Faculty Bibliography

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

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.

The group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 (PhCPN) and its functional cooperation with the cognate prefoldin were investigated. PhCPN existed as a homo-oligomer in a double-ring structure, which protected the citrate synthase of a porcine heart from thermal aggregation at 45 degrees C, and did the same on the isopropylmalate dehydrogenase (IPMDH) of a thermophilic bacterium, Thermus thermophilus HB8, at 90 degrees C. PhCPN also enhanced the refolding of green fluorescent protein (GFP), which had been unfolded by low pH, in an ATP-dependent manner. Unexpectedly, functional cooperation between PhCPN and Pyrococcus prefoldin (PhPFD) in the refolding of GFP was not observed. Instead, cooperation between PhCPN and PhPFD was observed in the refolding of IPMDH unfolded with guanidine hydrochloride. Although PhCPN alone was not effective in the refolding of IPMDH, the refolding efficiency was enhanced by the cooperation of PhCPN with PhPFD.

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.

The mouse homeobox gene Gbx2 is first expressed throughout the posterior region of the embryo during gastrulation, and becomes restricted to rhombomeres 1-3 (r1-3) by embryonic day 8.5 (E8.5). Previous studies have shown that r1-3 do not develop in Gbx2 mutants and that there is an early caudal expansion of the midbrain gene Otx2 to the anterior border of r4. Furthermore, expression of Wnt1 and Fgf8, two crucial components of the isthmic organizer, is no longer segregated to adjacent domains in Gbx2 mutants. In this study, we extend the phenotypic analysis of Gbx2 mutants by showing that Gbx2 is not only required for development of r1-3, but also for normal gene expression in r4-6. To determine whether Gbx2 can alter hindbrain development, we generated Hoxb1-Gbx2 (HG) transgenic mice in which Gbx2 is ectopically expressed in r4. We show that Gbx2 is not sufficient to induce r1-3 development in r4. To test whether an Otx2/Gbx2 interface can induce r1-3 development, we introduced the HG transgene onto a Gbx2-null mutant background and recreated a new Otx2/Gbx2 border in the anterior hindbrain. Development of r3, but not r1 and r2, is rescued in Gbx2-/-; HG embryos. In addition, the normal spatial relationship of Wnt1 and Fgf8 is established at the new Otx2/Gbx2 border, demonstrating that an interaction between Otx2 and Gbx2 is sufficient to produce the normal pattern of Wnt1 and Fgf8 expression. However, the expression domains of Fgf8 and Spry1, a downstream target of Fgf8, are greatly reduced in mid/hindbrain junction area of Gbx2-/-; HG embryos and the posterior midbrain is truncated because of abnormal cell death. Interestingly, we show that increased cell death and a partial loss of the midbrain are associated with increased expression of Fgf8 and Spry1 in Gbx2 conditional mutants that lack Gbx2 in r1 after E9.0. These results together suggest that cell survival in the posterior midbrain is positively or negatively regulated by Fgf8, depending on Fgf8 expression level. Our studies provide new insights into the regulatory interactions that maintain isthmic organizer gene expression and the consequences of altered levels of organizer gene expression on cell survival

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

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

Legionella pneumophila invades and replicates intracellularly in human and protozoan hosts. The bacteria use the Icm/Dot type IVB secretion system to translocate effectors that inhibit phagosome maturation and modulate host vesicle trafficking pathways. To understand how L. pneumophila modulates organelle trafficking in host cells, we carried out pathogen effector protein screening in yeast, identifying L. pneumophila genes that produced membrane trafficking [vacuole protein sorting (VPS)] defects in yeast. We identified four L. pneumophila DNA fragments that perturb sorting of vacuolar proteins. Three encode ORFs of unknown function that are translocated via the Icm/Dot transporter from Legionella into macrophages. VPS inhibitor protein (Vip) A is a coiled-coil protein, VipD is a patatin domain-containing protein, and VipF contains an acetyltransferase domain. Processing studies in yeast indicate that VipA, VipD, and VipF inhibit lysosomal protein trafficking by different mechanisms; overexpressing VipA has an effect on carboxypeptidase Y trafficking, whereas VipD interferes with multivesicular body formation at the late endosome and endoplasmic reticulum-to-Golgi body transport. Such differences highlight the multiple strategies L. pneumophila effectors use to subvert host trafficking processes. Using yeast as an effector gene discovery tool allows for a powerful, genetic approach to both the identification of virulence factors and the study of their function.