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PubMed1976

Ultrastructural cytochemistry of p-N,N-dimethylamino-beta-phenethylamine (DAPA) oxidation reactions.

The ultracytochemical localization of amine oxidase (AO) activity is demonstrated with a new substrate, p-N,N-dimethylamino-beta-phenethylamine (DAPA). DAPA was designed to yield a stronger reducing agent on oxidation by monoamine oxidase (MAO) than is obtained from the MAO substrate, tryptamine, upon oxidation. Thus MAO and possibly other oxidase(s) can be demonstrated with DAPA and the tetrazolium salt, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phthalhydrazidyl) tetrazolium chloride (BSPT). The latter is a nonosmiophilic tetrazolium salt which is reduced to an osmiophilic formazan. In addition, DAPA itself demonstrates AO activity ultracytochemically with and without BSPT. We speculate that either oxidative polymerization of DAPA or Schiff's base formation with protein after aldehyde formation is responsible for the latter reaction, which is made permanent for ultracytochemical localization by osmication at a later step. DAPA oxidation reaction products are demonstrated in guinea pig kidney, specifically in the endoplasmic reticulum, nuclear envelope and mitochondrial outer compartments and cristae. Differences in reaction product characteristics and localization in relation to formaldehyde fixation and the localization of reaction product in mitochondrial cristae, as well as outer compartments, suggest that DAPA oxidation is mediated through one or more MAOs and possible other oxidases.

Animals↗

PubMed1977

An improved method for the histochemical localization of glucose-6-phoshate dehydrogenase in animal and plant tissues.

An improved procedure is reported for the histochemical localization of glucose-6-phosphate dehydrogenase in fresh-frozen sections of animal and plant tissues. Used in this procedure are an incubation medium and an inert high-molecular-weight polyvinyl alcohol as a stabilizer to check enzyme diffusion. The incubation medium consists of 10 mg each of nicotinamide adenine dinucleotide phosphate and nitroblue tetrazolium chloride and 60 mg of glucose-6-phosphate per 20 ml of 22% polyvinyl alcohol solution in 0.2 M Tris-maleate buffer, pH 7.2. Use of this medium has given consistent results with the tissues tested. Because the use of an electron-transfer agent in the incubation medium often resulted in enzyme diffusion, its use in glucose-6-phosphate dehydrogenase localization is considered undersirable. Incubation time of 15 to 30 min for animal tissue and 20 to 45 min for plant tissue of 37degreesC was found to be optimal for producing the characteristic blue formazan at the reaction sites.

Adrenal Cortex↗

PubMed1977

Cytochemical demonstration of 5-formyl tetrahydrofolate cyclodehydrase and 5,10-methenyl tetrahydrofolate cyclohydrolase activity.

The enzyme 5-formyl tetrahydrofolate cyclodehydrase plays an important role in the conversion of 5-formyl tetrahydrofolate to 5,10-methenyl tetrahydrofolate. A second enzyme, cyclohydrolase, converts 5,10-methenyl tetrahydrofolate to 10-formyl tetrahydrofolate. These folate derivatives play a significant part in the biosynthesis of purines. A method has been devised for the cytochemical demonstration of 5-formyl tetrahydrofolate cyclodehydrase and 5,10-methenyl tetrahydrofolate cyclohydrolase activity which uses 5-formyl tetrahydrofolate or 5,10-methenyl tetrahydrofolate as substrate respectively, blocking possible interferences by other enzymes, and allows the nonenzymatic reduction of nitro-blue tetrazolium by 5,10-methenyl tetrahydrofolate formed by the action of the cyclodehydrase on the substrate 5-formyl tetrahydrofolate, and by 10-formyl tetrahydrofolate formed by the action of cyclohydrolase on the substrate 5,10-methenyl tetrahydrofolate, thus revealing intracellular sites of enzyme activity. The methods appear to show only intracellular localization of the blue formazan deposits of reduced tetrazolium. The distribution of positivity in cells of human blood and bone marrow is described.

Aminohydrolases↗

PubMed1977

Ultracytochemical demonstration of monoamine oxidase activity in nervous and non-nervous tissue of the rat.

