Effects Of Various Modulators Research Articles

Immune-metabolic genesis of pathological processes

This article deals with metabolic-immune processes at rest and under stress conditions, which, in turn, results in the development of immune-dependent and immune-associated disorders. The article analyzes study results and conclusions of various literature sources and experimental data in healthy individuals and patients suffering from non-specific inflammatory lung diseases; purulent-inflammatory diseases and their combinations, primary and secondary progressive multiple sclerosis in the acute stage and remission. Research studies investigated the impact of the type, stage, combination of diseases on the parameters of the immunologic and metabolic statuses, as well as their correlations. The authors also analyzed metabolic effects of immunomodulators. Based on the analysis of the literature and own clinical and experimental data, the authors identified the ability of metabolic factors to regulate immunological processes. A correlative analysis of examination results of the patients with various diseases helped detect the unity of the immune-metabolic mechanisms of pathology. The data on the therapeutic effect of various modulators through differentiated biochemical chains and vice versa – the metabolic effect through immunological mechanisms –were analyzed in the study. Thus, one can testify that there is the phenomenon of a mediated effect of some immunocorrectors on the reactivity through metabolic chains. The fact that a number of modulators and metabolics can simultaneously affect the biochemical and immunological parameters of patients proved the above phenomenon. There was revealed a significant correlation interaction of the immune-metabolic parameters with various types of purulent-inflammatory diseases, which proves the formation of a single mechanism of pathology.

Sustainable synthesis of modulated Fe-MOFs with enhanced catalyst performance for persulfate to degrade organic pollutants

In this study, four typical modulators (NH4OH(A), CH3COOH(B), CH3COONa(C) and CH3COONH4(D)) were applied to modulate the microwave-assisted synthesis of Fe-MOFs. The effects of various modulators on the yield, electrochemistry activity and PS activation capacity of prepared catalysts were systematically investigated. The ideal modulator was revealed as the 7.5 mM CH3COONH4. Contributed by the defects caused by the dual effects of CH3COONH4, Fe-MOFs-D-7.5/PS system showed excellent orange G (OG) degradation with high reaction stoichiometric efficiency (RSE) and desirable recycling performance. The main radicals should be SO4·− and O2·− which were confirmed by EPR and chemical quenchers. Furthermore, the frontier molecular orbital (FMO) theory and dual descriptor (DD) method were employed in predicting radical attacking sites of OG. According to the results of theoretical computations and experimental detection, degradation pathways of OG in Fe-MOFs-D-7.5/PS system were proposed. Similar to the function of the battery, this study gives new insight into the possible mediatory roles of Fe-MOFs-D-7.5 in PS activation by transferring the electrons between PS and the unsaturated metal sites (CUS). The Fe-MOFs-D-7.5/PS system is a promising process for environmental remediation.

A Comparison of Methods for Estimating the Activity of the ATP-Sensitive Potassium Channel in Mitochondria Based on the Effect of ATP

Abstract—Two methods for studying the functioning of the mitochondrial ATP-sensitive potassium channel, which plays an important role in cell physiology and especially in cardio- and neuroprotection, have been compared. It has been shown that the spectrophotometric method, which is most often used to study this channel and the influence of its modulators, has a number of limitations and mainly provides a qualitative assessment. A modification of this method is proposed, which makes it possible to obtain optical density changes recorded in a linear form that enables quantification of the change in the optical density and, as a consequence, allows a more accurate determination of the effect of various modulators. The concentration of ATP that causes half-maximal inhibition of the rate of potassium transport (ІС50_АТP) in the rat liver mitochondria has been determined by two different methods. The ІС50_АТP, as assessed by direct measurement with a potassium-selective microelectrode under 2,4-dinitrophenol-induced efflux of potassium, was 23 μM; it was 400 μM as assessed by the spectrophotometric method. The data we obtained suggest that recording of the 2,4-dinitrophenol-induced potassium efflux may be a more effective method for evaluating ATP-dependent transport of potassium ions in mitochondria.

