Quantitation of adsorption and conjugation of plasma proteins by residual glutaraldehyde in fixed collagenous tissue with radioiodinated plasma proteins

Residual glutaraldehyde (GA) in collagenous cardiovascular tissue prostheses after multiple saline rinses remains in the prostheses and accounts for adsorption and conjugation of a variety of plasma proteins. This may account for later beneficial or adverse effects. Human serum albumin (SA), gamma globulin (GG), and fibrinogen (FB) were iodinated with 125I using the iodogen-transfer technique. Bovine pericardium (PC) was fixed with 0.5% GA for 24 hr and rinsed to remove excess GA. Fresh and GA-fixed PC (FRPC, GAPC: 1 x 1 cm2), in triplicate, were incubated with 0.5-1.0 microCi of tracers in human, porcine, or bovine blood (2 ml) for a period of 0.5, 1, 2, and 3 hr and washed (5x) with saline. Maximum adsorbed proteins per unit weight of collagen (pmol/mg of PC, mean +/- SD) at 3 hr on FRPC and GAPC were quantified with a gamma counter. Fixed PC absorbed significantly more plasma proteins from blood than fresh PC. These conjugated plasma proteins are tightly bound to fixed PC. The adsorbed and conjugated plasma proteins for GAPC and FRPC have the same sequence: SA > GG > FB vs SA > GG > FB. Protein conjugation may affect the remodeling of collagenous cardiovascular tissue prostheses post implantation.     

Aspects of oligonucleotide and peptide sequencing with MALDI and electrospray mass spectrometry

Biopolymer sequencing with mass spectrometry has become increasingly important and accessible with the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Here we examine the use of sequential digestion for the rapid identification of proteolytic fragments, in turn highlighting the general utility of enzymatic MALDI ladder sequencing and ESI tandem mass spectrometry. Analyses were performed on oligonucleotides ranging in size from 2 to 50 residues, on peptides ranging in size from 7 to 44 residues and on viral coat proteins. MALDI ladder sequencing using exonuclease digestion generated a uniform distribution of ions and provided complete sequence information on the oligonucleotides 2-30 nucleic acid residues long. Only partial sequence information was obtained on the longer oligonucleotides. C-terminal peptide ladder sequencing typically provided information from 4 to 7 amino acids into the peptide. Sequential digestion, or endoprotease followed by exoprotease exposure, was also successfully applied to a trypsin digest of viral proteins. Analysis of ladder sequenced peptides by LCMS generated less information than in the MALDI-MS analysis and ESI-MS2 normally provided partial sequence information on both the small oligonucleotides and peptides. In general, MALDI ladder sequencing offered information on a broader mass range of biopolymers than ESI-MS2 and was relatively straightforward to interpret, especially for oligonucleotides.     

Regulation of an Escherichia coli/mammalian chimeric carbamoyl-phosphate synthetase

Carbamoyl-phosphate synthetase (CPSase) consists of a 120-kDa synthetase domain (CPS) that makes carbamoyl phosphate from ATP, bicarbonate, and ammonia usually produced by a separate glutaminase domain. CPS is composed of two subdomains, CPS.A and CPS.B. Although CPS.A and CPS.B have specialized functions in intact CPSase, the separately cloned subdomains can catalyze carbamoyl phosphate synthesis. This report describes the construction of a 58-kDa chimeric CPSase composed of Escherichia coli CPS.A catalytic subdomains and the mammalian regulatory subdomain. The catalytic parameters are similar to those of the E. coli enzyme, but the activity is regulated by the mammalian effectors and protein kinase A phosphorylation. The chimera has a single site that binds phosphoribosyl 5'-pyrophosphate (PRPP) with a dissociation constant of 25 microM. The dissociation constant for UTP of 0.23 mM was inferred from its effect on PRPP binding. Thus, the regulatory subdomain is an exchangeable ligand binding module that can control both CPS.A and CPS.B domains, and the pathway for allosteric signal transmission is identical in E. coli and mammalian CPSase. A deletion mutant that truncates the polypeptide within a postulated regulatory sequence is as active as the parent chimera but is insensitive to effectors. PRPP and UTP bind to the mutant, suggesting that the carboxyl half of the subdomain is essential for transmitting the allosteric signal but not for ligand binding.     

