Effect of short photoperiod on organ growth kinetics and serum hormone profile in pullets of domestic fowl, Gallus gallus domesticus

Effect of short photoperiod (SP; LD 6:18) treatment on serum hormone profile and growth rate of adrenal, thyroid, ovary, oviduct, liver and lymphoid organ was studied in rearing pullets (RIR breed) of 1 to day 90 old. Body weight and growth index of SP pullets were lesser as compared to pullets reared under LD 12:12. Except for ovary (recorded marginal increment), weights and growth indices of thyroid, adrenal and oviduct decreased under SP. Weight of liver and lymphoid organs was higher at 30 and 90 days, in SP pullets as compared to LD 12:12. Histometric data suggested that the transition from small to big follicles was slow in ovary of SP pullets, and also reduced follicular atresia was noted in SP pullets. Except for higher corticosterone level at 30 days and higher progesterone level at 30 and 60 days, relative levels of all the hormones at all other ages were lower in SP pullets. In general, the present observations suggested intraovarian changes in pullets exposed to SP.     

Retinal in the blood and liver of the fowl in relation to sex and maturity

1. Concentrations of retinal (vitamin A(1) aldehyde) in the plasma and liver of laying hens, mature cockerels, immature pullets and pullets undergoing sexual maturation have been measured. 2. The plasma of laying hens contained about 8mug. of retinal/100ml., about ten times that found in the plasma of mature cockerels and immature pullets. In laying hens that had received large doses of retinyl palmitate 8-4 weeks previously, the mean concentration of retinal was 18.3mug./100ml. of plasma. 3. The appearance of significant amounts of retinal in the plasma of maturing pullets coincided with hypertrophy of the oviduct, increase in concentration of plasma lipid and onset of egg-laying. 4. Retinal was present in the livers of all types of fowl examined and the concentrations, which ranged from 0.2 to 5.8mug./g. wet wt., were highly correlated (r=0.79) with the concentrations of liver retinyl esters, which ranged from 92 to 1530mug./g. wet wt.     

Regulation of avidin accumulation by prostaglandins in chick oviduct culture

Regulation of avidin accumulation by prostaglandins (PGs) and their inhibitors was studied in chick oviduct organ culture. Avidin was induced neither by progesterone nor PGF2 alpha in the oviduct of immature chicks. By progesterone and PGs, a high avidin synthesis was induced when the chicks received diethylstilbestrol (DES) for 7 days. Enhanced avidin production was observed by PGF2 alpha, PGE1 and PGE2, whereas PGA2 and PGB2 had a slight inhibitory effect and PGA1 and PGB1 had no effect on avidin production. PGF2 alpha was most effective at a concentration of 10-20 micrograms/ml. The effects of progesterone and PGF2 alpha were not additive. Mefenamic acid, at concentrations of 40 and 60 micrograms/ml, inhibited 50 and 85%, respectively, of the avidin synthesis induced by progesterone, whereas the inhibition of the total protein synthesis was only 20%, and this only by the higher concentration of the drug. Tolfenamic and meclofenamic acid were also inhibitory in the case of progestin-induced avidin synthesis. These studies indicate that the PGs (F2 alpha, E1 and E2) might be involved in the avidin induction in the chick differentiated oviduct. The specific inhibition of the progesterone-dependent avidin synthesis by the PG inhibitors suggests that PGs may be connected with the progesterone action in the oviduct. We propose that the avidin synthesis by the chick oviduct might be considered as a model system for studying PG effects on the synthesis of a specific protein.     

Radioimmunoassay of the A prostaglandins

Antibodies to the (PGA) prostaglandin A were produced in rabbits immunized with a conjugate of PGE2 covalently linked to (BSA) bovine serum albumin by reaction with carbodiimide reagent. A radioimmunoassay was developed using dextran-coated charcoal to separate the free from antibody bound PGA1-3H. The sensitivity of the method was found to be 100 picograms/ml of plasma. Ethyl acetate was used for extraction of plasma and the various classes of PGs were separated by silicic acid column chromatography. Recovery of PGA1-3H throughout the entire procedure was 65-75%. The antibody showed progressively decreasing affinity to PGA2, PGA1, PGE2, PGE1, PGB2, and PGF2alpha, respectively. The mean plasma PGA level in adult males (N=13) was found to be 1.39 + or - 0.55 ng/ml, and 1.62 + or - 0.52 ng/ml in adult females (N=7). Corresponding plasma and serum samples were found to give essentially similar results. Plasma PGA levels in adult males treated with indomethacin for rheumatoid arthritis were 0.18 + or - 0.15 ng/ml (P 0.001 in comparison with the normal adult males). This method is sufficiently sensitive, precise, and rapid to allow the routine estimation of the PGAs in biological samples.     

