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L M Roy

Publications and source records attributed to L M Roy.

11 recordsLinked to original sources
PubMed1999

The maize tha4 gene functions in sec-independent protein transport in chloroplasts and is related to hcf106, tatA, and tatB.

Proteins are translocated across the chloroplast thylakoid membrane by a variety of mechanisms. Some proteins engage a translocation machinery that is derived from the bacterial Sec export system and require an interaction with a chloroplast-localized SecA homologue. Other proteins engage a machinery that is SecA-independent, but requires a transmembrane pH gradient. Recently, a counterpart to this Delta pH mechanism was discovered in bacteria. Genetic studies revealed that one maize protein involved in this mechanism, HCF106, is related in both structure and function to the bacterial tatA and tatB gene products. We describe here the mutant phenotype and molecular cloning of a second maize gene that functions in the Delta pH mechanism. This gene, thylakoid assembly 4 (tha4), is required specifically for the translocation of proteins that engage the Delta pH pathway. The sequence of the tha4 gene product resembles those of the maize hcf106 gene and the bacterial tatA and tatB genes. Sequence comparisons suggest that tha4 more closely resembles tatA, and hcf106 more closely resembles tatB. These findings support the notion that this sec-independent translocation mechanism has been highly conserved during the evolution of eucaryotic organelles from bacterial endosymbionts.

Amino Acid Sequence↗
PubMed1998

A SecY homologue is required for the elaboration of the chloroplast thylakoid membrane and for normal chloroplast gene expression.

Results of in vitro and genetic studies have provided evidence for four pathways by which proteins are targeted to the chloroplast thylakoid membrane. Although these pathways are initially engaged by distinct substrates and involve some distinct components, an unresolved issue has been whether multiple pathways converge on a common translocation pore in the membrane. A homologue of eubacterial SecY called cpSecY is localized to the thylakoid membrane. Since SecY is a component of a protein-translocating pore in bacteria, cpSecY likely plays an analogous role. To explore the role of cpSecY, we obtained maize mutants with transposon insertions in the corresponding gene. Null cpSecY mutants exhibit a severe loss of thylakoid membrane, differing in this regard from mutants lacking cpSecA. Therefore, cpSecY function is not limited to a translocation step downstream of cpSecA. The phenotype of cpSecY mutants is also much more pleiotropic than that of double mutants in which both the cpSecA- and DeltapH-dependent thylakoid-targeting pathways are disrupted. Therefore, cpSecY function is likely to extend beyond any role it might play in these targeting pathways. CpSecY mutants also exhibit a defect in chloroplast translation, revealing a link between chloroplast membrane biogenesis and chloroplast gene expression.

Amino Acid Sequence↗
PubMed1996

Mos proto-oncogene function during oocyte maturation in Xenopus.

The function of the Xenopus c-mos proto-oncogene product (Mos(xe)) has been investigated during oocyte maturation. Experiments with a new antibody able to immunoblot Mos(xe) demonstrated the time course of MAP kinase (MAP K) activation in oocytes paralleled Mos(xe) accumulation, and in activated eggs the deactivation of MAP K paralleled the degradation of Mos(xe). Ablation of Mos synthesis by microinjection of antisense oligodeoxynucleotides abolished activation of MAP K by progesterone, but microinjection of GST-Mos fully restored both MAP K activation and germinal vesicle breakdown (GVBD). The Mos(xe) level at metaphase of Meiosis I (MI) was 2 - 3-fold less than that at metaphase of Meiosis II (MII), but MAP K activation was maximal at metaphase in both MI and MII. In the transition between MI and MII, both cyclin B and Mos(xe) levels rapidly declined in the presence of cycloheximide and injection of exogenous GST-Mos(xe) did not prevent degradation of either protein, although MAP K was activated. Microinjection of GST-Mos(xe) into oocytes was able to activate MAP K before GVBD and H1 kinase activation, and microinjection of constitutively-activated thiophosphorylated MAP K induced de novo synthesis of Mos(xe) before H1 kinase activation, suggesting the existence of a positive feedback loop between MAP K and Mos(xe) accumulation.

