Protein expression changes in the nucleus accumbens and amygdala of inbred alcohol-preferring rats given either continuous or scheduled access to ethanol.

Chronic ethanol (EtOH) drinking produces neuronal alterations within the limbic system. To investigate changes in protein expression levels associated with EtOH drinking, inbred alcohol-preferring (iP) rats were given one of three EtOH access conditions in their home-cages: continuous ethanol (CE: 24h/day, 7days/week access to EtOH), multiple scheduled access (MSA: four 1-h sessions during the dark cycle/day, 5 days/week) to EtOH, or remained EtOH-naïve. Both MSA and CE groups consumed between 6 and 6.5g of EtOH/kg/day after the 3rd week of access. On the first day of EtOH access for the seventh week, access was terminated at the end of the fourth MSA session for MSA rats and the corresponding time point (2300h) for CE rats. Ten h later, the rats were decapitated, brains extracted, the nucleus accumbens (NAcc) and amygdala (AMYG) microdissected, and protein isolated for 2-dimensional gel electrophoretic analyses. In the NAcc, MSA altered expression levels for 12 of the 14 identified proteins, compared with controls, with six of these proteins altered by CE access, as well. In the AMYG, CE access changed expression levels for 22 of the 27 identified proteins, compared with controls, with 8 of these proteins altered by MSA, as well. The proteins could be grouped into functional categories of chaperones, cytoskeleton, intracellular communication, membrane transport, metabolism, energy production, or neurotransmission. Overall, it appears that EtOH drinking and the conditions under which EtOH is consumed, differentially affect protein expression levels between the NAcc and AMYG. This may reflect differences in neuroanatomical and/or functional characteristics associated with EtOH self-administration and possibly withdrawal, between these two brain structures.

Effect of JP-8 jet fuel exposure on protein expression in human keratinocyte cells in culture.

Dermal exposure to jet fuel is a significant occupational hazard. Previous studies have investigated its absorption and disposition in skin, and the systemic biochemical and immunotoxicological sequelae to exposure. Despite studies of JP-8 jet fuel components in murine, porcine or human keratinocyte cell cultures, proteomic analysis of JP-8 exposure has not been investigated. This study was conducted to examine the effect of JP-8 administration on the human epidermal keratinocyte (HEK) proteome. Using a two-dimensional electrophoretic approach combined with mass spectrometric-based protein identification, we analyzed protein expression in HEK exposed to 0.1% JP-8 in culture medium for 24 h. JP-8 exposure resulted in significant expression differences (p<0.02) in 35 of the 929 proteins matched and analyzed. Approximately, a third of these alterations were increased in protein expression, two-thirds declined with JP-8 exposure. Peptide mass fingerprint identification of effected proteins revealed a variety of functional implications. In general, altered proteins involved endocytotic/exocytotic mechanisms and their cytoskeletal components, cell stress, and those involved in vesicular function.

Changes in gene and protein expression in magnetic field-treated human glioma cells.

Because few cancer studies have examined protein profiles and genetic regulation from a single carcinogen exposure, the objective of this study was to determine genetic change via microarray and to evaluate whether that change was a precursor to cellular protein changes. In separate but experimentally identical studies, human glioma SF767 cells were exposed for 3 h to 60-Hz magnetic fields (sham or 1.2 muT). Microarray results suggested that magnetic field treatment resulted in the up-regulation of 5 genes, whereas 25 genes were down-regulated. The mean abundance of 10 identified proteins was altered following 1.2 muT exposure relative to sham (3 increase, 7 decrease). These studies suggest a limited but complicated response in the glioma cells to the magnetic field treatment.

The effect of ageing and exercise on skeletal muscle function.

The contractile and selected biochemical properties of fast- and slow-twitch skeletal muscle were studied at 9, 18, and 28 months of age in sedentary and regularly exercised rats. The isometric twitch duration was prolonged with aging in both the fast- and slow-twitch muscle. This effect was primarily due to a prolonged one-half relaxation time (1/2RT), which developed late in life. Regular exercise tended to further prolong the twitch duration, particularly in the slow-twitch soleus. Surprisingly, twitch and tetanic tension (Po), peak rate of tension development and decline, and the maximal shortening velocity were all unaltered between 9 and 28 months of age. Furthermore, regular exercise (running or swimming) had little or no effect on these properties. The prolonged 1/2RT with aging could not be explained by a decreased rate of Ca2+ sequestration by the sarcoplasmic reticulum, as the rate of Ca2+ uptake measured in muscle homogenates was unaltered in any of the muscles studied between 9 and 28 months. The degree of muscle fatigue (decline in Po) with 30 min of contractile activity in the slow-twitch soleus was not affected by aging. However, lactate reached two-fold higher levels and glycogen fell to considerably lower levels in the muscles of the old rats. This suggests an increased glycolysis and glycogen utilization during contractile activity in aged rats.

