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PubMed1963

DENATURATION AND RENATURATION OF MALIC DEHYDROGENASE IN A CELL-FREE EXTRACT FROM A MARINE PSYCHROPHILE.

Burton, Sheril D. (Oregon State University, Corvallis), and Richard Y. Morita. Denaturation and renaturation of malic dehydrogenase in a cell-free extract from a marine psychrophile. J. Bacteriol. 86:1019-1024. 1963.-Malic dehydrogenase from a marine psychrophilic vibrio (PS 207) was found to be heat-sensitive at 30 C, the maximal growth temperature for the organism. Initial denaturation was reversible, with maximal renaturation occurring when the denatured enzyme was slowly cooled in the presence of mercaptoethanol, reduced nicotinamide adenine dinucleotide, and malate. No renaturation occurred when these compounds were added after slow cooling, or when the renaturation mixture was rapidly cooled. Mercaptoethylamine, cysteine, glutathione, or mercaptoacetic acid could not replace mercaptoethanol. The kinetics of denaturation and renaturation suggest the presence of several malic isozymes each with different heat labilities, or that these processes are occurring in several distinct steps.

Bacteria↗

PubMed1985

Cysteamine effects on monoamines, dopamine-beta-hydroxylase and the hypothalamic-pituitary axis.

Cysteamine (beta-mercaptoethylamine, MEA) is a naturally occurring sulfhydryl compound that depletes pituitary PRL, causes a reduction in brain and gut somatostatin (SRIF), and suppresses norepinephrine (NE) and epinephrine (EPI) synthesis by inhibition of dopamine-beta-hydroxylase (DBH). SRIF inhibits GH and TSH secretion, whereas, NE and EPI facilitate their release. The objectives of this investigation were to: (1) determine the dose-response and time course of DBH inhibition by MEA in vivo and in vitro, and correlate these findings with MEA tissue levels and (2) assess the function of SRIF and NE/EPI in regulation of episodic GH and TSH secretion using MEA. Animals were administered MEA (75-300 mg/kg, s.c.) and hypothalamic levels of dopamine (DA), NE, EPI, serotonin (5-HT) and MEA were measured by high-performance liquid chromatography (HPLC) and electrochemical detection. DBH activity was measured in vitro after exposure to MEA +/- N-ethylmaleimide (NEMI). Chronically cannulated rats were administered MEA (100 or 300 mg/Kg) and serial blood samples were removed in undisturbed animals, and after 30 min swimming stress. Cannulated rats with bilateral lesions of the ventromedial/arcuate nuclei (VMN/ARC) were administered MEA (150 mg/kg). MEA caused a dose-related decrease in NE/EPI nd in increase in DA at doses greater than or equal to 150 mg/kg. Tissue MEA was highest at 4 h (679 +/- 64 pM/mg tissue), but still measureable after 24 h. MEA inhibited DBH in vitro (95% inhibition at 10(-3) M); NEMI blocked inhibition. Stress-induced GH supression and corticosterone release were partially blocked by a low dose of MEA (100 mg/kg).(ABSTRACT TRUNCATED AT 250 WORDS)

3,4-Dihydroxyphenylacetic Acid↗

PubMed1993

Quantitative and qualitative characteristics of colony-forming unit-erythroid colonies in myelodysplastic syndrome patients.

The microcytofluorometrical method was applied to determine the relative hemoglobin (Hb) content in the bone marrow colony-forming unit-erythroid (CFU-E) colonies from 6 patients with myelodysplastic syndromes (MDS) and 10 healthy subjects. This method relies on a photochemical reaction, by which intracellular Hb is converted into fluorescent porphyrin using a 0.2 M mercaptoethylamine solution (an SH donor) and violet light (lambda = 405 nm). The relative Hb content was determined as a function of the intensity of emitted porphyrin fluorescence. The number of colonies identified by porphyrin fluorescence was smaller in MDS patients than in normal subjects. The relative Hb content was also lower in MDS patients than in normal subjects. In addition, the coefficient of variation of the relative Hb content in the CFU-E colonies was larger in MDS patients than in normal subjects. These findings suggest that colonies with low relative Hb content undergo impaired erythropoiesis and that the CFU-E colonies undergoing the impaired erythropoiesis are mixed with CFU-E colonies showing normal erythropoiesis in the bone marrow of MDS patients.

