Regulation of vaccinia viral infection was studied using three animal cell lines: KRC-7 (rat hepatoma), L929 (mouse fibroblast), and CV-1 (African green monkey kidney). KRC-7 is highly enriched in p67, a glycoprotein which protects eIF-2 alpha-subunit from phosphorylation by eIF-2 kinases. We report: (i) At 5 pfu per cell of the virus, KRC-7 is resistant to the virus. Other cells are sensitive. At 25 pfu per cell of the virus, KRC-7 is also sensitive to the virus. After productive viral infection, the cell extracts showed strong p67-DG activity and actively deglycosylated exogenous p67. After p67-deglycosylation, the cell extracts also phosphorylated eIF-2. (ii) The rate of synthesis of a major host protein (approximately 45 kDa) in infected L929 cells measured after 2 h of viral infection declined more than 50%. The rate declined thereafter. The rate of synthesis of host proteins in viral-resistant KRC-7 cells (infected with 5 pfu per cell of the virus) remained unchanged. The mechanism of resistance of KRC7 cells to vacinia virus at 5 pfu per cell of the virus was investigated. The p67 level in these cells was varied by growing the cells under different physiological conditions such as serum starvation and expression of p67-sense and p67-antisense DNA. At low p67 level in the cells, p67-DG is activated. This deglycosylates p67 and inactivates p67. This accompanies eIF-2 phosphorylation and shutoff of host protein synthesis. At high p67 level in the cells, activation of p67-DG is prevented. This prevents shut-off of host protein synthesis and viral growth.

Acetylglucosaminidase↗

PubMed1997

Viral infection. II. Hemin induces overexpression of p67 as it partially prevents appearance of an active p67-deglycosylase in baculovirus-infected insect cells.

The roles of p67-deglycosylase (p67-DG) in the regulation of protein synthesis in baculovirus-infected insect cells were studied. Like vaccinia viral infection, baculovirus infection of insect cells also induced the appearance of a p67-DG. However, p67-DG activity could not be detected because these cells do not contain a detectable level of p67. The baculovirus expression vector system (BEVS), however, promotes significant expression of cloned p67-cDNA. The expression of p67 was significantly enhanced by the addition of hemin to the growth medium. Maximum enhancement was observed at 5 microM hemin. Data suggest that hemin prevents the activation of latent p67-DG inside the cell and does not have any effect on p67 gene transcription. To gain a better understanding of the mechanism of p67-DG activation and hemin stimulation of p67 synthesis, we have now purified p67-DG from baculovirus-infected insect cells. We prepared antibodies against this protein. These antibodies reacted with a 105-kDa protein in cell extracts from the uninfected insect cells (Sf9), KRC-7, and L929 (animal cells). In addition, these antibodies reacted with an additional 60-kDa protein in the cell extracts of baculovirus-infected Sf9 cells and vaccinia virus-infected KRC-7 and L929 cells. Data are also presented to show that the antibodies against p67-DG reacted more efficiently (40%) with the 60-kDa protein in both hemin-deficient reticulocyte lysate and hemin-deficient baculovirus-infected cells. We suggest that hemin prevents the conversion of an inactive p67-DG into an active form possibly by covalent modification such as protein phosphorylation or protein glycosylation. The active form is more efficiently recognized by the p67-DG antibodies since these antibodies were prepared against the active form of p67-DG.

Acetylglucosaminidase↗

PubMed1997

p67 transcription regulates translation in serum-starved and mitogen-activated KRC-7 cells.

The regulation of protein synthesis was studied in KRC-7 cells (rat hepatoma) grown in complete medium, during serum starvation, and mitogen activation. Upon serum starvation, the cells lost almost completely p67 mRNA, p67 protein, and protein synthesis activity. After phorbol 12-myristate 13-acetate addition, the same serum-starved cells regained p67 mRNA, p67 protein, and protein synthesis activity. Also, the extracts from the serum-starved cells phosphorylated the eukaryotic initiation factor-2 (eIF-2) alpha-subunit. This eIF-2 alpha-subunit phosphorylation was not observed when the extracts from either the cells grown in complete medium or mitogen-activated cells were used (Gupta, S., Wu, S., Chatterjee, N., Ilan, J., Ilan, J., Osterman, J. C., and Gupta, N. K. (1995) Gene Expr. 5, 113-122). We now report the following. 1) The eIF-2 kinase activity was the same in the cells grown in complete medium, after serum starvation, and subsequent mitogen stimulation. However, the eIF-2 kinase in the cells grown in complete medium and also after mitogen activation of the serum-starved cells cannot phosphorylate eIF-2 alpha-subunit as these cells contain p67. After removal of endogenous p67 by p67 antibodies, the extracts from all these cells similarly phosphorylated exogenously added eIF-2. 2) None of the cell extracts showed p67 deglycosylase activity. 3) The p67 mRNA was synthesized in serum-starved cells by expression of a p67 cDNA. The appearance of p67 mRNA in the serum-starved cells was accompanied by the appearance of p67 protein. Also, the rates of protein synthesis in the serum-starved cells were restored nearly to the level observed in the confluent cells. The expression of p67 cDNA also significantly increased protein synthesis rates in the cells grown in complete medium and in mitogen-activated cells. These results show that the loss of protein synthesis activity in serum-starved cells was due to loss of p67 mRNA. The expressed p67 mRNA was stable in serum-starved cells. These results, therefore, suggest that the loss of p67 mRNA in serum-starved cells is due to loss of p67 transcription. The p67 transcription regulates translation.

