To investigate genetic contributors to congenital joint dislocation, we have characterised a consanguineous family segregating an autosomal recessive connective tissue disorder (lax skin and joints) with neurological abnormalities.

We performed a 400-microsatellite genome screen under a hypothesis of homozygosity for a common ancestral disease allele. Multipoint linkage analysis identified a 50 cM region at 10q23.1-23.4 with a peak logarithm of odds (LOD) score of Z = 2.99. Fine mapping refined this to a 14 Mb critical region which demonstrated homozygosity by descent exclusively in affected individuals with a maximal LOD of Z = 3.63. Eighty-five genes within this interval were prioritised for disease gene candidacy. One gene ALDH18A1, encoding ∆1-pyrroline-5-carboxylate synthase (P5CS), was focused on since a missense mutation causes progressive neurodegeneration, joint dislocations, lax skin and metabolic derangement in a consanguineous Algerian family. Sequencing of ALDH18A1 in an affected individual identified the transition, 2350C>T, predicting the substitution H784Y. Comparative sequence analysis showed H784 is conserved across all phyla within a novel C-terminal tail motif.

P5CS is a bifunctional enzyme that converts glutamate to proline and ornithine upstream of the urea cycle. Proline, a major constituent of connective tissue, has also been implicated in neurotransmission. Therefore, impaired synthesis could lead to abnormal joint, skin and brain development. However, in vivo enzymatic assays we performed suggest proline biosynthesis is not perturbed, correlating with the normal metabolic profiles found in affected individuals. A partial P5CS crystal structure is available and hints towards roles for H784 in P5CS dimerisation and modulation of an active site cleft. Moonlighting roles (i.e. the evolutionary adoption of an unrelated function by an enzyme) exist for other metabolic enzymes. P5CS may therefore possess additional uncharacterised functions. The pathogenic effect of H784Y on P5CS suggests novel roles for the C-terminal domain in P5CS stability or function within the cell.

Chronic wounds may in part result from a deficiency in crucial growth factors. To compensate for this deficiency the topical application of growth factors has been studied. Fibroblast growth factor 2 (FGF-2, or basic FGF) has been well studied for this purpose. The aim was to determine the concentration profiles of this growth factor within experimental wounds following topical administration in different formulations.

A dosage of 0.3 µg FGF-2 was incorporated into three formulations (solution, gel or dried gel film on Melolin™ backing). FGF-2 formulations or PBS was administered to punch biopsy wounds in rats (6 animals per group: total n = 72). At two, five or eight hours, the animals from each group were euthanised by CO2 inhalation and cervical dislocation. Wound tissue was dissected horizontally to surface granulation, subcutaneous fat, superficial muscle and deep muscle layers and the amount of FGF-2 at various wound depths was quantified via ELISA. Tissue FGF-2 concentrations were compared using a repeated measures ANOVA (Minitab Version 14.1).

The highest concentrations of FGF-2 were seen in the surface granulation tissue of rats two hours after receiving the solution formulation (2275 ± 787 pg/g; mean ± SD, > 1000 pg/g higher than control levels). Concentrations decreased with increasing tissue depth and were significantly greater than the PBS control in the surface granulation and subcutaneous fat layers (P < 0.05, ANOVA). There was a statistically significant difference in the mean FGF-2 levels with respect to formulation and time following application of the formulation (P < 0.05, ANOVA).

In conclusion, elevated FGF-2 could be measured in superficial wound tissues up to eight hours post-application of a solution. However, application of a comparable amount (0.3 µg FGF-2) in hypromellose gels or films did not give appreciable elevation of FGF-2 in wound tissues.

It has been suggested that homeostatic regulation of synaptic plasticity is required to maintain the overall strength of synaptic inputs to a cell within a dynamic range. This has been implemented in the Bienenstock, Cooper and Munro (BCM) computational model by θM, a cell-wide threshold for long-term potentiation (LTP) that is modulated by previous levels of postsynaptic cell firing. The aim of this research was to test the predictions of the model regarding θM for hippocampal LTP.