Adult↗

PubMed1997

The myelotoxicity of chloramphenicol: in vitro and in vivo studies: I. In vitro effects on cells in culture.

1 Chloramphenicol is used extensively in non-industrialized countries for the treatment of life-threatening infections because it is cheap and effective, despite its known hemotoxicity and linkage to fatal aplastic anaemia. It is important to define the mechanism of toxicity so that means can be devised to ameliorate the toxic effects in order to produce safer usage. 2 Chloramphenicol, at concentrations from 5 mM to 2 mM initiated apoptosis in dividing cells from a monkey kidney-derived cell line and in haematopoietic progenitor cells from human neonatal cord blood. 3 Growth of progenitor cells was suppressed at concentrations of chloramphenicol which would be considered less than therapeutic during patient treatment. 4 These effects could be ameliorated in progenitor cells by co-culture with the antioxidant mercaptoethylamine and in monkey kidney cells by co-culture with vitamin C. 5 This is the first report of apoptosis in chloramphenicol toxicity and suggests a possible link between a metabolic event i.e. the production of free radicals; a morphological effect, apoptosis; and a clinical effect, bone marrow suppression and aplastic anaemia.

Animals↗

PubMed1988

A comparison of the effectiveness of cysteamine and phosphocysteamine in elevating plasma cysteamine concentration and decreasing leukocyte free cystine in nephropathic cystinosis.

Cysteamine (beta-mercaptoethylamine, MEA) is currently used to treat children with nephropathic cystinosis. In this study MEA was compared to phosphocysteamine (MEAP), a phosphorothioester that tastes and smells better than MEA, with respect to its ability to elevate plasma MEA and deplete leukocytes of cystine. Studies were performed in six children with nephropathic cystinosis ranging in age from 2 to 10 yr. After equimolar oral doses of either MEA or MEAP plasma cysteamine was determined at various times for 6 h. MEA was determined by sodium borohydride reduction followed by high-performance liquid chromatography separation and electrochemical detection. Leukocyte cystine was measured before and 1 and 6 h after drug administration. Peak plasma MEA was obtained 30 min to 1 h after a dose and was not significantly different when MEA (48.6 +/- 10.7, mean +/- SD) or MEAP (54.1 +/- 20.2) was given. Significant plasma MEA concentrations were seen as early as 15 min after an oral dose, indicating rapid absorption. Analysis of vomitus indicated that hydrolysis of the phosphate group of MEAP occurs in the stomach. The percent decrease in leukocyte cystine content obtained with MEA administration (61.9%) was not significantly different from the decrease observed when MEAP was administered (65.3%). MEA and MEAP appear to be equally effective in their cystine-depleting properties.

Administration, Oral↗

PubMed1981

Somatostatin in rat tissues is depleted by cysteamine administration.

Administration of cysteamine (mercaptoethylamine) induces in rats severe perforating duodenal ulcers. Because the ulcerogenic properties of cysteamine are markedly reduced by treatment with somatostatin, we considered the possibility that cysteamine-induced duodenal ulcer might be mediated by depletion of tissue somatostatin, and thereby of its paracrine influences on gastrin and gastric acid secretion. To test this hypothesis, we measured the concentration of immunoreactive somatostatin (IR-somatostatin) in stomach and duodenal mucosa at intervals after administration of a single ulcerogenic dose (30 mg/kg by stomach tube). IR-somatostatin in these tissues fell rapidly to reach a minimum at 4 h (stomach 31%, duodenum 60% of control respectively). IR-somatostatin in hypothalamus and pancreas decreased gradually to a minimum at 7 h. Another duodenal ulcerogen, propionitrile (10 mg/100 g bw, s.c.) which is more toxic than cysteamine, and several stressful procedures including ether anesthesia, restraint and s.c. formalin did not lower stomach or duodenal IR-somatostatin. Gut, pancreas and hypothalamic VIP levels were not influenced by cysteamine. These findings suggest that cysteamine is a relatively specific depletor of tissue somatostatin. Because blood levels of somatostatin fell, and only trivial amounts of the peptide were found in the stomach lumen after cysteamine administration, it appears likely that this agent acts at the cellular level to cause breakdown of preformed somatostatin and/or to acutely reduce its synthesis.

