Lawrence E. Scheving, Ph.D.: chronobiology pioneer, "Full Bird" Colonel, and native son.
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Anatomy↗
Biomedical subjects
E R Burns
Publications and source records attributed to E R Burns.
Biological time and in vivo research: a field guide to pitfalls.
Biological rhythmicity is a fundamental characteristic of all life forms, from primitive bacteria to man. The molecular biology, genetics, and the neurobiology of the biological clock(s) are being elucidated. Daily (circadian) statistically significant fluctuations occur in all of the normal biological variables studied in the experimental animal and the human. Many researchers, however, are not aware of the negative impact biological rhythmicity can have on experimental design and/or data interpretation. This article serves not as a review, but as a "field guide" to the pitfalls that can occur when research is performed in the absence of an understanding of biological rhythmicity. The major topics discussed are: 1) data transfer from the diurnally in-active/resting/sleeping lab animal to the diurnally active human, 2) frequency of sampling, 3) free-running vs. synchronization, 4) alternating periods of resistance and susceptibility, 5) phase shifting of a rhythm, 6) the assumption that one mean +/- S.E. from control animals can be "stretched" across an experimental time span, and 7) plotting data on an "hours after treatment" format vs. a "time of day" format. The hope is that by avoiding the pitfalls, biological time will become an ally in the endeavor to understand human biology.
Animals↗
Diagnosis of the hemolytic state using serum levels of erythrocyte adenylate kinase.
Red cell hemolysis is classically diagnosed by a combination of nonspecific laboratory tests, including serum bilirubin, LDH, and the reticulocyte count. None of these tests alone or in combination has the specificity to reliably ascertain the presence of hemolysis. We have previously demonstrated that erythrocyte adenylate kinase (EAK) is a red cell specific enzyme released from damaged red cells. Its activity can be measured in serum by rapid electrophoresis or immunological methods and correlates linearly with the degree of hemolysis in vitro. We now report on a clinical study comparing EAK levels in patients with and without hemolysis. The clinical diagnosis of hemolysis was established in hospitalized patients with anemia by the combined elevation of the bilirubin, LDH, and reticulocyte count in the absence of liver disease and demonstrable blood loss. The normal range of serum EAK was determined in 30 healthy nonanemic voluntary blood donors and was 0-3.5 Units (mean = 0.5). In 25 patients with hemolytic anemia due to sickle cell disease, hemolytic transfusion reactions, or TTP, the mean EAK level was 62.4 with a range 0-298 Units (P < 0.001 compared to normals). Levels of EAK exceeded the normal range in 24 of 25 patients (96%). In a control group of 44 hospitalized patients with liver disease or myocardial infarction and no clinical evidence of hemolysis, the mean EAK level was 0.12 with a range of 0-3.2 (P = 0.1, NS compared to normals and P < 0.001 compared to patients with hemolysis). None of the control patients had EAK levels that exceeded the normal range. The diagnostic sensitivity of the EAK assay for hemolysis, as calculated according to Baye's algorithm, was 96%, with a specificity and accuracy of 97%. Measurement of serum EAK represents a highly sensitive and specific test for the diagnosis of hemolytic anemia.
Adenylate Kinase↗
Differentiation and resolution of erythrocyte and muscle adenylate kinase activities in serum by electrophoresis.
Adenylate kinase activity originating from erythrocytes has been shown to be distinct from muscle adenylate kinase or myokinase activity, until now considered to be identical enzyme activities. The two activities can be differentiated by electrophoretic fractionation, thus making it possible to quantify the erythrocyte adenylate kinase activity present in serum.
Adenylate Kinase↗
Renal vein thrombosis treated with thrombolytic therapy: case report and brief review.
