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Fatty acid synthase (FAS) regulates the production of fatty acids and plays a role in regulating apoptosis. Hoechst 33342-induced apoptosis in BC3H-1 myocytes was used as a model to explore intracellular changes in FAS protein (Western blot) and FAS mRNA (RT-PCR). Total lipid and individual phospholipid synthesis was inhibited by a lethal dose of Hoechst 33342 (20 microg/ml) while total lipid and phospholipid degradation ([1-14C]-acetate pulse chase method) were not. Hoechst 33342 at 20 microg/ml reduced the concentration of FAS protein, which was followed more than 6 hr later by a reduction in FAS mRNA. In conclusion, the inhibition of fatty acid synthesis induced by 20 microg/ml of Hoechst 33342 is attributed to the degradation of FAS protein by activated caspases rather than by inhibition of FAS enzyme activity or FAS mRNA synthesis.

Glycosphingolipids are ubiquitous membrane constituents that are subdivided in neutral or acidic fractions (gangliosides and sulfatides). Their analysis requires extraction and separation by thin-layer chromatography or high-performance liquid chromatography. Ganglioside composition changes occur in response to variations in cellular morphology and function. Glycosphingolipids are implicated in the pathogenesis of various diseases, including glycosphingolipidoses, peripheral neuropathies caused by anti-ganglioside antibodies, and secretory diarrhea. Gangliosides play a role in the induction of apoptosis. For example, ceramide-induced apoptosis is associated with increased synthesis of a ganglioside, GD3. Gangliosides are also potential diagnostic markers and therapeutic targets for cancer.

Abstract. Hoechst 33342's effects on apoptosis and mitochondrial membrane potential (delta psi) were investigated in a myelogenous leukemia cell line, HL-60. Delta psi was detected with 2 lipophilic cationic fluorochromes: 3,3'-dihexyloxacarbocyanine iodide [DiOC6(3)] or 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Mitochondrial mass was measured with nonyl acridine orange (NAO). Protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) depolarized mitochondria in control experiments. Cell viability was determined by propidium iodide uptake. Hoechst 33342 at 10-20 mg/L decreased fluorescence for DiOC6(3) at 0.5 hr. The fluorescence partially normalized at 3 hr and then progressively decreased at 5-24 hr, resulting in cell shrinkage and death. Mitochondrial mass decreased 40-70% by 1 hr and 70-90% at 24 hr. A lower concentration of Hoechst 33342, 5 mg/L, reduced the delta psi at 0.5 hr, but delta psi returned to control values after 3 hr. Mitochondrial mass decreased 30-40% and then partially normalized, and cell viability was > 92% at 24 hr. Protonophore carbonyl cyanide m-chlorophenylhydrazone lowered delta psi with little cell death. Thus, at high concentration, Hoechst 33342 induces depolarization of delta psi and subsequent apoptosis. Lack of apoptosis at low concentration of Hoechst 33342, despite depolarization of delta psi, indicates that mitochondrial membrane depolarization alone is insufficient to induce apoptosis.

Human cells contain deoxyribonucleic acid in mitochondria and nuclei. Human diseases may be caused by mutations in mitochondrial DNA, nuclear DNA or both. The volume of work performed in the diagnostic molecular pathology laboratory will continue to grow as more disease-related mutations are discovered. Many factors will influence the diagnostic molecular pathology laboratory in the 21st century, such as future clinical laboratory organization, amplification methods, specimen integrity, ethical guidelines and opportunities to expand service. In the evaluation of a patient suspected of a mitochondrial DNA mutation, care must be exercised in the selection of a primer for amplification and of the specimen to be examined for the mutation. The uneven distribution of normal and abnormal mitochondrial DNA within the various tissues (heteroplasmy) may result in a normal mitochondrial DNA sequence if the wrong tissue is examined. The presence of mitochondrial-like sequences (pseudogenes) within nuclear DNA may result in amplification of nuclear genes if generic primers are used to duplicate a mitochondrial DNA gene. Diabetes mellitus is a heterogeneous disease with mutations occurring in a variety of proteins leading to either prereceptor, receptor or postreceptor defects. In this example, the diagnostic molecular pathology laboratory may be asked to define the specific genotype a specific patient with this common phenotype may possess.

