Edward F. Rossomando

Tumor necrosis factor-α as a biochemical marker of pain and outcome in temporomandibular joints with internal derangements☆

OBJECTIVE Previous studies have demonstrated the presence of tumor necrosis factor-alpha (TNF) in human temporomandibular joint (TMJ) synovial fluid. The present study continues the investigation of the role of TNF in TMJs with internal derangements. MATERIALS AND METHODS Synovial fluid was obtained from 18 TMJs in 12 patients undergoing either arthroscopy (14 joints) or arthrotomy (four joints) for internal derangements. Standardized clinical data were collected preoperatively, intraoperatively, and postoperatively. RESULTS When pain on palpation was absent, the mean preoperative TNF level was 14 +/- 6 ng/mL. When pain on palpation was present, the mean TNF level was 42 +/- 39 ng/mL (significant difference at P = .05). When the surgical outcome was poor, the mean preoperative TNF level was 26 +/- 9 ng/mL. When the outcome was within the stated guidelines for a favorable result, the mean TNF level was 12 +/- 7 ng/mL (significant difference at P = .05). In addition, a significant reduction (P = .05) in TNF following joint lavage (preoperative, 48 ng/mL to postoperative, 7 ng/mL) was found. CONCLUSIONS The finding of a positive correlation between preoperative pain and TNF values suggests a biochemical basis for the origin of the pain associated with internal derangements. The relationship between preoperative TNF levels and surgical outcome suggests that the prognosis for surgery may be predicted by measuring biochemical markers of joint disease.

Orthodontic forces increase tumor necrosis factor α in the human gingival sulcus

The production of cytokines has been associated with the biology of tooth movement in animal populations. The purpose of this study was to measure tumor necrosis factor-α (TNF) directly in the human gingival sulcus before and after the application of an orthodontic force. To recover TNF from the sulcus, paramagnetic beads, coated with monoclonal antibodies for TNF, were introduced into the gingival sulcus of 50 teeth undergoing orthodontic tooth movement (by two force systems) in 20 patients. Retrieval was performed by a permanent magnetic device designed to fit the periodontal sulcus. The samples were taken before force application (controls), and at a fixed time after force application. The amount of immunoabsorbed TNF was quantified with an immunochemical assay. There was a greater than twofold increase in TNF recoverable from the gingival sulcus after application of orthodontic forces (mean of 12.9 ng vs 30.5 ng). A Students t test for paired samples demonstrated statistical significance at p < 0.01. We conclude that the quantity of paradental TNF, found in human gingival sulcus, is elevated during tooth movement. The source may be from the adjacent gingiva, but more likely the compressed periodontal ligament and the resorbing bone adjacent to the root surface. (AM J ORTHOD DENTOFAC ORTHOP 1995;108:519-24.)

Capillary electrophoresis: Separation and quantitation of reverse transcriptase polymerase chain reaction products from polio virus

In the present study reverse transcriptase (RT) polymerase chain reaction (PCR) products were generated from the RNA of polio virus. The products of the RT-PCR were analyzed by slab-gel electrophoresis (SGE) on 4% agarose gels, and capillary electrophoresis (CE). CE separations were performed in a coated capillary containing a linear polyacrylamide. Samples were injected hydrodynamically or electrokinetically. Detection of the RT-PCR products on CE was by UV absorbance at (254 nm) or by laser-induced fluorescence (LIF). While SGE resulted in adequate separation of 163 and 97 base pair RT-PCR products, separation of the 97, 71 and 53 base pair products was minimal. CE separations showed baseline resolution for all the above PCR products. Finally, it was possible to quantitate the amount of RT-PCR product by developing a standard curve showing a linear relationship between the amount of RNA used in the RT-PCR and the amount of product formed in the RT-PCR. These results suggest the greater resolution and enhanced sensitivity observed, together with the ease of quantitation, make CE a powerful alternative to SGE for the separation and quantitation of PCR products.

