Faculty Bibliography

Passenger proteins migrate from inner centromeres to the spindle midzone during late mitosis, and those described to date are essential both for proper chromosome segregation and for completion of cell cleavage. We have purified and cloned the human passenger protein TD-60, and we here report that it is a member of the RCC1 family and that it binds preferentially the nucleotide-free form of the small G protein Rac1. Using siRNA, we further demonstrate that the absence of TD-60 substantially suppresses overall spindle assembly, blocks cells in prometaphase, and activates the spindle assembly checkpoint. These defects suggest TD-60 may have a role in global spindle assembly or may be specifically required to integrate kinetochores into the mitotic spindle. The latter is consistent with a TD-60 requirement for recruitment of the passenger proteins survivin and Aurora B, and suggests that like other passenger proteins, TD-60 is involved in regulation of cell cleavage.

The analysis of structural brain asymmetry has been a focal point in anthropological theories of human brain evolution and the development of lateralized behaviors. While physiological brain asymmetries have been documented for humans and animals presenting with pathological conditions or under certain activation tasks, published studies on baseline asymmetries in healthy individuals have produced conflicting results. We tested for the presence of cerebral blood flow asymmetries in 7 healthy, sedated baboons using positron emission tomography, a method of in vivo autoradiography. Five of the 7 baboons exhibited hemispheric asymmetries in which left-sided flow was significantly greater than right-sided flow. Furthermore, the degree of asymmetry in 8 of 24 brain regions was found to be significantly correlated with age; older individuals exhibited a higher degree of asymmetry than younger individuals. Cerebral blood flow itself was uncorrelated with age, and differences between males and females were not significant.

The subventricular zone (SVZ) is the most active site for the production of new neurons in the adult mouse brain. Neural stem cells in the adult SVZ give rise to neuroblasts that travel via the rostral migratory stream (RMS) to the olfactory bulb, where they differentiate into interneurons. The enzyme telomerase has been identified in other population of stem cells and is necessary for the synthesis of telomeric DNA to prevent chromosomal shortening, end-to-end fusions, and apoptosis during successive rounds of cell division. However, previous studies have failed to detect telomerase in the adult mammalian brain. Here we demonstrate that telomerase is expressed by all brain regions shortly after birth, but becomes restricted to the SVZ and olfactory bulb in the adult mouse brain. Cultures of neural precursor cells or of migratory neuroblasts purified from the SVZ were each found to possess telomerase activity. After elimination of migrating neuroblasts and immature precursor cells in vivo by treatment with cytosine-beta-D-arabinofuranoside (Ara-C), telomerase activity was still detectable in the remaining SVZ, which includes a population of neural stem cells. Following withdrawal of Ara-C, telomerase activity subsequently increased with a time course that parallels regeneration of the SVZ network and RMS. Finally, intracranial surgery alone, which has previously been shown to increase the number of cells in the SVZ, produced higher telomerase levels in the SVZ. We conclude that telomerase is active in neural precursor cells of the adult mouse and suggest that its regulation is an important parameter for cellular proliferation to occur in the mammalian brain

Sexually dimorphic development of the gonad is essential for germ cell development and sexual reproduction. We have found that the Drosophila embryonic gonad is already sexually dimorphic at the time of initial gonad formation. Male-specific somatic gonadal precursors (msSGPs) contribute only to the testis and express a Drosophila homolog of Sox9 (Sox100B), a gene essential for testis formation in humans. The msSGPs are specified in both males and females, but are only recruited into the developing testis. In females, these cells are eliminated via programmed cell death dependent on the sex determination regulatory gene doublesex. Our work furthers the hypotheses that a conserved pathway controls gonad sexual dimorphism in diverse species and that sex-specific cell recruitment and programmed cell death are common mechanisms for creating sexual dimorphism.

