Identification of the glycosaminoglycan keratan sulfate in the prostatic secretory cell

BACKGROUND. Prostate secretory granules (PSG) represent the basic secretory unit of the prostate gland, containing many of its exocrine proteases. Recent analysis of intraluminal corpora amylacea, a proposed by-product of PSG secretion, detected sulfated glycosaminoglycans (GAG) possibly keratan sulfate (KS),indicating a secretory mechanism for GAG in the human prostate surface epithelial cell. METHODS. Immunostains using anti-KS and anti-prostate-specific antigen (PSA) were evaluated on 10 sequential radical prostatectomy specimens, three of which had received neoadjuvant antiandrogen therapy. Extracts of normal secretory tissue as well as a sample composed almost entirely of prostatic stroma were subjected to Western blot analysis, using the same antibody panel. RESULTS. Keratan sulfate secretion from the normal prostate epithelial cell has been confirmed and correlates, as does PSA, with the presence of cytoplasmic PSG. No such correlation exists in most adenocarcinomas or in benign epithelium after androgen ablation. Western blot analyses confirmed tissue immunostains and demonstrated a secretory proteoglycan of 70-95 kDa. CONCLUSIONS. Recognition of PSG heralds a novel secretory mechanism within the human prostate gland that is linked to the secretion of KS. The role of KS in normal prostate secretion remains unknown, although it appears downregulated in neoplastic and androgen-ablated cells. (C) 2000 Wiley-Liss, Inc.

X-ray absorption and phase contrast imaging to study the interplay between plant roots and soil structure

Plant performance is, at least partly, linked to the location of roots with respect to soil structure features and the micro-environment surrounding roots. Measurements of root distributions from intact samples, using optical microscopy and field tracings have been partially successful but are imprecise and labour-intensive. Theoretically, X-ray computed micro-tomography represents an ideal solution for non-invasive imaging of plant roots and soil structure. However, before it becomes fast enough and affordable or easily accessible, there is still a need for a diagnostic tool to investigate root/soil interplay. Here, a method for detection of undisturbed plant roots and their immediate physical environment is presented. X-ray absorption and phase contrast imaging are combined to produce projection images of soil sections from which root distributions and soil structure can be analyzed. The clarity of roots on the X-ray film is sufficient to allow manual tracing on an acetate sheet fixed over the film. In its current version, the method suffers limitations mainly related to (i) the degree of subjectivity associated with manual tracing and (ii) the difficulty of separating live and dead roots. The method represents a simple and relatively inexpensive way to detect and quantify roots from intact samples and has scope for further improvements. In this paper, the main steps of the method, sampling, image acquisition and image processing are documented. The potential use of the method in an agronomic perspective is illustrated using surface and sub-surface soil samples from a controlled wheat trial. Quantitative characterization of root attributes, e.g. radius, length density, branching intensity and the complex interplay between roots and soil structure, is presented and discussed.

Structural interpretation of mutations in phenylalanine hydroxylase protein aids in identifying genotype-phenotype correlations in phenylketonuria

Phenylalanine hydroxylase (PAH) is the enzyme that converts phenylalanine to tyrosine as a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. Over 300 mutations have been identified in the gene encoding PAH that result in a deficient enzyme activity and lead to the disorders hyperphenylalaninaemia and phenylketonuria. The determination of the crystal structure of PAH now allows the determination of the structural basis of mutations resulting in PAH deficiency. We present an analysis of the structural basis of 120 mutations with a 'classified' biochemical phenotype and/or available in vitro expression data. We find that the mutations can be grouped into five structural categories, based on the distinct expected structural and functional effects of the mutations in each category. Missense mutations and small amino acid deletions are found in three categories:'active site mutations', 'dimer interface mutations', and 'domain structure mutations'. Nonsense mutations and splicing mutations form the category of 'proteins with truncations and large deletions'. The final category, 'fusion proteins', is caused by frameshift mutations. We show that the structural information helps formulate some rules that will help predict the likely effects of unclassified and newly discovered mutations: proteins with truncations and large deletions, fusion proteins and active site mutations generally cause severe phenotypes; domain structure mutations and dimer interface mutations spread over a range of phenotypes, but domain structure mutations in the catalytic domain are more likely to be severe than domain structure mutations in the regulatory domain or dimer interface mutations.

