Faculty Bibliography

The voltage-gated sodium channel Na(v)1.8 produces a tetrodotoxin-resistant current and plays a key role in nociception. Annexin II/p11 binds to Na(v)1.8 and facilitates insertion of the channel within the cell membrane. However, the mechanisms responsible for removal of specific channels from the cell membrane have not been studied. We have identified a novel protein, clathrin-associated protein-1A (CAP-1A), which contains distinct domains that bind Na(v)1.8 and clathrin. CAP-1A is abundantly expressed in DRG neurons and colocalizes with Na(v)1.8 and can form a multiprotein complex with Na(v)1.8 and clathrin. Coexpression of CAP-1A and Na(v)1.8 in DRG neurons reduces Na(v)1.8 current density by approximately 50% without affecting the endogenous or recombinant tetrodotoxin-sensitive currents. This effect of CAP-1A is blocked by bafilomycin A1 treatment of transfected DRG neurons. CAP-1A thus is the first example of an adapter protein that links clathrin and a sodium channel and may regulate Na(v)1.8 channel density at the cell surface

Hedgehog signaling is required for formation and patterning of the anterior pituitary gland. However, the role of Hedgehog in pituitary precursor cell specification and subsequent placode formation is not well understood. We analyzed pituitary precursor cell lineages and find that pitx3 and distal-less3b (dlx3b) expression domains define lens and pituitary precursor positions. We show that pitx3 is required for pituitary pre-placode formation and cell specification, whereas dlx3b and dlx4b are required to restrict pituitary placode size. In smoothened mutant embryos that cannot transduce Hedgehog signals, median pituitary precursors are mis-specified and form an ectopic lens. Moreover, overexpression of sonic hedgehog (shh) blocks lens formation, and derivatives of lens precursors express genes characteristic of pituitary cells. However, overexpression of shh does not increase median pituitary placode size nor does it upregulate patched (ptc) expression in pituitary precursors during early somitogenesis. Our study suggests that by the end of gastrulation, pitx3-expressing cells constitute an equivalence domain of cells that can form either pituitary or lens, and that a non-Hedgehog signal initially specifies this placodal field. During mid-somitogenesis, Hedgehog then acts on the established median placode as a necessary and sufficient signal to specify pituitary cell types.

BACKGROUND: The impact of strict enforcement of Section 405 of the New York State Public Health Code to restrict resident work to eighty hours per week and the adoption of a similar policy by the Accreditation Council on Graduate Medical Education in 2002 for orthopaedic residency training have not been evaluated. Adoption of these rules has created accreditation as well as staffing problems and has generated controversy in the surgical training community. The purposes of this study were (1) to evaluate the attitudes of orthopaedic residents and attending surgeons toward the Code 405 work-hour regulations and the effect of those regulations on the perceived quality of residency training, quality of life, and patient care and (2) to quantify the effect of the work-hour restrictions on the actual number of hours worked. METHODS: We administered a thirty-four-question Likert-style questionnaire to forty-eight orthopaedic surgery residents (postgraduate years [PGY]-2 through 5) and a similar twenty-nine-question Likert-style questionnaire to thirty-nine orthopaedic attending surgeons. All questionnaires were collected anonymously and analyzed. Additionally, resident work hours before and after strict enforcement of the Code 405 regulations were obtained from resident time sheets. RESULTS: The average weekly work hours decreased from 89.25 to 74.25 hours for PGY-2 residents and from 86.5 to 73.25 hours for PGY-3 residents, and they increased from 61.5 to 68.5 hours for PGY-4 residents. Residents at all levels felt that they had increased time available for reading. There was general agreement between attending and resident surgeons that their operating experience had been negatively impacted. Senior residents thought that their education had been negatively affected, while junior residents thought that their operating experience in general had been negatively affected. Senior residents and attending surgeons felt that continuity of care had been negatively impacted. All agreed that quality of life for the residents had improved and that residents were more rested. CONCLUSIONS: On the basis of the survey data, the implementation of the new work-hour restrictions was found to result in a decrease in the number of hours worked per week for PGY-2 and PGY-3 residents and in an increase in work hours for PGY-4 residents. This could explain the definite difference between the attitudes expressed by the senior residents and those of the junior residents. Senior residents felt that their education was negatively impacted by the work rules, while junior residents expressed a more neutral view. However, senior residents did not believe that their operative experience was as negatively impacted as did junior residents. Although junior and senior residents and attending surgeons agreed that resident quality of life had improved, we were not able to determine whether this offset the perceived negative impact on education, continuity of care, and operative experience