Stimulation of Lipogenesis as Well as Fatty Acid Oxidation Protects against Palmitate-Induced INS-1 β-Cell Death

Saturated fatty acids are generally cytotoxic to β-cells. Accumulation of lipid intermediates and subsequent activation of lipid-mediated signals has been suggested to play a role in fatty acid-induced toxicity. To determine the effects of lipid metabolism in fatty acid-induced toxicity, lipid metabolism was modulated by up- and down-regulation of a lipogenic or fatty acid oxidation pathway, and the effects of various modulators on palmitate (PA)-induced INS-1 β-cell death were then evaluated. Treatment with the liver X receptor agonist T0901317 reduced PA-induced INS-1 cell death, regardless of its enhanced lipogenic activity. Furthermore, transient expression of a lipogenic transcription factor sterol regulatory element binding protein-1c (SREBP-1c) was also protective against PA-induced cytotoxicity. In contrast, knockdown of SREBP-1c or glycerol-3-phosphate acyltransferase 1 significantly augmented PA-induced cell death and reduced T0901317-induced protective effects. Conversely, T0901317 increased carnitine PA transferease-1 (CPT-1) expression and augmented PA oxidation. CPT-1 inhibitor etomoxir or CPT-1 knockdown augmented PA-induced cell death and reduced T0901317-induced protective effects, whereas the peroxisome proliferator-activated receptor (PPAR)-α agonist bezafibrate reduced PA-induced toxicity. In particular, T0901317 reduced the levels of PA-induced endoplasmic reticulum (ER) stress markers, including phospho-eukaryotic initiation factor-2α, phospho-C-Jun N terminal kinase, and CCAAT/enhancer-binding protein homologous protein. In contrast, knockdown of SREBP-1c or glycerol-3-phosphate acyltransferase 1 augmented PA-induced ER stress responses. Results of these experiments suggested that stimulation of lipid metabolism, including lipogenesis and fatty acid oxidation, protected β-cells from PA-induced lipotoxicity and that protection through enhanced lipogenesis was likely due to reduced ER stress.

The IKK‐NF‐κB pathway: a source for novel molecular drug targets in pain therapy?

Several studies indicate that the nuclear factor-kappa B (NF-kappaB) -activation cascade plays a crucial role not only in immune responses, inflammation, and apoptosis but also in the development and processing of pathological pain. Accordingly, a pharmacological intervention into this pathway may have antinociceptive effects and could provide novel treatment strategies for pain and inflammation. In this review we summarize the role of NF-kappaB in the nervous system, its impact on nociception, and several approaches that investigated the effects of various modulators of the classical I-kappaB-kinase-NF-kappaB signal transduction pathway in inflammatory nociception and neuropathic pain. The results indicate that NF-kappaB has an impact on nociceptive transmission and processing and that a number of substances that inhibit the NF-kappaB-activating cascade are capable of reducing the nociceptive response in different animal models. Therefore, a modulation of specific participants in the NF-kappaB signal transduction might exert a useful approach for the development of new painkillers.

Geranylgeranylated Proteins are Involved in the Regulation of Myeloma Cell Growth

Prenylation is essential for membrane localization and participation of proteins in various signaling pathways. This study examined the role of farnesylated and geranylgeranylated proteins in the regulation of myeloma cell proliferation. Antiproliferative and apoptotic effects of various modulators of farnesylated and geranylgeranylated proteins were investigated in myeloma cells. Depletion of geranylgeranylpyrophosphate inhibited myeloma cell proliferation through accumulation of cells in G(1) phase of the cell cycle and loss of cells in S phase. In contrast, depletion of farnesylpyrophosphate had no or only minor effects. Furthermore, inhibition of geranylgeranyl transferase I activity was more effective in reducing myeloma cell growth when compared with inhibition of farnesyl transferase activity. This indicates that protein geranylgeranylation is important for myeloma cell proliferation and cell cycle progression through G(1). Geranylgeranylated target proteins involved in the control of proliferation include GTPases, such as Rac-1, Cdc42, and RhoA. Inhibition of Rho, Rac, and Cdc42 GTPases by toxin B reduced proliferation, without affecting cell viability, whereas specific inhibition of Rho GTPases by C3 exoenzyme was without effect. This suggests a role for Rac and/or Cdc42 GTPases in myeloma cell growth. Rac-1 activity was found in all myeloma cell lines and was suppressed by the depletion of intracellular pools of geranylgeranylpyrophosphate, whereas interleukin-6 rapidly induced Rac-1 activation. Furthermore, dominant-negative Tat-Rac-1 reduced myeloma cell proliferation, whereas constitutively active Tat-Rac-1 enhanced proliferation. These results indicate that protein geranylgeranylation is essential for myeloma cell proliferation and suggest that Rac-1 is a regulator of myeloma cell growth.

