Expression of anaphylatoxin receptors on platelets in patients with coronary heart disease.

OBJECTIVE: Inhibition of components of the complement system or of its receptors has been postulated as a concept for primary and secondary prevention in atherosclerosis and was applied in clinical trials. Although the anaphylatoxin-receptors C3aR and C5aR are commonly associated with inflammatory cells, in vitro studies suggested their expression also on platelets. METHODS AND RESULTS: Expression levels of C3aR and C5aR were measured by flow cytometry in a collective of 302 patients with documented coronary artery disease (CAD) including patients with stable CAD (n = 152), unstable angina (n = 54), acute myocardial infarction (AMI; Non-ST elevation myocardial infarction, n = 70, ST elevation MI, n = 26) or healthy controls (n = 21). Patients with stable CAD, unstable angina or AMI had significantly higher expression of C5aR on platelets in comparison to healthy controls (MFI 14.68 (5.2), 14.56 (5.18) and 13.34 (4.52) versus 10.68 (3.1)); p < 0.001). In contrast, the expression of C3aR on platelets was significantly enhanced in patients with stable and unstable CAD but not in patients with AMI compared to controls. While there was a strong correlation between the soluble ligands of these receptors C3a and C5a, we observed only a weak correlation with their receptors on platelets. Similarly, agonist induced aggregation (MEA, ADP, and TRAP) showed only a weak correlation with the expression level of anaphylatoxin - receptors on platelets. Of note, the expression of both anaphylatoxin-receptors on platelets strongly correlated with platelet activation as assessed with the surface activation marker P-selectin (r = 0.47, p > 0.001 for C3aR, r = 0.76 for C5aR, p < 0.001). Likewise, we observed a positive correlation of C3aR with other molecules associated with platelet activation such as SDF-1. CONCLUSION: In summary, we observed a positive correlation between the expression of anaphylatoxin-receptors C3aR and C5aR with platelet activation in patients with CAD. Further investigations are needed to study the clinical and mechanistic relevance of these findings.

Interruption of classic CD40L-CD40 signalling but not of the novel CD40L-Mac-1 interaction limits arterial neointima formation in mice.

The co-stimulatory immune molecule CD40L figures prominently in a variety of inflammatory conditions including arterial disease. Recently, we made the surprising finding that CD40L mediates atherogenesis independently of its classic receptor CD40 via a novel interaction with the leukocyte integrin Mac-1. Here, we hypothesised that selective blockade of the CD40L-Mac-1 interaction may also retard restenosis. We induced neointima formation in C57/BL6 mice by ligation of the left carotid artery. Mice were randomised to daily intraperitoneal injections of either cM7, a small peptide selectively inhibiting the CD40L-Mac-1 interaction, scM7, a scrambled control peptide, or saline for 28 days. Interestingly, cM7-treated mice developed neointima of similar size compared with mice receiving the control peptide or saline as assessed by computer-assisted analysis of histological cross sections. These data demonstrate that the CD40L-Mac-1 interaction is not required for the development of restenosis. In contrast, CD40-deficient mice subjected to carotid ligation in parallel, developed significantly reduced neointimal lesions compared with respective wild-type controls (2872 ± 843 µm² vs 35469 ± 11870 µm²). Flow cytometry in CD40-deficient mice revealed reduced formation of platelet-granulocyte and platelet-inflammatory monocyte- aggregates. In vitro, supernatants of CD40-deficient platelet-leukocyte aggregates attenuated proliferation and increased apoptosis of smooth muscle cells. Unlike in the setting of atherosclerosis, CD40L mediates neointima formation via its classic receptor CD40 rather than via its recently described novel interaction with Mac-1. Therefore, selective targeting of CD40L-Mac-1 binding does not appear to be a favorable strategy to fight restenosis.

Blinks and saccades as indicators of fatigue in sleepiness warnings: looking tired?

