In vivo induction of heat shock proteins in the substantia nigra following L-DOPA administration is associated with increased activity of mitochondrial complex I and nitrosative stress in rats: regulation by glutathione redox state

Increasing evidence suggests a critical role for oxidative and nitrosative stress in the pathogenesis of most important neurodegenerative disorders. Parkinson's disease (PD) is a neurodegenerative disease characterized by a severe depletion in number of dopaminergic cells of the substantia nigra (SN). Administration of L-DOPA (LD) is the more effective treatment for patients with PD. However, the vast majority of patients suffer LD-related complications, which represent the major problem in the clinical management of PD. In the present study, LD administration to rats resulted in a significant dose-dependent increase in Hsp70 synthesis which was specific for the SN. The amount of 70 kDa protein increased after 6 h treatment reaching the maximal induction after 24-48 h. Induction of Hsp70 in the SN was associated with a significant increase in constitutive Hsc70 and mitochondrial Hsp60 stress proteins, and with increased expression of mitochondrial complex I whereas no significant changes were found in the activity of complex IV. In the same experimental conditions, a significant decrease in reduced glutathione was observed, which was associated with an increased content of oxidized glutathione content as well as nitric oxide (NO) synthase activity, NO metabolites and nitrotyrosine immunoreactivity. Interestingly, Hsp70 induction, iNOS up-regulation and nitrotyrosine formation have been confirmed also in SN and striatum of rats treated with LD and carbidopa, this latter being an inhibitor of the peripheral DOPA decarboxylase. Our data are in favor of the importance of the heat shock signal pathway as a basic mechanism of defense against neurotoxicity elicited by free radical oxygen and nitrogen species produced in aging and neurodegenerative disorders.     

Mechanical testing of cancellous bone from the femoral head: experimental errors due to off-axis measurements

The aim of this study was to verify whether a misalignment between the testing direction and the trabecular main direction has a significant effect on the compressive behaviour of cancellous bone. Ten cylindrical specimens were extracted from femoral heads with a misalignment to the trabecular main direction of approximately 20 degrees. Each specimen was paired with a specimen extracted aligned with the main direction of the trabeculae on the basis of the closest bone volume fraction, obtaining two groups, one 'aligned' and one 'misaligned'. The average off-axis angle was 6.1 degrees and 21.6 degrees for the 'aligned' and 'misaligned' group, respectively. The specimens underwent micro-tomographic analysis, compressive testing, micro-indentation testing and ashing. No significant differences were found in histomorphometric parameters, hardness and ash density between the two groups, whereas significant differences were found in Young's modulus and ultimate stress: both parameters, measured for the 'misaligned' group, were about 40% lower than those measured for the 'aligned' group. These results demonstrate a great effect of the angle between the testing direction and the main direction of the trabecular structure on the compressive behaviour of cancellous bone. This angle should be reduced as much as possible (in the present work the average value was 6.6+/-3.3 degrees), in any case measured, and always reported together with the mechanical parameters of cancellous bone.     

Critical requirement for cell cycle inhibitors in sustaining nonproliferative states

In adult vertebrates, most cells are not in the cell cycle at any one time. Physiological nonproliferation states encompass reversible quiescence and permanent postmitotic conditions such as terminal differentiation and replicative senescence. Although these states appear to be attained and maintained quite differently, they might share a core proliferation-restricting mechanism. Unexpectedly, we found that all sorts of nonproliferating cells can be mitotically reactivated by the sole suppression of histotype-specific cyclin-dependent kinase (cdk) inhibitors (CKIs) in the absence of exogenous mitogens. RNA interference-mediated suppression of appropriate CKIs efficiently triggered DNA synthesis and mitosis in established and primary terminally differentiated skeletal muscle cells (myotubes), quiescent human fibroblasts, and senescent human embryo kidney cells. In serum-starved fibroblasts and myotubes alike, cell cycle reactivation was critically mediated by the derepression of cyclin D-cdk4/6 complexes. Thus, both temporary and permanent growth arrest must be actively maintained by the constant expression of CKIs, whereas the cell cycle-driving cyclins are always present or can be readily elicited. In principle, our findings could find wide application in biotechnology and tissue repair whenever cell proliferation is limiting.     