Hepatic protein pattern alterations following perfluorodecanoic acid exposure in rats.

The effect after 8 days of 20 and 50 mg/kg in vivo exposure to perfluoro-n-decanoic acid (PFDA) on the protein pattern of rat liver whole homogenates was studied using high-resolution, large-scale two-dimensional polyacrylamide gel electrophoresis. PFDA exposure altered 13 proteins reproducibly in each of 5 samples tested. While most of the altered proteins remained unidentified, several known mitochondrial proteins were induced by PFDA as were others in a dose-related manner. Conversely, PFDA caused the reduction of albumin as well as several unknown proteins, also in a dose-related manner. Cytoskeletal proteins were unaffected by PFDA. The present results suggest that PFDA's toxic mechanism may involve more than membrane integration and lipid accumulation. Changes in the two-dimensional protein pattern indicate that selective protein synthetic or catabolic processes may undergo qualitative alterations after PFDA treatment as well.

Induction of enoyl-CoA hydratase by LD50 exposure to perfluorocarboxylic acids detected by two-dimensional electrophoresis.

The effect of in vivo exposure to perfluoro-n-octanoic and perfluoro-n-decanoic acids was examined in the rat liver by two-dimensional electrophoresis (2DE). Using nonequilibrium pH-gradient electrophoresis in the first dimension separation, proteins associated with the mitochondrial/peroxisomal cell fraction were observed and immunologically identified. Conspicuous inductions in peroxisomal enoyl-CoA hydratase and other proteins of the peroxisomal beta-oxidative pathway were observed following single-dose exposure to each compound. The abundance of the tentatively-identified mitochondrial equivalent, crotonase, was not altered by these intoxications. These results confirm previous observations of perfluorocarboxylic acid toxicity and support the use of 2D protein-pattern alterations in biomarker research. The ability to identify this type of alteration via 2DE, in association with specific toxic effects by chemically related compounds, may provide new and additional markers for chemical-induced tissue damage.

Differential protein expression during aging in ventricular myocardium of Fischer 344 x Brown Norway hybrid rats.

The aging heart undergoes well characterized structural changes associated with functional decline, though the underlying mechanisms are not understood. The aim of this study was to determine to what extent ventricular myocardial protein expression was altered with age and which proteins underwent protein nitration. Fischer 344 x Brown Norway F1 hybrid (FBN) rats of four age groups were used, 4, 12, 24, and 34 months. Differential protein expression was determined by 2-DE and proteins were identified by peptide mass fingerprinting. Altered protein nitration with age was assessed by immunoblotting. Over 1000 protein spots per sample were detected, and 255 were found to be differentially expressed when all aged groups were compared to young rats (4 months) (p0.05). A strong positive correlation between differential protein expression and increasing age (p=0.03, R(2)=0.997) indicated a progressive, rather than abrupt, change with age. Of 46 differentially expressed proteins identified, seventeen have roles in apoptosis, ten in hypertrophy, seven in fibrosis, and three in diastolic dysfunction, aging-associated processes previously reported in both human and FBN rat heart. Protein expression alterations detected here could have beneficial effects on cardiac function; thus, our data indicate a largely adaptive change in protein expression during aging. In contrast, differential protein nitration increased abruptly, rather than progressively, at 24 months of age. Altogether, the results suggest that differential myocardial protein expression occurs in a progressive manner during aging, and that a proteomic-based approach is an effective method for the identification of potential therapeutic targets to mitigate aging-related myocardial dysfunction.

Investigation of protein expression in magnetic field-treated human glioma cells.