Temperature-sensitive DNA polymerase mutants (dnaE) are protected from cell death on incubation at nonpermissive temperature by mutation in the cydA gene controlling cytochrome bd oxidase. Protection is observed in complex (Luria-Bertani [LB]) medium but not on minimal medium. The cydA mutation protects a thymine-deficient strain from death in the absence of thymine on LB but not on minimal medium. Both dnaE and Deltathy mutants filament under nonpermissive conditions. Filamentation per se is not the cause of cell death, because the dnaE cydA double mutant forms long filaments after 24 h of incubation in LB medium at nonpermissive temperature. These filaments have multiply dispersed nucleoids and produce colonies on return to permissive conditions. The protective effect of a deficiency of cydA at high temperature is itself suppressed by overexpression of cytochrome bo3, indicating that the phenomenon is related to energy metabolism rather than to a specific effect of the cydA protein. We propose that filamentation and cell death resulting from thymine deprivation or slowing of DNA synthesis are not sequential events but occur in response to the same or a similar signal which is modulated in complex medium by cytochrome bd oxidase. The events which follow inhibition of replication fork progression due to either polymerase inactivation, thymine deprivation, or hydroxyurea inhibition differ in detail from those following actual DNA damage.

An outbreak of aseptic meningitis occurred in the northern area of Jiangsu Province in China from January to July in 2003. A total of 1,681 cases were involved in this outbreak, and 99% of patients were <15 years of age. To identify the etiologic agent, 66 cerebrospinal fluid specimens were tested by cell culture. Eighteen showed an enteroviruslike cytopathic effect on MRC-5 human fetal diploid lung cells. An enterovirus primer-mediated reverse transcriptase-polymerase chain reaction, a standard neutralization assay, and sequencing of the complete capsid-encoding (VP1) gene identified the 18 isolates (FDJS03) as echovirus 30. At least a 10% difference was seen in nucleotide sequences of VP1 between FDJS03 isolates and other global strains of echovirus 30. Phylogenetic analysis based on complete sequences of VP1 was performed to further characterize the FDJS03 isolates. This report is the first to identify a distinct lineage of echovirus 30 as a probable cause of this outbreak.

AutoEM is a software package developed by Zhang et al. [J. Struct. Biol. 1356, 251] for semi-automated acquisition of cryo-electron micrographs from Tecnai series electron microscopes and is used frequently at the lowest level of automation. We report here on the new progress that we have made based on their preliminary work. A fourth low-dose state is created where the system can pre-select all the good holes in a grid square from a single CCD image taken at low magnification, making the system operative at much higher levels of automation. An additional control interface enables the operator to monitor the status of the program and the quality of the data, interact with the program, and direct the execution process according to intermediate results. When data acquisition is in progress, all useful information is automatically saved in certain text files which are easily accessible by a database. More detailed improvements and general advantages are illustrated and discussed. We have started to use the program to perform routine data collection. A number of applications show that the performance of the program is satisfactory and the quality of the micrographs and their power spectra acquired by the program is comparable to those manually collected under the same conditions

We report molecular genetic studies of three genes involved in early germ-line proliferation in Caenorhabditis elegans that lend unexpected insight into a germ-line/soma functional separation of autosomal/X-linked duplicated gene pairs. In a genetic screen for germ-line proliferation-defective mutants, we identified mutations in rpl-11.1 (L11 protein of the large ribosomal subunit), pab-1 [a poly(A)-binding protein], and glp-3/eft-3 (an elongation factor 1-alpha homolog). All three are members of autosome/X gene pairs. Consistent with a germ-line-restricted function of rpl-11.1 and pab-1, mutations in these genes extend life span and cause gigantism. We further examined the RNAi phenotypes of the three sets of rpl genes (rpl-11, rpl-24, and rpl-25) and found that for the two rpl genes with autosomal/X-linked pairs (rpl-11 and rpl-25), zygotic germ-line function is carried by the autosomal copy. Available RNAi results for highly conserved autosomal/X-linked gene pairs suggest that other duplicated genes may follow a similar trend. The three rpl and the pab-1/2 duplications predate the divergence between C. elegans and C. briggsae, while the eft-3/4 duplication appears to have occurred in the lineage to C. elegans after it diverged from C. briggsae. The duplicated C. briggsae orthologs of the three C. elegans autosomal/X-linked gene pairs also display functional differences between paralogs. We present hypotheses for evolutionary mechanisms that may underlie germ-line/soma subfunctionalization of duplicated genes, taking into account the role of X chromosome silencing in the germ line and analogous mammalian phenomena

