New preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for fatty acids and derivatives

A preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for carboxylic acids is described. 9-Anthraldehyde hydrazone is oxidized with an organic oxidant, N-chlorosuccinimide, in an organic solvent such as ethyl acetate to give ADAM, and then the reaction mixture is directly used as the reagent solution for the derivatization of carboxylic acids. Both the oxidation and the derivatization reaction are carried out at room temperature, and an aliquot of the derivatization mixture is directly injected into a chromatograph. 9-Anthrylmethyl ester derivatives formed from ADAM and various carboxylic acids are sufficiently separated on a reversed-phase column and are sensitively detected fluorometrically. The present method was applied to the high-performance liquid chromatographic determination of long and short chain fatty acids, keto acids, and hydroxy acids.     

Hemostatic effect of a heat-treated factor VIII concentrate (Haemate P) in von Willebrand's disease

A heat-treated factor VIII (F VIII) concentrate (Haemate P) has been administered to patients with various types of von Willebrand's disease (vWD). The 4 activities of F VIII/vWF as well as change in the multimeric structure of vWF were then studied. In 4 patients with type I vWF who were given a Ristocetin cofactor (Rcof) dose of 42-78 U/kg, there was a clear reduction of the bleeding time and an increase of F VIII: C, F VIII: Ag, Rcof and vWF: Ag for several hours. The recovery of Rcof. after 1 h was 50-75%. Although the multimeric composition of vWF in these patients was similar to that of normal plasma, the density of each multimer band was very low. After infusion, however, the density of all multimer bands increased for several hours, to decrease again after 24 h. In 4 patients with type II A vWD who received a dose of Rcof of 55-76 U/kg, the 4 activities of F VIII/vWF increased similarly as was the case in type I. All patients had only 3-4 smaller multimer bands. New larger and intermediate multimers appeared for several hours after infusion of the preparation. Two patients with type III vWD who received doses of Rcof of 52 and 65 U/kg showed also a similar increase in the 4 activities of F VIII/vWF after infusion. All the multimers lacking in these patients appeared for several hours after infusion.     

Immunocytochemical localization of the 27K beta-crystallin polypeptide in the mouse lens during development using a specific monoclonal antibody: implications for cataract formation in the Philly mouse

The Philly mouse develops a hereditary cataract about 5 weeks after birth. Although the causative agent is not known, data suggest that there is a correlation between cataract formation and the selective absence of a 27 kilodalton (27K) beta-crystallin lens polypeptide. The ontogeny of the 27K beta-crystallin polypeptide was examined in normal mice in order to evaluate its role in normal development and determine what impact its absence may have on the Philly mouse lens. A monoclonal antibody was used with the PAP method to immunocytochemically localize the 27K polypeptide in lenses of normal mice during development. beta-Crystallins detected with polyclonal antisera were found in differentiated fiber cells throughout the lens. In contrast, the 27K beta-crystallin polypeptide detected with a specific monoclonal antibody was not found in the fiber cells of the inner part of the lens (nucleus), but was specifically localized in the fiber cells of the outer part of the lens called the cortex. The polypeptide was found only in elongating and differentiated fiber cells and not in mitotically active epithelial cells. Although a minor component of the 2-day-old lens, the 27K polypeptide comprised a large portion of the 16-day-old lens including the anterior and posterior poles. These data show that the 27K polypeptide is a minor component of the embryonic lens, but becomes a major contributor to the postnatal lens. The 27K beta-crystallin lens polypeptide is abundant in the fiber cells of the normal postnatal mouse lens. The absence of the 27K polypeptide in the Philly mouse may contribute to the observed failure of fiber cells to differentiate in the Philly mouse after birth or may be deleterious in some other manner to normal lens development. The selective absence of the 27K beta-crystallin polypeptide, a defect which precedes cataract formation in the Philly mouse, is intriguing since it suggests a relationship between this major lens polypeptide and lens clarity.     

Difference in arginine vasopressin responsive cAMP production between apical and basolateral membrane of cultured renal epithelial cells (MDCK)

It has been reported that vasopressin (AVP)-sensitive renal epithelial cell line (MDCK) forms morphologically polarized monolayers when cultured on plates. We studied whether the AVP-responsive cAMP production system would be located solely on the basolateral surface of these cells as has already been shown on the renal tubules. We used two methods to overcome the inaccessibility to the basolateral surface of the cultured cell layer and to study the apical and basolateral surfaces separately. One was culture on collagen sheet and the other was on Millipore filters. Our experiments showed that MDCK cell increased adenosine 3':5'-cyclic monophosphate (cAMP) content prominently only when vasopressin was accessible to the basolateral surface. Accordingly, MDCK cells were shown to have the AVP-responsive cAMP production system predominantly on the basolateral surface of the cell membrane.     

