Apoptosis in the pathogenesis of renal disease with a focus on tubulointerstitial injury

Renal cell apoptosis is important not only in normal physiological conditions of the kidney but also in pathological processes. In normal renal development, it removes unwanted, damaged or harmful cells, and in the healthy adult kidney, it maintains cellular homeostasis by regulating the balance between cell proliferation and cell loss. The apoptotic process has now been described in the pathogenesis and prognosis of certain renal diseases with both beneficial and detrimental roles. It causes deletion of cells intrinsic to the kidney after, for example, toxic, ischaemic, immune or radiation damage, and this loss can be destructive and can cause significant reduction of renal function. In contrast, it can control and limit inflammatory processes in both the acute and chronic phases of renal disease. Information on the positive and negative outcomes of renal cell apoptosis, plus the thousands of publications on more general aspects of apoptosis mechanisms, have now presented real opportunities for the development of therapies that selectively delete or protect certain renal cell populations. This review will discuss some of the more general aspects of renal cell apoptosis and then concentrate on the detrimental or beneficial roles of apoptosis in the initiation, progression or resolution of selected, mainly tubulointerstitial, renal diseases.

Interacting roles of myofibroblasts, apoptosis and fibrogenic growth factors in the pathogenesis of renal tubulo-interstitial fibrosis

The interrelationship between myofibroblasts and fibrogenic growth factors in the pathogenesis of renal fibrosis is poorly defined. A temporal and spatial analysis of myofibroblasts, their proliferation and death, and presence of transforming growth factor-beta1 (TGF-beta1) and platelet-derived growth factor-B (PDGF-B) was carried out in an established rodent model in which chronic renal scarring and fibrosis occurs after healed renal papillary necrosis (RPN), similar to that seen with analgesic nephropathy. Treated and control groups (N = 6 and 4, respectively) were compared at 2, 4, 8 and 12 weeks. A positive relationship was found between presence of tubulo-interstitial myofibroblasts and development of fibrosis. Apoptotic myofibroblasts were identified in the interstitium and their incidence peaked 2 weeks after treatment. Levels of interstitial cell apoptosis and fibrosis were negatively correlated over time (r = -0.57, p < 0.01 ), suggesting that as apoptosis progressively failed to limit myofibroblast numbers, fibrosis increased. In comparison with the diminishing apoptosis in the interstitium, the tubular epithelium had progressively increasing levels of apoptosis over time, indicative of developing atrophy of nephrons. TGF-beta1 protein expression had a close spatial and temporal association with fibrosis and myofibroblasts, whilst PDGF-B appeared to have a closer link with populations of other chronic inflammatory cells such as infiltrating lymphocytes. Peritubular myofibroblasts were often seen near apoptotic cells in the tubular epithelium, suggestive of a paracrine toxic effect of factor/s secreted by the myofibroblasts. In vitro , TGF-beta1 was found to be toxic to renal tubular epithelial cells. These findings suggest an interaction between myofibroblasts, their deletion by apoptosis, and the presence of the fibrogenic growth factor TGF-beta1 in renal fibrosis, whereby apoptotic deletion of myofibroblasts could act as a controlling factor in progression of fibrosis.

Expression of a truncated Brca1 protein delays lactational mammary development in transgenic mice

To address the hypothesis that certain disease-associated mutants of the breast-ovarian cancer susceptibility gene BRCA1 have biological activity in vivo, we have expressed a truncated Brca1 protein (trBrca1) in cell-lines and in the mammary gland of transgenic mice. Immunofluorescent analysis of transfected cell-lines indicates that trBRCA1 is a stable protein and that it is localized in the cell cytoplasm. Functional analysis of these cell-lines indicates that expression of trBRCA1 confers an increased radiosensitivity phenotype on mammary epithelial cells, consistent with abrogation of the BRCA1 pathway. MMTV-trBrca1 transgenic mice from two independent lines displayed a delay in lactational mammary gland development, as demonstrated by altered histological profiles of lobuloalveolar structures. Cellular and molecular analyses indicate that this phenotype results from a defect in differentiation, rather than altered rates of proliferation or apoptosis. The results presented in this paper are consistent with trBrca1 possessing dominant-negative activity and playing an important role in regulating normal mammary development. They may also have implications for germline carriers of BRCA1 mutations.