Animals↗

PubMed1984

Cysteamine inhibition of bovine pituitary secretory granule prolactin immunoassayability and release.

Cysteamine [2-mercaptoethylamine (CySH)] displays a variety of neuroendocrine effects, the most potent being the depletion of immunoassayable tissue PRL. The present study used bovine adenohypophysial secretory granules to characterize this inhibition of measurable hormone (assayability). CySH decreased assayability in a dose-dependent manner at pH 7.4, with 50% inhibition observed close to 2 mM. Maximal inhibition was found between pH 6.0 and 6.5, whereas diminished (or no) inhibition occurred under alkaline conditions, depending on the buffer. In contrast, reduced glutathione (without CySH) increased assayability at pH 8-8.5, had little potency near neutrality, and inhibited assayability under acidic conditions. Electrophoretic studies under nondenaturing conditions demonstrated that CySH exposure of standard PRL resulted in additional charged species. With granules, CySH markedly decreased the staining of the major PRL band, no new bands were evident, and this effect was abolished by glutathione. Sodium dodecyl sulfate-electrophoretic patterns indicated that CySH resulted in higher apparent mol wt species of granule and standard PRL. This effect was nullified by mercaptoethanol. Never was there evidence for species smaller than monomeric. Depletion of PRL might involve interference with the conversion from oligomeric storage PRL to assayable PRL; 44-fold increases in PRL oligomer immunoactivity after alkali and thiol treatment were reduced to 6-fold increases when CySH was present. Reactions involved in production of assayable hormone appear relevant to secretion, since CySH also inhibited PRL release from granules, with maximal inhibition occurring under acidic conditions. Thus, CySH may be useful in investigating the physicochemical properties of tissue PRL and may also represent an approach to treatment of hyperprolactinemic states.

Animals↗

PubMed1987

Cysteamine-induced decrease of somatostatin in rat brain synaptosomes in vitro.

The mechanism of somatostatin depletion induced by cysteamine [2-mercaptoethylamine (CySH)] was studied in isolated nerve endings (synaptosomes) from rat brain in vitro. A dose-dependent reduction of somatostatin-like immunoreactivity (SLI) was observed which reached its maximal extent (41%) at a concentration of 300 microM CySH after 1-5 min. There was no release of somatostatin into the incubation medium. CySH at concentrations of up to 10 mM did not interfere in the RIA. Among a variety of compounds, structurally related to CySH 4-aminothiophenol, 2-aminothiophenol and N,N-dimethylaminothiol exhibited the highest efficacy in decreasing somatostatin (60%, 50%, 30%, respectively, at 10 mM and 10 min). The disulfide form of CySH cystamine and dimercaprol resulted in about 15% reduction after 10-min incubation, whereas taurine, alanine, cysteine, and mercaptoethanol were inactive. A saturable, sodium-dependent uptake process was found for the disulfide form of [35S]CySH cystamine [Michaelis-Menten constant (Km) = 18.6 microM, maximum velocity (Vmax) = 2.3 nmol/mg protein X 3 min) which was inhibited by cysteine (87% at 1 mM). [35S]CySH, at concentrations of 20 microM or less, was not stable in buffer solution. It underwent considerable nonenzymatic conversion into its dimeric form (60% at 37 C and 3 min), however it exhibited the same kinetic data for its uptake. Size exclusion HPLC of purified hypothalamic synaptosomes revealed a major SLI peak coeluting with synthetic somatostatin-14 and two minor peaks representing somatostatin-28 and a 13,000 mol wt protein. The three molecular forms of somatostatin were reduced to varied extent by CySH (somatostatin-14 by about 70%, somatostatin-28 by 15%, and the high mol wt form by 30%). Our experiments suggest that high affinity uptake of CySH may precede its action in decreasing somatostatin levels. Increased release or inhibition of synthesis of somatostatin have been excluded as possible mechanisms. It is suggested that SLI is equally affected in nerve endings and in perikarya.

