Importance and challenge of making an early diagnosis in LMNA-related muscular dystrophy.

OBJECTIVE: To identify the most useful clinical and histologic markers that facilitate early diagnosis in LMNA-related muscular dystrophy and to assess the usefulness of Western blotting (WB) for lamin A/C. METHODS: We analyzed the clinical and histologic features and WB results of all patients with laminopathies diagnosed in a research-based diagnostic service over 8 years. RESULTS: Although patients with congenital muscular dystrophy (MDCL) (n = 5) and Emery-Dreifuss muscular dystrophy (EDMD) (n = 5) had distinctive early clinical features, the lack of a suggestive clinical phenotype significantly delayed diagnosis in 2 of 3 patients with limb-girdle muscular dystrophy (LGMD) (n = 3). In addition, 6 of 20 muscle biopsy samples were considered nondystrophic, which contributed to delays in diagnosis in some patients. Neck extensor involvement (weakness or contractures) was the most consistent clinical sign, present in all patients. Reduced lamin A/C levels on WB were seen in 5 of 9 patients with laminopathies. CONCLUSION: Clinical features provide the best clues for diagnosing MDCL and EDMD early in the disease, and we urge clinicians to become familiar with those phenotypes. WB for lamin A/C may contribute to diagnosis but requires technical expertise, and results are normal in many individuals with LMNA mutations. Because of the survival benefit of early diagnosis and treatment, we recommend that LMNA gene sequencing be performed in all patients with undiagnosed congenital muscular dystrophy and neck extensor weakness, all patients with genetically undiagnosed LGMD, and those with suggestive clinical signs and nonspecific histologic abnormalities.

Growth of nanocrystals and thin films at the water-oil interface.

The use of the water-oil interface provides significant advantages in the synthesis of inorganic nanostructures. Employing the water-toluene interface, luminescent CdS nanocrystals have been obtained at a relatively modest temperature of 35 degrees C. The diameters of the particulates can be varied between 1.0 and 5.0 nm. In addition, we have devised a new method for transferring thin films at the water-toluene interface onto solid substrates. Using this method, thin films consisting of Au and Ag nanocrystals spread over very large areas (square centimetres) are obtained in a single step. These films are directly usable as ingredients of functional devices. We show this by constructing a working amine sensor based on films of Au nanocrystals. The materials obtained have been characterized by X-ray diffraction, scanning and transmission electron microscopy, absorption and emission spectroscopy and charge transport measurements.

Spinal muscular atrophy: molecular mechanisms.

Spinal muscular atrophy (SMA) is a relatively common autosomal recessive neuromuscular disorder characterised by muscle weakness and atrophy due to degeneration of motor neurons of the spinal cord and cranial motor nuclei. The clinical phenotype incorporates a wide spectrum. No effective treatment is currently available and patients may experience severe physical disability which is often life limiting. The most common type of SMA is caused by homozygous disruption of the survival motor neuron 1 (SMN1) gene by deletion, conversion or mutation and results in insufficient levels of survival motor neuron (SMN) protein in motor neurons. While diagnosis is usually achieved by genetic testing, an illustrative clinical case is described that highlights the molecular and diagnostic complexities. While there is an emerging picture concerning the function of the SMN protein and the molecular pathophysiological mechanisms underpinning the disease, a number of substantial issues remain unresolved. The selective vulnerability of the motor neuron and the site and timing of the primary pathogenesis are not yet determined. Utilising the organisation of the SMN genomic region, recent advances have identified a number of potential therapeutic targets. As such, this review incorporates discussion of the clinical manifestations, molecular genetics, diagnosis, mechanisms of disease pathogenesis and development of novel treatment strategies.

Changes in mouse granulosa cell gene expression during early luteinization.

Changes in gene expression during granulosa cell luteinization have been measured using serial analysis of gene expression (SAGE). Immature normal mice were treated with pregnant mare serum gonadotropin (PMSG) or PMSG followed, 48 h later, by human chorionic gonadotropin (hCG). Granulosa cells were collected from preovulatory follicles after PMSG injection or PMSG/hCG injection and SAGE libraries generated from the isolated mRNA. The combined libraries contained 105,224 tags representing 40,248 unique transcripts. Overall, 715 transcripts showed a significant difference in abundance between the two libraries of which 216 were significantly down-regulated by hCG and 499 were significantly up-regulated. Among transcripts differentially regulated, there were clear and expected changes in genes involved in steroidogenesis as well as clusters of genes involved in modeling of the extracellular matrix, regulation of the cytoskeleton and intra and intercellular signaling. The SAGE libraries described here provide a base for functional investigation of the regulation of granulosa cell luteinization.

