Invertebrate DNA metabarcoding reveals changes in communities across mine site restoration chronosequences

Invertebrate biomonitoring can reveal crucial information about the status of restoration projects; however, it is routinely underused because of the high level of taxonomic expertise and resources required. Invertebrate DNA metabarcoding has been used to characterize invertebrate biodiversity but its application in restoration remains untested. We use DNA metabarcoding, a new approach for restoration assessment, to explore the invertebrate composition from pitfall traps at two mine site restoration chronosequences in southwestern Australia. Invertebrates were profiled using two cytochrome oxidase subunit 1 assays to investigate invertebrate biodiversity. The data revealed differences between invertebrate communities at the two mines and between the different age plots of the chronosequences. Several characteristic taxa were identified for each age within the chronosequence, including springtails within the youngest sites (Order: Collembola) and millipedes within the oldest and reference sites (Order: Julida). This study facilitates development of a molecular “toolkit” for the monitoring of ecological restoration projects. We suggest that a metabarcoding approach shows promise in complementing current monitoring practices that rely on alpha taxonomy.     

Use of lithium and SB-415286 to explore the role of glycogen synthase kinase-3 in the regulation of glucose transport and glycogen synthase.

Glycogen synthase kinase 3 (GSK3) is inactivated by insulin and lithium and, like insulin, Li also activates glycogen synthase (GS) via inhibition of GSK3. Li also mimics insulin's ability to stimulate glucose transport (GT), an observation that has led to the suggestion that GSK3 may coordinate hormonal increases in GT and glycogen synthesis. Here we have used Li and SB-415286, a selective GSK3 inhibitor, to establish the importance of GSK3 in the hormonal activation of GT in terms of its effect on GS in L6 myotubes and 3T3-L1 adipocytes. Insulin, Li and SB-415286 all induced a significant inhibition of GSK3, which was associated with a marked dephosphorylation and activation of GS. In L6 myotubes, SB-415286 induced a much greater activation of GS (6.8-fold) compared to that elicited by insulin (4.2-fold) or Li (4-fold). In adipocytes, insulin, Li and SB-415286 all caused a comparable activation of GS despite a substantial differentiation-linked reduction in GSK3 expression ( approximately 85%) indicating that GSK3 remains an important determinant of GS activation in fat cells. Whilst Li and SB-415286 both inhibit GSK3 in muscle and fat cells, only Li stimulated GT. This increase in GT was not sensitive to inhibitors of PI3-kinase, MAP kinase or mTOR, but was suppressed by the p38 MAP kinase inhibitor, SB-203580. Consistent with this, phosphorylation of p38 MAP kinase induced by Li correlated with its stimulatory effect on GT. Our findings support a crucial role for GSK3 in the regulation of GS, but based on the differential effects of Li and SB-415286, it is unlikely that acute inhibition of GSK3 contributes towards the rapid stimulation of GT by insulin in muscle and fat cells.     

Qualitative and quantitative analysis of granules in atrial appendage cardiocytes in different physiological states

Summary Atrial appendage cardiocytes of mammals, including man, contain multiple cytoplasmic granules that vary in number in different physiological states. Using morphologic and comprehensive morphometric techniques, these granules were assessed in Sprague-Dawley rats following dehydration for 5 days, volume-loading by substituting 1% NaCl as drinking water for 7 days, unilateral nephrectomy plus volume-loading for 7 days, and in late term pregnant animals (18–20 days; term 21 days). Although principally located in the paranuclear region, granules were observed throughout the sarcoplasm. Cytological features indicative of synthetic activity and granule formation were readily apparent in all groups with the exception of pregnant rats where they were infrequently observed. Granule contents were released by exocytosis and observed in the right appendage of control, dehydrated and nephrectomy/volume-loaded groups and left appendage of volumeloaded animals. Exocytosis was not observed in pregnant animals. By point counting, the proportional volume of cardiocytes occupied by granules (V _v ) in controls was significantly greater for right than for left appendage (2.12±0.22% vs 1.29±0.16%; mean±SEM;p<0.05). A significantly similar difference was found for nephrectomy/volume-loaded animals. There was no significant difference inV _v for right appendage between the control and experimental groups; for left appendage there was a significant increase inV _v to 2.42±0.09% (p<0.05) for volume-loaded animals only. Estimation of the maximum diameter of granule profiles in control animals was 238±9 nm and 230±6 nm for right and left appendages, respectively. The profile diameters in the left appendages of dehydrated (202±9 nm) and pregnant (200±7 nm) animals were significantly (p<0.05) less than those of the control animals. The morphometric findings did not correlate with predictions based upon published biochemical data. In the course of this study, a previously unreported bimembranous, circular to ovoid structure was observed in the cardiocyte sarcoplasm of all animals; the nature and function of this structure is unknown.     

