Optimization of beta-quantification methods for high-throughput applications.

BACKGROUND: Risk of cardiovascular disease is assessed, in part, by laboratory measurement of the concentrations of several lipoproteins. beta-Quantification is a method of lipoprotein measurement that uses ultracentrifugation to partially separate lipoprotein classes. Although beta-quantification is used largely in clinical and basic research, methods have not been described to allow the analysis of a large number of small-volume specimens with a short turnaround time. We report two variations of the traditional 5-mL method used by the Lipid Research Clinics Program that overcome these shortcomings. METHODS: Two lower-volume modifications of the traditional 5-mL beta-quantification method were developed. The methods used either 1 or 0.23 mL of specimen and required substantially less time for analysis (20 and 6 h, respectively) than the 5-mL method (2.5 days). The goal was to develop ultracentrifugation methods such that the concentration of cholesterol in the bottom fraction, from which LDL-cholesterol concentration is calculated, agreed with the 5-mL method. Fresh serum specimens (n = 45) were analyzed by the three methods to determine comparability of the methods based on the recovery of cholesterol in the bottom fraction after ultracentrifugation. To evaluate intrarun precision, replicate specimens (n = 17) were analyzed in a single run for each method. This experiment also evaluated how quickly the fractions would remix after separation by ultracentrifugation. For the 1-mL method, accuracy of the measurement of LDL- and HDL-cholesterol concentrations and the interrun precision were established by analysis of frozen serum specimens provided by the CDC, which established target values for the pools using reference methods. RESULTS: No clinically significant differences in cholesterol concentrations in the bottom fraction were observed for the 1- and 0.23-mL methods, which had mean biases of 0.8% and 1.5% relative to the 5-mL method, respectively. Intra- and interrun variability was acceptable for each method, e.g., <1.8% for cholesterol in the bottom fraction. Ultracentrifuged specimens were stable for at least 4 h with no evidence of contamination of cholesterol in the bottom fraction. For comparison specimens provided by the CDC, the 1-mL method met the accuracy and precision goals of the National Cholesterol Education Program for the measurement of HDL- and LDL-cholesterol concentrations (goals: total error <13% and <12%, respectively), with total errors of 6.45% and 5.43%, respectively. CONCLUSIONS: Both the 1- and 0.23-mL beta-quantification methods are suitable substitutes for the traditional 5-mL method for use in clinical and basic research for the determination of LDL-cholesterol concentration. Both methods provide much higher throughput and require substantially less specimen volume. The 0.23-mL method can be performed in 1 day, but it is slightly less precise than the 1-mL method. In our laboratory setting, as many as 80 specimens are routinely processed per day using the 1-mL method.

Electrospray ionization/mass spectrometric analyses of human promonocytic U937 cell glycerolipids and evidence that differentiation is associated with membrane lipid composition changes that facilitate phospholipase A2 activation.

Upon differentiation, U937 promonocytic cells gain the ability to release a large fraction of arachidonate esterified in phospholipids when stimulated, but the mechanism is unclear. U937 cells express group IV phospholipase A(2) (cPLA(2)), but neither its level nor its phosphorylation state increases upon differentiation. A group VI PLA(2) (iPLA(2)) that is sensitive to a bromoenol lactone inhibitor catalyzes arachidonate hydrolysis from phospholipids in some cells and facilitates arachidonate incorporation into glycerophosphocholine (GPC) lipids in others, but it is not known whether U937 cells express iPLA(2). We confirm that ionophore A23187 induces substantial [(3)H]arachidonate release from differentiated but not control U937 cells, and electrospray ionization mass spectrometric (ESI/MS) analyses indicate that differentiated cells contain a higher proportion of arachidonate-containing GPC species than control cells. U937 cells express iPLA(2) mRNA and activity, but iPLA(2) inhibition impairs neither [(3)H]arachidonate incorporation into nor release from U937 cells. Experiments with phosphatidate phosphohydrolase (PAPH) and phospholipase D (PLD) inhibitors coupled with ESI/MS analyses of PLD-PAPH products indicate that differentiated cells gain the ability to produce diacylglycerol (DAG) via PLD-PAPH. DAG promotes arachidonate release by a mechanism that does not require DAG hydrolysis, is largely independent of protein kinase C, and requires cPLA(2) activity. This may reflect DAG effects on cPLA(2) substrate state.