Aminopeptidases↗

PubMed1997

Nine cases of Merkel cell tumour.

Merkel cell tumour is an aggressive neuroendocrine neoplasm arising in the dermis. Although only a few hundred cases have been reported worldwide, nine were seen in Nottingham between 1985 and early 1994. The patients were five women and four men age 63-88. One was the first Afro-Caribbean reported to have such a tumour. In no case was the diagnosis made clinically; histological and histochemical examination was necessary. Three of the patients died quickly with metastatic disease. The primary treatment is surgical excision. For advanced disease, radiotherapy is commonly beneficial.

Aged↗

PubMed1997

Neuroendovascular management of intracranial aneurysms.

Intracranial aneurysms are a common disease constituting a significant health problem worldwide. Aneurysmal subarachnoid hemorrhage, cerebral aneurysms, classification of aneurysms, and management of aneurysms are discussed.

Cerebrovascular Circulation↗

PubMed1996

Functional reorganization in thalamocortical networks: transition between spindling and delta sleep rhythms.

Thalamic reticularis, thalamocortical, and cortical cells participate in the 7-14-hz spindling rhythm of early sleep and the slower delta rhythms of deeper sleep, with different firing patterns. In this case study, showing the interactions of intrinsic and synaptic properties, a change in the conductance of one kind of cell effectively rewires the thalamocortical circuit, leading to the transition from the spindling to the delta rhythm. The two rhythms make different uses of the fast (GABAA) and slow (GABAB) inhibition generated by the thalamic reticularis cells.

Cerebral Cortex↗

PubMed1996

Angiography of cerebral aneurysms.

Magnetic resonance and computed tomographic angiography have been increasingly applied to the study of disease affecting the cerebral vasculature. Despite these advances, however, conventional cerebral angiography clearly remains the diagnostic gold standard and essential guide to any microneurosurgical or endovascular therapeutic decision concerning cerebral aneurysms. Detailed cerebral angiography is a dynamic study and is influenced by prior axial imaging. It should be tailored to the specific circumstance to obtain information required for selection of the most beneficial treatment.

Aneurysm, Ruptured↗

PubMed1995

Large scale isolation of expression vector cassette by magnetic triple helix affinity capture.

Explore the source record for details and available documents.

Base Composition↗

PubMed1994

Regulation of eIF-2 alpha-subunit phosphorylation in reticulocyte lysate.

An eIF-2 associated 67-kDa protein (p67) protects eIF-2 alpha-subunit from eIF-2 kinase(s) catalyzed phosphorylation and promotes protein synthesis in the presence of active eIF-2 kinase(s). p67 is a glycoprotein and contains multiple O-linked GlcNAc moieties. We have now studied the roles of hemin, p67, and the glycosyl residues on p67 in the regulation of eIF-2 alpha-subunit phosphorylation in reticulocyte lysates. The results are as follows: (i) Both hemin and p67 inhibited HRI (heme-regulated protein synthesis inhibitor) and dsI (double-stranded RNA activated protein synthesis inhibitor) catalyzed phosphorylation of eIF-2 alpha-subunit in vitro. However, only hemin, and not p67, inhibited casein kinase catalyzed phosphorylation of eIF-2 beta-subunit. (ii) Only p67, and not hemin, inhibited eIF-2 alpha-subunit phosphorylation by eIF-2 kinase(s) in reticulocyte lysate. Significant eIF-2 alpha-subunit phosphorylation was observed even in the presence of hemin when p67 in the reticulocyte lysate was removed by treatment with p67 antibodies. (iii) Reticulocyte lysate contains a p67-deglycosylase in latent form, and hemin prevents activation of this deglycosylase. In the absence of hemin, this p67-deglycosylase is activated. Once activated in the absence of hemin, the activated deglycosylase deglycosylates p67, even in the presence of hemin. This inactivates p67 and allows eIF-2 kinase to phosphorylate eIF-2 alpha-subunit and inhibit protein synthesis. Protein synthesis in reticulocyte lysate is thus regulated by two novel cascades of covalent modifications: protein deglycosylation leading to protein phosphorylation.

Acetylglucosaminidase↗

PubMed1994

Mechanism of action of an eukaryotic initiation factor-2 (eIF-2) associated 67 kDa glycoprotein (p67) and an eIF-2 kinase (dsI).

