Pi Form Research Articles

Flavor SU(3) symmetry in charmlessBdecays

QCD sum rules are used to estimate the flavour SU(3)-symmetry violation in two-body B decays to pions and kaons. In the factorizable amplitudes the SU(3)-violation manifests itself in the ratio of the decay constants f_K/f_pi and in the differences between the B->K, B_s->K and B->pi form factors. These effects are calculated from the QCD two-point and light-cone sum rules, respectively, in terms of the strange quark mass and the ratio of the strange and nonstrange quark-condensate densities. Importantly, QCD sum rules predict that SU(3) breaking in the heavy-to-light form factors can be substantial and does not vanish in the heavy-quark mass limit. Furthermore, we investigate the strange-quark mass dependence of nonfactorizable effects in the B->K pi decay amplitudes. Taking into account these effects we estimate the accuracy of several SU(3)-symmetry relations between charmless B-decay amplitudes.

Spectator interactions and factorization in B→π lν decay

We investigate the factorization of different momentum modes that appear in matrix elements for exclusive B meson decays into light energetic particles for the specific case of B -> pi form factors at large pion recoil. We first integrate out hard modes with virtualities of order m_b^2 (m_b being the heavy quark mass), and then hard-collinear modes with virtualities m_b Lambda (Lambda being the strong interaction scale). The resulting effective theory contains soft and collinear fields with virtualities Lambda^2. We prove a previously conjectured factorization formula for B -> pi form factors in the heavy quark limit to all orders in alpha_s, paying particular attention to `endpoint singularities' that might have appeared in hard spectator interactions.

Identification in Saccharomyces cerevisiae of a New Stable Variant of Alkyl Hydroperoxide Reductase 1 (Ahp1) Induced by Oxidative Stress

Yeasts lacking cytoplasmic superoxide dismutase (Cu,Zn-SOD) activity are permanently subjected to oxidative stress. We used two-dimensional PAGE to examine the proteome pattern of Saccharomyces cerevisiae strains lacking Cu,Zn-SOD. We found a new stable form of alkyl hydroperoxide reductase 1 (Ahp1) with a lower isoelectric point. This form was also present in wild type strains after treatment with tert-butyl hydroperoxide. In vitro enzyme assays showed that Ahp1p had lower specific activity in strains lacking Cu,Zn-SOD. We studied three mutants presenting a reduced production of the low pI variant under oxidative stress conditions. Two of the mutants (C62S and S59D) were totally inactive, thus suggesting that the acidic form of Ahp1p may only appear when the enzyme is functional. The other mutant (S59A) was active in vitro and was more resistant to inactivation by tert-butyl hydroperoxide than the wild type enzyme. Furthermore, the inactivation of Ahp1p in vitro is correlated with its conversion to the low pI form. These results suggest that in vivo during some particular oxidative stress (alkyl hydroperoxide treatment or lack of Cu,Zn-SOD activity but not hydrogen peroxide treatment), the catalytic cysteine of Ahp1p is more oxidized than cysteine-sulfenic acid (a natural occurring intermediate of the enzymatic reaction) and that cysteine-sulfinic acid or cysteine-sulfonic acid variant may be inactive.

A new autotuning algorithm for PID controllers using dead-beat format

A novel algorithm for PID controllers based on dead-beat control and fuzzy inference mechanism is presented in this paper. The proposition is an extension of the work by the authors where the PI form of the algorithm was presented. The inclusion of the derivative term makes the method suitable for application in all types of processes including the ones having high rate disturbances. The proposed algorithm seems to be a complete and generalized PID autotuner as can be seen by the simulated and experimental results. In all the cases the method shows substantial improvement over the controller tuned with Ziegler Nichol's formula and the PI controller proposed in R. Bandyopadhyay, D. Patranabis, A fuzzy logic based PI autotuner, ISA Transactions 37 (1998) 227–235.

