Coordination of murine limb muscle, skeleton and connective tissue development by Lmx1b

The underlying genetic defects of a congenital disease Nail-Patella Syndrome are loss-of-function mutations in the LMX1B gene. Lmx1b encodes a LIM-homeodomain transcription factor that is expressed specifically in the dorsal limb bud mesenchyme. Gain- and loss-of-function experiments suggest that Lmx1b is both necessary and sufficient to specify dorsal limb patterning. However, how Lmx1b coordinates patterning of the dorsal tissues in the limb, including muscle, skeleton and connective tissues, remains unknown. One possibility is that each tissue specifies its own pattern cell-autonomously, i.e., Lmx1b is expressed in tissues in which it functions and different tissues do not communicate with each other. Another possibility is that tissues that express Lmx1b interact with adjacent tissues and provide patterning information thereby directing the development of tissues non-cell-autonomously. Previous results showed that Lmx1b is expressed in limb connective tissue and skeleton, but is not expressed in muscle tissue. Moreover, muscles and muscle connective tissue are closely associated during development. Therefore, we hypothesize that Lmx1b controls limb muscle dorsal-ventral (DV) patterning through muscle connective tissue, but regulates skeleton and tendon/ligament development cell-autonomously. ^ To test this hypothesis, we first examined when and where the limb dorsal-ventral asymmetry is established during development. Subsequently, conditional knockout and overexpression experiments were performed to delete or activate Lmx1b in different tissues within the limb. Our results show that deletion of Lmx1b from whole limb mesenchyme results in all dorsal tissues, including muscle, tendon/ligament and skeleton, transforming into ventral structures. Skeleton-specific knockout of Lmx1b led to the dorsal duplication of distal sesamoid and metacarpal bones, but did not affect the pattern formation of other tissues, suggesting that Lmx1b controls skeleton development cell-autonomously. In addition, this skeleton-specific pattern alteration only occurs in distal limb tissues, not proximal limb tissues, indicating different regulatory mechanisms operate along the limb proximal-distal axis. Moreover, skeleton-specific ectopic expression of Lmx1b reveals a complementary skeletal-specific dorsalized phenotype. This result supports a cell-autonomous role for Lmx1b in dorsal-ventral skeletal patterning. This study enriched our understanding of limb development, and the insights from this research may also be applicable for the development of other organs. ^

Coordination of Family Preservation Services in a Rural Community: A Case Study

Family preservation programs designed to prevent the out-of-home placement of children depend on the coordination of services from multiple agencies. Little is known regarding how coordination occurs. This case study examined this issue. Information was sought from all workers who provided services to each of five families and 'from families' case records. Thirty-one workers were interviewed with a semi-structured interview schedule containing rating scales and questions with open-ended response formats. Case records were reviewed with a case record review form. Analyses of data revealed the following. Services were coordinated to a moderate degree but that coordination deteriorated over time. Workers elaborated how aspects of communities, human service agencies, workers, and families affected coordination. Implications of findings for future research were drawn.

Does the choice of bottle nipple affect the oral feeding performance of very-low-birthweight (VLBW) infants?

BACKGROUND: There is a continuous debate regarding the best bottle nipple to be used to enhance the bottle-feeding performance of a preterm infant. Aim: To verify that feeding performance can be improved by using the bottle nipple with the physical characteristics that enhance infants' sucking skills. METHODS: Ten "healthy" VLBW infants (941+/-273 g) were recruited. Feeding performance was monitored at two time periods, when taking 1-2 and 6-8 oral feedings/d. At each time and within 24 h, performance was monitored using three different bottle nipples offered in a randomized order. Rate of milk transfer (ml/min) was the primary outcome measure. The sucking skills monitored comprised stage of sucking, suction amplitude, and duration of the generated negative intraoral suction pressure. RESULTS: At both times, infants demonstrated a similar rate of milk transfer among all three nipples. However, the stage of sucking, suction amplitude, and duration of the generated suction were significantly different between nipples at 1-2, but not 6-8 oral feedings/d.CONCLUSION: We did not identify a particular bottle nipple that enhanced bottle feeding in healthy VLBW infants. Based on the notion that afferent sensory feedback may allow infants to adapt to changing conditions, we speculate that infants can modify their sucking skills in order to maintain a rate of milk transfer that is appropriate with the level of suck-swallow-breathe coordination achieved at a particular time. Therefore, it is proposed that caretakers should be more concerned over monitoring the coordination of suck-swallow-breathe than over the selection of bottle nipples.