Human pancreatic islets express mRNA species encoding two distinct catalytically active isoforms of group VI phospholipase A2 (iPLA2) that arise from an exon-skipping mechanism of alternative splicing of the transcript from the iPLA2 gene on chromosome 22q13.1.

An 85-kDa Group VI phospholipase A2 enzyme (iPLA2) that does not require Ca2+ for catalysis has recently been cloned from three rodent species. A homologous 88-kDa enzyme has been cloned from human B-lymphocyte lines that contains a 54-amino acid insert not present in the rodent enzymes, but human cells have not previously been observed to express catalytically active iPLA2 isoforms other than the 88-kDa protein. We have cloned cDNA species that encode two distinct iPLA2 isoforms from human pancreatic islet RNA and a human insulinoma cDNA library. One isoform is an 85-kDa protein (short isoform of human iPLA2 (SH-iPLA2)) and the other an 88-kDa protein (long isoform of human iPLA2 (LH-iPLA2)). Transcripts encoding both isoforms are also observed in human promonocytic U937 cells. Recombinant SH-iPLA2 and LH-iPLA2 are both catalytically active in the absence of Ca2+ and inhibited by a bromoenol lactone suicide substrate, but LH-iPLA2 is activated by ATP, whereas SH-iPLA2 is not. The human iPLA2 gene has been found to reside on chromosome 22 in region q13.1 and to contain 16 exons represented in the LH-iPLA2 transcript. Exon 8 is not represented in the SH-iPLA2 transcript, indicating that it arises by an exon-skipping mechanism of alternative splicing. The amino acid sequence encoded by exon 8 of the human iPLA2 gene is proline-rich and shares a consensus motif of PX5PX8HHPX12NX4Q with the proline-rich middle linker domains of the Smad proteins DAF-3 and Smad4. Expression of mRNA species encoding two active iPLA2 isoforms with distinguishable catalytic properties in two different types of human cells demonstrated here may have regulatory or functional implications about the roles of products of the iPLA2 gene in cell biologic processes.

Activation of Rho-dependent transcription termination by NusG. Dependence on terminator location and acceleration of RNA release.

There is a kinetic limitation to Rho function at the first intragenic terminator in the lacZ gene (tiZ1) which can be overcome by NusG: Rho can terminate transcription with slowly moving, but not rapidly moving, RNA polymerase unless NusG is also present. Here we report further studies with two other Rho-dependent terminators that are not kinetically limited (tiZ2 and lambda tR1) which show that the requirement for NusG depends on the properties of the terminator and its location in the transcription unit. NusG is also shown to increase the rate of Rho-mediated dissociation of transcription complexes arrested at a specific termination stop point in the tiZ1 region and the rates of dissociation with three different Rho factors and two different terminators correlated with their sensitivity to RNA polymerase elongation kinetics. These results suggest a model of NusG function which involves an alteration in the susceptibility of the transcription complex to Rho action which allows termination to occur within the short kinetic window when RNA polymerase is traversing the termination region.

Distinction among eight opiate drugs in urine by gas chromatography-mass spectrometry.

Opiates are commonly abused substances, and forensic urine drug-testing for them requires gas chromatographic-mass spectrometric (GC-MS) confirmation. There are also medical reasons to test urine for opiates, and confirmation procedures other than GC-MS are often used for medical drug-testing. A thin-layer chromatographic (TLC) method distinguishes morphine, acetylmorphine, hydromorphone, oxymorphone, codeine, dihydrocodeine, hydrocodone, and oxycodone in clinical specimens. In certain clinical circumstances, GC-MS confirmation is requested for opiates identified by TLC, but, to our knowledge, no previous report examines all of the above opiates in a single GC-MS procedure. We find that they can be distinguished by GC-MS analyses of trimethylsilyl (TMS) ether derivatives, and identities of 6-keto opiates can be further confirmed by GC-MS analysis of methoxime (MO)-TMS derivatives. Inclusion of deuterium-labeled internal standards permits identification of the opiates in urine at concentrations below the TLC cutoff level of 600 ng/ml, and the GC-MS assay is linear over a concentration range that spans that level. This GC-MS procedure has proved useful as a third-stage identification step in a medical drug-testing sequence involving prior immunoassay and TLC.

Mass spectrometric evidence that agents that cause loss of Ca2+ from intracellular compartments induce hydrolysis of arachidonic acid from pancreatic islet membrane phospholipids by a mechanism that does not require a rise in cytosolic Ca2+ concentration.

