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E Knight

Publications and source records attributed to E Knight.

At least 37 records · Page 2Linked to original sources
PubMed1994

Production and characterization of recombinant mouse brain-derived neurotrophic factor and rat neurotrophin-3 expressed in insect cells.

Bioactive brain-derived neurotrophic factor (BDNF) and neurotrophin-3 were produced using the baculovirus expression system and purified to homogeneity using ion-exchange and reversed-phase chromatography. Yields of purified neurotrophin-3 (300-500 micrograms/L) were similar to levels reported for baculovirus-expressed nerve growth factor (NGF), whereas initial yields of BDNF were significantly lower (20-50 micrograms/L). Improved production of BDNF (150-200 micrograms/L) was achieved by expressing BDNF from a chimeric prepro-NGF/mature BDNF construct using the Trichoplusia ni insect cell line. Tn-5B1-4. Examination of the distribution of BDNF protein from both the non-chimeric prepro-BDNF and the chimeric prepro-NGF/mature BDNF viruses in Sf-21- and Tn-5B1-4-infected cells suggests a specific deficiency in the Tn-5B1-4 cells in processing the nonchimeric precursor. In addition, the vast majority of the BDNF protein at 2 days after infection was intracellular and insoluble. N-terminal amino acid sequencing of purified recombinant BDNF and neurotrophin-3 demonstrated that the insect cells processed their precursors to the correct N-terminus expected for the mature protein. Bioactivity was characterized in vitro on primary neuronal cultures from the CNS and PNS.

Animals↗
PubMed1991

A human 15-kDa IFN-induced protein induces the secretion of IFN-gamma.

A 15,000 molecular weight protein (15-kDa), induced and secreted by human PBMC after treatment with IFN-alpha or -beta, was assessed for its ability to modulate cellular function. Although it had no effect on growth or 2'5'-A synthetase activity in Daudi, U-937, or HL-60 cells, when incubated with fresh human PBMC, LPS-induced monocyte cytotoxicity against WEHI-164 target cells was augmented. This stimulation was inhibited by both an antibody against TNF-alpha and a rabbit polyclonal antiserum to the 15-kDa protein. Furthermore, when the 15-kDa protein was added to PBMC an increase in GTP cyclohydrolase I activity, as assessed by neopterin secretion, resulted. Neopterin secretion by PBMC in response to the 15-kDa was increased in a dose-responsive manner up to more than sixfold over baseline, with a 15-kDa concentration of less than 10 ng/ml effective. The 15-kDa protein also stimulated indoleamine 2,3-dioxygenase (IDO) activity in fresh, human PBMC. Induction of neopterin secretion and IDO activity was inhibited by a polyclonal antiserum to 15-kDa. LPS-induced cytotoxic activity was not augmented by 15-kDa pretreatment of purified monocytes, indicating the need for the presence of a second cell population and the indirect action of the 15-kDa on the induction of monocyte activities. When PBMC or purified CD3+ cells, but not purified CD14+ cells, were incubated with the 15-kDa protein, secretion of a factor was induced that resulted in the induction of IDO activity in PMA-differentiated THP-1 cells. An antibody to IFN-gamma, but not IFN-alpha, inhibited the induction of IDO activity by this secreted factor. In addition, antiserum to the 15-kDa blocked the secretion of IFN-gamma from the CD3+ cells. Thus, a 15-kDa product of IFN-alpha- and IFN-beta-treated monocytes and lymphocytes can stimulate secretion of IFN-gamma from CD3+ cells.

Cell Division↗
PubMed1991

IFN-induced 15-kDa protein is released from human lymphocytes and monocytes.

The enhancement or inhibition of synthesis of specific proteins by IFN is believed to cause subsequent IFN-induced biological responses. The roles of most of these proteins in the biological responses induced by the IFNs, for example, inhibition of virus replication and inhibition of cell growth, remain largely unknown. Our recent research has focused on the induction and synthesis of an IFN-induced 15-kDa protein. In this report we show that human lymphocytes and monocytes, after treatment with IFN-beta, release into the medium an IFN-induced 15-kDa protein. At 24 h after induction of the 15-kDa protein in lymphocytes or monocytes, more than 50% of the total 15-kDa protein is in the medium. The human monocytic cell line THP-1 also releases 15-kDa protein into the medium after its induction by IFN-beta. An intracellular half-life of 12 h has been calculated for the 15-kDa protein in monocytes and THP-1 cells. The exocellular release of the 15-kDa protein by lymphocytes and monocytes suggests that 1) it may have an intercellular signaling role and 2) it may be an in vivo mediator of some of the biological responses induced by IFN.

Cell Communication↗
PubMed1991

An interferon-induced 16-kD protein is present in the membranes of interferon-sensitive but not in interferon-resistant mouse cells.

