Incorporation in vitro of labeled amino acids into bone marrow cell proteins

Nearly all experiments on the incorporation of labeled amino acids into tissue proteins in vitro have been done on tissues whose cell structure has been partially or completely disintegrated, e.g. tissue slices, segments, or homogenates. Since cell destruction reduces or abolishes the uptake of labeled amino acids (1), it seemed worth while to carry out studies on intact cells in vitro. Bone marrow cells were found to be suitable for this purpose. The labeled amino acids used were glycine-1-C14, L-leucine-1-C14, L-lysine-1-C14, and L-lysine-6-C14

The degradation of L-lysine in guinea pig liver homogenate: formation of alpha-aminoadipic acid

A summary of the little that is known of the metabolism of lysine in animals is as follows: it is indispensable in the diet, its α-amino group does not participate in reversible transamination reaction in vivo (2), neither the L nor D form is attacked by the appropriate amino acid oxidase, certain ε-nitrogen-substituted derivatives can replace lysine in the diet and their α-amino groups are oxidized by amino acid oxidases (3, 4), no α-nitrogen-substituted derivatives yet prepared can substitute for lysine in the diet (4-6)

Isolation of a peptide in guinea pig liver homogenate and its turnover of leucine

Leucine was synthesized with C14 in the carboxyl group. 10 mg. of the radioactive amino acid (DL) and 0.66 gm. (wet weight) of guinea pig liver homogenate were added to a reaction mixture containing 1.3 per cent of an amino acid mixture corresponding to the composition of casein and 0.005 M fumarate, all in a final volume of 4 ml. of isotonic saline solution(1) at pH 7.4. The reaction was carried out under oxygen for 6 hours at 38°

Alpha-aminoadipic acid: A product of lysine metabolism

As part of a study of protein and peptide metabolism lysine was synthesized with C14 in the ε position and resolved into the L and D isomers. 10 mg. of labeled lysine dihydrochloride (either L- or D-) and 0.66 gm. (wet weight) of guinea pig liver homogenate were added to a reaction mixture containing 1.3 per cent of an amino acid mixture corresponding to the composition of casein except for lysine and 0.01 M α-ketoglutarate, all in a final volume of 4 ml. of isotonic saline solution.(1) The reaction was carried out under oxygen for 6 hours at 38°

The incorporation of labeled lysine into the proteins of guinea pig liver homogenate

When C14-labeled lysine is incubated with guinea pig liver homogenate, α-aminoadipic, α-ketoadipic, and glutaric acids are formed from the lysine (1). These transformations were established by finding the radioactivity of the C14 tracer in the metabolic products. The homogenate proteins coagulated by boiling at pH 5 also contained radioactivity. The counts given by the proteins corresponded to about 0.02 to 0.03 per cent of that added as lysine; the extent of lysine incorporation into the proteins was of the same order of magnitude as Melchior and Tarver (2) had found after incubating S35-labeled methionine and Winnick et al. (3, 4) C14-labeled glycine with rat tissue homogenates. Yet we could not satisfy ourselves that the radioactivity remaining in the proteins in our experiments, although it persisted through exhaustive extraction, did not come from traces of adsorbed radioactive lysine. Some counts were found in the protein when the homogenate was boiled prior to incubation with isotopic lysine

Incorporation in vitro of labeled amino acids into proteins of rabbit reticuloytes

Continuing our work on the incorporation of labeled amino acids into proteins (1), we have begun a study of the incorporation in vitro of C14-labeled glycine, L-histidine, L-leucine, and L-lysine into the proteins of rabbit reticulocytes. In preliminary experiments the incorporation into the hemoglobin isolated from the reticulocytes was determined. But, after it was found that plasma contains factors accelerating amino acid incorporation, it was decided to proceed as rapidly as possible toward the identification of these factors; we have, therefore, measured incorporation into the total proteins of the reticulocytes, since isolation of the hemoglobin was time-consuming. The results obtained with hemoglobin and with the total proteins are essentially the same, indicating that the other proteins of the reticulocytes incorporate amino acids at approximately the same rate as hemoglobin

The uptake in vitro of C14-labeled glycine, L-leucine, and L-lysine by different components of guinea pig liver homogenate