Animals↗

PubMed1988

[Effect of cysteamine on vocalization responses by arterial algogenics in guinea pigs].

Effects of cysteamine (2-mercaptoethylamine) on vocalization responses, a parameter of nociceptive response, was studied in conscious guinea pigs. Intra-arterial injection of bradykinin (3 micrograms), acetylcholine (300 micrograms), capsaicin (3 micrograms) and vanillyl n-nonoylamide (3 micrograms, VNA) induced severe vocalization responses. Three hours after an administration of cysteamine (300 mg/kg, s.c.), a significant suppressive effect was observed for bradykinin and acetylcholine-evoked vocalization. A weak suppressive effect appeared in capsaicin and VNA-evoked vocalization, but there were not statistically significant changes on vocalization counts when compared with the value of the saline-treated animals. However, consecutive pretreatment of guinea pigs with VNA led to a complete suppression of capsaicinoids-evoked vocalization only. Cysteamine completely suppressed bradykinin, acetylcholine, capsaicin and VNA-evoked vocalization responses in VNA desensitized guinea pigs. These findings suggest that the mechanisms of bradykinin and acetylcholine-evoked vocalization response differ from that of capsaicinoids.

Acetylcholine↗

PubMed1985

Rapid depletion of somatostatin in isolated mouse pancreatic islets after treatment with cysteamine.

Cysteamine (CSH; beta-mercaptoethylamine) is known to deplete pancreatic somatostatin without affecting the insulin or glucagon content. It may therefore be useful for studies of intra-islet regulation of hormone release. In the present study injection of CSH (60 mg/kg body weight) to mice decreased the somatostatin content of their isolated pancreatic islets to 50% in 1 h and 30% in 4 h as compared to islets of non-injected controls. Exposure of isolated mouse islets to CSH (100 micrograms/ml) for either 0.5 h followed by incubation in control medium for 3.5 h, or continuously for 4 h, decreased the somatostatin content to about 40% of the controls. There was no change in the islet content of insulin or glucagon. Islets pretreated with CSH (100 micrograms/ml) for 1 h in vitro showed a decreased glucose stimulation of both oxygen consumption and glucose oxidation. Measurements of insulin release after a similar preincubation of the islets indicated an increased basal release and an attenuated glucose stimulation. It is concluded that CSH rapidly decreases islet somatostatin both in vivo and in vitro. This depletion may lead to a loss of tonic inhibition by islet somatostatin on basal insulin release. It is, however, more plausible that the increased basal insulin release reflected a direct effect of CSH on the islet beta-cells.

Animals↗

PubMed2000

Inhibitory effects of WR-2721 and cysteamine on tumor initiation in mammary glands of pregnant rats by radiation.

We evaluated the effect of WR-2721 [S-2-(3-aminopropylamino)-ethylphosphorothioic acid] and cysteamine (2-mercaptoethylamine) on the development of radiation-induced mammary tumors in rats. Pregnant rats were treated with WR-2721 or cysteamine 30 min prior to whole-body irradiation with gamma rays from a (60)Co source at a dose of 1.5 or 2.6 Gy. Additional pregnant rats were given saline and then exposed to gamma rays at a dose of 0, 1.5 or 2.6 Gy as a control. All rats were implanted with pellets of diethylstilbestrol, a tumor promoter, 1 month after termination of nursing and were observed for 1 year to detect palpable mammary tumors. No mammary tumors developed in the saline-injected nonirradiated rats. However, when rats were irradiated with 1.5 or 2. 6 Gy after saline treatment, the incidence of mammary tumors was high (71.4 and 92.3%, respectively). Administration of WR-2721 or cysteamine prior to irradiation with 1.5 Gy significantly decreased the tumor incidence (23.8 and 20.8%, respectively). Tumor prevention by either agent was less effective at the higher dose. The appearance of the first mammary tumor occurred later in rats treated with WR-2721 or cysteamine than in the control rats. An increasing rate of adenocarcinoma in the control group was observed with increasing dose from 1.5 Gy up to 2.6 Gy. However, the development of adenocarcinoma did not increase after pretreatment with WR-2721 or cysteamine in rats irradiated with 2.6 Gy. Many of the mammary tumors that developed in the control rats were of the ER(+)PgR(+) type. Administration of WR-2721 produced no tumors of the ER(+)PgR(+) type. Cysteamine treatment increased the development of ER-negative tumors. The serum concentration of progesterone was significantly higher in rats treated with WR-2721 or cysteamine than in the control rats. On the other hand, the estradiol-17beta concentration was reduced by treatment with WR-2721, but not significantly compared to the control. WR-2721 and cysteamine had no effect on the prolactin concentration of the irradiated rats. The results suggest that administration of WR-2721 or cysteamine prior to the irradiation has a potent preventive effect on theinitiation phase during mammary tumorigenesis.