The present study examines changes in a variety of oculomotoric variables as a function of increasing sleepiness in 129 participants, who have been passed through a broad range of subjective alertness. Up to now, spontaneous eye blinks are the most promising biosignal for in-car sleepiness warnings. Reviewing the current literature on eye movements and fatigue, experimental data are provided including additional indicative oculomotoric parameters; inter-individual differences in the experiments were also assessed. Here, self-rated alertness decreased over six steps on average and proved itself a reliable measurement. Regarding oculomotoric parameters, blink duration, delay of lid reopening, blink interval and standardised lid closure speed were identified as the best indicators of subjective as well as objective sleepiness. Saccadic parameters and fixation durations also showed specific changes with increasing sleepiness. Substantial inter-individual differences in all of these variables were illustrated. Oculomotoric parameters were linked to three different components of sleepiness while driving: a) deactivation; b) decreasing attention, resulting in disinhibition of spontaneous blinks and reflexive saccades; c) increasing attempts of self-activation. Finally, implications for the development of drowsiness detection devices were discussed.

Growth and function of isolated canine pancreatic ductal cells.

These studies investigated the growth characteristics and functional properties of isolated canine pancreatic ductal epithelial cells. Cells were isolated from the accessory pancreatic duct and cultured by using three conditions: on vitrogen-coated petri dishes with fibroblast conditioned medium (nonpolarized); in vitrogen-coated Transwells above a fibroblast feeder layer (polarized); or as organotypic rafts above a fibroblast-embedded collagen layer (polarized). Growth characteristics, transepithelial resistances, and carbonic anhydrase and cyclic adenosine monophosphate (AMP) responses were evaluated. Under polarized conditions, the cells grew as monolayers with columnar epithelial characteristics. The monolayers developed high transepithelial resistance and became impervious to the passage of horseradish peroxidase. Epithelial growth factor (EGF) (2 ng/ml) stimulated ductal cell growth and accelerated the formation of a high-resistance monolayer. Forskolin (10 microM) rapidly decreased transepithelial resistance. Carbonic anhydrase activity, which was lower in nonpolarized compared with polarized conditions, was stimulated by carbachol (175 microM). Secretin, however, did not stimulate carbonic anhydrase activity in these cells. Although secretin stimulated adenylyl cyclase activity in early-passage cells, this response was lost in later-passage cells. Both vasoactive intestinal polypeptide (VIP; 1 microM) and forskolin (10 microM) consistently increased adenylyl cyclase activity. Isolated canine pancreatic ductal epithelial cells proliferate in vitro, develop high-resistance epithelial monolayers, and respond to stimuli that activate adenylyl cyclase. These cells should provide a useful model for regulatory studies of ductal cell functions.

The inhibitory effect of genistein on the growth and metastasis of a transplantable rat accessory sex gland carcinoma.

BACKGROUND: A cell line (K1) derived from a carcinogen-induced accessory sex gland carcinoma was used to examine the effects of the soybean extract, genistein, on tumor growth and metastasis. METHODS: Male Lobund-Wistar rats were injected s.c. with 20 million K1 cells; genistein (50 mg/kg BW) or the vehicle was administered s.c. every 12 h for 31 days. RESULTS: Genistein significantly inhibited tumor growth. Compared with controls, fewer genistein-treated rats developed invasive tumors (11% vs. 44%) or lymph node metastases (44% vs. 89%). No lung metastases were found in genistein-treated animals in contrast to controls (0% vs. 44%). Estrogenic side effects were precipitated in genistein-treated rats, including decreased accessory sex gland complex weight, increased pituitary weight, decreased testis weight, and decreased (BW). Serum testosterone was undetectable and serum prostate-specific acid phosphatase activity was 38% lower in genistein-treated rats compared with controls. Genistein concentrations in the solid tumors (2 nmol/g) were one-third those in blood. CONCLUSIONS: These data suggest that genistein may be a useful chemotherapeutic agent to inhibit the growth and metastasis of accessory sex gland cancers, such as those derived from the prostate.

Current activities at the Centers for Disease Control and Prevention's National Diabetes Laboratory.

In 1997, the Centers for Disease Control and Prevention established the National Diabetes Laboratory in order to help prevent and treat type 1 diabetes. This state-of-the-art laboratory collaborates with research scientists and key national and international organizations throughout the world to identify and study risk factors for type 1 diabetes by developing measurements for glycosylated proteins, developing and evaluating technology for measuring genetic risk factors for the disease, and working to standardize autoantibody measurements. Developing improved technologies for diagnosing and managing diabetes and developing reference materials for properly calibrating and standardizing blood glucose meters are also critical aspects of the laboratory's work. In addition, the laboratory provides quality storage for valuable collections of biologics and other materials and facilitates sharing of specimens, associated epidemiologic data, and test results. Working with our partners in diabetes research, we are improving the diagnosis, treatment, and prevention of type 1 diabetes.