Characterization of mouse profilaggrin: Evidence for nuclear engulfment and translocation of the profilaggrin B-domain during epidermal differentiation

Filaggrin is a keratin filament associated protein that is expressed in granular layer keratinocytes and derived by sequential proteolysis from a polyprotein precursor termed profilaggrin. Depending on the species, each profilaggrin molecule contains between 10 and 20 filaggrin subunits organized as tandem repeats with a calcium-binding domain at the N-terminal end. We now report the characterization of the complete mouse gene. The structural organization of the mouse gene is identical to the human profilaggrin gene and consists of three exons with a 4 kb intron within the 5' noncoding region and a 1.7 kb intron separating the sequences encoding the calcium-binding EF-hand motifs. A processed pseudogene was found embedded within the second intron. The third and largest exon encodes the second EF-hand, a basic domain (designated the B-domain) followed by 12 filaggrin repeats and a unique C-terminal tail domain. A polyclonal anti-body raised against the conceptually translated sequence of the B-domain specifically stained keratohyalin granules and colocalized with a filaggrin antibody in granular layer cells. In upper granular layer cells, B-domain containing keratohyalin granules were in close apposition to the nucleus and, in some cells, appeared to be completely engulfed by the nucleus. In transition layer cells, B-domain staining was evident in the nucleus whereas filaggrin staining remained cytoplasmic. Nuclear staining of the B-domain was also observed in primary mouse keratinocytes induced to differentiate. This study has also revealed significant sequence homology between the mouse and human promoter sequences and in the calcium-binding domain but the remainder of the protein-coding region shows substantial divergence.

Detection of the initial infective stages of the protozoan parasite Marteilia sydneyi in Saccostrea glomerata and their development through to sporogenesis

DNA probes were used in in situ hybridisation on histological sections of oysters exposed for defined intervals to Marteilia sydneyi infection to reveal the early development of the parasite in the oyster host, Saccostrea glomerata. The initial infective stages enter through the palps and gills whereupon extrasporogonic proliferation results in the liberation of cells into surrounding connective tissue and haemolymph spaces. Following systemic dissemination, the parasite infiltrates the digestive gland and becomes established as a nurse cell beneath the epithelial cells ill a digestive tubule. Here, cell-within-cell proliferation results in the eventual liberation of daughter cells from the nurse cell into spaces between adjacent epithelial cells. None of these stages had previously been described. Proliferation is associated with host responses, including haemocytic infiltration of the connective tissue and diapedesis across tubule epithelia. The responses cease as sporogenesis begins. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Peroxisome proliferator-activated receptor alpha in the human breast cancer cell lines MCF-7 and MDA-MB-231

Peroxisome proliferator-activated receptor (PPAR) alpha is a ligand-activated transcription factor that has been linked with rodent hepatocarcinogenesis. It has been suggested that PPARalpha mRNA expression levels are an important determinant of rodent hepatic tumorigenicity. Previous work in rat mammary gland epithelial cells showed significantly increased PPARalpha mRNA expression in carcinomas, suggesting the possible role of this isoform in rodent mammary gland carcinogenesis. In this study we sought to determine whether PPARalpha is expressed and dynamically regulated in human breast cancer MCF-7 and MDA-MB-231 cells. Having established the presence of PPARalpha in both cell types, we then examined the consequence of PPARa activation, by its ligands Wy-14,643 and clofibrate, on proliferation. With real-time reverse transcriptase-polymerase chain reaction, we showed that PPARalpha mRNA was dynamically regulated in MDA-MB-231 cells and that PPARalpha activation significantly increased proliferation of the cell line. In contrast, PPARalpha expression in MCF-7 cells did not change with proliferation during culture and was present at significantly lower levels than in MDA-MB-231 cells. However, PPARalpha ligand activation still significantly increased the proliferation of MCF-7 cells. The promotion of proliferation in breast cancer cell lines following PPARalpha activation was in stark contrast to the effects of PPARgamma-activating ligands that decrease proliferation in human breast cancer cells. our results established the presence of PPARalpha in human breast cancer cell lines and showed for the first time that activation of PPARalpha in human breast cancer cells promoted proliferation. Hence, this pathway may be significant in mammary gland tumorigenesis. (C) 2002 Wiley-Liss, Inc.

