Acetate non-utilizing mutants of Arabidopsis: evidence that organic acids influence carbohydrate perception in germinating seedlings.

A phenotypic screen was employed to isolate Arabidopsis plants that are deficient in their ability to utilize or sense acetate. The screening strategy, based on resistance to the toxic acetate analogue monofluoroacetic acid, was adapted from one that has been used successfully to identify important metabolic and regulatory genes involved in acetate metabolism in fungi. Following conventions established from the fungal work, the mutants were called acn mutants for acetate non-utilization. Three highly resistant plant lines were the focus of genetic and physiological studies. Mutant acn1 appears to be a true acetate non-utilizing mutant, as it displays increased sensitivity to exogenous acetate. The progeny of the original acn2 mutant did not germinate, even in the presence of sucrose as an exogenous carbon source. The germination of seeds from the F3 generation depended on the sucrose concentration in the medium. Only a small proportion of seeds germinated in the absence of exogenous sucrose and in the presence of 100 mM sucrose, but up to 70% of seeds germinated on 20 mM sucrose. Mutant acn3 exhibited sensitivity to exogenous sucrose, showing significant chlorosis on medium containing 20 mM sucrose, but no chlorosis when grown in the absence of exogenous sucrose. This phenotype was alleviated if acetate was provided. The acn mutants demonstrate that disrupting organic acid utilization can have profound affects on carbohydrate metabolism.

Co-ordinate regulation of genes involved in storage lipid mobilization in Arabidopsis thaliana.

Molecular genetic approaches in the model plant Arabidopsis thaliana (Col0) are shedding new light on the role and control of the pathways associated with the mobilization of lipid reserves during oilseed germination and post-germinative growth. Numerous independent studies have reported on the expression of individual genes encoding enzymes from the three major pathways: beta-oxidation, the glyoxylate cycle and gluconeogenesis. However, a single comprehensive study of representative genes and enzymes from the different pathways in a single plant species has not been done. Here we present results from Arabidopsis that demonstrate the co-ordinate regulation of gene expression and enzyme activities for the acyl-CoA oxidase- and 3-ketoacyl-CoA thiolase-mediated steps of beta-oxidation, the isocitrate lyase and malate synthase steps of the glyoxylate cycle and the phosphoenolpyruvate carboxykinase step of gluconeogenesis. The mRNA abundance and enzyme activities increase to a peak at stage 2, 48 h after the onset of seed germination, and decline thereafter either to undetectable levels (for malate synthase and isocitrate lyase) or low basal levels (for the genes of beta-oxidation and gluconeogenesis). The co-ordinate induction of all these genes at the onset of germination raises the possibility that a global regulatory mechanism operates to induce the expression of genes associated with the mobilization of storage reserves during the heterotrophic growth period.

Promoter trapping of a novel medium-chain acyl-CoA oxidase, which is induced transcriptionally during Arabidopsis seed germination.

The first step of peroxisomal fatty acid beta-oxidation is catalyzed by a family of acyl-CoA oxidase isozymes with distinct fatty acyl-CoA chain-length specificities. Here we identify a new acyl-CoA oxidase gene from Arabidopsis (AtACX3) following the isolation of a promoter-trapped mutant in which beta-glucuronidase expression was initially detected in the root meristem. In acx3 mutant seedlings medium-chain acyl-CoA oxidase activity was reduced by 95%, whereas long- and short-chain activities were unchanged. Despite this reduction in activity lipid catabolism and seedling development were not perturbed. AtACX3 was cloned and expressed in Escherichia coli. The recombinant enzyme displayed medium-chain acyl-CoA substrate specificity. Analysis of beta-glucuronidase activity in acx3 revealed that, in addition to constitutive expression in the root axis, AtACX3 is also up-regulated strongly in the hypocotyl and cotyledons of germinating seedlings. This suggests that beta-oxidation is regulated predominantly at the level of transcription in germinating oilseeds. After the discovery of AtACX3, the Arabidopsis acyl-CoA oxidase gene family now comprises four isozymes with substrate specificities that encompass the full range of acyl-CoA chain lengths that exist in vivo.

Long-chain acyl-CoA oxidases of Arabidopsis.

