Penelope A. Koch

Biogenesis of erythrocyte membrane proteins in vivo studies in anemic rabbits

To study the process of red cell membrane protein synthesis we have followed the time course of [3-H]leucine appearance in total protein and individual peptides of the erythrocyte membrane following injection of the amino acid into phenylhydrazine-anemic rabbits. Multiple peripheral blood samples were taken from single animals over a 5-week period. Erythrocyte membrane proteins were separated by polyacrylamide gel electrophoresis in sodium dodecylsulfate and dithiothreitol; incorporation of radioactivity was determined by gel slicing and liquid scintillation spectrometry. Appearance of [3-H]leucine in circulating erythrocytes reached a peak at 1-3 days, with a steady decline thereafter. The radioactive amino acid appeared first in the lowest molecular weight peptides and last in the largest peptides; at the earliest time point (8 h), little radioactivity was observed in any of the four largest peptides present in the membranes (bands A, 1, 2 and 3). Certain smaller peptides (bands 4, 5 and 9) were the predominant species labeled at this time. By 24 h all peptides showed significant incorporation. With maturation of the red cells, label largely disappeared from bands A, 9 and several smaller peptides; this was confirmed by finding that the peptides are virtually absent from mature circulating erythrocytes. These data are interpreted as showing that red cell membrane proteins are synthesized asynchronously during the life cycle of the erythrocyte; the largest peptides are made predominantly in the earlier marrow stages of development, while certain of the smaller peptides are still being synthesized in the reticulocyte stage. Several membrane proteins appear to be specific to the reticulocyte and are lost during the process of cell maturation in the circulation.

Red cell maturation: Loss of a reticulocyte-specific membrane protein

Abstract A protein present in red cell ghost membranes from reticulocyterich blood disappears from the membrane in vivo as the reticulocytes mature. This protein, with an estimated molecular weight of 33,000, is not present in mature circulating red blood cells. It is not associated with the ribosomes present in the reticulocyte.

Role of a child advocate in the selection of donors for pediatric bone marrow transplantation

FROM A MEDICAL-LEGAL POINT OF VI[EW, pediatric bone marrow transplantation may be complicated when the sibling donor, as well as the recipient, is a minor. A conflict of interest arises when parents are asked to give informed consent for both donor and recipien t . Strongly motivated to cure their sick child, parents might not adequately consider the interests of the donor, their healthy child, who must undergo the risk of anesthesia and the pain Of marrow harvest. In 1976, prior to beginning a bone marrow transplantation program at the Childrens Hospital of Philadelphia, the transplant physicians requested the Administrative Judge of the Philadelphia Family Court to assist in developing a judicial procedure optimally responsive to the medical and legal interests of those involved.

Biogenesis of erythrocyte membrane proteins. In vitro studies with rabbit reticulocytes.

The capability of rabbit reticulocytes to synthesize red cell membrane proteins has been tested in vitro. Reticulocyte-rich blood from phenylhydrazine-treated rabbits was incubated in vitro in a complete amino acid medium containing ferrous salts, glucose, rabbit plasma and [3-H]leucine. Red cell ghost membranes were prepared by hypotonic lysis and leucine incorporation into hemoglobin and total membrane proteins determined. The pattern of incorporation into individual peptides was determined by polyacrylamide gel electrophoresis of labeled membranes on large (19 mm) gels which were then sliced into 1 mm sections; radioactivity was compared with densitometric tracings of Coomassie blue stained analytical (6 mm) gels. Incorporation of [3-H]leucine into both hemoglobin and membrane protein was linear over 1 h. Gel analysis of labeled membranes revealed that the amino acid was primarily incorporated into peptides with molecular weights of 90 000 or less; three peptides of molecular weights 90 000, 60 000 and 33 000 showed the highest specific activity. Synthesis of the four largest peptide species was negligible. Removable of ferrous salts inhibited synthesis of both globin and membrane protein equally (approx. 50%). However, puromycin and cycloheximide preferentially inhibited the synthesis of globin as compared to membrane proteins. Reticulocytes remain capable of synthesizing a number of membrane proteins; these results are consistent with studies of red cell membrane synthesis in anemic rabbits in vivo.

Absorption of oral aminoglycosides following bone marrow transplantation

Four patients with severe gastrointestinal reactions receiving oral “nonabsorbable” antibiotics for gut sterilization following bone marrow transplantation absorbed clinically significant amounts of aminoglycoside (gentamicin and/or tobramycin). Serum concentrations of 2.2, 2.6, 5.8, and 12.0 μg/ml were measured. Two of these patients had acute graft versus host reactions and two had severe mucositis following cytoreduction with intensive chemotherapy and irradiation. Nephrotoxicity occurred in the latter patients. One patient was studied in detail. Her hospital course and investigative results are presented. Four additional patients with mild gastrointestinal reactions following cytoreduction did not absorb gentamicin when their toxicity was maximal. Serum aminoglycoside determinations are necessary in patients receiving oral aminoglyclosides for gut sterilization following bone marrow transplantation if moderate to severe gastrointestinal reactions occur.

Delayed onset of gastrointestinal disease in the recipients of bone marrow transplants. A variant graft-versus-host reaction.

The report describes a subacute syndrome consisting of anorexia, mouth ulcers, abdominal pain, and diarrhea which occurred in three allogenic transplant recipients which appears to be distinct from the usual patterns of acute or chronic graft-versus-host disease (GVHD). The patient with myelomonocytic leukemia was treated with cyclophosphamide (60 mg/kg) i.v. 4 and 3 days before and total body X irradiation to 800 rad 1 day before transplantation. The radiation was administered from a 6-Mev source at least 3.5 m from the patient via opposing lateral fields. The patients were maintained in the protected environment until their absolute neutrophil counts (ANC) became greater than 1000/mm/sup 3/. All blood products were irradiated (1500 rad) prior to transfusion. Methotrexate was administered weekly for 100 days to prevent GVHD. (JMT)

868 SPLENIC HYPOFUNCTION OF PATIENTS UNDERGOING BONE MARROW TRANSPLANTATION (BMT)

Five recipients of BMT at the Childrens Hospital of Philadelphia developed late bacterial infections. This prompted us to review our clinical experience seeking evidence for splenic hypofunction in those and other patients (pts). Accordingly, we sought Howell-Jolly bodies (HJB) in peripheral blood smears of 29 pts; performed liver-spleen scans (LSS) in 9 pts with chronic graft-vs-host disease (CGVHD) and/or infection; and reviewed the spleens of 7 pts who died and had autopsies. In our series as a whole, 12 pts developed GVHD (5 acute; 2 chronic; 5 acute and chronic). 7/29 pts developed HJB; 4 of these had GVHD. Four pts receiving LSS showed decreased or absent splenic function; 3 had GVHD. The incidence of HJB or decreased function on LSS was not related to underlying diagnosis or preparative regimen. Lymphoid depletion was evident on all 7 spleens whose histopathology was reviewed: 2 showed total; 3 marked; and 2 moderately depleted lymphoid tissue. Our retrospective data suggest that splenic hypofunction is fairly common in the recipients of BMT, especially in pts with GVHD. Prospective studies performed at regular intervals after BMT will be necessary to define the true incidence of this condition and identify those pts who may need prophylactic antibiotic therapy.