A method is described for the ultrastructural localization of the enzyme monoamine oxidase based on the formation of an osmiophilic formazan from the tetrazolium salt (2-(2'-benzothiazolyl)-5-styrl-3-(4'-phthalhydrazidyl) tetrazolium chloride) (BSPT) and using tryptamine as substrate. It was tested in rat heart, liver, adrenal cortex and medulla, kidney, pineal gland, cerebral cortex and cerebellar cortex and was found to provide good ultrastructural preservation, high contrast of the specific, enzyme-dependent precipitate and improved sensitivity. Monoamine oxidase activity was found in the mitochondrial outer compartment, nuclear envelope and endoplasmic reticulum in all organs studied; it was also seen occasionally in plasma membranes of liver and heart. Similar localizations were observed in interstitial cells and capillary endothelial cells. The enzyme-dependent nature of the observed precipitate was verified by control experiments consisting of incubation of overfixed tissue, omission of substrate from the incubation medium, and incubation in the presence of a specific, nonreversible inhibitor, pargyline. These studies confirm and extend the results of cell fractionation studies using liver, demonstrate the widespread distribution of monoamine oxidase in tissues, and suggest that monoamine oxidase plays important functional roles in extraneuronal cells.

Adrenal Glands↗

PubMed1978

The two dimensional structure of tetrazolium-mucopolysaccharide complexes deduced from reactivity studies.

The formation of complexes between mucopolysaccharides and tetrazolium salts has been studied both by turbidimetry and nephelometry. The technique of laser nephelometry allows the detection of colloidal aggregates in systems which may, by turbidimetric methods, be ambiguous. The results indicate that binding of tetrazolium salts to polyanions can result in soluble as well as insoluble complexes; the monotetrazolium salts form soluble complexes and the ditetrazolium compounds form insoluble complexes. The insoluble complexes are stable at relatively low pH, and are disrupted during reduction to the formazan. Complex formation is decreased at high ratios of heparin to tetrazolium, and divalent cations, even at high concentration, do not precipitate mucopolysaccharides. It is concluded that stable ionic interactions with spatial charge separation are responsible for the cross linking of the mucopolysaccharides and the formaiton of large insoluble aggregates.

Chemical Phenomena↗

PubMed1981

Improved contrast in cytochemistry of dehydrogenases by scanning transmission electron microscopy.

A scanning transmission electron microscope (STEM) was used to examine ultrathin sections of rabbit white skeletal muscle. Lactic dehydrogenase (LDH) activity was localized in the tissue using the tetra-nitro blue tetrazolium (TNBT) method. For most specimens postfixation was omitted in order to avoid reoxidation and solubilization of the formazan by osmium tetroxide. The STEM image revealed sufficient contrast of the intracellular structures and apparently electron-dense reaction product in the sarcoplasmic reticulum and mitochondria. Substantially less contrast was obtained when the same areas were observed by conventional transmission electron microscopy (CTEM). In material postfixed with osmium tetroxide, although the tissue contrast was improved, the TNBT reaction product was focally leached out, exhibiting lower contrast than in unosmicated sections. These results indicate that the fine structural visualization of dehydrogenases with TNBT, the STEM technique as used in the present study is superior to that obtained by CTEM.

Animals↗

PubMed1984

Histochemical localization of NADP-dependent dehydrogenase activity with four different tetrazolium salts.

The properties of the four most commonly used tetrazolium salts, neotetrazolium, nitro blue tetrazolium (nitro-BT), tetranitro-BT, and 2-(2-benzothiazolyl-3-(4-phthalhydrazidyl)-5-styryl-te trazolium (BPST), have been compared for their effects on the localization of nicotinamide adenine dinucleotide phosphate (NADP)-dependent dehydrogenases under optimal incubation conditions in cryostat sections of rat liver. Glucose-6-phosphate dehydrogenase has been selected as an example of these dehydrogenases. It was found that the use of nitro-BT and tetranitro-BT, unlike neotetrazolium and BPST, in combination with an exogeneous electron carrier and azide, resulted in localization patterns in agreement with the sites of activity as determined by microchemical techniques. In the absence of an intermediate carrier the localization was very similar to that of NADPH cytochrome c (P450) reductase as demonstrated immunocytochemically. BPST did not properly localize dehydrogenase activity, most probably because of the redistribution of formazan, due to its lack of firm substantivity. Neotetrazolium reduction in nitrogen gave the localization pattern, both in the presence and absence of carrier, of the reductase, suggesting that the transfer of reducing equivalents from the exogenous electron carrier to neotetrazolium proceeds via cellular electron transport systems. The reduction of nitro-BT and tetranitro-BT via intermediate carriers was oxygen sensitive in parenchymal cells, but not in the non-parenchymal liver cells. This oxygen sensitivity could be blocked by azide. With neotetrazolium, oxygen inhibited both carrier-mediated and carrier-independent reactions, effects that were not reversed with azide. Possible mechanisms of action between oxygen, reduced carriers, and tetrazolium salts are discussed.