The latent TGF-beta binding protein (LTBP)-3 is an extracellular matrix (ECM) protein that binds the small latent complex (SLC) of TGF-beta. Disruption of the Ltbp-3 gene by homologous recombination in mice yields mutant animals that display multiple skeletal abnormalities. In addition, these mice have retarded growth. On an inbred 129 SvEv background, half of the Ltbp-3 mutant mice die between 3 and 4 weeks after birth. These mice show severe involution of the thymus and spleen and a sharp reduction in the numbers of CD4/CD8 double positive T-cells in the thymus. The thymus and spleen defect is caused by elevated corticosterone levels in the serum and can be reversed by injection of aminoglutethimide (AMG), an inhibitor of steroid synthesis. This result indicates that the thymus and spleen defect is a secondary defect due to high corticosterone levels probably induced by stress of unknown etiology

Duplex ultrasonography of the deep venous system of the lower extremities is often utilized in the diagnostic evaluation of total hip and knee arthroplasty patients suspected of pulmonary embolism in an attempt to identify the embolic source. A retrospective review of 135 patients who were clinically suspected of pulmonary embolism after 71 total knee arthroplasties and 64 total hip arthroplasties was performed. Of the 35 patients diagnosed with pulmonary embolism, 2 (5.7%) had deep venous thrombosis identified by duplex ultrasonography. The routine use of this imaging modality is not an effective strategy for identifying clinically significant deep venous thrombosis that leads to pulmonary embolism. A negative duplex ultrasound result should not preclude an extensive evaluation for pulmonary thrombosis in symptomatic patients

Enzymes involved in catecholamine synthesis are present in the highest concentration in the adrenal medulla, however they were found also in other, mainly nervous tissues. The aim of our study was to quantify the exact concentration of tyrosine hydroxylase (TH) and dopamine-ss-hydroxylase (DBH) mRNA in rat stellate ganglia under control conditions and at different intervals after exposure to immobilization stress (IMO). In rats immobilized once for 2h, we determined TH and DBH mRNA in different time intervals up to 22 h after the end of the stress stimulus. TH immunoreactive protein levels were also determined in stellate ganglia. TH and DBH mRNA levels were quantified by RT-competitive-PCR. In stellate ganglia, the concentration of TH mRNA was 17+/-1.6 amol/microg of total RNA, which is approximately 30-times lower than in the adrenal medulla. The concentration of DBH mRNA in the stellate ganglia was 2601+/-203 amol/microg of total RNA, which is the concentration similar to adrenal medulla, but is 150-times higher than concentration of TH mRNA in stellate ganglia. After a single 2-h immobilization the highest elevation of TH and DBH mRNA levels was measured 22 h after the termination of the stress stimulus. Repeated immobilization (7 days, 2h daily) did not produce further increase in TH and DBH mRNA levels compared to already elevated levels in adapted control group (immobilized for 6 days, 2h daily and decapitated 22 h later). Levels of TH protein were significantly changed only after the repeated immobilization. This study compared for the first time the precise amounts of TH and DBH mRNA in rat stellate ganglia under control conditions and after immobilization stress, and indicates large differences in their concentration. TH and DBH mRNA concentrations in stellate ganglia are markedly elevated for a prolonged period of time after termination of the stress stimuli.

