Apurinic and/or apyrimidinic endonuclease activity in ataxia telangiectasia cell extracts.

The possibility that the increased sensitivity of ataxia telangiectasia towards ionizing radiation is related to a DNA-repair deficiency has been examined further. When compared to unaffected controls, 6 lines of fibroblast cells derived from ataxia patients demonstrated a slightly reduced endonucleolytic activity (165 +/- 12 units vs. 214 +/- 28 units) towards apurinic and/or apyrimidinic sites as determined in a "nicking" assay.     

Selective increase in cytokeratin synthesis in cultured rat hepatocytes in response to hormonal stimulation

Addition of a combination of insulin, dexamethasone and EGF at seeding time to cultured rat hepatocytes in serum-free medium caused a selective increase in the biosynthesis of particular cytokeratin components. This increase was prominent during the first day in culture. No significant increases were detected in the absence of hormones or in the presence of either hormones added alone or in pairs, except in the case of insulin plus dexamethasone, which yielded an effect close to that obtained with the three factors. Interestingly, the latter condition also maintained a high level of albumin production over a 6-day period in culture.     

Variables affecting the selectivity and efficiency of retention of DNA fragments by E. coli RNA polymerase in the nitrocellulose-filter-binding assay

In this paper we characterize the effect of varying the solution conditions and filter-binding protocols on the extent and selectivity of DNA retention on nitrocellulose filters by DNA-binding proteins. These effects are illustrated by the binding interaction of Escherichia coli RNA polymerase with λ and T7 phage DNA restriction fragments. We present procedures which will help enhance the selective retention of some DNA restriction fragments over others. These include increasing the pH and salt concentration, decreasing the enzyme-to-DNA ratio, and including an appropriate washing step. Selective binding is not dependent on the presence of Mg²⁺. Although we only show data for RNA polymerase-DNA interactions, many of the principles discussed are likely to find practical applications in studying selective DNA-protein binding in general.     

Anti-insulin receptor serum mimics the developmental role of insulin in mouse mammary explants

A number of growth factors can maintain hormonally-responsive epithelium in murine mammary explants as well as insulin, but only insulin can promote the synthesis of casein and alpha-lactalbumin, in the presence of glucocorticoid and prolactin. Anti-insulin receptor serum can elicit these effects of insulin on milk protein gene expression. The anti-serum is unique in its ability to mimic the developmental role of insulin in murine mammary epithelium.     

Phosphoglycerate mutase in mammalian striated muscles: Subcellular localization and binding partners

Contrary to previously published data, we have found that in mammalian skeletal muscles, phosphoglycerate mutase (PGM) is organized in a regular, striated fashion within the sarcomere. In the absence of the enzyme effectors, PGM localizes mainly at the M-line, but under conditions typical for contracting muscle, the enzyme accumulates within the I-band of the sarcomere. Searching for muscle PGM binding partners, we have found that PGM interacts with several enzymes of triose phosphate metabolism. It might suggest that PGM is a central structural element of the muscle glycolytic complex located within the isotropic region of the sarcomere.

Structured summary

MINT-7034028: PGM (uniprotkb:P16290) physically interacts (MI:0218) with lactate dehydrogenase B (uniprotkb:P42123), lactate dehydrogenase A (uniprotkb:P04642), G3PDH (uniprotkb:P04797), aldolase (uniprotkb:P05065), Creatine kinase (uniprotkb:P07335), phPhosphoglycerate kinase (uniprotkb:P16617) and Enolase (uniprotkb:P04764) by pull down (MI:0096)     

Cloning and expression of the yeast galactokinase gene in an Escherichia coli plasmid.

This report describes the construction and isolation of a plasmid, derived from pBR322, which carries a BglII restriction fragment of DNA containing the galactokinase gene from Saccharomyces cerevisiae. This was accomplished by the following procedure: (1) Purified galactokinase mRNA, labelled with 125I, was hybridized to BglII digests of yeast DNA employing Southern's filter transfer technique to identify a restriction fragment containing the galactokinase gene. (2) This fragment was partially purified by agarose gel electrophoresis, ligated into the BamHI site of pBR322 and transformed into Escherichia coli to generate a clone bank containing the galactokinase gene. (3) This bank was screened by in situ colony hybridization with galactokinase mRNA resulting in the identification of a plasmid carrying this gene. This plasmid DNA hybridized with the galactokinase mRNA to the same extent in the presence of absence of a large excess of unlabelled mRNA from cells that were not induced for galactokinase synthesis, while the same amount of unlabelled galactose-induced mRNA reduced the hybridization by 95%. When this plasmid was introduced into an E. coli strain deleted for the galactose operon it caused the synthesis of low levels of yeast galactokinase activity.     