Animals↗

PubMed1990

Development of a quantitative histochemical method for determination of succinate dehydrogenase activity in autonomic neurons and its application to the study of aging in the autonomic nervous system.

An accurately validated method was developed for quantitative determination of succinate dehydrogenase (EC 1.3.99.1; SDH) activity in individual sympathetic neuron perikarya by microdensitometric measurement of an SDH-nitroblue tetrazolium-derived formazan final reaction product. Optimal incubation medium and reaction conditions were determined for measurement of reaction product in cryostat sections of rat superior cervical and celiac-superior mesenteric ganglia. The Beer-Lambert laws were verified for the ganglion tissue, and microdensitometric measurements (expressed as mean cell density readings; MCDR/min-1), characteristic of the Michaelis-Menten equation, enabled the results to be used for enzyme kinetic determinations of SDH activity. Km and Vmax values were obtained following Hans linear transformation of the readings. Between the ages of 6-24 months no significant variations in Km values were recorded, indicating an unchanged structure for SDH (overall mean Km = 0.083 +/- 0.055 mM). However, in both ganglia there were significant decreases (ranging from 43-54%) in Vmax values for SDH at 24 months. The overall mean Vmax value at 6 months was 4.01 +/- 0.61 (MCDR) and at 24 months was 2.07 +/- 0.76 (MCDR). This suggests that an overall decrease in metabolic activity takes place with age in sympathetic neurons of the rat superior cervical and celiac-superior mesenteric ganglia.

Aging↗

PubMed2006

Select estrogens within the complex formulation of conjugated equine estrogens (Premarin) are protective against neurodegenerative insults: implications for a composition of estrogen therapy to promote neuronal function and prevent Alzheimer's disease.

BACKGROUND: Results of the Women's Health Initiative Memory Study (WHIMS) raised concerns regarding the timing and formulation of hormone interventions. Conjugated equine estrogens (CEE), used as the estrogen therapy in the WHIMS trial, is a complex formulation containing multiple estrogens, including several not secreted by human ovaries, as well as other biologically active steroids. Although the full spectrum of estrogenic components present in CEE has not yet been resolved, 10 estrogens have been identified. In the present study, we sought to determine which estrogenic components, at concentrations commensurate with their plasma levels achieved following a single oral dose of 0.625 mg CEE (the dose used in the WHIMS trial) in women, are neuroprotective and whether combinations of those neuroprotective estrogens provide added benefit. Further, we sought, through computer-aided modeling analyses, to investigate the potential correlation of the molecular mechanisms that conferred estrogen neuroprotection with estrogen interactions with the estrogen receptor (ER). RESULTS: Cultured basal forebrain neurons were exposed to either beta-amyloid(25-35) or excitotoxic glutamate with or without pretreatment with estrogens followed by neuroprotection analyses. Three indicators of neuroprotection that rely on different aspects of neuronal damage and viability, LDH release, intracellular ATP level and MTT formazan formation, were used to assess neuroprotective efficacy. Results of these analyses indicate that the estrogens, 17alpha-estradiol, 17beta-estradiol, equilin, 17alpha-dihydroequilin, equilinen, 17alpha-dihydroequilenin, 17beta-dihydroequilenin, and Delta8,9-dehydroestrone were each significantly neuroprotective in reducing neuronal plasma membrane damage induced by glutamate excitotoxicity. Of these estrogens, 17beta-estradiol and Delta8,9-dehydroestrone were effective in protecting neurons against beta-amyloid25-35-induced intracellular ATP decline. Coadministration of two out of three neuroprotective estrogens, 17beta-estradiol, equilin and Delta8,9-dehydroestrone, exerted greater neuroprotective efficacy than individual estrogens. Computer-aided analyses to determine structure/function relationships between the estrogenic structures and their neuroprotective activity revealed that the predicted intermolecular interactions of estrogen analogues with ER correlate to their overall neuroprotective efficacy. CONCLUSION: The present study provides the first documentation of the neuroprotective profile of individual estrogens contained within the complex formulation of CEE at concentrations commensurate with their plasma levels achieved after an oral administration of 0.625 mg CEE in women. Our analyses demonstrate that select estrogens within the complex formulation of CEE contribute to its neuroprotective efficacy. Moreover, our data predict that the magnitude of neuroprotection induced by individual estrogens at relatively low concentrations may be clinically undetectable and ineffective, whereas, a combination of select neuroprotective estrogens could provide an increased and clinically meaningful efficacy. More importantly, these data suggest a strategy for determining neurological efficacy and rational design and development of a composition of estrogen therapy to alleviate climacteric symptoms, promote neurological health, and prevent age-related neurodegeneration, such as AD, in postmenopausal women.

