A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.      

The radioenhancement of two human head and neck squamous cell carcinomas by 2'-2' difluorodeoxycytidine (gemcitabine; dFdC) is mediated by an increase in radiation-induced residual chromosome aberrations but not residual DNA DSBs

PURPOSE: The present study aimed at investigating if 2'-2' difluorodeoxycytidine (dFdC) radioenhancement was mediated by an effect on induction and/or repair of radiation-induced DNA DSBs and chromosome aberrations in cells with different intrinsic radiosensitivity. METHODS: Confluent human head and neck squamous cell carcinoma cell lines designated SCC61 and SQD9 were treated with 5 microM dFdC for 3 or 24 h prior to irradiation. DNA DSBs induction and repair were analyzed by PFGE. Radiation-induced chromosome aberrations were examined with a FISH technique. RESULTS: In both cell lines, dFdC did not modify radiation-induced DNA DSBs in a dose range between 0 and 40 Gy. After a single dose of 40 Gy, dFdC affected neither the kinetic of repair nor the residual amount of DNA DSBs up to 4 h after irradiation. Whereas dFdC did not increase the induction of chromosome aberrations, after a single dose of 5 Gy, the percentage of aberrant cells and the number of aberrations per aberrant cells were significantly higher in combination with dFdC. CONCLUSION: Our data suggest that under experimental conditions yielding substantial radioenhancement, dFdC decreases the repair of genomic lesions inducing secondary chromosome breaks but has no effect on DNA DSBs repair as measured by PFGE.     

Proteins and saccharides of the sea urchin organic matrix of mineralization: characterization and localization in the spine skeleton

Properties of the echinoderm skeleton are under biological control, which is exerted in part by the organic matrix embedded in the mineralized part of the skeleton. This organic matrix consists of proteins and glycoproteins whose carbohydrate component is specifically involved in the control mechanisms. The saccharide moiety of the organic matrix of the spines of the echinoid Paracentrotus lividus was characterized using enzyme-linked lectin assays (ELLAs). O-glycoproteins, different types of complex N-glycoproteins, and terminal sialic acids were detected. Sialic acids are known to interact with Ca ions and could play an important role in the mineralization process. Some of the carbohydrate components detected by ELLAs as well as two organic matrix proteins (SM30 and SM50) were localized within different subregions of the spine skeleton using field-emission scanning electron microscopy. The mappings show that some of these components are not homogeneously distributed in the different skeletal subregions. For example, some N-glycoproteins were preferentially located in the putative amorphous subregion of the skeleton, whereas some O-glycoproteins were localized in the subregion where skeletal growth is inhibited. These results suggest that the biological control exerted on the skeletal properties can be partly modulated by local differences in the organic matrix composition.     

Mice deficient in small leucine-rich proteoglycans: novel in vivo models for osteoporosis,osteoarthritis, Ehlers-Danlos syndrome,muscular dystrophy,and corneal diseases

Small leucine-rich proteoglycans (SLRPs) are extracellular molecules that bind to TGFbetas and collagens and other matrix molecules. In vitro, SLRPs were shown to regulate collagen fibrillogenesis, a process essential in development, tissue repair, and metastasis. To better understand their functions in vivo, mice deficient in one or two of the four most prominent and widely expressed SLRPs (biglycan, decorin, fibromodulin, and lumican) were recently generated. All four SLRP deficiencies result in the formation of abnormal collagen fibrils. Taken together, the collagen phenotypes demonstrate a cooperative, sequential, timely orchestrated action of the SLRPs that altogether shape the architecture and mechanical properties of the collagen matrix. In addition, SLRP-deficient mice develop a wide array of diseases (osteoporosis, osteoarthritis, muscular dystrophy, Ehlers-Danlos syndrome, and corneal diseases), most of them resulting primarily from an abnormal collagen fibrillogenesis. The development of these diseases by SLRP-deficient mice suggests that mutations in SLRPs may be part of undiagnosed predisposing genetic factors for these diseases. Although the distinct phenotypes developed by the different singly deficient mice point to distinct in vivo function for each SLRP, the analysis of the double-deficient mice also demonstrates the existence of rescuing/compensation mechanisms, indicating some functional overlap within the SLRP family.     