Genes involved in the synthesis and degradation of matrix polysaccharide in Actinobacillus actinomycetemcomitans and Actinobacillus pleuropneumoniae biofilms

Biofilms are composed of bacterial cells embedded in an extracellular polysaccharide matrix. A major component of the Escherichia coli biofilm matrix is PGA, a linear polymer of N-acetyl-D-glucosamine residues in beta(1,6) linkage. PGA mediates intercellular adhesion and attachment of cells to abiotic surfaces. In this report, we present genetic and biochemical evidence that PGA is also a major matrix component of biofilms produced by the human periodontopathogen Actinobacillus actinomycetemcomitans and the porcine respiratory pathogen Actinobacillus pleuropneumoniae. We also show that PGA is a substrate for dispersin B, a biofilm-releasing glycosyl hydrolase produced by A. actinomycetemcomitans, and that an orthologous dispersin B enzyme is produced by A. pleuropneumoniae. We further show that A. actinomycetemcomitans PGA cross-reacts with antiserum raised against polysaccharide intercellular adhesin, a staphylococcal biofilm matrix polysaccharide that is genetically and structurally related to PGA. Our findings confirm that PGA functions as a biofilm matrix polysaccharide in phylogenetically diverse bacterial species and suggest that PGA may play a role in intercellular adhesion and cellular detachment and dispersal in A. actinomycetemcomitans and A. pleuropneumoniae biofilms.     

Leukotriene B4 omega-hydroxylase in rat liver microsomes: identification as a cytochrome P-450 that catalyzes prostaglandin A1 omega-hydroxylation, and participation of cytochrome b5

The omega-hydroxylation of leukotriene B4 (LTB4) by rat liver microsomes requires NADPH and molecular oxygen, suggesting that the hydroxylation is catalyzed by a cytochrome P-450 (P-450)-linked monooxygenase system. The reaction is inhibited by CO, and the inhibition is reversed by irradiation of light at 450 nm in a light-intensity-dependent manner. The extent of the reversal is strongly dependent on the wavelength of the light used, the 450-nm light is most efficient. The finding provides direct evidence for the identification of the LTB4 omega-hydroxylase as a P-450. The P-450 seems to be also responsible for prostaglandin A1 (PGA1) omega-hydroxylation, but not for lauric acid omega-hydroxylation. The LTB4 omega-hydroxylation is competitively inhibited by PGA1, but not affected by lauric acid. The Ki value for PGA1 of 38 microM agrees with the Km value for PGA1 omega-hydroxylation of 40 microM. LTB4 inhibits the PGA1 omega-hydroxylation by rat liver microsomes in a competitive manner with the Ki of 43 microM, which is consistent with the Km for the LTB4 omega-hydroxylation of 42 microM. An antiserum raised against rabbit pulmonary PG omega-hydroxylase (P-450p-2) inhibits slightly the omega-hydroxylations of LTB4 and PGA1, while it has stronger inhibitory effect on lauric acid omega-hydroxylation. In addition to NADPH-cytochrome P-450 reductase, cytochrome b5 appears to participate in the LTB4 omega-hydroxylating system, since the reaction is inhibited by an antibody raised against the cytochrome b5 as well as one raised against the reductase.     

Protective role of p21(Waf1/Cip1) against prostaglandin A2-mediated apoptosis of human colorectal carcinoma cells.

