Enteric neurons from Parkinson's disease patients display ex vivo aberrations in mitochondrial structure.

Based on autopsy material mitochondrial dysfunction has been proposed being part of the pathophysiological cascade of Parkinson's disease (PD). However, in living patients, evidence for such dysfunction is scarce. As the disease presumably starts at the enteric level, we studied ganglionic and mitochondrial morphometrics of enteric neurons. We compared 65 ganglia from 11 PD patients without intestinal symptoms and 41 ganglia from 4 age-matched control subjects. We found that colon ganglia from PD patients had smaller volume, contained significantly more mitochondria per ganglion volume, and displayed a higher total mitochondrial mass relative to controls. This suggests involvement of mitochondrial dysfunction in PD at the enteric level. Moreover, in PD patients the mean mitochondrial volume declined in parallel with motor performance. Ganglionic shrinking was evident in the right but not in the left colon. In contrast, mitochondrial changes prevailed in the left colon suggesting that a compensatory increase in mitochondrial mass might counterbalance mitochondrial dysfunction in the left colon but not in the right colon. Reduction in ganglia volume and combined mitochondrial morphometrics had both predictive power to discriminate between PD patients and control subjects, suggesting that both parameters could be used for early discrimination between PD patients and healthy individuals.

Impaired primary mouse myotube formation on crosslinked type I collagen films is enhanced by laminin and entactin.

In skeletal muscle, the stem cell niche is important for controlling the quiescent, proliferation and differentiation states of satellite cells, which are key for skeletal muscle regeneration after wounding. It has been shown that type I collagen, often used as 3D-scaffolds for regenerative medicine purposes, impairs myoblast differentiation. This is most likely due to the absence of specific extracellular matrix proteins providing attachment sites for myoblasts and/or myotubes. In this study we investigated the differentiation capacity of primary murine myoblasts on type I collagen films either untreated or modified with elastin, laminin, type IV collagen, laminin/entactin complex, combinations thereof, and Matrigel as a positive control. Additionally, increased reactive oxygen species (ROS) and ROCK signaling might also be involved. To measure ROS levels with live-cell microscopy, fibronectin-coated glass coverslips were additionally coated with type I collagen and Matrigel onto which myoblasts were differentiated. On type I collagen-coated coverslips, myotube formation was impaired while ROS levels were increased. However, anti-oxidant treatment did not enhance myotube formation. ROCK inhibition, which generally improve cellular attachment to uncoated surfaces or type I collagen, enhanced myoblast attachment to type I collagen-coated coverslips and -films, but slightly enhanced myotube formation. Only modification of type I collagen films by Matrigel and a combination of laminin/entactin significantly improved myotube formation. Our results indicate that type I collagen scaffolds can be modified by satellite cell niche factors of which specifically laminin and entactin enhanced myotube formation. This offers a promising approach for regenerative medicine purposes to heal skeletal muscle wounds. STATEMENT OF SIGNIFICANCE: In this manuscript we show for the first time that impaired myotube formation on type I collagen scaffolds can be completely restored by modification with laminin and entactin, two extracellular proteins from the satellite cell niche. This offers a promising approach for regenerative medicine approaches to heal skeletal muscle wounds.

The role of mitochondrial OXPHOS dysfunction in the development of neurologic diseases.

The development of neurologic disease is a complex and multi-faceted process. Several factors, such as physiology, environment and genetics may play key roles in the manifestation of the associated illnesses. During the past decades, it has become clear that, at the cellular level, mitochondria function as more than "just" an energy source for our cells and plays a significant role in such aspects as neuronal development, maintenance and degeneration. Malfunctions in mitochondrial respiration and ATP production may prove disastrous for our cells and neurons, ultimately resulting in apoptosis, neurodegeneration and consequently, neurodegenerative diseases.

Patient-derived fibroblasts indicate oxidative stress status and may justify antioxidant therapy in OXPHOS disorders.