Neurofilament heavy chain-like messenger RNA and protein are present in benign prostate and down-regulated in prostatic carcinoma.

Differences in gene expression between benign and malignant human prostate specimens were investigated using the differential display technique. RNA samples from paired benign and malignant areas microdissected from opposite sides of the same prostate gland were used for reverse transcription PCR. A 477-bp band was identified that was consistently present in benign prostate but absent or diminished in intensity in malignant tissue. This band was cloned, and the sequence demonstrated 99% identity with a region in the fourth exon of the human neurofilament heavy chain gene (NF-H). Northern blotting with a cDNA probe derived from this band confirmed the presence of a similarly sized message of approximately 3.9 kb in both prostate and brain, and reverse transcription PCR using primers specific to an upstream region of exon 4 confirmed NF-H-like mRNA expression in benign prostatic tissue. Immunostaining with a monoclonal antibody to NF-H showed a positive reaction in benign prostatic epithelial cells but complete absence of staining in prostatic cancer cells. These data demonstrate the presence of a NF-H-like gene product in normal prostatic epithelial cells that is down-regulated or absent in prostatic carcinomas.

Intravenous vs. intraprostatic administration of N-methyl-N-nitrosourea to induce prostate cancer in rats.

To develop an improved model of human prostate cancer, 16-wk-old Wistar rats were treated orally for 18 days with the antiandrogen, flutamide (50 mg/kg body weight [BW]/day), followed by 3 days of s.c. testosterone (100 mg/kg BW). There were the only treatments the control animals received (Group 1, n = 10). On the day after the third testosterone injection, N-methyl-N-nitorsourea (MNU) was administered via the tail vein at a dose of 50 mg/kg BW (Groups 2, n = 40 and 3, n = 20); in some rats, a second dose was delivered by the same route 22 wk later (Group 3). A smaller dose of MNU (15 mg/kg BW) was administered intraprostatically (Group 4, n = 20) to a fourth group. In Groups 2, 3, and 4, silastic capsules containing testosterone were implanted s.c. approximately every 6 wk beginning 1 wk post-MNU. Accessory sex gland tumors arose in MNU-treated rats in Group 2 (12/40, 30%). Group 3 (8/20, 40%), and Group 4 (8/20, 40%); 90% were macroscopic (25/28). There were no neoplasms in these organs in the control rats (Group 1, 0/10). These accessory sex gland neoplasms were adenocarcinomas or undifferentiated carcinomas which appeared to be derived from the prostate based on location and histological characteristics, although the size and spread of some of the tumors precluded definitive localization of the tissue of origin. The incidence of these neoplasms was similar in rats given a single dose of MNU intraprostatically or two doses of MNU i.v., but the animals treated intraprostatically maintained higher body weights and developed fewer extraneous tumors. The average (+/- SD) latent period for clinical or postmortem detection of prostate neoplasia after MNU was shortest in the rats given two i.v. doses (39 +/- 3 wk) compared with the single i.v. dose (45 +/- 6 wk) or an intraprostatic dose (56 +/- 7 wk). In 57% of the cases (16/28), the prostate tumors metastasized to distant sites. An activating point mutation was detected in codon 12 of the Ki-ras oncogene in the MNU-induced primary prostate tumors (8/10 examined), and metastases arising from these prostate tumors (2/3) but was absent in normal prostate tissue (0/6). This study demonstrates that two systemic doses of MNU increase the incidence and decrease the latency of prostate neoplasms compared with a single dose, and that a single dose of MNU injected intraprostatically induces prostate adenocarcinoma without many of the other tumors and weight loss typically found after i.v. administration.

Autoradiographic localization of beta-endorphin binding in the pancreas.