Stimulation of pancreatic islets with glucose induces phospholipid hydrolysis and accumulation of nonesterified arachidonic acid, which may amplify the glucose-induced Ca2+ entry into islet beta-cells that triggers insulin secretion. Ca2+ loss from beta-cell intracellular compartments has been proposed to induce both Ca2+ entry and events dependent on arachidonate metabolism. We examine here effects of inducing Ca2+ loss from intracellular sequestration sites with ionophore A23187 and thapsigargin on arachidonate hydrolysis from islet phospholipids. A23187 induces a decline in islet arachidonate-containing phospholipids and release of nonesterified arachidonate. A23187-induced arachidonate release is of similar magnitude when islets are stimulated in Ca2+-replete or in Ca2+-free media or when islets loaded with the intracellular Ca2+ chelator BAPTA are stimulated in Ca2+-free medium, a condition in which A23187 induces no rise in beta-cell cytosolic [Ca2+]. Thapsigargin also induces islet arachidonate release under these conditions. A23187- or thapsigargin-induced arachidonate release is prevented by a bromoenol lactone (BEL) inhibitor of a beta-cell phospholipase A2 (iPLA2), which does not require Ca2+ for catalytic activity and which is negatively modulated by and physically interacts with calmodulin by Ca2+-dependent mechanisms. Agents that cause Ca2+ loss from islet intracellular compartments thus induce arachidonate hydrolysis from phospholipids by a BEL-sensitive mechanism that does not require a rise in cytosolic [Ca2+], and a BEL-sensitive enzyme-like iPLA2 or a related membranous activity may participate in sensing Ca2+ compartment content.

Electrospray ionization mass spectrometric analyses of phospholipids from rat and human pancreatic islets and subcellular membranes: comparison to other tissues and implications for membrane fusion in insulin exocytosis.

Glucose-induced insulin secretion from pancreatic islets involves hydrolysis of arachidonic acid from phospholipids as an intermediary event. Accumulation of nonesterified arachidonate in islet membranes may influence both ion fluxes that trigger insulin secretion and fusion of secretory granule and plasma membranes. Recent findings indicate that plasmenylethanolamine species may also participate in fusion of such membranes, but high-performance liquid chromatographic (HPLC) and gas chromatographic/mass spectrometric (GC/MS) analyses of islet secretory granule phospholipids suggested that they contain little plasmenylethanolamine. Here, electrospray ionization mass spectrometry (ESI/MS) of intact phospholipid molecules is used to demonstrate that the most prominent components of all major glycerophospholipid headgroup classes in islets are arachidonate-containing species. Such species contribute the majority of the ESI/MS negative ion current from rat and human islet glycerophosphoethanolamine (GPE), and the fraction of GPE negative ion current contributed by plasmenylethanolamine species in rat islets is higher than that for rat liver or heart and similar to that for brain. The most prominent sn-2 substituent of plasmenylethanolamine species in brain is docosahexaenoate and in islets is arachidonate. Arachidonate-containing plasmenylethanolamine species are also prominent components of GPE from islet secretory granules and plasma membranes. Fusion of islet secretory granule and plasma membranes is demonstrated to be catalyzed by cytosolic components from insulinoma cells and rat brain with chromatographic similarities to a rabbit brain factor that specifically catalyzes fusion of plasmenylethanolamine-containing membranes.

Transcription termination factor Rho is essential for Micrococcus luteus.

The growth of Micrococcus luteus, a soil microorganism that belongs to the high-G+C gram-positive phylogenetic group, is prevented by bicyclomycin, an antibiotic that inhibits the activity of the M. luteus transcription termination factor Rho. A mutant that can grow in 0.3 mM bicyclomycin has a Rho that is insensitive to bicyclomycin and has the single amino acid residue change of Asp474 to Gly. These results indicate that the function of its Rho factor is essential for M. luteus and that growth of a gram-positive organism can be blocked by bicyclomycin.

Function of the novel subdomain in the RNA binding domain of transcription termination factor Rho from Micrococcus luteus.