Zeolite/rock phosphate - a novel slow release phosphorus fertiliser for potted plant production

A glasshouse study was undertaken to determine if the zeolite mineral clinoptilolite from an Australian deposit in combination with rock phosphate (RP) could significantly enhance the uptake of P by sunflowers. The zeolite/RP combination was intended to act as an exchange-fertiliser, with Ca2+ exchanging onto the zeolite in response to plant uptake of nutrient cations (NH4+ or K) enhancing the dissolution of the RP. A reactive RP (Sechura) and a relatively non-reactive RP (Duchess) were examined. Zeolite was used in Ca2+-, K+- and NH4+-saturated forms at ratios of 3.5:1 and 7:1 with RP; Ca2+-zeolite was considered the control, with exchange-induced dissolution possible from K+-and NH4+-zeolite, The zeolite/RP mixture was applied as a vertical band adjacent to the sunflower seedling. In addition, N was supplied as urea in an effort to determine if RP dissolution resulted from H+ release by nitrification. Phosphorus supply from the zeolite/RP system was compared with an available P source (KH2PO4). The experiment clearly demonstrated greatly enhanced plant uptake of P from RP when applied in combination with NH4-zeolite, though the P uptake was lower than that from the soluble P source. The zeolite/RP interaction was much more effective with the reactive R-P than the non-reactive material, Within the NH4+-zeolite/RP band, root proliferation was greatly increased, as would be expected in an exchange-fertiliser system. The K+-zeolite system did not produce a significantly greater yield than the Ca2+-zeolite control, probably because adequate K+ supply from the basal application reduced uptake within the zeolite/RP band, thus reducing the extent of exchange-induced dissolution. Nevertheless, increased root proliferation within the band was observed, implying that exchange-induced dissolution may also be possible from this system. The zeolite/RP system offers the considerable advantage of P release in response to plant demand and is unique in this regard. (C) 2002 Elsevier Science B.V. All rights reserved.

Fibroblast growth factor receptor 4 (FGFR4) expression in newborn murine calvaria and primary osteoblast cultures

Fibroblast growth factor receptor (FGFR) signalling is important in the initiation and regulation of osteogenesis. Although mutations in FGFR1, 2 and 3 genes are known to cause skeletal deformities, the expression of FGFR4 in bony tissue remains unclear. We have investigated the expression pattern of FGFR4 in the neonatal mouse calvaria and compared it to the expression pattern in cultures of primary osteoblasts. Immunohistochemistry demonstrated that FGFR4 was highly expressed in rudimentary membranous bone and strictly localised to the cellular components (osteoblasts) between the periosteal and endosteal layers. Cells in close proximity to the newly formed osteoid (preosteoblasts) also expressed FGFR4 on both the endosteal and periosteal surfaces. Immunocytochemical analysis of primary osteoblast cultures taken from the same cranial region also revealed high levels of FGFR4 expression, suggesting a similar pattern of cellular expression in vivo and in vitro. RT-PCR and Western blotting for FGFR4 confirmed its presence in primary osteoblast cultures. These results suggest that FGFR4 may be an important regulator of osteogenesis with involvement in preosteoblast proliferation and differentiation as well as osteoblast functioning during intramembranous ossification. The consistent expression of FGFR4 in vivo and in vitro supports the use of primary osteoblast cultures for elucidating the role of FGFR4 during osteogenesis.

Evaluating Australia's National Medicines Policy using geographical mapping

Background: There has been a proliferation of quality use of medicines activities in Australia since the 1990s. However, knowledge of the nature and extent of these activities was lacking. A mechanism was required to map the activities to enable their coordination. Aims: To develop a geographical mapping facility as an evaluative tool to assist the planning and implementation of Australia's policy on the quality use of medicines. Methods: A web-based database incorporating geographical mapping software was developed. Quality use of medicines projects implemented across the country was identified from project listings funded by the Quality Use of Medicines Evaluation Program, the National Health and Medical Research Council, Mental Health Strategy, Rural Health Support, Education and Training Program, the Healthy Seniors Initiative, the General Practice Evaluation Program and the Drug Utilisation Evaluation Network. In addition, projects were identified through direct mail to persons working in the field. Results: The Quality Use of Medicines Mapping Project (QUMMP) was developed, providing a Web-based database that can be continuously updated. This database showed the distribution of quality use of medicines activities by: (i) geographical region, (ii) project type, (iii) target group, (iv) stakeholder involvement, (v) funding body and (vi) evaluation method. At September 2001, the database included 901 projects. Sixty-two per cent of projects had been conducted in Australian capital cities, where approximately 63% of the population reside. Distribution of projects varied between States. In Western Australia and Queensland, 36 and 73 projects had been conducted, respectively, representing approximately two projects per 100 000 people. By comparison, in South Australia and Tasmania approximately seven projects per 100 000 people were recorded, with six per 100 000 people in Victoria and three per 100 000 people in New South Wales. Rural and remote areas of the country had more limited project activity. Conclusions: The mapping of projects by geographical location enabled easy identification of high and low activity areas. Analysis of the types of projects undertaken in each region enabled identification of target groups that had not been involved or services that had not yet been developed. This served as a powerful tool for policy planning and implementation and will be used to support the continued implementation of Australia's policy on the quality use of medicines.