DDT inhibition of Ca-Mg ATPase from peripheral nerves and muscles of lobster, Homarus americanus

Sensitivity of CaMg ATPase from axonic plasma membrane (APM) and sarcoplasmic reticulum (SR) of lobster, $\text{Homarus}\text{,}\text{americanus}$, to DDT was studied. The CaMg ATPase found in SR with the high Ca²⁺ affinity is sensitive to DDT while the portion of ATPase related to the low Ca²⁺ affinity site is not inhibited by DDT. Also, DDT is more inhibitory against the CaMg ATPase prepared from APM than the one obtained from SR. The relationship between inhibition of the CaMg ATPase by DDT in the axonic nerve membrane and $\text{in}\text{,}\text{vivo}$ poisoning symptoms of the nervous system is discussed.     

Hydrophobic Interaction between the SH2 Domain and the Kinase Domain Is Required for the Activation of Csk

The protein tyrosine kinase C-terminal Src kinase (Csk) is activated by the engagement of its Src homology (SH) 2 domain. However, the molecular mechanism required for this is not completely understood. The crystal structure of the active Csk indicates that Csk could be activated by contact between the SH2 domain and the β3-αC loop in the N-terminal lobe of the kinase domain. To study the importance of this interaction for the SH2-domain-mediated activation of Csk, we mutated the amino acid residues forming the contacts between the SH2 domain and the β3-αC loop. The mutation of the β3-αC loop Ala228 to glycine and of the SH2 domain Tyr116, Tyr133, Leu138, and Leu149 to alanine resulted in the inability of the SH2 domain ligand to activate Csk. Furthermore, the overexpressed Csk mutants A228G, Y133A/Y116A, L138A, and L149A were unable to efficiently inactivate endogenous Src in human embryonic kidney 293 cells. The results suggest that the SH2-domain-mediated activation of Csk is dependent on the binding of the β3-αC loop Ala228 to the hydrophobic pocket formed by the side chains of Tyr116, Tyr133, Leu138, and Leu149 on the surface of the SH2 domain.     

Functionality of nitrated acetylcholine receptor: the two-step formation of nitrotyrosines reveals their differential role in effectors binding

The presence of nitrotyrosines is associated with several neurodegenerative pathologies. We evaluated the functionality of the nicotinic acetylcholine receptor possessing nitrotyrosines. The spectrum of the nitrated receptor displays an absorption band characteristic of ortho-nitrophenol. The presence of carbamylcholine in the agonist site prevented the effect of nitration by tetranitromethane in some conditions. The nitration occurred with two discrete steps and pointed out the differential involvement of tyrosines in the binding of acetylcholine and neurotoxin. We concluded that at least two residues involved in agonist binding can be nitrated, which bring similar contributions to the binding energy of the neurotransmitter.     

Analysis of estrogen receptor alpha signaling complex at the plasma membrane

There is accumulating evidence that the estrogen receptor (ER) can transduce specific signals at the plasma membrane. We tried to clarify the biological function of ER as a signaling molecule by identifying proteins that interact with the membrane-localized ER. The activation function 1 and 2 (AF-1 and AF-2) domains of ERalpha with or without the membrane-targeting sequence were stably expressed in the breast cancer cell line, MCF-7. The level of tyrosine phosphorylation of AF-2 was significantly elevated by the membrane localization. By mass-spectrometry analysis, alpha- and beta-tubulins and heat shock protein 70 were identified as the AF-1-associated proteins. Of these, tubulins are associated only with membrane-targeted AF-1.     

PTTH-stimulated ERK phosphorylation in prothoracic glands of the silkworm, Bombyx mori: Role of Ca/calmodulin and receptor tyrosine kinase

Our previous studies showed that the prothoracicotropic hormone (PTTH) stimulated extracellular signal-regulated kinase (ERK) phosphorylation in prothoracic glands of Bombyx mori both in vitro and in vivo. In the present study, the signaling pathway by which PTTH activates ERK phosphorylation was further investigated using PTTH, second messenger analogs, and various inhibitors. ERK phosphorylation induced by PTTH was partially reduced in Ca²⁺-free medium. The calmodulin antagonist, calmidazolium, partially inhibited both PTTH-stimulated ERK phosphorylation and ecdysteroidogenesis, indicating the involvement of calmodulin. When the prothoracic glands were treated with agents that directly elevate the intracellular Ca²⁺ concentration [either A23187, thapsigargin, or the protein kinase C (PKC) activator, phorbol 12-myristate acetate (PMA)], a great increase in ERK phosphorylation was observed. In addition, it was found that PTTH-stimulated ecdysteroidogenesis was greatly attenuated by treatment with PKC inhibitors (either calphostin C or chelerythrine C). However, PTTH-stimulated ERK phosphorylation was not attenuated by the above PKC inhibitors, indicating that PKC is not involved in PTTH-stimulated ERK phosphorylation. A potent and specific inhibitor of insulin receptor tyrosine kinase, HNMPA-(AM)₃, greatly inhibited the ability of PTTH to activate ERK phosphorylation and stimulate ecdysteroidogenesis. However, genistein, another tyrosine kinase inhibitor, did not inhibit PTTH-stimulated ERK phosphorylation, although it did markedly attenuate the ability of A23187 to activate ERK phosphorylation. From these results, it is suggested that PTTH-stimulated ERK phosphorylation is only partially Ca²⁺- and calmodulin-dependent and that HNMPA-(AM)₃-sensitive receptor tyrosine kinase is involved in activation of ERK phosphorylation by PTTH.     