Hoechst 33342, a bisbenzimidazole dye, binds to adenine/thymine rich regions in the minor groove of deoxyribonucleic acid (DNA). This dye induces apoptosis in BC3H-1 myocytes. The mechanism of Hoechst 33342-induced apoptosis was investigated. Inhibitors of ribonucleic acid (RNA) synthesis, protein synthesis, and serine or cysteine proteases failed to prevent BC3H-1 myocyte death induced by Hoechst 33342. Apoptosis may be dependent on increased p53 expression. Hoechst 33342 had no effect on p53 expression in BC3H-1 myocytes. Lactate oxidation, a monitor of mitochondrial function, was altered by Hoechst 33342 in dose dependent manner. Also, nuclear extracts were used to assay endogenous topoisomerase I activity which was inhibited by Hoechst 33342 treatment of BC3H-1 myocytes. Therefore, Hoechst 33342 appears to initiate apoptosis in BC3H-1 myocytes by a pathway which is independent of de novo RNA and protein synthesis. However, the dye does initiate mitochondrial dysfunction and inhibition of nuclear topoisomerase I as two important steps in the apoptotic pathway.

Bisbenzimidazoles (Hoechst 33342 and Hoechst 33258) are cell permeable, adenine-thymine binding fluorescent dyes used to stain deoxyribonucleic acid (DNA) during the evaluation of cell cycle, induction of apoptosis by various ligands and cell viability by flow cytometry. These dyes inhibit topoisomerase I activity in vitro, like camptothecin. In this study, Hoechst 33342 is shown to induce apoptosis at concentration of 10 micrograms/mL or greater after 3 hours incubation in Dulbecco's Modified Eagle Medium characterized by rounded cell morphology, half-moon nuclei with condensed chromatin and a DNA fragmentation ladder of 180 base pair multiples. Hoechst 33258 at the same molarity or seven times greater molarity did not induce apoptosis. If the BC3H-1 myocytes were incubated in RPMI-1640 media, two times the concentration of Hoechst 33342 (20 micrograms/mL) was required to initiate apoptosis. Staining of unfixed cells with Hoechst 33342 may induce apoptosis in the absence of ligands. Therefore, Hoechst 33342 concentration and staining interval should be tested before ligands which may induce apoptosis are evaluated.

Nitric oxide is generated from L-arginine by the action of nitric oxide synthase, an enzyme encoded by three different genes. Nitric oxide is involved in an expanding number of phenomena. This involvement may be documented by direct detection using spectrophotometric or electrochemical methods or more often by indirect methods. Indirect methods for detection of nitric oxide effects include localization of nitric oxide synthase enzyme by immunochemistry or messenger ribonucleic acid (mRNA) by in situ hybridization, bioassays, inhibition of nitric oxide synthase activity, iron responsive element binding protein activity, and production of nitrate/nitrite, L-citrulline, or cyclic guanosine monophosphate (cGMP). Careful evaluation of potential pitfalls associated with these indirect methods of detecting nitric oxide effects prior to their use will prevent misinterpretation of results.

Reliably diagnosing endometriosis traditionally requires surgery. To evaluate a possible non-surgical method, a case-control series of unexplained infertility patients undergoing diagnostic laparoscopy were scored by clinical criteria and reactivity to human carbonic anhydrase II by Western blotting. The CA II autoantibodies were found in none of the fertile controls, 38 percent of infertile controls, 55 percent of stage 1, 50 percent of stage 2, 73 percent of stage 3, and 85 percent of stage 4 endometriosis patients. Advanced endometriosis was associated with more intense reactivity. Combining clinical and antibody scores for infertile groups showed a positive association with disease stage with positive predictive values of 76 to 95 percent, negative predictive values of 90 to 60 percent, and a likelihood ratio of 18.3. It is concluded by us that CA II immunoreactivity, clinical, and combined scores all identified stages 2 to 4 endometriosis patients. However, based on predictive values and likelihood ratios, the combined score is best at identifying endometriosis non-surgically.