Cells migrating to sites of tissue damage in response to the danger signal HMGB1 require NF-kappaB activation

Tissue damage is usually followed by healing, as both differentiated and stem cells migrate to replace dead or damaged cells. Mesoangioblasts (vessel-associated stem cells that can repair muscles) and fibroblasts migrate toward soluble factors released by damaged tissue. Two such factors are high mobility group box 1 (HMGB1), a nuclear protein that is released by cells undergoing unscheduled death (necrosis) but not by apoptotic cells, and stromal derived factor (SDF)-1/CXCL12. We find that HMGB1 activates the canonical nuclear factor kappaB (NF-kappaB) pathway via extracellular signal-regulated kinase phosphorylation. NF-kappaB signaling is necessary for chemotaxis toward HMGB1 and SDF-1/CXCL12, but not toward growth factor platelet-derived growth factor, formyl-met-leu-phe (a peptide that mimics bacterial invasion), or the archetypal NF-kappaB-activating signal tumor necrosis factor alpha. In dystrophic mice, mesoangioblasts injected into the general circulation ingress inefficiently into muscles if their NF-kappaB signaling pathway is disabled. These findings suggest that NF-kappaB signaling controls tissue regeneration in addition to early events in inflammation.     

Potomacanthus lobatus gen. et sp. nov., a new flower of probable Lauraceae from the Early Cretaceous (Early to Middle Albian) of eastern North America

A charcoalified fossil flower, Potomacanthus lobatus gen. et sp. nov., is described from the Early Cretaceous (Early to Middle Albian) Puddledock locality, Virginia, USA. Internal floral structure was studied using nondestructive synchrotron-radiation x-ray tomographic microscopy (SRXTM). The flower is bisexual and trimerous. The perianth consists of two whorls of tepals. The androecium has two whorls of fertile stamens. Anthers open by two distally hinged valves. The gynoecium consists of a single carpel that is plicate in the style and ascidiate in the ovary and contains a single pendant ovule. The fossil flower shares many similarities with flowers of extant Lauraceae and is unlike flowers of other families of Laurales. However, the fossil flower also differs in detail from all extant or fossil Lauraceae, particularly in configuration of the androecium. The new taxon, together with previously described but more fragmentary material from the Puddledock locality, provides the earliest fossil record of plants more closely related to Lauraceae than to any other extant family. It reveals several derived morphological characters that are potential synapomorphies among extant representatives of the family Lauraceae and contributes to the growing evidence for an early diversification of Laurales before the end of the Early Cretaceous.     

Crisponi syndrome is caused by mutations in the CRLF1 gene and is allelic to cold-induced sweating syndrome type 1

Crisponi syndrome is a severe autosomal recessive condition that is phenotypically characterized by abnormal, paroxysmal muscular contractions resembling neonatal tetanus, large face, broad nose, anteverted nares, camptodactyly, hyperthermia, and sudden death in most cases. We performed homozygosity mapping in five Sardinian and three Turkish families with Crisponi syndrome, using high-density single-nucleotide polymorphism arrays, and identified a critical region on chromosome 19p12-13.1. The most prominent candidate gene was CRLF1, recently found to be involved in the pathogenesis of cold-induced sweating syndrome type 1 (CISS1). CISS1 belongs to a group of conditions with overlapping phenotypes, also including cold-induced sweating syndrome type 2 and Stuve-Wiedemann syndrome. All these syndromes are caused by mutations of genes of the ciliary neurotrophic factor (CNTF)-receptor pathway. Here, we describe the identification of four different CRLF1 mutations in eight different Crisponi-affected families, including a missense mutation, a single-nucleotide insertion, and a nonsense and an insertion/deletion (indel) mutation, all segregating with the disease trait in the families. Comparison of the mutation spectra of Crisponi syndrome and CISS1 suggests that neither the type nor the location of the CRLF1 mutations points to a phenotype/genotype correlation that would account for the most severe phenotype in Crisponi syndrome. Other, still-unknown molecular factors may be responsible for the variable phenotypic expression of the CRLF1 mutations. We suggest that the syndromes can comprise a family of "CNTF-receptor-related disorders," of which Crisponi syndrome would be the newest member and allelic to CISS1.     