Transcriptional regulation of the human Fas (CD95) promoter

The coordination of the apoptotic program necessitates the timely expression of sensor, effector, and mediator molecules. Fas/CD95, a transmembrane receptor which tethers the cell-death machinery, triggers apoptosis to maintain immune homeostasis, tolerance, and surveillance. Dysregulation in Fas-mediated apoptosis, either from disproportionate expression or disruptions in the downstream signaling pathway, manifests in autoimmune disorders and certain malignant progression. ^ In this project, the transcriptional requirements underlying two modulators of Fas expression were investigated. In T-lymphocytes, activation results in potent Fas upregulation followed by an acquisition of sensitivity towards FasL-mediated apoptosis. Human fas promoter cloning and analysis have identified a cis-element critical for inducible Fas expression. EMSA studies using this region demonstrated a constitutive association with the transcription factor Sp1 and inducible NF-κB binding in response to activation. These interactions were mutually exclusive, as the rB/Sp1 element bound with recombinant Sp1 was readily displaced by increasing amounts of NF-κB p50. Thus, Fas upregulation by T-cell activation stimuli is dependent upon NF-κB binding at the fas promoter. ^ The capacity of Sp1 to direct basal Fas expression was examined through mutagenesis of several GC-rich regions within the core fas promoter. Reporter analysis of single or combinatorial mutant GC-box constructs revealed usage of a particular GC-element in moderating over 50% of basal fas transcription. Inducible expression was Sp1-independent, however, since activated Jurkat cells containing fas Sp1-mutant constructs retained equivalent reporter induction. Overall, a dual-level of transcriptional control exists in fas, where constitutive activity is monitored through Sp1 binding, whereas T-cell activation obligates NF κB transactivation. ^ In response to genotoxic damage, p53 modulates Fas levels partly by a transcription-dependent mechanism. Reconstitution of wild-type p53 in the hepatoma cell line Hep3B readily induced Fas transcription. Furthermore, fas promoter analysis identified an undescribed p53 responsive element which, when deleted, ablated p53-mediated reporter activity. Therefore, the pro-apoptotic function mediated by p53 is driven partially through the enhancement of Fas expression. ^ Altogether, events elicting Fas transcription may invoke single or overlapping mechanisms that converge at the level of promoter activity. Agents that enhance or attenuate these pathways may be therapeutically beneficial in modulating the expression and sensitivity towards Fas-dependent apoptosis. ^

Temporal and spatial regulation of cell division by the nucleoid in Escherichia coli

Selection of division sites and coordination of cytokinesis with other cell cycle events are critical for every organism to proliferate. In E. coli, the nucleoid is proposed to exclude division from the site of the chromosome (nucleoid occlusion model). We studied the effect of the nucleoid on timing and placement of cell division. An early cell division protein, FtsZ, was used to follow development of the division septum. FtsZ forms a ring structure (Z ring) at potential division sites. The dynamics of Z ring was visualized in live cells by fusing FtsZ with a green fluorescent protein (GFP). Emanating FtsZ-GFP polymers from the constricted septum or aggregates in daughter cells were also observed, probably representing the FtsZ depolymerization and immature FtsZ nucleation processes. We next examined the nucleoid occlusion model. Mutants carrying abnormally positioned chromosomes were employed. In chromosomal partition mutants, replicated chromosomes cannot segregate. The Z ring was excluded from midcell to the edge of the nucleoid. This negative effect of nucleoids was further confirmed in replication deficient dnaA mutants, in which only a single chromosome is present in the cell center. These results suggest that the nucleoid, replicating or not, inhibits division in the area where the chromosome occupies. In addition, increasing the level of FtsZ does not overcome nucleoid inhibition. Interestingly in anucleate cells produced by both mutants, the Z ring was localized in the central part of the cell, which indicates that the nucleoid is not required for FtsZ assembly. Relaxation of chromosomes by reducing the gyrase activity or disruption of protein translation/translocation did not abolish the division inhibition capacity of the nucleoid. However, preventing transcription did compromise the nucleoid occlusion effect, leading to formation of multiple FtsZ rings above the nucleoid. In summary, we demonstrate that nucleoids negatively regulate the timing and position of division by inhibiting FtsZ assembly at unselected sites. Relief of this inhibition at midcell is coincident with the completion of DNA replication. On the other hand, FtsZ assembly does not require the nucleoid. ^

The utility of N5 for gene therapy of human cancer

Cancer is a result of defects in the coordination of cell proliferation and programmed cell death. The extent of cell death is physiologically controlled by the activation of a programmed suicide pathway that results in a morphologically recognizable form of death termed apoptosis. Inducing apoptosis in tumor cells by gene therapy provides a potentially effective means to treat human cancers. The p84N5 is a novel nuclear death domain containing protein that has been shown to bind an amino terminal domain of retinoblastoma tumor suppressor gene product (pRb). Expression of N5 can induce apoptosis that is dependent upon its intact death domain and is inhibited by pRb. In many human cancer cells the functions of pRb are either lost through gene mutation or inactivated by different mechanisms. N5 based gene therapy may induce cell death preferentially in tumor cells relative to normal cells. We have demonstrated that N5 gene therapy is less toxic to normal cells than to tumor cells. To test the possibility that N5 could be used in gene therapy of cancer, we have generated a recombinant adenovirus engineered to express N5 and test the effects of viral infection on growth and tumorigenicity of human cancer cells. Adenovirus N5 infection significantly reduced the proliferation and tumorigenicity of breast, ovarian, and osteosarcoma tumor cell lines. Reduced proliferation and tumorigenicity were mediated by an induction of apoptosis as indicated by DNA fragmentation in infected cells. We also test the potential utility of N5 for gene therapy of pancreatic carcinoma that typically respond poorly to conventional treatment. Adenoviral mediated N5 gene transfer inhibits the growth of pancreatic cancer cell lines in vitro. N5 gene transfer also reduces the growth and metastasis of human pancreatic adenocarcinoma in subcutaneous and orthotopic mouse model. Interestingly, the pancreatic adenocarcinoma cells are more sensitive to N5 than they are to p53, suggesting that N5 gene therapy may be effective in tumors resistant to p53. We also test the possibilities of the use of N5 and p53 together on the inhibition of pancreatic cancer cell growth in vitro and vivo. Simultaneous use of N5 and RbΔCDK has been found to exert a greater extent on the inhibition of pancreatic cancer cell growth in vitro and in vivo. ^