Biomedical subjects

K R Pennypacker

Publications and source records attributed to K R Pennypacker.

50 records · Page 3Linked to original sources

PubMed1992

Constitutive expression of AP-1 transcription factors in the rat adrenal. Effects of nicotine.

The AP-1 proteins (c-fos and fos-related antigens and jun proteins) are transcription factors that are induced in most cells by various stimuli, but the expression of these proteins is low or undetectable in the basal state. Our data indicate that the rat adrenal gland contains a basally expressed elevated level of AP-1 proteins and mRNAs (40- and 35-kDa fos-related antigens, a 39-kDa c-jun protein, c-jun, junB, and junD mRNA), as well as high basal AP-1 DNA binding activity. Both the medulla and cortex contained equivalent levels of fos-related antigens and c-jun protein; however, the majority of AP-1 DNA binding was detected in the medulla. Handling of rats for several days prior to sacrifice failed to affect protein expression or binding; on the other hand, repeated injections of nicotine increased AP-1 DNA binding. A single nicotine injection 60 min prior to removing the adrenals caused a significant reduction in AP-1 DNA binding without any detectable changes in AP-1 protein expression. Pretreatment of adrenal nuclear extracts with alkaline phosphatase prior to incubation with the AP-1 oligomer decreased binding activity. Thus, unlike most tissues where AP-1 DNA binding activity is controlled by increased expression of AP-1 proteins, DNA binding in the rat adrenal gland appears to be controlled at the post-translational level, possibly through dephosphorylation.

Adrenal Glands↗

PubMed1992

A 35 kDa Fos-related antigen is co-localized with substance P and dynorphin in striatal neurons.

The rat striatum after dopamine denervation followed by repeated apomorphine treatment was examined for the co-expression of c-fos and Fos-related antigens with dynorphin, substance P and [Met5]enkephalin using Western blot and immunohistochemical techniques. Administration of apomorphine, a dopamine agonist, elevated the level of 35 kDa Fos-related antigen which co-localized with dynorphin and substance P, but not enkephalin, in striatal neurons.

Animals↗

PubMed1992

Apomorphine induction of AP-1 DNA binding in the rat striatum after dopamine depletion.

The expression of AP-1 transcription factors was assessed in the dopamine-depleted rat striatum over a 1 week period of repeated apomorphine injections. A single injection of apomorphine increased the expression of a 35 kDa Fos-related antigen and Jun proteins and their expression continued to increase until day 3 of repeated apomorphine treatment in dopamine-depleted striata. Apomorphine induces AP-1 transcription factors which may be involved in modulating gene expression in the striatum.

Animals↗

PubMed1992

Dopamine stimulates [3H]phorbol 12,13-dibutyrate binding in cultured striatal cells.

The effect of dopamine (DA) on the binding of [3H]phorbol 12,13-dibutyrate ([3H]PdBu) in cultured rat striatal cells was examined. DA maximally increased specific [3H]PdBu binding by 70 +/- 10%, an increase comparable to that observed with norepinephrine (NE). This finding suggests that DA activates protein kinase C in cultured striatal cells, because increases in [3H]PdBu binding reflect translocation of protein kinase C. Half-maximal stimulation was observed with 10(-6) M DA. The peak response was observed at 2-3 min after addition of 10(-4) M DA, but [3H]PdBu binding was still increased above basal at 30 min. DA was not acting via an adrenergic receptor. Prazosin (10(-6) M) blocked the response to NE, suggesting mediation by an alpha 1-adrenergic receptor, but had little effect on the response to DA. Conversely, the D1 receptor antagonist SCH-23390 (10(-6) M) blocked the response to DA, but only partially inhibited the response to NE. Morphine (10(-6) M) inhibited the response to DA by 46 +/- 14%, but did not affect significantly the response to NE. The DA effect on [3H]PdBu binding is apparently independent of the increase in cyclic AMP seen on D1 receptor activation. Forskolin, apomorphine, and the D1 agonist SKF-38393 all increased cyclic AMP in striatal cells, but were less effective than DA in stimulating [3H]PdBu binding. The D2 agonist quinpirole was ineffective in stimulating either cyclic AMP or [3H]PdBu binding.

