Effects of pathophysiological concentrations of albumin on NHE3 activity and cell proliferation in primary cultures of human proximal tubule cells

The progression of renal disease correlates strongly with hypertension and the degree of proteinuria, suggesting a link between excessive Na+ reabsorption and exposure of the proximal tubule to protein. The present study investigated the effects of albumin on cell growth and Na+ uptake in primary cultures of human proximal tubule cells (PTC). Albumin (1.0 mg/ml) increased cell proliferation to 134.1 +/- 11.8% (P < 0.001) of control levels with no change in levels of apoptosis. Exposure to 0.1 and 1.0 mg/ml albumin increased total Na-22(+) uptake to 119.1 &PLUSMN; 6.3% (P = 0.005) and 115.6 &PLUSMN; 5.3% (P < 0.006) of control levels, respectively, because of an increase in Na+/H+ exchanger isoform 3 (NHE3) activity. This was associated with an increase in NHE3 mRNA to 161.1 +/- 15.1% (P < 0.005) of control levels in response to 0.1 mg/ml albumin. Using confocal microscopy with a novel antibody raised against the predicted extracellular NH2 terminus of human NHE3, we observed in nonpermeabilized cells that exposure of PTC to albumin (0.1 and 1.0 mg/ml) increased NHE3 at the cell surface to 115.4 &PLUSMN; 2.7% (P < 0.0005) and 122.4 +/- 3.7% (P < 0.0001) of control levels, respectively. This effect was paralleled by significant increases in NHE3 in the subplasmalemmal region as measured in permeabilized cells. These albumin-induced increases in expression and activity of NHE3 in PTC suggest a possible mechanism for Na+ retention in response to proteinuria.

Temperature-dependent sex ratio in a bird

To our knowledge, there is, so far, no evidence that incubation temperature can affect sex ratios in birds, although this is common in reptiles. Here, we show that incubation temperature does affect sex ratios in megapodes, which are exceptional among birds because they use environmental heat sources for incubation. In the Australian brush-turkey Alectura lathami, a mound-building megapode, more males hatch at low incubation temperatures and more females hatch at high temperatures, whereas the proportion is 1 : 1 at the average temperature found in natural mounds. Chicks from lower temperatures weigh less, which probably affects offspring survival, but are not smaller. Megapodes possess heteromorphic sex chromosomes like other birds, which eliminates temperature-dependent sex determination, as described for reptiles, as the mechanism behind the skewed sex ratios at high and low temperatures. Instead, our data suggest a sex-biased temperature-sensitive embryo mortality because mortality was greater at the lower and higher temperatures, and minimal at the middle temperature where the sex ratio was 1 : 1.

Flavonoids in Australian Melaleuca, Guioa, Lophostemon, Banksia and Helianthus honeys and their potential for floral authentication

Flavonoids in Australian honeys from five botanical species (Melaleuca, Guioa, Lophostemon, Banksia and Helianthus) have been analyzed in relation to their floral origins. Tea tree (Melaleuca quinquenervia) and heath (Banksia ericifolia) honeys show a common flavonoid profile comprising myricetin (3,5,7,3',4',5'-hexahydroxyflavone), tricetin (5,7,3',4,5'-pentahydroxyflavone), querectin (3,5,7,3',4'-pentahydroxyflavone) and luteolin (5,7,3',4'-tetrahydroxyflavone), which was previously suggested as a floral marker for an Australian Eucalyptus honey (bloodwood or Eucalyptus intermedia honey). These honeys of various floral species can be differentiated by their levels of total flavonoids, being 2.12 mg/100 g for heath honey and 6.35 m/100 g for tea tree honey. In brush box (Lophostemon conferta) honey, the flavonoid profile comprising mainly tricetin, luteolin and quercetin is similar to that of another Eucalyptus honey (yellow box or Eucalyptus melliodora honey). These results indicate that the flavonoid profiles in some of the Australian non-Eucalyptus honeys may contain more or less certain flavonoids from Eucalyptus floral sources because of the diversity and extensive availability of Eucalyptus nectars for honeybee foraging yearly around or a possible cross contamination of the monofloral honeys during collection, transportation and/or storage. Further analyses are required to differentiate and/or verify the botanical sources of the flavonoids that contribute to the flavonoid profiles of these honeys, by restricting honey sampling areas and procedures, employing other complementary analytical methods (e.g. pollen analysis, sugar profile) and using materials (e.g. nectar) directly sourced from the flowering plant for comparative studies. In Australian crow ash (Guioa semiglauca) honey, myricetin, tricetin, quercetin, luteolin and an unknown flavonoid have been found to be the main flavonoids, which is characteristic only to this type of honey, and could thus be used as the floral marker, while in Australian sunflower (Helianthus annuus) honey, the content of total flavonoids is the smallest amount comparing to those in the other honeys analysed in this study. However, the flavonoid quercetin and the flavonoid profile mainly consisting of quercetin, quercetin 3,3'-dimethyl ether (5,7,4'-trihydroxy3,3'-dimethoxyflavone), myricetin and luteolin are characteristic only to this sunflower honey and could thus be used for the authentication.

