A novel insertional mutation in the prion protein gene: clinical and bio-molecular findings.

A young man, presenting with early onset of personality and behavioural changes followed by slowly progressive cognitive impairment associated with marked bi-parietal cerebral atrophy, was found to carry a novel seven extra-repeat insertional mutation in the prion protein gene (PRNP). In vitro, the mutated recombinant prion protein (PrP) showed biochemical properties that were consistent with pathological PrP variants. Our results further underline the heterogeneity of neurological pictures associated with insertional mutations of PRNP, indicating the diagnostic difficulties of sporadic cases with early-onset atypical dementia.

Charcot-Marie-Tooth disease with giant axons: a clinicopathological and genetic entity.

The authors report an Italian family with autosomal-dominant Charcot-Marie-Tooth disease (CMT) in which there were giant axons in the sural nerve biopsy. Linkage to the known CMT2 loci (CMT2A, CMT2B, CMT2D, CMT2F) and mutations in the known CMT2 genes (Cx32, MPZ, NEFL), GAN, NEFM, and CMT1A duplication/HNPP deletion were excluded. This family with CMT and giant axons has a pathologic and genetic entity distinct from classic CMT.

EGF-responsive rat neural stem cells: molecular follow-up of neuron and astrocyte differentiation in vitro.

Neural stem cells (NSCs) could be very useful for the "cell therapy" treatment of neurological disorders. For this reason basic studies aiming to well characterize the biology of NSCs are of great interest. We carried out a molecular and immunocytochemical analysis of EGF-responsive NSCs obtained from rat pups. After the initial growth of NSCs as floating neurospheres in EGF-containing medium, cells were plated on poly-L-ornithine-coated dishes either in the presence or absence of EGF. We followed cell differentiation and apoptosis for 21 days in vitro and analyzed the expression levels of some genes having a major role in these processes, such as pRB, pRB2/p130, p27, and p53. We observed that EGF impairs neuronal differentiation. Furthermore, in the absence of mitogens, apoptosis, which appeared to proceed through the "p53 network," was significantly lower than in the presence of EGF. The cyclin kinase inhibitor p27, while important for cell cycle exit, seemed dispensable for cell survival and differentiation.

Unusual manifestation of vertebral osteoid osteoma: case report.

We report the case of a 64 year-old man with a clinical history suggesting a low thoracic-cord involvement, in which an unexpected vertebral osteoid osteoma was discovered. The patient underwent MRI of the thoraco-lumbar spine, which included sagittal and axial T1-weighted images, and sagittal double-echo T2-weighted images. Subsequently, CT scan was carried out with 2-mm-thick axial sections, aimed at T10 vertebra. Magnetic resonance imaging disclosed an extra-axial mass at T10 level. Computed tomography scan suggested an osteoid osteoma of the tenth thoracic vertebra, involving the lamina with marked sclerosis and prevalently endocanalar extension. Histology following surgical resection confirmed the diagnosis. In the reported case CT scan provided the correct pre-operative diagnosis of osteoid osteoma despite its unusual clinical--anamnestic presentation. Magnetic resonance imaging was useful in establishing the relationship of the neoplasm with the spinal cord.

Increased expression of IGF-binding protein-5 in Duchenne muscular dystrophy (DMD) fibroblasts correlates with the fibroblast-induced downregulation of DMD myoblast growth: an in vitro analysis.

In DMD the progressive loss of muscle ability and concomitant increasing fibrosis might originate from, besides other causes, the fibroblast paracrine inhibition of satellite cell "growth." In this study we report that in myoblast/fibroblast coculture experiments, the presence of DMD fibroblasts negatively interfered with DMD myoblast growth to an extent directly proportional to the percentage of DMD fibroblasts present in the mixed-cell cultures. Moreover, the observation that media conditioned with proliferating DMD fibroblasts inhibited the growth of DMD myoblasts more seriously than did control fibroblast-conditioned media suggested a paracrine effect by diffusible factors. IGF-binding proteins could act as such diffusible factors; in fact, IGFBP-5 transcript increased threefold in DMD fibroblasts proliferating in DMD muscle extracts, whereas IGFBP-3 mRNA decreased. In addition, high levels of IGFBP-5 protein were detected in DMD fibroblast-conditioned media. In neutralizing IGFBP-5 in DMD fibroblast-conditioned media by means of specific antibodies, or inhibiting IGFBP-5 gene expression in DMD fibroblasts by means of oligo antisense, the fibroblast-conditioned media lost inhibitory power over DMD myoblast proliferation.