Analysis of Mouse Keratin 6a Regulatory Sequences in Transgenic Mice Reveals Constitutive, Tissue-Specific Expression by a Keratin 6a Minigene

The analysis of keratin 6 expression is complicated by the presence of multiple isoforms that are expressed constitutively in a number of internal stratified epithelia, in palmoplantar epidermis, and in the companion cell layer of the hair follicle. In addition, keratin 6 expression is inducible in interfollicular epidermis and the outer root sheath of the follicle, in response to wounding stimuli, phorbol esters, or retinoic acid. In order to establish the critical regions involved in the regulation of keratin 6a (the dominant isoform in mice), we generated transgenic mice with two different-sized mouse keratin 6a constructs containing either 1.3 kb or 0.12 kb of 5' flanking sequence linked to the lacZ reporter gene. Both constructs also contained the first intron and the 3' flanking sequence of mouse keratin 6a. Ectopic expression of either transgene was not observed. Double-label immunofluorescence analyses demonstrated expression of the reporter gene in keratin 6 expressing tissues, including the hair follicle, tongue, footpad, and nail bed, showing that both transgenes retained keratinocyte-specific expression. Quantitative analysis of beta -galactosidase activity verified that both the 1.3 and 0.12 kb keratin 6a promoter constructs produced similar levels of the reporter. Notably, both constructs were constitutively expressed in the outer root sheath and interfollicular epidermis in the absence of any activating stimulus, suggesting that they lack the regulatory elements that normally silence transcription in these cells. This study has revealed that a keratin 6a minigene contains critical cis elements that mediate tissue-specific expression and that the elements regulating keratin 6 induction lie distal to the 1.3 kb promoter region.

An overview of diversion strategies for Australian drug-related offenders

This paper provides a descriptive overview of options for diversion of drug-related offenders from the criminal justice system. Drug-related offences include drug offences (for example, possession of a prohibited substance); offences that are directly linked to intoxication (for example, drink-driving or assault); and offences committed to support drug use (for example, theft). After an offence has been detected by police, multiple opportunities for diversion occur throughout the criminal justice process. (a) Pre-arrest: when an offence is first detected, prior to a charge being laid. This is known as police diversion and includes fines, warnings and cautions, sometimes with educational information or referral to assessment and treatment. (b) Pre-trial: when a charge is made but before the matter is heard at court. Examples are treatment as a condition of bail, conferencing and prosecutor discretion. (c) Pre-sentence: a delay of sentence while assessment and treatment are sought. (d) Post-sentence: as part of sentencing, for example suspended sentences, drug courts, noncustodial sentences and circle sentencing. (e) Pre-release: prior to release from a sentence, on parole. Issues for diversion programmes include net widening, the ethics of coercion to treatment, the needs of families and intersectoral collaboration. A framework for diversion is presented in which increasingly treatment-focused and coercive diversion strategies are used as offenders' criminal careers and drug problems increase.

Identification of genes implicated in toxin production in the cyanobacterium Cylindrospermopsis raciborskii

Cylindrospermopsis raciborskii is a bloom-forming cyanobacterium found in both tropical and temperate climates which produces cylindrospermopsin, a potent hepatotoxic secondary metabolite. This organism is notorious for its association with a significant human poisoning incident on Palm Island, Australia, which resulted in the hospitalization of 148 people. We have screened 13 C. raciborskii isolates from various regions of Australia and shown that both toxic and nontoxic strains exist within this species. No association was observed between geographical origin and toxin production. Polyketide synthases (PKSs) and peptide synthetases (PSs) are enzymes involved in secondary metabolite biosynthesis in cyanobacteria. Putative PKS and PS genes from C. raciborskii strains AWT205 and CYPO2OB were identified by PCR using degenerate primers based on conserved regions within each gene. Examination of the strain-specific distribution of the PKS and PS genes in C. raciborskii isolates demonstrated a direct link between the presence of these two genes and the ability to produce cylindrospermopsin. Interestingly, the possession of these two genes was also linked. They were also identified in an Anabaena bergii isolate that was demonstrated to produce cylindrospermopsin. Taken together, these data suggest a likely role for these determinants in secondary metabolite and toxin production by C. raciborskii. (C) 2001 John Wiley & Sons, Inc.

Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of human melanoma is regulated by Smac/DIABLO release from mitochondria

In previous studies we have shown that the sensitivity of melanoma cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)induced apoptosis was determined largely by the level of expression of death receptor TRAIL receptor 2 on the cells. However, approximately one-third of melanoma cell lines were resistant to TRAIL, despite expression of high levels of TRAIL receptor 2. The present studies show that these cell lines had similar levels of TRAIL-induced activated caspase-3 as the TRAIL-sensitive lines, but the activated caspase-3 did not degrade substrates downstream of caspase-3 [inhibitor of caspase-activated DNase and poly(ADP-ribose) polymerase]. This appeared to be due to inhibition of caspase-3 by X-linked inhibitor of apoptosis (XIAP) because XIAP was bound to activated caspase-3, and transfection of XIAP into TRAIL-sensitive cell lines resulted in similar inhibition of TRAIL-induced apoptosis. Conversely, reduction of XIAP levels by overexpression of Smac/ DIABLO in the TRAIL-resistant melanoma cells was associated with the appearance of catalytic activity by caspase-3 and increased TRAIL-induced apoptosis. TRAIL was shown to cause release of Smac/DIABLO from mitochondria, but this release was greater in TRAIL-sensitive cell lines than in TRAIL-resistant cell lines and was associated with downregulation of XIAP levels. Furthermore, inhibition of Smac/DIABLO release by overexpression of Bcl-2 inhibited down-regulation of XIAP levels. These results suggest that Smac/DIABLO release from mitochondria and its binding to XIAP are an alternative pathway by which TRAIL induces apoptosis of melanoma, and this pathway is dependent on the release of activated caspase-3 from inhibition by XIAP and possibly other inhibitor of apoptosis family members.

Relationship of interleukin-6 and tumor necrosis factor-alpha with muscle mass and muscle strength in elderly men and women: The health ABC study

Background. A decline in muscle mass and muscle strength characterizes normal aging. As clinical and animal studies show it relationship between higher cytokine levels and low muscle mass, the aim of this study was to investigate whether markers, of inflammation are associated with muscle mass and strength in well-functioning elderly persons. Methods. We Used baseline data (1997-1998) of the Health, Aging, and Body Composition (Health ABC) Study on 3075 black and white men and women aged 70-79 years. Midthigh muscle cross-sectional area (computed tomography), appendicular muscle mass (dual-energy x-ray ab absorptiometry). isokinetic knee extensor strength (KinCom). and isometric inip strength were measured. plasma levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were assessed by enzyme-linked immunosorbent assay (ELISA). Results. Higher cytokine levels were generally associated with lower muscle mass and lower muscle strength. The most consistent relationship across the gender and race groups was observed for IL-6 and grip strength: per SD increase in IL-6, grip strength was 1.1 to 2.4 kg lower (p < .05) after adjustment for age, clinic Site. health status, medications, physical activity. smoking. height. and body fat. Ail overall measure of elevated cytokine level was created by combining the levels of IL-6 and TNF-alpha. With the exception of white men, elderly persons having high levels of IL-6 (> 1.80 pg/ml) as well as high levels of TNF-alpha (> 3.20 pg/ml) had a smaller muscle area, less appendicular mass. a lower knee extensor strength. and a lower grip strength compared to those with low levels of both cytokines. Conclusions. Higher plasma concentrations of IL-6 and TNF-alpha are associated with lower muscle mass and lower muscle strength in well-functioning older men and women. Higher cytokine levels. as often observed in healthy older persons. may contribute to the loss Of muscle mass and strength that accompanies aging.