The impact of prolonged fasting during aestivation on the structure of the small intestine in the green-striped burrowing frog, Cyclorana alboguttata

The effects of short-term fasting and prolonged fasting during aestivation on the morphology of the proximal small intestine and associated organs were investigated in the green-striped burrowing frog, Cyclorana alboguttata (Anura: Hylidae). Animals were fasted for 1 week while active or for 3-9 months during aestivation. Short-duration fasting (1 week) had little effect on the morphology of the small intestine, whilst prolonged fasting during aestivation induced marked enteropathy including reductions in intestinal mass, length and diameter, longitudinal fold height and tunica muscularis thickness. Enterocyte morphology was also affected markedly by prolonged fasting: enterocyte cross-sectional area and microvillous height were reduced during aestivation, intercellular spaces were visibly reduced and the prevalence of lymphocytes amongst enterocytes was increased. Mitochondria and nuclei were also affected by 9 months of aestivation with major disruptions to mitochondrial cristae and increased clumping of nuclear material and increased infolding of the nuclear envelope. The present study demonstrates that the intestine of an aestivating frog responds to prolonged food deprivation during aestivation by reducing in size, presumably to reduce the energy expenditure of the organ.

Use of community genetic screening to prevent HFE-associated hereditary haemochromatosis

HFE-associated hereditary haemochromatosis is a recessive, iron-overload disorder that affects about one in 200 north Europeans and that can be easily prevented. However, genetic screening for this disease is controversial, and so we assessed whether such screening was suitable for communities. Cheek-brush screening for the Cys282Tyr HFE mutation was offered to individuals in the workplace. Outcomes were assessed by questionnaires before and after testing. 11307 individuals were screened. We recorded no increase in anxiety. in individuals who were homozygous for the Cys282Tyr mutation or non-homozygous. Self-reported tiredness before testing was significantly higher in homozygous participants than in non-homozygous participants (chi(2) test, p=0.029). Of the 47 homozygous individuals identified, 46 have taken steps to treat or prevent iron accumulation. Population genetic screening for HFE-associated hereditary haemochromatosis can be practicable and acceptable.

Phenolic acids in Australian Melaleuca, Guioa, Lophostemon, Banksia and Helianthus honeys and their potential for floral authentication

Eight phenolic acids and two abscisic acid isomers in Australian honeys from five botanical species (Melaleuca, Guioa, Lophostemon, Banksia and Helianthus) have been analyzed in relation to their botanical origins. Total phenolic acids present in these honeys range from 2.13 mg/100 g sunflower (Helianthus annuus) honey to 12.11 mg/100 g tea tree (Melaleuca quinquenervia) honey, with amounts of individual acids being various. Tea tree honey shows a phenolic profile of gallic, ellagic, chlorogenic and coumaric acids, which is similar to the phenolic profile of an Australian Eucalyptus honey (bloodwood or Eucalyptus intermedia honey). The main difference between tea tree and bloodwood honeys is the contribution of chlorogenic acid to their total phenolic profiles. In Australian crow ash (Guioa semiglauca) honey, a characteristic phenolic profile mainly consisting of gallic acid and abscisic acid could be used as the floral marker. In brush box (Lophostemon conferta) honey, the phenolic profile, comprising mainly gallic acid and ellagic acid, could be used to differentiate this honey not only from the other Australian non-Eucalyptus honeys but also from a Eucalyptus honey (yellow box or Eucalyptus melliodora honey). However, this Eucalyptus honey could not be differentiated from brush box honey based only on their flavonoid profiles. Similarly, the phenolic profile of heath (Banksia ericifolia) honey, comprising mainly gallic acid, an unknown phenolic acid (Phl) and coumaric acid, could also be used to differentiate this honey from tea tree and bloodwood honeys, which have similar flavonoid profiles. Coumaric acid is a principal phenolic acid in Australian sunflower honey and it could thus be used together with gallic acid for the authentication. These results show that the HPLC analysis of phenolic acids and abscisic acids in Australian floral honeys Could assist the differentiation and authentication of the honeys. © 2005 Elsevier Ltd. All rights reserved.