Renal vein thrombosis (RVT) can occur as a complication of the nephrotic syndrome. We present the case of a young woman with systemic lupus erythematosus with nephrotic syndrome and bilateral RVT with extension of the thrombus into the vena cava to the level of the right atrium and multiple pulmonary emboli. She was treated acutely with streptokinase, with complete resolution of the thrombi. In general, anticoagulation is the mainstay of therapy for RVT. Review of the literature reveals that thrombolytic therapy can be used safely and appears to have been reserved for those patients with the most severe disease or the more grave prognosis. we feel that thrombolytic therapy is warranted in the presence of bilateral RVT with acute renal failure, massive clot size with high risk of acute embolic events, or recurrent pulmonary emboli, in the absence of overriding contraindications.
Adolescent↗
The temporal relationships of synthesis and phosphorylation in stress proteins 70 and 90 in aged caloric restricted rats exposed to bleomycin.
A single intraperitoneal injection of the human therapeutic drug bleomycin (BL) was administered to three groups of male Fischer 344 rats at time 0, and the incorporation of [35S]methionine ("synthesis") and phosphorylation patterns of stress proteins (sps/hsps) from bone marrow cells were analyzed over time by two-dimensional electrophoresis and fluorography. Two groups of rats, young ad libitum (Y/AL--3 months) and old ad libitum (O/AL--28 months), had free access to rat chow, and a third group of old rats (O/CR--28 months) were maintained on a caloric restricted intake (60% of the AL diet). The administration of BL in Y/AL, O/AL and O/CR animals activated the 35S-labeling of sp 90 which reached a peak at 4 hours. Labeling of sp 90 was significantly greater in Y/AL compared to O/AL, and the incorporation pattern of O/CR was intermediate to Y/AL and O/AL animals. All labeling of sp 90 in each group had disappeared by 10 hours after BL administration. Stress protein 70x (inducible form) in these three animal groups displayed a similar pattern of 35S-incorporation, but the amount of labeling was less than that of sp 90. No labeling of sp 70x remained by 13 hours after BL administration. Phosphorylation ([32P] phosphate incorporation) of sp 90 reached a maximum level at 2 hours in all animals, and 32P-labeling in Y/AL was significantly increased over O/AL and O/CR with an intermediate level found in O/CR animals. The turnover rate (phosphorylation/dephosphorylation) of sp 90 induced by BL was significantly suppressed and temporarily extended in O/AL as compared with O/CR, which implied that CR not only increased incorporation of sp 90, but also enhanced a utilization of the phosphate pool very similar to that seen in Y/AL animals.
Aging↗
Circadian variations of gastrointestinal peptides and cell proliferation in rats: effects of adrenalectomy.
The hypothalamo-pituitary-adrenal axis appears to play an important role in regulating the circadian fluctuations of brain-gut peptides, as well as the cell cycle of the gastrointestinal mucosa. Since dexamethasone treatment tended to restore circadian fluctuations lost to adren-x, the influence of adrenal glucocorticoids in the coordination of the rhythms of regulatory peptides and cell cycle kinetics appears to be substantial.
Adrenalectomy↗
Clinical impact of chemotherapy-induced thrombocytopenia in patients with gynecologic cancer.
PURPOSE AND METHODS: This retrospective analysis of 501 patients with gynecologic cancer treated with chemotherapy evaluates the relationship between platelet count and clinical bleeding, as well as the clinical effects of platelet transfusion therapy. Thrombocytopenic patients were divided into six groups according to platelet counts, and major or minor bleeding manifestations were documented. Thrombocytopenia was defined as a platelet count less than 100,000/microL. RESULTS: Thrombocytopenia occurred in 182 (36.3%) patients over 808 of 1,546 chemotherapy cycles (52%). No intracranial or life-threatening bleeding occurred in any patient. The majority of patients (139 [76.4%]) had no clinical bleeding. Minor bleeding, such as purpura, occurred in 34 patients (18.7%) and 44 cycles (5.4%). Major bleeding occurred in nine patients (4.9%) and 10 cycles (1.3%). Five major bleeding events occurred in 49 patients with platelet counts between 0 and 10,000/microL. Forty-three of these patients received platelet transfusions. Thirty-eight of 43 transfused patients (88.3%) had no bleeding. Of the remaining five patients, two were transfused prophylactically with no effect. Three major bleeding events occurred in patients with platelet counts that ranged from 11,000 to 20,000/microL, but these were due to chronic instrumentation or trauma. In patients with platelet counts more than 20,000/microL, major bleeding occurred only from necrotic metastatic lesions. Random-donor platelet transfusions provided inconsistent increments in platelet counts. Overall, 27.5% of patients achieved the expected increase in platelet number based on units of platelet concentrate transfused. The use of single-donor or human leukocyte antigen (HLA)-matched platelets did not provide greater increments in those patients who were refractory to random-donor platelets. CONCLUSION: Platelet counts > or = 10,000/microL are not associated with spontaneous major bleeding. Prophylactic platelet transfusions in patients with gynecologic malignancies and chemotherapy-induced thrombocytopenia should be limited to those with platelet counts < or = 10,000/microL, provided they are not bleeding and have no major anatomic or pathophysiologic precursors of bleeding.