Adenocarcinoma↗

PubMed1988

Effects of cysteamine on blood pressure: possible mediation through vasopressin release.

Cysteamine (beta-mercaptoethylamine, CSH) has been reported to have various effects on the neuroendocrine system. Reports indicate CSH decreases pituitary oxytocin (OT) without affecting pituitary vasopressin (VP). However, preliminary studies from our laboratory strongly indicate that CSH has an effect on VP release. Experiments were conducted with dibenzyline-treated, urethane-anesthetized, male Sprague-Dawley (SD) rats. Rats were injected with 4 mU of standard VP and 4 mg/100 g of CSH. Administration of VP resulted in an increase in mean arterial pressure (MAP) of 23.5 +/- 3.2 mm Hg. Administration of CSH resulted in a consistent, immediate decrease in MAP of 13.0 +/- 2.0 mm Hg prior to an increase of 21.0 +/- 2.6 mm Hg. The effects due to VP and CSH were strikingly different; the CSH-induced MAP rise took longer to peak and to return to baseline. Both the VP- and CSH-induced MAP rise were markedly inhibited by a prior administration of a specific VP antagonist d(CH2)5[Tyr(Me)]AVP. In addition, the typical increase in MAP observed in SD rats following CSH administration was substantially reduced when the same dose was administered in homozygous diabetes insipidus (HODI) rats. The data presented here strongly suggest that CSH-induced MAP elevation is due to the release of VP from the pituitary gland.

Animals↗

PubMed1975

Thiol adducts of ethacrynic acid: a correlation of the rate of liberation of ethacrynic acid with the onset and magnitude of the diuretic response.

It is thought that a derivative of ethacrynic acid (EA) must possess an intact alpha, beta-unsaturated ketone group in order to be capable of eliciting a diuretic response. The 2,3-dimercapto-1-propanol and the cysteine adducts of ethacrynic acid lack such a functional group and still have diuretic activity, especially the cysteine adduct. An in vitro study showed that various thiol adducts of EA liberate EA and the accompanying thiol at a rate that is primarily dependent on the nature of the functional groups present in the thiol portion of the adduct. When the thiol adducts of EA were injected into dogs, the cysteine and mercaptoethylamine hydrochloride adducts which rapidly release EA under specific in vitro conditions were as effective as EA in producing a diuretic response. The onset of action was also similar to that of EA. The thiosalicylic acid adduct of EA releases the accompanying thiol at an intermediate rate in vitro and was less effective than EA in a small dose (3.3 mumol/kg) and the peak response to it was slower to develop. Other adducts that release EA and the accompanying thiol slowly in vitro either produce a very weak response which takes considerable time to develop or are completely devoid of diuretic activity. Thus, the onset and magnitude of the diuretic response produced by various thiol adducts of EA (with the possible exception of the cysteine adduct) are governed primarily by the rate of in vivo release of EA.

Analysis of Variance↗

PubMed1992

Inhibition of ubiquitin-protein ligase (E3) by mono- and bifunctional phenylarsenoxides. Evidence for essential vicinal thiols and a proximal nucleophile.