Adenosine Triphosphate↗

PubMed2002

Pharmacological and ischemic preconditioning of the human myocardium: mitoK(ATP) channels are upstream and p38MAPK is downstream of PKC.

BACKGROUND: These studies investigate the role of mitoK(ATP) channels, protein kinase C (PKC) and Mitogen activated protein kinase (p38MAPK) on the cardioprotection of ischemic (IP) and pharmacological preconditioning (PP) of the human myocardium and their sequence of activation. RESULTS: Right atrial appendages from patients undergoing elective cardiac surgery were equilibrated for 30 min and then subjected to 90 min of simulated ischemia followed by 120 min reoxygenation. At the end of each protocol creatinine kinase leakage (CK U/g wet wt) and the reduction of MTT to formazan dye (mM/g wet wt) were measured. Similar protection was obtained with alpha1 agonist phenylephrine, adenosine and IP and their combination did not afford additional cardioprotection. Blockade of mitoK(ATP) channels with 5-hydroxydecanoate, PKC with chelerythrine, or p38MAPK with SB203580 abolished the protection of IP and of PP. In additional studies, the stimulation of mitoK(ATP) channels with diazoxide or activation of PKC with PMA or p38MAPK with anisomycin induced identical protection to that of IP and PP. The protection induced by diazoxide was abolished by blockade of PKC and by blockade of p38MAPK. Furthermore, the protection induced by PMA was abolished by SB203580 but not by 5-hydroxydecanoate, whereas the protection induced by anisomycin was unaffected by either 5-hydroxydecanoate or chelerythrine. CONCLUSIONS: Opening of mitoK(ATP) channels and activation of PKC and p38MAPK are obligatory steps in the signal transduction cascade of IP and PP of the human myocardium with PKC activation being downstream of the opening of mitoK(ATP) channels and upstream of p38MAPK activation.

Adrenergic alpha-1 Receptor Antagonists↗

PubMed2006

A dietary supplement improves outcome in an experimental influenza model in old mice.

Twenty-month-old Swiss mice were allocated into three groups: (A) control; (B) infected group; and (C) infected but treated with 5 mg of the phytocompound MMT. Mice were infected intranasally with 30 microL of 75 HA viral units. MMT markedly blunted the nasal signs of virus infection and the febrile response. Formazan-positive cells, lung and plasma lipoperoxides, and TNF-alpha in lung tissue increased during viral infection, but improvement was seen in the MMT-treated group (P < 0.05). MMT also normalized SOD, catalase activities, and ascorbic acid and determined a significant decrease of lung but not nasal viral titer, although nasal inflammatory infiltrate dropped significantly. MMT has potential clinical applications with and has an excellent safety profile even in old animals.

Administration, Oral↗

PubMed1986

Staining of eosinophils with nitroblue tetrazolium in patients with chronic granulomatous disease.