Prostaglandin A2 (PGA2) suppresses tumor growth in vivo, is potently antiproliferative in vitro, and is a model drug for the study of the mammalian stress response. Our previous studies using breast carcinoma MCF-7 cells suggested that p21(Waf1/Cip1) induction enabled cells to survive PGA2 exposure. Indeed, the marked sensitivity of human colorectal carcinoma RKO cells to the cytotoxicity of PGA2 is known to be associated with a lack of a PGA2-mediated increase in p21(Waf1/Cip1) expression, inhibition of cyclin-dependent kinase activity, and growth arrest. To determine if cell death following exposure to PGA2 could be prevented by forcing the expression of p21(Waf1/Cip1) in RKO cells, we utilized an adenoviral vector-based expression system. We demonstrate that ectopic expression of p21(Waf1/Cip1) largely rescued RKO cells from PGA2-induced apoptotic cell death, directly implicating p21(Waf1/Cip1) as a determinant of the cellular outcome (survival versus death) following exposure to PGA2. To discern whether p21(Waf1/Cip1)-mediated protection operates through the implementation of cellular growth arrest, other growth-inhibitory treatments were studied for the ability to attenuate PGA2-induced cell death. Neither serum depletion nor suramin (a growth factor receptor antagonist) protected RKO cells against PGA2 cytotoxicity, and neither induced p21(Waf1/Cip1) expression. Mimosine, however, enhanced p21(Waf1/Cip1) expression, completely inhibited RKO cell proliferation, and exerted marked protection against a subsequent PGA2 challenge. Taken together, our results directly demonstrate a protective role for p21(Waf1/Cip1) during PGA2 cellular stress and provide strong evidence that the implementation of cellular growth arrest contributes to this protective influence.     

Relative turnover numbers of maize endosperm and potato tuber ADP-glucose pyrophosphorylases in the absence and presence of 3-phosphoglyceric acid

Adenosine diphosphate glucose pyrophosphorylase (AGPase; EC 2.7.7.27) synthesizes the starch precursor, ADP-glucose. It is a rate-limiting enzyme in starch biosynthesis and its activation by 3-phosphoglyceric acid (3PGA) and/or inhibition by inorganic phosphate (Pi) are believed to be physiologically important. Leaf, tuber and cereal embryo AGPases are highly sensitive to these effectors, whereas endosperm AGPases are much less responsive. Two hypotheses can explain the 3PGA activation differences. Compared to leaf AGPases, endosperm AGPases (i) lack the marked ability to be activated by 3PGA or (ii) they are less dependent on 3PGA for activity. The absence of purified preparations has heretofore negated answering this question. To resolve this issue, heterotetrameric maize ( Zea mays L.) endosperm and potato ( Solanum tuberosum L.) tuber AGPases expressed in Escherichia coli were isolated and the relative amounts of enzyme protein were measured by reaction to antibodies against a motif resident in both small subunits. Resulting reaction rates of both AGPases are comparable in the presence but not in the absence of 3PGA when expressed on an active-protein basis. We also placed the potato tuber UpReg1 mutation into the maize AGPase. This mutation greatly enhances 3PGA sensitivity of the potato AGPase but it has little effect on the maize AGPase. Thirdly, lysines known to bind 3PGA in potato tuber AGPase, but missing from the maize endosperm AGPase, were introduced into the maize enzyme. These had minimal effect on maize endosperm activity. In conclusion, the maize endosperm AGPase is not nearly as dependent on 3PGA for activity as is the potato tuber AGPase.     

A novel Method for the selective recovery and purification of γ‐polyglutamic acid from Bacillus licheniformis fermentation broth

Microbially produced gamma‐polyglutamic acid (γ‐PGA) is a commercially important biopolymer with many applications in biopharmaceutical, food, cosmetic and waste‐water treatment industries. Owing to its increasing demand in various industries, production of γ‐PGA is well documented in the literature, however very few methods have been reported for its recovery. In this paper, we report a novel method for the selective recovery and purification of γ‐PGA from cell‐free fermentation broth of Bacillus licheniformis. The cell‐free fermentation broth was treated with divalent copper ions, resulting in the precipitation of γ‐PGA, which was collected as a pellet by centrifugation. The pellet was resolubilized and dialyzed against de‐ionized water to obtain the purified γ‐PGA biopolymer. The efficiency and selectivity of γ‐PGA recovery was compared with ethanol precipitation method. We found that 85% of the original γ‐PGA content in the broth was recovered by copper sulfate‐induced precipitation, compared to 82% recovery by ethanol precipitation method. Since ethanol is a commonly used solvent for protein precipitation, the purity of γ‐PGA precipitate was analyzed by measuring proteins that co‐precipitated with γ‐PGA. Of the total proteins present in the broth, 48% proteins were found to be co‐precipitated with γ‐PGA by ethanol precipitation, whereas in copper sulfate‐induced precipitation, only 3% of proteins were detected in the final purified γ‐PGA, suggesting that copper sulfate‐induced precipitation offers better selectivity than ethanol precipitation method. Total metal content analysis of the purified γ‐PGA revealed the undetectable amount of copper ions, whereas other metal ions detected were in low concentration range. The purified γ‐PGA was characterized using infrared spectroscopy. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