Mechanism of regulation of eIF-2 alpha-subunit phosphorylation by dsI and p67 was studied. The results are as follows: (1) At low dsI concentration, p67 protected equimolar concentration of eIF-2. (2) At high dsI concentration, dsI efficiently phosphorylated eIF-2 alpha-subunit even when equimolar concentrations of both p67 and eIF-2 were present. Significantly increased p67 concentration was necessary to protect eIF-2 alpha-subunit at high dsI concentration. (3) dsI was also phosphorylated as it phosphorylated eIF-2 alpha-subunit. p67 inhibited both eIF-2 alpha-subunit and dsI phosphorylation similarly. (4) Although the [32P]-labelled dsI formed during the reaction could be effectively chased upon subsequent addition of excess unlabelled eIF-2 and ATP, the [32P] labelled eIF-2 formed under identical conditions, retained most of the radioactivity. (5) dsI coimmunoprecipitated with three subunit eIF-2 and p67 inhibited this coimmunoprecipitation reaction. It has been proposed: Three subunit eIF-2 and free p67 are in equilibrium with eIF-2 bound to p67 and, eIF-2.p67 complex is resistant to dsI phosphorylation. Activated dsI is already phosphorylated. At high concentration, dsI(P) can bind to free three subunit eIF-2 and form eIF-2.dsI(P) complex. dsI(P) in this complex then transfers its phosphoryl residue to eIF-2 and forms eIF-2 alpha(P) in an irreversible reaction. In a subsequent reaction, unphosphorylated dsI is autophosphorylated using [gamma 32P]-ATP and the cycle continues. Inhibition of eIF-2 alpha-subunit phosphorylation by p67 blocks this phosphorylation cycle and consequent dsI phosphorylation.

Aminopeptidases↗

PubMed1993

Characteristics of the eukaryotic initiation factor 2 associated 67-kDa polypeptide.

A eukaryotic initiation factor 2 (eIF-2) associated 67-kDa polypeptide (p67) protects the eIF-2 alpha-subunit from eIF-2 kinase(s) catalyzed phosphorylation, and this promotes protein synthesis in the presence of active eIF-2 kinase(s), [Datta, B., et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3324-3328]. This report presents the results of studies related to characteristics of p67 action and the mechanism of p67:eIF-2 interaction: (1) p67 antibodies inhibited protein synthesis in hemin-supplemented rabbit reticulocyte lysates, and such inhibition was reversed by preincubation of the antibodies, specifically with p67. (2) p67 inhibited HRI- and dsI-catalyzed phosphorylations of the eIF-2 alpha-subunit and histones, but it did not inhibit casein kinase catalyzed phosphorylation of the eIF-2 beta-subunit. (3) p67 bound specifically to the eIF-2 gamma-subunit. p67 co-immunoprecipitated with the eIF-2 subunits when a p67/eIF-2 mixture was treated with p67 or eIF-2 subunit antibodies and protein A agarose. However, when eIF-2 was preincubated specifically with the eIF-2 gamma-subunit antibodies, subsequent co-immunoprecipitation of p67 with the eIF-2 subunits was completely inhibited. Similarly, preincubation of p67 and p67 antibodies prevented subsequent p67 binding to eIF-2. Preincubation of eIF-2, with either eIF-2 alpha- or beta-subunit antibodies, had no effect on p67 co-immunoprecipitation with the eIF-2 subunits. (4) p67:eIF-2 interaction is necessary for p67 activity to protect the eIF-2 alpha-subunit from eIF-2 kinase(s) catalyzed phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Casein Kinases↗

PubMed1991

Syphilitic meningitis causing paraparesis in an HIV-negative woman.

Syphilitic meningitis, which can occur near the time of secondary syphilis, is frequently asymptomatic. There has been one recent report of an HIV-positive patient who developed syphilitic polyradiculopathy following a recent history of secondary syphilis. We describe an HIV-negative woman in whom paraparesis occurred secondary to syphilitic meningitis. Complete recovery followed a course of high-dose intravenous penicillin therapy, emphasizing the treatable nature of this cause of paraparesis.

Adult↗

PubMed1991

Single-photon emission computed tomography in distal field hypoperfusion.

To assess the ability of technetium-99m hexamethylpropyleneamineoxime single-photon emission computed tomography (SPECT) imaging to differentiate distal field hypoperfusion from other stroke mechanisms, 24 patients with acute cerebral ischemia were studied. SPECT scans were read by two physicians according to a preestablished set of criteria for distal field hypoperfusion. SPECT patterns read as "probable" or "definite" for distal field hypoperfusion were found in 42% (10/24); of these, 80% (8/10) had ipsilateral carotid occlusion or high-grade stenosis. Severe carotid stenosis was found in 43% (6/14) with SPECT scans negative for distal field hypoperfusion (Fisher exact test [1-tailed] p = 0.0796). The results suggest that a distal field hypoperfusion pattern on SPECT may identify patients with hemodynamically significant large vessel disease.

Arterial Occlusive Diseases↗