Human interferon-alpha (IFN-alpha) or IFN-beta has been shown to induce a 17-kD membrane protein in human cells which when eluted from SDS gels inhibited the multiplication of cells of different human cell lines. We show herein that mouse IFN-alpha/beta induces a 16-kD membrane protein in L1210 and Friend erythroleukemia cells sensitive to IFN-alpha/beta, (but not in the derived IFN-alpha/beta-resistant cell lines) as well in primary and monolayer cultures of mouse embryonic fibroblasts and adult mouse hepatocytes, and in suspensions of spleen cells. In addition, IFN-alpha/beta enhanced the expression of an 11-kD membrane protein which could be shown by immunoprecipitation to be beta 2-microglobulin. Anticell proliferation activity was not recovered from the 16-kD fraction of the SDS gels.

Animals↗
PubMed1991

Clumsiness in children--do they grow out of it? A 10-year follow-up study.

The question of whether problems of motor co-ordination in early childhood recede with age has rarely been addressed. This paper reports the findings from a follow-up study of 17 children, identified by their teachers as having poor motor co-ordination at age six. Now age 16, these children and their matched controls completed a battery of assessments. The results suggest that the majority of children still have difficulties with motor co-ordination, have poor self-concept and are experiencing problems of various kinds in school. However, there are individual differences in the extent to which the children have learned to cope with their continuing difficulties over the years.

Achievement↗
PubMed1989

A 15-kD interferon-induced protein and its 17-kD precursor: expression in Escherichia coli, purification, and characterization.

Using recombinant DNA technology, a 15-kD interferon (IFN)-induced protein and its 17-kD precursor have been expressed in Escherichia coli to obtain sufficient quantities of each protein for the investigation of their biological roles. Both the 15-kD and 17-kD proteins have been purified to homogeneity and crystallized. The recombinant 15-kD protein has an identical reversed-phase HPLC elution profile to that of the native 15-kD protein purified from human cells. Furthermore, the recombinant 15-kD and 17-kD proteins have identical amino- and carboxy-terminal amino acid sequences to those predicted from the DNA sequence. The native and recombinant 15-kD proteins give identical tryptic peptide maps, and the recombinant 17-kD protein gives only one additional tryptic peptide. We conclude that the recombinant 17-kD and 15-kD proteins are identical to the 17-kD precursor and the 15-kD stable product synthesized in human cells in response to IFN stimulation. In addition, we have demonstrated that the recombinant 17-kD precursor protein can be converted to the 15-kD protein by cytoplasmic extracts of human cells.

Amino Acid Sequence↗
PubMed1988

A 15-kDa interferon-induced protein is derived by COOH-terminal processing of a 17-kDa precursor.

An interferon-induced 15-kDa protein is synthesized from a precursor of higher molecular weight; the precursor contains 165 amino acids (17 kDa), whereas the stable product (15 kDa) contains 156 amino acids. The stable 15-kDa form is derived from the precursor 17-kDa form by the removal of eight amino acids from the COOH terminus and the methionine from the NH2 terminus. The existence of the precursor 17-kDa protein can be demonstrated after brief periods of in vivo labeling with [35S]methionine and by translation of mRNA in vitro.

Amino Acid Sequence↗
PubMed1988

Hepatic responses to edible gums during refeeding of starved rats.

The ability of edible gums to depress total liver lipids and activities of two hepatic enzymes (glucose-6-phosphate dehydrogenase and NADP-linked malic enzyme) was examined during the refeeding of 2 d starved rats. Gums were fed as 4% of dry ingredients, occasionally with added water, in otherwise identical high glucose, nutritionally adequate diets. Feeding of xantham gum for 1 or 2 d decreased the rise in two enzyme activities and in total liver lipids, but after 4 or 7 d only total liver lipids were affected. Agar, which is insoluble at room temperature, was effective only when incorporated in the diet as a stiff gel. Guar, carrageenan, karaya and pectin reduced the change in at least one hepatic parameter, but acacia and gum ghatti, which impart little viscosity to water, were without effect. The ability of seven gums to form semisolid gels at low concentrations was measured and appeared to be associated with the variable effectiveness of those gums in influencing lipid contents and enzyme activities of the liver.

Animals↗
PubMed1987

A membrane protein from IFN-beta-treated Daudi cells causes a cessation in cell growth.

An interferon-induced 17 kDa protein has been partially purified from the membranes of interferon-beta-treated Daudi cells. A fraction containing the 17 kDa protein purified 200 fold causes an inhibition of growth of Daudi, Namalva and Hela cells. The same fraction purified from the membranes of untreated cells causes no inhibition of cell growth. This interferon-beta-induced protein is located on the exterior of Daudi cells for it can be labeled with [125I] iodine catalyzed by lactoperoxidase. These results suggest that interferons induce a cell surface protein whose role is to cause an inhibition of cell growth.

Cell Division↗
PubMed1987

A growth inhibitory protein secreted by human diploid fibroblasts. Partial purification and characterization.