Animals↗

PubMed1990

Identification of calmodulin-binding proteins.

We have outlined and partially characterized a series of biotinylated calmodulin derivatives that may be useful in the study of calmodulin-binding protein expression, physical points of calmodulin-target interaction, and proteolytic mapping of related calmodulin-binding proteins. Biotinylated calmodulins offer several advantages as probes of protein-protein interactions. First, biotinylation can be directed to different amino acid residues. Second, biotinylation can be carried out under mild, near-physiological conditions, reducing the likelihood that conditions of protein modification would destroy biological function. Third, biotinylated proteins are stable, and reagents needed for their preparation and detection are relatively inexpensive. Fourth, the sensitivity of avidin-chromogenic enzyme systems is approaching that of radioactivity, with the added advantage that chromogens can be visualized in a relatively short time with respect to autoradiography. However, as with any protein modification procedure, one must be cautious when interpreting the results obtained with biotinylated proteins. For calmodulin-binding proteins, some interactions are impaired by modification of specific lysyl residues. On the other hand, interaction of biotinylated calmodulin with phosphodiesterase occurs, but this interaction may obscure recognition of the biotin residue by avidin. One approach to circumvent this problem is to have a series of site-directed biotinylated proteins available for use as outlined in this chapter. The choice of which agent to use is determined by the primary sequence of the protein of interest and whether any information is available concerning the effects of chemical modification on structure (i.e., acetylation experiments, modification of free sulfhydryls). In the absence of such information, an empirical approach can be taken. Photobiotin affords an easy means for biotinylation of proteins; however, the sites of modification are not always predictable. NHS-biotin derivatives are readily available and are relatively easy to use. Finally, one may wish to biotinylate the protein while liganded to its normal interacting molecule, in the case of calmodulin, calcium ion is the obvious choice. However, calmodulin could also be biotinylated while bound to a specific binding protein such as calcineurin. The latter method may be of use in determination of changes in reactivities of specific amino acid residues subsequent to binding. Finally, it may prove advantageous to biotinylate genetically engineered calmodulin, yeast calmodulin, or plant calmodulin to further define calmodulin-target protein interactions. Thus, the use of biotinylated calmodulin derivatives may offer insights into a range of structural and functional questions relevant to regulation of specific calmodulin-binding proteins.

Alkaline Phosphatase↗

PubMed1989

Changes in expression of tyrosine hydroxylase immunoreactivity in human SMS-KCNR neuroblastoma following retinoic acid or phorbol ester-induced differentiation.

Human SMS-KCNR cells differentiated in response to either retinoic acid or phorbol esters; differentiated cells extended numerous, complex neurites and showed reduced proliferation. Tyrosine hydroxylase (TH) immunoreactivity was measured in this cell line following treatment with retinoic acid (1-10 microM), 12-O-tetradecanoyl-phorbol-13-acetate (TPA; 16-160 nM), or combinations of these agents. After 21 days of treatment with either TPA or retinoic acid (RA), TH immunoreactivity was measured in this using densitometric scans of Western blots, was doubled relative to untreated or serum-deprived SMS-KCNR cultures. Increases in TH immunoreactivity could be detected after 6 days of treatment. Treatment with RA for 3 days followed by phorbol esters for an additional 3 days resulted in a 3-fold increase in TH immunoreactivity at day 6; reversing the order of drug treatment did not have this effect. Treatment of cultures with the divalent cationophore A23187 caused treated cells to retract neurites; expression of TH immunoreactivity was decreased relative to drug-treated and control cultures. These results suggest that retinoic acid treatment may 'prime' SMS-KCNR cells for the subsequent effects of phorbol esters, and indicate that the patterns of biochemical differentiation induced by TPA or RA are different.

Cell Differentiation↗

PubMed1989

Expression of calmodulin-dependent phosphodiesterase, calmodulin-dependent protein phosphatase, and other calmodulin-binding proteins in human SMS-KCNR neuroblastoma cells.