Biglycan knockout mice: new models for musculoskeletal diseases

Biglycan is a Class I Small Leucine Rich Proteoglycans (SLRP) that is localized on human chromosome Xq28-ter. The conserved nature of its intron-exon structure and protein coding sequence compared to decorin (another Class I SLRP) indicates the two genes may have arisen from gene duplication. Biglycan contains two chondroitin sulfate glycosaminoglycan (GAG) chains attached near its NH₂ terminus making it different from decorin that has only one GAG chain. To determine the functions of biglycan in vivo, transgenic mice were developed that were deficient in the production of the protein (knockout). These mice acquire diminished bone mass progressively with age. Double tetracycline-calcein labeling revealed that the biglycan deficient mice are defective in their capacity to form bone. Based on this observation, we tested the hypothesis that the osteoporosis-like phenotype is due to defects in cells critical to the process of bone formation. Our data shows that biglycan deficient mice have diminished capacity to produce marrow stromal cells, the bone cell precursors, and that this deficiency increases with age. The cells also have reduced response to tranforming growth factor- (TGF-), reduced collagen synthesis and relatively more apoptosis than cells from normal littermates. In addition, calvaria cells isolated from biglycan deficient mice have reduced expression of late differentiation markers such as bone sialoprotein and osteocalcin and diminished ability to accumulate calcium judged by alizerin red staining. We propose that any one of these defects in osteogenic cells alone, or in combination, could contribute to the osteoporosis observed in the biglycan knockout mice. Other data suggests there is a functional relationship between biglycan and bone morphogenic protein-2/4 (BMP 2/4) action in controlling skeletal cell differentiation. In order to test the hypothesis that functional compensation can occur between SLRPs, we created mice deficient in biglycan and decorin. Decorin deficient mice have normal bone mass while the double biglycan/decorin knockout mice have more severe osteopenia than the single biglycan indicating redundancy in SLRP function in bone tissue. To further determine whether compensation could occur between different classes of SLRPs, mice were generated that are deficient in both biglycan (class I) and fibromodulin, a class II SLRP highly expressed in mineralizing tissue. These doubly deficient mice had an impaired gait, ectopic calcification of tendons and premature osteoarthritis. Transmission electron microscopy analysis showed that like the decorin and biglycan knockouts, they have severely disturbed collagen fibril structures. Biomechanical analysis of the affected tendons showed they were weaker compared to control animals leading to the conclusion that instability of the joints could be the primary cause of all the skeletal defects observed in the fibromodulin/biglycan knockout mice. These studies present important new animal models for musculoskeletal diseases and provide the opportunity to characterize the network of signals that control tissue integrity and function through SLRP activity. Published in 2003.     

Ultrastructure of sea urchin calcified tissues after high-pressure freezing and freeze substitution

The improvements brought by high-pressure freezing/freeze substitution fixation methods to the ultrastructural preservation of echinoderm mineralized tissues are investigated in developing pedicellariae and teeth of the echinoid Paracentrotus lividus. Three freeze substitution (FS) protocols were tested: one in the presence of osmium tetroxide, one in the presence of uranyl acetate, and the last in the presence of gallic acid. FS in the presence of osmium tetroxide significantly improved cell ultrastructure preservation and should especially be used for ultrastructural studies involving vesicles and the Golgi apparatus. With all protocols, multivesicular bodies, suggested to contain Ca(2+), were evident for the first time in skeleton-forming cells. FS in the presence of gallic acid allowed us to confirm the structured and insoluble character of a part of the organic matrix of mineralization in the calcification sites of the tooth, an observation which modifies the current understanding of biomineralization control in echinoderms.     

Ultrastructure and cytochemistry of the early calcification site and of its mineralization organic matrix in Paracentrotus lividus (Echinodermata: Echinoidea)

 The ultrastructure and cytochemistry of skeleton formation sites prior to mineralization are described for the first time in echinoderms. These early sites are intracellular vacuoles located in syncytial pseudopodia of skeleteton-forming cells. They contain a mineralization organic matrix, which shows a calcium-binding ability and is framed in a tridimensional structure made of concentric layers bridged by radial threads. This organic matrix presents repetitive structures which could be implicated in mineralization control. Both the tridimensional organization of the organic matrix and its framing, before mineralization starts, question the current theories which suggest that the echinoderm organic matrix is soluble at the onset of mineralization and adsorbs on the forming crystal. Accepted: 19 March 1998     

The Prognostic Significance of Metabolic Response Heterogeneity in Metastatic Colorectal Cancer

Background

Tumoral heterogeneity is a major determinant of resistance in solid tumors. FDG-PET/CT can identify early during chemotherapy non-responsive lesions within the whole body tumor load. This prospective multicentric proof-of-concept study explores intra-individual metabolic response (mR) heterogeneity as a treatment efficacy biomarker in chemorefractory metastatic colorectal cancer (mCRC).