Full-length cDNAs coding for two distinct acyl-CoA oxidases were isolated by screening an Arabidopsis cDNA library. The genes for the two acyl-CoA oxidases have been termed AtACX1 and AtACX2. AtACX1 encodes a peptide of 664 amino acids possessing a molecular mass of 74.3 kDa. AtACX2 encodes a peptide of 691 amino acids in length with a molecular mass of 77.5 kDa. Peroxisomal targeting signals were identified in the primary sequences. AtACX1 has a putative PTS1, whereas AtACX2 has a characteristic PTS2. Expression of AtACX1 and AtACX2 in Escherichia coli gave active enzymes for enzymatic and biochemical analysis. AtACX1 was active with both medium-and long-chain saturated fatty acyl-CoAs and showed maximal activity with C14-CoA. Activity with mono-unsaturated acyl-CoAs was slightly higher than with the corresponding saturated acyl-CoA. AtACX2 was active with long-chain acyl-CoAs and showed maximal activity with C18-CoA. AtACX2 activity with mono-unsaturated acyl-CoAs was approximately twice as high as with the corresponding saturated acyl-CoA. Both enzymes have an apparent Km of approximately 5 microM with the preferred substrate. Northern analysis was conducted to determine the expression patterns of AtACX1 and AtACX2 during germination and in various tissues of a mature plant. The two genes showed generally similar expression profiles and steady-state mRNA levels in seedlings and mature tissues, but subtle differences were observed. Enzymatic analyses of plant extracts revealed that AtACX1 and AtACX2 are members of a family that includes acyl-CoA oxidases specific for shorter-chain acyl-CoAs. Through expression of antisense constructs of the individual genes, we were able to decrease long-chain oxidase activity only in antisense AtACX1 plants. Seedlings with long-chain oxidase activity reduced down to 30% of wild-type levels germinated and established normally; however, reduced root growth appeared to be a general feature of antisense AtACX1 plants.

No induction of beta-oxidation in leaves of Arabidopsis that over-produce lauric acid.

Leaves from transgenic Brassica napus L. plants engineered to produce lauric acid show increased levels of enzyme activities of the pathways associated with fatty acid catabolism (V.A. Eccleston and J.B. Ohlrogge, 1998, Plant Cell 10: 613-621). In order to determine if the increases in enzyme activity are mirrored by increases in the expression of genes encoding enzymes of beta-oxidation, which is the major pathway of fatty acid catabolism in plants, the medium-chain acyl-acyl carrier protein (ACP) thioesterase MCTE from California bay (Umbellularia california) was over-expressed under the control of the cauliflower mosaic virus 35S promoter in Arabidopsis thaliana (L.) Heynh. Arabidopsis was the most suitable choice for these studies since gene expression could be analyzed in a large number of independent MCTE-expressing lines using already well-characterized beta-oxidation genes. Levels of MCTE transcripts in leaves varied widely over the population of plants analyzed. Furthermore, active MCTE was produced as determined by enzymatic analysis of leaf extracts of MCTE-expressing plants. These plants incorporated laurate into triacylglycerol of seeds, but not into lipids of leaves as shown by gaschromatographic analysis of total fatty acid extracts. The expression levels of the beta-oxidation and other genes that are highly expressed during developmental stages involving rapid fatty acid degradation were measured. No significant difference in gene expression was observed among MCTE-expressing plants and transgenic and non-transgenic controls. To eliminate the possibility that post-translational mechanisms are responsible for the observed increases in enzyme activity acyl-CoA oxidase activity was also measured in leaves of MCTE-expressing plants using medium and long chain acyl-CoA substrates. No significant increases in either medium- or long-chain acyl-CoA oxidase activities were detected. We conclude that endogenous beta-oxidation is sufficient to account for the complete degradation of laurate produced in rosette leaves of Arabidopsis expressing MCTE.

Identification of women with T1-T2 breast cancer at low risk of positive axillary nodes.