Oxidative phosphorylation disorders are often associated with increased oxidative stress and antioxidant therapy is frequently given as treatment. However, the role of oxidative stress in oxidative phosphorylation disorders or patients is far from clear and consequently the preventive or therapeutic effect of antioxidants is highly anecdotic. Therefore, we performed a systematic study of a panel of oxidative stress parameters (reactive oxygen species levels, damage and defense) in fibroblasts of twelve well-characterized oxidative phosphorylation patients with a defect in the POLG1 gene, in the mitochondrial DNA-encoded tRNA-Leu gene (m.3243A>G or m.3302A>G) and in one of the mitochondrial DNA-encoded NADH dehydrogenase complex I (CI) subunits. All except two cell lines (one POLG1 and one tRNA-Leu) showed increased reactive oxygen species levels compared with controls, but only four (two CI and two tRNA-Leu) cell lines provided evidence for increased oxidative protein damage. The absence of a correlation between reactive oxygen species levels and oxidative protein damage implies differences in damage prevention or correction. This was investigated by gene expression studies, which showed adaptive and compensating changes involving antioxidants and the unfolded protein response, especially in the POLG1 group. This study indicated that patients display individual responses and that detailed analysis of fibroblasts enables the identification of patients that potentially benefit from antioxidant therapy. Furthermore, the fibroblast model can also be used to search for and test novel, more specific antioxidants or explore ways to stimulate compensatory mechanisms.

Transcriptional changes in OXPHOS complex I deficiency are related to anti-oxidant pathways and could explain the disturbed calcium homeostasis.

Defective complex I (CI) is the most common type of oxidative phosphorylation disease, with an incidence of 1 in 5000 live births. Here, whole genome expression profiling of fibroblasts from CI deficient patients was performed to gain insight into the cell pathological mechanism. Our results suggest that patient fibroblasts responded to oxidative stress by Nrf2-mediated induction of the glutathione antioxidant system and Gadd45-mediated activation of the DNA damage response pathway. Furthermore, the observed reduced expression of selenoproteins, might explain the disturbed calcium homeostasis previously described for the patient fibroblasts and might be linked to endoplasmic reticulum stress. These results suggest that both glutathione and selenium metabolism are potentially therapeutic targets in CI deficiency.

Development of microsatellite markers in Gonystylus bancanus (Ramin) useful for tracing and tracking of wood of this protected species.

Ten polymorphic microsatellite markers have been developed for Gonystylus bancanus (Ramin), a protected tree species of peat swamp forests in Malaysia and Indonesia. Eight markers were also shown to be polymorphic in other Gonystylus species. The markers will enable assessing the amount of genetic variation within and among populations and the degree of population differentiation, such that donor populations can be selected for reforestation projects. They may be used for tracing and tracking of wood in the production chain, so that legal trade in this Convention on International Trade in Endangered Species of Wild Fauna and Flora-protected timber species, derived from specifically described origins, can be distinguished from illegally logged timber.

Phytanic acid impairs mitochondrial respiration through protonophoric action.

Refsum disease is a rare, inherited neurodegenerative disorder characterized by accumulation of the dietary branched-chain fatty acid phytanic acid in plasma and tissues caused by a defect in the alphaoxidation pathway. The accumulation of phytanic acid is believed to be the main pathophysiological cause of the disease. However, the exact mechanism(s) by which phytanic acid exerts its toxicity have not been resolved. In this study, the effect of phytanic acid on mitochondrial respiration was investigated. The results show that in digitonin-permeabilized fibroblasts, phytanic acid decreases ATP synthesis, whereas substrate oxidation per se is not affected. Importantly, studies in intact fibroblasts revealed that phytanic acid decreases both the mitochondrial membrane potential and NAD(P)H autofluorescence. Taken together, the results described here show that unesterified phytanic acid exerts its toxic effect mainly through its protonophoric action, at least in human skin fibroblasts.

Sleep apnea in patients with myasthenia gravis.

To assess the prevalence of obstructive sleep apnea (OSA) in myasthenia gravis, the authors identified patients at risk of OSA using the multivariable apnea prediction index. OSA was diagnosed with polysomnography. The prevalence of OSA was 36% compared to an expected prevalence of 15 to 20% in the general population. When including the presence of daytime sleepiness (OSA syndrome), the prevalence was 11% compared to 3% in the general population.