NaSi-1 and Sat-1: structure, function and transcriptional regulation of two genes encoding renal proximal tubular sulfate transporters

Sulfate (SO42-) is an important anion regulating many metabolic and cellular processes. Maintenance Of SO42- homeostasis occurs in the renal proximal tubule via membrane transport proteins. Two SO42- transporters that have been characterized and implicated in regulating serum SO42- levels are: NaSi- 1, a Na+-SO4 (2-) cotransporter located at the brush border membrane and Sat-1, a SO4 (2-) -anion exchanger located on the basolateral membranes of proximal tubular cells. Unlike Sat-1, for which very few studies have looked at regulation of its expression, NaSi- 1 has been shown to be regulated by various hormones and dietary conditions in vivo. To study this further, NaSj- I (SLC13A1) and Sat- I (SLC26A1) gene structures were determined and recent studies have characterized their respective gene promoters. This review presents the current understanding of the transcriptional regulation of NaSj- I and Sat- 1, and describes possible pathogenetic implications which arise as a consequence of altered SO(4)(2-)homeostasis. (c) 2005 Elsevier Ltd. All rights reserved.

Mechanisms of inducible resistance against Bacillus thuringiensis endotoxins in invertebrates

Abstract Resistance in insect pests against the endotoxin of Bacillus thuringiensis (Berliner) (Bt) is a major threat to the usefulness of this biopesticide, both used as traditional formulations and in transgenic crops. A crucial requirement for the development of successful resistance management strategies is a molecular understanding of the nature and inheritance of resistance mechanisms. This information can be used to design management strategies that will delay or counteract Bt resistance. The best known Bt resistance mechanism is inactivation of brush border membrane receptors. This type of resistance has a largely recessive mode of inheritance, which has enabled the design of resistance management approaches involving high dose and refuge strategies. Recent observations suggest that other resistance mechanisms are possible, including a mechanism that sequesters the toxin in the gut lumen through inducible immune reactions. The elevated immune status associated with tolerance to the toxin can be transmitted to subsequent generations by a maternal effect, which has implications for resistance management in the field. The high dose/refuge strategy may not be appropriate for the management of these alternative resistance mechanisms and other strategies have to be developed if inducible dominant resistance or tolerance mechanisms occur frequently in the field.

Renal structural and functional repair in a mouse model of reversal of ureteral obstruction

The end point of immune and nonimmune renal injury typically involves glomerular and tubulointerstitial fibrosis. Although numerous studies have focused on the events that lead to renal fibrosis, less is known about the mechanisms that promote cellular repair and tissue remodeling. Described is a model of renal injury and repair after the reversal of unilateral ureteral obstruction (UUO) in male C57b1/6J mice. Male mice (20 to 25 g) underwent 10 d of UUO with or without 1, 2, 4, or 6 wk of reversal of UUO (R-UUO). UUO resulted in cortical tubular cell atrophy and tubular dilation in conjunction with an almost complete ablation of the outer medulla. This was associated with interstitial macrophage infiltration; increased hydroxyproline content; and upregulated type I, III, IV, and V collagen expression. The volume density of kidney occupied by renal tubules that exhibited a brush border was measured as an assessment of the degree of repair after R-UUO. After 6 wk of R-UUO, there was an increase in the area of kidney occupied by repaired tubules (83.7 +/- 5.9%), compared with 10 d UUO kidneys (32.6 +/- 7.3%). This coincided with reduced macrophage numbers, decreased hydroxyproline content, and reduced collagen accumulation and interstitial matrix expansion, compared with obstructed kidneys from UUO mice. GFR in the 6-wk R-UUO kidneys was restored to 43 to 88% of the GFR in the contralateral unobstructed kidneys. This study describes the regenerative potential of the kidney after the established interstitial matrix expansion and medullary ablation associated with UUO in the adult mouse.

