Nuclear poly(ADP-ribose) activity is a therapeutic target in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating and fatal motor neuron disease. Diagnosis typically occurs in the fifth decade of life and the disease progresses rapidly leading to death within ~ 2-5 years of symptomatic onset. There is no cure, and the few available treatments offer only a modest extension in patient survival. A protein central to ALS is the nuclear RNA/DNA-binding protein, TDP-43. In > 95% of ALS patients, TDP-43 is cleared from the nucleus and forms phosphorylated protein aggregates in the cytoplasm of affected neurons and glia. We recently defined that poly(ADP-ribose) (PAR) activity regulates TDP-43-associated toxicity. PAR is a posttranslational modification that is attached to target proteins by PAR polymerases (PARPs). PARP-1 and PARP-2 are the major enzymes that are active in the nucleus. Here, we uncovered that the motor neurons of the ALS spinal cord were associated with elevated nuclear PAR, suggesting elevated PARP activity. Veliparib, a small-molecule inhibitor of nuclear PARP-1/2, mitigated the formation of cytoplasmic TDP-43 aggregates in mammalian cells. In primary spinal-cord cultures from rat, Veliparib also inhibited TDP-43-associated neuronal death. These studies uncover that PAR activity is misregulated in the ALS spinal cord, and a small-molecular inhibitor of PARP-1/2 activity may have therapeutic potential in the treatment of ALS and related disorders associated with abnormal TDP-43 homeostasis.

The influence of atmospheric pressure on landfill methane emissions.

Landfills are the largest source of anthropogenic methane (CH4) emissions to the atmosphere in the United States. However, few measurements of whole landfill CH4 emissions have been reported. Here, we present the results of a multi-season study of whole landfill CH4 emissions using atmospheric tracer methods at the Nashua, New Hampshire Municipal landfill in the northeastern United States. The measurement data include 12 individual emission tests, each test consisting of 5-8 plume measurements. Measured emissions were negatively correlated with surface atmospheric pressure and ranged from 7.3 to 26.5 m3 CH4 min(-1). A simple regression model of our results was used to calculate an annual emission rate of 8.4 x 10(6) m3 CH4 year(-1). These data, along with CH4 oxidation estimates based on emitted landfill gas isotopic characteristics and gas collection data, were used to estimate annual CH4 generation at this landfill. A reported gas collection rate of 7.1 x 10(6) m3 CH4 year(-1) and an estimated annual rate of CH4 oxidation by cover soils of 1.2 x 10(6) m3 CH4 year(-1) resulted in a calculated annual CH4 generation rate of 16.7 x 10(6) m3 CH4 year(-1). These results underscore the necessity of understanding a landfill's dynamic environment before assessing long-term emissions potential.

Phosphorylation of the vesicle-tethering protein p115 by a casein kinase II-like enzyme is required for Golgi reassembly from isolated mitotic fragments.

Coat protein I (COPI) transport vesicles can be tethered to Golgi membranes by a complex of fibrous, coiled-coil proteins comprising p115, Giantin and GM130. p115 has been postulated to act as a bridge, linking Giantin on the vesicle to GM130 on the Golgi membrane. Here we show that the acidic COOH terminus of p115 mediates binding to both GM130 and Giantin as well as linking the two together. Phosphorylation of serine 941 within this acidic domain enhances the binding as well as the link between them. Phosphorylation is mediated by casein kinase II (CKII) or a CKII-like kinase. Surprisingly, the highly conserved NH(2)-terminal head domain of p115 is not required for the NSF (N-ethylmaleimide-sensitive fusion protein)-catalyzed reassembly of cisternae from mitotic Golgi fragments in a cell-free system. However, the ability of p115 to link GM130 to Giantin and the phosphorylation of p115 at serine 941 are required for NSF-catalyzed cisternal regrowth. p115 phosphorylation may be required for the transition from COPI vesicle tethering to COPI vesicle docking, an event that involves the formation of trans-SNARE [corrected] (trans-soluble NSF attachment protein [SNAP] receptor) complexes.

A complex of mammalian ufd1 and npl4 links the AAA-ATPase, p97, to ubiquitin and nuclear transport pathways.

The AAA-ATPase, p97/Cdc48p, has been implicated in many different pathways ranging from membrane fusion to ubiquitin-dependent protein degradation. Binding of the p47 complex directs p97 to act in the post-mitotic fusion of Golgi membranes. We now describe another binding complex comprising mammalian Ufd1 and Npl4. Yeast Ufd1p is required for ubiquitin-dependent protein degradation whereas yeast Npl4p has been implicated in nuclear transport. In rat liver cytosol, Ufd1 and Npl4 form a binary complex, which exists either alone or bound to p97. Ufd1/Npl4 competes with p47 for binding to p97 and so inhibits Golgi membrane fusion. This suggests that it is involved in another cellular function catalysed by p97, the most likely being ubiquitin-dependent events during mitosis. The fact that the binding of p47 and Ufd1/Npl4 is mutually exclusive suggests that these protein complexes act as adapters, directing a basic p97 activity into different cellular pathways.

