Cloning and gastrointestinal expression of rat hephaestin: relationship to other iron transport proteins

The membrane-bound ceruloplasmin homolog hephaestin plays a critical role in intestinal iron absorption. The aims of this study were to clone the rat hephaestin gene and to examine its expression in the gastrointestinal tract in relation to other genes encoding iron transport proteins. The rat hephaestin gene was isolated from intestinal mRNA and was found to encode a protein 96% identical to mouse hephaestin. Analysis by ribonuclease protection assay and Western blotting showed that hephaestin was expressed at high levels throughout the small intestine and colon. Immunofluorescence localized the hephaestin protein to the mature villus enterocytes with little or no expression in the crypts. Variations in iron status had a small but nonsignificant effect on hephaestin expression in the duodenum. The high sequence conservation between rat and mouse hephaestin is consistent with this protein playing a central role in intestinal iron absorption, although its precise function remains to be determined.

Protein targeting to the plasma membrane of adult skeletal muscle fiber: An organized mosaic of functional domains

The plasma membrane of differentiated skeletal muscle fibers comprises the sarcolemma, the transverse (T) tubule network, and the neuromuscular and muscle-tendon junctions. We analyzed the organization of these domains in relation to defined surface markers, beta -dystroglycan, dystrophin, and caveolin-3, These markers were shown to exhibit highly organized arrays along the length of the fiber. Caveolin-3 and beta -dystroglycan/dystrophin showed distinct, but to some extent overlapping, labeling patterns and both markers left transverse tubule openings clear. This labeling pattern revealed microdomains over the entire plasma membrane with the exception of the neuromuscular and muscle-tendon junctions which formed distinct demarcated macrodomains. Our results suggest that the entire plasma membrane of mature muscle comprises a mosaic of T tubule domains together with sareolemmal caveolae and beta -dystroglycan domains. The domains identified with these markers were examined with respect to targeting of viral proteins and other expressed domain-specific markers, We found that each marker protein was targeted to distinct microdomains, The macrodomains were intensely labeled with all our markers. Replacing the cytoplasmic tail of the vesicular stomatitis virus glycoprotein with that of CD4 resulted in retargeting from one domain to another. The domain-specific protein distribution at the muscle cell surface may be generated by targeting pathways requiring specific sorting information but this trafficking is different from the conventional apical-basolateral division. (C) 2001 Academic Press.

Functional heterogeneity within rhodamine123(lo) Hoechst33342(lo/sp) primitive hemopoietic stem cells revealed by pyronin Y

Objective. The aim of this study was to determine the function of primitive hematopoietic stem cells (PHSC) at phases G(0) and G(1) of the cell cycle. Materials and Methods. A combination of supravital dyes rhodamine123 (Rh), Hoechst33342 (Ho), and pyronin (PY) was used to isolate the G(0) and G(1) subsets of PHSC. A competitive repopulation assay was used to evaluate their in vivo function. Results. We confirmed that the Rh(lo)Lin(-)Kit(+)Sca-1(+) PHSC were relatively quiescent when compared with the more mature Rh(hi)Lin(-)Kit(+)Sca-1 HSC and Rh(hi)Lin(-)Kit(+)Sca-1(-) progenitors. In addition, cells with Rh(lo)Lin(-)Kit(+)Sca-1(+), Rh(lo)Ho(lo)Lin(-)Sca-1(+), or Rh(lo)Ho(sp)Lin(-)Sca-1(+) phenotypes identified the same cell population. We further subfractionated the Rh(lo)Ho(lo/sp)Lin(-)Sca-1(+) PHSC using PY into PYlo and PYhi subsets. Limiting dilution analysis revealed that the frequency of long-term in vivo competitive repopulating units (CRU) of the (PYRhHolo/sp)-Rh-lo-Ho-lo PHSC was 1 in 10 cells, whereas there was at least a three-fold lower frequency in those isolated at the G(1) phase (PYhi) We found a dose-dependent PY-mediated cytotoxicity that at moderate concentration affected most of the murine hematopoietic compartment but spared the early HSC compartment. Conclusion. Our data confirm that the HSC compartment is hierarchically ordered on the basis of quiescence and further extend this concept to PY-mediated cytotoxicity. PY supravital dye can be used to reveal functional heterogeneity within the (RhHolosp)-Ho-lo PHSC population but is of limited use in dissecting the relatively more mature hematopoietic stem/progenitor cell population. (C) 2001 International Society for Experimental Hematology. Published by Elsevier Science Inc.

Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration

We have investigated the expression and function of the isoforms of laminin bearing the alpha(5) chain, i.e. laminin-10/11 in neonatal and adult human skin. By immunostaining human skin derived from a variety of anatomic sites, we found that the laminin-alpha(5) chain is expressed abundantly in the basement membrane underlying the interfollicular epidermis and the blood vessels in the dermis. Interestingly, while the expression level of the well-studied laminin-5 isoform did not change significantly with age, laminin-10/11 (a5 chain) appeared to decrease in the basement membrane underlying the epidermis, in adult skin. In contrast, the levels of laminin-10/11 in the basement membrane underlying blood vessels remained unchanged in neonatal vs. adult skin. Importantly, in vitro cell adhesion assays demonstrated that laminin-10/11 is a potent adhesive substrate for both neonatal and adult keratinocytes and that this adhesion is mediated by the alpha(3)beta(1), and alpha(6)beta(4) integrins. Adhesion assays performed with fractionated basal keratinocytes showed that stem cells, transit amplifying cells and early differentiating cells all adhere to purified laminin-10/11 via these receptors. Further, laminin-10/11 provided a proliferative signal for neonatal foreskin keratinocytes, adult breast skin keratinocytes, and even a human papillomavirus type-18 transformed tumorigenic keratinocyte cell line in vitro. Finally, laminin-10/11 was shown to stimulate keratinocyte migration in an in vitro wound healing assay. These results provide strong evidence for a functional role for laminin-10/11 in epidermal proliferation during homeostasis, wound healing and neoplasia.

Functional recycling of C2 domains throughout evolution: A comparative study of synaptotagmin, protein kinase C and phospholipase C by sequence, structural and modelling approaches

The C2 domain is one of the most frequent and widely distributed calcium-binding motifs. Its structure comprises an eight-stranded beta-sandwich with two structural types as if the result of a circular permutation. Combining sequence, structural and modelling information, we have explored, at different levels of granularity, the functional characteristics of several families of C2 domains. At the coarsest level,the similarity correlates with key structural determinants of the C2 domain fold and, at the finest level, with the domain architecture of the proteins containing them, highlighting the functional diversity between the various subfamilies. The functional diversity appears as different conserved surface patches throughout this common fold. In some cases, these patches are related to substrate-binding sites whereas in others they correspond to interfaces of presumably permanent interaction between other domains within the same polypeptide chain. For those related to substrate-binding sites, the predictions overlap with biochemical data in addition to providing some novel observations. For those acting as protein-protein interfaces' our modelling analysis suggests that slight variations between families are a result of not only complementary adaptations in the interfaces involved but also different domain architecture. In the light of the sequence and structural genomic projects, the work presented here shows that modelling approaches along with careful sub-typing of protein families will be a powerful combination for a broader coverage in proteomics. (C) 2003 Elsevier Ltd. All rights reserved.

