The rockefeller university

Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.

Protein phosphorylation is one of the most common and extensively studied posttranslational modifications (PTMs). Compared to the O-phosphorylation of Ser, Thr, and Tyr residues, our understanding of histidine phosphorylation is relatively limited, particularly in higher eukaryotes, due to technical difficulties stemming from the intrinsic instability and isomerism of phosphohistidine (pHis). We report the design and synthesis of stable and nonisomerizable pHis analogues. These pHis analogues were successfully utilized in solid-phase peptide synthesis and semi-synthesis of histone H4. Significantly, the first antibody that specifically recognizes pHis was obtained using the synthetic peptide as the immunogen.

The presence of 5-methylcytosine (5-mC) in DNA is a vital epigenetic mark in vertebrates. While the enzymes responsible for methylating DNA in vertebrates have been identified, the means by which this mark can be removed are still unclear. Recently, it has been shown that activation-induced cytidine deaminase (AID) contributes to the demethylation of DNA in certain systems. This enzyme has been intensely studied in its role as a key driver of antibody diversification in B cells, but recent observations from early development in zebrafish and mice as well as heterokaryons point to a role beyond immunology. This review takes stock of the reports linking AID and related deaminases to DNA demethylation, and describes the many important questions left to be answered in this field.

Handed asymmetry in organ shape and positioning is a common feature among bilateria, yet little is known about the morphogenetic mechanisms underlying left-right (LR) organogenesis. We utilize the directional 360 degrees clockwise rotation of genitalia in Drosophila to study LA-dependent organ looping. Using time-lapse imaging, we show that rotation of genitalia by 360 degrees results from an additive process involving two ring-shaped domains, each undergoing 180 degrees rotation. Our results show that the direction of rotation for each ring is autonomous and strictly depends on the LR determinant myosin ID (MyoID). Specific inactivation of MyoID in one domain causes rings to rotate in opposite directions and thereby cancels out the overall movement. We further reveal a specific pattern of apoptosis at the ring boundaries and show that local cell death is required for the movement of each domain, acting as a brake-releaser. These data indicate that organ looping can proceed through an incremental mechanism coupling LR determination and apoptosis. Furthermore, they suggest a model for the stepwise evolution of genitalia posture in Diptera, through the emergence and duplication of a 180 degrees LR module.

Accumulation of beta-amyloid (A beta) and loss of synapses are hallmarks of Alzheimer's disease (AD). How synaptic activity relates to A beta accumulation and loss of synapses is a current topic of major interest. Synaptic activation promotes A beta secretion, and chronic reduction of synaptic activity reduced A beta plaques in an AD transgenic mouse model. This suggested beneficial effects of reducing synaptic activity in AD. We now show that reduced synaptic activity causes detrimental effects on synapses and memory despite reducing plaques using two different models of chronic synaptic inhibition: deafferentation of the barrel cortex and administration of benzodiazepine. An interval of prolonged synaptic inhibition exacerbated loss of synaptophysin compared with synaptically more active brain in AD transgenic but not wild-type mice. Furthermore, an interval of benzodiazepine treatment, followed by a washout period, exacerbated memory impairment in AD transgenic mice. Exacerbation of synaptic and behavioral abnormalities occurred in the setting of reduced A beta plaques but elevated intraneuronal A beta immunoreactivity. These data support beneficial effects of synaptic activation on A beta-related synaptic and behavioral impairment in AD.

Objective To test the hypothesis that predisposition to childhood herpes simplex virus (HSV) type 1 encephalitis (HSE) may be determined in part by human genetic factors. Study design A genetic epidemiologic survey of childhood HSE (onset at age 3 months to 15 years) over a 20-year period (1985-2004) was conducted throughout France (comprising 29 university hospital neuropediatric centers). A total of 85 children fulfilled the diagnostic criteria for inclusion. Family and personal histories were obtained by face-to-face interview for 51 patients. Results No familial cases of HSE were identified in our survey; however, a high proportion (20%) of the children interviewed had a relevant family history: parental consanguinity (12% of patients), early-onset herpetic keratitis in a first-degree relative (6%), or both (2%). The narrow window of high susceptibility to HSE before age 3 years (62% of patients) further indicates that predisposition to HSE is tightly age-dependent. Conclusions This survey suggests that childhood HSE, although sporadic, may result from Mendelian predisposition (from autosomal recessive susceptibility in particular), at least in some children. There likely is incomplete penetrance, however, which may reflect, at least in part, the impact of age at the time of HSV-1 infection. (J Pediatr 2010; 157:623-9).

