Characterization of the epitope recognized by a mAb that reacts differentially with murine suppressor T cells

Although reliable antibodies are available that distinguishhuman suppressor T (T_s) cells from CTL and other T cells, feware available for murine T_s cells. We have developed a mAb (984D4.6.5)that, in the presence of complement, depletes alloantigen-specificT_s cells but not CTL. This antibody recognizes activated TT_scells but not their precursors. In these studies, flow cytometricanalysis demonstrates that 984D4.6.5 reacts with several T_scell hybridomas, cloned T_h cell lines and WEHI-3 (a myelomonocytictumor cell line). Reactivity was not detected with BW5147, T_hcell hybridomas, cloned T_h cells, CTL lines and hybridomas,B cell lines, thymocytes, splenocytes, bone marrow cells nora variety of tumor cells. Among 984D4.6.5 positive lines, expressionis heterogeneous and the number of cells expressing high levelsof the epitope is increased when the hybridomas are maintainedat a relatively high cell density. Neuriminidase and pronasedeplete the epitope recognized by mAb 984D4.6.5. Protein synthesisand glycosylation inhibitors also reduce expression of thisepitope. These observations suggest that the epitope recognizedby 984D4.6.5 is a carbohydrate linked to a polypeptide. Thisantibody was tested by ELISA for binding to a large panel ofcarbohydrates and glycollpids coupled to BSA. The only one thatbound 984D4.6.5 was LS tetrasaccharide c (NeuNAc2-6Galpß1-4GIcNAcß1-3GaIß1-4Glc),an O-linked carbohydrate. Comparative analysis shows that boththe sequence and the linkage of these sugars are essential tothe reactivity with the 984D4.6.5 antibody. This epitope isexpressed by a glycoprotein of-200 kDa, as shown by Westernblots. The identity of this glycoprotein remains to be determined,but indirect evidence suggests that it is not CD45.     

Ca-EGTA affects the relationship between [Ca2+] and tension in α-toxin permeabilized rat anococcygeus smooth muscle

Summary The relationship between calcium concentration ([Ca²⁺]) and force in smooth muscle can be studied by permeabilizing the sarcolemma and bathing the preparation in a mock intracellular solution. Normally [Ca²⁺] is set in these solutions using the Ca²⁺ chelator EGTA in the concentration range of 4–10 mm. This study shows that lowering total EGTA concentration ([EGTA]_t) below 10 mm depresses Ca²⁺-activated force generated in 0.1 m Ca²⁺. The observed threshold for the effect of EGTA_t is 0.2 mm, and the effect is maximal at approximately 10 mm. BAPTA, another Ca²⁺ chelator, also produces this effect. Tension production in smooth muscle is controlled by acto-myosin interaction. This in turn is mediated by the relative activities of myosin light chain kinase (MLCK) and phosphatase (MLCP). Inhibiting MLCP with Microcystin LR (10 m), an increase [EGTA_t] from 0.2 mm to 10 mm still enhanced force. This suggests that EGTA promotes phosphorylation of myosin by the activation of MLCK and not by inhibition of MLCP.     

Studies on the stability of trimelamol, a carbinolamine-containing antitumor drug

The stability of trimelamol (N2,N4,N6-trimethylol-N2,N4,N6-trimethylmelamine) a synthetic carbinolamine-containing antitumor drug, has been studied. Two major degradation pathways have been characterized and a unified mechanism proposed to rationalize the chemistry involved. One degradation pathway involves the consecutive loss of hydroxymethylene units by elimination of formaldehyde until the parent trimethylmelamine (4) results. An HPLC method was used to obtain kinetic data for the loss of trimelamol and to monitor the order of appearance of three degradation products. This pathway was shown to follow first-order kinetics at all pH values studied at both 18 and 37 degrees C. The second pathway involves the coupling of two trimelamol molecules via a methylene bridge to form bis(trimelamol) (6) which had been previously referred to in the literature as a "polymer". This reaction is acid catalyzed and temperature dependent. Bis(trimelamol) is virtually water insoluble and adheres strongly to glass surfaces. Finally, t1/2 values have been determined for trimelamol in aqueous solution at different temperatures, and the kinetics of formation of degradation products has been studied over a period of 30 h under a variety of conditions of pH and temperature. The data reported here are relevant to both the formulation and clinical administration of trimelamol, and may contribute to an understanding of mechanism of action and future analogue development studies.     