Functional analysis of neurotransmission at β2-laminin deficient terminals

beta2-Laminin is important for the formation of neuromuscular junctions in vertebrates. Previously, we have inactivated the gene that encodes for beta2-laminin in mice and observed predominantly prejunctional structural defects. In this study, we have used both intra- and extracellular recording methods to investigate evoked neurotransmission in beta2-laminin-deficient mice, from postnatal day 8 (P8) through to day 18(P18). Our results confirmed that there was a decrease in the frequency of spontaneous release, but no change in the postjunctional response to such release. Analysis of evoked neurotransmission showed an increase in the frequency of stimuli that failed to elicit an evoked postjunctional response in the mutants compared to litter mate controls, resulting in a 50% reduction in mean quantal content at mutant terminals. Compared to littermate controls, beta2-laminin-deficient terminals showed greater synaptic depression when subjected to high frequency stimulation. Furthermore, the paired pulse ratio of the first two stimuli was significantly lower in beta2-laminin mutant terminals. Statistical analysis of the binomial parameters of release showed that the decrease in quantal content was due to a decrease in the number of release sites without any significant change in the average probability of release. This suggestion was supported by the observation of fewer synaptic vesicle protein 2 (SV2)-positive varicosities in beta2-laminin-deficient terminals and by ultrastructural observations showing smaller terminal profiles and increased Schwann cell invasion in beta2-laminin mutants; the differences between beta2-laminin mutants and wild-type mice were the same at both P8 and P18. From these results we conclude that beta2-laminin plays a role in the early structural development of the neuromuscular junction. We also suggest that transmitter release activity may act as a deterrent to Schwarm cell invasion in the absence of beta2-laminin.

Patients with chronic neck pain demonstrate altered patterns of muscle activation during performance of a functional upper limb task

Study Design. Cross-sectional study. Objective. This study compared neck muscle activation patterns during and after a repetitive upper limb task between patients with idiopathic neck pain, whiplash-associated disorders, and controls. Summary of Background Data. Previous studies have identified altered motor control of the upper trapezius during functional tasks in patients with neck pain. Whether the cervical flexor muscles demonstrate altered motor control during functional activities is unknown. Methods. Electromyographic activity was recorded from the sternocleidomastoid, anterior scalenes, and upper trapezius muscles. Root mean square electromyographic amplitude was calculated during and on completion of a functional task. Results. A general trend was evident to suggest greatest electromyograph amplitude in the sternocleidomastoid, anterior scalenes, and left upper trapezius muscles for the whiplash-associated disorders group, followed by the idiopathic group, with lowest electromyographic amplitude recorded for the control group. A reverse effect was apparent for the right upper trapezius muscle. The level of perceived disability ( Neck Disability Index score) had a significant effect on the electromyographic amplitude recorded between neck pain patients. Conclusions. Patients with neck pain demonstrated greater activation of accessory neck muscles during a repetitive upper limb task compared to asymptomatic controls. Greater activation of the cervical muscles in patients with neck pain may represent an altered pattern of motor control to compensate for reduced activation of painful muscles. Greater perceived disability among patients with neck pain accounted for the greater electromyographic amplitude of the superficial cervical muscles during performance of the functional task.

Dissecting the EphA3/ephrin-A5 interactions using a novel functional mutagenesis screen

The EphA3 receptor tyrosine kinase preferentially binds ephrin-A5, a member of the corresponding subfamily of membrane-associated ligands. Their interaction regulates critical cell communication functions in normal development and may play a role in neoplasia. Here we describe a random mutagenesis approach, which we employed to study the molecular determinants of the EphA3/ephrin-A5 recognition. Selection and functional characterization of EphA3 point mutants with impaired ephrin-A5 binding from a yeast expression library defined three EphA3 surface areas that are essential for the EphA3/ephrin-A5 interaction. Two of these map to regions identified previously in the crystal structure of the homologous EphB2-ephrin-B2 complex as potential ligand/receptor interfaces. In addition, we identify a third EphA3/ephrin-A5 interface that falls outside the structurally characterized interaction domains. Functional analysis of EphA3 mutants reveals that all three Eph/ephrin contact areas are essential for the assembly of signaling-competent, oligomeric receptor-ligand complexes.

The Mre11 complex and ATM: a two-way functional interaction in recognising and signaling DNA double strand breaks

Mutations in components of the Mre 11/Rad50/Nbs1 complex give rise to genetic disorders characterized by neurological abnormalities, radiosensitivity, cell cycle checkpoint defects, genomic instability and cancer predisposition. Evidence exists that this complex associates with chromatin during DNA replication and acts as a sensor of double strand breaks (dsbs) in DNA after exposure to radiation. A series of recent reports provides additional support that the complex senses breaks in DNA and relays this information to ATM, mutated in ataxia-telangiectasia (A-T), which in turn activates pathways for cell cycle checkpoint activation. Paradoxically members of the Mre11 complex are also downstream of ATM in these pathways. Here, Lavin attempts to make sense of this sensing mechanism with reference to a series of recent reports on the topic. (C) 2004 Elsevier B.V. All rights reserved.