Measurement of urinary steroid profile in patients with adrenal tumor as a screening method for carcinoma

Results of measurement of urinary steroid metabolite profile using gas chromatographic analysis in eight patients with adrenocortical tumors, i.e. 3 adenomas with Cushing's Syndrome, one adenoma with virilization, one adenoma without clinical manifestations, one carcinoma with Cushing's syndrome and virilization, one carcinoma with Cushing's syndrome and feminization, and one carcinoma without endocrinological symptoms, are reported. A unique pattern dominated by 5 beta and 11 beta-hydroxy steroid metabolites was confirmed in five patients with Cushing's syndrome consisting of three cases with adenomas and two with carcinomas. Excessive 3 alpha, 17 alpha, 21-trihydroxy-5 beta-pregnan-20-one (tetrahydro-11-deoxycortisol, THS) and delta 5-pregnene-3 beta, 11 alpha, 20 alpha-triol (delta 5-pregnenetriol) values were found in all three carcinomas including a nonfunctional carcinoma. These findings would strongly suggest the tumor to be a carcinoma, although excessive excretion of THS and delta 5-pregnenetriol was detected in one patient with a large adenoma associated with virilization. One patient with carcinoma was responsive to ACTH stimulation while the remainder show almost no response to exogenous ACTH. Urinary steroid profiling using gas chromatographic analysis, especially the values for THS and delta 5-pregnenetriol, appears to be a useful method to use in detecting these steroid metabolic characteristics in patients with adrenocortical carcinoma.     

Turkey muscle acylphosphatase: purification and comparative studies

Turkey muscle acylphosphatase is strongly bound to anti-(horse muscle acylphosphatase ) antibodies covalently linked to an agarose resin. This permits use of an affinity chromatography step in the purification, which increased the final yield and allowed us to isolate three different molecular forms of the enzyme. Form 1 is a mixed disulfide between the polypeptide chain and glutathione; form 3 is an S-S dimer of the polypeptide chain present in form 1, while form 2, present in a very low amount, consists of a polypeptide chain quite similar in aminoacid composition to that found in form 1. The three molecular forms show very similar kinetic parameters. The comparison of these molecular forms with those isolated from horse muscle showed similar kinetic properties but different structural features.     

Analysis of apolipoproteins in high density lipoproteins by high performance liquid chromatography

A simple and rapid method for the analysis of apolipoproteins in high density lipoprotein (HDL) by high performance liquid chromatography (HPLC) has been developed (Kinoshita et al. (1983) J. Biochem. 94, 615-617). With this method, using a sodium phosphate buffer containing 0.1% sodium dodecyl sulfate (SDS) as an eluent, apolipoproteins can be analyzed from a very small amount of HDL fraction without delipidation using organic solvents. Separation profiles of apolipoproteins by this method were examined using several techniques. The elution pattern monitored by A280 can give precise quantitative as well as qualitative information about size-distribution of apolipoproteins, except for the apo C group. Moreover, separation of apo E from apo A-I was found to be improved by column elongation.     

Specific cytolysis of fresh tumor cells by an autologous killer T cell line derived from an adult T cell leukemia/lymphoma patient

Cytotoxic T cells (Tc) derived from one patient with adult T cell leukemia/lymphoma (ATL) killed fresh autologous lymphoma cells in vitro. The Tc were induced from peripheral blood leukocytes (PBL) of this patient during remission by multiple in vitro stimulations with an autologous ATLV-bearing cell line (ILT) that was previously established by cloning of PBL in the presence of interleukin 2 (IL 2). PBL from eight other ATL patients were stimulated in the same manner, and responder cells from a patient in remission also showed cytotoxicity specific for ATL virus (ATLV)-bearing cells. Fresh lymphoma cells were obtained in relapse and were used as target cells for the autologous Tc induced. They became susceptible to the Tc within 4 hr of in vitro incubation, and their susceptibility increased with incubation time for at least 12 hr. ATLV antigens on the cell surface of these lymphoma cells, however, were not detected by radioimmunoassay during these incubation periods, but were detectable after 16 hr of incubation. In addition, cytotoxicity against lymphoma cells was completely inhibited by autologous ILT cells used as "cold" target competitor cells. These findings indicate that the target antigen of the Tc was expressed on both autologous ILT cells and lymphoma cells, and it may be different from ATLV antigens detected by serologic methods. In addition, the data suggested allogeneic restriction of the Tc in that the preferentially killed allogeneic ATLV-bearing cells share several HLA antigens.     

Phorbol myristate acetate-stimulated release of cyclooxygenase products in rat pleural cells: derivatization of prostaglandins with 9-anthryldiazomethane for fluorometric determination by high performance liquid chromatography

White cells were collected from the wash of rat pleural cavity after exsanguination. The incubation mixture of the pleural cells with 1 microM phorbol myristate acetate (PMA) was extracted with acidified ethanol and purified with a Sep-pak C18. The resultant fraction containing prostaglandins (PG) and thromboxane (TX) was allowed to react with 9-anthryldiazomethane (ADAM). After removing contaminants and degraded reagent by silica gel Sep-pak, samples were applied to reversed phase high performance liquid chromatography of octadecylsilyl silica gel and monitored by a fluorescent detector. ADAM derivatives of the authentic PGD2, PGE2, PGF2 alpha, 6-keto-PGF1 alpha, 6-keto-PGE1, TXB2, 15-keto-PGE2, 13,14-dihydro-15-keto-PGF2 alpha and 13,14-dihydro-15-keto-PGE2 showed linear regression lines of peak heights within a range of 0.5-25 ng. By using this method PGD2, 6-keto-PGF1 alpha and TXB2 were detected in the incubation mixture of the rat pleural cells with PMA. The result clarified the origin of these PGs and TX found in the exudate of rat pleurisy induced by PMA. ADAM method for HPLC with a help of clean-up by Sep-pak could be a useful tool for detection of a series of arachidonate metabolites in biological materials.