In this study, we establish that the tyrosine kinase Syk is essential for osteoclast function in vitro and in vivo. Syk−/− osteoclasts fail to organize their cytoskeleton, and, as such, their bone-resorptive capacity is arrested. This defect results in increased skeletal mass in Syk−/− embryos and dampened basal and stimulated bone resorption in chimeric mice whose osteoclasts lack the kinase. The skeletal impact of Syk deficiency reflects diminished activity of the mature osteoclast and not impaired differentiation. Syk regulates bone resorption by its inclusion with the αvβ3 integrin and c-Src in a signaling complex, which is generated only when αvβ3 is activated. Upon integrin occupancy, c-Src phosphorylates Syk. αvβ3-induced phosphorylation of Syk and the latter's capacity to associate with c-Src is mediated by the immunoreceptor tyrosine-based activation motif (ITAM) proteins Dap12 and FcRγ. Thus, in conjunction with ITAM-bearing proteins, Syk, c-Src, and αvβ3 represent an essential signaling complex in the bone-resorbing osteoclast, and, therefore, each is a candidate therapeutic target.

Leukocytes and leukocyte-derived micro-particles contain low levels of tissue factor (TF) and incorporate into forming thrombi. Although this circulating pool of TF has been proposed to play a key role in thrombosis, its functional significance relative to that of vascular wall TF is poorly defined. We tested the hypothesis that leukocyte-derived TF contributes to thrombus formation in vivo. Compared to wild-type mice, mice with severe TF deficiency (ie, TF / , hTF-Tg , or "low-TF") demonstrated markedly impaired throm-bus formation after carotid artery injury or inferior vena cava ligation. A bone marrow transplantation strategy was used to modulate levels of leukocyte-derived TF. Transplantation of low-TF marrow into wild-type mice did not suppress arterial or venous thrombus formation. Similarly, transplantation of wild-type marrow into low-TF mice did not accelerate thrombosis. In vitro analyses revealed that TF activity in the blood was very low and was markedly exceeded by that present in the vessel wall. Therefore, our results suggest that thrombus formation in the arterial and venous macrovasculature is driven primarily by TF derived from the blood vessel wall as opposed to leuko-cytes. (Blood. 2005;105:192-198)

Summary We examined the mechanism by which M-CSF regulates the cytoskeleton and function of the osteoclast, the exclusive bone resorptive cell. We show that binding of M-CSF to its receptor c-Fms generates a signaling complex comprising phosphorylated DAP12, an adaptor containing an immunoreceptor tyrosine-based activation motif (ITAM) and the non-receptor tyrosine kinase Syk. c-Fms tyrosine 559, the exclusive binding site of c-Src, is necessary for regulation of DAP12/Syk signaling. Deletion of either of these molecules yields osteoclasts that fail to reorganize their cytoskeleton. Retroviral transduction of null precursors with wild type or mutant DAP12 or Syk reveals that the SH2 domain of Syk and the ITAM tyrosine residues and transmembrane domain of DAP12 mediate M-CSF signaling. Our data provide genetic and biochemical evidence that uncovers, an epistatic signaling pathway linking the receptor tyrosine kinase c-Fms to the immune adaptor DAP12 and the cytoskeleton.

SummaryDue to exciting advances in molecular biology, the laboratory mouse has become an important and frequently used model for studying thrombosis. This article reviews several experimental approaches that have been used to study arterial, venous, and microvascular thrombosis in mice. The advantages and limitations of different models are examined. Related topics of mouse anesthesia, phlebotomy, and in vitro hemostasis testing are also reviewed.

Background Capillary malformation is a cutaneous vascular anomaly that is present at birth, darkens over time, and can cause overgrowth of tissues beneath the stain. The lesion is caused by a somatic activating mutation in GNAQ. In a previous study we were unable to identify a GNAQ mutation in patients with a capillary malformation involving an overgrown lower extremity. We hypothesized that mutations in GNA11 or GNA14, genes closely related to GNAQ, also may cause capillary malformations. Methods Human capillary malformation tissue obtained from 8 patients that had tested negative for GNAQ mutations were studied. Lesions involved an extremity (n=7) or trunk (n=1). Droplet digital PCR (ddPCR) was used to detect GNA11 or GNA14 mutant cells (p.Arg183) in the specimens. Single molecule molecular inversion probe sequencing (smMIP-seq) was performed to search for other mutations in GNA11. Mutations were validated by sublconing and sequencing amplimers. Results We found a somatic GNA11 missense mutation (c.547C>T; p.Arg183Cys) in 3 patients with a diffuse capillary malformation of an extremity. Mutant allelic frequencies ranged from 0.3%–5.0%. GNA11 or GNA14 mutations were not found in 5 affected tissues or in unaffected tissues (white blood cell DNA). Conculsions GNA11 mutations are associated with extremity capillary malformations causing overgrowth. Pharmacotherapy that affects GNA11 signaling may prevent the progression of capillary malformations.