Autoradiographic localization of 125I-labeled beta-endorphin binding in the rabbit pancreas demonstrated specific binding in the pancreatic islet cells. Binding was inhibited by (1) nonradioactive beta-endorphin, (2) the opioid antagonist naloxone, (3) the mu receptor agonists morphine and [D-Ala2, (Me)Phe4, Gly(ol)5]enkephalin, (4) the delta receptor agonist [D-penicillamine2, D-penicillamine5]-enkephalin, (5) the mu and delta agonist met-enkephalin and (6) the delta and kappa agonist dynorphin. Specific binding was not clearly demonstrable in the acinar portion of the rabbit pancreas. The binding characteristics of 125I-beta-endorphin in the pancreatic islets were comparable with those of mu and delta opioid receptors in the rabbit brain. In the pancreas, beta-endorphin binding appeared to be concentrated in discrete areas in the islets. Combined immunohistochemistry and autoradiography demonstrated that beta-endorphin binding was primarily concentrated in the glucagon-containing alpha and somatostatin-containing delta cells, but was also found in the insulin-containing beta cells to a lesser extent. Given the intraislet location of the opioid binding sites, and our previous finding of immunoreactive beta-endorphin in the pancreatic beta cells and the inhibitory effect of beta-endorphin on insulin secretion, it appears that beta-endorphin may serve a paracrine or autocrine function in the regulation of pancreatic hormone secretion.

Effects of agents that inhibit cellular iron incorporation on bladder cancer cell proliferation.

Agents that interfere with cellular iron (Fe) incorporation inhibit tumor cell proliferation, including metals that bind to transferrin (Tf) such as gallium (Ga) or indium (In) and Fe chelators such as desferrioxamine (DFO). Ga nitrate is effective in the treatment of metastatic bladder cancer and these patients exhibit evidence for interference with Fe metabolism. We show here that bladder cancer cell proliferation in vitro is dependent on Tf-Fe. Concentrations of DFO that can be readily achieved in vivo inhibit cellular proliferation even in the presence of physiologic concentrations of Tf-Fe. Inhibition of proliferation by Tf-Ga is associated with decreased cellular Fe incorporation. However, when a physiologic concentration of Tf-Fe is added to an equimolar concentration of Tf-Ga, significant Fe incorporation is evident despite inhibition of proliferation. Thus, besides interference with Fe incorporation, Ga may also interfere with intracellular Fe distribution and/or directly inhibit an Fe- (or non-Fe-) requiring process necessary for cellular proliferation. DFO followed sequentially by Tf-Ga results in marked potentiation of inhibition of proliferation. The effects of this combination appear to be related to both interference with Fe metabolism and increased Ga uptake. This sequential combination may be useful in the treatment of bladder cancer.

Neuroblastoma sensitivity to growth inhibition by deferrioxamine: evidence for a block in G1 phase of the cell cycle.

Iron (Fe) is known to be necessary for cellular proliferation. Previous studies have suggested that neuroblastoma cells appear to be relatively sensitive to growth inhibition by a specific Fe chelator, deferrioxamine (DFO), in vitro. Also, DFO has been recently used for the treatment of neuroblastoma patients. In this paper we demonstrate that neuroblastoma cell proliferation in vitro is extremely sensitive to inhibition by DFO as compared to another cell line with almost identical growth kinetics. Neuroblastoma cells treated with DFO adapt appropriately to Fe chelation as measured by marked upregulation of transferrin receptor mRNA, increased functional transferrin receptor, and decreased cellular ferritin concentration. Further studies that quantitated cellular incorporation of 59Fe from added transferrin-59Fe in the presence of DFO indicated that neuroblastoma cells were more sensitive to inhibition of Fe incorporation by the chelator as compared to the other cell line. Neuroblastoma cells treated with DFO showed a consistent arrest in the G1 phase of the cell cycle. For cells taken from the "resting" state this block occurred before the vast majority of cells had entered S or G2-M phases of the cell cycle. Further evidence that neuroblastoma cells were arrested before the G1-S interface was provided when cells inhibited by DFO and released into aphidicolin exhibit arrest at the G1-S interface, whereas release from aphidicolin into DFO resulted in entry into S phase. Also, DFO-treated cells exhibited a decrease in both p34cdc2 immunoreactive protein as well as kinase activity. The results of these latter studies strongly indicate evidence for a Fe requirement for malignant cell proliferation before the onset of DNA synthesis. Our results also provide a basis for further studies that will better define a therapeutic approach to patients with neuroblastoma utilizing DFO treatment.

Immunocytochemical localization and endogenous synthesis of apolipoprotein E in testicular Leydig cells.