Regulation of albumin endocytosis by PSD95/Dlg/ZO-1 (PDZ) scaffolds - Interaction of Na+-H+ exchange regulatory factor-2 with ClC-5

The constitutive reuptake of albumin from the glomerular filtrate by receptor-mediated endocytosis is a key function of the renal proximal tubules. Both the Cl- channel ClC-5 and the Na+-H+ exchanger isoform 3 are critical components of the macromolecular endocytic complex that is required for albumin uptake, and therefore the cell-surface levels of these proteins may limit albumin endocytosis. This study was undertaken to investigate the potential roles of the epithelial PDZ scaffolds, Na+-H+ exchange regulatory factors, NHERF1 and NHERF2, in albumin uptake by opossum kidney ( OK) cells. We found that ClC-5 co-immunoprecipitates with NHERF2 but not NHERF1 from OK cell lysate. Experiments using fusion proteins demonstrated that this was a direct interaction between an internal binding site in the C terminus of ClC-5 and the PDZ2 module of NHERF2. In OK cells, NHERF2 is restricted to the intravillar region while NHERF1 is located in the microvilli. Silencing NHERF2 reduced both cell-surface levels of ClC-5 and albumin uptake. Conversely, silencing NHERF1 increased cell-surface levels of ClC-5 and albumin uptake, presumably by increasing the mobility of NHE3 in the membrane and its availability to the albumin uptake complex. Surface biotinylation experiments revealed that both NHERF1 and NHERF2 were associated with the plasma membrane and that NHERF2 was recruited to the membrane in the presence of albumin. The importance of the interaction between NHERF2 and the cytoskeleton was demonstrated by a significant reduction in albumin uptake in cells overexpressing an ezrin binding-deficient mutant of NHERF2. Thus NHERF1 and NHERF2 differentially regulate albumin uptake by mechanisms that ultimately alter the cell-surface levels of ClC-5.

Restricted mating dispersal and strong breeding group structure in a mid-sized marsupial mammal (Petrogale penicillata)

Ecological genetic studies have demonstrated that spatial patterns of mating dispersal, the dispersal of gametes through mating behaviour, can facilitate inbreeding avoidance and strongly influence the structure of populations, particularly in highly philopatric species. Elements of breeding group dynamics, such as strong structuring and sex-biased dispersal among groups, can also minimize inbreeding and positively influence levels of genetic diversity within populations. Rock-wallabies are highly philopatric mid-sized mammals whose strong dependence on rocky terrain has resulted in series of discreet, small colonies in the landscape. Populations show no signs of inbreeding and maintain high levels of genetic diversity despite strong patterns of limited gene flow within and among colonies. We used this species to investigate the importance of mating dispersal and breeding group structure to inbreeding avoidance within a 'small' population. We examined the spatial patterns of mating dispersal, the extent of kinship within breeding groups, and the degree of relatedness among brush-tailed rock-wallaby breeding pairs within a colony in southeast Queensland. Parentage data revealed remarkably restricted mating dispersal and strong breeding group structuring for a mid-sized mammal. Breeding groups showed significant levels of female kinship with evidence of male dispersal among groups. We found no evidence for inbreeding avoidance through mate choice; however, anecdotal data suggest the importance of life history traits to inbreeding avoidance between first-degree relatives. We suggest that the restricted pattern of mating dispersal and strong breeding group structuring facilitates inbreeding avoidance within colonies. These results provide insight into the population structure and maintenance of genetic diversity within colonies of the threatened brush-tailed rock-wallaby.

Significant patterns of population genetic structure and limited gene flow in a threatened macropodid marsupial despite continuous habitat in southeast Queensland, Australia

Many endangered species worldwide are found in remnant populations, often within fragmented landscapes. However, when possible, an understanding of the natural extent of population structure and dispersal behaviour of threatened species would assist in their conservation and management. The brush-tailed rock-wallaby (Petrogale penicillata), a once abundant and widespread rock-wallaby species across southeastern Australia, has become nearly extinct across much of the southern part of its range. However, the northern part of the species' range still sustains many small colonies closely distributed across suitable habitat, providing a rare opportunity to investigate the natural population dynamics of a listed threatened species. We used 12 microsatellite markers to investigate genetic diversity, population structure and gene flow among brush-tailed rock-wallaby colonies within and among two valley regions with continuous habitat in southeast Queensland. We documented high and signifcant levels of population genetic structure between rock-wallaby colonies embedded in continuous escarpment habitat and forest. We found a strong and significant pattern of isolation-by-distance among colonies indicating restricted gene flow over a small geographic scale (< 10 km) and conclude that gene flow is more likely limited by intrinsic factors rather than environmental factors. In addition, we provide evidence that genetic diversity was significantly lower in colonies located in a more isolated valley region compared to colonies located in a valley region surrounded by continuous habitat. These findings shed light on the processes that have resulted in the endangered status of rock-wallaby species in Australia and they have strong implications for the conservation and management of both the remaining 'connected' brush-tailed rock-wallaby colonies in the northern parts of the species' range and the remnant endangered populations in the south.