BACKGROUND AND OBJECTIVES: The diagnostic and therapeutic significance of axillary dissection has been questioned. We sought to define a subgroup of patients with early-stage breast cancer who are at low risk for positive axillary nodes. METHODS: Between 1970 and 1995, 1,598 women with stage I and II breast cancer underwent level I-II axillary dissection with a minimum of 10 nodes removed. The following factors were examined in univariate analysis for predicting positive nodes: race, method of detection, location of the primary tumor, age, menopausal status, obesity, ER status, PR status, pathologic tumor size, lymphatic vascular invasion, tumor grade, and histology. RESULTS: Four hundred and forty-five of the 1,598 patients (27.8%) had histologically positive axillary nodes. Significant factors in univariate analysis for positive nodes included: tumor size, lymphatic vascular invasion, grade, method of detection, primary tumor location, and age. The only group of women with a 0% risk of axillary nodes were those in whom the pathologic tumor size was < or = 5 mm and mammographically detected. A 5-10% risk of positive axillary nodes was identified in women with (1) pathologic tumor size 6-10 mm, mammographically detected, and age < or = 40 years, and (2) tubular carcinoma < or = 10 mm. Tumors detected on physical examination with or without mammography and women < or = 40 years had a significantly increased risk of nodes. In multivariate analysis lymphatic vascular invasion (P < 0.001), method of detection (P = 0.026), location (P = 0.01), and pathologic tumor size (P = 0.002) were significant predictors of positive axillary lymphadenopathy. CONCLUSIONS: The decision to forego an axillary dissection should be considered in (1) tumors mammographically detected and < or = 5 mm (2) mammographically detected, pathologic size 6-10 mm, age > 40 and (3) tubular carcinoma < or = 10 mm. All other groups had a > 10% risk of nodes and may benefit from axillary dissection.

Higher-plant medium- and short-chain acyl-CoA oxidases: identification, purification and characterization of two novel enzymes of eukaryotic peroxisomal beta-oxidation.

Medium- and short-chain acyl-CoA oxidases were identified in and subsequently purified from dark-grown maize plantlets. The oxidase showing preference for medium-chain fatty acyl-CoAs (C10-C14) was purified to homogeneity. The oxidase showing preference for short-chain fatty acyl-CoAs (C4-C8) was purified over 150-fold. Various catalytic properties confirmed these enzymes to be true acyl-CoA oxidases. They produced trans-2-enoyl-CoA and H2O2 from the saturated acyl-CoA, as verified by various independent assay techniques. They also exhibited FAD-dependent activity; i.e. removal of loosely bound FAD by gel filtration markedly reduced activity, which could be restored upon re-addition of FAD. They showed apparent Km values between 2 and 10 microM for the acyl-CoA substrate giving maximal activity, no activity with the corresponding free fatty acid, high pH optima (8.3-8.6) and a peroxisomal subcellular location. The medium-chain acyl-CoA oxidase was determined to be a monomeric protein with a molecular mass of 62 kDa. The short-chain acyl-CoA oxidase was shown to have a native molecular mass of 60 kDa, but exhibited a labile multimeric structure, as indicated by the elution of multiple peaks of activity during several chromatographic steps, and ultimately by the purification of a subunit of molecular mass 15 kDa. The medium- and short-chain acyl-CoA oxidases were demonstrated to be distinct from the maize equivalent of the cucumber glyoxysomal long-chain acyl-CoA oxidase previously purified and characterized [Kirsch, Loffler and Kindl (1986) J. Biol. Chem. 261, 8570-8575]. The maize long-chain acyl-CoA oxidase was partially purified to permit determination of its substrate specificity; it showed activity with a broad range of acyl-CoAs of chain length greater than C8, and maximal activity with C16. The implications of the existence of multiple acyl-CoA oxidases in the regulation of plant peroxisomal beta-oxidation are discussed.

Tacrolimus, a new immunosuppressant--a review of the literature.

OBJECTIVE: To review the clinical pharmacology, pharmacokinetics, adverse effects, therapeutic uses, and current status of tacrolimus. DATA SOURCES: Data from scientific literature were identified by a MEDLINE search. The data were extracted, evaluated, and summarized for presentation. Experiences from studies evaluating tacrolimus in the form of articles, abstracts, letters to the editor, or proceedings were considered for inclusion. STUDY SELECTION: Open and controlled clinical and animal trials were reviewed in evaluating the pharmacology, pharmacokinetics, and adverse effects of tacrolimus. DATA EXTRACTION: Data from animal and human studies published in the English literature were evaluated. DATA SYNTHESIS: Tacrolimus is an 822-kDa macrolide antibiotic that has potent immunosuppressive properties. The mechanism of action is similar to that of cyclosporine in that it ultimately blocks the production of interleukin 2, thereby inhibiting further T-lymphocyte proliferation. Tacrolimus is metabolized solely in the liver and the metabolites are primarily excreted in the bile. The elimination half-life of tacrolimus is approximately 8.5 h, and is prolonged in hepatic dysfunction. Tacrolimus has shown efficacy in the prophylaxis of allograft rejection in both animals and human clinical trials, and has been used effectively to rescue patients who have exhibited refractory rejection failing cyclosporine prophylaxis. Adverse effects requiring tacrolimus dosage adjustment include nephrotoxicity, neurotoxicity, alterations in glucose metabolism, and infection or susceptibility to malignancy. CONCLUSIONS: To date, trials comparing tacrolimus with cyclosporine are not available in the literature; however, tacrolimus appears to be useful in rescuing grafts, particularly liver grafts that fail cyclosporine-based immunosuppression. Direct comparisons with cyclosporine are needed to define the role of tacrolimus as primary transplant therapy.