Apolipoprotein E (apoE) is synthesized by a wide variety of tissues, including those involved in steroidogenesis; its function in most extrahepatic tissues is unknown. Significant amounts of apoE mRNA have been detected in the testis, but the cellular origin of this material has not yet been determined. The purpose of the present study was to determine whether the steroidogenic cells of the testis synthesize apoE. We localized apoE in the testis of mice by an avidin-biotin peroxidase technique using a cross-reactive anti-rat apoE antibody. ApoE immunoreactivity was strongest in interstitial cells but was also diffusely localized throughout the seminiferous tubules. Acute treatment of mice with hCG diminished apoE immunoreactivity in the testis. A murine Leydig tumor cell line (I-10 cells) also demonstrated apoE immunoreactivity, suggesting that at least one source of interstitial apoE is the Leydig cell. Normal Leydig cells were subsequently isolated from control and hCG-treated mice using Percoll density gradients. Isolated hepatocytes, I-10 cells, and Leydig cells (with or without hCG in vitro) were incubated in the presence of [35S]methionine. A 35S-labeled protein of approximately 33-35 kDa was immunoprecipitated from the cells and media of all three types of preparations using whole antiserum or affinity-purified antibody. Preincubating the antibodies with apoE-containing murine very low-density lipoprotein or purified rat apoE eliminated these bands. Leydig cell apoE synthesis and secretion were decreased by hCG treatment in vivo and/or in vitro. These data suggest that apoE is synthesized by normal and transformed Leydig cells and may play a role in sterol transport in the testis.

Treatment with gallium nitrate: evidence for interference with iron metabolism in vivo.

Gallium, when bound to transferrin, has been previously shown to cause tumor cell cytotoxicity by preventing cellular uptake of transferrin bound iron in vitro. Patients treated with constant infusion gallium nitrate for carcinoma show a rise in serum iron within 6 hr of the start of treatment. Serum iron returns to baseline by 24 hr post-infusion. Atomic analysis of iron and gallium content of Sephadex G-150 fractions of treatment sera indicate that about an equimolar amount of gallium and iron are associated with transferrin. These gallium and iron concentrations result in inhibition of transferrin mediated iron uptake in vitro, and in vivo allow for > 90% saturation of transferrin with metal. All seven patients who completed two courses of gallium therapy exhibited hypochromic microcytic anemia (mean fall in hemoglobin 3.5 grams %). Evidence for red cell iron depletion was confirmed by an increase (mean 3.3-fold) in zinc protoporphyrin levels. Since transferrin receptor increases on gallium treated iron requiring cells in vitro, we assessed cell surface transferrin receptor on peripheral blood lymphocytes by measuring fluorescent transferrin receptor antibody binding. A population of highly transferrin receptor positive cells peaks at 48 hr into the infusion. DNA analysis as well as double staining indicate the majority of transferrin receptor positive cells are unstimulated B lymphocytes. These studies provide the first documentation that constant infusion gallium treatment results in significant interference with iron metabolism and evidence for tissue iron depletion in vivo. These changes may correlate with therapeutic effects of gallium such as tumor response.

Localization of beta-endorphin in rabbit pancreatic islets.

To determine whether beta-endorphin might play a physiological role in regulating insulin secretion, we examined the rabbit pancreas for an endogenous source of beta-endorphin. Specific antibodies to beta-endorphin were used together with anti-insulin, anti-glucagon, and anti-somatostatin in single-labeled and double-labeled indirect immunofluorescence and in sequential avidin-biotin-peroxidase immunohistochemistry. Light microscopic examination of rabbit islets revealed beta-endorphin immunoreactivity primarily in the beta cells. Approximately 97% of the cells exhibiting beta-endorphin immunoreactivity contained immunoreactive insulin; whereas many, but not all, of the insulin-containing cells contained immunoreactive beta-endorphin. Strongly beta-endorphin-positive cells were usually weakly insulin-positive; and strongly insulin-positive cells were often beta-endorphin-negative. The beta-endorphin-positive cells that were insulin-negative were identified as immunoreactive glucagon- or somatostatin-containing cells. The intensity of the immunoreactive beta-endorphin appeared to be more heterogeneous than the staining intensities of other hormones. Together with the previously reported data on the effects of beta-endorphin on pancreatic hormone secretion, the results of this investigation suggest that beta-endorphin may play an important autocrine or paracrine role in the pancreatic islets.

Transferrin-independent iron uptake supports B lymphocyte growth.