There are two categories of autoantibodies to specific enzymes: immunoglobulin-complexed enzymes and circulating autoantibodies directed to enzymes in tissue or tissues. Immunoglobulin-complexed enzymes may result in elevated serum enzyme activity. They are found more frequently in elderly patients and have limited clinical significance. Immunoglobulin association with the enzyme must be demonstrated to distinguish this macroenzyme from other high molecular weight enzyme complexes. Autoantibodies to specific enzymes or regulators of enzyme activity do possess specific disease associations. The titers or presence of these autoantibodies may predict morbidity or response to therapy. These autoantibodies may be detected by Western blotting, enzyme-linked immunosorbent assays, tissue immunofluorescence, radioimmunoassay, immunoprecipitation flow cytometry or inhibition of enzyme activity. For example, anti-pyruvate dehydrogenase inhibits the activity of purified enzyme, but not relatively intact mitochondrial preparations. Most evidence suggests that the production of autoantibodies to specific enzymes represents an epiphenomenon secondary to tissue damage rather than a primary event in the pathogenetic pathway.

Blockade of adenosine receptors by 3-isobutyl-1-methylxanthine or degradation of endogenous adenosine with adenosine deaminase increased the phosphatidylcholine concentration in isolated rat adipocyte plasma membranes, an effect which was suppressed by the phosphatidylethanolamine methyltransferase inhibitor, S-adenosyl-L-homocysteine, and reversed by the adenosine analogue, N6-(L-phenylisopropyl)-adenosine. For example, the addition of N6-(L-phenylisopropyl)-adenosine to adenosine deaminase pretreated plasma membranes rapidly lowered the concentration of phosphatidylcholine by 171 nmol/mg at 30 seconds compared to control. Insulin-induced stimulation of phospholipid methylation in membranes treated with 3-isobutyl-1-methylxanthine or adenosine deaminase was achieved only after the addition of N6-(L-phenylisopropyl)-adenosine. These results suggest that adenosine receptor occupancy inhibits phospholipid methylation, is required for insulin stimulation of phospholipid methylation, and may perhaps activate a phosphatidylcholine-specific phospholipase C or phospholipase D.

Tyrphostins inhibit tyrosine kinases and have little effect on the activity of serine/threonine kinases. Pyruvate dehydrogenase kinase inactivates pyruvate dehydrogenase by phosphorylating serine residues within the multienzyme complex. This serine/theronine kinase represents a new family of protein kinases, and one (tyrphostin 47) of two tyrphostins tested appeared to activate the pyruvate dehydrogenase kinase as determined by [1-14C]-lactate oxidation to 14CO2. Experiments designed to determine if the tyrphostins altered pyruvate dehydrogenase activity in mitochondria prepared from rat epididymal adipocytes using [1-14C]-pyruvate as the substrate demonstrated a dose dependent increase in enzyme activity in the presence of tyrphostin 47, but not in tyrphostin 23. This apparent stimulation of pyruvate dehydrogenase activity was attributed to tyrphostin 47's ability to nonenzymatically decarboxylate [1-14C]-pyruvate, the substrate for the pyruvate dehydrogenase assay. Neither tyrphostin directly altered pyruvate dehydrogenase kinase activity. Therefore, assays utilizing [1-14C]-pyruvate and tyrphostin 47 are subject to analytical interference.