The Aspartyl Replacement of the Active Site Histidine in Histidine-containing Protein, HPr, of the Escherichia coliPhosphoenolpyruvate:Sugar Phosphotransferase System Can Accept and Donate a Phosphoryl Group: SPONTANEOUS DEPHOSPHORYLATION OF ACYL-PHOSPHATE AUTOCATALYZES AN INTERNAL CYCLIZATION

The active site residue, His(15), in histidine-containing protein, HPr, can be replaced by aspartate and still act as a phosphoacceptor and phosphodonor with enzyme I and enzyme IIA(glucose), respectively. Other substitutions, including cysteine, glutamate, serine, threonine, and tyrosine, failed to show any activity. Enzyme I K(m) for His(15) --> Asp HPr is increased 10-fold and V(max) is decreased 1000-fold compared with wild type HPr. The phosphorylation of Asp(15) led to a spontaneous internal rearrangement involving the loss of the phosphoryl group and a water molecule, which was confirmed by mass spectrometry. The protein species formed had a higher pI than His(15) --> Asp HPr, which could arise from the formation of a succinimide or an isoimide. Hydrolysis of the isolated high pI form gave only aspartic acid at residue 15, and no isoaspartic acid was detected. This indicates that an isoimide rather than a succinimide is formed. In the absence of phosphorylation, no formation of the high pI form could be found, indicating that phosphorylation catalyzed the formation of the cyclization. The possible involvement of Asn(12) in an internal cyclization with Asp(15) was eliminated by the Asn(12) --> Ala mutation in His(15) --> AspHPr. Asn(12) substitutions of alanine, aspartate, serine, and threonine in wild type HPr indicated a general requirement for residues capable of forming a hydrogen bond with the Nepsilon(2) atom of His(15), but elimination of the hydrogen bond has only a 4-fold decrease in k(cat)/K(m).

Contractile properties of rabbit psoas muscle fibres inhibited by beryllium fluoride.

The structure of truncated, recombinant Dictyostelium myosin motor domain complexed with Mg.ADP and slowly dissociating analogues of Pi has previously been characterized as two main states (S1-MgADP plus BeFx vs. A1F4- or Vi). The BeFx bound state is thought to mimic the weak actin-binding M.ATP complex, while the states with A1F4- or Vi bound mimic the M.ADP.Pi state. While the effects of A1F4- and Vi on fibre mechanics have been previously described (Chase et al., 1994, 1993), the effects of BeFx have not been characterized in detail. At pCa 4.5 (12 degrees C), we measured (i) steady-state isometric tension, (ii) stiffness (KS; 1 kHz sinusoids), and (iii) unloaded shortening velocity (Vu; slack test) in single skinned muscle fibres from rabbit psoas. Results were compared when tension was inhibited with either BeFx or 2,3-butanedione-monoxime (BDM) or modulated by altering myoplasmic [Ca2+]. With 3 mM total fluoride, 1 mM BeFx inhibited both tension and KS by approximately 50% (compared to 7-10 mM BDM and 50-100 microM A1F4-). Increasing [BeFx] to 10 mM further reduced tension to approximately 15% P0, but had little further effect on KS; with BDM and altered [Ca2+], KS scaled more proportionately with tension. Inhibition of tension and KS by BeFx was more rapidly reversible, compared with slow recovery from tension inhibition with A1F4- or Vi. Vu exhibited a complex dependence on [BeFx], being relatively unaffected by concentrations < or = 1 mM, and becoming inhibited steeply for [BeFx] above this level. With BDM, Vu co-varied more directly with force. Our results suggest that BeFx may induce a different cross-bridge state in fibres than do A1F4- or Vi, but all three analogues of Pi form complexes that mimic crossbridge states that follow ATP hydrolysis.

Gibberellin-photoaffinity labelling of two polypeptides in plant plasma membranes.

Two polypeptides of M(r) 68 kDa and 18 kDa were gibberellin (GA)-photoaffinity labelled in vitro in plasma membrane preparations from oat (Avena sativa L.) aleurone and from leaves and stems of wild-type and GA-sensitivity mutants of different species. Labelling of these polypeptides could be competed by biologically active, but not by inactive, GAs, indicating the likely biological significance of these interactions. On 2-dimensional gels the radiolabelled polypeptides were each resolved as one intensely labelled low abundance spot with a slightly lower pl form adjacent to it. There was a strong pH dependency for both labelling events, which correlated well with pH values at which GA are known to be most biologically active. A semi-dwarf GA-sensitivity mutant of sweet pea (Lathyrus odoratus L.), lb, showed reduced photoaffinity labelling of both polypeptides compared with the wild type, Lb. In the GA-insensitive Arabidopsis thaliana mutant gai, the level of labelling was the same as in wild type, GAI. This is the first report of GA-binding proteins in plant plasma membranes. Some preliminary sequence data are given for one of the labelled polypeptides. We discuss these mutants and consider their possible roles in GA perception or action.