Membrane traffic: do cones mark sites of fission?

Membrane fission occurs in eukaryotic cells whenever a vesicle is produced or a larger subcellular compartment is divided into smaller discrete units. Recent evidence suggests this fission event is promoted by enzymes that generate phosphatidic acid and thereby cause a distortion of the lipid bilayer.

The amino-terminal domain of the golgi protein giantin interacts directly with the vesicle-tethering protein p115.

Giantin is thought to form a complex with p115 and Golgi matrix protein 130, which is involved in the reassembly of Golgi cisternae and stacks at the end of mitosis. The complex is involved in the tethering of coat protomer I vesicles to Golgi membranes and the initial stacking of reforming cisternae. Here we show that the NH(2)-terminal 15% of Giantin suffices to bind p115 in vitro and in vivo and to block cell-free Golgi reassembly. Because Giantin is a long, rod-like protein anchored to the membrane by its extreme COOH terminus, these results support the idea of a long, flexible tether linking vesicles and cisternae.

Diode-pumped 214.8-nm Nd:YAG /Cr4+:YAG microchip laser system for the detection of NO.

A passively Q-switched 214.8-nm Nd:YAG/Cr(4+):YAG microchip laser system for the detection of NO was designed, constructed, and tested. The system uses the fifth harmonic of the 1.074-microm transition in Nd:YAG to detect NO by laser-induced fluorescence. A significant challenge was the development of an environmentally stable coating to provide the necessary discrimination between the 1.074-microm laser line and the stronger transition at 1.064 microm. The exact position of the fifth-harmonic frequency was determined by use of NO fluorescence excitation spectra to be 46556 +/- 1.5 cm(-1). With a pulse energy of approximately 50 nJ of fifth-harmonic light, we observed a detection sensitivity for NO of approximately 15 parts per billion by volume in a simple, compact optical system.

An NSF function distinct from ATPase-dependent SNARE disassembly is essential for Golgi membrane fusion.

The precise biochemical role of N-ethylmaleimide-sensitive factor (NSF) in membrane fusion mediated by SNARE proteins is unclear. To provide further insight into the function of NSF, we have introduced a mutation into mammalian NSF that, in Drosophila dNSF-1, leads to temperature-sensitive neuroparalysis. This mutation is like the comatose mutation and renders the mammalian NSF temperature sensitive for fusion of postmitotic Golgi vesicles and tubules into intact cisternae. Unexpectedly, at the temperature that is permissive for membrane fusion, this mutant NSF binds to, but cannot disassemble, SNARE complexes and exhibits almost no ATPase activity. A well-charaterized NSF mutant containing an inactivating point mutation in the catalytic site of its ATPase domain is equally active in the Golgi-reassembly assay. These data indicate that the need for NSF during postmitotic Golgi membrane fusion may be distinct from its ATPase-dependent ability to break up SNARE pairs.

GRASP55, a second mammalian GRASP protein involved in the stacking of Golgi cisternae in a cell-free system.

We have identified a 55 kDa protein, named GRASP55 (Golgi reassembly stacking protein of 55 kDa), as a component of the Golgi stacking machinery. GRASP55 is homologous to GRASP65, an N-ethylmaleimide-sensitive membrane protein required for the stacking of Golgi cisternae in a cell-free system. GRASP65 exists in a complex with the vesicle docking protein receptor GM130 to which it binds directly, and the membrane tethering protein p115, which also functions in the stacking of Golgi cisternae. GRASP55 binding to GM130, could not be detected using biochemical methods, although a weak interaction was detected with the yeast two-hybrid system. Cryo-electron microscopy revealed that GRASP65, like GM130, is present on the cis-Golgi, while GRASP55 is on the medial-Golgi. Recombinant GRASP55 and antibodies to the protein block the stacking of Golgi cisternae, which is similar to the observations made for GRASP65. These results demonstrate that GRASP55 and GRASP65 function in the stacking of Golgi cisternae.

A role for the vesicle tethering protein, p115, in the post-mitotic stacking of reassembling Golgi cisternae in a cell-free system.

During telophase, Golgi cisternae are regenerated and stacked from a heterogeneous population of tubulovesicular clusters. A cell-free system that reconstructs these events has revealed that cisternal regrowth requires interplay between soluble factors and soluble N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) attachment protein receptors (SNAREs) via two intersecting pathways controlled by the ATPases, p97 and NSF. Golgi reassembly stacking protein 65 (GRASP65), an NEM-sensitive membrane-bound component, is required for the stacking process. NSF-mediated cisternal regrowth requires a vesicle tethering protein, p115, which we now show operates through its two Golgi receptors, GM130 and giantin. p97-mediated cisternal regrowth is p115-independent, but we now demonstrate a role for p115, in conjunction with its receptors, in stacking p97 generated cisternae. Temporal analysis suggests that p115 plays a transient role in stacking that may be upstream of GRASP65-mediated stacking. These results implicate p115 and its receptors in the initial alignment and docking of single cisternae that may be an important prerequisite for stack formation.