The nitroblue tetrazolium (NBT) test is the primary screening test for chronic granulomatous disease (CGD). Neutrophils and other phagocytic cells readily reduce NBT to blue formazan after oxidative stimulation whereas CGD cells remain colorless. In the present study purified eosinophil populations were obtained from CGD patients and normals by exposing peripheral blood to the peptide N-formyl-methionyl-leucyl-phenylalanine and then centrifuged over a discontinuous Percoll gradient. The eosinophils were then incubated in 0.1% NBT (37 degrees C, 15 min) with either phorbol myristate acetate or buffer alone (HEPES with calcium and magnesium). Blue staining characteristic of NBT reduction occurred in the phorbol myristate acetate-stimulated eosinophils of half (4/8) of the CGD patients tested. The staining was most intense between the nuclear lobes of the eosinophil with little staining near the cell periphery. The staining pattern was present in 75.5 +/- 5.3% of the purified eosinophils in those patients in which the phenomenon occurred and was reproducible in the same patients over a 6-month period. Eosinophils from normal individuals tested with NBT and phorbol myristate acetate showed intense staining over the entire cell cytoplasm as did normal neutrophils. Purified CGD eosinophils that did show the staining pattern were not able to produce superoxide (as measured by cytochrome C reduction) when stimulated by phorbol myristate acetate indicating the staining was probably not related to superoxide production. Patients with CGD have a mild eosinophilia (4.6 +/- 0.7% in 11 patients at the National Institutes of Health) and eosinophil staining may account for the small number of positive NBT cells reported in some patients.

Adolescent↗

PubMed1999

Plant polyphenols inhibit benzoyl peroxide-induced superoxide anion radical production and diacylglyceride formation in murine peritoneal macrophages.

Naturally occurring plant polyphenols, which include ellagic acid (EA), tannic acid (TA), caffeic acid (CA), and ferulic acid (FA), were tested for their superoxide anion radical (SOR)-scavenging activities. SOR were produced by interaction of the tumor promoter benzoyl peroxide (BPO) with murine peritoneal macrophages in vitro. The levels of SOR were assessed microscopically by counting the number of formazan-positive cells per 250 cells produced by the reduction of nitro blue tetrazolium. BPO at a concentration of 15 micrograms/1.85 x 10(6) cells/0.5 ml induced maximum formation of SOR in resident and thioglycollate-elicited cells. All the tested polyphenols were able to inhibit the formation of SOR induced by the tumor promoter to a variable degree. Inhibition of BPO-induced SOR formation by polyphenols was in the following order: FA > TA > CA > EA. BPO stimulated the accumulation of diacylglycerol (DAG) in resident and elicited macrophages with concurrent release of choline equivalents from macrophages. Polyphenols inhibited DAG accumulation, which paralleled the inhibition of choline equivalent release. FA was observed to be the most effective and EA the least effective inhibitor of SOR formation, DAG accumulation, and release of choline equivalents. It is likely that inhibition of SOR formation might be due to some interference in the cellular lipid metabolism and phospholipid equivalent deacylation and choline release.

Animals↗

PubMed1992

Sensitive colorimetric bioassays for insulin-like growth factor (IGF) stimulation of cell proliferation and glucose consumption: use in studies of IGF analogs.