Transcription termination factor Rho from Micrococcus luteus, a high G + C Gram-positive bacterium, contains an unusual extra sequence within its RNA binding domain that is rich in Arg, Glu, and Asp residues and deficient in hydrophobic residues. To determine the role of this extra sequence, we compared the biochemical properties of a variant lacking nearly all the extra sequence, des(60-300) Rho, to that of wild-type M. luteus Rho. The two forms had very similar properties except that the des(60-300) Rho was unable to terminate transcription with Escherichia coli RNA polymerase at the promoter proximal sites used by the wild-type Rho on a lambda cro DNA template but could cause termination at more distal sites and did cause termination at proximal sites when ITP replaced GTP in the reaction mixture. The RNA binding properties of the two forms of this Rho with normal and inosine-substituted RNAs were found to correlate fully with their termination properties. These results indicate that the arginine-rich extra sequence is directly involved in the selection of the termination site and support the hypothesis that the sequence is present in M. luteus Rho to facilitate its binding to M. luteus transcripts, which are likely to have a high degree of base-paired secondary structure because of their high proportion of G residues.

Characterization of an unusual Rho factor from the high G + C gram-positive bacterium Micrococcus luteus.

A transcription termination factor (Rho) was purified from the Gram-positive bacterium Micrococcus luteus, and the complete gene sequence was determined. The M. luteus Rho polypeptide has 690 residues, which is 271 residues more than its homolog from Escherichia coli. Most of the additional residues compose a highly charged, hydrophilic segment that is inserted in a non-conserved region between two conserved regions of the RNA-binding domain of the known Rho homolog proteins. This segment extends from residues 49 to 311 and includes a stretch of 238 residues that contain no hydrophobic side chains. Biochemical studies indicate that the M. luteus protein is very similar to E. coli Rho in terms of its RNA-dependent NTPase activity and its sensitivity to the Rho-specific inhibitor bicyclomycin. However, the M. luteus protein has a less stringent RNA cofactor specificity. It also acts to terminate RNA transcription with E. coli RNA polymerase on the lambda cro DNA template, but at much earlier termination stop points than those recognized by E. coli Rho. Thus, the M. luteus protein functions as a true Rho factor, but with a different specificity than that of E. coli Rho. We propose that this altered specificity is consistent with its need to function on transcripts that have a high content of G + C residues.

Endocytosis and degradation of bovine apo- and holo-lactoferrin by isolated rat hepatocytes are mediated by recycling calcium-dependent binding sites.

We characterized endocytosis of iron-saturated (holo) and iron-depleted (apo) 125I-labeled bovine lactoferrin (Lf) by isolated rat hepatocytes. Hepatocytes ingested both Lf forms--determined by EGTA/dextran sulfate removal of surface-bound Lf--at maximal endocytic rates of 1.85 and 1.52 fmol cell-1 min-1 for 125I-apo-Lf and 125I-holo-Lf, respectively. First-order endocytic rate constants (37 degrees C) for 125I-apo-Lf and 125I-holo-Lf were 0.276 and 0.292 min-1, respectively. Regardless of Lf's iron content, hyperosmotic media (approximately 500 mmol/kg) inhibited Lf uptake by approximately 90%, indicating endocytosis of both Lf forms was primarily clathrin-dependent. Endocytosis of both Lf forms was not altered significantly in the presence of excess iron chelator desferrioxamine or rat holo-transferrin, or by cycloheximide treatment. Fluorescein isothiocyanate- and cyclohexanedione-modified Lf competed fully with native Lf for binding and endocytosis, indicating that, unlike human Lf, modification of lysine or arginine residues does not block the interaction of bovine Lf with cells. After binding Lf at 4 degrees C, cells at 37 degrees C internalized approximately 90% of Lf bound to Ca(2+)-dependent sites but not Lf bound to Ca(2+)-independent sites. Following uptake, hepatocytes released acid-soluble (degraded) products of 125I-Lf biphasically at 37 degrees C, an initial rapid phase within the first 20 min--more pronounced with 125I-holo-Lf--followed by a sustained linear release of 298 and 355 molecule equiv cell-1 min-1 for 125I-apo-Lf and 125I-holo-Lf, respectively. At 4 degrees C, both digitonin-permeabilized and intact cells bound approximately 1.1 x 10(6) 125I-Lf molecules to Ca(2+)-dependent sites per cell, indicating that hepatocytes do not contain a sizeable intracellular pool of these sites. Moreover, cells retained > 70% of Ca(2+)-dependent sites on the surface during sustained Lf endocytosis. Thus, these Lf binding sites recycle during endocytosis at an estimated 4-5 min/circuit.