Specific binding of the cAMP receptor protein of Escherichia coli to the lactose operon promoter

The nitrocellulose filter binding assay has been used to study effects of pH, temperature, ionic strength and magnesium ions on the specific binding of the cyclic adenosine 3',5'-monophosphate (cAMP) receptor protein (CAP) to the promoter of the lactose (lac) operon of Escherichia coli. The pH has a significant effect on binding with the greatest amount of specific binding appearing at pHs near 7 with a gradual decrease in binding as the pH is increased to 8. Specific binding was observed at temperatures of 22 degrees C and 37 degrees C but not at 4 degrees C. The specific binding was also found to be a function of the concentration of magnesium acetate and potassium chloride, being dependent on the specific cation present, the total ionic strength, and the concentration of the CAP protein. All binding decreases as the ionic strength, increases, but this decrease occurs at a lower ionic strength in magnesium acetate than in potassium chloride. In a double label experiment the filter assay demonstrates that the cAMP-CAP complex preferentially binds to the wild-type lac promoter in the presence of a lac promoter mutated at the CAP binding site. Based on these results and comparisons with other experiments reported in the literature, buffer conditions that approximate the physiological state of a cell appear to be best for studying the interaction between CAP and the lactose promoter in vitro.     

Transcription termination: sequence and function of the rho-independent tL3 terminator in the major leftward operon of bacteriophage lambda

For cloning, assaying the function and sequencing terminators, we have constructed the pD12 plasmid, in which the late promotor p'R of phage lambda controls the expression of the galK gene of the pK03 plasmid of McKenney et al. (1981). The lambda tL3 terminator region was cloned in this plasmid between the promoter and the galK gene, and found to be 90-94% effective in preventing galactokinase expression in both rho+ and rho- hosts. Is is also active in vitro, both in the presence or absence of the rho factor. The termination point is located at 4320 bp to the left of the SL startpoint of the PL-RNA, just downstream of gene exo. We have sequenced 356 bp of the hitherto uncharted lambda DNA to the right of the TaqI cut, which in turn is 110 bp to the right of the b522 deletion at 63.9% lambda. The tL3 terminator has several features common to other rho-independent termination sequences, including an 81% G+C-rich region of 2X8-bp symmetry ("stem") with a 5-bp intervening "loop", partially overlapping and followed by a sequence transcribed into the pyrimidine-rich CCUUUCU-OH 3' terminus of the RNA. The termination point that follows the last U was determined by the S1 mapping technique.     

DNA polymerase and thymidine kinase activities in MC-29 virus-induced transplantable hepatoma and the effect of cytostatic treatment on these activities

The activity of DNA polymerases and thymidine kinase was compared in the MC-29 leukosis virus-induced transplantable hepatoma and in the livers of rats treated with cyclophosphamide (CP), cytosine-arabinoside (ara-C) and 5-fluoro-uracil (5-FU). The specific activity of DNA polymerase was twenty times greater in the MC-29 leukosis virus-induced hepatoma, while thymidine kinase was only 3–5 times greater than in the liver.All three enzymes showed Michaelis-Menten kinetics in their substrate and template saturation curves. The template utilization of DNA polymerases from hepatoma and from liver was compared. Both had higher activities on a poly(dA) · poly(dT) template at pH 8.0, than on DNA at pH 7.5. After chromatography on a phosphocellulose column two polymerases were separated. The first peak eluted by 0.15 M KCl preferred DNA as template (polymerase α). The second eluted by 0.5 M KCl worked better on poly(dA) · poly(dT) (polymerase β). Thymidine kinase was eluted by 0.25 M KCl. Inhibition by N-ethylmaleimide (NEM) showed the polymerase α to be sensitive and the polymerase β to be resistant to the sulfhydryl blocking agent; similar to the respective enzymes of other eukaryotic cells. The specific acitivity of DNA polymerase decreased after CP treatment at 6 h and 72 h and after ara-C treatment at 72 h. The specific activities of thymidine kinase were not changed significantly in response to the drug administrations.