Proteomic and functional analysis of the mitotic Drosophila centrosome

Regulation of centrosome structure, duplication and segregation is integrated into cellular pathways that control cell cycle progression and growth. As part of these pathways, numerous proteins with well‐established non‐centrosomal localization and function associate with the centrosome to fulfill regulatory functions. In turn, classical centrosomal components take up functional and structural roles as part of other cellular organelles and compartments. Thus, although a comprehensive inventory of centrosome components is missing, emerging evidence indicates that its molecular composition reflects the complexity of its functions. We analysed the Drosophila embryonic centrosomal proteome using immunoisolation in combination with mass spectrometry. The 251 identified components were functionally characterized by RNA interference. Among those, a core group of 11 proteins was critical for centrosome structure maintenance. Depletion of any of these proteins in Drosophila SL2 cells resulted in centrosome disintegration, revealing a molecular dependency of centrosome structure on components of the protein translation machinery, actin‐ and RNA‐binding proteins. In total, we assigned novel centrosome‐related functions to 24 proteins and confirmed 13 of these in human cells.     

The biology of Bracon celer as a parasitoid of the olive fruit fly

A series of laboratory experiments was conducted on a colony of Bracon celer Szépligeti (Hymenoptera: Braconidae) reared on the olive fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae). Female B. celer preferentially probe and oviposit into olives containing late third-instar fly larvae. The parasitoid develops as a solitary, ectoparasitic idiobiont. Mean development time (oviposition to adult eclosion) at 22 °C was, for females, 36±1 (SE) days, and for males, 34±1 days. The mean longevity of adult female wasps when provided honey and water was significantly greater than when they were provided water alone, or nothing. The females produced an average of 9.7±7.2 progeny during their lifetimes, but production levels in the insectary colony suggested that this level of fecundity was artificially low and could be improved. The discrepancy may be a consequence of constraints on oviposition behavior imposed by the experimental design. The results are discussed with respect to insectary production methods and the potential use of B. celer as a biological control agent for olive fly in California.     

Fine needle aspiration biopsy and immunostaining findings in an aggressive inflammatory myofibroblastic tumor of the lung: a case report

BACKGROUND: Inflammatory myofibroblastic tumors (IMTs) can vary from benign pseudosarcomatous tumors to low grade sarcomas. To date, fine needle aspiration (FNA) findings of lung IMTs, especially in the aggressive form, have not been fully described. Here we present FNA biopsy findings in conjunction with immunohistochemical studies in a case of primary and recurrent pulmonary IMT. CASE: A 22-year-old man first presented with a left lung mass and 4.5 years later with a recurrent mass. Preoperative computed tomography-guided FNA was performed on both tumors. FNA cytologic smears of both specimens consisted of scant, distorted spindle cells suggestive of a spindle cell lesion but were insufficient for further classification. Needle core biopsies as well as touch imprints were performed during the FNA procedures. The imprints revealed abundant, well-preserved spindle cells with mild to moderate atypia and intermixed lymphocytes and plasma cells. The spindle cells in both specimens were immunoreactive for vimentin and smooth muscle actin and were negative for pancytokeratin, desmin, CD34 and c-kit. Thirty percent of the tumor cells were positive for p53. The findings were compatible with those of IMT. Histologic examination of the surgically resected initial and recurrent masses confirmed the diagnosis of lMT. CONCLUSION: The cytologic findings of pulmonary IMT in FNA specimens are suggestive of, although not specific for, IMT. Immunohistochemical studies can assist in the diagnosis by excluding other spindle cell lesions. Cytologic atypia and p53 immunoreactivity may be indicators of aggressive IMTs.     

Functional outcome of pannexin-deficient mice after cerebral ischemia

Pannexin (Px, Panx) channels have been implicated in several physiological and pathological processes. We recently studied the potential contribution of pannexins in ischemic brain damage using Px1^-/- Px2^-/- mice and provided evidence that (1) the release of IL-1β and hemichannel function in astrocytes are, in contrast to published data, not affected by the absence of Px1 and Px2, (2) channel function in neurons lacking Px1 and Px2 is impaired and (3) Px1^-/- Px2^-/- mice had a better functional outcome and smaller infarcts than wild-type mice when subjected to ischemic stroke. Here, we further investigate the neurological outcome of wild-type and pannexin double-knockout mice 48 h after permanent occlusion of the distal middle cerebral artery (MCAO). Pannexin double-knockout mice (Px1^-/- Px2^-/-) were less impaired in parameters such as exploration, anxiety, sensorimotor function and behavioral symmetry.     