We have established two in vitro bioassay systems for quantification of insulin-like growth factor (IGF) bioactivity. The first assay was used to quantitate mitogenic activity and the second was used to quantitate metabolic activity. Both assays use BALB/c 3T3 fibroblasts grown under serum-free conditions; detection of bioactivity in assays was performed colorimetrically and did not require the use of radioisotopes. The mitogenic bioassay, which requires 48 h for detection, quantitates changes in cell number and provides an index for determining the mitogenic activity of growth factors. Changes in cell number were measured by the enzymatic reduction of exogenously added MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide] to MTT-formazan by mitochondrial enzymes, which was directly correlated to cell number. The metabolic bioassay, which requires 22 h for detection, measures glucose consumption by detecting changes from the initial glucose concentration of conditioned medium after addition of various growth factors. When appropriate standards were established for these bioassays, they demonstrated a high level of reproducibility (coefficients of variation were 0.085-0.096 for the mitogenic bioassay and 0.120-0.191 for the metabolic bioassay). Both assays can be performed in 96-well microtiter plates, without the use of radioisotopes, or the limitations of conventional glucose, amino acid, or thymidine incorporation studies. In initial experiments for assay specificity, epidermal growth factor had no measurable effect in either assay. However, IGF-I, IGF-II, and insulin demonstrated effects on both metabolism and mitogenesis. In the case of the mitogenic bioassay, the maximum mitogenic activation by these growth factors was approximately 180% of control, and these factors demonstrated parallel sigmoidal dose-response curves, ranging from 0.02-2 ng/ml for IGF-I and from 2-200 ng/ml for both IGF-II and insulin. In the metabolic bioassay, in contrast to the mitogenic bioassay, insulin showed a dose-response curve whose shape was different from those of IGF-I and IGF-II. IGF-I and IGF-II stimulated glucose consumption in dose-dependent ranges of 0.02-3 ng/ml and 0.4-40 ng/ml, respectively. However, the dose-response effect of insulin was wider, ranging from 0.1-2000 ng/ml. When these assays were used to measure the bioactivity of IGF analogs, a des(1-3)-IGF-I, which has decreased affinity for IGF binding proteins, demonstrated activity equivalent to IGF-I, a [Leu27]IGF-II, which has markedly diminished affinity for the type 1 IGF receptor, exhibited approximately 0.07% of the potency of IGF-I and 1% of the potency of IGF-II.

3T3 Cells↗

PubMed1992

Interleukin-1 is both morphogenic and cytotoxic to cultured rat ovarian cells: obligatory role for heterologous, contact-independent cell-cell interaction.

An increasing body of information now suggests that intraovarian interleukin-1 (IL-1) may play an intermediary role in the ovulatory process. Given that follicular rupture inevitably requires marked tissue remodeling and possibly cell death, we set out to examine the morphogenic potential of IL-1 under in vitro circumstances. Treatment of freshly plated whole ovarian dispersates from immature rats with any one of several batches of IL-1 (10 ng/ml) for up to 96 h produced marked time-dependent morphological alterations, including cellular retraction, rounding, clumping, aggregation, blebbing, swelling, and, ultimately, irreversible detachment. Evidence of (asynchronous) cell death consisted of reduced total cell number, diminished cellular protein content, enhanced cellular release of lactic dehydrogenase, failure to exclude trypan blue, and attenuated reduction of the tetrazolium dye 3-[4,5-dimethylthiazol-2-y]2,5-diphenyltetrazolium bromide to spectrophotometrically detectable formazan. Comparable results were obtained when using established day 4 cultures, arguing against a possible critical action of IL-1 at the time of plating. Dose-response curves revealed IL-1 beta (EC50, 0.2-0.4 ng/ml) to be substantially more potent than IL-1 alpha (EC50, 2.7-2.8 ng/ml). Importantly, the concurrent provision of an IL-1 beta-directed polyclonal antibody yielded complete immunoneutralization of the IL-1 beta effect, arguing against the possible involvement of a non-IL-1 contaminant. An unrelated polyclonal antiserum raised against insulin-like growth factor-I was without effect. IL-1 action proved relatively specific, in that tumor necrosis factor-alpha (10 ng/ml), a putative cytotoxic principle, as well as IL-1-inducible ILs (IL-2 and -6; 100 U/ml) were without effect. Although minimally effective at the level of the isolated granulosa or theca-interstitial cell, IL-1 proved highly potent in heterologous (but not homologous), contact-dependent and independent cocultures of these somatic cell types, strongly suggesting obligatory cell-cell cooperation. These observations further indicate that IL-1 action is indirect and may require the induction of an intermediary soluble principle to serve as the final effector. Taken together, these findings indicate that relatively low concentrations of IL-1 (beta >> alpha), possible of somatic ovarian cell or resident ovarian macrophage origin, are capable of exerting specific dose- and time-dependent (immunoneutralizable) morphogenic as well as cytotoxic effects at the level of ovarian cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Enhancement of growth hormone bioactivity by zinc in the eluted stain assay system.