Trivalent arsenoxides bind to vicinal thiol groups of proteins. We showed previously that the simplest trivalent arsenoxide, inorganic arsenite, inhibits ubiquitin-dependent protein degradation in rabbit reticulocyte lysate (Klemperer, N.S., and Pickart, C.M. (1989) J. Biol. Chem. 264, 19245-19242). We now show that, relative to arsenite, phenylarsenoxides are 10-165-fold more potent inhibitors of protein degradation in the same system (K0.5 for inhibition by p-aminophenylarsenoxide was 3.5-20 microM, depending on the substrate). In the ubiquitin-dependent proteolytic pathway, covalent ligation of ubiquitin to protein substrates targets the latter for degradation. In certain cases, specificity in ubiquitin-substrate conjugation depends critically upon the properties of ubiquitin-protein ligase or E3. Among other effects, p-aminophenylarsenoxide decreased the steady-state level of ubiquitinated human alpha-lactalbumin; this is a substrate which is acted upon directly by ubiquitin-protein ligase-alpha (E3-alpha). This finding suggests that phenylarsenoxides (unlike arsenite) inhibit E3. Several other lines of evidence confirm this conclusion. 1) A complex of E3-alpha and the 14-kDa ubiquitin-conjugating (E2) isozyme binds to phenylarsenoxide-Sepharose resin, with the E3 component of the complex mediating binding. 2) p-Aminophenylarsenoxide inhibited isolated E3 (K0.5 approximately 50 microM); inhibition was readily reversed by addition of dithiothreitol (which contains a competing vicinal thiol group), but not by beta-mercaptoethylamine (a monothiol). 3) A bifunctional phenylarsenoxide (bromoacetylaminophenylarsenoxide) rapidly and irreversibly inactivated E3; bromoacetyl aniline, which lacks an arsenoxide moiety, did not inhibit E3. These results suggest that E3 possesses essential vicinal thiol groups and that there is a reactive nucleophile proximal to the vicinal thiol site. The bifunctional phenylarsenoxide should be a useful tool for probing the relationship between structure and function in E3. As expected from prior results with arsenite, p-aminophenylarsenoxide was also a potent inhibitor of the turnover of ubiquitin-(human) alpha-lactalbumin conjugates.

Animals↗

PubMed1991

[Qualitative and quantitative evaluation of CFU-E colonies in myelodysplastic syndromes].

Hemoglobin (Hb) synthesis in colony-forming-unit erythroid (CFU-E) colonies from the bone marrow of 10 normal subjects and 6 patients with myelodysplastic syndromes (MDS) was studied using microcytofluorometry. Hb content was determined utilizing a photochemical reaction in which the intracellular Hb is converted into fluorescent porphyrin. Briefly, the CFU-E in plasma clots was assayed according to Tepperman's method. Bone-marrow mononuclear cells were then dispensed onto petri dishes containing the plasma clots. The cells in plasma clots were cultured for seven days and then air-dried. The samples were then fixed with pure methanol. Thereafter, the dishes were exposed to ultraviolet light (lambda = 405 nm) in the presence of an SH-donor (0.2 M mercaptoethylamine hydrochloride). The intensity of porphyrin fluorescence was measured using a microcytofluorometer. When the photochemical reaction was carried out for 50 min, the intensity of fluorescence was found to be proportional to the mean corpuscular Hb (MCH) level, suggesting that the intracellular Hb level can be determined as a function of the intensity of this fluorescence. The number of CFU-E colonies in MDS patients was smaller than that in normal subjects. In addition, intracolony Hb level was markedly lower in the MDS cases than in normal subjects. Our findings suggest that the maturation process of CFU-E is impaired in MDS patients.

Adult↗

PubMed1991

Inhibition of cell colony formation by selenite: involvement of glutathione.