Antineoplastic Agents↗
Comparative studies of synthesis, phosphorylation, DNA binding and proteolytic characteristics of a novel protein during phases of the mouse spleen cell cycle.
1. Cultured mouse spleen cells were exposed to the mitogen Concanavalin A followed by isoproterenol, and nuclei were electronically sorted from seven partitions of the cell cycle. 2. Several nuclear proteins, including stress proteins, which were cell-cycle-stage specific, were elicited by isoproterenol as determined by micro-electrophoresis and fluorography. 3. Two novel S-phase proteins (X0 and X') demonstrated differing synthesis and phosphorylation patterns during the cell-cycle phases. 4. X' showed DNA binding characteristics and proteolytic properties (hydrolyzing X0 or beta-galactosidase); both proteins were cell-cycle regulated.
Animals↗
Paradoxic effect of multiple mild coagulation factor deficiencies on the prothrombin time and activated partial thromboplastin time.
Single coagulation factor deficiencies predictably prolong the prothrombin time (PT) and activated partial thromboplastin time (APTT) at levels below 35% of normal activity. Acquired coagulopathies generally are characterized by multiple coagulation factor deficiencies. The effect was studied of such combined deficiencies on the PT/APTT using plasma from patients congenitally deficient in specific factors and pooled normal plasma. The PT begins to lengthen when individual factor levels fall below 25%. The APTT becomes prolonged when the levels of Factor V fall below 45%; the levels of Factors II and XI fall below 40%; and the levels of Factors I, V, VII, VIII, IX, and XII fall below 25% of normal. When plasma samples containing 50% activity of a single factor and 100% of all other factors were prepared by mixing the congenitally deficient plasma samples with the normal pool, the resulting mixtures had normal PT and APTT values. However, when two of these 50% factor-deficient plasmas were combined so that the mixture contained 75% activity of two coagulation factors and 100% of all other factors, the resulting PT and APTT were prolonged over the clotting times of either 50% factor-deficient plasma. Similar findings were obtained in patients with mild factor reductions caused by warfarin treatment. These data indicate that prolongations of the PT and APTT in disorders of coagulation affecting multiple factors represent less of a reduction in factor levels than is generally appreciated. This may explain the poor clinical correlation between abnormalities in these test results and clinical bleeding in acquired disorders of hemostasis.
Blood Coagulation Factors↗
A nuclear protein associated with lethal heat shock of HL-60 cells.
The responses to stress in living cells are well known. Thermal stress causes decreased protein synthesis as well as rapid induction of heat shock proteins (hsps), or alternately termed stress proteins (sps). The exposure of cultured promyelocytic leukemia cells (HL-60) to a 45 degrees C lethal heat shock for 1 h elicited synthesis and phosphorylation of a polypeptide M(r) 48,000 and pI 7.5 (p 48) as visualized by two-dimensional polyacrylamide gel ultra-microelectrophoresis. p 48, which was not observed at sublethal temperatures (39 and 41 degrees C), was synthesized during all phases of the cell cycle but was phosphorylated only in G0 + G1 and S-phases. The appearance of p 48 was marked by a concomitant and reciprocal reduction in hsps or sps 70 and 90. Distinct protease V8 fragment maps of p 48, hsps 70 and 90 in conjunction with immunochemical determination indicated vast differences in their primary structures. These facts suggest that p 48 was not formed from coalesced breakdown products of hsps 70 or 90. Western blotting showed that p 48 possessed the same immunochemical determinants as two other proteins with the same molecular mass but different isoelectric points. In an association assay, p 48 was shown to bind with actins and hsp 90 from HL-60 nuclei.