Glutamate transporter localization does not correspond to the temporary functional recovery and late degeneration after acute ocular ischemia in rats

Purpose: To determine whether the localization of retinal glutamate transporters is affected by retinal ischaemia and whether their ability to transport glutamate decreases with the progression of ischemic retinal and optic nerve degeneration. Methods: Retinal ischemia was induced in rats by acutely increasing the intraocular pressure (IOP, 110 mmHg/60 min). Reperfusion was permitted for periods up to 60 days post-ischemia. Functional evaluation was performed by monitoring the pupil light reflexes (PLRs) and electroretinograms (flash, flicker ERG and oscillatory potentials). Glutamate transporter localization and D-aspartate (glutamate analogue) uptake were assessed by immunohistochemistry. Results: Intense immunoreactivity for the retinal glutamate transporters (GLAST, GLT1, EAAC1 and EAAT5) was observed at all time points after the insult, despite severe retinal degeneration. D-aspartate was also normally accumulated in the ischemic retinas. Ten days post-operatively the PLR ratio (ratio = indirect/direct PLR = 34 +/- 7(.)5%) was significantly less than the pre-operative value (pre-op = 76(.)7 +/- 2 (.)6%, p < 0(.)05). However, 25 and 35 days post-operatively PLR ratios did not differ significantly from pre-operative values (44(.)4 +/- 6(.)9 and 53(.)8 +/- 9(.)6%, p > 0(.)05). Forty-five and 60 days post-operatively the PLR ratio declined again and was significantly lower than the pre-operative value (33(.)8 + 8(.)7 and 26(.)2 + 8(.)9%, p < 0(.)05). Statistical analysis revealed that all tested ERG components had significantly higher values at 32, but not at 42 and 58 days post-operatively when compared to the first time point recorded post-operatively (10 days). Conclusions: While retinal glutamate transport is compromised during an acute ischemic insult, consequent retinal recovery and degeneration are not due to a change in the excitatory amino acid transporter localization or D-aspartate (glutamate analogue) uptake. Rat retina and optic nerve are capable of spontaneous, but temporary, functional recovery after an acute ischemic insult. (C) 2004 Elsevier Ltd. All rights reserved.

Personality disorders in the community: Results from the Australian National Survey of Mental Health and Well-Being Part III - Relationships between specific type of personality disorder, Axis 1 mental disorders and physical conditions with disability and health consultations

Background The aims of this study were threefold. First, to ascertain whether personality disorder (PD) was a significant predictor of disability (as measured in a variety of ways) over and above that contributed by Axis I mental disorders and physical conditions. Second, whether the number of PD diagnoses given to an individual resulted in increasing severity of disability, and third, whether PD was a significant predictor of health and mental health consultations with GPs, psychiatrists, and psychologists, respectively, over the last 12 months. Method Data were obtained from the National Survey of Mental Health and Wellbeing, conducted between May and August 1997. A stratified random sample of households was generated, from which all those aged 18 and over were considered potential interviewees. There were 10 641 respondents to the survey, and this represented a response rate of 78%. Each interviewee was asked questions indexing specific ICD-10 PD criteria. Results Five measures of disability were examined. It was found that PD was a significant predictor of disability once Axis I and physical conditions were taken into account for four of the five disability measures. For three of the dichotomously-scored disability measures, odds ratios ranged from 1.88 to 6.32 for PD, whilst for the dimensionally-scored Mental Summary Subscale of the SF-12, a beta weight of -0.17 was recorded for PD. As regards number of PDs having a quasi-linear relationship to disability, there was some indication of this on the SF-12 Mental Summary Subscale and the two role functioning measures, and less so on the other two measures. As regards mental consultations, PD was a predictor of visits to GPs, psychiatrists and psychologists, over and above Axis I disorders and physical conditions. Conclusion The study reports findings from a nationwide survey conducted within Australia and as such the data are less influenced by the selection and setting bias inherent in other germane studies. However, it does support previous findings that PD is a significant predictor of disability and mental health consultations independent of Axis I disorders and physical conditions.