I n this study, we establish that the tyrosine kinase Syk is essential for osteoclast function in vitro and in vivo. Syk −/− osteoclasts fail to organize their cytoskeleton, and, as such, their bone-resorptive capacity is arrested. This defect results in increased skeletal mass in Syk −/− embryos and dampened basal and stimulated bone resorption in chimeric mice whose osteoclasts lack the kinase. The skeletal impact of Syk defi ciency refl ects diminished activity of the mature osteoclast and not impaired differentiation. Syk regulates bone resorption by its inclusion with the αvβ3 integrin and c-Src in a signaling complex, which is generated only when αvβ3 is activated. Upon integrin occupancy, c-Src phosphorylates Syk. αvβ3-induced phosphorylation of Syk and the latter's capa city to associate with c-Src is mediated by the immunoreceptor tyrosine-based activation motif (ITAM) proteins Dap12 and FcRγ. Thus, in conjunction with ITAM-bearing proteins, Syk, c-Src, and αvβ3 represent an essential signaling complex in the bone-resorbing osteoclast, and, therefore, each is a candidate therapeutic target.

Methylomicrobium buryatense 5GB1 is an obligate methylotroph which grows on methane or methanol with similar growth rates. It has long been assumed that the core metabolic pathways must be similar on the two substrates, but recent studies of methane metabolism in this bacterium suggest that growth on methanol might have significant differences from growth on methane. In this study, both a targeted metabolomics approach and a 13C tracer approach were taken to understand core carbon metabolism in M. buryatense 5GB1 during growth on methanol and to determine whether such differences occur. Our results suggest a systematic shift of active core metabolism in which increased flux occurred through both the Entner-Doudoroff (ED) pathway and the partial serine cycle, while the tricarboxylic acid (TCA) cycle was incomplete, in contrast to growth on methane. Using the experimental results as constraints, we applied flux balance analysis to determine the metabolic flux phenotype of M. buryatense 5GB1 growing on methanol, and the results are consistent with predictions based on ATP and NADH changes. Transcriptomics analysis suggested that the changes in fluxes and metabolite levels represented results of posttranscriptional regulation. The combination of flux balance analysis of the genome-scale model and the flux ratio from 13C data changed the solution space for a better prediction of cell behavior and demonstrated the significant differences in physiology between growth on methane and growth on methanol. IMPORTANCE One-carbon compounds such as methane and methanol are of increasing interest as sustainable substrates for biological production of fuels and industrial chemicals. The bacteria that carry out these conversions have been studied for many decades, but gaps exist in our knowledge of their metabolic pathways. One such gap is the difference between growth on methane and growth on methanol. Understanding such metabolism is important, since each has advantages and disadvantages as a feedstock for production of chemicals and fuels. The significance of our research is in the demonstration that the metabolic network is substantially altered in each case and in the delineation of these changes. The resulting new insights into the core metabolism of this bacterium now provide an improved basis for future strain design.

Abnormal cold sensitivity is a common feature of a range of neuropathies. In the murine somatosensory system, multiple aspects of cold sensitivity are dependent on TRPM8, both short term and in response to peripheral nerve injury. The specialized nature of cold-sensitive afferents and the restricted expression of TRPM8 render it an attractive target for the treatment of cold hypersensitivity. This current study examines the effect of a novel TRPM8 antagonist (M8-An) in naive and spinal nerveligated rats through behavioral and in vivo electrophysiological approaches. In vitro, M8-An inhibited icilin-evoked Ca 21 currents in HEK293 cells stably expressing human TRPM8 with an IC 50 of 10.9 nM. In vivo, systemic M8-An transiently decreased core body temperature. Deep dorsal horn recordings were made in vivo from neurons innervating the hind paw. M8-An inhibited neuronal responses to innocuous and noxious cooling of the receptive field in spinal nerve-ligated rats but not in naive rats. No effect on neuronal responses to mechanical and heat stimulation was observed. In addition, M8-An also attenuated behavioral responses to cold but not mechanical stimulation after nerve ligation without affecting the uninjured contralateral response. The data presented here support a contribution of TRPM8 to the pathophysiology of cold hypersensitivity in this model and highlight the potential of the pharmacological block of TRPM8 in alleviating the associated symptoms.