Modulators of urinary stone formation.

Urine contains compounds that modulate the nucleation, growth and aggregation of crystals as well as their attachment to renal epithelial cells. These compounds may function to protect the kidneys against: 1, the possibility of crystallization in tubular fluid and urine, which are generally metastable with respect to calcium salts, 2, crystal retention within the kidneys thereby preventing stone formation and 3, possibly against plaque formation at the nephron basement membrane. Since oxalate is the most common stone type, the effect of various modulators on calcium oxalate (CaOx) crystallization has been examined in greater details. Most of the inhibitory activity resides in macromolecules such as glycoproteins and glycosaminoglycans while nucleation promotion activity is most likely sustained by membrane lipids. Nephrocalcin, Tamm-Horsfall protein, osteopontin, urinary prothrombin fragment 1, and bikunin are the most studied inhibitory proteins while chondroitin sulfate (CS), heparan sulfate (HS) and hyaluronic acid (HA) are the best studied glycosaminoglycans. Crystallization modulating macromolecules discussed here are also prominent in cell injury, inflammation and recovery. Renal epithelial cells on exposure to oxalate and CaOx crystals produce some of the inflammatory molecules such as monocyte chemoattractant protein-1 (MCP-1) with no apparent role in crystal formation. In addition, macrophages surround the CaOx crystals present in the renal interstitium. These observations indicate a close relationship between inflammation and nephrolithiasis.

Non-coordinate Regulation of Endogenous Epithelial Sodium Channel (ENaC) Subunit Expression at the Apical Membrane of A6 Cells in Response to Various Transporting Conditions

In many epithelial tissues in the body (e.g. kidney distal nephron, colon, airways) the rate of Na(+) reabsorption is governed by the activity of the epithelial Na(+) channel (ENaC). ENaC activity in turn is regulated by a number of factors including hormones, physiological conditions, and other ion channels. To begin to understand the mechanisms by which ENaC is regulated, we have examined the trafficking and turnover of ENaC subunits in A6 cells, a polarized, hormonally responsive Xenopus kidney cell line. As previously observed by others, the half-life of newly synthesized ENaC subunits was universally short ( approximately 2 h). However, the half-lives of alpha- and gamma-ENaC subunits that reached the apical cell surface were considerably longer (t(12) > 24 h), whereas intriguingly, the half-life of cell surface beta-ENaC was only approximately 6 h. We then examined the effects of various modulators of sodium transport on cell surface levels of individual ENaC subunits. Up-regulation of ENaC-mediated sodium conductance by overnight treatment with aldosterone or by short term incubation with vasopressin dramatically increased cell surface levels of beta-ENaC without affecting alpha- or gamma-ENaC levels. Conversely, treatment with brefeldin A selectively decreased the amount of beta-ENaC at the apical membrane. Short term treatment with aldosterone or insulin had no effect on cell surface amounts of any subunits. Subcellular fractionation revealed a selective loss of beta-ENaC from early endosomal pools in response to vasopressin. Our data suggest the possibility that trafficking and turnover of individual ENaC subunits at the apical membrane of A6 cells is non-coordinately regulated. The selective trafficking of beta-ENaC may provide a mechanism for regulating sodium conductance in response to physiological stimuli.