Structural studies of the adrenal zona glomerulosa and renal juxtaglomerular apparatus in pregnant sheep

The ultrastructural changes in the adrenal zona glomerulosa and renal juxtaglomerular apparatus have been examined during normal pregnancy in sheep. As pregnancy progressed, increasing numbers of cells in the adrenal zona glomerulosa displayed mitochondria with straight tubular "rod-like" structures replacing their normal lamelliform cristae; groups of cells showing these mitochondrial changes were predominantly located in the middle and superficial regions of the zona glomerulosa, but at all stages remained interspersed with cells with apparently normal mitochondria. In the same animals, the renal juxtaglomerular index was raised, reflecting an increase in renin storage, and juxtaglomerular myoepithelioid cells showed increased numbers of cytoplasmic granules, but no apparent increase in granular endoplasmic reticulum and Golgi profiles; there were no distinguishing morphological changes in juxtaglomerular peripolar cells. These findings provide morphologic evidence of stimulation of the adrenal zona glomerulosa in association with increased juxtaglomerular renin storage during pregnancy. The mitochondrial changes observed in an increasing proportion of cells in the zona glomerulosa closely resemble those seen in sodium-depleted animals, and may reflect the altered steroidogenic capacity of the adrenal gland in pregnant sheep. The finding of groups of cells displaying altered mitochondria lying next to cells with normal mitochondria suggests the presence of cells with different sensitivities to stimuli for aldosterone production or may indicate the presence of different cell types in the zona glomerulosa responding to different stimuli.     

Cerebral osmoregulation of renal sodium excretion--a response analogous to thirst and vasopressin release

Studies in sheep have shown that renal excretion of sodium may be under osmoregulatory control. When sheep become dehydrated, or are infused intravenously with hypertonic saline, they increase renal Na excretion in addition to secreting vasopressin and developing a thirst. These natriuretic, antidiuretic, and dipsogenic responses to dehydration and hypertonicity can be greatly reduced by lowering the cerebrospinal fluid NaCl concentration or by prior ablation of tissue in the anterior wall of the third ventricle. Lowering of cerebrospinal fluid NaCl concentration also prevents postprandial natriuresis which normally occurs in association with a postprandial increase in plasma Na concentration and tonicity. We propose that there is a cerebral osmoregulatory control of Na excretion which may interact with volume influences from the cardiovascular system to regulate renal Na output. The effector mechanism from brain to kidney mediating such cerebral control of Na excretion is probably hormonal.     

Blood pressure and metabolic effects of 18-oxo-cortisol in sheep

18-Oxo-cortisol (18-oxo-F) has been isolated from the urine of subjects with primary aldosteronism. This study examines the pressor, mineralocorticoid and glucocorticoid effects of 18-oxo-F in conscious sheep--a well studied species for the assessment of the pressor effect of steroid hormones. 18-oxo-F (24 mg/day i.v. for 5 days, n = 3) increased mean arterial pressure MAP (64 +/- 2 mmHg control and 75 +/- 6 mmHg on day 5 P less than 0.001). There was no change in heart rate. Plasma [K+] decreased from a control of 4.3 +/- 0.1 mmol/l control to 2.9 +/- 0.3 mmol/l on day 5 (P less than 0.001). Urinary Na+ excretion decreased on the first infusion day (233 +/- 18 mmol/day control and 124 +/- 20 mmol/day on infusion day 1 P less than 0.001). Urinary K+ excretion was reduced on days 1, 4 and 5 of the infusion. Thus in sheep, 18-oxo-F increased blood pressure associated with in vivo evidence of mineralocorticoid activity.