Identification of glutathione S-transferase p-1 as the class pi form dominantly expressed in mouse hepatic adenomas.

To clarify which of the two genes for pi class glutathione S‐transferases (GSTs) (p‐1 and p‐2) is dominantly expressed in mouse hepatic adenomas, the relative mRNA levels were examined by means of the reverse transcription‐polymerase chain reaction (RT‐PCR). Hepatic adenomas were induced in male and female B6C3F1 mice by diethylnitrosamine treatment. Northern blot analysis revealed that pi class mRNA levels were decreased in adenomas of male mice, but increased in those of females, with reference to the respective surrounding non‐adenoma tissues. In contrast to the marked sex difference in surrounding tissues, pi class GST mRNA levels in adenomas were almost the same in both males and females. To evaluate p‐1 and p‐2 mRNA levels separately, the products of RT‐PCR employing primers common for both cDNAs were digested with the endonuclease BanI (specific for p‐2) and then resolved by electrophoresis. The p‐1 mRNA was thus found to be dominant in adenomas of both female and male mice. The p‐2 mRNA levels were increased in the lesions as compared with those in the surrounding non‐adenoma tissues. Recombinant p‐1 and p‐2 proteins were expressed in Escherichia coli. Unlike p‐1, the p‐2 protein did not show any significant activity towards 1‐chloro‐2,4‐dinitrobenzene and did not bind to S‐hexylglutathione‐Sepharose despite immunological cross‐reactivity. The dominant pi class form in adenomas could also be identified as p‐1 by its binding to S‐hexylglutathione‐Sepharose. Single radial immunodiffusion analyses confirmed that the p‐1 protein levels were in line with the mRNA findings, i.e., 1.9±0.3 mg/g adenoma as compared to 6.5±1.2 mg/g non‐adenoma tissue for males and 2.2±0.6 mg/g as compared to 0.7±0.2 mg/g for females. The results thus indicated that the change of pi class forms in adenomas is caused mainly by alteration in the p‐1 level and the contribution of p‐2 is minimal.

Conformal symmetry on the light cone and nonleading twist distribution amplitudes of a massive vector meson

A complete set of asymptotic three particle light cone distribution amplitudes of twist 3 and 4 for a transversely polarized massive vector meson built out of massless current quarks is constructed. The method used is based on a modified conformal projectors technique which allows to handle kinematical corrections due to a finite hadron mass. Consequences of our finding for the \rho -meson hard diffractive electroproduction and \gamma \rho \pi form factor are discussed. Our results may imply a breakdown of OPE for some exclusive processes beyond the leading twist level.

Putative Folding Pathway of Insulin-like Growth Factor-I

Insulin-like growth factor-1 (IGF-I) has three disulfide bonds and refolding of the fully reduced molecule generates varying ratios of correctly (PII) and incorrectly (PI) folded forms via several intermediates. All of the intermediates have the disulfide bond between Cys18 and 61 formed, indicating that formation of this disulfide is the first step in refolding. In order to further understand the refolding pathway, two intermediate forms, PIII with the additional disulfide Cys(6/47) formed and PIIIawith Cys(6/48) formed, were isolated. The oxidation of the remaining Cys48 and 52 in PIII and Cys47 and 52 in PIIIawould lead to PI and PII, respectively; however, air oxidation of these resulted in a rapid reshuffling into other intermediates as well as folding into the fully oxidized forms, and this occurred whether refolding was started with PIII or PIIIa. When oxidation occurred in the presence of an excess of oxidized glutathione, the predominant species generated were various glutathione adducts regardless of the initial intermediate form, indicating that formation of the last disulfide bond is not a favorable process relative to disulfide exchange when excess disulfides from oxidized glutathione are present. Interestingly, if 80 μmcopper sulfate, an oxidant, is added to the refolding buffer, PIII resulted in formation of the PI form alone, whereas PIIIaresulted in the PII form alone. It was concluded from these results that the intermediate forms of IGF-1 can rapidly reshuffle between different disulfide structures, and that formation of the last disulfide bond is not as favorable a process as the conversion to other intermediates. The oxidation to form the last disulfide bond in PIII or PIIIais accelerated and hence the interconversion to other intermediates is kinetically minimized only in the presence of copper sulfate. It appears, therefore, that the two intermediate forms, PIII and PIIIa, are the precursors of the corresponding fully oxidized forms, but their conversions are not energetically a favorable process.

