Proteomic analysis of normal and malignant prostate tissue to identify novel proteins lost in cancer

BACKGROUND. Alterations of important protein pathways, including loss of prostate secretory granules, and disruption of the prostatic secretory pathway have been identified as early events in malignancy. In this study, proteomics was used to map the differences in protein expression between normal and malignant prostate tissues and to identify and analyze differentially expressed proteins in human prostate tissue with particular regard to the proteins lost in malignancy. METHODS. Small quantities of normal and malignant prostate tissue were taken fresh from 34 radical prostatectomy cases. After histological examination, proteins were solubilized from selected tissues and separated using two-dimensional electrophoresis. Using image analysis, the proteome of normal and malignant tissues were mapped and differentially expressed proteins (present in normal and absent in malignant tissue) were identified and subsequently analyzed using peptide mass finger printing and N-terminal sequencing. Western blotting and immunohistochemistry were performed to examine expression profiles and tissue localization of candidate proteins. RESULTS. Comparison of protein maps of normal and malignant prostate were used to identify 20 proteins which were lost in malignant transformation, including prostate specific antigen (PSA), alpha-l antichymotrypsin (ACT), haptoglobin, and lactoylglutathione lyase. Three of the 20 had not previously been reported in human prostate tissue (Ubiquitin-like NEDD8, calponin, and a follistatin-related protein). Western blotting confirmed differences in the expression profiles of NEDD8 and calponin, and immunohistochemistry demonstrated differences in the cellular localization of these two proteins in normal and malignant prostate glands. CONCLUSIONS. The expression of NEDD8, calponin, and the follistatin-related protein in normal prostate tissues is a novel finding and the role of these important functional proteins in normal prostate and their loss or reduced expression in prostate malignancy warrants further investigations. (C) 2002 Wiley-Liss, Inc.

Four cases of equine polysaccharide storage myopathy in the United Kingdom

This paper describes four cases of equine polysaccharide storage myopathy which were confirmed by histological examination of muscle biopsy specimens. The horses were of mixed breeding, with warmblood and thoroughbred dominating. They all had recurrent episodes of rhabdomyolysis, indicated by clinical signs and increased plasma levels of muscle enzymes. They were managed conservatively and have continued athletic careers despite their disease.

An optimized model for rat liver perfusion studies

Conditions which influence the viability, integrity, and extraction efficiency of the isolated perfused rat liver were examined to establish optimal conditions for subsequent work in reperfusion injury studies including the choice of buffer, use of oncotic agents, hematocrit, perfusion flow rate, and pressure. Rat livers were perfused with MOPS-buffered Ringer solution with or without erythrocytes. Perfusates were collected and analyzed for blood gases, electrolytes, enzymes, radioactivity in MID studies, and lignocaine in extraction studies. Liver tissue was sampled for histological examinations, and wet:dry weight of the liver was also determined. MOPS-buffered Ringer solution was found to be superior to Krebs bicarbonate buffer, in terms of pH control and buffering capacity, especially during any prolonged period of liver perfusion. A pH of 7.2 is chosen for perfusion since this is the physiological pH of the portal blood. The presence of albumin was important as an oncotic agent, particularly when erythrocytes were used in the perfusate. Perfusion pressure, resistance, and vascular volume are how-dependent and the inclusion of erythrocytes in the perfusate substantially altered the flow characteristics for perfusion pressure and resistance but not vascular volume. Lignocaine extraction was relatively flow-independent. Perfusion injury as defined by enzyme release and tissue fine structure was closely related to the supply of O-2. The optimal conditions for liver perfusion depend upon an adequate supply of oxygen. This can be achieved by using either erythrocyte-free perfusate at a how rate greater than 6 ml/min/g liver or a 20% erythrocyte-containing perfusate at 2 ml/min/g. (C) 1996 Academic Press, Inc.

Ethanol feeding enhances inflammatory cytokine expression in lipopolysaccharide-induced hepatitis

