A simple method to change geometrical proportions of muscles by means of a tibial lengthening osteotomy in the rat

A method is designed to change geometrical proportions of muscles in the rat. After a tibial osteotomy, the application of a stainless steel prosthesis resulted in an elongation of about 8% of the tibia. In 23 out of 28 operated rats a correct application of the prosthesis was maintained till the rats were sacrificed. The operation caused hardly any muscle tissue damage. No disuse of the manipulated leg was observed in the operated rats.     

Characterization of Nicotine-Induced Desensitization of Evoked Dopamine Release from Rat Striatal Synaptosomes

Abstract: Nicotine has been shown to stimulate neurotransmitter release from brain tissue by acting on presynaptic receptors. In this study, the ability of nicotine pretreatment to produce functional desensitization was investigated in rat striatal synaptosomes in which the release of [³H]dopamine was measured with an in vitro superfusion system. Pretreatment of synaptosomes with low concentrations of l -nicotine resulted in a decrease in the ability of a subsequent nicotine challenge to evoke [³H]dopamine release. The IC₅₀ for nicotine-induced desensitization was found to be 12 n M with a maximum inhibition of >90% at 300 n M . Nicotine pretreatment did not affect the release evoked by amphetamine, veratridine, or 15 m M K⁺. The onset of nicotine-induced desensitization occurred with a t _1/2 of 43 s at 30 n M nicotine. The temperature dependence of onset yielded a Q10 of 1.2.Recovery from desensitization was slower ( t _1/2 = 4.33 min), and both the onset and recovery appeared to follow a single first-order process. Several intermittent schedules of nicotine treatment were found to be effective at inducing and maintaining desensitization. The results of this study show that nonstimulating concentrations of nicotine can produce a complete functional desensitization of subsequent nicotine-induced neurotransmitter release.     

Directed evolution reveals the mechanism of HitRS signaling transduction in Bacillus anthracis

Two component systems (TCSs) are a primary mechanism of signal sensing and response in bacteria. Systematic characterization of an entire TCS could provide a mechanistic understanding of these important signal transduction systems. Here, genetic selections were employed to dissect the molecular basis of signal transduction by the HitRS system that detects cell envelope stress in the pathogen Bacillus anthracis. Numerous point mutations were isolated within HitRS, 17 of which were in a 50-residue HAMP domain. Mutational analysis revealed the importance of hydrophobic interactions within the HAMP domain and highlighted its essentiality in TCS signaling. In addition, these data defined residues critical for activities intrinsic to HitRS, uncovered specific interactions among individual domains and between the two signaling proteins, and revealed that phosphotransfer is the rate-limiting step for signal transduction. Furthermore, this study establishes the use of unbiased genetic selections to study TCS signaling and provides a comprehensive mechanistic understanding of an entire TCS.     

Regional and local impact on species diversity – from pattern to processes

The impact of regional factors (such as speciation or dispersal) on the species richness in local communities (S_L) has received increasing attention. A prominent method to infer the impact of regional factors is the comparison of species richness in local assemblages (S_L) with the total number of species in the region (S_R). Linear relations between S_R and S_L have been interpreted as an indication of strong regional influence and weak influence of interactions within local communities. We propose that two aspects bias the outcome of such comparisons: (1) the spatial scale of local and regional sampling, and (2) the body size of the organisms. The impact of the local area reflects the scales of ecological interactions, whereas the ratio between local and regional area reflects the inherent moment of autocorrelation. A proposed impact of body size on the relation is based on the high dispersal and high abundance of small organisms. We predict strongest linearity between S_R and S_L for large organisms, for large local areas (less important ecological interactions) and for sampling designs where the local habitat area covers a high proportion of the regional area (more important autocorrelation). We conducted a meta-analysis on 63 relations obtained from the literature. As predicted, the linearity of the relationship between S_L and S_R increased with the proportion of local to regional sampling area. In contrast, neither the body size of the organisms nor the local area itself was significantly related to the relation between S_L and S_R. This indicated that ecological interactions played a minor role in the shape of local to regional richness plots, which instead was mainly influenced by the sampling design. We found that the studies published so far were highly biased towards larger organisms and towards high similarity between the local and regional area. The proposed prevalence of linear relationships may thus be an artefact and plots of S_L to S_R are not a suitable tool with which to infer the strength of local interactions.     