A chromatographic and spectroscopic study of photodegraded metronidazole in aqueous solution

Preparative reversed-phase HPLC has been used to isolate a yellow photodegradation (2) product from aqueous metronidazole (1) solutions buffered with citrate:phosphate. Compound 2 can be further degraded into colorless materials by light, heat, or the addition of the nonaqueous solvents. These products have been characterized by UV, IR, proton NMR, mass spectroscopy, and melting point determination. It is proposed that the initial yellow degradation product (2) is an "excimer ion-pair" formed by the stabilization of 1 in its first electronic excited state by the citrate molecule.     

Comparison of total alkaline phosphatase and three assays for bone-specific alkaline phosphatase in childhood and adolescence

We compared serum levels of total alkaline phosphatase (TAP) and bone-specific alkaline phosphatase (BAP) as determined by three different assays (lectin affinity electrophoresis, immunoradiometric assay, enzyme-linked immunosorbent assay) in subjects aged 5–20 years suffering from X-linked hypophosphatemic rickets ( n = 14), chronic renal failure ( n = 10) and chronic cholestatic liver disease ( n = 16). Results were compared to controls of the same age and were expressed as standard deviation scores (SDS). TAP correlated significantly with BAP ( r > 0.9 for each assay; p <0.001) in controls. In children with cholestatic diseases, TAP (median SDS + 2.0) was elevated, but BAP, as measured by the electrophoretic assay, was within the reference range for most patients (median SDS: -0.4; p = 0.003 for the difference between the median SDS of TAP and BAP). In contrast, results for BAP as determined by the two immunoassays were not significantly different from TAP in any of the three patient groups ( p > 0.05 in each group for both assays). In this study, the two immunoassays did not have a detectable advantage over lectin affinity electrophoresis in the determination of BAP.     

Isolation and characterization of propagable cell lines (HUNC) from the androgen-sensitive Dunning R3327H rat prostatic adenocarcinoma

The Dunning H rat prostate tumor (R3327H) is a widely used experimental model of human prostatic adenocarcinoma (CaP). The Dunning H tumor has been characterized as androgen-sensitive, androgen-receptor (AR) positive, prostate-specific antigen and prostatic acid phosphatase (PAP) positive. To date, the tumor has been maintained by serial passage in vivo because of the lack of an in vitro cell line that retains the characteristics of the in vivo tumor. The objective of the present study was to establish a propagable cell line from R3327H adenocarcinoma that maintained androgen sensitivity and expression of AR, PSA and PAP. Tissue harvested from an in vivo R3327H tumor was dissociated with collagenase and placed into Richter's improved media (with supplements). A cytokeratin-positive epithelial cell line (HUNC- E) and a vimentin-positive stromal cell line (HUNC-S) were generated from the primary culture, subcultured continuously for >300 days, and passaged >50 times. Survival of the HUNC-E cell line in vitro depended on several media supplements, including nicotinamide, insulin, transferrin, selenium and epidermal growth factor (EGF). HUNC-E cells expressed AR and produced PSA and PAP throughout the culture period, as confirmed by immunocytochemistry and Western blot analyses. Addition of 14 nM testosterone (T) or dihydrotestosterone (DHT) to HUNC-E cells, stimulated DNA synthesis as well as anchorage-independent growth and PSA production, which demonstrated the androgen-sensitive nature of the cells in vitro. When HUNC-E and HUNC-S cells were combined in a 3:1 ratio and introduced subcutaneously into syngeneic male hosts, tumors formed in 2/3 animals with an average latency of 7 months. RT-PCR and immunocytochemical characterization of the HUNC cell lines revealed that the cells expressed several growth factors and their cognate receptors, including HGF, TGF-alpha and the TGF-betas, indicating the establishment of potential autocrine loops in the neoplastic cells. The HUNC-E and HUNC-S CaP cell lines, which retain the characteristics of the epithelial and stromal components of the in vivo R3327H tumor, will allow a more thorough and informative molecular and biological analysis of prostatic adenocarcinoma.