Nucleotide Availability in Maize (Zea mays L.) Root Tips (Estimation of Free and Protein-Bound Nucleotides Using 31P-Nuclear Magnetic Resonance and a Novel Protein-Ligand-Binding Assay).

Sequestration of nucleotides in cells through protein binding could influence the availability of nucleotides and free energy for metabolic reactions and, therefore, affect rates of physiological processes. We have estimated the proportion of nucleotides bound to proteins in maize (Zea mays L.) root tips. Binding of nucleoside mono- and diphosphates to total root-tip protein was studied in vitro using high-performance liquid chromatography and a new ligand-binding technique. We estimate that approximately 40% of the ADP, 65% of the GDP, 50% of the AMP, and virtually all the GMP in aerobic cells are bound to proteins. In hypoxic cells, free concentrations of these nucleotides increase proportionately much more than total intracellular concentrations. Little or no binding of CDP, UDP, CMP, and UMP was observed in vitro. Binding of nucleoside triphosphate (NTP) to protein was estimated from in vivo 31P-nuclear magnetic resonance relaxation measurements. In aerobic root tips most (approximately 70%) of the NTP is free, whereas under hypoxia NTP appears predominantly bound to protein. Our results indicate that binding of nucleotides to proteins in plant cells will significantly influence levels of free purine nucleotides available to drive and regulate respiration, protein synthesis, ion transport, and other physiological processes.

Vancomycin redistribution: dosing recommendations following high-flux hemodialysis.

Although increased vancomycin clearance has been reported with highly permeable hemodialysis membranes (such as polysulfone), failure to consider post-dialysis redistribution could lead to unnecessary dosage supplementation. In protocol 1, twelve hemodialysis patients admitted for vascular access thrombectomy received 15 mg/kg of vancomycin as surgical prophylaxis. Post-operatively, patients underwent high-flux hemodialysis (HFHD) for two hours using a Fresenius F-80 polysulfone dialyzer (QB = 417 +/- 49, QD = 800 ml/min). Vancomycin's intradialytic clearance increased 13-fold compared to the patient's endogenous clearance (120 +/- 59 vs. 9 +/- 8 ml/min, respectively) yet dialysate recovery indicated that only 17% of body stores were removed (179 +/- 70 mg). Although serum vancomycin levels decreased 33% during HFHD, vancomycin levels increased in all patients following dialysis and the post-rebound values reached 87% of the pre-dialysis concentration. In protocol 2, eight outpatients receiving maintenance HFHD with a F-80 dialyzer (Kt/V = 1.29 +/- 0.08) were given 20 mg/kg of vancomycin immediately following dialysis on Monday; pre- and post-levels were measured during the next three dialysis treatments. The predialysis serum vancomycin levels were > 7.5 micrograms/ml (9.7 +/- 1.0 micrograms/ml; range 8.0 to 11.0) in all patients the following Monday. Thus, vancomycin clearance is increased during HFHD, but redistribution post-HD minimizes changes in serum levels. We recommend a 20 mg/kg i.v. loading dose and subsequent doses of 15 mg/kg every seven days; to account for individual variability, weekly vancomycin levels should be drawn before dialysis.

Selective shunt in the management of variceal bleeding in the era of liver transplantation.