Cardiac assessment in childhood carriers of Duchenne and Becker muscular dystrophies.

Cardiac disease in adult female carriers of the X-linked dystrophinopathies, Duchenne and Becker muscular dystrophies, is a well-recognised entity. A single study has reported a 15% incidence of cardiac abnormalities in female carriers under 16 years. Our study aims, clinically and with electrocardiograph and echocardiograph, to determine the incidence of cardiac abnormality in young girls who are proven carriers of X-linked dystrophinopathies. Twenty-three girls aged 6.2-15.9 years were assessed. All had normal cardiac examination. None had electrocardiograph abnormalities consistent with dystrophic cardiomyopathy. Left ventricular fractional shortening ranged from 33 to 55% (normal>28%). Septal thickness, posterior wall thickness and wall thickness ratio were within normal limits. No cardiac abnormalities have been demonstrated in young girls who are proven carriers of X-linked dystrophinopathies in our study. This has important implications for planning timing of carrier determination and cardiac assessment.

Spatial localization of transplanted hemopoietic stem cells: inferences for the localization of stem cell niches.

The spatial distribution of subpopulations of hemopoietic progenitor cells following syngeneic transplantation was investigated at the single-cell level. The location of infused hemopoietic progenitor cells within the femoral bone marrow of nonablated recipients was determined by 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester labeling of cells and in situ fixation by perfusion. Analysis performed over 15 hours after infusion demonstrated that the spatial distribution of transplanted marrow cells is not a random process. Although the majority of cells enter the bone marrow from the central marrow vessels, the subsequent localization within the bone marrow varied according to their phenotype. Candidate "stem cells" demonstrated selective redistribution and were significantly enriched within the endosteal region, whereas mature terminally differentiated and lineage-committed cells selectively redistributed away from the endosteal region and were predominantly in the central marrow region. Together, these data strongly support historical evidence of the presence of endosteal hemopoietic stem cell niches.

Clinical rhabdomyolysis.

Although rhabdomyolysis is an uncommon disorder, especially in children, it can present as a severe, life-threatening event. Paediatricians need to be aware of this important, probably underdiagnosed disorder in order to implement appropriate early treatment. In this report, seven children with rhabdomyolysis of both forms, endogenous and exogenous, are presented. Despite comprehensive 'up-to-date' investigations being performed on paediatric patients with endogenous (often recurrent) rhabdomyolysis, the majority of these patients' underlying disorders will remain undiagnosed. Overall, these patients usually have a very good prognosis even if repeated, severe life-threatening episodes occur. It is recommended that a regimen of early therapy with fluids and sodium bicarbonate be instituted in all patients with rhabdomyolysis.

A Huntington's disease CAG expansion at the murine Hdh locus is unstable and associated with behavioural abnormalities in mice.

Huntington's disease (HD) is a dominant disorder characterized by premature and progressive neurodegeneration. In order to generate an accurate model of the disease, we introduced an HD-like mutation (an extended stretch of 72-80 CAG repeats) into the endogenous mouse Hdh gene. Analysis of the mutation in vivo reveals significant levels of germline instability, with expansions, contractions and sex-of-origin effects in evidence. Mice expressing full-length mutant protein display abnormal social behaviour in the absence of acute neurodegeneration. Given that psychiatric changes, including irritability and aggression, are common findings in HD patients, our data are consistent with the hypothesis that some clinical features of HD may be caused by pathological processes that precede gross neuronal cell death. This implies that effective treatment of HD may require an understanding and amelioration of these dysfunctional processes, rather than simply preventing the premature death of neurons in the brain. These mice should facilitate the investigation of the molecular mechanisms that underpin the pathway from genotype to phenotype in HD.

Spontaneous abdominal hematoma in dermatomyositis.

Dermatomyositis is associated with a number of systemic manifestations and diseases. We present 2 patients with dermatomyositis, aged 11 and 50 years, who developed acute abdominal pain, both a result of spontaneous hemorrhage. Hemorrhage was detectable by physical examination in one and on computed tomography scan of the abdomen in the other. Both patients made a full recovery with supportive treatment. While the cause of the hemorrhage was uncertain, in 1 patient massive calcinosis of the abdominal wall was present, and trauma may have been the precipitant. Spontaneous abdominal hematoma is a cause of acute abdominal pain in patients with dermatomyositis, and surgery may be avoided if the diagnosis is recognized.

Clinical and neurophysiological features of tick paralysis.

