Modification of the hepatotoxicity of D-galactosamine in the rat by cycloheximide.

The effect of cycloheximide (1.5 mg/kg), a potent inhibitor of protein biosynthesis, on D-galastosamine (375 mg/kg)-induced hepatic necrosis and hepatic triglyceride accumulation was studied in rats. Serum transaminase levels, 24 hr after D-galactosamine administration, were significantly reduced in animals treated simultaneously or 4 hr before D-galactosamine with cycloheximide, when compared to animals given D-galactosamine alone. Transaminase levels in rats given cycloheximide 4 hr after D-galactosamine were not reduced. Histological grading of hepatocyte necrosis showed a similar pattern of protection in the pretreated and simultaneously treated groups. Hepatic triglycerides were significantly reduced only in the latter group. Fatality 48 hr after D-galactosamine administration was significantly less common in rats pretreated with cycloheximide when compared to rats given D-galactosamine without cycloheximide, and surviving animals in the cycloheximide pretreated group had a lower serum transaminase level, a lower necrosis score, and a reduced hepatic triglyceride level. These data are consistent with the concept that protein synthesis is important in the pathogenesis of D-galactosamine-induced hepatotoxicity.     

Inferred expression regulator activities suggest genes mediating cardiometabolic genetic signals

Expression QTL (eQTL) analyses have suggested many genes mediating genome-wide association study (GWAS) signals but most GWAS signals still lack compelling explanatory genes. We have leveraged an adipose-specific gene regulatory network to infer expression regulator activities and phenotypic master regulators (MRs), which were used to detect activity QTLs (aQTLs) at cardiometabolic trait GWAS loci. Regulator activities were inferred with the VIPER algorithm that integrates enrichment of expected expression changes among a regulator’s target genes with confidence in their regulator-target network interactions and target overlap between different regulators (i.e., pleiotropy). Phenotypic MRs were identified as those regulators whose activities were most important in predicting their respective phenotypes using random forest modeling. While eQTLs were typically more significant than aQTLs in cis, the opposite was true among candidate MRs in trans. Several GWAS loci colocalized with MR trans-eQTLs/aQTLs in the absence of colocalized cis-QTLs. Intriguingly, at the 1p36.1 BMI GWAS locus the EPHB2 cis-aQTL was stronger than its cis-eQTL and colocalized with the GWAS signal and 35 BMI MR trans-aQTLs, suggesting the GWAS signal may be mediated by effects on EPHB2 activity and its downstream effects on a network of BMI MRs. These MR and aQTL analyses represent systems genetic methods that may be broadly applied to supplement standard eQTL analyses for suggesting molecular effects mediating GWAS signals.     

Scaleup of spinfilter perfusion bioreactor for mammalian cell retention

A spinning cylindrical filter, known as a spinfilter, permits the mammalian cell bioreactor operation at high perfusion rates leading to very high cell densities (10(7) mL(-1)). Filter screens with openings (25 mum) slightly larger than the average cell size have been used to retain single cells in suspension over a long period of operation without clogging. We have previously shown why it is necessary to optimize the rotational speed of the spinfilter in order to achieve efficient cell retention and avoid potential screen clogging. Effects of bulk mixing and perfusion rate on screen fouling and cell retention were also investigated. Based on this analysis, in this article, we suggest strategies for scaleup of spinfilters. Experimental data from 12- and 175-L (working volume) bioreactors is shown in support of the scaleup analysis. (c) 1994 John Wiley & Sons, Inc.     

Characterization of the 70S Ribosome from Rhodopseudomonas palustris using an integrated "top-down" and "bottom-up" mass spectrometric approach

We present a comprehensive mass spectrometric approach that integrates intact protein molecular mass measurement ("top-down") and proteolytic fragment identification ("bottom-up") to characterize the 70S ribosome from Rhodopseudomonas palustris. Forty-two intact protein identifications were obtained by the top-down approach and 53 out of the 54 orthologs to Escherichia coli ribosomal proteins were identified from bottom-up analysis. This integrated approach simplified the assignment of post-translational modifications by increasing the confidence of identifications, distinguishing between isoforms, and identifying the amino acid positions at which particular post-translational modifications occurred. Our combined mass spectrometry data also allowed us to check and validate the gene annotations for three ribosomal proteins predicted to possess extended C-termini. In particular, we identified a highly repetitive C-terminal "alanine tail" on L25. This type of low complexity sequence, common to eukaryotic proteins, has previously not been reported in prokaryotic proteins. To our knowledge, this is the most comprehensive protein complex analysis to date that integrates two MS techniques.     

Nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator, an ABC transporter, catalyze adenylate kinase activity but not ATP hydrolysis

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter family. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain. Previous biochemical reports suggest NBD1 is a site of stable nucleotide interaction with low ATPase activity, whereas NBD2 is the site of active ATP hydrolysis. It has also been reported that NBD2 additionally possessed adenylate kinase (AK) activity. Knowledge about the intrinsic biochemical activities of the NBDs is essential to understanding the Cl(-) ion gating mechanism. We find that purified mouse NBD1, human NBD1, and human NBD2 function as adenylate kinases but not as ATPases. AK activity is strictly dependent on the addition of the adenosine monophosphate (AMP) substrate. No liberation of [(33)P]phosphate is observed from the gamma-(33)P-labeled ATP substrate in the presence or absence of AMP. AK activity is intrinsic to both human NBDs, as the Walker A box lysine mutations abolish this activity. At low protein concentration, the NBDs display an initial slower nonlinear phase in AK activity, suggesting that the activity results from homodimerization. Interestingly, the G551D gating mutation has an exaggerated nonlinear phase compared with the wild type and may indicate this mutation affects the ability of NBD1 to dimerize. hNBD1 and hNBD2 mixing experiments resulted in an 8-57-fold synergistic enhancement in AK activity suggesting heterodimer formation, which supports a common theme in ABC transporter models. A CFTR gating mechanism model based on adenylate kinase activity is proposed.     

Neuronal non-CG methylation is an essential target for MeCP2 function

1. Download : Download high-res image (148KB)
2. Download : Download full-size image

     

Volatile anesthetics selectively alter [3H]ryanodine binding to skeletal and cardiac ryanodine receptors.

The effect of clinical concentrations of volatile anesthetics on ryanodine receptors of cardiac and skeletal muscle sarcoplasmic reticulum was evaluated using [3H]ryanodine binding. At 2 volume percent, halothane and enflurane stimulated binding to cardiac SR by 238% and 204%, respectively, while isoflurane had no effect. In contrast, halothane and enflurane had no effect on [3H]ryanodine binding to skeletal ryanodine receptors, while isoflurane produced a significant stimulation. These results suggest that volatile anesthetics interact in a site-specific manner with ryanodine receptors of cardiac or skeletal muscle to effect Ca2+ release-channel gating.     

Lack of Stereoselectivity in Ability of Nicotine to Release Dopamine from Rat Synaptosomal Preparations

Both the naturally occurring (-)-isomer and the synthetic (+)-isomer of nicotine caused release of 3H from a crude synaptosomal fraction of rat brain preincubated with [3H]dopamine. The isomers were equipotent in producing this response, which was concentration-dependent, a significant effect on the fractional release of dopamine being observed at 10(-4) M nicotine. The effect did not appear to be the result of synaptosomal damage, as levels of the intrasynaptosomal marker lactate dehydrogenase did not increase in the supernatant. Nicotine-induced release was inhibited by removal of external Ca2+ and by the presence in vitro of pempidine (230 microM). Neither hexamethonium (500 microM) in vitro nor the chronic administration of (-)-nicotine in vivo had any effect on the nicotine-induced release of [3H]dopamine. It is concluded that nicotine exerts this effect via a presynaptic nicotinic receptor of the "ganglionic" type, but that this receptor differs from that in the periphery by showing a relative lack of stereospecificity. There is no evidence of a functional "down regulation" in this receptor on chronic exposure to nicotine in vivo.     

Summer diet of beluga whales inferred by fatty acid analysis of the eastern Beaufort Sea food web

Beluga whales (Delphinapterus leucas) are the most abundant odontocetes in Arctic waters and are thus thought to influence food web structure and function. The diet of the Beaufort Sea beluga population is not well known, partly due to the inherent difficulty of observing feeding behaviour in Arctic marine cetaceans. To determine which prey items are critical to the Beaufort Sea beluga diet we first examine and describe the Mackenzie Delta and Beaufort Sea food web using fatty acid analyses. Fatty acid profiles effectively partitioned prey items into groups associated with their habitat and feeding ecology. Next, the relative contribution of various prey items to beluga diet was investigated using fatty acids. Finally, beluga diet variability was examined as a function of body size, a known correlate of habitat use. Beluga appeared to feed predominantly on Arctic cod (Boreogadus saida) collected from near shore and offshore regions. Size related dietary differences suggested larger sized beluga preferred offshore Arctic cod given the shared high levels of long chain monounsaturates, whereas smaller sized beluga appeared to feed on prey in their near shore habitats that included near shore Arctic cod. The presence of Arctic cod groups in shallow near shore and deep offshore habitats may facilitate the behavioural segregation of beluga habitat use as it relates to their size and resource requirements. Given Arctic cod are a sea ice associated fish combined with the accelerated sea ice loss in this region, beluga whales may need to adapt to new dietary regimes.