This study reports the Emory experience with 147 distal splenorenal shunts (DSRS) and 110 orthotopic liver transplants (OLT) between January 1987 and December 1991. The purpose was to clarify which patients with variceal bleeding should be treated by DSRS versus OLT. Distal splenorenal shunts were selected for patients with adequate or good liver function. Orthotopic liver transplant was offered to patients with end-stage liver disease who fulfilled other selection criteria. The DSRS group comprised 71 Child's A, 70 Child's B, and 6 Child's C patients. The mean galactose elimination capacity for all DSRS patients was 330 +/- 98 mg/minute, which was significantly (p less than 0.01) above the galactose elimination capacity of 237 +/- 82 mg/minute in the OLT group. Survival analysis for the DSRS group showed 91% 1-year and 77% 3-year survival, which was better than the 74% 1-year and 60% 3-year survivals in the OLT group. Variceal bleeding as a major component of end-stage disease leading to OLT had significantly (p less than 0.05) poorer survival (50%) at 1 year compared with patients without variceal bleeding (80%). Hepatic function was maintained after DSRS, as measured by serum albumin and prothrombin time, but galactose elimination capacity decreased significantly (p less than 0.05) to 298 +/- 97 mg/minute. Quality of life, measured by a self-assessment questionnaire, was not significantly different in the DSRS and OLT groups. Hospital charges were significantly higher for OLT (median, $113,733) compared with DSRS ($32,674). These data support a role for selective shunt in the management of patients with variceal bleeding who require surgery and have good hepatic function. Transplantation should be reserved for patients with end-stage liver disease. A thorough evaluation, including tests of liver function, help in selection of the most appropriate therapeutic approach.

Prevalence of invasive cytomegalovirus disease with administration of muromonab CD-3 in patients undergoing orthotopic liver transplantation.

OBJECTIVE: To assess the association of cytomegalovirus (CMV) disease with the administration of muromonab CD-3 (OKT-3) in patients undergoing liver transplant; specifically, to assess the risk of OKT-3 use as an agent for rejection prophylaxis and as an agent for therapy of rejection. DESIGN: Retrospective review of medical records. STUDY POPULATION: 83 liver transplant recipients (43 men, 40 women) with a mean age of 41.5 years (range 16-62). DATA EXTRACTION: The medical record for each liver transplant recipient was reviewed and analyzed for the following variables: (1) preoperative recipient CMV serology, (2) donor CMV serology, (3) incidence of invasive CMV disease, (4) administration of OKT-3, (5) postoperative administration time of OKT-3, and (6) the relationship between the administration of OKT-3 and the prevalence of invasive CMV disease. RESULTS: OKT-3 was administered to 34 of 83 (40.9 percent) liver remainder received OKT-3 as rejection rescue. All patients received OKT-3 5 mg iv for 14 days. Seventeen of the 34 patients receiving OKT-3 (50 percent) developed invasive CMV disease; 58.8 percent of the patients (20/34) receiving OKT-3 were given the agent within the first 14 postoperative days. Sixteen of these 20 patients (80 percent) developed invasive CMV disease. One of 14 patients (7.1 percent) who received OKT-3 after the first 14 postoperative days developed invasive CMV disease. Of those patients 94 percent (16/17) received OKT-3 in the first 14 postoperative days. This prevalence differed significantly from those receiving OKT-3 after the 14th postoperative day and those who did not receive OKT-3 at any time during their hospital course. CONCLUSIONS: The patients who received early administration of OKT-3 in our study had a greater risk of invasive CMV disease than did those who received OKT-3 later in the hospital course.

Contribution of Malate and Amino Acid Metabolism to Cytoplasmic pH Regulation in Hypoxic Maize Root Tips Studied Using Nuclear Magnetic Resonance Spectroscopy.

(31)P-, (13)C-, and (15)N-nuclear magnetic resonance spectroscopy were used to determine the roles of malate, succinate, Ala, Asp, Glu, Gln, and gamma-aminobutyrate (GABA) in the energy metabolism and regulation of cytoplasmic pH in hypoxic maize (Zea mays L.) root tips. Nitrogen status was manipulated by perfusing root tips with ammonium sulfate prior to hypoxia; this pretreatment led to enhanced synthesis of Ala early in hypoxia, and of GABA at later times. We show that: (a) the ability to regulate cytoplasmic pH during hypoxia is not significantly affected by enhanced Ala synthesis. (b) Independent of nitrogen status, decarboxylation of Glu to GABA is greatest after several hours of hypoxia, as metabolism collapses. (c) Early in hypoxia, cytoplasmic malate is in part decarboxylated to pyruvate (leading to Ala, lactate, and ethanol), and in part converted to succinate. It appears that activation of malic enzyme serves to limit cytoplasmic acidosis early in hypoxia. (d) Ala synthesis in hypoxic root tips under these conditions is due to transfer of nitrogen ultimately derived from Asp and Gln, present in oxygenated tissue. We describe the relative contributions of glycolysis and malate decarboxylation in providing Ala carbons. (e) Succinate accumulation during hypoxia can be attributed to metabolism of Asp and malate; this flux to succinate is energetically negligible. There is no detectable net flux from Glc to succinate during hypoxia. The significance of the above metabolic reactions relative to ethanol and lactate production, and to flooding tolerance, is discussed. The regulation of the patterns of metabolism during hypoxia is considered with respect to cytoplasmic pH and redox state.