SEC14-like protein 1 interacts with cholinergic transporters

Trafficking of the vesicular acetylcholine transporter (VAChT) to synaptic vesicles has the potential to regulate storage and release of acetylcholine. We used the C-terminal tail of the vesicular acetylcholine transporter as bait for the screening of a brain cDNA library by yeast-two hybrids. Here we report an interaction uncovered in this screening with SEC14L1, a mammalian SEC14-like protein that may function as a phospholipid transfer protein. The interaction of VAChT and SEC14L1 occurred through the GOLD domain found in the latter and was confirmed in mammalian cells. In addition, we also found that SEC14L1 co-immunoprecipitates with the high affinity choline transporter (CHT1), but not with synaptophysin or synaptotagmin. In cultured cells SEC14L1 was predominantly found in the cytosol with little or no localization in defined organelles. In contrast, overexpression of VAChT or CHT1 with SEC14L1 recruited the latter to large intracellular organelles similar to vesicles or vesicle aggregates. Finally, we find that overexpression of SEC14L1 modestly decreases high affinity choline transport activity. We suggest that interaction of cholinergic transporters with proteins containing the GOLD domain may be relevant for transporter function.     

Molecular,cellular and histological changes in skin from a larval to an adult phenotype during bony fish metamorphosis

Developmental models for skin exist in terrestrial and amphibious vertebrates but there is a lack of information in aquatic vertebrates. We have analysed skin epidermal development of a bony fish (teleost), the most successful group of extant vertebrates. A specific epidermal type I keratin cDNA (hhKer1), which may be a bony-fish-specific adaptation associated with the divergence of skin development (scale formation) compared with other vertebrates, has been cloned and characterized. The expression of hhKer1 and collagen 1α1 in skin taken together with the presence or absence of keratin bundle-like structures have made it possible to distinguish between larval and adult epidermal cells during skin development. The use of a flatfish with a well-defined larval to juvenile transition as a model of skin development has revealed that epidermal larval basal cells differentiate directly to epidermal adult basal cells at the climax of metamorphosis. Moreover, hhKer1 expression is downregulated at the climax of metamorphosis and is inversely correlated with increasing thyroxin levels. We suggest that, whereas early mechanisms of skin development between aquatic and terrestrial vertebrates are conserved, later mechanisms diverge. This work was carried out within the project “Arrested development: The Molecular and Endocrine Basis of Flatfish Metamorphosis” (Q5RS-2002-01192) with financial support from the Commission of the European Communities. It does not necessarily reflect the Commission’s views and in no way anticipates its future policy in this area. This project was further supported by Pluriannual funding to CCMAR by the Portuguese Science and Technology Council. M.A. Campinho was sponsored by the Portuguese Ministry of Science (grant no. SFRH/BD/6133/2001).     

Aberrant 5' splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization

Despite a growing number of splicing mutations found in hereditary diseases, utilization of aberrant splice sites and their effects on gene expression remain challenging to predict. We compiled sequences of 346 aberrant 5′splice sites (5′ss) that were activated by mutations in 166 human disease genes. Mutations within the 5′ss consensus accounted for 254 cryptic 5′ss and mutations elsewhere activated 92 de novo 5′ss. Point mutations leading to cryptic 5′ss activation were most common in the first intron nucleotide, followed by the fifth nucleotide. Substitutions at position +5 were exclusively G>A transitions, which was largely attributable to high mutability rates of C/G>T/A. However, the frequency of point mutations at position +5 was significantly higher than that observed in the Human Gene Mutation Database, suggesting that alterations of this position are particularly prone to aberrant splicing, possibly due to a requirement for sequential interactions with U1 and U6 snRNAs. Cryptic 5′ss were best predicted by computational algorithms that accommodate nucleotide dependencies and not by weight-matrix models. Discrimination of intronic 5′ss from their authentic counterparts was less effective than for exonic sites, as the former were intrinsically stronger than the latter. Computational prediction of exonic de novo 5′ss was poor, suggesting that their activation critically depends on exonic splicing enhancers or silencers. The authentic counterparts of aberrant 5′ss were significantly weaker than the average human 5′ss. The development of an online database of aberrant 5′ss will be useful for studying basic mechanisms of splice-site selection, identifying splicing mutations and optimizing splice-site prediction algorithms.