Faculty Bibliography

Current treatments for damaged articular cartilage (i.e., shaving the articular surface, perforation or abrasion of the subchondral bone, and resurfacing with periosteal and perichondrial resurfacing) often produce fibrocartilage, or hyaline-appearing repair that is not sustained over time (Henche 1967, Ligament and Articular Cartilage Injuries. Springer-Verlag, New York, NY, pp. 157-164; Insall 1974, Clin. Orthop. 101: 61-67; Mitchell and Shepard 1976, J. Bone Joint Surg. [Am.] 58: 230-233; O'Driscoll et al. 1986, J. Bone Joint Surg. [Am.] 68: 1017-1035; 1989, Trans. Orthop. Res. Soc. 14: 145; Kim et al. 1991, J. Bone Joint Surg. [Am.] 73: 1301-1315). Autologous chondrocyte transplantation, although promising, requires two surgeries, has site-dependent and patient age limitations, and has unknown long-term donor site morbidity (Brittberg et al. 1994, N Engl. J. Med. 331: 889-895; Minas 2003, Orthopedics 26: 945-947; Peterson et al. 2003, J. Bone Joint Surg. Am. 85-A(Suppl. 2): S17-S24). Osteochondral allografts remain a widely used method of articular resurfacing to delay arthritic progression. The present study compared the histological response to four types of osteochondral implants in a rabbit model: autograft, frozen, freeze-dried, and fresh implants. Specimens implanted in the femoral groove were harvested at 6 and 12 weeks. Results showed similar restoration of the joint surface regardless of implant type, with a trend toward better repair at the later timepoint. As has been observed in other studies (Frenkel et al. 1997, J. Bone Joint Surg. 79B: 281-286; Toolan et al. 1998, J. Biomed. Mater. Res. 41: 244-250), each group in this study had at least one specimen in which a healthy-appearing surface on the implant was not well-integrated with host tissues. Although the differences were not statistically significant, freeze-dried implants at both timepoints had the best histological scores. The osteochondral grafts tested successfully restored the gross joint surface and congruity. At 12 weeks, no significant differences were observed between the various allografts and autologous osteochondral grafts

In recent years, investigators have made great progress in delineating developmental pathways of several lymphoid and myeloid lineages and in identifying transcription factors that establish and maintain their fate. However, the developmental branching points between these two large cell compartments are still controversial, and little is known about how their diversification is induced. Here, we give an overview of determinants that play a role at lymphoid-myeloid junctures, in particular transcription factors and cytokine receptors. Experiments showing that myeloid lineages can be reversibly reprogrammed into one another by transcription factor network perturbations are used to highlight key principles of lineage commitment. We also discuss experiments showing that lymphoid-to-myeloid but not myeloid-to-lymphoid conversions can be induced by the enforced expression of a single transcription factor. We close by proposing that this asymmetry is related to a higher complexity of transcription factor networks in lymphoid cells compared with myeloid cells, and we suggest that this feature must be considered when searching for mechanisms by which hematopoietic stem cells become committed to lymphoid lineages

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has revolutionized the study of protein localization and dynamics. GFP fusions permit analysis of proteins in living cells and offer distinct advantages over conventional immunofluorescence. Among these are lower background, higher resolution, robust dual color colocalization, and avoidance of fixation artifacts. In the case of Ras and Rho family proteins, GFP fusions have allowed breakthroughs in the understanding of how CAAX proteins are targeted to specific cell membranes and how signaling at different membranes can result in different cellular responses. GFP-tagged Rho proteins have also been informative in analyzing the interactions with the cytosolic chaperone, RhoGDI. The major disadvantages of studying GFP fusion proteins is that they are generally overexpressed relative to endogenous proteins, and the GFP tag can, in principle, affect protein function. Fortunately, in the case of Ras and Rho family proteins, a GFP tag at the N terminus seems to have little effect on protein targeting and function. Nevertheless, it is prudent to confirm GFP fusion protein data with the study of the endogenous protein. This chapter describes the tagging of Rho proteins with GFP and the analysis of GFP-Rho protein localization by epifluorescence and confocal microscopy. It further describes methods of analyzing endogenous Rho proteins as confirmation of data acquired using GFP-Rho fusion proteins. These techniques will be useful for anyone studying Rho protein function and are widely applicable to many cell types and signal transduction systems

Helicases and translocases are proteins that use the energy derived from ATP hydrolysis to move along or pump nucleic acid substrates. Single molecule manipulation has proved to be a powerful tool to investigate the mechanochemistry of these motors. Here we first describe the basic mechanical properties of DNA unraveled by single molecule manipulation techniques. Then we demonstrate how the knowledge of these properties has been used to design single molecule assays to address the enzymatic mechanisms of different translocases. We report on four single molecule manipulation systems addressing the mechanism of different helicases using specifically designed DNA substrates: UvrD enzyme activity detection on a stretched nicked DNA molecule, HCV NS3 helicase unwinding of a RNA hairpin under tension, the observation of RecBCD helicase/nuclease forward and backward motion, and T7 gp4 helicase mediated opening of a synthetic DNA replication fork. We then discuss experiments on two dsDNA translocases: the RuvAB motor studied on its natural substrate, the Holliday junction, and the chromosome-segregation motor FtsK, showing its unusual coupling to DNA supercoiling.