The intuitive basis of implicature: relevance theoretic implicitness versus gricean implying

The notion of implicature was first introduced by Paul Grice (1967, 1989), who defined it essentially as what is communicated less what is said. This definition contributed in part to the proliferation of a large number of different species of implicature by neo-Griceans. Relevance theorists have responded to this by proposing a shift back to the distinction between "explicit" & "implicit" meaning (corresponding to "explicature" & "implicature," respectively). However, they appear to have pared down the concept of implicature too much, ignoring phenomena that may be better treated as implicatures in their overgeneralization of the concept of explicature. These problems have their roots in the fact that explicit & implicit meaning intuitively overlap & thus do not provide a suitable basis for distinguishing implicature from other types of pragmatic phenomena. An alternative conceptualization of implicature based on the concept of "implying" with which Grice originally associated his notion of implicature is thus proposed. From this definition, it emerges that implicature constitutes something else inferred by the addressee that is not literally said by the speaker. Instead, it is meant in addition to what the speaker literally says & is consequently defeasible like all other types of pragmatic phenomena. 1 Figure, 60 References. Adapted from the source document

Expression of Heparan Sulphate N-deacetylase/N-sulphotransferase by Vascular Smooth Muscle Cells

Heparan sulphate is an important mediator in determining vascular smooth muscle cell (SMC) phenotype. The sulphation pattern of the heparan sulphate chains is critical to their function. We have examined the initial step in the biosynthesis of the sulphated domains mediated by the enzyme heparan sulphate N-deacetylase/N-sulphotransferase (NDST). Rabbit aortic SMC in primary culture exhibited NDST enzyme activity and expressed NDST-1 in their Golgi apparatus, with maximal expression in SMC 2 days after dispersal in primary culture confirmed by Western blot analysis. Endothelial cells, macrophages and fibroblasts expressed NDST-1 but had generally less intense staining than SMC, although SMC expression decreased with culture. The uninjured rat aorta also showed widespread expression of NDST-1. After balloon de-endothelialisation, NDST-1 could not be detected in SMC of the neointima in the early stages of neointimal formation, but was re-expressed at later time points (after 12 weeks). In human coronary arteries, SMC of the media and the diffuse intimal thickening expressed NDST-1, while SMC in the atherosclerotic plaque were negative for NDST-1. We conclude that SMC may regulate their heparan sulphate sulphation at the level of expression of the enzyme heparan sulphate NDST in a manner related to their phenotypic state.

Nuclear weapons as obstacles to international security

This article argues that nuclear weapons serve no useful purpose in military calculations; moreover, their continued retention invites the dangers of further proliferation and of accidental use. They are thus defined here as obstacles to, rather than as facilitators of, international security. Seven reasons are presented to support this contention, including an assessment of the moral implications and the strategic limitations of nuclear weapons. Despite these limitations, and the recent commitments made by the nuclear weapon states to eliminate their arsenals, nuclear weapons remain central to the strategic doctrines of these states. Several reasons are put forward to explain why this retention continues, including the unchanging nature of strategic mindsets, the presence of vested interests, and now, in the case of the USA at least, a renewed reliance on nuclear weapons, regardless of how appropriate and effective such a strategy might be against emerging terrorist or `rogue state' threats.

Dietary supplementation with vitamin E modulates avian intestinal immunity

The effect of dietary vitamin E on immunoglobulin A (IgA) antibody production, which acts as the first line of defence at the intestinal mucosa, has not been evaluated in chickens. In the present study the impact of the inclusion of supplementary levels of vitamin E to the diet, on total and antigen-specific IgA antibody titres, T-cell subsets and Ia+ cells, was assessed. From hatching, chickens received a maize-based diet which was supplemented with either 25, 250, 2500 or 5000 mg dl-alpha-tocopherol acetate/kg. Primary immunisation with tetanus toxoid (T. toxoid) emulsified in a vegetable oil-in-water adjuvant was administered by the intraperitoneal route at 21 d of age. At 35 d of age all birds received an oral booster vaccination of T. toxoid. Significantly higher total IgA antibody titres were present in the day 42 intestinal scrapings of birds receiving the 5000 mg/kg vitamin E-supplemented diet (VESD) (P=0.05) and a notable increase was observed in birds receiving the 250 mg/kg VESD (P=0.06). At days 21 and 42 total serum IgA antibody titres of birds receiving the 250 mg/kg VESD was significantly higher (P