The immunohistochemical localization of drug-metabolizing enzymes in prostate cancer.

The major groups of enzymes involved in activating and detoxifying therapeutic drugs, not least several anti-cancer drugs, include the cytochromes P450 (P450s), epoxide hydrolase, and glutathione S-transferases (GSTs). The expression of these enzymes in malignant tumours is one possible mechanism of anti-cancer drug resistance. This study has investigated the presence, cellular localization, and distribution of drug-metabolizing enzymes in prostate cancer. The P450 subfamilies CYP1A, CYP2C, and CYP3A were present in 63, 25, and 61 per cent of tumours, respectively. Epoxide hydrolase was identified in 96 per cent of tumours. GST-alpha and GST-mu were expressed in 29 and 41 per cent of tumours, respectively, while there was no immunoreactivity for the pi form of GST. The absence of GST-pi in prostate cancer contrasts with the frequent expression of GST-pi observed in other types of malignant tumour. In non-neoplastic prostatic epithelium, there was expression of CYP1A, CYP2C, epoxide hydrolase, and the different forms of GST, while there was no apparent immunoreactivity for CYP3A.

ELISA procedures for the quantitation of glutathione transferases in the urine

The proximal portion of the human kidney tubular system contains the alpha form, while the distal portion contains the pi form of glutathione transferase. These cytoplasmic proteins are released into the urine under pathological conditions, and an ELISA procedure has been developed for their quantitation. Optimal conditions with respect to concentrations of antibody and antigen and incubation times were determined. The procedure developed can detect as little as 0.5 ng enzyme per ml urine, even in the presence of high concentrations of other proteins. No cross reaction between these two isoenzymes or with a number of other proteins in the urine was observed. Antibodies interacted with these antigens in urine samples in the same manner as they interacted with the purified proteins. Storage of samples without loss of antigen required the presence of low concentrations of detergent, such as Tween 20, which both stabilized the enzymes and prevented their adsorption to the walls of the plastic tubes. The results indicate that increased urinary levels of these two enzyme proteins, as determined by the ELISA procedure, are useful markers for tubular damage.

Proliferative capacity of oligodendrocytes in the demyelinating twitcher spinal cord.

The proliferative capacity of oligodendrocytes was investigated in the spinal white matter of the twitcher mouse, a murine model of a genetic demyelinating disease globoid cell leukodystrophy (GLD), in which degeneration of oligodendrocytes due to metabolic perturbation has been well documented. In normal mice at 30 and 45 days of age, proliferating cells labeled with 5-bromo-2'-deoxyuridine (BrdU) were scarce, and the majority of BrdU-labeled cells did not immunostain with antibodies for oligodendrocytes, astrocytes, or microglia/macrophages. Only a few cells with markers for oligodendrocytes, carbonic anhydrase (CA), or the Pi form of glutathione-S-transferase (Pi), were labeled with BrdU. In the twitcher spinal cord, total numbers of BrdU-labeled cells were almost 6 times that of the normal littermate mice at 30 days of age, and 28 times at 45 days of age. However, this increase was largely due to an increase of cells labeled with F4/80, a marker for the microglia/macrophages. CA or Pi positive cells only constituted less than 10% of all labeled cells. With progression of demyelination from 30-45 days, total numbers of CA positive or Pi positive oligodendrocytes decreased, but percentages of cells double-labeled with BrdU and CA or Pi remained fairly constant. The results indicated that oligodendrocytes proliferated, to some extent, in the twitcher despite the genetic metabolic defect, and their decrease in number with progression of disease was not due to declined proliferation but rather cellular degeneration as the result of an intrinsic metabolic perturbation.

Adrenocorticotrophic-hormone-dependent regulation of a mu-class glutathione transferase in mouse adrenocortical cells.