G-protein-coupled receptors (GPCRs) are the largest protein superfamily in the human genome; they comprise 30% of current drug targets and regulate diverse cellular signaling responses. The role of endosomal trafficking in GPCR signaling regulation is gaining substantial consideration. However, this process remains difficult to study due to the inability to distinguish among many individual receptors, simultaneously trafficking within multiple endosomal pathways. Here we show accurate measurement of the internalization and endosomal trafficking of single groups of serotonin (5-hydroxytryptamine, 5-HT) receptors using single quantum dot (QD) probes and quantitative colocalization. We demonstrate that the presence of a QD tag does not interfere with 5-HT receptor internalization or endosomal recycling. Direct measurements show simultaneous trafficking of the 5-HT1A receptor in two distinct endosomal recycling pathways. Single-molecule imaging of endosomal trafficking will significantly impact the understanding of cellular signaling and provide powerful tools to elucidate the actions of GPCR-targeted therapeutics.

Glucocorticoid hormones are secreted in response to stimuli that activate the hypothalamo-pituitary-adrenocortical (HPA) axis and self-regulate through negative feedback. Negative feedback that occurs on a rapid time scale is thought to act through nongenomic mechanisms. In these studies, we investigated fast feedback inhibition of HPA axis stress responses by direct glucocorticoid action at the paraventricular nucleus of the hypothalamus (PVN). Local infusion of dexamethasone or a membrane-impermeant dexamethasone-BSA conjugate into the PVN rapidly inhibits restraint-induced ACTH and corticosterone release in a manner consistent with feedback actions at the cell membrane. The dexamethasone fast feedback response is blocked by the cannabinoid CB1 receptor antagonist AM-251, suggesting that fast feedback requires local release of endocannabinoids. Hypothalamic tissue content of the endocannabinoid 2-arachidonoyl glycerol is elevated by restraint stress, consistent with endocannabinoid action on feedback processes. These data support the hypothesis that glucocorticoid-induced fast feedback inhibition of the HPA axis is mediated by a nongenomic signaling mechanism that involves endocannabinoid signaling at the level of the PVN. (Endocrinology 151: 4811-4819, 2010)

The U1 small nuclear RNA (U1 snRNA) as a component of the minor U2-dependent spliceosome recognizes 5' splice sites (5'ss) containing GT as the canonical dinucleotide in the intronic positions +1 and +2. The c 165+1G>T germline mutation in the 5'ss of exon 2 of the Fanconi anemia C (FANCC) gene commonly predicted to prevent correct splicing was identified in nine FA patients from three pedigrees. RT-PCR analysis of the endogenous FANCC mRNA splicing pattern of patient-derived fibroblasts revealed aberrant mRNA processing, but surprisingly also correct splicing at the TT dinucleotide, albeit with lower efficiency. This consequently resulted in low levels of correctly spliced transcript and minute levels of normal posttranslationally processed FANCD2 protein, indicating that this naturally occurring TT splicing might contribute to the milder clinical manifestations of the disease in these patients. Functional analysis of this FANCC 5'ss within splicing reporters revealed that both the noncanonical TT dinucleotide and the genomic context of FANCC were required for the residual correct splicing at this mutant 5'ss. Finally, use of lentiviral vectors as a delivery system to introduce expression cassettes for TT-adapted U1 snRNAs into primary FANCC patient fibroblasts allowed the correction of the DNA-damage-induced G2 cell-cycle arrest in these cells, thus representing an alternative transcript-targeting approach for genetic therapy of inherited splice-site mutations.