Raji, a malignant B-lymphocyte cell line containing Epstein-Barr virus genomic elements, has been conditioned to proliferate optimally in transferrin (Tf)-free medium containing a very low concentration of an iron salt. We provide evidence that an Tf-independent iron uptake system is physiologically important for maintaining the growth of these cells. The data show that Raji cells take up iron from iron salts using a relatively high-capacity, low-affinity, temperature- and calcium-dependent uptake system. The apparent capacity of this system increases when: (1) cells are cultured in Tf-free medium containing high concentrations of iron salt as opposed to medium containing Tf; and (2) when the iron salt concentration of Tf-free medium is lowered to about 1.6 mumol/L. Cellular iron uptake also increases when a maximum number of cells are in S and G2 and M cell phases of the cell cycle. The cells are sensitive to growth inhibition by the addition of deferoxamine. This evidence supports the hypothesis that certain malignant lymphocytes, under iron deplete conditions, fulfill an iron requirement for proliferation by an adaptation such as Tf-independent iron uptake.

Sources of cholesterol for testosterone biosynthesis in murine Leydig cells.

The sources of cholesterol for testosterone production were investigated in freshly isolated murine Leydig cells. In vitro stimulation with human CG (hCG) (0.2 IU/ml) caused a 75-fold increase in testosterone production. Leydig cells contained approximately equal amounts of free and esterified cholesterol (7.8 vs. 8.7 micrograms/mg protein). The total cholesterol content of cells stimulated for 4 h with hCG was significantly decreased compared with unstimulated cells (8.4 vs. 17.6 micrograms/mg protein); both free and esterified cholesterol decreased by about 50%. In unstimulated Leydig cells incubated with [14C]acetate for 12 h, the majority of incorporated [14C] was found in free and esterified cholesterol, whereas, in the hCG-stimulated cells, 80% of incorporated 14C was in testosterone. The activity of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase after 4 h in hCG-stimulated cells was 20% higher than in unstimulated cells (115.5 vs. 84.4 pmol/mg protein.min). However, by 6 h, HMG-CoA reductase activity doubled in the hCG-stimulated compared with unstimulated cells. By 12 h, HMG-CoA reductase activity in hCG-stimulated cells was 4 times the preincubation level and 8 times the 12-h level in unstimulated cells. HMG-CoA reductase activity induced by hCG was blocked by aminoglutethimide, an inhibitor of the cholesterol side-chain cleavage enzyme. Lovastatin, a potent inhibitor of HMG-CoA reductase, had no effect on unstimulated or hCG-stimulated testosterone production during a 12-h incubation. Murine high density lipoproteins (mHDL) increased HMG-CoA reductase activity in both unstimulated (29%) and hCG-stimulated (20%) cells. During a 6 h incubation, mHDL increased hCG-stimulated testosterone production by 20%, but had no effect on unstimulated testosterone production. These results suggest that murine Leydig cells store enough cholesterol and cholesteryl esters to support testosterone production for at least 12 h in vitro. Although mHDL does not have a major stimulatory effect on testosterone biosynthesis, it may be involved in the regulation of de novo cholesterol synthesis.

Beta-endorphin inhibits insulin secretion from isolated pancreatic islets.

