Efficient micro-recovery and guanidination of peptides directly from MALDI target spots

A method is presented for the recovery and subsequent guanidination of tryptic peptides from samples previously spotted on a matrix-assisted laser desorption/ionization (MALDI) target. The procedure is shown to have applicability to both in-solution and in-gel digests, yielding improved confidence in protein identification and sequence coverage in all instances. Recovery from the plate is essentially quantitative, with no residual analyte observed on the target spot. The technique is rapid, simple, and has extended applicability to other processing steps, including (but not limited to) derivatization for specific peptide studies or enzymatic treatment for subsequent profiling of posttranslational modifications. This method circumvents the failure of an initial analysis to generate suitable information and is particularly relevant for the analysis of precious samples.     

The nuclear death domain protein p84N5 activates a G2/M cell cycle checkpoint prior to the onset of apoptosis

In contrast to extracellular signals, the mechanisms utilized to transduce nuclear apoptotic signals are not well understood. Characterizing these mechanisms is important for predicting how tumors will respond to genotoxic radiation or chemotherapy. The retinoblastoma (Rb) tumor suppressor protein can regulate apoptosis triggered by DNA damage through an unknown mechanism. The nuclear death domain-containing protein p84N5 can induce apoptosis that is inhibited by association with Rb. The pattern of caspase and NF-kappaB activation during p84N5-induced apoptosis is similar to p53-independent cellular responses to DNA damage. One hallmark of this response is the activation of a G(2)/M cell cycle checkpoint. In this report, we characterize the effects of p84N5 on the cell cycle. Expression of p84N5 induces changes in cell cycle distribution and kinetics that are consistent with the activation of a G(2)/M cell cycle checkpoint. Like the radiation-induced checkpoint, caffeine blocks p84N5-induced G(2)/M arrest but not subsequent apoptotic cell death. The p84N5-induced checkpoint is functional in ataxia telangiectasia-mutated kinase-deficient cells. We conclude that p84N5 induces an ataxia telangiectasia-mutated kinase (ATM)-independent, caffeine-sensitive G(2)/M cell cycle arrest prior to the onset of apoptosis. This conclusion is consistent with the hypotheses that p84N5 functions in an Rb-regulated cellular response that is similar to that triggered by DNA damage.     

The genetics of stamenoid petal production in oilseed rape (Brassica napus) and equivalent variation in Arabidopsis thaliana

 The agronomic potential of a Brassica napus variant with petalless flowers was compromised by an associated detrimental change in leaf morphology. Genetic analysis demonstrated the cosegregation of genes controlling both morphologies. Two STAP loci controlling the production of flowers with stamenoid petals were mapped to homoeologous locations in the genome of B. napus. The STAP loci were probably duplicate genes because they exhibited an epistatic interaction such that only plants homozygous for recessive stap alleles at both loci expressed the variant phenotype. The CURLY LEAF (CLF) gene of Arabidopsis thaliana pleiotropically influences both flower and leaf morphologies. The cloned CLF gene of Arabidopsis was homologous to a polymorphic B. napus locus coincident with one of the B. napus STAP loci. The possibility that CLF is a candidate gene for STAP suggests that the variant stap alleles of B. napus exert pleiotropic effects over both flower and leaf morphologies. Received: 26 August 1996 / Accepted: 20 September 1996     

Mutation of Npr2 Leads to Blurred Tonotopic Organization of Central Auditory Circuits in Mice

Tonotopy is a fundamental organizational feature of the auditory system. Sounds are encoded by the spatial and temporal patterns of electrical activity in spiral ganglion neurons (SGNs) and are transmitted via tonotopically ordered processes from the cochlea through the eighth nerve to the cochlear nuclei. Upon reaching the brainstem, SGN axons bifurcate in a stereotyped pattern, innervating target neurons in the anteroventral cochlear nucleus (aVCN) with one branch and in the posteroventral and dorsal cochlear nuclei (pVCN and DCN) with the other. Each branch is tonotopically organized, thereby distributing acoustic information systematically along multiple parallel pathways for processing in the brainstem. In mice with a mutation in the receptor guanylyl cyclase Npr2, this spatial organization is disrupted. Peripheral SGN processes appear normal, but central SGN processes fail to bifurcate and are disorganized as they exit the auditory nerve. Within the cochlear nuclei, the tonotopic organization of the SGN terminal arbors is blurred and the aVCN is underinnervated with a reduced convergence of SGN inputs onto target neurons. The tonotopy of circuitry within the cochlear nuclei is also degraded, as revealed by changes in the topographic mapping of tuberculoventral cell projections from DCN to VCN. Nonetheless, Npr2 mutant SGN axons are able to transmit acoustic information with normal sensitivity and timing, as revealed by auditory brainstem responses and electrophysiological recordings from VCN neurons. Although most features of signal transmission are normal, intermittent failures were observed in responses to trains of shocks, likely due to a failure in action potential conduction at branch points in Npr2 mutant afferent fibers. Our results show that Npr2 is necessary for the precise spatial organization typical of central auditory circuits, but that signals are still transmitted with normal timing, and that mutant mice can hear even with these deficits.