Field excitatory postsynaptic potentials (fEPSPs) were recorded in response to stimulation of the Schaffer collaterals in area CA1 of acute hippocampal slices from 6-7-week-old male Sprague-Dawley rats. Priming stimulation of one pathway significantly reduced the level of LTP induced 30 min later by 100 Hz stimulation of a second (heterosynaptic) pathway (mean ± SEM, 12 ± 5%, n = 6, P < 0.05, unpaired t-test) compared with control LTP (27 ± 5%, n = 8). In accord with predictions of the BCM model of cell-wide changes in θM, priming stimulation of synapses on the basilar dendrites significantly inhibited LTP induced at synapses on the apical dendrites (16 ± 4%, n = 6, P < 0.05) compared with control LTP (30 ± 3%, n = 6). Contrary to the predicted role of cell-firing in changes of θM, hyperpolarising cells (by direct current injection) during priming to completely prevent somatic action potentials, did not prevent the priming effect (control LTP: 40 ± 10%, n = 5; primed LTP: -6 ± 6%, n = 5, P < 0.05).

These results confirm that synaptic plasticity is homeostatically regulated by the cell-wide history of activity. However, postsynaptic cell firing does not mediate this regulatory process.

Tumour necrosis factor-α (TNFα) is an important pro-inflammatory cytokine that is released from activated immune cells. An important part of the TNFα response is modulation of gene expression, predominantly via nuclear factor-κB (NF-κB) and activator protein-1 (AP1) transcription factors. Several key TNFα responses have been linked to the intracellular accumulation of redox-active species (RAS). A major source of intracellular RAS is the mitochondrial respiratory chain. We have found that a mitochondrially targeted antioxidant delays TNFα-induced transcription factor activation. In the current study we have investigated whether mitochondrial RAS participate in TNFα-induced gene expression.

The human monocyte-like leukaemic U937 cell line was used as a model system to investigate TNFα-induced redox-mediated gene expression by microarray analysis. Total RNA was isolated from U937 cells treated with either 5 ng/mL human TNFα, 2 μM mitoE (a mitochondrially targeted version of vitamin E), or a combination of both for 45 and 120 min. Total RNA was reverse transcribed to cDNA with poly-dT anchored oligo primers and applied to human 20K oligo arrays as biological triplicates.

MitoE was found to repress the TNFα-induced expression of a subset of genes by approximately 50%, including inhibitory κB-a (IκBa), inhibitory κB-z (IκBz), monocyte chemoattractant protein-1 (MCP-1) and manganous superoxide dismutase (MnSOD), which are known targets of NF-κB. MitoE was also found to repress the basal expression of a small number of genes by two-fold, including MCP-1. qRT-PCR or western blotting has confirmed the effect of mitoE on TNF-induced and basal expression of MCP-1, IκBa and MnSOD.

This study suggests that TNFα-induced RAS production from the mitochondria contributes to the induction of NF-κB-mediated gene expression, and that RAS from the mitochondria help maintain gene expression in resting cells. These findings lend weight to the emerging idea that strict modulation of intracellular RAS levels is an important element of cellular signalling.

The epithelial sodium channel (ENaC) is located at the apical membrane of polarised epithelia and mediates transport of sodium ions into cells. Tight control of ENaC function is essential for maintaining sodium homeostasis, blood volume and pressure. Controlling the number of active channels present at the cell surface is important for regulating ENaC activity. The neural precursor cell expressed, developmentally downregulated gene 4 (Nedd4) family of proteins (e.g. Nedd4-2) ubiquitinate ENaC and decrease its cell surface expression. The activity of Nedd4-2 is modulated by serum and glucocorticoid regulated kinase 1 (SGK1), which phosphorylates Nedd4-2 and increases cell surface expression of ENaC. COMMD1 is a recently identified ENaC binding partner, and negative regulator of channel activity. Other studies suggest that COMMD1 is also involved in intracellular protein trafficking and ubiquitin-dependent protein degradation. In this project we aim to characterise the interaction between ENaC and COMMD1, and identify the mechanism(s) by which COMMD1 down-regulates ENaC activity.

Glutathione S-transferase (GST) pull-down and coimmunoprecipitation assays were used to identify the binding interface between COMMD1 and ENaC. Cell surface biotinylation and ENaC ubiquitination assays were developed to investigate if COMMD1 affects the cell surface expression and ubiqitination of ENaC respectively.

The results showed that the conserved C-terminal COMM domain in COMMD1 is essential for binding to ENaC. The binding site for COMMD1 in bENaC was located at its N-terminal domain. COMMD1 down-regulated ENaC by increasing ubiquitin modification of ENaC and removing ENaC from the cell surface. COMMD1 also bound to SGK1 and prevented Nedd4-2 mediated degradation of SGK1.

It is suggested that COMMD1 might affect the interaction between SGK1 and Nedd4-2 and this pathway is likely to be involved in the COMMD1-mediated ubiquitination and down-regulation of ENaC activity.