Selenium is an essential trace element that has been shown to have anticarcinogenic activity. One mechanism that has been proposed for this activity is a cytotoxic effect of selenium on tumor cells. As a means of assessing its cytotoxicity, we have examined the effect of selenite on tumor cell viability, using as an assay the ability of the cells to form colonies. We have found that brief exposure of HeLa cells to micromolar concentrations of selenite resulted in significant inhibition of colony formation, indicating that this is an assay for selenite cytotoxicity that is more sensitive than those that have been employed previously. In order to investigate the involvement of cellular glutathione in selenite cytotoxicity, we treated cells with buthionine sulfoximine (BSO) before selenite exposure. This treatment, which resulted in a 7-fold reduction in the level of intracellular glutathione, also caused a significant decrease in the inhibitory effect of selenite on colony formation. However, when cells were exposed to selenite that had previously been reacted with glutathione, the BSO-induced decrease in cytotoxicity was eliminated. In contrast, reaction of selenite with other sulfhydryl compounds, such as cysteine and mercaptoethylamine, did not restore its potency in BSO-treated cells. The simplest explanation for these results is that, for selenite to exert its inhibitory effect, it must react with intracellular glutathione to form the selenodiglutathione derivative.

Buthionine Sulfoximine↗

PubMed1989

[Megakaryocyte ploidy in patients with myelodysplastic syndrome--by microcytofluorometry with DAPI staining after removal of Wright-Giemsa staining].

In the myelodysplastic syndrome (MDS) patients the ploidy distribution of megakaryocyte DNA was rarely reported. We applied DAPI (4',6-diamidino-2-phenylindole) staining for measuring nuclear DNA content in megakaryocytes which have been morphologically identified on the Wright-Giemsa stained smear in 8 normal subjects and 12 MDS patients. Briefly, megakaryocytes morphologically examined on a Wright-Giemsa stained smear were photographed, and were located. We then removed the Wright-Giemsa stains by immersing it in 50% ethanol, 37 degrees C for 1 hour and 100% methanol, 37 degrees C for 1 hour. The DAPI staining was performed in DAPI solution (DAPI 0.01 mg/ml, pH 7.4 Tris-EDTA-2 Na buffer solution and 0.01 mol 2-mercaptoethylamine hydrochloride were mixed at the ratio of 0.5: 98.5: 1.0) for more than 30 min. The amount of nuclear DNA in the megakaryocyte previously identified was measured by cytofluorometry. The population of megakaryocyte in the normal subjects was the largest in the 16N, and in the 10 cases of the 12 MDS patients was the largest in the 8N, in the other 2 cases was the largest in the 4N. These results represent the development of the megakaryocyte nucleus in the MDS patients may be disturbed.

Adult↗

PubMed1989

Highly sensitive enzyme immunoassay system: determination of nervous system-related proteins at the level of single neurons.

We established a highly sensitive sandwich-type enzyme immunoassay system which was applicable to the measurement of macromolecular antigens at the level of single cells. The assay system was composed of a solid-phase (polystyrene ball, 3.2 mm in diameter) with immobilized monospecific antibodies and the same antibodies labeled with beta-D-galactosidase from Escherichia coli. The antibody IgG was purified from antisera by means of immunoaffinity chromatography. It was then digested with pepsin to obtain the F(ab')2 fragments of antibodies. The antibody F(ab')2 fragments were immobilized noncovalently on polystyrene balls. Other aliquots of antibody F(ab')2 fragments were reduced with 2-mercaptoethylamine, and the resulting Fab' fragments (which contained one thiol group in each molecule) were coupled with beta-D-galactosidase in a two-step procedure by use of N,N'-o-phenylenedimaleimide. The assay system was highly sensitive, and amol (1 x 10(-18) mol) levels of antigens were detected. The high sensitivity of the present assay was the result of the following factors. (1) A very small amount (1 amol) of the galactosidase was determined using conventional laboratory instruments by measuring its activity with a fluorogenic substrate, 4-methylumbelliferyl-beta-D-galactoside. (2) The nonspecific binding of labeled antibodies to the polystyrene ball solid-phase (blank value of the assay) was low. (3) Antibodies could be coupled selectively with galactosidase, which brings about an increase in the efficiency of the immunoreaction of the labeled antibodies and a decrease in the nonspecific binding of the label on the solid-phase in the assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