The change in transmembrane potential of rat adipocytes was measured using the fluorescent probe 3,3'-diethylthiadicarbocyanine iodide, diS-C2-(5). The method was calibrated by altering the potassium ion concentration while keeping the sum of potassium and sodium ions at a constant concentration of 153 mM (Bailey et al: Bioelectrochem. Bioenergetics 21:333-42, 1989). Two insulin-mimetic agents, phospholipase C from Clostridium perfringens and concanavalin A, induced a dose dependent hyperpolarization of rat epididymal adipocytes, like insulin. Removal of endogenous adenosine with adenosine deaminase or adenosine receptor blockade with isobutylmethylxanthine following the initiation of insulin-induced hyperpolarization resulted in depolarization. These same agents induced hyperpolarization of -6 to -8 mV when added without insulin. The replacement of adenosine with its analogue, N6-phenylisopropyladenosine, plus insulin depolarized the cells toward the transmembrane potential established by insulin, -2.0 mV. These studies suggest that adenosine receptor occupancy is required to maintain insulin-induced hyperpolarization.

To assess mitochondrial function (pyruvate dehydrogenase [PDH] activity), cells were grown in the appropriate media to confluence, rinsed and incubated in glucose free media containing 25 microM L-lactate and [1-14C]-D,L-lactate. Lactate oxidation was measured as the amount of lactate oxidized in nmol of 14CO2 generated per mg of protein per minute. Basal activity varied with cell number and the cell type studied: fibroblast 2.26 +/- 0.01; Chinese hamster ovary (CHO) 42 +/- 0.4; BC3H-1 52 +/- 2.1 nmol per mg per minute. The CHO cells screened for PDH activity decreased their dependence on lactate as a substrate in the presence of 5mM glucose by 60 percent. Increasing the cold lactate concentration diluted the labelled lactate available for pyruvate oxidation in a dose dependent manner. The mitochondrial inhibitor rotenone (25 microM) decreased assay activity by > 75 percent in CHO and BC3H-1 cells. The lactate oxidation assay was shown to be sensitive enough to measure insulin stimulation of PDH in a dose dependent manner with maximum activity occurring at concentrations between 1 microU per ml and 100 microU per ml.

Enzymic procedures have been developed for the specific determination of three phospholipids in an effort to make the estimation of the phospholipids of amniotic fluid more sound analytically. Apart from the actual determination of these analytes (lecithin, sphingomyelin, and phosphatidylglycerol), these enzymic procedures facilitate the evaluation of a number of the basic premises and procedural steps involved with the traditional procedures for the evaluation of fetal lung maturity. To this end, the changes are reported in sphingomyelin concentrations with gestational age. Although lacking in sufficient clinical data to assign "cut-off" values as yet, the enzymic procedures seem to correlate well with the existing procedures and are analytically accurate and precise.

A useful laboratory test for the differentiation of liver, bone, and intestinal alkaline phosphatase (ALP) isoenzymes in serum is presented. Electrophoresis in polyacrylamide gel is performed with untreated serum as well as with serum incubated at 56 degrees C for 10 min. The heating step denatures bone isoenzyme which may obscure the liver ALP band when present in large amounts. Visualization of ALP activity is accomplished by the use of buffered p-toluidinium 5-bromo-4-chloro-indolyl phosphate and magnesium ions. In serum of patients with cholestatic liver disease, the occurrence of large molecular weight liver cell membrane fragments which contain ALP activity is postulated. These ALP-containing fragments occur at the origin of the electrophoretogram, unable to penetrate the small pore separation gel. Abnormalities involving ALP isoenzymes, such as bone isoenzyme arising from increased osteoblastic activity, may be detected. Intestinal isoenzyme, normally present in small amounts in some subjects of blood groups B or O, may be elevated in certain liver diseases, such as cirrhosis. By the use of this method the routine question of whether an ALP found to be increased in a screening procedure is due to liver or bone abnormality may be answered. In addition, the occurrence of abnormal ALP bands arising from cholestatic conditions and the occurrence of abnormal amounts of intestinal isoenzyme may also be detected.