Intraoperative evaluation of skin flap viability has primarily been dependent on clinical judgment. The purpose of this study was to determine whether an orthogonal polarization spectral imaging device could be used to accurately predict viability of random-pattern skin flaps. Orthogonal polarization spectral imaging is a newly developed technique that visualizes the microcirculation using reflected light without the use of fluorescent dyes and allows for noninvasive real-time observation of functional microvascular networks. In Sprague-Dawley rats (n = 24), three types of random skin flaps were designed with unknown zones of viability (n = 8 per group). After flap elevation, the skin flaps were evaluated by both clinical examination and orthogonal polarization spectral imaging. Areas of the flap determined to be nonviable by clinical examination were measured and marked. Orthogonal polarization spectral imaging was subsequently performed, and areas of the skin flap with stasis (i.e., cessation of red blood cell movement) in the dermal microcirculation on orthogonal polarization spectral imaging were measured and marked. The skin flaps were then secured in place. Flaps were evaluated on a daily basis for clinical signs of ischemia and necrosis. On postoperative day 7, the total amount of random skin flap necrosis was measured and recorded. Clinical examination of the random skin flaps significantly underestimated the actual amount of eventual flap necrosis, and as result was a very poor predictor of flap necrosis. By contrast, assessment of microcirculatory stasis using the orthogonal polarization spectral imaging device correlated well with the subsequent development of necrosis in all groups. In the three groups, the average amount of flap necrosis predicted by clinical examination deviated from actual necrosis by approximately 2 to 4 cm. However, the amount that orthogonal polarization spectral imaging differed from actual necrosis was 0.1 to 0.3 cm. Therefore, orthogonal polarization spectral imaging was an excellent predictor of eventual flap necrosis and much more accurate than clinical observation (p < 0.001). Intraoperative evaluation of axial and random pattern flap viability has traditionally been based on clinical examination as no other reliable, convenient test currently exists. The authors demonstrated that an orthogonal polarization spectral imaging device accurately predicts zones of necrosis in random pattern flaps by directly visualizing cessation of microcirculatory flow. Intraoperative stasis in the dermal microcirculation correlated precisely with subsequent flap necrosis. Orthogonal polarization spectral imaging was significantly more accurate than clinical examination, which consistently underestimated flap necrosis. The orthogonal polarization spectral imaging technique may have value in the intraoperative assessment of skin flap perfusion such as that required after skin-sparing mastectomy

To test the hypothesis that the phosphatidylinositol 3-kinase (PI3 kinase)/protein kinase Akt signaling pathway is involved in nitric oxide (NO)-induced endothelial cell migration and angiogenesis, we treated human and bovine endothelial cells with NO donors, S-nitroso-L-glutathione (GSNO) and S-nitroso-N-penicillamine (SNAP). Both GSNO and SNAP increased Akt phosphorylation and activity, which were blocked by cotreatment with the PI3 kinase inhibitor wortmannin. The mechanism was due to the activation of soluble guanylyl cyclase because 8-bromo-cyclic GMP activated PI3 kinase and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ) blocked NO-induced PI3 kinase activity. Indeed, transfection with adenovirus containing endothelial cell NO synthase (eNOS) or protein kinase G (PKG) increased endothelial cell migration, which was inhibited by cotransfection with a dominant-negative mutant of PI3 kinase (dnPI3 kinase). In a rat model of hind limb ischemia, adenovirus-mediated delivery of human eNOS cDNA in adductor muscles resulted in time-dependent expression of recombinant eNOS, which was accompanied by significant increases in regional blood perfusion and capillary density. Coinjection of adenovirus carrying dnPI3 kinase abolished neovascularization in ischemic hind limb induced by eNOS gene transfer. These findings indicate that NO promotes endothelial cell migration and neovascularization via cGMP-dependent activation of PI3 kinase and suggest that this pathway is important in mediating NO-induced angiogenesis.

The nicotinic acetylcholine receptor (nAChR) carries two binding sites for snake venom neurotoxins. alpha-Bungarotoxin from the Southeast Asian banded krait, Bungarus multicinctus, is a long neurotoxin which competitively blocks the nAChR at the acetylcholine binding sites in a relatively irreversible manner. Low angle x-ray diffraction was used to generate electron density profile structures at 14-A resolution for Torpedo californica nAChR membranes in the absence and presence of alpha-bungarotoxin. Analysis of the lamellar diffraction data indicated a 452-A lattice spacing between stacked nAChR membrane pairs. In the presence of alpha-bungarotoxin, the quality of the diffraction data and the lamellar lattice spacing were unchanged. In the plane of the membrane, the nAChRs packed together with a nearest neighbor distance of 80 A, and this distance increased to 85 A in the presence of toxin. Electron density profile structures were calculated in the absence and presence of alpha-bungarotoxin, revealing a location for the toxin binding sites. In native, fully-hydrated nAChR membranes, alpha-bungarotoxin binds to the nAChR outer vestibule and contacts the surface of the membrane bilayer.