Plasma steroids and steroid-binding capacity in male semelparous dasyurid marsupials (Phascogale tapoatafa) that survive beyond the breeding season in captivity

The semelparous dasyurids display a unique life history, in that all males die within a few weeks of the completion of the breeding season. Studies of several semelparous species have revealed that the male die-off is stress-related, and accompanied by increased plasma androgen and cortisol levels and decreased corticosteroid binding capacity, resulting in suppression of immune and inflammatory responses. This study examines the endocrine profile of male brush-tailed phascogales (Phascogale tapoatafa) that survive beyond the breeding season in captivity. Plasma cortisol, corticosteroid binding globulin and albumin levels were monitored in both males and females and steroid partitioning calculated. Captive males surviving beyond the breeding season did not show the elevation in plasma cortisol and decrease in corticosteroid binding capacity reported in wild males. Plasma albumin concentrations also remained constant during the sampling period. These data indicate that captive males do not undergo the same stress response described in wild populations. (c) 2006 Elsevier Inc. All rights reserved.

A longitudinal controlled study of factors associated with mutans streptococci infection and caries lesion initiation in children 21 to 72 months old

Purpose: The purpose of this longitudinal study was to determine factors associated with mutans streptococci (MS) infection and development of caries lesions in a group of children 21 to 72 months old. Methods: The 63 caries-free children, recruited since birth, were divided into: (1) a study group of 24 children who were colonized with MS; and (2) a control group of 39 children without MS. The children were recalled every 3 months for approximately 24 months, and their social, medical, and dental histories were updated. At each recall, the teeth were checked for presence or absence of plaque, enamel hypoplasia, and caries lesions, and their MS status was assessed using a commercial test kit. Results: MS infection was associated with: (1) visible plaque (P < .01); (2) enamel hypoplasia (P < .05); (3) commencement of tooth-brushing after 12 months of age (P < .05); (4) lack of parental assistance with tooth-brushing (P < .025); and (5) increased hours of child care/school (P < .05). Four children (20%) were colonized at an age range of 21 to 36 months, 9 (45%) at 37 to 48 months, and 7 (35%) at 49 to 72 months (P < .001). Eight children who developed caries lesions: (1) had more hypoplastic teeth (P < .001); (2) ate sugar-containing snacks (P < .05); and (3) did not brush regularly with chlorhexidine gel (P < .01) compared to those who remained free of caries lesions. Conclusions: Lack of oral hygiene, consumption of sugar-containing snacks, and enamel hypoplasia are significant factors for both MS infection and caries lesion initiation.

Adenocarcinoma of colon differentiating as dome epithelium of gut-associated lymphoid tissue

Aims: An early adenocarcinoma of the ascending colon was confined to a mass of gut-associated lymphoid tissue (GALT). The first description of an adenocarcinoma of colon differentiating as dome epithelium is presented. Methods and results: A plaque-like carcinoma was identified opposite the ileocaecal valve in an asymptomatic 56-year-old man with a family history of colorectal cancer. Malignant epithelium was confined to a mass of GALT filling but limited to the submucosa, Characterization of the neoplasm was undertaken by means of mucin histochemistry, immunohistochemistry, electron microscopy and assessment of DNA microsatellite instability status. The malignant epithelium comprised well differentiated columnar cells with a microvillous brush border and expressing MUC1, but no goblet cells or expression of MUC2. The demonstration of focal clusters of intraepithelial B-lymphocytes supported the presence of functioning M-cells within the malignant neoplasm. The cancer was DNA microsatellite stable despite the finding of tumour infiltrating lymphocytes. Conclusions: There is evidence for the origin of colorectal neoplasia from dome epithelium in both experimental models and microreconstruction studies of early adenomas in nonpolypotic human colorectal mucose, It is suggested that the lymphocyte-rich subset of colorectal cancer that expresses MUC1 but not MUC2 may be differentiating as dome epithelium of gut-associated lymphoid tissue.