Cell Cycle↗
Preoperative laboratory assessment of hemostasis for orthognathic surgery.
Platelet count, prothrombin time, and activated partial thromboplastin time provide a baseline to evaluate patients with known coagulopathy, as well as present an opportunity to diagnose disease in previously symptom free patients. Current hematologic management of patients with Von Willebrand's disease uses heated Factor VIII that allows patients to undergo orthognathic surgery without significant risk of disease transmission from banked blood products.
Adult↗
Prevention of growth of Yersinia enterocolitica in blood by polyester fiber filtration.
The ability of polyester white cell-reduction blood filters to prevent the growth of Yersinia enterocolitica in units of donated blood was studied. Sixteen units of freshly drawn blood were inoculated with 10, 50, 100, or 150 colony-forming units (CFU) per mL of a clinical isolate of Y. enterocolitica (serotype O:3). The units were subsequently fractionated into red cell concentrate and resuspended in AS-1 or AS-3 solution. One-half of the red cell concentrates in each solution were filtered within 15 hours of phlebotomy and stored for 42 days. The remaining units served as unfiltered controls. Bacterial growth was monitored by weekly cultures and, on the last storage day, by the presence of endotoxin and the formation of methemoglobin. One hundred twelve primary cultures (560 plates) were performed. Units collected in AS-1 and filtered remained sterile when initially inoculated with 50 CFU or less. Filtered units spiked with 100 CFU or less and collected in AS-3 remained sterile throughout their shelf life. All unfiltered units supported bacterial growth and the formation of endotoxin and methemoglobin. The filtration of freshly donated blood proves to limit the growth of Y. enterocolitica in red cell components.
Blood Gas Analysis↗
The high mobility group of nuclear proteins as biomarkers of age and caloric restriction in rats.
The quantitative levels and phosphorylation states of the high mobility group (HMG) of proteins were investigated in bone marrow, brain, heart, kidney, liver, pancreas, spleen, testis and thymus of three groups of male Fischer 344 rats. Two groups of rats, young ad libitum (Y/AL - 1 1/2 mo.) and old ad libitum (O/AL - 28 mo.), had free access to rat chow, and a third group of old rats were maintained on a caloric restricted intake (O/CR - 28 mo.). The quantities of HMGs 1,2,14 and 17 were significantly reduced in O/AL rats compared with Y/AL rats in all tissues examined, and in many cases, the amount of HMGs of O/CR rats were increased by varying degrees from O/AL animals. In G2-phase nuclei of bone marrow, spleen and testis, phosphorylation of HMG proteins was reduced significantly in O/AL rats, but was enhanced in O/CR animals (especially HMG14). These levels of HMGs in O/CR animals, altered by age and diet dependent factors, reflect a condition which is more reminiscent of Y/AL than O/AL animals.
Aging↗
Treatment of septic thrombocytopenia with immune globulin.