Animals↗
PubMed1993

Requirement for Cdk2 in cytostatic factor-mediated metaphase II arrest.

The unfertilized eggs of vertebrates are arrested in metaphase of meiosis II because of the activity of cytostatic factor (CSF). Xenopus CSF is thought to contain the product of the Mos proto-oncogene, but other proteins synthesized during meiosis II are also required for arrest induced by CSF. In Xenopus oocytes, ablation of synthesis of cyclin-dependent kinase 2 (Cdk2) during meiosis resulted in absence of the metaphase II block, even though the Mosxe protein kinase was fully active at metaphase. Introduction of purified Cdk2 restored metaphase II arrest, and increasing the amount of Cdk2 during meiosis I (when Mosxe is present) led to metaphase arrest at meiosis I. These data indicate that metaphase arrest is a result of cooperation between a proto-oncogene kinase and a cyclin-dependent kinase and illustrate the interaction of a cell growth regulator with a cell cycle control element.

Animals↗
PubMed1992

A cdc2-related kinase oscillates in the cell cycle independently of cyclins G2/M and cdc2.

The Eg1 gene in Xenopus laevis is related in sequence to the cdc2+ gene. We show here that the Eg1 gene product (cdk2) possesses histone H1 protein kinase activity and binds to PSTAIR antibodies as well as to Sepharose beads linked to the 13-kDa product of the suc 1 gene (p13suc1). Eg1 protein kinase is active only in an Mr approximately 200,000 complex with other proteins but is not associated with any of the three known Xenopus mitotic cyclins or with any newly synthesized protein in egg extracts that exhibit cell cycle oscillations in vitro. The protein kinase activity of Eg1 oscillates in the mitotic cell cycle, being high in M-phase and low in interphase. Hyperactivation of cdc2 kinase by the addition of cyclin A has no effect on the activity or oscillatory behavior of Eg1. Inhibition of cdc2 kinase activation by emetine or RNase treatment of oscillating extracts does not inhibit the activation of Eg1 but does block deactivation normally seen during exit from mitosis. These results indicate that Eg1 is regulated by a cell cycle clock independently of cyclin and cdc2 kinase.

Amino Acid Sequence↗
PubMed1991

Epidermal growth factor treatment of A431 cells alters the binding capacity and electrophoretic mobility of the cytoskeletally associated epidermal growth factor receptor.

The epidermal growth factor (EGF) receptor interacts with structural elements of A431 cells and remains associated with the cytoskeleton following extraction with nonionic detergents. Extraction of cells with 0.15% Triton X-100 resulted in detection of only approximately 40% of the EGF binding sites on the cytoskeleton. If the cells were exposed to EGF prior to extraction, approximately twofold higher levels of low-affinity EGF binding sites were detected. The difference in number of EGF binding sites was not a consequence of differences in numbers of EGF receptors associated with the cytoskeleton; equal amounts of 35S-labeled receptor were immunoprecipitated from the cytoskeletons of both control and EGF-treated cells. The effect of EGF pretreatment on binding activity was coincident with a change in the mobility of the receptor from a doublet of Mr approximately 160-180 kDa to a single sharp band at 180 kDa. The alteration in receptor mobility was not a simple consequence of receptor phosphorylation in that the alteration was not reversed by alkaline phosphatase treatment, nor was the shift produced by treatment of the cells with phorbol ester. The two EGF receptor species demonstrated differential susceptibility to V8 proteinase digestion. The EGF-induced 180 kDa species was preferentially digested by the proteinase relative to the 160 kDa species, indicating that EGF binding results in a conformational change in the receptor. The EGF-mediated preservation of binding activity and altered conformation may be related to receptor oligomerization.

Carcinoma, Squamous Cell↗
PubMed1991

Activation of p34cdc2 kinase by cyclin A.