Three distinct molecular surfaces in ephrin-A5 are essential for a functional interaction with EphA3

Eph receptor tyrosine kinases (Ephs) function as molecular relays that interact with cell surface-bound ephrin ligands to direct the position of migrating cells. Structural studies revealed that, through two distinct contact surfaces on opposite sites of each protein, Eph and ephrin binding domains assemble into symmetric, circular heterotetramers. However, Eph signal initiation requires the assembly of higher order oligomers, suggesting additional points of contact. By screening a random library of EphA3 binding-compromised ephrin-A5 mutants, we have now determined ephrin-A5 residues that are essential for the assembly of high affinity EphA3 signaling complexes. In addition to the two interfaces predicted from the crystal structure of the homologous EphB2 center dot ephrin-B2 complex, we identified a cluster of 10 residues on the ephrin-A5 E alpha-helix, the E-F loop, the underlying H beta-strand, as well as the nearby B - C loop, which define a distinct third surface required for oligomerization and activation of EphA3 signaling. Together with a corresponding third surface region identified recently outside of the minimal ephrin binding domain of EphA3, our findings provide experimental evidence for the essential contribution of three distinct protein-interaction interfaces to assemble functional EphA3 signaling complexes.

Identification of two evolutionarily conserved and functional regulatory elements in intron 2 of the human BRCA1 gene

Cross-species comparative genomics is a powerful strategy for identifying functional regulatory elements within noncoding DNA. In this paper, comparative analysis of human and mouse intronic sequences in the breast cancer susceptibility gene (BRCA1) revealed two evolutionarily conserved noncoding sequences (CNS) in intron 2, 5 kb downstream of the core BRCA1 promoter. The functionality of these elements was examined using homologous-recombination-based mutagenesis of reporter gene-tagged cosmids incorporating these regions and flanking sequences from the BRCA1 locus. This showed that CNS-1 and CNS-2 have differential transcriptional regulatory activity in epithelial cell lines. Mutation of CNS-1 significantly reduced reporter gene expression to 30% of control levels. Conversely mutation of CNS-2 increased expression to 200% of control levels. Regulation is at the level of transcription and shows promoter specificity. Both elements also specifically bind nuclear proteins in vitro. These studies demonstrate that the combination of comparative genomics and functional analysis is a successful strategy to identify novel regulatory elements and provide the first direct evidence that conserved noncoding sequences in BRCA1 regulate gene expression. (c) 2005 Elsevier Inc. All rights reserved.

From complexity to category: Responding to diagnostic uncertainties of autistic spectrum disorders

Objective: Recent data from Education Queensland has identified rising numbers of children receiving diagnoses of autistic spectrum disorder (ASD). Faced with funding diagnostic pressures, in clinical situations that are complex and inherently uncertain, it is possible that specialists err on the side of a positive diagnosis. This study examines the extent to which possible overinclusion of ASD diagnosis may exist in the presence of uncertainty and factors potentially related to this practice in Queensland. Methods: Using anonymous self-report, all Queensland child psychiatrists and paediatricians who see paediatric patients with development/behavioural problems were surveyed and asked whether they had ever specified an ASD diagnosis in the presence of diagnostic uncertainty. Using logistic regression, elicited responses to the diagnostic uncertainty questions were related to other clinical- and practice-related characteristics. Results: Overall, 58% of surveyed psychiatrists and paediatricians indicated that, in the face of diagnostic uncertainty, they had erred on the side of providing an ASD diagnosis for educational ascertainment and 36% of clinicians had provided an autism diagnosis for Carer's Allowance when Centrelink diagnostic specifications had not been met. Conclusion: In the absence of definitive biological markers, ASD remains a behavioural diagnosis that is often complex and uncertain. In response to systems that demand a categorical diagnostic response, specialists are providing ASD diagnoses, even when uncertain. The motivation for this practice appears to be a clinical risk/benefit analysis of what will achieve the best outcomes for children. It is likely that these practices will continue unless systems change eligibility to funding based on functional impairment rather than medical diagnostic categories.