Effects of scopolamine on delayed-matching-to-sample and paired associates tests of visual memory and learning in human subjects: comparison with diazepam and implications for dementia.

Two experiments examined dose-related effects of 200, 400 and 600 micrograms scopolamine (n = 24, s.c.) and 5 and 10 mg diazepam (n = 6, PO) on parallel tests of visual memory and learning taken from the CANTAB battery. Scopolamine significantly impaired accuracy of performance on a delayed matching to sample test of visual recognition memory in a dose- and delay-dependent manner, but had only marginal decremental effects on a test of visuospatial paired associates learning. Scopolamine significantly lengthened decision times in a visual search matching to sample task at the 400 and 600 micrograms doses, without significantly affecting accuracy. The drug also impaired performance on tests of spatial (on accuracy and response time measures) and pattern (on response time only) memory. Most of the deleterious effects on scopolamine were removed by covariance analyses with indices of subjective sedation, but the effects of delayed matching accuracy and latency remained. By contrast, diazepam significantly impaired paired associates learning but affected delayed matching to sample in a delay-independent manner. These results suggest that scopolamine can produce selective deficits in tests of short-term visual recognition memory which do not depend on overall impairments in arousal and which contrast with deficits in visual associative learning produced by diazepam. They have implications for the pharmacological modelling of dementia and memory disorders in man and for the neurochemical substrates of the short-term recognition memory and associative learning for visual stimuli.

Tunable infrared laser detection of pyrolysis products of explosives in soils.

A research program involving two applications of tunable infrared laser differential absorption spectroscopy (TILDAS) with multipass, long-path absorption cells to the detection of explosives contamination in soils is reported. In the first application, sensitive, specific real-time species concentration measurements by TILDAS have led to new understanding of the processes involved in explosives detection by the heating of contaminated soils and the quantification of the resulting pyrolysis gases. In the second, we present results of our calculations of the properties of astigmatic off-axis resonator absorption cells, which show that useful TILDAS path lengths can be achieved inside a cone penetrometer probe.

Determination of atmospheric methyl bromide by cryotrapping-gas chromatography and application to soil kinetic studies using a dynamic dilution system.

Methyl bromide (CH(3)Br) is considered to be a major source of stratospheric Br, which contributes to the destruction of ozone. It is therefore necessary to understand the natural sinks of this compound and to accurately measure ambient mixing ratios. Methodology is described for the measurement of atmospheric CH(3)Br by cryotrapping-gas chromatography and its application to soil kinetics. A 2-propanol/dry ice cryotrap was used to preconcentrate CH(3)Br in standard and air samples, with subsequent detection using a gas chromatograph equipped with an O(2)-doped electron capture detector (GC-ECD). The GC-ECD cryotrapping method had a detection limit of 0.23 pmol of CH(3)Br. This is equivalent to the amount of CH(3)Br in a 500 mL sample of ambient air at the estimated northern hemisphere atmospheric mixing ratio of 11 parts per trillion by volume (pptv). A dynamic dilution system was developed to produce mixing ratios of CH(3)Br ranging between 4 and 1000 pptv. Calibrated mixing ratios of CH(3)Br produced with the dilution system were used to determine soil uptake kinetics employing a dynamic soil incubation method.

Joint space measurement in hand radiographs using computerized image analysis.

OBJECTIVE: To compare computerized joint space (JS) measurements with conventional joint space narrowing (JSN) scores in patients with mild rheumatoid arthritis. METHODS: Serial paired hand and wrist radiographs from 34 patients with classic rheumatoid arthritis were evaluated. Purpose-written software automatically measured the JS on test images and standard clinical hand radiographs; JSN was scored "blind" by 6 observers. RESULTS: The software proved reliable. JS values differed significantly (men > women; metacarpophalangeal > proximal interphalangeal joints), declining with disease duration more than with age; JSN scores correlated poorly and varied more. CONCLUSION: Computerization permits sensitive JS measurement and should be of benefit in studies of early joint disease.

Preparing for networking information systems in health care organizations.

Because the health care administrator faces unprecedented demands concerning information, well-designed and carefully selected information systems have become a critical factor in the viability and survival of health care institutions. Local area network systems are having a significant impact in this regard. Among their advantages are flexibility, dependability, low cost, and modularity. Health care facilities, have found networking to be a simple, cost-effective solution to collecting, processing, and distributing large volumes of data. The keys to a successful implementation recommended in this article should assist health care professionals once they have made the hard decision to use a local area network to process their day-to-day transactions.