Therapy of the Kasabach-Merritt syndrome with cryoprecipitate plus intra-arterial thrombin and aminocaproic acid.

A patient with the Kasabach-Merritt syndrome with disseminated intravascular coagulopathy and congestive heart failure due to a giant hepatic cavernous hemangioma achieved thrombosis of his tumor with a combination of cryoprecipitate plus intra-arterial thrombin and epsilon aminocaproic acid. This was documented by an absence of Indium 111-labelled platelet sequestration, marked decrease in thrombin:antithrombin complex generation, and normalization of platelet count and fibrinogen. Interventional angiography with aminocaproic acid and thrombin is advocated in patients whose tumors are amenable to such an approach so as to avoid a systemic antifibrinolytic state.

Muromonab CD-3: a review of its pharmacology, pharmacokinetics, and clinical use in transplantation.

Muromonab CD-3 (OKT-3) is a monoclonal antibody that is highly effective in the treatment of acute rejection in solid organ transplants. Due to its monoclonal nature, each molecule is identical because it is derived from a single antibody-producing clone. OKT-3 is administered only by intravenous injection and has a harmonic half-life of approximately 18 hours. It binds specifically to the CD-3 complex, which is involved in antigen recognition and cell stimulation, on the surface of T lymphocytes. Immediately after administration CD-3-positive T lymphocytes are abruptly removed from the circulation. The route of metabolism for OKT-3 is not clear; it may be removed by opsonization by the reticuloendothelial system when bound to T lymphocytes, or by human antimurine antibody production. The agent has been effective in reversing corticosteroid-resistant acute rejection in renal, liver, and cardiac transplant recipients. Its use in pancreatic and bone marrow recipients is inconclusive. OKT-3 has a considerable number of initial side effects, and some life-threatening reactions may occur. This drug should not be administered to any patient who is greater than 3% usual body weight because of the potential for the development of severe pulmonary edema. OKT-3 may also be associated with a high rate of infection, especially of the viral type. The usual dose is 5 mg administered as an intravenous bolus over 2-4 minutes daily for 10-14 days. Approximately 85% of patients treated with OKT-3 develop reactive human antimurine antibodies that, over time, may lead to tachyphylaxis and neutralization of the murine antibody OKT-3. OKT-3 is potent immunosuppressive agent and is an important prototype of future monoclonal antibodies.

A hypercoagulable state follows orthotopic liver transplantation.

Orthotopic liver transplantation may be associated during the postoperative period with hepatic artery thrombosis, a catastrophic occurrence generally necessitating emergency retransplantation. To assess the contribution of the coagulation mechanism to this complication, the levels of procoagulant and anticoagulant proteins were followed in 41 liver transplant patients during the first 10 postoperative days. The mean activities of all procoagulant factors reach normal values on day 1 except for factors V and VII, which achieve normal activity by day 3. Supernormal levels of factor VIII activity and antigen are noted (peak values on day 5 of 334% +/- 113% and 481% +/- 260%, respectively). The anticoagulant proteins show delayed recovery, with deficient antithrombin III levels seen in 81% of patients on day 3 and 57% on day 5. Similarly, proteins C and S are subnormal in 24% and 21%, respectively on day 3, and 20% and 10%, respectively, on day 5. During this period, elevated levels of thrombin/antithrombin complexes are encountered, reflecting in vivo activation of the coagulation mechanism. Activated thrombin is, therefore, being generated at a time when a decrease in the major regulatory anticoagulant proteins exists. These data suggest an imbalance between the hemostatic and thrombotic mechanisms and indicate a sustained prothrombotic state that may contribute to the risk for hepatic artery thrombosis. Using a regimen of low-dose heparin and fresh frozen plasma infusion, no thromboses have been seen in 65 consecutive liver transplants.