Age and ethnicity affect the risk and outcome of focal segmental glomerulosclerosis

In patients with proteinuria, African-American (AA) ethnicity is reported to be a risk factor for focal segmental glomerulosclereosis (FSGS) and its progression to end-stage renal disease (ESRD). We reviewed our single-center experience to determine the probability of FSGS and its progression to ESRD based on ethnicity and age at presentation in children with proteinuria with or without nephrotic syndrome. Proteinuria without systemic disease or acute glomerulonephritis was the presenting feature in 17% (236/1,403) of children in the renal patient database of Texas Children's Hospital, Baylor College of Medicine. Histopathological diagnoses were established in 107 of 236 patients (45%). FSGS was identified in 65 patients, accounting for 28% of all patients with proteinuria and 61% of patients who underwent renal biopsy. FSGS was more prevalent in AA (45%) than in non-AA patients (22%) (P=0.001), and AA patients with FSGS were older at presentation (12.7+/-4.4 years) than non-AA patients (5.6+/-4.6 years) (P<0.001). Among patients who underwent renal biopsy, increasing age at presentation increased the probability of having FSGS in AA but not non-AA patients (P=0.04). Five-year actuarial renal survival of FSGS was worse in AA (8%) than in non-AA patients (31%) (P=0.01). These data suggest an increased risk and worse outcome of FSGS in AA compared with non-AA children.     

Separation of the enantiomers of ibuprofen and its major phase I metabolites in urine using capillary electrophoresis

This report describes a simple, rapid, automated microassay for measuring in vitro changes of oxidative burst of phagocytes following challenge with metals for orthopedic devices. The production of reactive oxygen species (ROS) by polymorphonuclear leukocytes (PMNs) was measured using 2',7'-dichlorofluorescin-diacetate (DCFH-DA) as fluorescent probe. DCFH-DA enters the cells and is oxidized by ROS to fluorescent DCF. The DCF generated was directly proportional to ROS produced intracellularly: The fluorescence intensity was read and converted to an index of ROS production by cells. In our experimental system, granulocytes (PMNs) were isolated from normal human blood and seeded in microplates. To verify if metals could influence ROS production, chromium, cobalt, nickel, molybdenum, titanium, aluminum, and vanadium prepared as aqueous extracts in phosphate-buffered saline were tested onto PMNs using phorbolmyristate acetate (PMA) as positive control. Molybdenum, aluminum, and vanadium increased ROS generation by PMNs, while signals not different from unstimulated PMNs were recorded for chromium, cobalt, nickel, and titanium. The DCFH-DA microplate-based assay provides an in vitro tool for the detection of oxygen-reactive species generated by PMNs as a response to metals.     

Rapid multiplex analysis for the factor V Leiden and prothrombin G20210A mutations associated with hereditary thrombophilia

Thromboembolic episodes are common events and affect approximately one in 1,000 persons annually. Pulmonary embolism alone accounts for 50,000 to 100,000 deaths per year in the United States with > 50% of those being elderly persons. Resistance to activated protein C is the most common inherited disorder associated with hereditary thrombophilia. A missense mutation has been identified in the gene coding for coagulation factor V (codon 506) which renders this procoagulant factor resistant to inactivation by activated protein C resulting in an increased risk for venous thrombosis. Recently, a second polymorphism was identified in the prothrombin gene (factor II) which is also associated with increased risk for venous thrombosis. Because of the high prevalence of these two mutations in the general population as well as in specific patient populations, the ability readily to detect these two mutations must be feasible. In this study, we evaluated 303 patients for the prothrombin mutatin (G20210A) which were previously tested for the factor V mutation using established polymerase chain reaction-mediated restriction fragment length polymorphism assays. In these patients, 30 (9.9%) were found to be heterozygous for the factor V Leiden mutation with no homozygous mutants identified. Twenty individuals (6.6%) were heterozygous for the prothrombin G20210A mutation, and we identified two individuals (0.66%) who were homozygous for the 20210A allele. Of the total 303 individuals screened, two were double heterozygotes for both the factor V Leiden and the prothrombin gene mutations. We also describe a multiplex polymerase chain reaction-mediated restriction fragment length polymorphism assay for detecting both mutations in a single-tube double-enzyme digestion reaction making identification of these two mutations easily achievable.     

Phase I trial of weekly scheduling and pharmacokinetics of titanocene dichloride in patients with advanced cancer

PURPOSE: To determine the maximum-tolerated dose (MTD) and the dose-limiting toxicities (DLTs) of a weekly schedule of titanocene dichloride (TD) and to define the pharmacokinetics of titanium in plasma and urine. PATIENTS AND METHODS: Twenty patients with a median age of 58 years received 83 courses of TD. TD was given as 1-hour infusion at escalating doses from 70 to 185 mg/m2/wk. Pharmacokinetic analysis was performed in eight patients for total plasma titanium (TPTi) and in three patients for ultrafiltrable titanium (UFTi). RESULTS: At the fifth dose level (185 mg/m2/wk), a variety of DLTs were seen in five patients: fatigue in three, bilirubinemia in one, and hypokalemia in two. A further six patients were treated at 140 mg/m2; only one had dose-limiting creatinine elevation and this dose was therefore defined as the MTD. No myelosuppression or alopecia were observed. One patient with adenocarcinoma of unknown primary had a minor response. Pharmacokinetic analysis showed that TPTi maximum concentration (Cmax) values were linear with dose and elimination of TPTi was triphasic with a long terminal half-life (t1/2; median, 165 hours; range, 89 to 592). Between 7% and 24.3% of the total of administered titanium was eliminated in urine over the first 24 hours. In contrast, UFTi elimination was described by a one-compartment model with a t1/2 of 0.41 hours; peak levels of UFTi were 5.2% +/- 2.5% those of TPTi. CONCLUSION: The MTD of TD given on a weekly schedule is 140 mg/m2, with cumulative, but reversible creatinine and bilirubin elevation being the DLTs.     