Evolution of local recruitment and its consequences for marine populations

Advantages of dispersal on the scales that are possible in a long pelagic larval period are not apparent, even for benthic species. An alternative hypothesis is that wide dispersal may be an incidental byproduct of an ontogenetic migration from and then back to the parental habitat. Under this hypothesis, the water column is a better habitat than the bottom for early development. Because the parental area is often an especially favorable habitat for juveniles and adults, selection may even favor larval retention or larval return rather than dispersal. Where larval capabilities and currents permit, a high percentage of recruits may then be produced from local adults. Expected consequences of a high proportion of local recruitment are stronger links between stock and recruitment, greater vulnerability to recruitment overfishing and local modifications of habitat, greater local benefits from fishery reserves, and possibly more localized adaptation within populations. Export of some larvae is consistent with a high proportion of retained or returning larvae, could stabilize populations linked by larval exchange, and provide connectivity between marine reserves. Even a small amount of larval export could account for the greater gene flow, large ranges, and long evolutionary durations seen in species with long pelagic larval stages.

Variable expression of mental retardation, autism, seizures, and dystonic hand movements in two families with an identical ARX gene mutation

Two families, originally diagnosed as having nonsyndromic X-linked mental retardation (NSXLMR), were reviewed when it was shown that they had a 24-bp duplication (428-45 1dup(24bp)) in the ARX gene [Stromme et al., 2002: Nat Genet 30:441-445]. This same duplication had also been found in three other families: one with X-linked infantile spasms and hypsarrhythmia (X-linked West syndrome, MIM 308350) and two with XLMR and dystonic movements of the hands (Partington syndrome, MIM 309510). On review, manifestations of both West and Partington syndromes were found in some individuals from both families. In addition, it was found that one individual had autism and two had autistic behavior, one of whom had epilepsy. The degree of mental retardation ranged from mild to severe. A GCG trinucleotide expansion (GCG)10+7 and a deletion of 1,517 by in the ARX gene have also been found in association with the West syndrome, and a missense mutation (1058C >T) in a family with a newly recognized form of myoclonic epilepsy, severe mental retardation, and spastic paraplegia [Scheffer et al., 2002: Neurology, in press]. Evidently all these disorders are expressions of mutations in the same gene. It remains to be seen what proportions of patients with infantile spasms, focal dystonia, autism, epilepsy, and nonsyndromic mental retardation are accounted for by mutations in the ARX gene. (C) 2002 Wiley-Liss, Inc.

Altered kinetics and benzodiazepine sensitivity of a GABA(A) receptor subunit mutation [gamma(2)(R43Q)] found in human epilepsy

The gamma-aminobutyric acid type A (GABA(A)) receptor mediates fast inhibitory synaptic transmission in the CNS. Dysfunction of the GABA(A) receptor would be expected to cause neuronal hyperexcitability, a phenomenon linked with epileptogenesis. We have investigated the functional consequences of an arginine-to-glutamine mutation at position 43 within the GABA(A) gamma(2)-subunit found in a family with childhood absence epilepsy and febrile seizures. Rapid-application experiments performed on receptors expressed in HEK-293 cells demonstrated that the mutation slows GABA(A) receptor deactivation and increases the rate of desensitization, resulting in an accumulation of desensitized receptors during repeated, short applications. In Xenopus laevis oocytes, two-electrode voltage-clamp analysis of steady-state currents obtained from alpha(1)beta(2)gamma(2) or alpha(1)beta(2)gamma(2)(R43Q) receptors did not reveal any differences in GABA sensitivity. However, differences in the benzodiazepine pharmacology of mutant receptors were apparent. Mutant receptors expressed in oocytes displayed reduced sensitivity to diazepam and flunitrazepam but not the imiclazopyricline zolpidem. These results provide evidence of impaired GABA(A) receptor function that could decrease the efficacy of transmission at inhibitory synapses, possibly generating a hyperexcitable neuronal state in thalamocortical networks of epileptic patients possessing the mutant subunit.

