 2g, the DLP lower bounds reach Wolf's upper bound on state complexity and thus are trivially tight. We begin by showing that for about half of the remaining values of m the DLP bounds cannot be tight. In these cases, we give a lower bound on the absolute state complexity of C-L(D,mQ(infinity)), which improves the DLP lower bound. Next we give a good coordinate order for C-L(D,mQ(infinity)). With this good order, the state complexity of C-L(D,mQ(infinity)) achieves its DLP bound (whenever this is possible). This coordinate order also provides an upper bound on the absolute state complexity of C-L(D,mQ(infinity)) (for those values of m for which the DLP bounds cannot be tight). Our bounds on absolute state complexity do not meet for some of these values of m, and this leaves open the question whether our coordinate order is best possible in these cases. A straightforward application of these results is that if C-L(D,mQ(infinity)) is self-dual, then its state complexity (with respect to the lexicographic coordinate order) achieves its DLP bound of n /2 - q(2)/4, and, in particular, so does its absolute state complexity.

Patchy populations in stochastic environments: Critical number of patches for persistence

We introduce a model for the dynamics of a patchy population in a stochastic environment and derive a criterion for its persistence. This criterion is based on the geometric mean (GM) through time of the spatial-arithmetic mean of growth rates. For the population to persist, the GM has to be greater than or equal to1. The GM increases with the number of patches (because the sampling error is reduced) and decreases with both the variance and the spatial covariance of growth rates. We derive analytical expressions for the minimum number of patches (and the maximum harvesting rate) required for the persistence of the population. As the magnitude of environmental fluctuations increases, the number of patches required for persistence increases, and the fraction of individuals that can be harvested decreases. The novelty of our approach is that we focus on Malthusian local population dynamics with high dispersal and strong environmental variability from year to year. Unlike previous models of patchy populations that assume an infinite number of patches, we focus specifically on the effect that the number of patches has on population persistence. Our work is therefore directly relevant to patchily distributed organisms that are restricted to a small number of habitat patches.