The clinical and neurophysiological findings in six Australian children with generalized tick paralysis are described. Paralysis is usually caused by the mature female of the species Ixodes holocyclus. It most frequently occurs in the spring and summer months but can be seen at any time of year. Children aged 1-5 years are most commonly affected. The tick is usually found in the scalp, often behind the ear. The typical presentation is a prodrome followed by the development of an unsteady gait, and then ascending, symmetrical, flaccid paralysis. Early cranial nerve involvement is a feature, particularly the presence of both internal and external ophthalmoplegia. In contrast to the experience with North American ticks, worsening of paralysis in the 24-48 h following tick removal is common and the child must be carefully observed over this period. Death from respiratory failure was relatively common in the first half of the century and tick paralysis remains a potentially fatal condition. Respiratory support may be required for > 1 week but full recovery occurs. This is slow with several weeks passing before the child can walk unaided. Anti-toxin has a role in the treatment of seriously ill children but there is a high incidence of acute allergy and serum sickness. Neurophysiological studies reveal low-amplitude compound muscle action potentials with normal motor conduction velocities, normal sensory studies and normal response to repetitive stimulation. The biochemical structure of the toxin of I. holocyclus has not been fully characterized but there are many clinical, neurophysiological and experimental similarities to botulinum toxin.

Dynamic changes in NADPH-diaphorase staining reflect activity of nitric oxide synthase: evidence for a dopaminergic regulation of striatal nitric oxide release.

In fixed tissue, neuronal NADPH-diaphorase staining results from nitric oxide synthase (NOS) activity. Neuronal NOS only synthesizes nitric oxide once activated by the binding of Ca2+/calmodulin. We show here that neuronal NADPH-diaphorase staining is also dependent on Ca2+/calmodulin, implying that only activated NOS is detected. In addition, in bovine pulmonary endothelial cells, carbachol and bradykinin dramatically and rapidly increase the intensity of NADPH-diaphorase staining. Furthermore, administration of MK801, an NMDA antagonist, decreases neuronal NADPH-diaphorase staining. This suggests that the intensity of the NADPH-diaphorase staining is related to the level of enzyme activation at the moment of tissue fixation. The potential of exploiting this observation to detect cellular activation of NOS is illustrated by the observations that the intensity of NADPH-diaphorase staining in rat striatal neurones is decreased following systemic treatment with the D1-like dopamine receptor antagonist SCH23390, and increased by the D2-like antagonist eticlopride. These results therefore provide strong evidence that the NADPH-diaphorase reaction can be used to monitor NOS activity at a cellular level of resolution, and reveal a dopaminergic regulation of NOS activity in the striatum mediated by D1-like and D2-like dopamine receptors.

A role for hippocampal opioids in long-term functional plasticity.

In the granule cells of the hippocampus, glutamate coexists with opioid peptides derived from the proenkephalin and prodynorphin genes. The functional significance of this coexistence has been unclear but recent evidence suggests that the dynorphins and enkephalins play a crucial role in regulating the efficiency of neurotransmission at granule-cell synapses. Together with evidence that the level of opioid activity in this pathway can change dramatically according to the physiological or pathological state of the tissue, this information focuses attention on granule-cell opioids as primary mediators of hippocampal plasticity.

N-methyl-D-aspartate and nitric oxide regulate the expression of calcium/calmodulin-dependent kinase II in the hippocampal dentate gyrus.

Injection of small volumes of N-methyl-D-aspartate (NMDA) or Sin-1 molsidomine (a nitric oxide releasing agent) onto the dendrites of granule cells in the hippocampal dentate gyrus leads to changes in the level of expression of a number of genes. There is a fall in prodynorphin mRNA levels with a corresponding increase in proenkephalin mRNA levels. Similar changes in opioid gene expression occur following the induction of long-term potentiation (LTP). We report here that at short time periods (1-6 h) after injections of NMDA or sin-1 molsidomine, there is an increase in the levels of the mRNA encoding the alpha subunit of Ca2+/calmodulin-dependent protein kinase II (CaMKII alpha), consistent with a report of elevated CaMKII alpha mRNA in postsynaptic neurons in the CA1 region of the hippocampus following LTP induction [54]. However, we also report that 24 h after injection of NMDA or sin-1, there is a dramatic decrease in CaMKII alpha mRNA levels in the vicinity of the injection. This effect is specific for CaMKII alpha mRNA, in that many other mRNA species are not affected, and occurs in the dendritic population of CaMKII alpha mRNA as well as in the pool of mRNA in the granule cell bodies. The effect is blocked by an inhibitor of cGMP-dependent protein kinase. The biphasic regulation of CaMKII alpha mRNA may be of considerable functional importance for the long-term response of granule cells to local stimulation of NMDA receptors or NO release.