We previously reported phenotypic changes in human breast cancer cells following low-level magnetic field (MF) exposure. Here proteomic methods were used to investigate the biochemical effect of MF exposure in SF767 human glioma cells. Protein alterations were studied after exposure to 1.2 microTesla (microT) MF [12 milliGauss (mG), 60 Hertz (Hz)] +/- epidermal growth factor (EGF). SF767 cells were exposed for 3 h to sham conditions (<0.2 microT ambient field strength) or 1.2 microT MF (+/-EGF; 10 ng/ml). Solubilized protein fractions (sham; 1.2 microT; sham + EGF; 1.2 microT + EGF) were loaded for electrophoresis by 2D-PAGE and stained using a colloidal Coomassie blue technique to resolve and characterize the proteins. Protein patterns were compared across groups via Student's t-test using PDQUEST software. Cell profiles revealed significant alterations in the spot density of a subset of treated cells. Automated spot excision and processing was performed prior to peptide mass fingerprinting proteins of interest. Fifty-seven proteins from the detectable pool were identified and/or found to differ significantly across treatment groups. The mean abundance of 10 identified proteins was altered following 1.2 microT exposure. In the presence of EGF six proteins were altered after low magnetic field treatment by increasing (4) or decreasing (2) in abundance. The results suggest that the analysis of differentially expressed proteins in SF767 cells may be useful as biomarkers for biological changes caused by exposure to magnetic fields.

Soleus muscle atrophy in rats induced by cast immobilization: lack of effect by anabolic steroids.

To test the hypothesis that an anabolic steroid such as nandrolone decanoate (ND) will ameliorate or abolish disuse-mediated alterations resulting from five weeks of hindlimb immobilization, female Sprague-Dawley rats were divided into six groups: control, control + ND, shortened-immobilized, shortened-immobilized + ND, stretched-immobilized, and stretched-immobilized + ND. Immobilization was accomplished by wrapping hindlimbs with plaster of Paris. Nandrolone decanoate in sesame oil was administered via weekly intraperitoneal injection (7 mg/kg) while nontreated rats received equivalent volumes of the vehicle. After five weeks selected morphometric, biochemical, and mechanical parameters were examined in the slow-twitch soleus muscle (SOL). Muscle wet weight fell from a control value of 145 +/- 19 mg to 70 +/- 8 mg (p less than .05) in the immobilized-shortened group where ND had no effect (81 +/- 11 mg). Muscle stretch alone prevented weight loss (159 +/- 31 mg). The addition of ND resulted in significant SOL hypertrophy (200 +/- 42 mg), p less than .05. Immobilization resulted in a significant shift in muscle protein distribution toward sarcoplasmic protein, a change unaltered by ND but abolished by stretch. Muscle strength, as indicated by peak tetanic tension, fell 45% (p less than .05) as a result of shortened immobilization. Nandrolone decanoate had no effect on this condition, although the ameliorating effect of stretch was abolished when in combination with ND treatment. The elevation observed in maximal shortening velocity (shortened-immobilized) was unaffected by ND alone, but abolished by stretch with or without ND treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Proteomics: applications and opportunities in preclinical drug development.

Advances in DNA sequencing and the near-term availability of whole genome sequences for several pharmaceutically relevant organisms promise to dramatically alter the breadth and scale of high-throughput proteomic studies. The substantial amount of literature is available in the public domain, demonstrate the potential of proteomics in the preclinical phases of pharmaceutical development. Over the next few years, it is anticipated that functional genomics and proteomics will have major impacts on the clinical phases of drug development. Expected benefits are earlier proof-of-concept studies in man and increased efficiency of clinical trials through the availability of biologically relevant markers for drug efficacy and safety.

Comparative 2D-electrophoretic mapping of human and rodent hepatic stress proteins as potential biomarkers.

Toxicologic studies in rodents demonstrate that two-dimensional polyacrylamide gel electrophoresis of proteins (2DE) is very useful in the detection and evaluation of chemical toxicity by providing information regarding cellular status at the molecular level. Identification of a set of specific biomarkers of exposure or effect, with a proclivity for both a particular rodent and human target tissue, is required for development of an electrophoretically based testing system. In this regard, stress proteins, such as the heat shock and glucose-regulated proteins (Hsp and Grp), are appropriate candidates. The present investigation was undertaken to identify these stress proteins on conventional two-dimensional electrophoretic gel patterns of human and rat liver homogenates. The following stress proteins were identified, their x, y coordinate positions mapped, and abundances determined, and these data statistically analyzed and compared: Hsp25, Hsp32, Hsp60, Hsc70, Hsp70, Hsp90, Grp75, Grp78, Grp94, protein disulfide isomerase (PDI), and ER-60. With the exception of Hsp25 and Hsp32, the stress proteins examined were constitutively expressed at detectable levels in both unstressed human and rat liver; in virtually identical patterns. Based on our results, the human hepatic 2DE stress protein pattern seems well-suited to toxicologic screening particularly in in vitro applications and via extrapolations from rodent exposures.

Toxicant-induced alterations in two-dimensional electrophoretic patterns of hepatic and renal stress proteins.