Elevated concentrations of plasma tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 and IL-6 have been detected in patients with alcoholic hepatitis and have been implicated in the pathogenesis of hepatocyte necrosis. The present study used a rat model to conduct a detailed histological and biochemical examination of the expression of various pro-inflammatory cytokines and associated liver pathology in ethanol-potentiated lipopolysaccharide (LPS)-induced liver injury. Male Wistar rats were pair-fed either the control or ethanol-containing (36% of caloric intake as ethanol) form of the Lieber-DeCarli liquid diet for 6 weeks. Liver injury was induced by the i.v. injection of LPS (1 mu g/g bodyweight), with animals being killed at O, 1, 3, 6, 12 and 24 h after injection. At the later time points, plasma transaminase and transpeptidase activities were significantly elevated in ethanol-fed LPS-treated rats compared with control-fed LPS-treated animals. At these times after LPS treatment, hepatocytes in ethanol-fed animals displayed fatty change and necrosis with an associated neutrophil polymorph infiltrate. Time course analysis revealed that plasma TNF-alpha (1-3 h post-LPS) and IL-6 (3 h post-LPS) bioactivity was significantly elevated in ethanol-fed compared with control-fed animals. No difference was seen in plasma IL-1 alpha concentration (maximal in both groups 6 h post-LPS). The expression of TNF-alpha, IL-1 alpha, IL-1 beta and IL-6 mRNA were elevated between 1 and 6 h post-LPS in the livers of both control and ethanol-fed rats. However, ethanol-fed LPS-treated animals exhibited significantly higher maximal expression of IL-1 and IL-6 mRNA. Comparison of the appearance of cytokine mRNA and plasma bioactivity indicated an effect of ethanol feeding on post-transcriptional processing and/or the kinetics of the circulating cytokines. Elevated levels of both hepatic cytokine mRNA expression and the preceding plasma cytokines are presumably a necessary prerequisite for hepatic injury seen in this model and, therefore, possibly for the damage seen in human alcoholics. Further studies using this model may lead to significant advances in our understanding of the pathogenic mechanisms of alcoholic liver disease in humans.

Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia

Henneguya lesteri n. sp, (Myxosporea) is described from sand whiting, Sillago analis, from the southern Queensland coast of Australia. H. lesteri displays a preference for the pseudobranchs and is typically positioned along the afferent blood vessels, displacing the adjoining lamellae and disrupting their normal array, The plasmodia appeared as whitish-hyaline, elliptical cysts (mean dimensions 230 x 410 mum) attached to the oral mucosa lining of the hyoid arch on the inner surface of the operculum. Infections of the gills were also found, in which the plasmodia were spherical, averaged 240 x 240 mum in size and were located on the inner hemibranch margin. The parasites lodged in the gill filament crypts and generated a mild hyperplastic response of the branchial epithelium, In histological sections, the plasmodium wall and adjoining ectoplasm appeared as a finely granulated, weakly eosinophilic layer, Ultrastructurally, this section of the host-parasite interface contained an intricate complex of pinocytotic channels. H. lesteri is polysporic, disporoblastic and pansporoblast forming. Sporogenesis is asynchronous, with the earliest developmental stages aligned predominantly along the plasmodium periphery, and maturing sporoblasts and spores toward the center. Ultrastructural details of sporoblast and spore development are in agreement with previously described myxosporeans. The mature spore is drop-shaped, length (mean) 9.1 mum, width 4.7 mum, thickness 2.5 mum, and comprises 2 polar capsules positioned closely together, a binucleated sporoplasm and a caudal process of 12.6 mum. The polar capsules are elongated, 3.2 x 1.6 mum, with 4 turns of the polar filament. Mean length of the everted filament is 23.2 mum, Few studies have analyzed the 18S gene-of marine Myxosporea. In fact, H. lesteri is the first marine species of Henneguya to be characterized at the molecular level: we determined 1966 bp of the small-subunit (18S) rDNA, The results indicated that differences between this and the hitherto studied freshwater Henneguya species are greater than differences among the freshwater Henneguya species.

Differential perturbation of neuronal and glial glutamate transport systems in retinal ischaemia

Glutamate is the major excitatory neurotransmitter in the retina and is removed from the extracellular space by an energy-dependent process involving neuronal and glial cell transporters. The radial glial Muller cells express the glutamate transporter, GLAST, and preferentially accumulate glutamate. However, during an ischaemic episode, extracellular glutamate concentrations may rise to excitotoxic levels. Is this catastrophic rise in extracellular glutamate due to a failure of GLAST? Using immunocytochemistry, we monitored the transport of the glutamate transporter substrate, D-aspartate, in the retina under normal and ischaemic conditions. Two models of compromised retinal perfusion were compared: (1) Anaesthetised rats had their carotid arteries occluded for 7 days to produce a chronic reduction in retinal blood flow. Retinal function was assessed by electroretinography. D-aspartate was injected into the eye for 45 min, Following euthanasia, the retina was processed for D-aspartate. GLAST and glutamate immunocytochemistry. Although reduced retinal perfusion suppresses the electroretinogram b-wave, neither retinal histology, GLAST expression, nor the ability of Muller cells to uptake D-aspartate is affected. As this insult does not appear to cause excitotoxic neuronal damage, these data suggest that GLAST function and glutamate clearance are maintained during periods of reduced retinal perfusion. (2) Occlusion of the central retinal artery for 60 min abolishes retinal perfusion, inducing histological damage and electroretinogram suppression. Although GLAST expression appears to be normal. its ability to transport D-aspartate into Muller cells is greatly reduced. Interestingly, D-aspartate is transported into neuronal cells, i.e. photoreceptors, bipolar and ganglion cells. This suggests that while GLAST is vitally important for the clearance of excess extracellular glutamate, its capability to sustain inward transport is particularly susceptible to an acute ischaemic attack. Manipulation of GLAST function could alleviate the degeneration and blindness that result from ischaemic retinal disease. (C) 2001 Elsevier Science Ltd, All rights reserved.