粪产碱杆菌青霉素G酰化酶在枯草芽孢杆菌中的表达、纯化及其性质

利用PCR技术克隆了粪产碱杆菌 (Alcaligenesfaecalis,CICCAS1.76 7)青霉素G酰化酶 (pencillinGacylase ,PGA)基因 (GenBank登录号AF4 5 5 35 6 )。通过构建工程菌E .coli(pETAPGA) ,该酶在大肠杆菌中获得了表达 ,表达产物分泌到周质空间。进一步构建的工程菌B .subtilis (pMAPGA)和B .subtilis(pBAPGA)实现了该酶的胞外分泌表达。分泌表达的最高表达量为 6 5 3u/L ,比野生型A .faecalis表达量高 10 9倍。表达产物经硫酸铵分级沉淀和DEAE SepharoseCL 6B两步纯化 ,纯度提高 86倍 ,活力回收率达到 81% ,纯化后的PGA活力为 1.4 6 9u/mg。研究表明 ,PGA家族成员中只有粪产碱杆菌PGA和巨大芽孢杆菌PGA可以在枯草芽孢杆菌中分泌表达。与巨大芽孢杆菌PGA相比 ,粪产碱杆菌PGA的最适pH值为 8.0 ,最适温度为 6 0°C ,而且在有机溶剂中具有更强的稳定性。该酶在水相中具有较低的头孢氨苄合成活力。本研究为粪产碱杆菌PGA的获得提供了新的途径。     

Roles of pgaABCD genes in synthesis, modification, and export of the Escherichia coli biofilm adhesin poly-beta-1,6-N-acetyl-D-glucosamine

The linear homopolymer poly-beta-1,6-N-acetyl-D-glucosamine (beta-1,6-GlcNAc; PGA) serves as an adhesin for the maintenance of biofilm structural stability in diverse eubacteria. Its function in Escherichia coli K-12 requires the gene products of the pgaABCD operon, all of which are necessary for biofilm formation. PgaC is an apparent glycosyltransferase that is required for PGA synthesis. Using a monoclonal antibody directed against E. coli PGA, we now demonstrate that PgaD is also needed for PGA formation. The deletion of genes for the predicted outer membrane proteins PgaA and PgaB did not prevent PGA synthesis but did block its export, as shown by the results of immunoelectron microscopy (IEM) and antibody adsorption assays. IEM also revealed a conditional localization of PGA at the cell poles, the initial attachment site for biofilm formation. PgaA contains a predicted beta-barrel porin and a superhelical domain containing tetratricopeptide repeats, which may mediate protein-protein interactions, implying that it forms the outer membrane secretin for PGA. PgaB contains predicted carbohydrate binding and polysaccharide N-deacetylase domains. The overexpression of pgaB increased the primary amine content (glucosamine) of PGA. Site-directed mutations targeting the N-deacetylase catalytic activity of PgaB blocked PGA export and biofilm formation, implying that N-deacetylation promotes PGA export through the PgaA porin. The results of previous studies indicated that N-deacetylation of beta-1,6-GlcNAc in Staphylococcus epidermidis by the PgaB homolog, IcaB, anchors it to the cell surface. The deletion of icaB resulted in release of beta-1,6-GlcNAc into the growth medium. Thus, covalent modification of beta-1,6-GlcNAc by N-deacetylation serves distinct biological functions in gram-negative and gram-positive species, dictated by cell envelope differences.     

Colocalization of polyol-metabolizing enzymes and immunological detection of fructated proteins in the female reproductive system of the rat

The expression of aldose reductase (AR) and sorbitol dehydrogenase (SDH), which, in concert, catalyze the conversion of glucose to fructose via sorbitol, in the rat ovary, oviduct, and uterus, was investigated by immunohistochemical and biochemical analyses. The activities and protein levels of AR and SDH were higher in the ovary than in the oviduct and uterus. A strong immunoreactivity to the anti-AR antibody was observed in granulosa cells and epithelia of the oviduct, endometrium, and endometrial glands, and virtually the same tissues were strongly stained with the anti-SDH antibody. The application of an anti-fructated lysine antibody, which detects an adduct of fructose with the epsilon-amino group of lysine in proteins, in this study detected marked staining mainly in the egg and luminal surface of the oviductal epithelia. Collectively, these data indicate that fructose is produced by coordinately expressed AR and SDH in the egg and epithelia of the oviduct and suggest that the resulting sorbitol and fructose can be used as energy sources for spermatozoa motility during the fertilization process. The abundance of AR compared with SDH suggests that it also plays an additional role in the reproductive system, which might include a source of reducing power and protection against toxic carbonyl compounds.     