Concentration dependent effects of dextran on the physical properties of acid milk gels

The effect of dextran from Leuconostoc mesenteroides (DEX₅₀₀), added to milk prior to acidification with glucono-δ-lactone (GDL) or Streptococcus thermophilus DSM20259, was studied with respect to polysaccharide concentration. The incorporation of 5–30 g/kg DEX₅₀₀ significantly affected gelation behavior. Increasing DEX₅₀₀ concentrations resulted in a linear increase of gel stiffness (GDL gels: R² = 0.96; microbial acidification: R² = 0.94; P < 0.05) and 30 g/kg DEX₅₀₀ resulted in a 2-fold higher stiffness compared to gels without polysaccharide. The respective stirred gels depicted a significant reduction in syneresis, which decreased from 30.4% (0 g/kg DEX₅₀₀) to 22.0% (30 g/kg DEX₅₀₀) for chemically acidified gels after 1 d of storage. Physical characteristics of DEX₅₀₀ in aqueous solution were helpful to explain its behavior in the complex system milk.     

Longitudinal Analysis of Osteogenic and Angiogenic Signaling Factors in Healing Models Mimicking Atrophic and Hypertrophic Non-Unions in Rats

Impaired bone healing can have devastating consequences for the patient. Clinically relevant animal models are necessary to understand the pathology of impaired bone healing. In this study, two impaired healing models, a hypertrophic and an atrophic non-union, were compared to physiological bone healing in rats. The aim was to provide detailed information about differences in gene expression, vascularization and histology during the healing process. The change from a closed fracture (healing control group) to an open osteotomy (hypertrophy group) led to prolonged healing with reduced mineralized bridging after 42 days. RT-PCR data revealed higher gene expression of most tested osteogenic and angiogenic factors in the hypertrophy group at day 14. After 42 days a significant reduction of gene expression was seen for Bmp4 and Bambi in this group. The inhibition of angiogenesis by Fumagillin (atrophy group) decreased the formation of new blood vessels and led to a non-healing situation with diminished chondrogenesis. RT-PCR results showed an attempt towards overcoming the early perturbance by significant up regulation of the angiogenic regulators Vegfa, Angiopoietin 2 and Fgf1 at day 7 and a further continuous increase of Fgf1, -2 and Angiopoietin 2 over time. However µCT angiograms showed incomplete recovery after 42 days. Furthermore, lower expression values were detected for the Bmps at day 14 and 21. The Bmp antagonists Dan and Twsg1 tended to be higher expressed in the atrophy group at day 42. In conclusion, the investigated animal models are suitable models to mimic human fracture healing complications and can be used for longitudinal studies. Analyzing osteogenic and angiogenic signaling patterns, clear changes in expression were identified between these three healing models, revealing the importance of a coordinated interplay of different factors to allow successful bone healing.     