Intracellular calcium buffering shapes calcium oscillations in Xenopus melanotropes.

The pituitary melanotrope cell of Xenopus laevis displays cytosolic Ca2+ oscillations that arise for the interplay between the burst-like openings of voltage-operated Ca2+ channels and Ca2+-extrusion mechanisms. We have previously shown that Ca2+-extrusion rates increase with increases in [Ca2+]i, suggesting that Ca2+ itself plays a role in shaping the Ca2+ oscillations. The purpose of the present study was to test this hypothesis by manipulating the intracellular Ca2+ buffering capacity of the cell and determining the consequences of such manipulations for the shape of the Ca2+ oscillations. We manipulated the cytosolic buffering capacity by loading the fast Ca2+ chelator BAPTA into cells. During loading the [Ca2+]i was dynamically imaged with confocal laser scanning microscopy. The basal [Ca2+]i was reduced with BAPTA loading and this reduction was associated with lower Ca2+-extrusion rates, a broadening of the Ca2+ oscillations and declined oscillation frequencies. Short loading periods of the buffer led to new, stable patterns of Ca2+ signaling and to reduced but stable levels of peptide secretion. We propose that the cytosolic Ca2+ buffer capacity, and thus by inference the profile of intracellular Ca2+ buffering proteins, is an important factor in setting the frequency and shape of Ca2+ oscillations.

Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity.

A novel anti-human DR5 monoclonal antibody, TRA-8, induces apoptosis of most tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-sensitive tumor cells both in vitro and in vivo. In contrast to both the membrane-bound form of human TRAIL, which induced severe hepatitis in mice, and the soluble form of human TRAIL, which induced apoptosis of normal human hepatocytes in vitro, TRA-8 did not induce significant cell death of normal human hepatocytes. However, both primary hepatocellular carcinoma cells and an established liver cancer cell line were highly susceptible to the killing mediated by TRA-8. We show here that elevated levels of cell-surface expression of DR5 and increased susceptibility to DR5-mediated apoptosis are characteristics of malignant tumor cells. In contrast, DR5 alone is not sufficient to trigger apoptosis of normal hepatocytes. Therefore, selective, specific targeting of DR5 with an agonistic antibody might be a safe and effective strategy for cancer therapy.

Membrane-initiated Ca(2+) signals are reshaped during propagation to subcellular regions.

An important aspect of Ca(2+) signaling is the ability of cells to generate intracellular Ca(2+) waves. In this study we have analyzed the cellular and subcellular kinetics of Ca(2+) waves in a neuroendocrine transducer cell, the melanotrope of Xenopus laevis, using the ratiometric Ca(2+) probe indo-1 and video-rate UV confocal laser-scanning microscopy. The purpose of the present study was to investigate how local Ca(2+) changes contribute to a global Ca(2+) signal; subsequently we quantified how a Ca(2+) wave is kinetically reshaped as it is propagated through the cell. The combined kinetics of all subcellular Ca(2+) signals determined the shape of the total cellular Ca(2+) signal, but each subcellular contribution to the cellular signal was not constant in time. Near the plasma membrane, [Ca(2+)](i) increased and decreased rapidly, processes that can be described by a linear and exponential function, respectively. In more central parts of the cell slower kinetics were observed that were best described by a Hill equation. This reshaping of the Ca(2+) wave was modeled with an equation derived from a low-pass RC filter. We propose that the differences in spatial kinetics of the Ca(2+) signal serves as a mechanism by which the same cellular Ca(2+) signal carries different regulatory information to different subcellular regions of the cell, thus evoking differential cellular responses.

Minimal model for intracellular calcium oscillations and electrical bursting in melanotrope cells of Xenopus laevis.