We have identified and partially purified a growth inhibitor protein secreted by human diploid fibroblast cells. This protein is not secreted constitutively but only after induction with the double stranded hetero duplex polyriboinosinic:polyribocytidylic acid. The growth inhibitory activity has been purified 3,800-fold and has an estimated molecular mass of 12,000 daltons. The protein will inhibit the growth in culture of human diploid fibroblast cells, human cells derived from tumors, and mouse L cells. Although interferon-beta is secreted with the growth inhibitory protein, the partially purified growth inhibitory protein has no antiviral activity, and its activity is not neutralized by antibodies to interferon-alpha, interferon-beta, and interferon-gamma. We believe this growth inhibitory activity to reside in a newly defined protein and have named it fibroblast-derived growth inhibitor.

Cycloheximide↗
PubMed1987

Acute nephropathy induced by gold sodium thiomalate: alterations in renal heme metabolism and morphology.

Gold compounds are used clinically in rheumatoid arthritis therapy. Acute renal toxicity is observed in some patients receiving chrysotherapy. The present study addresses morphofunctional and biochemical changes in rat kidneys during the first 8 days following a single ip injection of gold sodium thiomalate (AuTM), one of the gold compounds presently in clinical use. Compared to controls, AuTM pretreatment resulted in increased urine output and elevated serum creatinine and urea nitrogen concentrations. Also, by Day 8, treated rats had decreased body weights and increased kidney weights. Postmortem examination on Day 1 showed pale and mottled kidneys and diffusely pale inner cortex. Microscopically, there was severe coagulative necrosis of the proximal tubular epithelium. Epithelial regeneration was prominent by Day 4 and was nearly complete by Day 8. The regenerating epithelium was hyperplastic with basophilic cytoplasm and pleomorphic nuclei. Alterations in renal heme biosynthesis and drug metabolism paralleled the morphologic changes. The activity of delta-aminolevulinic acid dehydratase and benzo[a]pyrene hydroxylase were inhibited on Days 1, 2, and 4 following AuTM administration. Decreases in monooxygenase activity were accompanied by decreases in renal cytochrome P-450 levels. In contrast, renal microsomal heme oxygenase activity was elevated 9.5-fold on Day 1 and 2.5-fold on Day 2. By Day 8, all renal enzymatic activities assayed for were similar to those obtained with untreated rats.

Animals↗
PubMed1987

Interferon-induced transcription of a gene encoding a 15-kDa protein depends on an upstream enhancer element.

A human gene encoding an interferon-induced 15-kDa protein has been isolated from a genomic library. The gene appears to be single-copy and is composed of two exons, the first of which contains the ATG translation initiation codon. In vitro nuclear run-on assays showed that the transcription rate of the gene is stimulated after interferon treatment. To analyze transcriptional regulatory sequences, we constructed recombinant plasmids for use in transient transfection assays of HeLa cells. Constructs containing 115 nucleotides 5' to the transcription initiation site were found to be fully inducible by interferon. Assays of deletion mutants identified a critical element for interferon induction located between -115 and -96, just upstream of the "CCAAT box." Moreover, a DNA fragment including this region can confer interferon inducibility on a heterologous promoter (thymidine kinase) when cloned in either orientation upstream of the gene or downstream of the gene. These are properties characteristic of an enhancer element that is active only after treatment with interferon. This regulatory sequence may be shared by a group of interferon-induced genes, since a very similar sequence is present within the functional region near the RNA start site of another interferon-induced gene.

Amino Acid Sequence↗
PubMed1987

Regulation of c-myc RNA and its proteins in Daudi cells by interferon-beta.

It has been shown previously that interferons (IFNs) -alpha and -beta cause a reduction in the steady-state level of poly(A) c-myc RNA in the Burkitt lymphoma, Daudi. In this report we show that the c-myc RNA reduction is not mediated by simple changes in the poly-adenylation of either nascent or existing c-myc transcripts, since similar reductions of c-myc sequences were observed in poly(A) and total cellular RNA preparations from IFN-beta-treated cells. Furthermore, the first exon of c-myc RNA in Daudi cells contains several mutations, suggesting that the germ line configuration of the first exon is not essential for the IFN-beta-mediated regulation. The c-myc RNA reduction was also detected in cells whose protein synthesis was inhibited by more than 95% with cycloheximide or emetine. We surmise that neither sustained nor IFN-induced protein synthesis is required for the c-myc RNA regulation. Antisera raised against either the carboxy- or amino-terminal c-myc peptides precipitate in Daudi cells proteins of 66,000 and 63,000 daltons. In cells treated with IFN-beta, the amounts of these proteins are reduced by 46-74% which is in agreement with the reduction detected at the level of c-myc RNA.

Burkitt Lymphoma↗
PubMed1987

Orthodeoxia: a new finding in interstitial fibrosis.

Orthodeoxia--arterial desaturation accentuated by the upright position and improved by recumbency--has been described earlier with true pulmonary vascular shunts and intracardiac shunts. This phenomenon has been described in some parenchymal lung diseases, but has not been reported with interstitial fibrosis. We describe 2 patients with predominantly basal interstitial fibrosis, disabling dyspnea, and severe hypoxemia who demonstrated this positional oxygenation change. Large or surgically correctable arteriovenous malformations (AVM), or intracardiac shunts, were not demonstrated in either patient. This finding has important implications for oxygen prescription and the explanation of positional dyspnea.

Aged↗