Calmodulin (CaM)-dependent enzymes, such as CaM-dependent phosphodiesterase (CaM-PDE), CaM-dependent protein phosphatase (CN), and CaM-dependent protein kinase II (CaM kinase II), are found in high concentrations in differentiated mammalian neurons. In order to determine whether neuroblastoma cells express these CaM-dependent enzymes as a consequence of cellular differentiation, a series of experiments was performed on human SMS-KCNR neuroblastoma cells; these cells morphologically differentiate in response to retinoic acid and phorbol esters [12-O-tetradecanoylphorbol 13-acetate (TPA)]. Using biotinylated CaM overlay procedures, immunoblotting, and protein phosphorylation assays, we found that SMS-KCNR cells expressed CN and CaM-PDE, but did not appear to have other neuronal CaM-binding proteins. Exposure to retinoic acid, TPA, or conditioned media from human HTB-14 glioma cells did not markedly alter the expression of CaM-binding proteins; 21-day treatment with retinoic acid, however, did induce expression of novel CaM-binding proteins of 74 and 76 kilodaltons. Using affinity-purified polyclonal antibodies, CaM-PDE immunoreactivity was detected as a 75-kilodalton peptide in undifferentiated cells, but as a 61-kilodalton peptide in differentiated cells. CaM kinase II activity and subunit autophosphorylation was not evident in either undifferentiated or neurite-bearing cells; however, CaM-dependent phosphatase activity was seen. Immunoblot analysis with affinity-purified antibodies against CN indicated that this enzyme was present in SMS-KCNR cells regardless of their state of differentiation. Although SMS-KCNR cells did not show a complete pattern of neuronal CaM-binding proteins, particularly because CaM kinase II activity was lacking, they may be useful models for examination of CaM-PDE and CN expression. It is possible that CaM-dependent enzymes can be used as sensitive markers for terminal neuronal differentiation.

Avidin↗

PubMed1988

Calmodulin-binding proteins in human Y-79 retinoblastoma and HTB-14 glioma cell lines.

The Y-79 human retinoblastoma cell line has been used as a model system for studying differentiation of primitive neuroectodermal cells into either glial-like (glial fibrillary acidic protein positive) or neuron-like (neuron-specific enolase-positive) cells. To determine whether Y-79 retinoblastoma cells express neuronotypic calmodulin-binding proteins, Y-79 cells were either treated with butyrate or dibutyryl cyclic AMP (dbcAMP) in serum-containing medium or were maintained in serum-free media. Using a biotinylated calmodulin blot overlay technique, we found that Y-79 cells treated with dbcAMP or butyrate expressed low levels of membrane-bound calmodulin-binding proteins of 150, 147, 127, and 126 kilodaltons (kDa); butyrate-treated cells also expressed a calmodulin-binding peptide of 135 kDa. Since butyrate treatment of Y-79 cells induces the expression and the secretion of interphotoreceptor retinoid-binding protein (IRBP, 140 kDa), we tested the hypothesis that the calmodulin-binding protein of 135 kDa induced by butyrate treatment was IRBP. Purified bovine IRBP did not bind calmodulin; further, the 135-kDa calmodulin binding protein was not immunoreactive with antisera directed against IRBP. Since dbcAMP and butyrate induce some glial-like characteristics in Y-79 cells, we compared the calmodulin-binding protein pattern in these cells with that seen in human HTB-14 glioma cells. The HTB-14 line did not express calmodulin-binding proteins, even after treatments with agents that induce morphologic change in these cells. Thus, we conclude that Y-79 cells express membrane-bound calmodulin-binding proteins, but in a pattern different from that seen with adult, differentiated neurons or from human HTB-14 glioma cells.

Biotin↗

PubMed1986

Hormonal basis for sexual dimorphism of the sound-producing apparatus of the oyster toadfish.