The role of dogs in transmission of gastrointestinal parasites in a remote tea-growing community in northeastern India

The prevalence and risk factors associated with canine gastrointestinal parasitic zoonoses and the role of dogs in the mechanical transmission of human Ascaris infection was examined in three tea estates in Assam, India. Nearly all (99%) dogs harbored one or more zoonotic species of gastrointestinal parasites, with hookworm infection being most common (94%). Parasitic stages presumed to be host-specific for humans such as Ascaris spp. (31%), Trichuris trichiura (25%), and Isospora belli (2%) were also recovered from dog feces. A polymerase chain reaction-linked restriction fragment length polymorphism technique was used to differentiate the species of Ascaris eggs in dog feces. The results of this study demonstrate the role of the dog as a significant disseminator and environmental contaminator of Ascaris lumbricoides in communities where promiscuous defecation by humans occurs.

Late Quaternary Milankovitch-scale climatic change and variability and its impact on monsoonal Australasia

Four pollen and charcoal records derived from marine cores around the northern perimeter of Australia are examined to provide a regional picture of patterns, causes and impacts of climate change over the last 100-300 ka. The availability of radiocarbon dates and oxygen isotope records for the cores provides primary chronological control. Spectral analysis of components of these records demonstrates an overall importance of Milankovitch frequencies with clear glacial-interglacial cyclicity dominated by variation in precipitation. In addition, a number of pollen taxa, as well as charcoal particles, exhibit a 30 ka frequency that is considered, from its relationship with biomass burning and with results of past modelling, to reflect changes in the intensity of El Nino-Southern Oscillation (ENSO) variability. Pollen components of all records show a decline, frequently stepwise, in more fire-sensitive vegetation and its replacement with more fire-tolerant vegetation. There is some evidence that this trend is linked to an onset or general increase in ENSO activity and perhaps also to variation in monsoon activity dating from about 300 ka BP that was caused by changes to oceanic circulation within the Indonesian region. The trend may have accelerated within the last 45 ka due to burning by indigenous people. (C) 2003 Elsevier B.V. All rights reserved.

Choking deaths: the role of antipsychotic medication

Background An increased risk of choking associated with antipsychotic medication has been repeatedly postulated. Aims To examine this association in a large number of cases of choking deaths. Method Cases of individuals who had died because of choking were linked with a case register recording contacts with public mental health services. The actual and expected rates of psychiatric disorder and the presence of psychotropic medication in post-mortem blood samples were compared. Results The 70 people who had choked to death were over 20 times more likely to have been treated previously for schizophrenia. They were also more likely to have had a prior organic psychiatric syndrome. The risk for those receiving thioridazine or lithium was. respectively, 92 times and 30 times greater than expected. Other antipsychotic and psychotropic drugs were not over-represented. Conclusions The increased risk of death in people with schizophrenia may be a combination of inherent predispositions and the use of specific antipsychotic drugs. The increased risk of choking in those with organic psychiatric syndromes is consistent with the consequences of compromised neurological competence. Declaration of interest None.

The secondary vascular system of Actinopterygii: interspecific variation in origins and investment

Vascular casts of 3 species of Chondrichthyes, 1 of Dipnoi, 1 of Chondrostei and 14 species of the Teleostei were examined by light and scanning electron microscopy in order to give a qualitative and quantitative analysis of interarterial anastomoses (iaas) that indicate the presence (or absence) of a secondary vascular system (SVS). Anastomoses were found to originate from a variety of different primary blood vessels, many of which have not been previously identified as giving rise to secondary vessels. Segmental arteries derived from the dorsal aorta and supplying body musculature were major sites of origin of the SVS, although there was considerable variation in where, in the hierarchy of arterial branching, the anastomoses occurred. The degree of investment in a SVS was species specific, with more active species having a higher degree of secondary vascularisation. This difference was quantified using an absolute count of iaas between Anguilla reinhardtii and Trachinotus baillonii. A range of general features of the SVS is also described. No evidence of iaas was found on the coeliac, mesenteric or renal circulation in any species. Evidence of iaas was lacking in the dipnoan and chondrichthyan species examined, suggesting that a SVS is restricted to Actinopterygii. The presence and distribution of a SVS does not appear to be exclusively linked to phylogenetic position, but rather to the physiological adaptation of the species.