To insure an adequate supply of nutrients, omnivores choose among available food sources. This process is exemplified by the well-characterized innate aversion of omnivores to otherwise nutritious foods of imbalanced amino acid content. We report that brain-specific inactivation of GCN2, a ubiquitously expressed protein kinase that phosphorylates translation initiation factor 2 alpha (eIF2alpha) in response to intracellular amino acid deficiency, impairs this aversive response. GCN2 inactivation also diminishes phosphorylated eIF2alpha levels in the mouse anterior piriform cortex following consumption of an imbalanced meal. An ancient intracellular signal transduction pathway responsive to amino acid deficiency thus affects feeding behavior by activating a neuronal circuit that biases consumption against imbalanced food sources.

OBJECTIVE: Telomeres are DNA repeats which cap and protect chromosome ends, facilitate homologue pairing and chiasmata formation during early meiosis, and shorten with cell division and exposure to reactive oxygen to mediate aging. Early germ cells contain telomerase, a reverse transcriptase which adds telomeres to 3-prime DNA ends, but telomerase activity declines in oocytes, fixing telomere length earlier during development. Experimentally induced telomere shortening in mice disrupts meiosis, impairs chiasmata formation, halts embryonic cell cycles, and promotes apoptosis in embryos, a phenotype which mimics reproductive senescence in women. Ethical constraints limit study of human embryos to nondestructive assays, such as morphologic evaluation under transmission optics, but cytoplasmic fragmentation is a reliable marker of apoptosis. STUDY DESIGN: Study design consisted of observational study of effect of telomere length in human eggs on cytoplasmic fragmentation, and on other morphologic features of preimplantation embryos. To test the hypothesis that telomere shortening triggers apoptosis in human embryos, we evaluated telomere length as a predictor of cytoplasmic fragmentation in embryos from women undergoing in vitro fertilization. RESULTS: Telomere length negatively predicted fragmentation in day 3 preimplantation embryos, after controlling for patient age and basal follicle stimulating hormone level. Telomere length did not predict other features of preimplantation embryo morphology. CONCLUSION: The finding that telomere length in human eggs predicts cytoplasmic fragmentation in embryos provides evidence that telomere shortening induces apoptosis in human preimplantation embryos, consistent with a telomere theory of reproductive senescence in women

OBJECTIVE: To develop a method to evaluate spindle dynamics in living oocytes and in karyoplasts during the initial stages of activation and after pharmacological disruption of cytoskeleton. DESIGN: Morphological study using a novel microscope. SETTING: Translational research laboratory at marine biological laboratory. ANIMAL(S): Six-week-old CD-1 or B6C3F1 mice superovulated with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG). INTERVENTION(S): Spindles of living oocytes and karyoplasts were imaged at 5-10 minute intervals using the Pol-Scope during the initial stages of oocyte activation and after pharmacological disruption of cytoskeleton. MAIN OUTCOME MEASURE(S): Assessment of spindle dynamics using Pol-Scope imaging. RESULT(S): During oocyte activation, spindle mid-region birefringence increased, followed by spindle rotation and second polar body extrusion in both intact oocytes and karyoplasts. Activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate failed to induce spindle activation in 60% of living oocytes and caused spindle disruption in some oocytes. Inhibition of PKC by a myristoylated PKC pseudosubstrate inhibited metaphase II release in most oocytes evaluated (86.7%). Cytochalasin D inhibited only spindle rotation and separation. Nocodazole disrupted spindles in less than 5 minutes after administration. CONCLUSION(S): Pol-Scope imaging allows investigation at near real time of spindle dynamics during activation of living oocytes. Spindles also showed evidence of activation even in karyoplasts. The procedure may be useful for detecting functional spindle aberrations in living oocytes. Further studies are needed to determine whether spindle dynamics predict clinical outcome