We investigated the endocrine basis for sexual dimorphism of the sound-producing apparatus (swimbladder and attached sonic muscles) in the oyster toadfish Opsanus tau by implanting steroid pellets in gonadectomized females and males. In females, testosterone and dihydrotestosterone caused, respectively, 32.3 and 31.5% increase (P less than 0.0001), and estradiol 17 beta caused 11.3% increase (P less than 0.0104) in swimbladder weight compared with controls. In males, testosterone caused 17.7% (P less than 0.028), dihydrotestosterone 24.0% (P less than 0.0008), and estradiol 10.8% (N.S.) increase from controls. Swimbladder weight gains occurred despite the fact that these fish refused food and did not gain weight. In a final experiment on fed females, testosterone and 11-keto-testosterone caused a similar increase (16.6 and 18.9%, respectively) compared with controls. These results indicate that swimbladders in adults of both sexes retain the ability to respond to steroid treatments and suggest that sexual differences in bladder size are controlled without a critical period by hormonal concentrations within the fish.

Air Sacs↗

PubMed1985

Effects of gonadal steroids on catecholamine levels in the brain of the oyster toadfish.

We studied the effects of gonadol steroid implants on catecholamine levels in the brain of ovariectomized female toadfish (Opsanus tau L.). Control values for dopamine (DA), norepinephrine (NE) and epinephrine (EPI) in the brain were 2181 +/- 226, 1112 +/- 80 and 6.5 +/- 2.02 ng/g, respectively. Compared to controls, 17 beta-estradiol decreased brain levels of DA to 547 +/- 261, and dihydrotestosterone decreased DA to 444 +/- 290 ng/g (P less than 0.001). Furthermore, dihydrotestosterone increased brain levels of EPI to 15.0 +/- 4.7 ng/g. The steroids had no effect on brain levels of NE. These results suggest a role for both androgens and estrogens in regulating brain levels of DA and EPI in toadfish.

Animals↗

PubMed1985

Sexual differences and steroid-induced changes in metabolic activity in toadfish sonic muscle.

Since the male oyster toadfish (Opsanus tau) is more active in sound production than the female, we hypothesized that sonic muscles of the male are biochemically specialized to perform more work. In order to categorize the muscle biochemically and test for sexual differences, we measured the activity of two anaerobic enzymes, 3-phosphoglyceraldehyde dehydrogenase (3PG) and lactic dehydrogenase (LDH), and two aerobic enzymes, malate dehydrogenase (MDH) and glutamic oxaloacetic transaminase (GOT). Males exhibited greater 3PG and GOT activity than females (p less than 0.05). Both MDH and LDH showed little activity in either sex. High 3PG and low LDH levels indicate a sustained level of glycolysis, with pyruvate shuttled into aerobic metabolism, and high GOT activity indicates a high level of aerobic metabolism. From this and other data, we conclude that toadfish sonic muscle can be classified as fast-twitch oxidative glycolytic or fast-twitch fatigue resistant. The endocrine basis for these sexual differences was examined by implanting steroid pellets into ovariectomized females. Testosterone induced a doubling of 3PG activity (p less than 0.02), and dihydrotestosterone induced an eight-fold increase (p less than 0.0005) in GOT concentration over controls. The steroids had no effect on LDH and MDH activities. Hormones, therefore, trigger one of the fundamental sexual differences underlying toadfish communication, namely a difference in metabolism, providing the male with the capacity for increased sound production.

Animals↗

PubMed1985

A rapid and sensitive method for detection and quantification of calcineurin and calmodulin-binding proteins using biotinylated calmodulin.

Purified bovine brain calmodulin was biotinylated with biotinyl-epsilon-aminocaproic acid N-hydroxysuccinimide. Biotinylated calmodulin was used to detect and quantify calmodulin-binding proteins following both protein blotting and slot-blot procedures by using alkaline phosphatase or peroxidase coupled to avidin. When purified bovine brain calcineurin, a calmodulin-dependent protein phosphatase, was immobilized on nitrocellulose slot blots, biotinylated calmodulin bound in a calcium-dependent saturable manner; these blots were then quantified by densitometry. Biotinylated calmodulin was able to detect as little as 10 ng of calcineurin, and the binding was competitively inhibited by addition of either native calmodulin or trifluoperazine. When biotinylated calmodulin was used to probe protein blots of crude brain cytosol and membrane preparations after gel electrophoresis, only protein bands characteristic of known calmodulin-binding proteins (i.e., calmodulin-dependent protein kinase, calcineurin, spectrin) were detected with avidin-peroxidase or avidin-alkaline phosphatase procedures. Purified calcineurin was subjected to one- and two-dimensional gel electrophoresis and protein blotting; as expected, only the 61-kDa calmodulin-binding subunit was detected. When the two-dimensional protein blot was incubated with biotinylated calmodulin and detected with avidin-alkaline phosphatase, several apparent forms of the 61-kDa catalytic subunit were detected, consistent with isozymic species of the enzyme. The results of these studies suggest that biotinylated calmodulin can be used as a simple, sensitive, and quantifiable probe for the study of calmodulin-binding proteins.