Protective effect of prostaglandin A2 against menadione-induced cell injury in cultured porcine aorta endothelial cells

Prostaglandin A2 (PGA2) stimulates the biosynthesis of gamma-glutamylcysteine synthetase and elevates glutathione (GSH) contents in cultured mammalian cells. To clarify the importance of gamma-glutamylcysteine synthetase induction in the defence of endothelial cells against oxidative stress, the effect of PGA2 on menadione (2-methyl-1,4-naphthoquinone)-induced cell injury was examined. Incubation of porcine aorta endothelial cells with menadione produced marked loss of cellular GSH and protein sulfhydryl groups, followed by leakage of lactic dehydrogenase (LDH) into the culture medium. The LDH leakage and modification of protein thiol was, however, completely prevented by pretreatment of the cells with PGA2. The protective effect of PGA2 was more potent than that of cysteine delivery agents such as methionine, N-acetylcysteine or 2-oxo-4-thiazolidine carboxylic acid (OTC). The results suggest that cellular GSH plays an important role in the defence against oxidative stress, and induction of gamma-glutamylcysteine synthetase is effective for protecting vascular endothelial cells.     

Inhibition of vesicular stomatitis virus replication by prostaglandin A1 in Aedes albopictus cells

Cyclopentenone prostaglandins (PGs) exhibit antiviral activity against RNA and DNA viruses in mammalian cell lines, and this effect has been associated with the induction of a heat shock protein (hsp70). We investigated the effect of prostaglandin A1 (PGA1) on the replication of vesicular stomatitis virus (VSV) in Aedes albopictus (mosquito) cells. PGA1 was found to inhibit VSV replication dose dependently. Virus yield was reduced to 50% (3 microg PGA1/ml) and to 95% with 8 microg PGA1/ml. Even with the dramatic reduction of virus production observed in cells treated with PGA1, VSV-specific protein synthesis was unaltered. Treatment of cells with PGA1 (5 microg/ml) stimulated the synthesis of a polypeptide identified as a heat-shock protein (hsp) by immunoblot analysis. PGA1 induced hsp70 synthesis in uninfected cells. However, in VSV-infected cells the induction of hsp70 by PGA1 was reduced. This is the first report of antiviral effects of PGs affecting the replication of VSV in a mosquito cell line.     

Poly-γ-d-glutamic acid and protective antigen conjugate vaccines induce functional antibodies against the protective antigen and capsule of Bacillus anthracis in guinea-pigs and rabbits

Anthrax is a lethal infectious disease caused by the spore-forming Bacillus anthracis . The two major virulence factors of B. anthracis are exotoxin and the poly-γ- d -glutamic acid (PGA) capsule. The three components of the exotoxin, protective antigen (PA), lethal factor and edema factor act in a binary combination, which results in massive edema and organ failure in the progress of anthrax disease. The antiphagocytic PGA capsule disguises the bacilli from immune surveillance and allows unimpeded growth of bacilli in the host. Because PA can elicit a protective immune response, it has been a target of the anthrax vaccine. In addition to PA, efforts have been made to include PGA as a component of the anthrax vaccine. In this study, we report that PA–PGA conjugates induce expressions of anti-PA, anti-PGA and toxin-neutralizing antibodies in guinea-pigs and completely protect guinea-pigs against a 50 × LD₅₀ challenge with fully virulent B. anthracis spores. Polyclonal rabbit antisera produced against either PA or ovalbumin conjugated to a PGA-15mer offer a partial passive protection to guinea-pigs against B. anthracis infection, indicating that anti-PGA antibodies play a protective role. Our results demonstrate that PA–PGA conjugate vaccines are effective in the guinea-pig model, in addition to the previously reported mouse model.     

Additional stabilization of penicillin G acylase-agarose derivatives by controlled chemical modification with formaldehyde.