The effects of ionic zinc (Zn2+) on human (h) GH bioactivity have been examined using a lactogenic bioassay. The potencies of pituitary-derived hGH (IRP 80/505), recombinant 22K hGH (IRP 88/624), pituitary-derived human PRL (IRP 84/500), and a recombinant methionyl 20-kilodalton variant of hGH in the presence of selected concentrations of ZnCl2 were investigated with an eluted stain assay that uses Nb2 rat lymphoma cells. This precise colorimetric bioassay is based upon the reduction of a yellow tetrazolium salt, 3-[4,5-dimethyl-thiazol-2-yl]2,5-di-phenyl-tetrazolium bromide, to its purple formazan by lactogen-activated Nb2 cells. Zinc (6-100 microM) enhanced the bioactivity of low doses (< 0.045 nM) of both pituitary-derived and recombinant 22K hGH, although the magnitude of enhancement was considerably less than might have been anticipated from previous binding studies (13). Higher concentrations of pituitary-derived hGH (> 0.045 nM) were inhibited by Zn2+. The bioactivity of recombinant methionyl 20K hGH was greatly enhanced by zinc (3-100 microM). In contrast to hGH, the bioactivity of hPRL was not potentiated by Zn2+. These discriminatory effects of Zn2+ when stimulating via the lactogenic receptor are in concordance with the results of previous radioligand binding studies (13). The striking enhancement of 20K hGH lactogenic bioactivity was observed at Zn2+ concentrations within the physiological range for normal human serum (5-20 microM).

Animals↗

PubMed1998

Growth factor effects on apoptosis of rat gastric enterochromaffin-like cells.

Enterochromaffin-like (ECL) cells are histamine-containing endocrine cells in the gastric epithelium that show increased density during chronic atrophic gastritis. The current study determined cell number and apoptosis of isolated rat ECL cells in response to several growth factors. Isolated ECL cells from fundic mucosa (enrichment >90%) were grown in serum-free medium over 2-5 days. Cell number was determined by mitochondrial formazan production; apoptosis was measured by Tdt-mediated dUTP nick end labeling reaction and DNA fragmentation-based enzyme-linked immunosorbent assay. Immunocytochemistry and RT-PCR demonstrated the presence of epidermal growth factor receptor, neuronal growth factor receptor (type 1), and fibroblast growth factor (FGF) receptor (type 1). Gastrin (EC50, approximately 2 pM), transforming growth factor-alpha (TGF alpha; 10-30 ng/ml), and basic FGF (bFGF; 1-10 ng/ml) increased the total number of cultured ECL cells. bFGF augmented the gastrin (1 pM)-induced response. Beta-neuronal growth factor (10 ng/ml) and bFGF (2 ng/ml) decreased the programed death of ECL cells. Interleukin-1beta (100 pg/ml, 24 h) stimulated apoptosis 2- to 3-fold in ECL cells, and simultaneous incubation with TGF alpha (20 ng/ml) or bFGF (2 ng/ml) significantly inhibited this effect. ECL cells express specific receptors for gastrin, epidermal growth factor, neuronal growth factor, and FGF. bFGF prolonged ECL cell survival by inhibiting spontaneous apoptosis. Our data further indicate that TGF alpha and bFGF increase ECL cell number by inhibiting cytokine-induced programed cell death.

Animals↗

PubMed2004

Cytotoxic activity of camptothecin and paclitaxel in newly established continuous human medullary thyroid carcinoma cell lines.

At the time of diagnosis, more than one quarter of patients with medullary thyroid carcinoma (MTC) has distant metastases. Only few of these patients can be cured by surgery. Standard chemotherapy is characterized by low response rates and short response time. The establishment of eight human MTC cell lines provides a new basis for in vitro investigation of cytotoxic drugs. Camptothecin (CPT) and paclitaxel, which never have been investigated in the treatment of MTC, were tested for their cytotoxic profile in comparison with the clinically ineffective dacarbazine. Eight MTC cell lines were established from seven patients with MTC. IC(50) values were calculated from dose-response relationships using cell counts and a formazan dye assay (WST-1). IC(50) values were 3.5 +/- 1.2 nmol/liter for CPT and 8.2 +/- 1.9 nmol/liter for paclitaxel. Dacarbazine showed no reduction of cell proliferation at concentrations 10-fold higher than clinically achievable. Given peak plasma concentrations of 65 +/- 20 nmol/liter for CPT and 1 micro mol/liter for paclitaxel, these promising in vitro results provide a basis for the performance of clinical trials in patients with advanced MTC.

Antineoplastic Agents, Alkylating↗