Phenotypic characterisation of rat striatal neurones in primary culture.

The aim of this study was to determine to what extent the neuronal phenotypes present in primary cultures of rat striatal neurones correspond to those present in vivo. A large percentage of cultured striatal neurones contained relatively high levels of proenkephalin mRNA. In addition, a high level of expression was found for the prosomatostatin mRNA. Protachykinin mRNA and proneuropeptide Y mRNA were also expressed, but at a comparatively low level. No prodynorphin mRNA could be detected. Considerable numbers of neurones were also found to express NADPH-diaphorase activity, while a smaller number of neurones were positive for acetylcholinesterase. The NADPH-diaphorase and the acetylcholinesterase could be detected both in cell bodies, and in neuronal processes contacting groups of neighbouring neurones. Since nitric oxide does not require synaptic specialisations to exert its intercellular actions, this provides strong evidence that NADPH-positive neurones communicate with other cells in primary culture. These observations demonstrate that when striatal neurones are grown in primary culture, a range of neurochemical phenotypes are present which correspond closely to those present in the mature striatum in vivo. Together with the evidence for cell-cell interactions, this suggests that primary striatal cultures will provide a suitable model to study the molecular mechanisms controlling striatal function.

Selective regulation of dendritic MAP2 mRNA levels in hippocampal granule cells by nitric oxide.

Application of NMDA, or agents releasing nitric oxide (NO), onto the dendrites of hippocampal granule cells increased the levels of the mRNA encoding MAP2, a cytoskeletal component induced during periods of neurite outgrowth. Furthermore, local increases in the hybridisation signal in the molecular layer, representing dendritic MAP2 mRNA, occurred independently of changes in MAP2 mRNA levels in the cell body layer. The selective modulation of MAP2 mRNA in dendrites reveals a mechanism allowing a sustained stimulation of dendritic outgrowth to be confined to those regions of a neuron's dendritic arbour local to glutamate receptor stimulation.

Induction of c-fos gene expression is not responsible for increased proenkephalin mRNA levels in the hippocampal dentate gyrus following NMDA stimulation.

The increased levels of proenkephalin mRNA in the dentate gyrus following hippocampal stimulation have been assumed to be a consequence of the transient induction of c-fos. Injection of 50 microM NMDA in vivo onto the dendrites of a small number of granule cells causes a pronounced but highly localised elevation in proenkephalin mRNA levels 24 h later, whereas vehicle has no effect. In contrast, there is a widespread induction of c-fos mRNA throughout the dentate gyrus, 45 min after injection of either vehicle or NMDA, suggesting that induction of c-fos expression is unrelated to the subsequent, anatomically discrete, increase in proenkephalin mRNA levels.

Nitric oxide alters proenkephalin and prodynorphin gene expression in hippocampal granule cells.

Application of N-methyl-D-aspartate on to the dendrites of hippocampal granule cells dramatically decreased prodynorphin messenger RNA levels in the affected cells while increasing proenkephalin messenger RNA levels. Sin-1 molsidomine (an agent which releases nitric oxide) and 8-bromo-cyclic GMP were similarly effective, and the actions of sin-1 molsidomine were blocked by inhibition of cyclic GMP-dependent protein kinase. Since, in this region, dynorphins act to inhibit potentiation of synaptic transmission, while enkephalins have excitatory effects, this switch in opioid gene expression is likely to have a prolonged effect on the efficiency of the mossy fibre synapses. In addition, the results demonstrate a powerful role for nitric oxide in the long-term regulation of hippocampal excitability.

zif/268 does not mediate increases in proenkephalin mRNA levels after NMDA receptor stimulation.

THE immediate-early gene zif/268 is induced in the hippocampal dentate gyrus by stimuli leading to long-term potentiation (LTP), and is though then to alter the rate of transcription of other genes involved in hippocampal plasticity, such as the proenkephalin (Penk) gene. Injection of 50 microM NMDA in vivo on to the granule cells of the dentate gyrus results in a highly localized increase in Penk mRNA levels 24 h later, while vehicle has no effect. In contrast, there is a widespread induction of zif/268 mRNA throughout the dentate gyrus, after injection of either NMDA or vehicle. This suggests that zif/268 induction in the hippocampal dentate gyrus is not sufficient to increase Penk gene expression.