Functional clam cyclin A and B proteins have been produced using a baculovirus expression system. Both cyclin A and B can induce meiosis I and meiosis II in Xenopus in the absence of protein synthesis. Half-maximal induction occurs at 50 nM for cyclin A and 250 nM for cyclin B. Addition of 25 nM cyclin A to activated Xenopus egg extracts arrested in the cell cycle by treatment with RNase or emetine activates cdc2 kinase to the normal metaphase level and stimulates one oscillatory cell cycle. High levels of cyclin A cause marked hyperactivation of cdc2 kinase and a stable arrest at the metaphase point in the cell cycle. Kinetic studies demonstrate the concentration of cyclin A added does not affect the 10 min lag period required for kinase activation or the timing of maximal activity, but does control the rate of deactivation of cdc2 kinase during exit from mitosis. In addition, exogenous clam cyclin A inhibits the degradation of both A- and B-type endogenous Xenopus cyclins. These results define a system for investigating the biochemistry and regulation of cdc2 kinase activation by cyclin A.

Animals↗
PubMed1990

The cyclin B2 component of MPF is a substrate for the c-mos(xe) proto-oncogene product.

Previous studies from this laboratory have shown that purified MPF from Xenopus eggs contains cyclin B2 complexed with cdc2 kinase. The activation of MPF during oocyte maturation is known to require expression of the c-mos(xe) proto-oncogene. We show here that immunoprecipitates of either v-mos from Moloney murine sarcoma virus-transformed NIH 3T3 cells or c-mos from Xenopus eggs phosphorylate cyclin B2 in vitro. Phosphopeptide analysis reveals a pattern similar to that observed with cdc2 kinase. Moreover, ablation of c-mos(xe) from oocytes by antisense oligonucleotide injection reduces the rate of cyclin B2 phosphorylation in oocyte extracts by 40%. These results suggest that the mechanism of activation of MPF by c-mos(xe) involves phosphorylation of the cyclin component.

Animals↗
PubMed1989

Characterization of the epidermal growth factor receptor associated with cytoskeletons of A431 cells.

Epidermal growth factor receptors (EGF-R) have been shown to be associated with the detergent-insoluble cytoskeleton of A431 cells, where they retained both a functional ligand-binding domain and tyrosine kinase activity. In the present study we have characterized the tyrosine kinase and ligand binding activities of this cytoskeletally associated EGF-R. The tyrosine kinase activity of the cytoskeletally associated EGF-R was stimulated by EGF treatment of intact cells as evidenced by increased autophosphorylation and phosphorylation of the exogenous substrate angiotensin II (AII). The kinetic behavior of the EGF-R associated with cytoskeletons of EGF-treated cells was similar to that of purified receptors. The stimulation of the receptor kinase activity required EGF treatment of intact cells prior to Triton extraction. If cytoskeletons were prepared from untreated cells and then incubated with EGF, there was no stimulation of the detergent-insoluble receptor kinase activity, indicating that the immobilized receptor was unable to undergo EGF-stimulated activation. Comparison of peptide maps from soluble and cytoskeletally associated EGF-R revealed qualitatively similar patterns; however, they are distinguished by a prominent 46 kD band in digests of the cytoskeletal EGF-R. Saturable binding of 125I-EGF to A431 cytoskeletons prepared from adherent and suspended cells demonstrated the presence of specific receptors on the cytoskeleton. High-affinity EGF-R were preferentially retained upon detergent extraction of adherent cells, whereas both low- and high-affinity receptors were solubilized from the cytoskeletons of suspended cells. Suspension of cells resulted in the solubilization of an additional 15% of the EGF-R to that solubilized in adherent cells, indicating that EGF-R can reversibly associate with the structural elements of the cell.

Adenosine Triphosphate↗
PubMed1986

Anesthetic consideration in dogs with traumatic myocarditis.

The trauma patient is commonly encountered in veterinary practice. Traumatic myocarditis has often been overlooked due to its delayed onset and preoccupation with other traumatic problems. The traumatic heart is very sensitive and in itself can cause death. Often a trauma patient requires surgery and a proper anesthetic protocol must be used which does not increase the incidence of cardiac arrhythmias. A review of anesthetic management including preanesthetic medications, induction drugs and techniques and maintenance with inhalation anesthesia reveals the need for careful selection of medications. Isoflurane was shown to be beneficial in reducing the incidence of undesirable responses of these patients.

Anesthesia↗