We have reported (1) that L-lysine labeled with C14 can be incorporated into the proteins of guinea pig liver homogenate under two different conditions. In the one case the enzyme used was the whole homogenate, the optimum pH was near 6.2, there was an obligatory requirement of calcium, and the incorporation was independent of oxygen. This set of conditions is designated below as the “acid calcium” condition. In the other case the enzyme system was the precipitate obtained by centrifuging the homogenate diluted 15-fold with Ringer’s solution at 2500 X g, the optimum pH was near to 7.3, the reaction was accelerated a little by calcium but the presence of calcium was not obligatory, and the incorporation was a little less under nitrogen than under oxygen. This set of conditions is designated below as the “alkaline” condition

A peptide fraction in liver

We reported in a preliminary communication (1) the isolation of a peptide fraction from guinea pig liver. The following points of interest appeared at once: many different amino acids were obtained on hydrolysis; the peptide fraction contained most of the indispensable amino acids, which indicated that it probably is important in protein metabolism; when guinea pig liver homogenate was incubated with C14-labeled glycine, leucine, or lysine, these were rapidly incorporated into this peptide fraction, which is further evidence that it is metabolically active; the peptide fraction had not been described hitherto; a fraction containing one or more large peptides can be separated from so complex a mixture as liver homogenate by starch chromatography

Cerebral edema in a patient following cytoreductive surgery and hyperthermic intraoperative intraperitoneal chemoperfusion

BACKGROUND: Cytoreductive surgery and intraoperative, intraperitoneal hyperthermic chemoperfusion (HIPEC) is increasingly used to treat peritoneal surface metastases. We describe a fatal case of cerebral edema in a patient with appendiceal carcinoma and an underlying seizure disorder who underwent cytoreductive surgery and HIPEC. CASE PRESENTATION: A case of fatal postoperative cerebral edema is presented in a patient with an underlying seizure disorder and recurrent mucinous adenocarcinoma of the appendix. The patient was treated with cytoreductive surgery and intraoperative intraperitoneal hyperthermic chemoperfusion. The details and implications of this complication are discussed. CONCLUSION: The recognition of this potential complication is important for physicians performing cytoreductive surgery and HIPEC. Special caution should be taken when patients with seizure disorders are being considered for this treatment

Chemokine CXCL12 activates dual CXCR4 and CXCR7-mediated signaling pathways in pancreatic cancer cells

<p>Abstract</p> <p>Background</p> <p>Previously assumed to be a select ligand for chemokine receptor CXCR4, chemokine CXCL12 is now known to activate both CXCR4 and CXCR7. However, very little is known about the co-expression of these receptors in cancer cells.</p> <p>Methods</p> <p>We used immunohistochemistry to determine the extent of co-expression in pancreatic cancer tissue samples and immunoblotting to verify expression in pancreatic cancer cell lines. In cell culture studies, siRNA was used to knock down expression of CXCR4, CXCR7, K-Ras and β-arrestin -2 prior to stimulating the cells with CXCL12. Activation of the mitogen-activated protein kinase pathway (MAPK) was assessed using both a Raf-pull down assay and western blotting. The involvement of the receptors in CXCL12-mediated increases in cell proliferation was examined via an ATP-based proliferation assay.</p> <p>Results</p> <p>First, we discovered frequent CXCR4/CXCR7 co-expression in human pancreatic cancer tissues and cell lines. Next, we observed consistent increases in ERK1/2 phosphorylation after exposure to CXCL12 or CXCL11, a CXCR7 agonist, in pancreatic cancer cell lines co-expressing CXCR4/CXCR7. To better characterize the receptor-mediated pathway(s), we knocked down CXCR4 or CXCR7, exposed the cells to CXCL12 and examined subsequent effects on ERK1/2. We observed that CXCR7 mediates the CXCL12-driven increase in ERK1/2 phosphorylation. Knockdown of CXCR4 expression however, decreased levels of K-Ras activity. Conversely, KRAS knockdown greatly reduced CXCL12-mediated increases in ERK1/2 phosphorylation. We then evaluated the role of β-arrestin-2, a protein directly recruited by chemokine receptors. We observed that β-arrestin-2 knockdown also inhibited increases in ERK1/2 phosphorylation mediated by both CXCR4 and CXCR7. Finally, we investigated the mechanism for CXCL12-enhanced cell proliferation and found that either receptor can modulate cell proliferation.</p> <p>Conclusions</p> <p>In summary, our data demonstrate that CXCR4 and CXCR7 are frequently co-expressed in human pancreatic cancer tissues and cell lines. We show that β-arrestin-2 and K-Ras dependent pathways coordinate the transduction of CXCL12 signals. Our results suggest that the development of therapies based on inhibiting CXCL12 signaling to halt the growth of pancreatic cancer should be focused at the ligand level in order to account for the contributions of both receptors to this signaling pathway.</p