A. W. Campbells Australian career: 1905-1937

The Australian Alfred Walter Campbell (1868-1937) is remembered as one of the two chief pioneers of the study of the cytoarchitectonics of the primate cerebral cortex. He had worked in Britain carrying out neuroanatomical and neuropathological research for almost two decades before his famous monograph on Histological Studies on the Localisation of Cerebral Function appeared in 1905. In that year he returned to his native Australia and practiced for over 30 years in Sydney as a neurologist rather than a neuropathologist, publishing mainly clinical material though he was involved in the investigation of the epidemic of Australian X disease, a viral encephalitis. His abrupt change in both the nature and the location of his career at a time when he was well established in Britain appears to have been a consequence of his marriage and the need to provide for a family. His simultaneous apparent abandonment of research seems not to have really been the case. As judged from the contents of a paper presented to a local medical congress in Sydney in 1911, it appears that, in Australia, Campbell did carry out a major comparative anatomical and histological investigation of the possibility of localization of function in the cerebellar cortex. He never published this work in detail. His investigation let him to conclude that no such localization of function existed, a view contrary to the then topical interpretation of Bolk (1906), but one in accordance with Gordon Holmes' views a decade later. Campbell's circumstances in Sydney, his extremely reticent nature and the essentially negative outcome of his investigation probably explain his failure to make his study more widely known.

Maternal antecedents for cerebral palsy in extremely preterm babies: A case-controlled study

The study aimed to identify significant antenatal risk factors for cerebral palsy (CP) among extremely preterm infants with a matched case-control design. Infants born between 1989 and 1996 at 24 to 27 weeks' gestation who survived to hospital discharge were evaluated: 30 with a proven diagnosis of CP at 2 years corrected for prematurity and 120 control children matched for gestational age without CP. Information on maternal obstetric risk factors and medication was obtained. Matched analyses were performed and odds ratios (OR) and 95% confidence intervals (CI) were calculated. An antenatal diagnosis of intrauterine growth restriction was associated with an increased risk of CP (OR 6.6; 95% CI 1.8 to 25.2), while maternal administration of corticosteroids was associated with a reduced risk of CP (OR 0.4; 95% CI 0.1 to 0.98). A high rate of placental histopathology was achieved but no relation between clinical or histological chorioamnionitis or funisitis and CP was demonstrated. Maternal preeclampsia was not associated with a statistically significant reduction in the risk of CP. It is concluded that a reduced risk of CP in extremely preterm infants is associated with the antenatal use of corticosteroids.

Significance of serum early pregnancy factor concentrations during pregnancy and embryonic development in Sminthopsis macroura (Spencer) (Marsupialia : Dasyuridae)

Marsupial pregnancy differs from that in eutherians in duration, placentation and hormonal profile so much so that maternal recognition of pregnancy may not occur in polyovular marsupials. However, a comparison of gravid and non-gravid uteri reveals differences indicative of histological and physiological adaptations to pregnancy. In the present study, the hypothesis that embryo-maternal signalling occurs in polyovular marsupials was tested by examining serum from non-pregnant and pregnant Sminthopsis macroura for the presence of early pregnancy factor (EPF), a serum protein secreted by the ovary in response to the presence of a newly fertilized egg in the oviduct. EPF is detectable in the serum of pregnant, but not in non-pregnant, females in all eutherians studied to date. In the present study, EPF was detected in S. macroura serum by the rosette inhibition test during the first 9 days of the 10.7 day gestation period in this marsupial. However, EPF was not detected on day 10, just before parturition, or in non-pregnant or preovulatory animals. Immunohistochemical analysis of ovaries from gravid and non-gravid animals demonstrates that EPF is found in the capillaries, interstitial spaces and secretory cells of the corpus luteum. It is concluded that the spatiotemporal pattern of EPF activity described strongly indicates that maternal recognition of pregnancy in marsupials is mediated, at least in part, by EPF. Because the endocrinological milieu is the same in pregnant and non-pregnant marsupials, the possibility of using marsupials as an experimental system for studying EPF function unconfounded by hormonal effects is presented.