Cocktail modulator mixtures for overcoming multidrug resistance in renal cell carcinoma

Objectives. To determine the effect of various modulators on intracellular drug accumulation in renal cell carcinoma (RCC) tumor cells and the modulation of cytotoxicity of various chemotherapeutic drugs on the native RCC cell line and acquired intrinsic multidrug resistance (MDR) sublines because MDR is a major obstacle to effective chemotherapy of RCC. Methods. The cytotoxity of adriamycin to RCC 8701 and its MDR subline was analyzed. Fourteen MDR modulators, including calcium antagonists, protein kinase C inhibitor, glutathione transferase inhibitor, protein/peptide synthesis inhibitors, respiratory chain inhibitors, uncoupling reagent, adenosine triphosphate synthesis inhibitor, and ionophores, were examined for their MDR-reverse activity using the microplate tetrazolium test. Results. The intracellular adriamycin concentration significantly increased and reached maximum 4 hours after simultaneous treatment with calcium antagonists, tamoxifen, and oligomycin. The result demonstrated that verapamil, quinidine, tamoxifen, and oligomycin had an additive effect on the cytotoxicity of adriamycin and vinblastine against RCC8701 and RCC8701/ADR800 tumor cells. RCC8701/ADR800 tumor cells were more sensitive to modulator enhancement than native cells. The enhancement was related to the dosage and treatment duration of the modulators. Further trials on simultaneous additions to cocktail mixtures of the above four modulators showed no additive or synergistic effect on cytotoxicity against RCC8701/ADR800 tumor cells. Conclusions. Calcium antagonists and tamoxifen and oligomycin can individually be an effective chemotherapy adjunct for overcoming the native drug resistance or acquired MDR in RCC. Combination regimens, however, need more study regarding timing of administration, dosage, and frequency of modulators.

Role of the cyclic adenosine monophosphate-dependent protein kinase in the control of meiotic resumption in bovine oocytes cultured with thecal cell monolayers.

It has long been known that intracellular levels of cAMP are implicated in the process of meiotic resumption in bovine oocytes. The present study was undertaken to evaluate the effects of various modulators of cAMP in a coculture system with thecal cell monolayers that have been shown to promote meiotic arrest (germinal vesicle [GV] stage) in bovine oocytes. Ovaries were obtained from a slaughterhouse, and oocytes were collected by puncture of 1- to 5-mm follicles. Cumulus-oocyte complexes (COC) were cultured for 18 h in tissue culture medium 199 supplemented with 10% fetal calf serum and various modulators. When H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinole-sulfonamide) , a protein kinase A inhibitor, was used alone, a significant increase (p < 0.05) in the percentage of COC maintained in GV stage was observed. In the coculture system with thecal cell monolayers that maintained oocytes at the GV stage, H-89 significantly (p < 0.05) decreased this percentage. When oocytes were denuded of their cumulus cells, H-89 (50 microM) alone did not significantly increase the percentage of oocytes maintained at the GV stage. In addition, denuded oocytes were not significantly maintained at the GV stage when cocultured with thecal cell monolayers. In the absence of thecal cell monolayers, a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (Bx), and an activator of adenylate cyclase, forskolin (Fk), significantly increased (p < 0.05) the percentage of denuded oocytes maintained at the GV stage. In contrast, Bx-Fk were not effective in maintaining COC at the GV stage. The inhibitory effect of H-89 on COC was significantly increased (p < 0.05) by Bx-Fk. Using thecal cell monolayers with Bx-Fk, the percentage of COC maintained at the GV stage was not significantly decreased. These results suggest that oocytes were maintained in meiotic arrest by inhibition of cAMP-dependent protein kinase by H-89 in cumulus cells. It seems that inhibitory factors originating from thecal cells are regulated by a cAMP-dependent protein kinase. In turn, inhibitory factors released from thecal cells may regulate a cAMP-dependent protein kinase in cumulus cells.

Purification and molecular characterization of glycylglycine endopeptidase produced by Staphylococcus capitis EPK1.