Defective growth in vitro of Duchenne Muscular Dystrophy myoblasts: the molecular and biochemical basis.

As the molecular basis of Duchenne Muscular Dystrophy (DMD) was being discovered, increasing focus was placed on the mechanisms of progressive failure of myoregeneration. In this study, we propose a pathogenesis model for DMD, where an autocrine growth factor release of TGF-beta1-from necrotic myofibers-could contribute to the increasing loss of muscle regeneration. In fact, we report evidence that DMD myoblasts reduce their proliferation rate, in time and later cultures; in connection with this, we observed TGF-beta1 increase in conditioned media of DMD myoblasts, able to control the myoblast growth by reducing fusion and differentiation of DMD satellite cells.

Differentiation and apoptosis of neuroblastoma cells: role of N-myc gene product.

To clarify the role and function of the N-myc product in cell differentiation and apoptosis, we used the antisense oligonucleotide technique to inhibit N-myc gene expression in neuroblastoma cells with different phenotypes: intermediate (I) and neuronal (N), or Schwann-glia (S), respectively. The results suggest that N-myc operates along different pathways. Inhibiting N-myc gene expression either results in suppression of cell proliferation or in induction of differentiation and/or apoptosis.

Tissue transglutaminase-catalyzed formation of high-molecular-weight aggregates in vitro is favored with long polyglutamine domains: a possible mechanism contributing to CAG-triplet diseases.

To investigate possible biochemical mechanisms underlying the "toxic gain of function" associated with polyglutamine expansions, the ability of guinea pig liver tissue transglutaminase to catalyze covalent attachments of various polyamines to polyglutamine peptides was examined. Of the polyamines tested, spermine is the most active substrate, followed by spermidine and putrescine. Formation of covalent cross links between polyglutamine peptides and polyamines yields high-M(r) aggregates--a process that is favored with longer polyglutamines. In the presence of tissue transglutaminase, purified glyceraldehyde-3-phosphate dehydrogenase (a key glycolytic enzyme that binds tightly to the polyglutamine domains of both huntingtin and dentatorubral-pallidoluysian atrophy proteins) is covalently attached to polyglutamine peptides in vitro, resulting in the formation of high-M(r) aggregates. In addition, endogenous glyceraldehyde-3-phosphate dehydrogenase of a Balb-c 3T3 fibroblast cell line overexpressing human tissue transglutaminase forms cross-links with a Q60 polypeptide added to the cell homogenate. Possibly, expansion of polyglutamine domains (thus far known to occur in the gene products associated with at least seven neurodegenerative diseases) leads to increased/aberrant tissue transglutaminase-catalyzed cross-linking reactions with both polyamines and susceptible proteins, such as glyceraldehyde-3-phosphate dehydrogenase. Formation of cross-linked heteropolymers may lead to deposition of high-M(r) protein aggregates, thereby contributing to cell death.

Antisense oligonucleotides and myotonin gene expression in C2 mouse cells.

By describing the behavior of myotonin mRNA levels, from the quiescent to the differentiated state in C2 mouse myoblasts, we produced evidence bearing on the role of myotonin gene product in the control of cell growth and differentiation. To study the role of myotonin in myotonic dystrophy (DM) pathogenesis, we developed a suitable cellular model where myotonin gene expression was modulated by phosphorothioate antisense oligonucleotides in C2 cultured cells. Furthermore, an isoform of the gene product, similar to that described in humans and not yet described in the mouse, was found.

CTG repeat number in the nonaffected allele of myotonic dystrophy patients is not critical for disease expression.

To investigate whether unusual allele segregation might explain the dominant negative effect of the expanded allele for myotonic dystrophy on myotonin protein kinase mRNA metabolism, which is suggested to cause the disease, we determined the number of CTG repeats at the DM locus in the nonaffected alleles of 64 DM (dystrophia myotonia) patients. The relative distribution was then compared with the distributions obtained from alleles of the normal parents and normal siblings of DM patients. Comparison was also made with the allele distribution of normal subjects from the same geographic area. It appears that the CTG repeat number of the nonaffected allele in DM patients is not critical for the expression of the disease.