The Synthesis of Hadacidin: Sodium Cyanoborohydride Reduction of α-Oximinoic Acids

Abstract The natural product Hadacidin (N-formyl-N-hydroxy-glycine) has been isolated from various fungi and was found to inhibit the growth of bacteria, tumor cells, and plant and animal tissue1. Specifically, the drug has been shown to be an inhibitor of adenylosuccinate synthetase, consequently blocking purine synthesis de novo as well as purine salvage via either the purine nucleoside cycle or hypoxanthine salvage. Due to this inhibitory activity hadacidin was studied as a chemotherapeutic agent; however, it was found to be only weakly active against various carcinomas2 and therefore has not been made commercially available. There is a current resurgence of interest in the drug due to its ability to affect the growth and development of various organs and organisms in a very specific manner3. This communication presents a simple and rapid synthetic route to the naturally occurring antibiotic, that may find general applicability in the synthesis of a variety of N-hydroxylated amino acid compounds which ...

Effects of Sugars on Glycosidase Secretion in Dictyostelium discoideum

SUMMARY: When exponentially growing Dictyostelium discoideum amoebae were harvested from growth medium and suspended in a nutrient-free phosphate buffer, the cells released 20 to 30% of their total β-N-acetylglucosaminidase and α-mannosidase activities. This release was inhibited by sodium azide but stimulated (to a level of 90% release) by various saccharides when added to the phosphate buffer at millimolar concentrations. Various salts, amino acids and nucleotides were unable to stimulate the release process. Non-metabolizable sugars were more stimulatory than metabolizable sugars, but no clear distinction related to structure, ability to be transported or ability to be metabolized was found to account directly for the varying effects of the sugars on glycosidase release. The rate and amount of glycosidase release was dependent on the sugar concentration but not on the cell density. The results presented suggest that sugars stimulate a specific release of a variety of lysosomal enzymes and the relationship of this release to D. discoideum morphogenesis is discussed.

Immunomagnetic separation of tumor necrosis factor α: I. Batch procedure for human temporomandibular fluid

A batch separation procedure has been developed for retrieval of tumor necrosis factor (TNF) alpha from the microliter volumes of fluid isolated from the human temporomandibular joint (TMJ). Paramagnetic beads coated with monoclonal antibodies for TNF were used. The beads, and bound TNF, were recovered from solution with the aid of a magnetic field. The amount of bead-bound TNF was quantified using an immuno-based assay developed in this laboratory called the cluster assay. The cluster assay was specific for TNF and linear up to 10 ng. Using these methods we found that TMJ fluid contained 0.2-4.2 ng per 100 microliters of fluid with a mean value of 1.9 ng and a standard deviation of 1.1 ng. This study demonstrates the utility of batch immunomagnetic separation for the concentration and purification of proteins, and the cluster assay for quantification of proteins from microliter volumes of body fluids.

A fluorometric-high-performance liquid chromatographic assay procedure for several enzymatic activities using formycin analogs of adenosine 5′-mono, 5′-tri, and cyclic 3′,5′-monophosphate as substrates☆

The present study describes the synthesis of formycin 5′-triphosphate (FoTP), formycin 5′-monophosphate (FoMP), and formycin 3′,5′-cyclic monophosphate (cFoMP) from formycin A (FoA). These compounds, analogs of ATP, AMP, cAMP, and adenosine, respectively, are all fluorescent and differ chemically from the adenosine compounds by the reversal of the carbon atom at position 8 and the nitrogen at position 9 of the purine ring. Both FoMP and cFoMP were synthesized by chemical procedures from FoA while FoTP was made from FoMP enzymatically. All the analogs could be separated from each other using a high-performance liquid chromatographic (hplc) reverse-phase isocratic system that includes a μBondapak C-18 column as stationary phase and a solution of 0.01 m KH2PO4 adjusted to pH 5.5 with NaOH containing methanol as a mobile phase. At a flow rate of 2 ml/min, FoTP had a retention time of about 1 min followed by FoMP (2 min), cFoMP (3.5 min), and finally FoA (5.5 min). The analogs were detected by fluorometry using an excitation wavelength of 300 nm and an emission wavelength above 320 nm. This detection system proved to be more sensitive than absorbtion spectroscopy and as little as 2 pmol of the compounds could be measured. The analogs, together with the hplc system, were used to develop fluorometric (nonradioactive) assays for several enzymes including 3′5′-cyclic nucleotide phosphodiesterase (PDase), ATP pyrophosphohydrolase, and alkaline phosphatase. With these enzymes, the conversion of cFoMP to FoMP, FoTP to FoMP, and FoMP to FoA, respectively, could be followed. The conversion of FoA to formycin B (FoB), an analog of inosine, was also followed. The intracellular PDase activity isolated from the eukaryotic microorganism Dictyostelium discoideum was studied in some detail, and an apparent Km of 5 μm and Vmax of 0.1 nmol/min/mg protein were obtained for the enzyme at pH 7.5 and 30°. These values are compared to those in the literature. A number of advantages of this fluorometric-hplc assay procedure are discussed, including the facts that it offers an increase in sensitivity over other spectrophotometric assays and is at least equivalent to radiochemical assays currently in use.

