Effective fiber hypertrophy in satellite cell-depleted skeletal muscle

An important unresolved question in skeletal muscle plasticity is whether satellite cells are necessary for muscle fiber hypertrophy. To address this issue, a novel mouse strain (Pax7-DTA) was created which enabled the conditional ablation of >90% of satellite cells in mature skeletal muscle following tamoxifen administration. To test the hypothesis that satellite cells are necessary for skeletal muscle hypertrophy, the plantaris muscle of adult Pax7-DTA mice was subjected to mechanical overload by surgical removal of the synergist muscle. Following two weeks of overload, satellite cell-depleted muscle showed the same increases in muscle mass (approximately twofold) and fiber cross-sectional area with hypertrophy as observed in the vehicle-treated group. The typical increase in myonuclei with hypertrophy was absent in satellite cell-depleted fibers, resulting in expansion of the myonuclear domain. Consistent with lack of nuclear addition to enlarged fibers, long-term BrdU labeling showed a significant reduction in the number of BrdU-positive myonuclei in satellite cell-depleted muscle compared with vehicle-treated muscle. Single fiber functional analyses showed no difference in specific force, Ca(2+) sensitivity, rate of cross-bridge cycling and cooperativity between hypertrophied fibers from vehicle and tamoxifen-treated groups. Although a small component of the hypertrophic response, both fiber hyperplasia and regeneration were significantly blunted following satellite cell depletion, indicating a distinct requirement for satellite cells during these processes. These results provide convincing evidence that skeletal muscle fibers are capable of mounting a robust hypertrophic response to mechanical overload that is not dependent on satellite cells.     

Differing activities of homeostatic chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis

Despite their widespread expression, the in vivo recruitment activities of CCL19 (EBV-induced molecule 1 ligand chemokine) and CXCL12 (stromal cell-derived factor 1) have not been established. Furthermore, although CXCL13 (B lymphocyte chemoattractant) has been shown to induce lymphoid neogenesis through induction of lymphotoxin (LT)alpha1beta2, it is unclear whether other homeostatic chemokines have this property. In this work we show that ectopic expression in pancreatic islets of CCL19 leads to small infiltrates composed of lymphocytes and dendritic cells and containing high endothelial venules and stromal cells. Ectopic CXCL12 induced small infiltrates containing few T cells but enriched in dendritic cells, B cells, and plasma cells. Comparison of CCL19 transgenic mice with mice expressing CCL21 (secondary lymphoid tissue chemokine) revealed that CCL21 induced larger and more organized infiltrates. A more significant role for CCL21 is also suggested in lymphoid tissues, as CCL21 protein was found to be present in lymph nodes and spleen at much higher concentrations than CCL19. CCL19 and CCL21 but not CXCL12 induced LTalpha1beta2 expression on naive CD4 T cells, and treatment of CCL21 transgenic mice with LTbetaR-Fc antagonized development of organized lymphoid structures. LTalpha1beta2 was also induced on naive T cells by the cytokines IL-4 and IL-7. These studies establish that CCL19 and CXCL12 are sufficient to mediate cell recruitment in vivo and they indicate that LTalpha1beta2 may function downstream of CCL21, CCL19, and IL-2 family cytokines in normal and pathological lymphoid tissue development.     

Regiospecific and conformationally restrained analogs of melphalan and dl-2-NAM-7 and their affinities for the large neutral amino acid transporter (system LAT1) of the blood–brain barrier

Regiospecific and conformationally restrained analogs of melphalan and dl-2-NAM-7 have been synthesized and their affinities for the large neutral amino acid transporter (LAT1) of the blood–brain barrier have been determined to assess their potential for accessing the CNS via facilitated transport. Several analogs had K_i values in the range 2.1–8.5 μM with greater affinities than that of either l-phenylalanine (K_i = 11 μM) or melphalan (K_i = 55 μM), but lower than dl-2-NAM-7 (K_i = 0.08 μM). The results indicate that regiospecific positioning of the mustard moiety on the aromatic ring in these analogs is very important for optimal affinity for the large neutral amino acid transporter, and that conformational restriction of the dl-2-NAM-7 molecule in benzonorbornane and indane analogs leads to 25- to 60-fold loss, respectively, in affinity.     