A novel staphylolytic enzyme, ALE-1, acting on Staphylococcus aureus, was purified from a Staphylococcus capitis EPK1 culture supernatant. The optimal pH range for staphylolytic activity was 7 to 9. ALE-1 contains one Zn2+ atom per molecule. Analysis of peptidoglycan fragments released by ALE-1 indicated that the enzyme is a glycylglycine endopeptidase. The effects of various modulators were determined, and we found that o-phenanthroline, iodoacetic acid, diethylpyrocarbonate, and Cu2+ reduced the staphylolytic activity of ALE-1. beta-Casein, elastin, and pentaglycine were poor substrates for ALE-1. Molecular cloning data revealed that ALE-1 is composed of 362 amino acid residues and is synthesized as a precursor protein which is cleaved after Ala at position 35, thus producing a mature ALE-1 of 35.6 kDa. The primary structure of mature ALE-1 is very similar to the proenzyme form of lysostaphin. It has the modular design of an N-terminal domain of tandem repeats of a 13-amino-acid sequence fused to the active site containing C-terminal domain. Unlike lysostaphin, ALE-1 does not undergo processing of the N-terminal repeat domain in broth culture. ale-1 is encoded on the plasmid. Protein homology search suggested that ALE-1 and lysostaphin are members of the novel Zn2+ protease family with a homologous 38-amino-acid-long motif, Tyr-X-His-X(11)-Val-X(12/20)-Gly-X(5-6)-His.

Estradiol-induced Down-regulation of estrogen receptor. Effect of various modulators of protein synthesis and expression

Incubation of MCF-7 cells with estradiol (E 2) down-regulates estrogen receptor (ER) resulting in a progressive reduction of the capacity of cells to concentrate selectively [ 3H]E 2. Scatchard plot analysis failed to detect any transformation of residual receptors into peptides of lower binding affinity. [ 3H]Estrone gave an identical ER disappearance pattern with an ER half-life comprised between 2 and 3 h. A similar value was established by incubating the cells with [ 3H]tamoxifenaziridine ([ 3H]TAZ) for 1 h before the addition of excessive unlabeled E 2 which induced ER-down regulation and impeded any further labeling of the residual receptors. Submission of the [ 3H]TAZ labeled cell extracts to SDS-PAGE revealed no progressive emergence of low molecular weight cleavage products of the receptor (< 67 kDa). Two inhibitors of protein kinases, H-7 at 40 μM and H-89 at 20 μM, failed to block the E 2-induced ER down-regulation. On the contrary, the protein phosphatases 1 and 2A inhibitor, okadaic acid, was effective with concentrations higher than 0.1 μM indicating that a dephosphorylation mechanism was involved in this phenomenon. Cycloheximide (CHX) also significantly reduced the receptor decrease at concentrations higher than 1μM. G-C specific intercalating agents [actinomycin D (AMD) and chromomycin A 3 at 1 μM] also prevented ER disappearance; ethidium bromide (EB) and quinacrine were ineffective. AMD and CHX operated immediately after their addition to the medium indicating an inhibitory acttion on the synthesis of an RNA and/or a peptide with high turnover rate involved in ER decline. Moreover, AMD produced its suppressive effects under conditions impeding any labeling of newly synthetized receptors (i.e. [ 3H]TAZ with an excess of unlabeled E 2) rejecting the possibility of an increasing ER production which may partially hamper its disappearance. Finally, E 2-induced ER mRNA down-regulation was similarly abolished by AMD while EB and CHX were devoid of effect.

PHOTOSENSORY TRANSDUCTION IN CILIATES. IV. MODULATION OF THE PHOTOMOVEMENT RESPONSE OF Blepharisma japonicum BY cGMP

The effect of various modulators of cytoplasmic guanosine 3',5'-cyclic monophosphate (cGMP) level on the step-up photophobic responses in Blepharisma japonicum has been investigated to clarify the possible role of cGMP in the mechanism of photosensory signal transduction. Membrane-permeable analogs of cGMP, 8-bromo-guanosine 3',5'-cyclic monophosphate or dibutyryl cGMP, caused a marked dose-dependent prolongation of the latency for the photophobic response, resulting in inhibition of the photophobic response in Blepharisma japonicum. A similar effect was observed when cells were treated with 3'-isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, and pertussis toxin, a G-protein activity modulator. The G-protein activator, fluoroaluminate, and 6-anilino-5,8-quinolinedione (LY 83583), an agent which effectively lowers the cytoplasmic cGMP level, significantly enhanced the photoresponsiveness of these ciliates to visible light stimuli. These results suggest that cellular cGMP serves as a signal modulator in the photophobic response of Blepharisma japonicum.