We have tested the effect of chemical modifications with formaldehyde on the activity/stability of immobilized derivatives of the enzyme penicillin G acylase (PGA). These derivatives were previously stabilized through enzyme-support multipoint covalent attachment. We carried out very different chemical treatments of our derivatives by testing the effect of different variables which control the intensity and the nature of these amine-formaldehyde reactions. The variables tested were: formaldehyde concentration, pH, time, and temperature. We also developed a colorimetric titration of the free amine groups on immobilized PGA in order to evaluate the extension of the reaction between formaldehyde and the amine groups of the enzyme. As a consequence of these studies, we have been able to get additional stabilizations of our previously stabilized-immobilized derivatives: e.g. a factor of 24-fold was achieved in terms of stabilization against irreversible thermal inactivation. The integrated effect of additional chemical modification plus previous multipoint covalent attachment has allowed us to prepare PGA derivatives which are 50,000 more thermostable than native PGA as well as most of the commercial PGA derivatives.     

Immunologically distinct binding molecules for progesterone and RU38486 in the chick oviduct cytosol

[3H]Progesterone and [3H]RU38486 binding in the chick oviduct cytosol is associated with macromolecules which sediment as 8 S and 4 S moieties, respectively, in molybdate-containing 5-20% sucrose gradients. The [3H]progesterone binding could be displaced by excess progesterone, but not by RU38486. Conversely, the [3H]RU38486 binding was able to compete with RU38486 but not by excess progesterone. A preparation containing antibodies against chick oviduct progesterone receptor recognized only the [3H]progesterone-receptor complex but not the 4 S, [3H]RU38486 binding component of the chick cytosol. In the calf uterus cytosol, [3H]R5020 (a synthetic progestin) and [3H]RU38486 were associated with 8 S molecules and the peaks of radioactivity were displaceable upon preincubation with radionert steroids. In addition, the complexes were recognized by antibodies to chick oviduct progesterone receptor. Our data suggest that in the chick oviduct cytosol, RU38486 does not bind to progesterone receptor, but interacts with an immunologically distinct macromolecule.     

Neuropharmacology of amide derivatives of P-GABA.

Three lipophilic amide derivatives of phthaloyl-GABA (P-GABA), namely gamma-phthalimido N-amyl butyramide (PGA), gamma-phthalimido-N-hexylbutyramide (PGH) and gamma-phthalimido N-phenylbutyramide (PGP), were synthesized and evaluated for their hypnotic and anticonvulsant activities in mice. Both PGA and PGH showed moderate hypnotic activity but PGP had no such action. Picrotoxin (0.08 mg/kg) a non-specific GABA antagonist completely abolished the hypnotic action of PGA in subconvulsive doses. Bicuculline (0.04 mg/kg) a GABAA antagonist, 3-mercaptopropionic acid (6 mg/kg) a GAD inhibitor at subconvulsive doses failed to neutralise the hypnotic action of PGA. On the other hand, PGA showed significant protection only against picrotoxin-induced convulsions, but was inactive against other convulsants tested. PGP which has no hypnotic activity, and has a mild anticonvulsant action in all the models except picrotoxin. A definite correlation was observed between the brain GABA and the hypnotic activity of PGA. However the present data indicate that the hypnotic and anticonvulsant activities are mediated probably through different brain GABA-ergic mechanisms.     

Prostaglandin A1 inhibits rotenone-induced apoptosis in SH-SY5Y cells

The degeneration of nigral dopamine neurons in Parkinson's disease (PD) reportedly involves a defect in brain mitochondrial complex I in association with the activation of nuclear factor-kappaB (NF-kappaB) and caspase-3. To elucidate molecular mechanisms possibly linking these events, as well as to evaluate the neuroprotective potential of the cyclopentenone prostaglandin A1 (PGA1), an inducer of heat shock proteins (HSPs), we exposed human dopaminergic SH-SY5Y cells to the complex I inhibitor rotenone. Dose-dependent apoptosis was preceded by the nuclear translocation of NF-kappaB and then the activation of caspase-3 over the ensuing 24 h. PGA1 increased the expression of HSP70 and HSP27 and protected against rotenone-induced apoptosis, without increasing necrotic death. PGA1 blocked the rotenone-induced nuclear translocation of NF-kappaB and attenuated, but did not abolish, the caspase-3 elevation. Unexpectedly, the caspase-3 inhibitor, Ac-DEVD.CHO (DEVD), at a concentration that completely prevented the caspase-3 elevation produced by rotenone, failed to protect against apoptosis. These results suggest that complex I deficiency in dopamine cells can induce apoptosis by a process involving early NF-kappaB nuclear translocation and caspase-3 activation. PGA1 appears to protect against rotenone-induced cell death by inducing HSPs and blocking nuclear translocation of NF-kappaB in a process that attenuates caspase-3 activation, but is not mediated by its inhibition.