Methods

Standardized FDG-PET/CT was performed at baseline and after the first cycle of combined sorafenib (600mg/day for 21 days, then 800mg/day) and capecitabine (1700 mg/m²/day administered D1-14 every 21 days). MR assessment was categorized according to the proportion of metabolically non-responding (non-mR) lesions (stable FDG uptake with SUVmax decrease <15%) among all measurable lesions.

Results

Ninety-two patients were included. The median overall survival (OS) and progression-free survival (PFS) were 8.2 months (95% CI: 6.8–10.5) and 4.2 months (95% CI: 3.4–4.8) respectively. In the 79 assessable patients, early PET-CT showed no metabolically refractory lesion in 47%, a heterogeneous mR with at least one non-mR lesion in 32%, and a consistent non-mR or early disease progression in 21%. On exploratory analysis, patients without any non-mR lesion showed a significantly longer PFS (HR 0.34; 95% CI: 0.21–0.56, P-value <0.001) and OS (HR 0.58; 95% CI: 0.36–0.92, P-value 0.02) compared to the other patients. The proportion of non-mR lesions within the tumor load did not impact PFS/OS.

Conclusion

The presence of at least one metabolically refractory lesion is associated with a poorer outcome in advanced mCRC patients treated with combined sorafenib-capecitabine. Early detection of treatment-induced mR heterogeneity may represent an important predictive efficacy biomarker in mCRC.

Trial Registration

ClinicalTrials.gov NCT01290926     

Fibromodulin-deficient mice display impaired collagen fibrillogenesis in predentin as well as altered dentin mineralization and enamel formation.

To determine the functions of fibromodulin (Fmod), a small leucine-rich keratan sulfate proteoglycan in tooth formation, we investigated the distribution of Fmod in dental tissues by immunohistochemistry and characterized the dental phenotype of 1-day-old Fmod-deficient mice using light and transmission electron microscopy. Immunohistochemistry was also used to compare the relative protein expression of dentin sialoprotein (DSP), dentin matrix protein-1 (DMP 1), bone sialoprotein (BSP), and osteopontin (OPN) between Fmod-deficient mice and wild-type mice. In normal mice and rats, Fmod immunostaining was mostly detected in the distal cell bodies of odontoblasts and in the stratum intermedium and was weaker in odontoblast processes and predentin. The absence of Fmod impaired dentin mineralization, increased the diameter of the collagen fibrils throughout the whole predentin, and delayed enamel formation. Immunohistochemistry provides evidence for compensatory mechanisms in Fmod-deficient mice. Staining for DSP and OPN was decreased in molars, whereas DMP 1 and BSP were enhanced. In the incisors, labeling for DSP, DMP 1, and BSP was strongly increased in the pulp and odontoblasts, whereas OPN staining was decreased. Positive staining was also seen for DMP 1 and BSP in secretory ameloblasts. Together these studies indicate that Fmod restricts collagen fibrillogenesis in predentin while promoting dentin mineralization and the early stages of enamel formation.     

Selection against genetic defects in conservation schemes while controlling inbreeding

We studied different genetic models and evaluation systems to select against a genetic disease with additive, recessive or polygenic inheritance in genetic conservation schemes. When using optimum contribution selection with a restriction on the rate of inbreeding (ΔF) to select against a disease allele, selection directly on DNA-genotypes is, as expected, the most efficient strategy. Selection for BLUP or segregation analysis breeding value estimates both need 1–2 generations more to halve the frequency of the disease allele, while these methods do not require knowledge of the disease mutation at the DNA level. BLUP and segregation analysis methods were equally efficient when selecting against a disease with single gene or complex polygene inheritance, i.e. knowledge about the mode of inheritance of the disease was not needed for efficient selection against the disease. Smaller schemes or schemes with a more stringent restriction on ΔF needed more generations to halve the frequency of the disease alleles or the fraction of diseased animals. Optimum contribution selection maintained ΔF at its predefined level, even when selection of females was at random. It is argued that in the investigated small conservation schemes with selection against a genetic defect, control of ΔF is very important.