Design,synthesis and biological evaluation of piperazino-enaminones as novel suppressants of pro-Inflammatory cytokines

Infection triggers the release of pro-inflammatory cytokines (TNF-alpha and IL-6). Over-production, however, cause tissue injury seen in severe asthma. The ability of enaminone E121 to reduce pro-inflammatory cytokines in our laboratory encouraged further examination of its structural scaffold. Piperazino-enaminones were designed by incorporating n-arylpiperazine motif into the aromatic enaminone. Four possible modifications were explored systematically. Synthesis was accomplished by amination of the corresponding methyl/ethyl 2,4-dioxo-6-(substituted)cyclohexane-carboxylate.. Sixteen novel compounds were synthesized. Biological activity was tested in J774 macrophages stimulated with lipopolysaccharides. The release of cytokines was measured via ELISA. Four compounds significantly suppressed TNF-alpha and IL-6 release in dose-dependent manner.     

Regulation of Dendrite and Spine Morphogenesis and Plasticity by Catenins

The appropriate regulation of dendrite, spine, and synapse morphogenesis in neurons both during and after development is critical for the formation and maintenance of neural circuits. It is becomingly increasingly clear that the cadherin–catenin cell adhesion complex, a complex that has been widely studied in epithelia, regulates neuronal morphogenesis. More interestingly, the catenins, cytosolic proteins that bind to cadherins, regulate multiple aspects of neuronal morphogenesis including dendrite, spine, and synapse morphogenesis and plasticity, both independent of and dependent on their ability to bind cadherins. In this review, we examine some of the more recent and exciting studies that implicate individual catenins in various aspects of neuronal morphogenesis and plasticity.     

Dissociation of Telomere Dynamics from Telomerase Activity in Human Thyroid Cancer Cells

Prevention of telomere erosion through acquisition of telomerase activity is thought to be an essential mechanism in most human cancer cells for avoidance of cellular senescence and crisis. It has been generally assumed that once telomerase has been activated, no further telomere shortening should ensue. We show here, however, that a much more complex pattern of telomere dynamics can exist in telomerase-positive immortal cancer cells. Using a panel of subclones derived from a human thyroid cancer cell line, K1E7, we found that some clones show persistent decline in mean telomere restriction fragment (TRF) length by up to 2 kb over 450 population doublings (pd), despite sustained high telomerase activity (as assessed by thein vitro“TRAP” assay). TRF length subsequently stabilized at around 5 kb, but with no corresponding increase in telomerase activity. One clone showed an even more unexpected biphasic time course, with the mean TRF length initially increasing by 1.5 kb over 90 pd, before “plateauing” and then returning over a similar period to its original value, again without any correlation to TRAP activity. Such dissociations between telomere dynamics and telomerase activity support the existence of additional controls on telomere length in the intact cell. Our observations are consistent with current negative-feedback models of telomere length regulation by telomere binding proteins and these cell lines should prove useful experimental tools for their further evaluation.     

Complete Genome Sequence of Francisella tularensis Subspecies holarctica FTNF002-00

Francisella tularensis subspecies holarctica FTNF002-00 strain was originally obtained from the first known clinical case of bacteremic F. tularensis pneumonia in Southern Europe isolated from an immunocompetent individual. The FTNF002-00 complete genome contains the RD₂₃ deletion and represents a type strain for a clonal population from the first epidemic tularemia outbreak in Spain between 1997–1998. Here, we present the complete sequence analysis of the FTNF002-00 genome. The complete genome sequence of FTNF002-00 revealed several large as well as small genomic differences with respect to two other published complete genome sequences of F. tularensis subsp. holarctica strains, LVS and OSU18. The FTNF002-00 genome shares >99.9% sequence similarity with LVS and OSU18, and is also ∼5 MB smaller by comparison. The overall organization of the FTNF002-00 genome is remarkably identical to those of LVS and OSU18, except for a single 3.9 kb inversion in FTNF002-00. Twelve regions of difference ranging from 0.1–1.5 kb and forty-two small insertions and deletions were identified in a comparative analysis of FTNF002-00, LVS, and OSU18 genomes. Two small deletions appear to inactivate two genes in FTNF002-00 causing them to become pseudogenes; the intact genes encode a protein of unknown function and a drug:H⁺ antiporter. In addition, we identified ninety-nine proteins in FTNF002-00 containing amino acid mutations compared to LVS and OSU18. Several non-conserved amino acid replacements were identified, one of which occurs in the virulence-associated intracellular growth locus subunit D protein. Many of these changes in FTNF002-00 are likely the consequence of direct selection that increases the fitness of this subsp. holarctica clone within its endemic population. Our complete genome sequence analyses lay the foundation for experimental testing of these possibilities.