Compartmentation of Nucleotides in Corn Root Tips Studied by P-NMR and HPLC.

Corn (Zea mays L.) root tips were subjected to different conditions so that nucleotide levels varied over a wide range. Levels of nucleotides in corn root tips were measured using (31)P nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography. Results indicate: (a) Similar amounts of NTP and sugar nucleotides were observed by in vivo NMR and in extracts. In contrast, a significant amount of NDP observed in root tip extracts was not detected by in vivo NMR. Thus, for a given sample, [NTP]/[NDP] ratios determined in vivo by (31)P-NMR are always higher than ratios observed in extracts, deviating by approximately 4-fold at the highest ratios. The NMR-invisible pool of NDP appeared quite metabolically inert, barely changing in size as total cell NDP changed. We conclude that NDP in corn root tips is compartmented with respect to NMR visibility, and that it is the NMR-visible pool which responds dynamically to metabolic state. The NMR-invisible NDP could either be immobilized (and so have broad, undetectable NMR signals), or be complexed with species that cause the chemical shift of NDP to change (so it does not contribute to the NMR signal of free NDP), or both. (b) (31)P-NMR cannot distinguish between bases (A, U, C, and G) of nucleotides. HPLC analysis of root tip extracts showed that the relative amount of each base in the NTP and NDP pools was quite constant in the different samples. (c) In extracts, for each of the nonadenylate nucleotides, [NTP]/[NDP] was linearly proportional to [ATP]/[ADP], indicating near equilibrium in the nucleoside diphosphokinase (NDPK) reaction. However, the apparent equilibrium constants for the phosphorylation of GDP and UDP by ATP were significantly lower than 1, the true equilibrium constant for the NDPK reaction. Thus, for a given sample, [ATP]/[ADP] approximately [CTP]/[CDP] > [UTP]/[UDP] > [GTP]/[GDP]. This result suggests that the different NDPs in corn root tips do not have equal access to NDPK.

Comparison of whole-blood cyclosporine levels measured by radioimmunoassay and fluorescence polarization in patients post orthotopic liver transplant.

This study compared the analysis of whole blood cyclosporine concentrations measured by fluorescence polarization immunoassay (FPIA) and radioimmunoassay (RIA) polyclonal and monoclonal procedures. Fifteen orthotopic liver transplant patients with a mean age of 39 +/- 11.06 years were included in the study. One hundred thirteen levels were analyzed using FPIA, RIA polyclonal, and RIA monoclonal procedures. There was no difference statistically in comparing FPIA and RIA polyclonal results (p greater than 0.05). There was a statistical difference between FPIA and RIA monoclonal results p = 0.0001). With use of least squares simple linear regression analysis, FPIA results showed good correlation with RIA polyclonal results (R2 = 0.87). Poor correlation was shown between FPIA and RIA monoclonal results (R2 = 0.51). In this study population, FPIA produced results 2.5% higher than the RIA polyclonal procedure.

Drug-induced toxicities associated with high-dose cytosine arabinoside infusions.

The frequency and significance of central nervous system (CNS), ocular, and dermatologic toxicities associated with high-dose cytosine arabinoside (HDARA-C) infusions was evaluated. Patients were selected from one of three Southeastern Cancer Study Group protocols using HDARA-C 2-3 g/m2 body surface area (BSA) and their medical records were reviewed to identify and document the frequency of the toxicities. Those exhibiting CNS toxicity were compared across age, sex, race, previous standard-dose ARA-C or HDARA-C therapy, and infusion rate for toxicity occurrence. Statistical analysis was performed using Fisher's exact test with p less than 0.05. Of the 53 patients evaluated, 37.7% exhibited CNS, 37.7% ocular, and 45.3% dermatologic toxicities. Of the risk factors evaluated, only increasing age and previous ARA-C therapy approached statistical significance. The CNS toxicities associated with HDARA-C are clinically significant since permanent damage may result. Ocular and dermatologic toxicities usually resolve without medical intervention when HDARA-C therapy is discontinued. Further study is necessary to determine appropriate prophylaxis for these toxicities.