Three different forms of glutathione transferase (GST) have been resolved in the two mouse adrenal tumour cell lines Y1 and Kin 8. Two of these belong to the mu and pi classes respectively. The third form is so far unidentified. In the Y1 cells, the levels of the mu form (mGTmu1) and the unidentified form, are both down-regulated in the presence of adrenocorticotrophic hormone (ACTH) while the pi form is unaffected. The Kin 8 cell line is derived from Y1 cells and harbours a defect in the cyclic AMP (cAMP)-dependent protein kinase, making it refractory to cAMP-dependent regulation of several enzymes. The GST levels in this cell line were unaffected by ACTH. Also, the steady-state levels of mGTmu1 mRNA were much lower in Y1 cells treated with forskolin (which activates adenylate cyclase) compared with control cells, but there was no difference in mGTmu1 mRNA levels between control and forskolin-treated Kin 8 cells. This indicates that the ACTH-dependent regulation of the mu class GST is pre-translational and that a functional cAMP-dependent protein kinase is required for the regulation. We have further shown that the difference in mRNA steady-state levels between control and forskolin-treated Y1 cells is abolished when transcription is inhibited by actinomycin D. In light of the stability of mGTmu1 mRNA, it would appear most likely that actinomycin D inhibits the transcription of short-lived factors which regulate the turn-over of mGTmu1 transcripts in response to changes in intracellular cAMP levels.

Glutathione transferases in the urine: sensitive methods for detection of kidney damage induced by nephrotoxic agents in humans.

With the aid of immunohistochemical methods the localization of the various isoenzymes of glutathione S-transferase was investigated. The alpha isoenzyme was present solely in the proximal tubular cells of the human kidney, while the pi form was restricted to the distal convoluted tubules, the thin loop of Henle, and the collecting ducts. Damage to the epithelial cell membranes results in the increased excretion of these enzymes with the urine. The alpha and pi isoenzymes have been isolated in a highly purified form and used for the production of polyclonal antisera. Subsequently, radioimmunological and ELISA techniques were developed for quantitation of these proteins in the urine; the methods exhibited a high specificity and were sufficiently sensitive to determine nanogram quantities or less. Disease affecting tubular function, cyclosporine A treatment, administration of nephrotoxic antibiotics, and exposure to cadmium all resulted in characteristic changes in the pattern of the glutathione transferase isoenzymes present in urine. Such effects were seen also in patients who had previously been exposed to nephrotoxic agents, but in whom conventional tests for kidney function were apparently normal. Thus, it appears that radioimmunologic or immunochemical quantitation of alpha and pi forms of the enzyme can be used as sensitive and relatively simple markers for the early detection of toxic effects with respect to the renal tubuli.

Cellular sources of glutathione S-transferase P in primary cultured rat hepatocytes: localization by in situ hybridization.

Hepatocytes in vivo express Alpha and Mu but not Pi forms of glutathione S-transferase (GST). GST P (a fetal Pi form) appears in rat hepatocytes after 2 days in primary culture, which suggests that hepatocytes may undergo dedifferentiation [Abramovitz, Ishigaki and Listowsky (1989) Hepatology 9, 235-239]. However, in this and other studies, primary rat hepatocyte cultures were shown by immunohistochemistry to contain significant numbers of lipocytes (Ito cells). Freshly isolated lipocytes contained GST activity when assayed with chlorodinitrobenzene (680 nmol/min per mg), and expression of Alpha, Mu and Pi forms of GST was detected by Western-blot analysis. Expression of GST P persisted during culture of the lipocytes. In situ hybridization of the cultured cells was performed to define whether hepatocytes, lipocytes or both expressed the enzyme. Lipocytes in culture contained abundant GST P transcripts. Hepatocytes contained no GST P transcripts after 12 h in culture, and after 24 h, only a few hepatocytes expressed this enzyme. After 48 h in culture all hepatocytes contained GST P transcripts, and the number of transcripts continued to increase up until 72 h. Therefore, in freshly isolated preparations of hepatocytes and early in hepatocyte culture, measurable levels of GST P protein or message appeared to reflect the presence of lipocytes. After 48 h in culture almost all of the GST P reflected expression by the hepatocytes. Lipocytes constitutively expressed Alpha-, Mu- and Pi-class GSTs and had significant intracellular levels of GSH (5.2 nmol/mg of protein). Lipocytes are capable therefore of detoxifying a number of injurious compounds.