Apoptosis is one of the host cell’s responses in its fight against virus infection. Therefore, many viruses have developed strategies to circumvent apoptosis; one of these includes the expression of Bcl-2 homologs. The Bcl-2 family of proteins are regulators of the mitochondrial pathway of apoptosis. It has been proposed that the pro-apoptotic family members are activated by various apoptotic signals and thereupon induce mitochondrial apoptosis, while the anti-apoptotic family members prevent this process by inhibiting the activity of their pro-apoptotic counterparts. We have shown that orf virus can express an inhibitor of apoptosis, ORFV125, which shows some similarities to the cellular anti-apoptotic protein Bcl-2. However, the mechanism by which ORFV125 inhibits apoptosis is still unknown. The present study investigates whether ORFV125 inhibits the activation of the pro-apoptotic Bcl-2 proteins Bax and Bak.

TK143B cells stably expressing either ORFV125, Bcl-2 or the empty-vector were incubated with 50 µM caspase inhibitor (Z-VAD-FMK), added 1 h before UV-C treatment (80 J/m2). Eight hours after UV irradiation cells were stained with anti-Bax or -Bak antibodies and visualised by fluorescence microscopy. The antibodies used recognise an N-terminal epitope, which is exposed only when the proteins are activated by an apoptotic stimulus. While the empty-vector cell line showed a substantial number of cells expressing active Bax (30 ± 3%, mean ± SD, n = 3), almost no active Bax was detected in cells expressing either ORFV125 (0.6 ± 0.1%, P < 0.001, ANOVA multiple comparison test) or Bcl-2 (2 ± 0.6%, P < 0.001). A similar result was obtained for the activation of Bak, showing that ORFV125 can fully inhibit the activation of both proteins in a manner comparable with Bcl-2.

These results suggest that ORFV125 acts in a Bcl-2-like manner, and supports our predictions that ORFV125 may be a distant member of the Bcl-2 family.

Gonadotropin-releasing hormone (GnRH) neurons are the principal regulators of reproductive function and are strongly modulated by estrogen (E2). In addition to transcriptional effects of E2, rapid non-transcriptional actions of E2 have also been demonstrated to occur in GnRH neurons. The aim of this study was to evaluate the rapid effects of E2 on intracellular calcium concentration ([Ca2+]i) in adult GnRH neurons.

Calcium imaging experiments were undertaken using acute brain slices from transgenic mice (n = 68) in which the genetically-encoded calcium indicator ratiometric-pericam is expressed selectively in GnRH neurons. GnRH neurons were tested with: (1) 1-100 nM E2, both in pericam and pericam x estrogen receptor-β (ER-β) knockout mice; (2) the selective ER-α agonist 3,17-dihydroxy-19-nor-17α-pregna-1,3,5(10)-triene-21,16α-lactone (16α-LE2) and (3) a membrane-impermeable, bovine serum albumin conjugate of E2 (E2-6-BSA).

In 13 of 27 GnRH neurons exhibiting low frequency spontaneous [Ca2+]i transients, treatment with 100 nM E2 increased the frequency of the transients (P < 0.001, one-way ANOVA, Tukey post-hoc test); lower doses were not effective. In 9 of 11 GnRH neurons showing high frequency spontaneous [Ca2+]i transients, 1 nM E2 significantly reduced the frequency of the transients (P < 0.01, one-way ANOVA, Tukey post-hoc test). ER-β knockout mice did not respond to E2 differently to wild-type mice (n = 16, two-way ANOVA). Ten of 20 silent neurons were stimulated by 100 nM 16α-LE2. E2-6-BSA reproduced the inhibitory (1 nM, n = 3), but not the stimulatory (100 nM, n = 6) effect of E2.

In summary, these results show that E2 produces two opposite effects on [Ca2+]i; a stimulatory effect mediated by ER-α, and an inhibitory effect mediated by a membrane receptor. These data provide evidence in favour of the rapid control of adult GnRH neurons by E2. This might be involved in generating the pulsatile activity of the GnRH system.

Altered electromyographic (EMG) patterns of trunk, gluteal and thigh muscles have been found in groups of subjects with lumbopelvic or knee disorders and their presence may contribute towards injury prolongation or recurrence. This study aimed to investigate whether differences in EMG patterns of selected muscles exist when comparing subjects with a recent hamstring injury (HI) and control subjects during a weight-bearing task.

Sixteen sportsmen with a recent clinically diagnosed HI were compared to an uninjured control group (CG) of 18 men. Surface EMG activity was recorded from the gluteus maximus, gluteus medius, biceps femoris (BF), medial hamstring (MH), and the quadriceps muscles of the weight-bearing leg during contralateral hip flexion. Muscle onsets were expressed relative to the start of the anticipatory postural adjustments seen in force platform data.