Regulation of prolactin receptor expression by the tumour promoting phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate in human breast cancer cells

In both the normal and malignant human breast, cellular sensitivity to the proliferative and differentiative activities of the lactogenic hormones is conferred by expression of the prolactin receptor (PRLR). The PRLR is regulated by steroid hormones; however, recent findings have suggested that PRLR may also be regulated by protein kinase C. To examine this possibility we have studied the effect of various modulators of PKC activity on PRLR binding activity and gene expression in five PRLR positive human breast cancer cell lines. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), a tumour promoter and modulator of PKC activity, decreased PRLR binding activity in all cell lines examined. In MCF-7 cells, 10 nM TPA caused a 70% loss of PRLR mRNA after 12 h, paralleled 3 h later by a comparable loss of cell surface PRLR. Mezerein, a non-phorbol ester modulator of PKC activity and 1,2-dioctanoyl-sn-glycerol, a permeant analogue of the endogenous activator of PKC, also reduced PRLR binding activity and gene expression in a time- and concentration-dependent manner. Cycloheximide failed to abrogate the TPA-induced decline in PRLR mRNA levels, indicating that this process was not dependent upon continuing protein synthesis. No change in the stability of PRLR mRNA was observed during 24 h of TPA treatment and TPA reduced the rate of PRLR gene transcription within 3 h of treatment. These results demonstrate that modulators of PKC activity reduce PRLR binding activity and gene expression, implicating this signal transduction pathway in PRLR regulation.

The effect of subunit composition of rat brain GABA A receptors on channel function

Different combinations of cloned rat brain subunit isoforms of the GABA A receptor channel were expressed in Xenopus oocytes. The voltage-clamp technique was then used to measure properties of the GAGA-induced membrane currents and to study the effects of various modulators of the GABAA receptor channel (diazepam, DMCM, pentobarbital, and picrotoxin). This approach was used to obtain information on the minimal structural requirements for several functional properties of the ion channel. The combination α5β2γ2 was identified as the minimal requirement reproducing consensus properties of the vertebrate GABAA receptor channel, including cooperativity of GABA-dependent channel gating gating with a K a in the range of 10 μM, modulation by various drugs acting at the benzodiazepine binding site, picrotoxin sensitivity, and barbiturate effects.

The effect of non-NMDA antagonists and phorbol esters on excitatory amino acid stimulated inositol phosphate formation in rat cerebral cortex

The effects of various modulators of excitatory amino acid stimulated inositol phosphate (IP) formation were investigated in slices of rat cerebral cortex. Both quisqualic acid (Quis) and ibotenic acid (Ibo) stimulated IP formation in a dose-dependent manner. Quis (0.3mM) stimulated the rapid formation of inositol bis, tris and tetrakis-phosphates, with a slower linear rise in the monophosphate which plateaued after 20 min. The responses to both Ibo (0.3 mM) and Quis (0.3 mM) were dose-dependently inhibited by phorbol dibutyrate (PDBu); the Ibo response was particularly sensitive to PDBu, with a 90% inhibition of the response at 1 ?M and an IC(50) of 200 nM; Quis stimulated IP formation was less sensitive with a 50% inhibition observed at 10 ?M PDBu. PMA (1 ?M) and dioctanoylglycerol (30 ?M) also inhibited Ibo-stimulated IP formation though 4-?-phorbol (10 ?M) was ineffective. The inhibition by PDBu was reversed with staurosporine (10 ?M) and polymyxin (10 ?M), and both protein kinase C inhibitors potentiated the Ibo response in the absence of PDBu. The putative Quis-kainate receptor antagonists 6,7-dinitroquinoxaline-2,3-dione and 6-cyano-7-nitroquinoxaline-2,3-dione did not inhibit the responses to either Quis or Ibo at a concentration of 100 ?M. Both responses were, however, inhibited by dl-2-amino-4-phosphonobutyric acid and O- phospho- l -serine (IC(50)s were 1-2 mM), and also by the putative Quis receptor agonist dl-?-amino-3-hydroxy-5-methyl-4-isoxalone propionic acid (AMPA; IC(50) 10?M). Our data suggest that excitatory amino acid stimulated IP formation in rat cerebral cortex is via a site distinct from previously defined amino acid receptors.