Recent studies in this laboratory and by others suggest that two-dimensional polyacrylamide gel electrophoresis of proteins (2-DE) possesses significant utility in the detection of chemical toxicity and in providing information regarding toxic mechanism. After having identified a set of specific heat-shock and glucose-regulated proteins whose expression in rodent liver and kidney is highly conserved and constitutive, we compared the effect of in vivo exposure to perfluoro-n-octanoic acid and perfluoro-n-decanoic acid on their expression. The following stress proteins were identified, their x, y coordinate positions mapped, and abundance statistically analyzed and compared: hsp32, hsp60, hsc70, hsp70, hsp90, grp75, grp94, protein disulfide isomerase (PDI), and ER60. We report here that the stress response to perfluorocarboxylic acids is tissue-, toxicant-, and stress protein class-specific and dose-related. Furthermore, because nearly all of the proteins studied were constitutively expressed at detectable levels in both liver and kidney, the 2-DE stress protein pattern may be suitable to future toxicologic screening applications.

Effect of hindlimb immobilization on the fatigability of skeletal muscle.

The soleus (SOL) and extensor digitorum longus (EDL) muscles of the rat were studied in situ (33.5 degrees C) after 6 wk of disuse atrophy produced by hindlimb immobilization (IM). IM resulted in depressed peak twitch (Pt) and tetanic (Po) tension as well as a decreased rate of tension development (+dP/dt) and decline (-dP/dt) in the slow-twitch SOL. The fast-twitch EDL was affected to a lesser extent, because only Po (g . cm-2) and Po,-dP/dt (g . cm-2 . ms-1) were depressed after IM. Twitch duration, as measured by contraction time and one-half relaxation time, was shortened in the SOL and prolonged in the EDL. In both the fast and slow muscles 30 min of electrical stimulation resulted in a significant decline in Po. Relative to the prefatigued Po, the atrophied and control muscles showed a similar fatigue pattern. This occurred in spite of lower ATP and glycogen concentrations and higher lactate levels in the atrophied muscles. Our results indicate significant impairment of contractile function following IM in fast and slow muscles with preferential alterations in the slow SOL. In addition, despite a lower resting capacity and an increased dependence on glycolysis, contractile properties of atrophied muscles differed little from control muscles in their pattern of response to 30 min of electrical stimulation.

Charge modification in rodent hepatic Grp78/BiP following exposure to structurally diverse peroxisome proliferators.

This investigation was conducted to determine the comparative effect of structurally diverse peroxisome proliferators (PP) on the two-dimensional protein pattern of rat liver whole homogenates. Perfluoro-n-decanoic acid (PFDA), perfluoro-n-octanoic acid (PFOA), clofibrate, and di(2-ethylhexyl)phthalate(DEHP) are all known to cause the proliferation of hepatic peroxisomes and the induction of peroxisomal beta-oxidative and microsomal omega-oxidative enzymes. To detect potential differences between these compounds with regard to the liver, we examined the unique patterns of protein alteration produced by in vivo exposure to them. Following exposure to various doses, whole liver homogenates were prepared and separated by two-dimensional gel electrophoresis (2DE) using the ISO-DALT System. Stained gels were digitized and protein patterns analyzed using the Kepler 2D Gel Analysis System. Immunoglobulin heavy-chain binding protein (BiP), also known as 78 kD glucose regulated protein (Grp78), was identified immunologically and by comigration of recombinant Grp78. BiP is a luminal endoplasmic reticular (ER) protein that functions in the assembly and folding of nascent proteins as they enter the ER. The present results suggest a selective posttranslational modification of BiP following PFDA exposure. Single-dose exposure to PFDA was associated with a notable charge-modification of BiP that persists up to 30 days. PFOA, clofibrate, and DEHP had less effect in this regard. Our data suggest the likely nature of this PFDA-associated protein modification is associated with protein-phosphorylation. These results document the unique nature of PFDA's hepatotoxicity with respect to classic peroxisome proliferators and support the utility of 2D gel analysis in toxicity testing.

Effect of thyrotoxicosis on sarcoplasmic reticulum in rat skeletal muscle.