A minimal model is presented to explain changes in frequency, shape, and amplitude of Ca2+ oscillations in the neuroendocrine melanotrope cell of Xenopus Laevis. It describes the cell as a plasma membrane oscillator with influx of extracellular Ca2+ via voltage-gated Ca2+ channels in the plasma membrane. The Ca2+ oscillations in the Xenopus melanotrope show specific features that cannot be explained by previous models for electrically bursting cells using one set of parameters. The model assumes a KCa-channel with slow Ca2+-dependent gating kinetics that initiates and terminates the bursts. The slow kinetics of this channel cause an activation of the Kca-channel with a phase shift relative to the intracellular Ca2+ concentration. The phase shift, together with the presence of a Na+ channel that has a lower threshold than the Ca2+ channel, generate the characteristic features of the Ca2+ oscillations in the Xenopus melanotrope cell.

Prospects for autoimmune disease: Research advances in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a common chronic inflammatory disease associated with progressive destruction of diarthrodial joints, substantial morbidity and economic burden, and a shortened lifespan. Significant progress has been made in understanding the pathogenesis of RA, and increasingly effective therapies have been introduced, including anti-tumor necrosis factor alpha agents. Advances made in the past quarter century will pale in comparison to those anticipated for the next 25 years, including delineation of the genetic basis of disease susceptibility and severity, genetic definition of disease subtypes that differ in severity and response to therapy, and prompt initiation of effective individualized treatment based on genetic and environmental assessment. Reconstructive surgery will become increasingly unnecessary and the morbidity, economic burden, and mortality due to RA will be reduced substantially.

The challenges of integration in health care research.

The team approach in neuroscience patient care results in health care professionals interacting on a daily basis. However, the collaboration of health care disciplines in research is limited. The barrier to research integration among disciplines may be related to the diverse views of the quantitative and qualitative paradigms. Health care research is challenged by both the variety of health professionals and differing research paradigms. Patients will benefit from integrated health care research with a coordinated research agenda and efficient use of resources.

Evaluation of portable bladder ultrasound: accuracy and effect on nursing practice in an acute care neuroscience unit.

The purpose of this evaluation was to determine the accuracy of a portable ultrasound instrument in assessing bladder volume in an acute care neuroscience population and the effect of ultrasound assessment on nursing practice in an acute care neuroscience unit. In a 6-week prospective evaluation, 105 paired ultrasound measurements were performed by 45 nurses on 30 patients suspected to be retaining urine. Sixty-seven catheterizations were performed, and volumes were compared with corresponding ultrasound readings. The first ultrasound volume readings slightly underestimated the catheterized volumes, but the volumes from the first ultrasound readings and the catheterized volumes were highly correlated. Volume readings from a second ultrasound, the average of the first and second ultrasound readings, and the higher of the two ultrasound readings did not add to the ability of the ultrasound instrument to predict catheterized volumes. Patient age and gender did not change the relationship between ultrasound and catheterized volumes. The ultrasound assessment changed nursing practice in 51% of the instances; the most common change (32%) was that nurses did not catheterize the patient. The ultrasound assessment did not change nursing practice in 49% of the instances; the most common reason (41%) was that the ultrasound confirmed the need to catheterize the patient. The instrument was therefore judged to be an accurate and reliable tool that changed nursing practice in an acute care neuroscience unit.

Species relationships in Lactuca s.l. (Lactuceae, Asteraceae) inferred from AFLP fingerprints.

An AFLP data set comprising 95 accessions from 20 species of Lactuca s.l. (sensu lato) and related genera was generated using the primer combinations E35/M48 and E35/M49. In phenetic analyses of a data subset, clustering with UPGMA based on Jaccard's similarity coefficient resulted in the highest cophenetic correlation, and the results were comparable to those of a principal coordinates analysis. In analyses of the total data set, phenetic and cladistic analyses showed similar tree topologies for the well-supported parts of the trees. The validity of cladistic analysis of AFLP data is discussed. The results do not support a distinction among the serriola-like species L. sativa, L. serriola, L. dregeana, and L. altaica, which is in line with previous results. Therefore, we postulate that these species are conspecific. The serriola-like species L. aculeata occupies a clearly separate position, making it an ideal outgroup for studies of the closest relatives of L. sativa. The subsect. Lactuca as a group is well supported by our data, but the positions of L. saligna and L. virosa relative to the serriola-like species remain unclear. The close relationship between the sect. Mulgedium species L. tatarica and L. sibirica is corroborated by the present AFLP results and by additional crossability data.