The pathogenesis of oral lichen planus

Both antigen-specific and non-specific mechanisms may be involved in the pathogenesis of oral lichen planus (OLP). Antigen-specific mechanisms in OLP include antigen presentation by basal keratinocytes and antigen-specific keratinocyte killing by CD8(+) cytotoxic T-cells. Non-specific mechanisms include mast cell degranulation and matrix metalloproteinase (MMP) activation in OLP lesions. These mechanisms may combine to cause T-cell accumulation in the superficial lamina propria, basement membrane disruption, intra-epithelial T-cell migration, and keratinocyte apoptosis in OLP. OLP chronicity may be due, in part, to deficient antigen-specific TGF-beta1-mediated immunosuppression. The normal oral mucosa may be an immune privileged site (similar to the eye, testis, and placenta), and breakdown of immune privilege could result in OLP and possibly other autoimmune oral mucosal diseases. Recent findings in mucocutaneous graft-versus-host disease, a clinical and histological correlate of lichen planus, suggest the involvement of TNF-alpha, CD40, Fas, MMPs, and mast cell degranulation in disease pathogenesis. Potential roles for oral Langerhans cells and the regional lymphatics in OLP lesion formation and chronicity are discussed. Carcinogenesis in OLP may be regulated by the integrated signal from various tumor inhibitors (TGF-beta1, TNF-alpha, IFN-gamma, IL-12) and promoters (MIF, MMP-9). We present our recent data implicating antigen-specific and non-specific mechanisms in the pathogenesis of OLP and propose a unifying hypothesis suggesting that both may be involved in lesion development. The initial event in OLP lesion formation and the factors that determine OLP susceptibility are unknown.

Clinicopathological significance of the 'keloid-like' collagen and myxoid stroma in advanced rectal cancer

Aim: To establish the histological categorization of fibrotic stroma which reflects the biological behaviour of advanced rectal cancer. Methods and results: Six hundred and twenty-seven surgically resected cases of advanced rectal carcinoma were examined. We histologically categorized fibrotic stroma in the invasive frontal region into three groups: type A, multiple fine and mature fibres were stratified into layers: type B, broad bands of eosinophilic hyalinized collagen ('keloid-like' collagen) were intermingled: type C, myxoid stroma. Type A stroma was observed in 63% of patients, type B stroma in 25%, type C stroma in 12%.. The incidence of type A stroma decreased in accordance with Dukes stage (98% in Dukes A: 73% in B: 41%, in C1: 29% in C2) and conversely, there was an increase of C type (0%, in Dukes A; 4%, in B: 20% in C1: 54% in C2). Stroma type had a significant correlation with long-term survival (80% of 5-year survival in type A stroma: 54% in type B: 26% in type C). Based on multivariate analysis. it was found that the stromal pattern had independent prognostic value, together with nodal involvement. growth pattern. and lymphocyte infiltration. Conclusions: Tumour fibrotic stroma may play an important role as a regulator of neoplastic behaviour. Pathological categorization of the fibrotic stroma is helpful for predicting the prognostic outcome of patients with rectal carcinoma.

Oral lichen planus: Causes, diagnosis and management

Oral lichen planus (OLP) is a chronic inflammatory disease of unknown etiology. In this paper we review the clinical and histological features of OLP, process of OLP diagnosis, causes of OLP, management of OLP patients and medical treatment of OLP lesions. Approximately 0.2 per cent OLP patients develop intra-oral carcinoma each year compared with approximately 0.005 per cent Australian adults. Possible mechanisms of increased oral cancer risk in OLP patients are presented. The aims of current OLP therapy are to eliminate mucosal erythema and ulceration, alleviate symptoms and reduce the risk of oral cancer. Patient education may improve the outcomes of OLP therapy and further reduce the risk of oral cancer in OLP patients. Although OLP may be diagnosed clinically, appropriate specialist referral is required for: (i) histological diagnosis; (ii) assessment of causative/exacerbating factors, associated diseases and oral cancer risk; (iii) patient education and management; (iv) medical treatment; and (v) long-term review and re-biopsy as required.

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.

Development of Trichogramma australicum Girault (Hymenoptera : Trichogrammatidae) in Helicoverpa (Lepidoptera : Noctuidae) host eggs

Morphological studies of development of the egg parasitoid Trichogramma australicum Girault in the cotton bollworm, Helicoverpa armigera (Hubner), were conducted to provide benchmarks for assessing developmental rates in both natural hosts and artificial diets. Observations of living embryos and histological sections show that embryos proceed rapidly through cleavage and blastoderm formation and show a characteristic pinching or rotation 8 h after deposition. Eggs progressively increase in volume, primarily by increasing in diameter at the widest point. At 29 rectangle 1 C the duration of the egg stage is 22-24 h, the larval stage 27 h, the prepupal stage 50-52 h, and pupa 85 h. Larvae undergo dramatic shape changes as they ingest food but do not show signs of larval moults, reinforcing observations that there is only one larval instar. Criteria for staging the embryonic and postembryonic development in natural hosts will be used for future studies aimed at developing and refining artificial diets for Trichogramma.