DNA polymerase of Dictyostelium discoideum

Only one molecular weight species of DNA polymerase was found in different developmental stages of the eukaryotic microorganism Dictyostelium discoideum. The molecular weight of this DNA polymerase is estimated to be about 127 000 by sucrose gradient centrifugation. The enzyme is present in all stages of growth and development, including dormant spores. All DNA polymerase activity is lost upon incubation of the crude extract with N-ethylmaleimide. The reaction properties, molecular weight and N-ethylmaleimide sensitivity of the D. discoideum DNA polymerase are similar to those of the DNA polymerase-alpha from mammalian sources.

Intermediary purine-metabolizing enzymes from the cytosol of Dictyostelium discoideum monitored by high-performance liquid chromatography☆

The use of high-performance liquid chromatography to identify and quantitate five purine-metabolizing enzymes from a partially purified subcellular fraction of the eucaryotic microorganism Dictyostelium discoideum is described. All HPLC separations were carried out in an isocratic manner using reverse-phase C18 as the stationary phase. The mobile phase consisted of a phosphate buffer with either methanol or acetonitrile as cosolvent, and optimal separation conditions were attained by varying the organic concentration or the pH of the buffer or by employing paired-ion chromatographic techniques. Substrates and products were detected at either 254 nm for the purines or 295 nm for the formycin analogs. An adenosine kinase activity was identified, and it was demonstrated that formycin A (FoA) could be substituted for adenosine as the phosphate acceptor, yielding FoAMP as the product. With FoA as the substrate an apparent Km of 18.2 microM and an apparent Vmax of 32.4 mmol min-1 mg-1 were observed for the activity. A purine-nucleoside phosphorylase activity was found to cleave adenosine to adenine and ribosylphosphate. FoA was not found to be a substrate for this activity due to the unusual formycin C-glycosyl bond which was not hydrolyzed by enzymes or chemically with either HCl or NaOH. An adenylate deaminase activity was found to be present in the cytosolic S-100 of cells harvested during the onset of development, and this deaminase activity was greatly stimulated by ATP. With FoAMP as the substrate, an apparent Km of 236 microM and Vmax of 2.78 mumol min-1 mg-1 were observed. The deamination of FoAMP could be inhibited by the addition of the natural substrate AMP. An apparent Ki value of 136 microM was determined from initial rate data. An adenylosuccinate synthetase activity was observed to have a Km value for GTP, IMP, and aspartic acid of 23, 34, and 714 microM, respectively. The formycin analog FoIMP was not a substrate with this activity but was a competitive inhibitor of IMP. Finally hypoxanthine-guanine phosphoribosyltransferase was found to have Km and Vmax values for hypoxanthine of 55.5 microM and 34.3 nmol-1 min-1 mg-1. When guanine was used as the substrate, the rate of nucleotide formation was 50% that with hypoxanthine as the substrate. The advantages of using HPLC to examine the interconnecting activities of a multienzyme complex in subcellular fractions are discussed, including the increased sensitivity obtained by using formycin analogs in the assay procedures.(ABSTRACT TRUNCATED AT 400 WORDS)

Developmental changes in membrane-bound enzymes of Dictyostelium discoideum detected by concanavalin A-Sepharose affinity chromatography.

Abstract Plasma membrane-enriched fractions isolated from Dictyostelium discoideum at early stages of development were detergent extracted and subjected to affinity chromatography on a concanavalin A-Sepharose column. Alkaline phosphatase, 5′-nucleotidase, and cAMP phosphodiesterase activities were totally bound to the column when logarithmically growing cells were examined. As the cells entered development, however, a progressive decrease in the ability of these activities to bind to the affinity column was evident.