Endogenous production of nitric oxide and effects of nitric oxide and superoxide on melanotrope functioning in the pituitary pars intermedia of Xenopus laevis.

Previous studies have focused on the immunohistochemical detection of a nitric oxide (NO)-cyclic 3',5'-monophosphate (cGMP) pathway in the brain and pituitary of the aquatic toad Xenopus laevis. We here investigate the endogenous production and possible involvement of NO signaling in the regulation of melanotrope cell activity in the pituitary pars intermedia of this amphibian. Using immunohistochemical staining of cultured cells with a polyclonal antiserum against inducible NO synthase (iNOS), immunoreactivity was observed both in melanotropes and in stellate-shaped cells. Part of these stellate-shaped cells is characterized as folliculo-stellate cells by their capacity of beta-Ala-Lys-N(epsilon)-AMCA uptake. Using chemiluminescence detection we demonstrate the presence of NO and reaction products like nitrite (NO(-)(2)) or peroxynitrite (ONOO(-)) in the incubation medium of cultured melanotropes. Bacterial lipopolysaccharide (LPS) stimulates the generation of NO and reaction products, the effect of which was blocked by S-methyl-l-thiocitrulline hydrochloride, a potent general NOS inhibitor. With [(3)H]lysine incorporation and a superfusion technique, it is shown that peptide release from melanotropes is stimulated by administration of superoxide dismutase (SOD), which was added to the superfusion medium to prevent scavenging of NO by superoxide anions. Pretreating the cells with the general NOS inhibitor l-nitroarginine methyl ester for 48 h attenuated the SOD-induced stimulation, but did not affect the stimulation by sodium nitroprusside (SNP) or 3-morpholinylsydnoneimine chloride (SIN-1), whereas hemoglobin blocked the combined effect of SOD plus NO donors. The soluble guanylate cyclase inhibitor 1H-[1,2, 4]oxadiazolo[4,3a]-quinoxaline-1-one did not inhibit but even significantly potentiated the effect of NO donors on peptide release without affecting the SOD-induced stimulation of peptide release. In addition to the previously described neuronal NOS (nNOS) immunoreactivity in nerve fibers in the pars intermedia of Xenopus, the present data reveal iNOS and nNOS as potential sources of endogenous NO production in cultured cells of the pars intermedia. Our study shows that also in nonmammalian vertebrates endogenous NO production may be physiologically relevant under conditions where protection against oxidative damage is needed. The endocrine cells of the pars intermedia themselves, as well as the folliculo-stellate cells, under such conditions may dispose of a protective mechanism against oxidative stress. The sensitivity of the endogenous NO production to LPS suggests that NO may also play a role during systemic inflammation.

The natural history of multiple sclerosis: a geographically based study: 8: familial multiple sclerosis.

We have examined the demographics and long-term outcome of 1044 patients with sporadic and familial multiple sclerosis in a population-based cohort from London, Ontario. The mean follow-up was 25 years in duration, and by this time most patients had reached the unambiguous endpoint scores of the Kurtzke disability status scale (DSS), DSS 6, 8 or 10. An affected family member was identified in 19.8% of the total population, and this subgroup was further divided arbitrarily into the following three groups by the type and number of relatives affected: (i) first degree only; (ii) first degree plus others; (iii) second or third degree. The outcome in these groups was compared with that for those patients who, at a mean 25 year follow-up, had no relatives known to be affected. Familial cases closely resembled those remaining sporadic in both demographics and outcome, although onset in the most heavily loaded families was earlier and male/female ratio was greater. The times to DSS 6, 8 and 10 did not differ significantly when sporadic, familial and familial subgroups were compared. These results provide no clinical support for viewing familial multiple sclerosis as distinct from the sporadic form. The observed recurrence rate for siblings in a strictly defined epidemiological sample was 3.5%, much as projected. These results validate the recurrence risks which have previously been derived from age-corrected data for these first-degree relatives.