Thrombocytopenia frequently complicates systemic infection and results from multiple possible mechanisms. We and others have demonstrated that platelet-associated IgG (PAIgG) levels are elevated in the majority of patients with septic thrombocytopenia. Corticosteroids may be undesirable as a treatment for thrombocytopenia for patients with severe infection because of their potential for suppressing the immune response. We hypothesized that septic thrombocytopenia is, in most cases, an immune disorder analogous to idiopathic thrombocytopenic purpura (ITP) which might respond to intravenous gamma-globulin as a treatment for increasing the platelet count in this disorder. Intravenous immune globulin (IVIG), 400 mg/kg daily for 3 days, was administered in a randomized double-blind placebo-controlled trial. Twenty-nine patients who developed thrombocytopenia during a documented, septic episode were studied. Patients with disseminated intravascular coagulation (DIC), hypersplenism, or drugs known to cause thrombocytopenia were excluded. Elevated PAIgG levels were documented in 52% of evaluable patients. Mean platelet counts in the IVIG group rose from 43K at study entry to 178K (411% rise) by Day 9. In the placebo group platelets rose from 51K to 125K (261% rise; P = 0.02). Seventy-seven percent of the IVIG group had a minimum peak rise of 35K, vs 56% of the placebo group. Three patients in the placebo group had a serious bleeding episode, vs one in the IVIG group. The use of IVIG to treat septic thrombocytopenia not associated with DIC leads to a more rapid, more sustained, and greater increase in platelet count than placebo. Its use is recommended in the septic patient who is bleeding or is likely to need invasive or surgical procedures.
Blood Platelets↗
Synergistic effects of endothelin-1 (ET-1) and transforming growth factor alpha (TGF-alpha) or epidermal growth factor (EGF) on DNA replication and G1 to S phase transition.
The cooperative cell kinetic actions of ET-1 with TGF-alpha or EGF in normal rat kidney fibroblasts (NRK-49F) and KNRK cells (Kirsten MSV transformed) were analyzed by [3H]-thymidine incorporation assay and flow cytometry. A marked synergistic effect of TGF-alpha and ET-1 (or EGF and ET-1) on DNA synthesis and G1 to S transition was observed in NRK cells; 15-20% S for TGF-alpha and 12% S for ET-1 alone but 45-50% S in combination. There was no detectable effect on cell cycle kinetics by TGF-alpha (1 ng/ml) or EGF (1 ng/ml) plus ET-1 (1 ng/ml) in KNRK cells treated for 22 hours. Insulin, insulin-like growth factor I (IGF-I), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), and transforming growth factor beta (TGF-beta) were also tested and found to have no significant synergistic effects on ET-1 actions. Our findings suggest that the combination of TGF-alpha (EGF) and ET-1 is an important part of an intricate network which coordinates progression of G1 to S phase in normal cells.
Animals↗
A rare-event analysis model for quantifying white cells in white cell-depleted blood.
An analysis model to detect and quantify white cells (WBCs) in red cell concentrates (RBCC) drawn from units of blood that are highly depleted of WBCs is described. WBC detection is performed by fluorescence analysis of 50 microL of RBCC labeled with propidium iodide, a DNA/RNA fluorophore. Quantification is performed by regression analysis of standard dilutions of RBCC in substantially WBC-free red cells. This RBCC diluent is obtained by filtration of blood through a new medium. The method proves to be precise (CV = 7%), efficient (+/- 30 min/aliquot), and linear (r = 0.99) to 6 log10 WBC depletion of the native product. The current technique is preferable to those suggested previously, such as ficoll concentration, which requires the sacrifice of the unit of blood for counting purposes, and to earlier fluorescence analysis techniques that do not employ WBC-free red cell diluents. The latter do not monitor extremely low concentrations of WBCs because they lack adequate signal-to-noise discrimination. The sensitivity of the described method allows for monitoring of WBC depletion procedures with greater efficiency than is currently available commercially.
Blood Component Removal↗
Improvement in transfusion safety using a new blood unit and patient identification system as part of safe transfusion practice.
A new patient and blood unit identification system designed to confirm the identity of crossmatched blood products and that of the intended recipient was evaluated. Six hundred seventy-two red cell concentrates were transfused to 312 patients. Participating hospital personnel and patients were interviewed regarding the use and benefit of this unique system, which incorporates a "lock-box" approach to the identification process. The product and procedure were accepted unanimously and enthusiastically, and three potential mistransfusions were avoided by use of the system during the limited period of observation. This type of approach to the identification process affords greater security than conventional practices and minimally burdens staff.
Blood Transfusion↗