Intravenous administration of small doses of beta-endorphin causes immediate suppression of basal and glucose-stimulated insulin secretion in normal rabbits. The purpose of the present study was to determine if beta-endorphin directly inhibits glucose-stimulated insulin secretion from rabbit pancreatic islets. Islets were isolated from male New Zealand White rabbits and perifused for 1 h with medium containing 100 mg/dl glucose (M100) followed by a 1-h challenge with medium containing 300 mg/dl glucose (M300) with or without beta-endorphin and/or the specific opioid antagonist naloxone. Samples were collected every 5 min during the last 30 min of the baseline perifusion with M100 and during the 1-h challenge with the stimulatory concentration of glucose (M300). Total insulin secretion for each 1-h period was calculated by adding the areas under the curves for twice the 30-min baseline period and for the 1-h challenge period. The mean +/- SE area for the control islets during perifusion with M100 was 5.9 +/- 0.8 microU/islet.h. M300 stimulated a 4.2-fold increase in the amount of insulin secreted (24.5 +/- 3.6 microU/islet.h). The stimulated rate of insulin release was sustained throughout the 1-h test period with M300, averaging 0.42 +/- 0.02 microU insulin/islet.min. beta-Endorphin inhibited glucose-stimulated insulin secretion in a concentration-dependent manner. Maximal suppression of insulin secretion to a level well below the baseline secretion rate was produced by 300 nM beta-endorphin (1.9 +/- 0.3 microU/islet.h). The first 15 min of glucose-stimulated insulin secretion was 6 times less sensitive to the inhibitory effect of beta-endorphin than was the next 45 min. The concentrations of beta-endorphin causing 50% inhibition of glucose-stimulated insulin secretion (IC50) for the 5- to 15-, 20- to 60-, and 5- to 60-min intervals were 1.96, 0.35, and 0.57 nM, respectively. Naloxone (3 microM) had no effect on glucose-stimulated insulin secretion, but partially antagonized the inhibitory effect of 30 nM beta-endorphin (10.2 +/- 2.9 microU/islet.h naloxone plus beta-endorphin vs. 2.6 +/- 1.1 microU/islet.h beta-endorphin; P less than 0.05). These data demonstrate that beta-endorphin, at low concentrations, has a direct inhibitory effect on insulin secretion, and they support the idea that a naloxone-sensitive beta-endorphin-binding component is present in pancreatic islets.

Polypeptide composition and an immunological analysis of DNA methyltransferases from different species.

The cross-reactivity of the monoclonal anti-human placental DNA methyltransferase antibody M2B10 with DNA methyltransferases isolated from other species was investigated. This antibody immunoprecipitates DNA methyltransferases from mammalian cells, i.e., human placenta, mouse P815 cells, and rat liver cells. No cross-reactivity is observed with DNA methyltransferases from wheat germ and with bacterial DNA methyltransferases HpaII and EcoRI. The mammalian enzymes are characterized by polypeptides of molecular mass 150-190 kDa. Polypeptides smaller than 190 kDa are presumably generated by proteolysis of the native 190-kDa DNA methyltransferase. Trypsin digestion of the 190-kDa polypeptide isolated from mouse cells results in progressive appearance of DNA methyltransferase polypeptides of 150-190, 110, 100, and 52-60 kDa.

Effects of estradiol on estrogen receptor, progesterone receptor, and tyrosinase in hamster melanoma transplanted into athymic mice.

Nuclear estrogen binding was characterized in HM-1, a malignant hamster melanoma cell line transplanted into male and female athymic mice following acute, subchronic, and chronic injection of estradiol. Nuclear binding was saturable, of high affinity (10(10) M-1) and readily soluble in low salt buffer. Saturation analyses revealed that [3H]estradiol in excess of 5.0 nM apparently bound to a second class of lower affinity (10(9) M-1), higher capacity cytosol sites. Enzyme-linked immunoassay with a specific monoclonal antibody (H222 Sp gamma) directed against the human estrogen receptor protein was in excellent agreement (r = 0.93) with values obtained using hydroxyapatite to separate bound from free ligand. Nuclear estrogen receptor content in HM-1 cells was increased maximally 1 h after acute s.c. injection of a low dose (0.1 microgram) of estradiol. The increase in nuclear receptor content was accompanied by an apparent rapid reduction in cytosol binding. Subchronic (3 days) and chronic exposure (35 days) to estradiol also produced a significant, dose-related increase in tumor nuclear estrogen receptor content. Cytosol binding for progestin was low (less than or equal to 2 fmol) to absent in HM-1 xenografts not exposed to estradiol. Subchronic and chronic exposure to estradiol induced a dose-related, specific, high affinity (10(9) M-1) cytosol binding protein for progestin(s) in HM-1 xenografts carried in male and female athymic mice. In contrast, progestin binding to nuclear receptor was not increased in estrogen-primed animals, nor did acute injection of progesterone (100 micrograms s.c.) increase the amount of saturable, high affinity (10(9) M-1) nuclear progestin receptor in control or estradiol-primed athymic mice. In contrast to the induction of progestin binding, tyrosinase activity was not altered by a similar exposure to estradiol when assayed at a saturating concentration of tyrosine. These observations suggest that the estrogen receptor in HM-1 cells may be functional but that pigmentary changes observed in mammals following chronic exposure to estradiol may not be mediated by a direct effect on the rate limiting enzyme of melanin synthesis.