Targeting peptide nucleic acid-protein conjugates to structural features within duplex DNA

URF13 is the product of a mitochondrial-encoded gene (T-urf13) found only in maize plants containing the Texas male-sterile cytoplasm (cms-T), and it is thought to be responsible for both cytoplasmic male sterility and the susceptibility of cms-T maize to the fungal pathogens Bipolaris maydis race T and Phyllosticata maydis. Mitochondria isolated from cms-T maize are uniquely sensitive to pathotoxins (T-toxin) produced by these fungi and to methomyl (a commercial insecticide). URF13 acts as a receptor that specifically binds T-toxin to produce hydrophilic pores in the inner mitochondrial membrane. When expressed in Escherichia coli cells, URF13 also forms hydrophilic pores in the plasma membrane if exposed to T-toxin or methomyl. Topological studies established that URF13 contains three membrane-spanning alpha-helices, two of which are amphipathic and can contribute to pore formation. Chemical cross-linking of URF13 was used to demonstrate the existence of URF13 oligomers in cms-T mitochondria and E. coli cells. The ability of the carboxylate-specific reagent, N,N'-dicyclohexycarbodiimide, to cross-link URF13 was used in conjunction with site-directed mutagenesis to establish that the URF13 tetramer has a central core consisting of a four-alpha-helical bundle which undergoes a conformational change after interaction with T-toxin or methomyl. Overall, the experimental evidence indicates that URF13 functions as a ligand-gated, pore-forming T-toxin receptor in cms-T mitochondria.     

Simultaneous hydrolysis and fermentation of pulp mill primary clarifier sludge

Bioconversion of sludge from the primary clarifier of a sulphite pulping operation to ethanol offers a number of advantages over conventional disposal options. The amount of material which must be disposed of is reduced while, at the same time, salable and environmentally friendly fuel-ethanol is produced. In this study, primary clarifier sludge (PCS) was shown to be hydrolysed to produce fermentable sugars at a rate proportional to enzyme loading. Initial (1 hour) hydrolysis rates as high as 12.6 g reducing sugar/L · h were observed at an initial enzyme loading of 10 filter paper units (FPU)/g. Hydrolysis was inhibited by spent sulphite liquor (SSL), an inhibition which could be completely overcome by fermenting the SSL to remove sugars. Surfactants were found to only marginally improve the production of sugars. To reduce the deleterious effects of end product inhibition, single stage simultaneous hydrolysis and fermentation (SHF) was carried out using cellulase enzymes and Saccharomyces cerevisiae.     

Computerized cyclic voltammetric detection after HPLC of the antineoplastic agents etoposide,teniposide, adriamycin and its metabolite adriamycinol in urine samples

A computerized electrochemical detection system for application after HPLC, provided with a cyclic voltammetric oxidative and reductive module, is described for the on-line qualitative determination of electroactive antineoplastic agents and metabolites in urine samples, collected from cancer patients, following intravenous administration.The application of two cyclic voltammetric detection modes provides an insight into both oxidative and reductive electrode reactions of compounds, passing the detector and the occurrence of (it)reversible chemical and electrochemical processes at the electrode surface. In this way, redox properties of drugs and metabolites characteristic of their molecular structure, can be established, which may provide information related to their (enzymatic) bioactivation.In the cyclic voltammetric mode, the system permits automatic detection of a compound in the cell, recording, storage and plotting of voltammograms and calculation of the retention times, the half-wave potentials and the peak potentials of each scan of all individual compounds. For routine use, storage of 68 voltammograms on-line is sufficient for the analysis of biological samples in clinical-pharmacological research. Special attention has been paid to automatic, multi-reference-point component detection.Based on their concentrations in urine, the oxidative cyclic voltammetric mode, using a glassy carbon electrode, permits the determination of etoposide and teniposide, whereas the reductive cyclic voltammetric mode, with a static mercury drop electrode, permits the determination of adriamycin and its metabolite adriamycinol.