[Ambulatory monitoring of arterial pressure in the diagnosis and therapeutic evaluation of dysautonomic diseases: observations in a case of Shy-Drager syndrome]. / Il monitoraggio dinamico della pressione arteriosa nella diagnosi e valutazione terapeutica delle affezioni disautonomiche: osservazioni in un caso di sindrome di Shy-Drager.

Shy-Drager syndrome is a very rare disease affecting the autonomic nervous system. Usefulness of beta-adrenergic chronic therapy has already been focused in these cases. Ambulatory blood pressure monitoring may help to study the cardiocirculatory adaptation in Shy-Drager syndrome patients.

Progressive rubella panencephalitis. Follow-up EEG study of a case.

Progressive rubella panencephalitis is a very rare slow virus disease of the nervous system. The authors present a case, concerning a young man, aged 20 years, died 11 months after the onset of the disease. The following peculiarities of the case are emphasized: 1) the clinical symptomatology and the evolution (myoclonus, lack of cerebellar impairment) could suggest the diagnosis of SSPE; 2) the EEG recordings showed epileptiform abnormalities, long latency diffuse periodic complexes and--during interferon therapy and simultaneously with a temporary clinical improvement--the appearance of short latency anterior periodic complexes.

Regenerated EDL muscle of rats requires innervation to maintain AChE molecular forms.

Extensores digitorum longi of rats, infarcted and denervated by different surgical procedures, were used to analyze by biochemical and cytochemical methods the acetylcholinesterase (AChE) changes during muscle degeneration, regeneration, and early or delayed reinnervation. Biochemical tests showed that the regenerating muscle produces globular AChE forms (36% of controls) and small amounts of A12 (16S) asymmetric form (5% of controls); at the end of the regeneration, innervation and electromechanical function are required for the complete recovery of globular forms, and are absolutely critical to prevent A12 (16S) disappearance. Cytochemical observations showed that, unlike nicotinic receptor, AChE deposited at the neuromuscular junction before ischemic necrosis is protected from breakdown, as is the basal lamina of muscle fibers. Taken together, these observations contribute to the understanding of the factors that play a critical role in muscle repair and are, therefore, of clinical relevance.

Muscle-type MM creatine kinase is specifically bound to sarcoplasmic reticulum and can support Ca2+ uptake and regulate local ATP/ADP ratios.

Highly purified fractions of sarcoplasmic reticulum (SR) were prepared from chicken pectoralis muscles (Saito, A., Seiler, S., Chu, A., and Fleischer, S. (1984) J. Cell Biol. 99, 875-885) and analyzed for the presence of creatine kinase (CK). Vesicles derived from longitudinal SR contained 0.703 +/- 0.428 IU of CK/mg of (SR) protein. Immunogold localization of muscle-type MM-CK on ultrathin cryosections of muscle, after removal of soluble CK, revealed relatively strong in situ labeling of M-CK remaining bound to the M band as well as to the SR membranes. In addition, purified SR vesicles were also labeled by anti-M-CK antibodies, and the peripheral labeling was similar to that observed with anti-Ca2(+)-ATPase antibodies. Only some particulate CK enzyme was released from isolated SR membranes by EDTA/low salt buffer, and CK was resistant to extraction by 0.6 M KCl. Thus, some of the MM-CK present in muscle displays strong associative behavior to the SR membranes. The SR-bound CK was sufficient to support, in the presence of phosphocreatine plus ADP, a significant portion of the maximal in vitro Ca2+ uptake rate. The ATP regeneration potential of SR-bound CK was similar to the rate of Ca2(+)-stimulated ATP hydrolysis of isolated SR vesicles. Thus, CK bound to SR may be physiologically relevant in vivo for regeneration of ATP used by the Ca2(+)-ATPase, as well as for regulation of local ATP/ADP ratios in the proximity of the Ca2+ pump and of other ATP-requiring reactions in the excitation-contraction coupling pathway.

Histopathological heterogeneity and cytopathological similarity of findings in different muscles of two brothers affected by rigid spine syndrome.