SDS polyacrylamide gel electrophoresis of proteins.

Probably the most widely used of techniques for analyzing mixtures of proteins is SDS polyacrylamide gel electrophoresis. In this technique, proteins are reacted with the anionic detergent, sodium dodecylsulfate (SDS, or sodium lauryl sulfate) to form negatively charged complexes. The amount of SDS bound by a protein, and so the charge on the complex, is roughly proportional to its size. Commonly, about 1.4 g SDS is bound per 1 g protein, although there are exceptions to this rule. The proteins are generally denatured and solubilized by their binding of SDS, and the complex forms a prolate elipsoid or rod of a length roughly proportionate to the protein's molecular weight. Thus, proteins of either acidic or basic pI form negatively charged complexes that can be separated on the bases of differences in charges and sizes by electrophoresis through a sieve-like matr ix of polyacrylamide gel.

Glial cell abnormalities in the CNS of the carbonic anhydrase II deficient mutant mouse

The Car-2 n/Car-2 n mutant mouse is entirely lacking in carbonic anhydrase II (CA), which is normally found in myelin and glial cells in the CNS. CNS tissue from CA-deficient mutant mice was examined to see whether abnormalities could be detected which might help in understanding the function(s) of CA in the normal brain. Analyses of myelin yields and myelin proteins showed no differences between mutants and normal littermates. To visualize the oligodendrocytes and astrocytes in tissue sections, immunocytochemical staining was performed using antibodies against the Pi form of glutathione-S-transferase and glial-fibrillary acidic protein, respectively. In both gray and white matter from the mutants' brains the oligodendrocytes appeared to be shrinking and, possibly, degenerating. Hypertrophy of astrocytes occurred in the white matter, and the astrocytes in gray matter appeared swollen. It is suggested that imbalances in the HCO − 3 CO 2 ratios in various glial-cell compartments may produce abnormal distributions of water and ions in the brains of the CAII-deficient mice.

Characterization of Glutathione S-Transferase in Cultured Human Keratinocytes

The glutathione S-transferase activity and isozymic composition of cultured human keratinocytes were characterized. Keratinocytes were grown in culture and harvested at different stages of differentiation. Glutathione S-transferase activity was found in the soluble cell fraction but not in the microsomal cell fraction. The glutathione S-transferase specific activity of the soluble cell fraction was found to increase as the keratinocytes differentiated in culture. All of the enzymatic activity was found to reside with a single isozymic form that was concluded to be the pi form of the enzyme based on substrate specificity, sensitivity to inhibitors, molecular weight, and reactivity towards antibodies raised to alpha, mu, and pi forms of the enzyme. It is concluded that all of the isozymic forms of glutathione S-transferase noted in whole skin, with the exception of pi, are of extra-keratinocyte origin.

A Pi Form of Glutathione‐S‐Transferase Is a Myelin‐and Oligodendrocyte‐Associated Enzyme in Mouse Brain

The pi form of glutathione-S-transferase (GST), previously found to be oligodendrocyte associated, has also been found in myelin. In the brains of adult mice, immunocytochemical staining for a pi form of GST was observed in myelinated fibers, as well as oligodendrocytes. In contrast, and as previously found in rats, positive immunostaining for mu forms occurred in astrocytes and not in oligodendrocytes or myelinated fibers. Double immunofluorescence staining strengthened the conclusion that pi was in oligodendrocytes and myelin in mouse brains. The results of enzyme assays performed on brain homogenates and purified myelin indicated that GST specific activities in mouse brain myelin were almost as high (0.8-fold) as those in mouse brain homogenates. Low, but reproducible, GST activities were also observed in myelin purified from rat brains, in which pi had been demonstrated in oligodendrocytes and mu in astrocytes. The pi form was also stained by the immunoblot technique in myelin purified from mouse brain. It was concluded that pi is a myelin associated, as well as oligodendrocyte associated, enzyme in mouse brain, and possibly also in rat brain. This is the first report of localization of GSTs in mouse brain and of GST in myelin.