Electron microscopy visualization of DNA-protein complexes formed by Ku and DNA ligase IV

The repair of DNA double-stranded breaks (DSBs) is essential for cell viability and genome stability. Aberrant repair of DSBs has been linked with cancer predisposition and aging. During the repair of DSBs by non-homologous end joining (NHEJ), DNA ends are brought together, processed and then joined. In eukaryotes, this repair pathway is initiated by the binding of the ring-shaped Ku heterodimer and completed by DNA ligase IV. The DNA ligase IV complex, DNA ligase IV/XRRC4 in humans and Dnl4/Lif1 in yeast, is recruited to DNA ends in vitro and in vivo by an interaction with Ku and, in yeast, Dnl4/Lif1 stabilizes the binding of yKu to in vivo DSBs. Here we have analyzed the interactions of these functionally conserved eukaryotic NHEJ factors with DNA by electron microscopy. As expected, the ring-shaped Ku complex bound stably and specifically to DNA ends at physiological salt concentrations. At a ratio of 1 Ku molecule per DNA end, the majority of DNA ends were occupied by a single Ku complex with no significant formation of linear DNA multimers or circular loops. Both Dnl4/Lif1 and DNA ligase IV/XRCC4 formed complexes with Ku-bound DNA ends, resulting in intra- and intermolecular DNA end bridging, even with non-ligatable DNA ends. Together, these studies, which provide the first visualization of the conserved complex formed by Ku and DNA ligase IV at juxtaposed DNA ends by electron microscopy, suggest that the DNA ligase IV complex mediates end-bridging by engaging two Ku-bound DNA ends.     

Four independent mutations in the feline fibroblast growth factor 5 gene determine the long-haired phenotype in domestic cats

To determine the genetic regulation of "hair length" in the domestic cat, a whole-genome scan was performed in a multigenerational pedigree in which the "long-haired" phenotype was segregating. The 2 markers that demonstrated the greatest linkage to the long-haired trait (log of the odds > or = 6) flanked an estimated 10-Mb region on cat chromosome B1 containing the Fibroblast Growth Factor 5 (FGF5) gene, a candidate gene implicated in regulating hair follicle growth cycle in other species. Sequence analyses of FGF5 in 26 cat breeds and 2 pedigrees of nonbreed cats revealed 4 separate mutations predicted to disrupt the biological activity of the FGF5 protein. Pedigree analyses demonstrated that different combinations of paired mutant FGF5 alleles segregated with the long-haired phenotype in an autosomal recessive manner. Association analyses of more than 380 genotyped breed and nonbreed cats were consistent with mutations in the FGF5 gene causing the long-haired phenotype in an autosomal recessive manner. In combination, these genomic approaches demonstrated that FGF5 is the major genetic determinant of hair length in the domestic cat.     

Aging-associated reductions in AMP-activated protein kinase activity and mitochondrial biogenesis

Recent studies have demonstrated a strong relationship between aging-associated reductions in mitochondrial function, dysregulated intracellular lipid metabolism, and insulin resistance. Given the important role of the AMP-activated protein kinase (AMPK) in the regulation of fat oxidation and mitochondrial biogenesis, we examined AMPK activity in young and old rats and found that acute stimulation of AMPK-alpha(2) activity by 5'-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and exercise was blunted in skeletal muscle of old rats. Furthermore, mitochondrial biogenesis in response to chronic activation of AMPK with beta-guanidinopropionic acid (beta-GPA) feeding was also diminished in old rats. These results suggest that aging-associated reductions in AMPK activity may be an important contributing factor in the reduced mitochondrial function and dysregulated intracellular lipid metabolism associated with aging.