Differential Down-regulation of Protein Kinase C Isozymes

Types I, II, and III protein kinase C have been shown to be products of, respectively, gamma, beta, and alpha genes of this enzyme family (Huang, F. L., Yoshida, Y., Nakabayashi, H., Knopf, J. L., Young, W. S., III, and Huang, K.-P. (1987) Biochem. Biophys. Res. Commun. 149, 946-952). Incubation of the highly purified rat brain protein kinase C isozymes with trypsin (kinase/trypsin (w/w) = 100) under identical conditions results in a preferential degradation of types I and II enzymes, whereas the type III enzyme was relatively resistant to tryptic proteolysis. Degradation of the type III enzyme by trypsin could be facilitated with the addition of Ca2+, phosphatidylserine, and dioleoylglycerol; none of these components alone was effective. Limited proteolysis of the three protein kinase C isozymes generated distinctive fragments for each isozyme, indicating that each isozyme has different trypsin-sensitive sites. Tryptic digestion of the type III protein kinase C was used as a model to determine the effects of various modulators on protein kinase C degradation. While Ca2+ and phosphatidylserine together were sufficient to convert the type III protein kinase C from a trypsin-insensitive to a -sensitive form, addition of dioleoylglycerol greatly reduced the Ca2+ requirement for such a conversion. Among the various phospholipids tested, in the presence of either dioleoylglycerol or phorbol ester, phosphatidylserine, cardiolipin, and phosphatidic acid were the most effective, and phosphatidylcholine and phosphatidylethanolamine were the least effective in supporting the digestion of type III protein kinase. Other acidic phospholipids, such as lysophosphatidylserine and phosphatidylinositol, were also effective in supporting the degradation in the presence of phorbol ester but not in the presence of dioleoylglycerol. The relevance of these proteolytic reactions to physiological responses was assessed with phorbol ester on rat basophilic leukemia RBL-2H3 cells, which contained both types II and III protein kinase C. Immunoblot analysis with the isozyme-specific antibodies revealed that phorbol ester induced a faster degradation of type II than that of type III isozyme in these cells. The results demonstrate that the various protein kinase C isozymes have different susceptibilities to proteolysis in vitro, when tested with trypsin, as well as to endogenous proteases in intact cells.

Differential effects of cations and guanyl nucleotides on agonist and antagonist binding to rat adrenal and uterine angiotensin II receptors

The effects of various modulators (cations, Gpp(NH)p) of hormone-receptor interaction were tested on agonist ([ 125I]angiotensin II) and antagonist ( 125I-[Sar 1,Ala 8]angiotensin II) binding to membrane particles from the rat adrenal zona glomerulosa and uterine smooth muscle. The two radioiodinated peptides labeled the same population of binding sites. Sodium ion (140 mM) induced a 2 fold increase in the affinity of adrenal angiotensin II receptors for the agonist (K a = 2.15 nM −1, vs. 1.01 nM −1 for controls), but decreased antagonist binding by reducing the number of available receptors by up to 50% in both adrenal and uterine membrane particles. Potassium ion only inhibited antagonist binding. Calcium and magnesium ions (0–10 mM) increased agonist binding and decreased antagonist binding to adrenal and uterine angiotensin II receptors, an effect mediated by changes in both affinity and number of receptors for the two peptides. The non-hydrolyzable GTP analog, Gpp(NH)p (10 −9 - 10 −4 M) decreased the affinity of angiotensin II receptors for the agonist by up to 50%, but did not affect antagonist binding to the receptor. Thus, there were marked differences in the sensitivity of agonist and antagonist peptides of angiotensin II to the modulatory effect of cations and guanyl nucleotides on ligand-receptor interaction. It is suggested that these differences may be important in determining the activatory/inhibitory properties of angiotensin peptides.