The effect of thyrotoxicosis on the capacity of fragmented sarcoplasmic reticulum (FSR) and crude homogenate (CH) to sequester Ca2+ was determined in rat muscle for the slow-twitch type I soleus (SOL), the fast-twitch type IIA deep region of the vastus lateralis (DVL),and the fast-twitch type IIB superficial region of the vastus lateralis (SVL). The maximal rate of Ca2+ uptake (Vmax) and Km were determined in both the CH and FSR preparations, and the total Ca2+ uptake capacity of the CH was determined. In the slow SOL, thyrotoxicosis increased the Vmax (8.20 +/- 0.96 vs. 15.70 +/- 0.92 mumol Ca2+ . g wet muscle-1 . min-1) and the total Ca2+ uptake (17.62 +/- 1.30 vs. 27.13 +/- 2.16 mumol Ca2+ . g wet muscle-1) of the CH preparation. Thyrotoxicosis increased the FSR yield 2.3-fold in the slow-twitch SOL; however, the kinetic characteristics (Vmax and Km) of these vesicles were not altered. Thyrotoxicosis had no effect on the CH and FSR preparations in either the type IIA or type IIB sample. These results can be explained by a thyroid hormone-mediated increase in the quantity of the sarcoplasmic reticulum in type I muscle and suggest no effect on the hormone on the qualitative nature of the Ca2+-enzyme interaction.

Hindlimb immobilization: length-tension and contractile properties of skeletal muscle.

The effect of hindlimb immobilization (IM) on the contractile properties of fast and slow skeletal muscle was studied in rats following various periods of IM ranging from 1 to 42 days; muscle atrophy, muscle, fiber, and sarcomere length, and the length-tension characteristics were determined after 42 days of IM. The slow-twitch soleus (SOL), the fast-twitch extensor digitorum longus (EDL), and the fast-twitch superficial region of the vastus lateralis (SVL) all showed rapid atrophy following the onset of IM, reaching a new reduced steady-state weight by day 21. After 42 days of IM the passive tension (g) and active twitch tension (g/cm2) plotted vs. muscle length (cm) were shifted to the left for the slow-twitch SOL, indicating a decreased extensibility compared with control muscles. The peak tetanic tension of the slow SOL declined to 47% of the control level of 2,893 +/- 125 g/cm2, whereas the fast EDL maintained 72% of its initial force of 4,392 +/- 229 g/cm2, and the fast SVL was unaltered by IM. Peak twitch tension and peak rate of tension development and decline fell rapidly in the slow SOL while remaining relatively unaltered in the fast-twitch muscles. Surprisingly, maximal isotonic shortening velocity was elevated in both fast and slow muscles with IM. These results indicate that IM produces muscle atrophy in fast as well as slow skeletal muscle and, in addition, causes fiber type-specific changes in the contractile properties.

Acid phosphatase and protease activities in immobilized rat skeletal muscles.

The effect of hind-limb immobilization on selected lysosomal enzyme activities was studied in rat hind-limb muscles composed primarily of type I, IIA, or IIB fibers. Following immobilization, acid protease and acid phosphatase both exhibited significant (P less than 0.05) increases in their activity per unit weight in all three fiber types. Acid phosphatase activity increased at day 14 of immobilization in the three muscles and returned to control levels by day 21. Acid protease activity also changed biphasically, displaying a higher and earlier rise than acid phosphatase. The pattern of change in acid protease, but not acid phosphatase, closely parallels observed muscle wasting. The present data therefore demonstrate enhanced proteolytic capacity of all three fiber types early during muscular atrophy. In addition, the data suggest a dependence of basal hydrolytic and proteolytic activities and their adaptive response to immobilization on muscle fiber composition.

Effect of disuse on sarcoplasmic reticulum in fast and slow skeletal muscle.

The effect of 6 wk of hindlimb immobilization on rat skeletal muscle sarcoplasmic reticulum (SR) was determined in the slow-twitch, type I soleus (SOL), the fast-twitch, type IIA deep region of the vastus lateralis (DVL), and the fast-twitch, type IIB superficial region of the vastus lateralis (SVL). Immobilization produced a significant decline in the Ca2+ uptake rate (Vmax) of SR vesicles from the slow SOL (0.930 +/- 0.116 to 0.365 +/- 0.071 mumol Ca2+ . mg-1 . min-1), while the SR Vmax increased in the fast SVL (2.763 +/- 0.133 to 5.209 +/- 0.687) and was unaltered in the DVL. Vesicles from the fast SVL and DVL also exhibited a higher total Ca2+ uptake capacity following immobilization. An evaluation of the time course of the immobilization-mediated effect revealed an increased Ca2+ uptake capacity in all three samples after 1 wk. In the SOL total Ca2+ uptake returned to control level after 2 wk, while in the fast-twitch muscles the higher capacities were maintained. The Ca2+-stimulated SR ATPase activity was not altered in any of the muscles studies, although the total SR ATPase activity increased twofold in the slow SOL.