Animals↗

PubMedDate not supplied

Methamphetamine-induced changes in AP-1 binding and dynorphin in the striatum: correlated, not causally related events?

Activation of D1 dopamine (DA) receptors in the striatum increases the expression of the opioid neuropeptide, dynorphin (DYN). While cAMP is generally accepted as a second messenger in this signal transduction pathway, the role of Fos/FRA proteins and AP-1 binding in mediating D1 receptor-induced changes in DYN expression remains uncertain. In this study, DYN peptide and mRNA levels, as well as Fos/FRA proteins and AP-1 DNA binding activity, were measured in individual animals following acute challenge with the psychostimulant drug methamphetamine (METH). METH caused an initial decrease in striatal levels of DYN A, reflecting increased peptide release. Six hours postinjection, DYN mRNA levels were significantly elevated by METH as compared to vehicle-injected controls. At the same time, these drugs increased the expression of Fos/FRA-ir proteins, in particular the 35- and 40-kDa molecular weight species, and increased binding to the AP-1 DNA element. Analyses of the time course of METH's effects revealed that DYN mRNA levels, Fos/FRA proteins and AP-1 binding activity showed variable increases by 3 h but all were significantly elevated above control levels by 6 h posttreatment. The D1-specific antagonist, SCH 23390, completely blocked the METH-induced changes in DYN peptide and mRNA levels while a D2 receptor antagonist (sulpiride) had little or no effect. These data indicate that stimulant drugs such as METH increase the expression of DYN and AP-1 factors in the striatum via a D1 receptor-mediated mechanism. However, the finding that AP-1 binding merely paralleled, but did not precede, the increase in DYN expression, as would be expected if it were mediating increased gene transcription, suggests these may be correlative, not causally related events.

Adrenergic Uptake Inhibitors↗

PubMedDate not supplied

Acetaminophen-induced hepatotoxicity is associated with early changes in NF-kB and NF-IL6 DNA binding activity.

Nuclear transcription factors, such as NF-kB and NF-IL6, are believed to play an important role in regulating the expression of genes that encode for products involved in tissue damage and inflammation and, thus, may represent early biomarkers for chemical toxicities. In the present study changes in DNA binding activity of these factors were examined in livers of mice administered hepatotoxic doses of acetaminophen (APAP). NF-kB and NF-IL6 DNA binding occurred constitutively in control mouse liver. However, within 4 hr following administration of hepatotoxic doses of APAP, their binding activities were transiently lost and is in contrast to AP-1 transcription factor where activation occurs under similar conditions. These changes corresponded with increased release of inflammatory mediators (IL-6, serum amyloid A) and increased levels of enzymatic markers of hepatocyte damage. Similarly, treatment of mice with gadolinium chloride, an inhibitor of Kupffer cell activation and known to protect against APAP-induced hepatotoxicity, reduced the observed pathophysiological response in the liver while altering the APAP-associated changes in NF-kB DNA binding activity. NF-kB was found predominantly in parenchymal and endothelial cells and was composed primarily of relatively inactive p50 homodimer subunits in control liver. Taken together, these studies suggest that hepatotoxicity is associated with early and complex changes in DNA binding activities of specific transcription factors. In particular, NF-kB and NF-IL6 may serve as negative regulators of hepatocyte-derived inflammatory mediators and is analogous to that previously observed in certain other cell systems such as B lymphocytes.

Acetaminophen↗