Dynamic Interaction of PTPμ with Multiple Cadherins In Vivo

There is a growing body of evidence to implicate reversible tyrosine phosphorylation as an important mechanism in the control of the adhesive function of cadherins. We previously demonstrated that the receptor protein tyrosine phosphatase PTPμ associates with the cadherin–catenin complex in various tissues and cells and, therefore, may be a component of such a regulatory mechanism (Brady-Kalnay, S.M., D.L. Rimm, and N.K. Tonks. 1995. J. Cell Biol. 130:977– 986). In this study, we present further characterization of this interaction using a variety of systems. We observed that PTPμ interacted with N-cadherin, E-cadherin, and cadherin-4 (also called R-cadherin) in extracts of rat lung. We observed a direct interaction between PTPμ and E-cadherin after coexpression in Sf9 cells. In WC5 cells, which express a temperature-sensitive mutant form of v-Src, the complex between PTPμ and E-cadherin was dynamic, and conditions that resulted in tyrosine phosphorylation of E-cadherin were associated with dissociation of PTPμ from the complex. Furthermore, we have demonstrated that the COOH-terminal 38 residues of the cytoplasmic segment of E-cadherin was required for association with PTPμ in WC5 cells. Zondag et al. (Zondag, G., W. Moolenaar, and M. Gebbink. 1996. J. Cell Biol. 134: 1513–1517) have asserted that the association we observed between PTPμ and the cadherin–catenin complex in immunoprecipitates of the phosphatase arises from nonspecific cross-reactivity between BK2, our antibody to PTPμ, and cadherins. In this study we have confirmed our initial observation and demonstrated the presence of cadherin in immunoprecipitates of PTPμ obtained with three antibodies that recognize distinct epitopes in the phosphatase. In addition, we have demonstrated directly that the anti-PTPμ antibody BK2 that we used initially did not cross-react with cadherin. Our data reinforce the observation of an interaction between PTPμ and E-cadherin in vitro and in vivo, further emphasizing the potential importance of reversible tyrosine phosphorylation in regulating cadherin function.     

Mechanisms of Functional and Physical Genome Reduction in Photosynthetic and Nonphotosynthetic Parasitic Plants of the Broomrape Family

Nonphotosynthetic plants possess strongly reconfigured plastomes attributable to convergent losses of photosynthesis and housekeeping genes, making them excellent systems for studying genome evolution under relaxed selective pressures. We report the complete plastomes of 10 photosynthetic and nonphotosynthetic parasites plus their nonparasitic sister from the broomrape family (Orobanchaceae). By reconstructing the history of gene losses and genome reconfigurations, we find that the establishment of obligate parasitism triggers the relaxation of selective constraints. Partly because of independent losses of one inverted repeat region, Orobanchaceae plastomes vary 3.5-fold in size, with 45 kb in American squawroot (Conopholis americana) representing the smallest plastome reported from land plants. Of the 42 to 74 retained unique genes, only 16 protein genes, 15 tRNAs, and four rRNAs are commonly found. Several holoparasites retain ATP synthase genes with intact open reading frames, suggesting a prolonged function in these plants. The loss of photosynthesis alters the chromosomal architecture in that recombinogenic factors accumulate, fostering large-scale chromosomal rearrangements as functional reduction proceeds. The retention of DNA fragments is strongly influenced by both their proximity to genes under selection and the co-occurrence with those in operons, indicating complex constraints beyond gene function that determine the evolutionary survival time of plastid regions in nonphotosynthetic plants.     

Mutations in SLC33A1 cause a lethal autosomal-recessive disorder with congenital cataracts, hearing loss, and low serum copper and ceruloplasmin

Low copper and ceruloplasmin in serum are the diagnostic hallmarks for Menkes disease, Wilson disease, and aceruloplasminemia. We report on five patients from four unrelated families with these biochemical findings who presented with a lethal autosomal-recessive syndrome of congenital cataracts, hearing loss, and severe developmental delay. Cerebral MRI showed pronounced cerebellar hypoplasia and hypomyelination. Homozygosity mapping was performed and displayed a region of commonality among three families at chromosome 3q25. Deep sequencing and conventional sequencing disclosed homozygous or compound heterozygous mutations for all affected subjects in SLC33A1 encoding a highly conserved acetylCoA transporter (AT-1) required for acetylation of multiple gangliosides and glycoproteins. The mutations were found to cause reduced or absent AT-1 expression and abnormal intracellular localization of the protein. We also showed that AT-1 knockdown in HepG2 cells leads to reduced ceruloplasmin secretion, indicating that the low copper in serum is due to reduced ceruloplasmin levels and is not a sign of copper deficiency. The severity of the phenotype implies an essential role of AT-1 in proper posttranslational modification of numerous proteins, without which normal lens and brain development is interrupted. Furthermore, AT-1 defects are a new and important differential diagnosis in patients with low copper and ceruloplasmin in serum.