There were no significant differences for muscle onsets for the injured versus uninjured sides (HI group) and the preferred versus non-preferred sides (CG, P > 0.05, paired t-tests). In the HI group, onsets of BF and MH of the injured side, and onsets of MH of the uninjured side, were significantly earlier when compared to the CG bilateral average (mean difference ± SEM, injured BF 208.1 ± 75.0 ms, P < 0.01; injured MH 114.0 ± 44.5 ms, P < 0.02, uninjured MH 103.3 ± 49.3 ms, P < 0.05, ANCOVA controlling for age and activity level). There were no between-group differences for the gluteal and quadriceps muscles onsets, and the uninjured BF.

The earlier onset of the hamstring muscles in preparation for single leg stance of the injured and uninjured leg of the HI group in comparison to the bilateral average of the CG suggests an alteration in the motor control of these muscles. These changes may be an important factor to be considered in the rehabilitation of hamstring injuries.

The ankyrin repeat is a commonly observed motif that mediates interactions between cellular proteins. Almost all chordopoxviruses encode multiple ankyrin repeat proteins of unknown function. We have identified a potential F-box-like domain at the C-terminus of most poxviral ankyrin repeat proteins. Cellular F-box proteins function as adaptors in the multisubunit ubiquitin ligase S-phase kinase associated protein 1 (Skp1), Cullin1 (Cul1), F-box protein (SCF1) complex of the ubiquitin-proteasome system. F-box proteins recruit substrate proteins to the SCF1 complex for polyubiquitination and proteasomal degradation. The interaction between F-box proteins and the SCF1 component Skp1 is mediated by the F-box domain. We tested the five ankyrin proteins of the parapoxvirus Orf virus for their ability to interact with Skp1.

Co-immunoprecipitation was used to determine possible interactions of the five Orf virus ankyrin repeat proteins with the SCF1 components Skp1, Cul1, and (Ring-box 1) Rbx1. Each of the orf virus proteins was transiently expressed in human embryonic kidney 293 cells and then immunoprecipitated. The samples were analyzed by western blotting showing that each orf virus ankyrin protein co-precipitated endogenous Skp1, Cul1 and Rbx1. An F-box deletion construct of one orf virus ankyrin/F-box protein did not co-precipitate any SCF1 components demonstrating that the interaction of the full-length protein is F-box-dependent. The co-precipitated SCF1 complexes retained their intrinsic activity in an in vitro non-specific poly-ubiquitination assay.

The results indicate that the large class of poxviral ankyrin proteins function as F-box proteins. The extensive number of poxviral ankyrin/F-box proteins suggests cellular proteins from multiple pathways could be targeted. These poxviral F-box proteins could target the cellular anti-viral response or other cellular physiological processes whose manipulation could enhance viral survival and replication.

Many developing countries have introduced policies encouraging the use of generic medicines in order to improve access to affordable essential medicines. Successful implementation of these policies requires acceptance by all stakeholders, including the consumer. The present, qualitative, study explores South African consumers’ perceptions regarding generic medicines and their impact on their medicine purchasing behaviour.

Data were collected through focus group discussions (n = 12 groups) conducted in three cities within South Africa during December 2005 to January 2006. Key informants were purposively sampled according to their socio-economic status. Key informants recruited other participants through snowball sampling, yielding a total of 72 participants. During the discussions participants were asked whether they would select between brands of paracetamol (i.e. PanadoR, innovator brand; PacimolR, generic) to treat a headache. A second scenario required them to select between brands of amoxicillin (i.e. AmoxilR, innovator brand; MoxypenR, generic) for treatment of an infection. Discussions were tape-recorded and transcribed. Content analysis of the transcriptions was undertaken by the first author and reviewed jointly by the research team for confirmation.

Across all income and age groups, participants selected the original paracetamol for their headache. For amoxicillin, participants relied on the prescriber to decide which product to use. They agreed with generic substitution provided the prescriber supported this. Participants felt that cheaper generic products were of inferior quality. They reported they would pay higher prices to obtain the original medicines to treat their minor ailments, and that they would rely on the advice of their doctor and pharmacist for the prescription medicines.

Governments have to ensure that adequate information campaigns, which target consumers and healthcare providers, accompany implementation of policies for generic medicines. This is needed to achieve success in the overall goal of improving access to affordable, quality medicines.