Studies on the properties of cellulase enzyme from Aspergillus terreus GN1

Biotechnology and BioengineeringVolume 24, Issue 3 p. 737-742 Communictions to the EditorFree Access Studies on the properties of cellulase enzyme from Aspergillus terreus GN1 S. K. Garg, S. K. Garg Dairy Microbiology Division National Dairy Research Institute Karnal 132001, IndiaSearch for more papers by this authorS. Neelakantan, S. Neelakantan Dairy Microbiology Division National Dairy Research Institute Karnal 132001, IndiaSearch for more papers by this author S. K. Garg, S. K. Garg Dairy Microbiology Division National Dairy Research Institute Karnal 132001, IndiaSearch for more papers by this authorS. Neelakantan, S. Neelakantan Dairy Microbiology Division National Dairy Research Institute Karnal 132001, IndiaSearch for more papers by this author First published: March 1982 https://doi.org/10.1002/bit.260240316Citations: 22AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 L. Berghem and L. G. Pettersson, Eur. J. Biochem., 37, 21 (1973). 2 G. Halliwell and M. Riaz, Arch. Mikrobiol., 78, 295 (1971). 3 K. Selby and G. C. Maitland, Biochem. J., 104, 716 (1967). 4 T. M. Wood and S. I. McCrae, Biochem. J., 128, 1183 (1972). 5 M. Mandels and E. T. Reese, Dev. Ind. Microbiol., 5, 5 (1964). 6 G. Boretti, L. Garafano, P. Montecucchi, and C. Spolla, Arch. Mikrobiol., 92, 189 (1972). 7 R. K. Sedha, H. K. Tewari, and D. S. Chahal, Indian J. Microbiol., 18(2), 93 (1978). 8 M. Mandels and J. Weber, Adv. Chem. Ser., 95, 391 (1969). 9 S. K. Garg and S. Neelakantan, J. Food Sci. Technol., 18(2), 64 (1981). 10 S. K. Garg and S. Neelakantan, Biotechnol. Bioeng., 23, 1653 (1981). 11 W. C. Haynes, L. J. Wickerham, and C. W. Hesseltine, Appl. Microbiol., 3, 31 (1965). 12 S. K. Garg and S. Neelakantan, Biotechnol. Bioeng., 24, 109 (1982). 13 O. H. Lowry, N. J. Rosbrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 14 G. S. Miller, Anal. Chem., 31, 426 (1959). 15 M. Mandels, R. Andreotti, and C. Roche, Biotechnol. Bioeng. Symp., 6, 21 (1976). 16 P. L. Hurst, J. Nielsen, P. A. Sullivan, and M. G. Shepherd, Biochem. J., 165, 33 (1977). 17 A. E. Clarke, and B. A. Stone, Biochem. J., 96, 793 (1965). 18 G. Okada, J. Biochem., 80, 910 (1976). Citing Literature Volume24, Issue3March 1982Pages 737-742 ReferencesRelatedInformation

Heterocyclic polycyclic aromatic hydrocarbon carcinogenesis: 7H-dibenzo[ c,g]carbazole metabolism by microsomal enzymes from mouse and rat liver

The metabolism of dibenzo[ c,g]carbazole (DBC), was studied in vitro using microsomal fractions of mouse and rat liver from animals, which were treated with 3-methylcholanthrene (MC). The separation of extractable metabolites by high pressure liquid chromatography (HPLC) and thinlayer chromatography (TLC) as well as identification of most of them by nuclear magnetic resonance, mass spectrometry and comparison with synthetically obtained products are described. The microsomes of both species produced the same twelve compounds of which the following have been identified: five monohydroxylated derivatives (phenols), the product of further oxidation of one of them, and a dihydrodiol. The 5-OH-DBC (60% including its spontaneously-formed dimer) and the 3-OH-DBC (14%) are the main metabolites. Three minor metabolites cochromatographed with synthetically prepared 2-OH-DBC, 4-OH-DBC and 6-OH-DBC. The dihydrodiol detectable in small quantity (4–6%) was tentatively identified as 3,4-dihydroxy-3,4-dihydro-DBC by the sensitivity of its formation to very low concentrations of the inhibitor of microsomal epoxide hydrolase, 1,1,1-trichloropropene oxide, by its molecular ion and major fragment in mass spectrometry and by its dehydration product 3-OH-DBC. No other dihydrodiols were detected. The qualitative and quantitative effects of various modulators of metabolism (enzyme inhibitors, apparently homogeneous epoxide hydrolase, glutathione, supernatant fraction) were investigated. The results are discussed with respect to possible ultimate carcinogens.