The pathological changes in muscles biopsied from 2 brothers with rigid spine syndrome are reported. The findings ranged from marked fascicular atrophy and fibrosis to hypotrophy of small groups of fibres and vacuolation in most fibres. The presence of vacuoles and deposits of accumulated material seemed to be common to all the biopsies. These findings, compared with those reported in the literature, confirmed the histopathological heterogeneity of this syndrome but proposed also the hypothesis that similar elementary lesions of muscle fibres can account for the initiation of the pathological process, developing asynchronously in different muscles because of their different activity.

Phenotype heterogeneity among hemizygotes in a family biochemically screened for adrenoleukodystrophy.

We report on two clinically, neurologically normal relatives of a boy affected by adrenoleukodystrophy (ALD); they were found repeatedly to have the biochemical defect of an ALD hemizygote. The assay consisted in the determination of very-long-chain fatty acids in lyophilized and reconstituted plasma. While no evidence of neurologic disease (leukodystrophy or myeloneuropathy) was present in these hemizygotes, adrenocortical insufficiency provoking compensatory high ACTH release was found in both. These findings should be taken into consideration when counseling families in which cases with clinically expressed ALD are represented in several generations.

Innervation is required to stabilize and amplify creatine kinase activity in regenerated extensor digitorum longus muscles of rats.

Neural control of creatine kinase (CK, adenosine 5'-triphosphate creatine phosphotransferase: EC 2.7.3.2) was investigated by measuring enzymatic activity and isoenzymatic representation of CK in extensor digitorum longus (EDL) muscles of adult rats during and after regeneration. Experimental models were ischemized EDLs, reversibly or permanently denervated. Results showed that, during regeneration, CK in muscle fibers was likewise modified in both reversibly and permanently denervated EDLs. After regeneration a clear dichotomy was observed between the regenerated EDLs which were innervated and recovering CK activity, and those in which innervation was prevented and were rapidly losing activity. Further to investigate the neural influence on CK turnover, merely denervated age-matched EDLs were analysed and found to lose CK activity rapidly. The major conclusions are that during regeneration muscle CK is autonomously expressed, but following regeneration neural influence becomes an absolute requirement for stabilization and amplification of CK.

ConA and WGA receptors in rat skeletal muscles. Changes in density, distribution and composition following denervation.

The differential binding of fluorescein-labeled ConA, WGA and UEA to cryostatic sections of control and of 8 days denervated muscles is described. Receptor sites for ConA and WGA appear to be very abundant in skeletal muscles and their abundance seems to increase following denervation. In contrast, concentration of receptor sites for UEA is below the sensitivity of the method. Differences in the distribution of the binding sites for ConA and WGA, apparent in untreated sections, were further analysed following predigestions by collagenase, hyaluronidase or neuraminidase. The most important difference among lectins appeared to be the preferential binding of ConA to the surface of muscle fibres and of WGA to connective tissue. By comparing results in control and denervated muscles a clear change of the effects of neuraminidase on WGA binding was evident following denervation. The binding of fluorescein-labeled ConA and WGA to untreated and to predigested cryostatic sections of skeletal muscle is a sensitive and simple histochemical method which can disclose precocious changes in composition of glycoconjugates following denervation and, what might be useful, in other experimental or pathological conditions.

High sensitivity method for fluorofore detection in gradient polyacrylamide slab gels through excitation by laser light: application to glycoproteins stained with concanavalin A-fluorescein isothiocyanate.

An easy-to-assemble apparatus for the laser-light excitation of fluorofores in polyacrylamide gels is described. The assemblage is made up of a continuous-wave ion-argon laser with adjustable power output, a beam diffuser, appropriate filters to block excitation light, and a photographic camera. With this setup a minimum 20-fold increase of sensitivity was obtained for fluorofore detection in polyacrylamide gels as compared to the more conventional uv-light excitation using a commercial preparation of Con A-FITC (concanavalin A-fluorescein isothiocyanate) as reference molecule in the gel. The same apparatus, used to analyze the Con A-positive glycoproteins contained in serum Cohn fraction IV separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed a number of fluorescent components in a wide range of relative intensities while uv-light excitation showed none. Acrylamide concentration in the gel is critical, since a working limit of between 10 and 12% has been found, above which the diffusion of Con A-FITC in the gel, necessary to label glycoprotein bands, is hampered. The system described here also permits the optimization of detection of minor components not otherwise observable by conventional light excitation, because light power, angle of incidence, and beam divergence can be adapted to analyze specific areas of the sample gel.