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The combined use of electrospray ionization run in so-called "native mode" with top-down mass spectrometry (nTDMS) is enhancing both structural biology and discovery proteomics by providing three levels of information in a single experiment: the intact mass of a protein or complex, the masses of its subunits and non-covalent cofactors, and fragment ion masses from direct dissociation of subunits that capture the primary sequence and combinations of diverse post-translational modifications (PTMs). While intact mass data are readily deconvoluted using well-known software options, the analysis of fragmentation data that result from a tandem MS experiment - essential for proteoform characterization - is not yet standardized. In this tutorial, we offer a decision-tree for the analysis of nTDMS experiments on protein complexes and diverse bioassemblies. We include an overview of strategies to navigate this type of analysis, provide example data sets, and highlight software for the hypothesis-driven interrogation of fragment ions for localization of PTMs, metals, and cofactors on native proteoforms. Throughout we have emphasized the key features (deconvolution, search mode, validation, other) that the reader can consider when deciding upon their specific experimental and data processing design using both open-access and commercial software.

The pathological consequences resulting from deregulation of the apoptotic program include cancer (too little apoptosis) or diseases of cell deprivation, such as Alzheimer's (too much apoptosis). We have identified an additional pathology whereby cells reaching the earliest stage of chromatin cleavage have the potential to suppress apoptotic execution and survive. One specific cleavage event associated with this process is restricted to a location within the mixed lineage leukemia (MLL) gene at 11q23. The site of cleavage is consistent with the location where large, approximately 50 kbp loops of supercoiled DNA are attached to the nuclear matrix. Cells modified by this process generate MLL translocations, as shown by inverse PCR, that survive for days to weeks but which have no known relationship with clinical disease. Using a specific approach, cells stimulated by anti-CD95 antibody, a potent stimulator of the apoptotic program, facilitated creation of the MLL-AF9 fusion gene. Further, this rearrangement, which is commonly observed in patients with AML linked to exposure to cytotoxic agents, was efficiently transcribed in cells that were able to undergo cell division. These data are discussed in the context of benzene and benzene metabolite toxicity that impacts the process of apoptosis and is known to lead to leukemic disease.

The purpose of this study was to examine whether improvement in motor function could be demonstrated in old rats, and to see if GDNF affected post-synaptic DA function. Aged (20 month old) versus young rats were tested following GDNF treatment for postural control by using an inclined balance beam and a wire grip strength test. Rats were also examined electrophysiologically for spontaneous striatal cell firing rate alone and in the presence of DA receptor agonists, and histologically for the intensity of striatal TH staining, and number of DA containing nigral cells. Behavior was significantly improved in the aged animals who received central GDNF infusions, although the extent of improvement was less than what has been observed in 16-month-old rats. There was no effect of GDNF treatment in the aged animals on spontaneous firing rate in the striatum, or on the post synaptic response to locally applied D(1) and D(2) receptor family agonists. However, there was an effect of age alone on firing rate, and on the response to locally applied SKF 38393 and quinpirole. By using unbiased cell counting we observed no age-related decline in the number of TH positive cells in the substantia nigra. There was no effect of GDNF on the number of TH positive cells in the substantia nigra in either young or aged rats, although there were morphological improvements in DA neurons of the GDNF treated aged rats. These results replicate earlier studies showing an effect of age on striatal firing rate and dopamine receptor function, and suggest that the GDNF mediated improvement in behavior may be located other than post synaptically within the striatum.

UpI is a basic protein, with molecular mass (approximately 28 kDa) and a pI > 9.4, isolated from the sea anemone Urticina piscivora. It is a potent cardiac stimulatory protein with the partial amino acid sequence D1ENEN5LYGPN10ENKAK15AKDLT20AGASY25LTKEA30GCTKL35QAGCT40MYQAY45N [1]. The toxic effects of UpI and the crude extract from which it was isolated have been investigated on three tumour cell lines: KB, L1210, and HEL 299 cells. UpI, however, was less potent on each cell line than the crude extract. Since previous experiments had shown extracts of U. piscivora to be haemolytic on erythrocytes of rat, guinea pig and dog, the haemolytic action of UpI was investigated. It was found to be a potent haemolysin on erythrocytes of rat, guinea pig, dog, pig and human, causing haemolysis on erythrocytes of each species tested at concentrations as low as 10(-10) M. Haemolysis was inhibited in a concentration-dependent manner by the phospholipid sphingomyelin but not cholesterol. Using scanning electron microscopy, it is now being shown that UpI produces significant structural damage to membranes of erythrocytes from rat and guinea pig. It proved to be a potent ichthyotoxin. These data suggest that sea anemone toxin not only possess different pharmacological activities but that UpI, one of the active constituents, could be responsible for the different pharmacological effects exhibited by the crude extract.

Previous work has demonstrated that direct injections of methylnaloxonium (MN), a relatively lipophobic quaternary opiate antagonist, in the area of the nucleus raphe pontis (RPn) significantly attenuated alfentanil-induced rigidity. It was hypothesized that other hindbrain sites, particularly the other raphe nuclei, might play a role in this rigidity. Therefore, a study was performed in which 57 rats, divided into four groups, were implanted with chronic guide cannulae directed at brain sites anterior, lateral, or posterior to the RPn. After each animal was pretreated with intracerebral injections of MN, alfentanil (0.5 mg/kg) was administered subcutaneously. Electromyographic activity was recorded from the gastrocnemius muscle as a measure of hindlimb rigidity. Each animal was subsequently injected at 4 to 5 day intervals with MN two additional times at sites 1 and 2 mm deeper, respectively, than the initial injection. Data were thus obtained on animals treated with either MN or saline at 3 successive histologically identified sites which were either anterior, lateral or posterior to the RPn. The administration of MN into two specific sites in the region just lateral to the nucleus raphe pontis significantly [F(1,38) = 18.68 and 5.02 respectively, p less than 0.05] reversed the rigidity produced by systemic alfentanil administration. There was a weak effect of MN injections anterior to the RPn but this could not be localized to any one site. These results suggest that discrete brainstem regions involved in opiate action can be sensitively and selectively identified by direct intracranial injections of a lipophobic opiate antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)