We report a long-term survival case of Arima syndrome requiring hemodialysis. The patient, now 25 years of age, was hypotonic at birth. She was diagnosed with Dandy-Walker syndrome at an early month of age when she underwent posterior cranial fossa cystectomy and vermian agenesis was confirmed. With some delay in psychomotor development, she showed the development of language comprehension and meaningful speech and started to walk without aid at the age of 7 years. Polycystic kidneys were found at 11 years, and Arima syndrome was diagnosed at 16 years when she presented herself to our hospital with rupture of esophageal varices. With progressive deterioration of renal function, she was placed on chronic hemodialysis at 23 years. She presented short stature, right blepharoptosis and telecanthus on physical examination; pancytopenia, liver dysfunction and renal failure on laboratory studies agenesis of cerebellar vermis on magnetic resonance imaging reduced amplitude of electroretinographic response, and retinal pigmentary changes under funduscopy. Hemodialysis was initiated uneventfully except that nafamostat mesilate was used as anticoagulant because of her bleeding tendency. Arima syndrome, also known as cerebro-oculo-hepato-renal syndrome, is a disorder characterized by cerebellar vermis aplasia and other clinical features such as profound psychomotor retardation, severe visual impairment, characteristic facial appearance with blepharoptosis, hepatic fibrosis and progressive renal insufficiency. The clinical findings of our patient were consistent with Arima syndrome though her psychomotor retardation and visual impairment were relatively moderate as compared with those previously reported. As most patients with Arima syndrome may die of uremia in their early teens, dialysis therapy should be considered to improve the patient's survival and quality of life depending on the severity of psychomotor retardation and other systemic disorders.

Two aspects of the mechanisms by which iron is absorbed by the intestine were studied in the Caco2 cell model, using 59Fe(II)-ascorbate. Data showing the importance of vesicular processes and cycling of apotransferrin (apoTf) to uptake and overall transport of Caco2 cell monolayers (or basolateral 59Fe release) were obtained by comparing effects of: a) adding apoTf to the basal chamber; b) adding vesicular transport inhibitors; or c) cooling to 4 degrees C. These showed that apoTf may be involved in as much as half of Fe transfer across the basolateral membrane, and that vesicular processes may also play a role in non-apoTf-dependent Fe transport. Studies were initiated to examine potential interactions of other metal ions with Fe(II) via DMT1. Kinetic data showed a single, saturable process for uptake of Fe(II) that was pH dependent and had a Km of 7 microM. An excess of Mn(II) and Cu(I) over Fe(II) of 200: 1 (microM: microM) in 1 mM ascorbate markedly inhibited Fe uptake. The kinetics were not competitive. Km increased and Vmax decreased. We conclude that vesicular transport, involving endo- and exocytosis at both ends of the enterocyte, is a fundamental aspect of intestinal iron absorption and that DMT1 may function as a transporter not just for divalent but also for monovalent metal ions.

A microneedle array has been fabricated and applied to the measurement of transdermal skin potentials in human subjects. Potential changes were recorded in the vicinity of superficial wounds, confirming the generation of a lateral electric field in human skin. The measured electric field decays with distance from the wound edge, and is directed towards the wound. The measurement of endogenous fields in skin is a prelude to the study of the therapeutic efficacy of applied electric fields to chronic non-healing wounds

Following absorption, lead can concentrate in bodily compartments where it disrupts cellular processes and can result in detrimental health consequences. The concentration and impact of lead within follicular fluid has not been characterized and we used inductively coupled plasma mass spectroscopy (ICP-MS) to determine lead levels in blood and follicular fluid from nine patients undergoing in vitro fertilization (IVF) treatment. Lead levels within follicular fluid were found to be significantly higher in non-pregnant patients compared to pregnant patients suggesting that elevated concentrations of the environmental toxicant lead adversely affect female reproduction

Neuropsychologists have clearly implicated the hippocampus in the consolidation of memory, particularly episodic memory, the mental replay of past experiences. When recorded from behaving animals, by far the most obvious firing pattern of the primary neurons of the hippocampus is the place field: a cell tends to fire only when the animal's head is in a particular part of its environment. It seems reasonable to suspect that the primary firing correlate of the primary neurons of a structure should underlie the primary function of that structure as revealed by behavioral lesion experiments. However, we are currently still at a loss to explain how the firing of hippocampal neurons contributes to hippocampal function. This review seeks to examine the commonalities between place cells and episodic memory, and posits that an analogy can be made between the stabilization of place fields and the consolidation of memory.